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MECHANISTIC AND CLINICAL ASPECTS OF PROSTATE SPECIFIC ANTIGEN EXPRESSION IN
NON-PROSTATIC TISSUES
Nosratollah Zarghami
A Thesis subrnitted in conformity with the requirements for the Degree of Doctor of Philosophy
Graduate Department of Clinical Biochemistry University of Toronto
OCopyright by Nosratollah Zarghami 1997
National Libraiy u*m of Canada Bibliothèque nationale du Canada
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MECHANISTIC AND CLINICAL ASPECTS OF PROSTATE SPECIFIC ANTIGEN EXPRESSION IN NON-PROSTATIC
TISSUES
Nosratollah Zarghami Ph.D. 1997
Department of Clinical Biochemistry University of Toronto
Abstract
Prostate specific antigen (PSA) is a 30 KDa serine protease which shares significant protein
and gene sequence homology with pancreatic-rend kallikrein and glandular kallikrein. Prostate
specific antigen, as its name denotes, was initially discovered in seminal plasma and extracts from
the prostate gland. Since the breast is a steroid hormone regulated organ and the knowledge that
PSA is regulated by androgens in the prostate, 1 hypothesize that the expression of PSA in breast
tissue is under the regulation of the steroid hormone receptor system. In this thesis, I have devised
appropriate experiments and tissue culture systems to study the reguiation of PSA in breast tissues
and ce11 lines.
The results from our studies on the PSA regulation in non-prostatic tissues yielded the
following :
1. We have developed reverse transcription-polymerase chah reaction (RT-PCR) methods for
detecting prostate-specific antigen (PSA) mRNA expression in tumors. Our data
. . 11
demonstrate that there is good agreement between presence of PSA protein and PSA mRNA
in breast tumors and that these tests give complementary information.
2. Based on clinical observations, we have developed a tissue culture system to study the
regulation of PSA gene in breast cancer. We have show that the breast cancer ce11 lines T-
47D and BT-474 produce PSA when stimulated by androgens, progestins and
glucocorticoids/ mineralocorticoids but not estrogens.
3 . Cyclical changes of PSA concentration during the menstmal cycle of normal women were
monitored and we found that PSA levels in serum are highest dunng the mid-late follicular
phase, drop continuously with a half-life of 5-6 days between the late follicular phase and
mid-cycle and reach a minimum during the mid luteal phase.
4. Our studies indicate that the PSA gene is expressed in prirnary lung tumors. The identity
of the PSA gene expression was venfied using PSA immunoassay, imrnunohistochernistry,
RT-PCR, Southem blot hybridization with a specific PSA RNA probe and DNA
sequencing. By cloning and sequencing of aberrant PCR products, we were able to isolate
a 450 bp unique sequence not previously deposited in GenBank.
Acknowledgements
1 would like to express rny gratitude and appreciation to Drs. Eleftherios Diamandis,
Reinhold Vieth, and Cliff Lingwood for their guidance, advice, encouragement, and support, and
Dr. Elefiherios Diamandis for his supervision of this research project and detailed review of the
dissertation. I would also like to th& Drs. Donald Sutherland, Dionyssios Katsaros, Mario
D'Costa, Edward Sauter, Linda Giudice, and The Canadian Red Cross Blood Transfùsion Service
for providing human specimens, usehl information, helpfùl discussion, advice, technical support
and help. Finally, I would like to specially thank Linda Grass, Dr. He Yu, Michael Levesque,
Katerina Angelopoulou and Lisene Santos for their generous help and contributions to this research
project.
Table of Contents
CHAPTER PAGE
CHAPTER 1. Introduction
CELAPTER 2. Literature Review 2- 1. A histoncal Perspective of PSA 2-2. Structural and Molecular Features of PSA 2-3. The PSA Gene 2-4. Regulation of PSA Production
2-4- 1. Positive Control Mechanisms 2-4-2. Negative Control Mechanisms
2-5. Effects of Growth Factors on PSA Gene 2-6. Biological Function of PSA 2-7. Moiecular Foms of PSA 2-8. PSA in Non-Prostatic Tissues
2-8- I . Penurethral Glands 2-8-2. Breast
2-8-2- 1 Breast Cancer Tissue 2-8-2-2 Breast Cancer Cells 2-8-2-3 Other Bremi Tissues 2 -8 -24 Breast Fhr ids 2-8-2-5 PSA Regdation and Fzinction in Breast Tissue 2-8-2- 6 Chical Applications
2-8-3. Arnniotic Fluid 2-8-4. Female Semm 2-8-5. Ovary, Endometrium and Other Tissues 2-8-6. Other Cancers
2-9. S teroid Hormone Receptors 2-9- 1. Mechanisms of Gene Regulation 2-9-2. Clinical Application of Estrogen & Progesterone Receptors
in Breast Cancer 2-9-3. Steroid Hormone Receptors and Cancer
CEMPTER 3. Hypothesis
CHAPTER 4. Objectives
CHAPTER 5. Materials and Methods 5- 1. Preparation of PSA cDNA
5- 1- 1. Transformation Procedure 5-2. Positive and Negative Control Ce11 Lines 5-3. Preparation of Tumor Cytosol Extracts
5-4. PSA Measurements 50 5-5. High Performance Liquid Chromatography (HPLC) 5 1 5-6. Total Protein Determination 52 5-7. Isolation of Total RNA 52 5-8. Reverse Transcriptase 53 5-9. Oligonucleotide Prirners 54 5- 10. PCR Protocol 54 5- 1 1. Nested Primer PCR 55 5- 12. Labelling of PSA cDNA Probe 5 5 5- 13. RNA Labelling by In-Vitro Transcription 56 5- 14. Direct Incorporation of DIG-dUTP Dunng PCR 56 5- 1 5. Detection Limit of PCR 57 5- 1 6. Gel Electrophoresis, Southem Blotting and Hybridization 57 5- 1 7. Detection Protocol 58 5- 18. Cloning of PCR Products 59 5 - 1 9. DNA Sequencing 59 5-20. Tissue Culture Systern 59
5-2 O- 1. Compoundî 59 5-20-2. Cell LNies 60 5-20-3. CeII Culture Procedure 65 5-20- 4. Stimulation wzth Steroid Compoirndr 65 5-20-5. Stimzilution with Semm Sampies and Goriadotropin Hormones 66 5-20-6. Blochg Experiments 66 5-20- 7. Kineîic Ewperiments 67
5-2 1 . Lysis Procedure 67 5-22. Measurement of Steroid Hormone Receptors 67 5-23. Measurement of Hormones in Serurn 68 5-24. Irnmunohistochemistiy 68 5-25. Subject Selection 69
3-25 1. Breast Tissue Specimens 69 5-25-2. Serum Specimens Ob tained Dtiring Menstmal Cycle 69 5-25-3. L ung Cancer Patients 70
5-25-3- 1. Case Study 70 5-25-3-2. Lzrng Ttimor Tissues 71
5-26. Ethical issues 7 1 5-27. Statistical Analysis 73
CHAPTER 6. Results 6- 1. Optimization of PCR Protocol 6-2. Determinhg Limits of Detection Methods
6 - 2 4 Ethidium Bromide Stuining 6-2-2. Nested PCR 6-2-3. Direct DIG-dUTP Incorporation 6 - 2 4 Souîhern BIot Hybridization
6-3. PSA Gene Expression in Breast Tumors 6-4. Steroid Hormone Regulation of PSA Gene Expression in Breast Cancer
6-4- 1. Kinelics of PSA Ekpresszon 6 - 4 2 Induction of PSA Produciion by Steroid Homones 6-4-3. Detemination of Dose-Response Stimulatory Effecls of
Steroid Hormones 6- 4-4. Blocking Steroid Hormone Receptors
6-5. Variation of PSA Protein During the Menstmal Cycle 6-6. Expression of PSA Gene in Lung Tissue 6-7. Frequency of PSA mRNA Expression in Lung Tumors
CEMPTER 7. Discussion 7- 1. Detection of PSA Protein and rnRNA in Breast Turnors 7-2. Steroid Hormone Regulation of PSA Gene in Breast Cancer 7-3. PS A Production Dunng the Menstrual Cycle 7-4. Expression of PSA Gene in Lung Tissue 7-5. Frequency of PSA mRNA Expression in Lung Tumors 7-6. Conclusions
CHAPTER 8. Concluding Remarks and Future Directions
CEIAPTER 9. References
vii
List of Figures
Page Figure
Figure 1- 1
Figure 1-2
Figure 2-2- 1
Figure 2-9- 1
Figure 6-2- 1 - 1
Figure 6-2-2- 1
Figure 6-2-3 - 1
Figure 6-3 - 1.
Figure 6-4- 1 - 1.
Figure 6-4- 1-2.
Figure 6-4- 1-3.
Figure 6-4- 1-4
Figure 6-4-2- 1.
Figure 6-4-3- 1.
Figure 6-5- 1
Figure 6-5-2
Figure 6-5-3
Mechanism of action of steroid hormones 2
Structural requirements for androgen regulation of gene transcription 3
Homology of the human tissue kallikrein gene farnily 11
Generai stmctural and fùnctional organization of nuclear receptors and concsensus responsive elements 35
RT-PCR of PSA niRNA and P -actin rnRNA 76
Nested primer PCR 78
Direct incorporation of DIG-dUTP in PCR reaction 79
Detection of PSA mRNA by hybridization 83
RT-PCR of PSA rnRNA extracted fiom T47D cells 86
Appearance of PSA rnRNA and PSA protein inside and outside 87
Representative chromatograrz of the PSA cDNA sequence 89
Complete sequence of the 6 16 nucleotide region of the PSA cDN A 90
PSA concentration in T-47D ce11 line tissue culture supernatant after stimulation with various compounds 92
Dose-response expenment of six representative steroids 94
Senim PSA and progesterone dunng menstrual cycle of M.T 99
Semm PSA and progesterone during menstmal cycle of C.J and L.S 1 O0
Semm progesterone levels during the menstrual cycle and its stimulation effect 102
Figure 6-5-4
Figure 6-6- 1
Figure 6-6-2
Figure 6-6-3
Figure 6-6-4
Figure 6-6-5
Figure 6-6-6
Figure 6-7- 1
Figure 6-7-2
Figure 6-7-3
RT-PCR of PSA mRNA and actin obtained fiom T-47D stimulate with semm of voIunteer M.M 1 03
Molecular weight identitication of PSA in the lung tissue extract 105
RT-PCR of RNA extracted fiom various tissues 1 06
Representative chromatograrn of the PSA cDNA sequence of the lung tissue 1 07
Complete sequence of the PSA cDNA sequence isolated fiom lung tissue 108
Imrnunohistochemical localization of PSA in lung tissue 110
Production of PSA by BT-474 cells d e r stimulation with becIomet hasone 112
RT-PCR for PSA mRNA extracted fiom lung tumors 115
RT-PCR for PSA mRNA extracted from lung tumors with aberrant bands 116
Sequence of unique 450 bp PCR product 118
List of Tables
Tabte Page
Table 2-2-1
Table 2-7- 1
TabIe 5-20- 1 - 1
Table 5-20-2- 1
Table 5-35-3-2- 1
Tab te 6-2-4- 1
Table 6-3- 1
Table 6-4- 1
Table 6-4-2- 1
Table 6-4-4- 1
Table 6-6- 1
Human tissue kallikrein gene family
Nomenclature of hK proteins
List of steroid/cornpounds tested for PSA production
Ce11 lines tested for PSA production in tissue culture system
Clinicopathologicai findings in primary lung tumors
Detection of PSA mRNA with various RT-PCR protocols
Detection of PSA protein and PSA mRNA in pnmary breast tumors
Steroid hormone receptor levels in different cell lines
Regulation of PSA gene expression by various compounds in the breast carcinoma ce11 Iine T-47D
Blocking of PSA production by vanous compounds
Cornparison of DNA sequence of PCR product found in lung tissue with sequence deposited in GenBank
Table 6-7- 1 Distribution of PSA protein in lung tumor cytosols 113
List of Abbreviations
AR
ARE
ApoD
ALP
BPH
dNTP
DIG-dUTP
DMSO
ER
EGF
EDTA
FGF
FSH
GR
hGK- 1 (hKLK2)
hKLK 1
hKLK3
IGF-1
IGF-II
IGFBPs
IGFBP-3
androgen receptor
androgen response element
apolipoprotein D
alkaline phosphatase
benign prostatic hyperplasia
deoxynucleoside triphosphate
digoxigenin-labelled deoxyuridine triphosphate
dirnethylsulfoxide
estrogen receptor
epidemal growth factor
ethylenediaminetetraacetic acid
fibroblast growth factor
follicle stimulating hormone
glucocorticoid hormone receptor
human glandular kallikrein 1
tissue kallikrein
human kallikreid (PSA)
insulin-like growth factor-1
insulin-like growth factor-II
insulin-like growth factor binding proteins
insulin-like growth factor binding protein-3
LB
LH
MR
PMSF
PR
PI
PBS
PKC
RA
RAR
RT-PCR
SSC
SDS
TR-FIA
TPA
TGF-a
TGF- P
TR
VDR
Luria-Bertani
Iuteinizing hormone
rnineralocorticoid hormone receptor
phenylrnethylsulphonyl fluoride
progesterone receptor
isoelectric point
p hosp hate-buffered saline
protein kinase C
retinoic acid
retinoic acid receptor
reverse transcription-polymerase chain reaction
saline sodium citrate
sodium dodecyl sulfate
time-resoived imunofluorometric assay
tumor-promoting phorbol ester
transforming growth factor alpha
transforming growth factor beta
thyroid hormone receptor
vitamin D receptor
1
CHAPTER 1. Introduction
Steroid hormone receptors belong to a superfamily of ligand-inducible transcription factors
that regulate hormone-responsive genes. Steroid hormone responsive tissues can be found in the
breast, prostate, endometriun, uterus, ovaries, and other diverse tissues. These tissues have steroid
hormone receptors that are present as inactive proteins, but once they associate with their cognate
hormone, they become activated, bind to specific regions of the DNA, and regulate hormone-
responsive genes. The current mode1 for steroid hormone action (Figure 1-1) starts with the passive
diffùsion of steroids into the cell. The steroid then binds noncovalently and with high affinity to the
steroid hormone receptor in the cytoplasm. Ligand binding causes the receptor to undergo a process
called activation or transformation in which a conformational change in the receptor is thought to
occur. The receptor dissociates frorn the nonsteroid-binding subunits with which it is normally
associated, unmasking the DNA binding domain of the receptor protein. The steroid-receptor
complex then translocates to the nucleus where it binds to specific DNA sequences called steroid
hormone-regdatory elements (HREs) such as androgen-regulatory elements (ARES). M e r binding
to HRE, the steroid-receptor complex alters the transcription rate of specific genes near or in which
the ARE is located. A typical androgen-regulatory responsive gene is shown in Figure 1-2. The
.4RE, shown with the consensus DNA sequence, is located in the 5' regulatory region of the gene
where an adrogen receptor bound to the ARE can interact with transcription factors that bind to
other regulatory elements, such as the TATA and CAAT boxes, which are also present in this
region. In this fashion, androgens increase or decrease the amount of messenger RNA and
ultimately the level of protein that is synthesized fiom these genes, and thus alter cellular functions.
The presence of such receptors in tumors is considered a good prognostic indicator since the tumor
Figu 1-1. R d t of hormone in s i~oid ceceptoi u t i ~ a t i a i . Progestcron i ~ c p t œ trisu in a tranuripion>lly inad* 8-102 cnnplct ia association wi'th k a t hdc pmteim hsp90, hg70, rd w56 in the Jwm of homsrr. Addition d hamne (Hl chnaes tk anformation d che tuepta (dbwcd by dissœiation of M. -. r fm of hsps, hich c m be *id rher u l t nd AlP u u m t , still rcquircr hamonc br mnscriptiavl xtinücn. hamnt bdcd recepa & d r to its mponw t * m n t (HU). kcom p h p h o y l a t d (m. a*ta 6- tmmaiptiorr (rcpceimted by an a m W .
CONSENSUS ELEMENTS
ENHANCER PROMOTER ELEMENTS ELEMENTS
,
GGTAC AXWTTTCT
Fig e 1-2. Structurai requirements for androgen regulation of gene transcription.
i
ARE ARE CAAT TATA -1 . BOX BOX T T
TRAKSCWPTION TERMTNATlON START SITE S I E
GENE - 3- r -
h - -
4
will iikely respond to hormonal treatrnent with hormone antagonias which block the receptors and
ultimate turnor proliferation. However, not al1 receptor positive tumors will respond to endocrine
treatrnent and this finding was proposed as an indication that in sorne tumors the receptors are
inactive or defective, or post-receptor defects may be present. In the prostate, for example, cells
have androgen receptors (AR) that respond to the hormone dihydrotestosterone. This mechanisrn
" up-regdates" the gene that produces prostate-specific antigen (PS A). In the breast, estrogen
regulated genes include pS2, cathepsin-D, and the progesterone receptor. Apo D is another recent
known biochernical marker that is up-regulated by androgens in human breast cancer ce11 lines.
Apo D protein is present in seminai fluid and gene expression is also under the regdatory function
of androgen receptor. Expression of pepsinogen C, an aspartyl proteinase enzyme, is also under
the control of steroid hormones in the breast. Studies on the hormonal regdation of pepsinogen C
in breast cancer cells have revealed that androgens, glucocorticoids and to a lesser extent
progesterone, are able to induce the expression of this gene in breast cancer ce11 lines. The
measurement of steroid hormone receptor-inducible proteins is one of the ways to test for the
presence of active receptor molecules in the tumor cells. The inducible proteins may have better
prognostic significance than the receptors thernselves. In this project we examined the mechanisrn
of production and clinical aspects of prostate specific antigen (PSA) in breast and lung tissues.
Prostate specific antigen (PSA) has become an important rnarker in clinical medicine over
the last decade. Serum PSA is a routine marker of prostatic disease, especially for prostate
adenocarcinorna, since the first methods becarne available for in-vitro testing in the rnid- 1980s.
Over the past few years, our understanding of the molecular structure, physiology, and
pathophysiology ofthis protein has grown irnmensely: PSA is no longer simply an "antigenNdefined
5
by a specific antibody but rather, a protein with a biological role, evolutionary history and clinical
significance.
PSA is a 33 kDa single chain glycoprotein and a senne protease with chymotrypsin-like
enzymatic activity. Until recently, PSA was thought to be produced exclusively by the prostate
epithelial cells lining the acini and ducts of the prostate gland and to be absent fiom al1 female
tissues and fluids. PSA is present at high concentrations in seminal plasma and used as a marker
for screening, diagnosis, and monitoring of prostate cancer (1 -3). Immunohistochernical detection
of PSA in tumors of non-prostatic ongin has been reported as a rare event (4). Since the recent
description by Our group of the production of PSA protein by breast tumors (5 ), we have
demonstrated that PSA is present in 30-40% of femaie breast tumors (6) , more rarely in other
tumors (7), in normal breast tissue (8), and in biological fluids including breast rnilk (9), breast cyst
fluid (lO), breast discharge fluid and amniotic fluid (1 1). Some female breast tumors produce very
high levels of PS A while othen do not express any PSA.
Association analysis between PSA levels and levels of progesterone (PR) and estrogen
receptors (ERS) in female breast tumors, clearly demonstrated that PSA was associated with the
presence of the PR, but not the ER. Survival analysis reveaied that patients with breast tumors
producing PSA live longer and relapse less fkquently than patients with tumors that do not produce
PSA. Thus, PSA is a new favourable prognostic indicator in female breast cancer (12).
The regdation of this glycoprotein in-vivo has important clinical application. There is
increasing evidence that transcription of the prostate specific antigen (PSA) gene is androgen
regulated. The PSA prornoter contains a sequence which is closely related to the ARE (androgen
response element) consensus sequence. This sequence is a high af£inity androgen binding site and
6
acts as a fùnctional ARE in transfected LNCaP cells (13). PSA gene regulation has been studied
extensively in the LNCaP prostate cancer ceU line and has been found to be positively regulated
by a number of steroid hormones, such as R5020, estradiol, and epitestosterone ( 1 3). However, the
androgen receptor in this ce11 line has been found to have a point mutation which leads to a relaxed
atflluty for steroid hormones and, as such, the LNCaP ce11 line may not be the ideal cell mode1 with
which to study the regulation of this gene.
The growth and differentiation of the mammary gland is a result of complex interaction of
a number of endocrine hormones which act alone and in combination. The development and
progression of breast cancer is also strongly influenced by hormonal status, yet the molecular
mechanisms underlying the complex pattern of multihomone regulation of marnrnary epithelium
are not well understood. Given the fact that PSA is a steroid hormone-regulated protein in the
prostate, we thought that PSA may indeed be expressed in non-prostatic tissues. Particularly, its
presence in breast cancer patients may reflect the existence of a complete hormone receptor
pathway and that understanding of its fùnction and regulation in this tissue may have importance
in breast cancer research both in basic and clinical applications. For these considerations, we were
prompted to examine the possibility that production of PSA protein and PSA mRNA by breast
cancer cell lines could be mediated by homonal factors and their related receptors.
CHAPTER 2. Literature Review
2-1. A Historieal Perspective of PSA
PSA is a normal constituent of prostate secretions. Very few other tissues or organs have
been demonstrated to produce this protein. Its history and biochemistry have been extensively
reviewed (1 4). In brief, PSA was initially discovered in seminal plasma and was named gamma-
seminoprotein by a group of Iapanese scientists who were searching for a sernen marker in the late
1960s (1 5). Over the past few years another group of researchers isolated a similar protein from
semen and called it protein E, based on its electrophoretic mobility (16). In 1978, Sensabaugh
characterized this protein biochernically with the use of an immunoelectrophoretic method and
named it as p30 according to its molecular weight (17). Because of its NI-vitro stability,
immunogenecity, and tissue specificity, p30 was considered to be a usefûi marker in criminai and
forensic investigations ( 1 8).
PSA was found in prostate by Wang et al in 1977 (19) and was later isolated from this tissue
by the sarne research group (20). Since this protein was identified only in prostatic tissue at that
time, it was named prostate-specific antigen. Wang et al also demonstrated that PSA was present
in seminai plasma and that it was similar to the PSA they found in the prostate (2 1). Now, although
it has not been universally accepted that PSA is the only protein under discussion, consensus has
been reached that gamma-seminoprotein, protein E,, p30, and PSA are the same protein (22). PSA
was later identified in male serum by the same group who found PSA in the prostate (23).
Over the last few years, a nurnber of reports have challenged the absolute tissue specificity
of PSA. However, many groups have reported immunoreactivity for PSA in a variety of normal and
neoplastic tissue types. These include rare apocnne sweat gland carcinoma (24), apocnne breast
8
carcinorna (24), salivary gland neoplasm (4), colon carcinomas (25)- pancreatic acinar ce11
carcinoma (26), pnmary ovmian carcinoma (27), adenocarcinorna of paraurethrai gland (28,29),
biliary tract carcinomas (25), and bladder carcinoma (30). Moreover, PSA has been
immunohistochernically demonstrated in normal axillary and perineal apocrine sweat glands (24),
in addition to the paraurethral (3 1) and perianal glands (32). In most cases, PSA positivity was
obtained when polyclonal antibodies were used in imrnunohistochernistry. However, none of these
tumors stained positive when monoclonal PSA antibodies were used, and it was concluded that the
positive staining was due to cross-reactivity of the polyclonal antibodies.
The isolated reports that challenged the absolute tissue specificity of PSA did not attract
much interest, because of their rarity. However, with newly developed ultrasensitive imrnunological
assays for PSA, our group recently reported that PSA is frequently present in extracts of normal,
hyperplastic and cancerous breast tissues (5-8), in female discharge fluid and in breast rnilk (9).
PSA was also found in some ovarian tumor extracts (28), in endometrium (33)- and in many other
tumor types (7).
2-2. Structural and Molecular Features of PSA
PSA is a single-chah glycoprotein of approximately 33 kDa. It has been initiaily
demonstrated that PSA has 240 amino acid residues (34), howwer, based on sequence of its mature
protein is believed to consist of 237 amino acids (35,36). The PSA polypeptide chah has five
interna1 disulphide bonds and these bonds allow saminai PSA to have two different rnolecular
foms. One has a complete and intact structure known as native form; the other has a cleavage at
lysine-lysine bond located between amino acid residues 145 and 146 and is called "nicked".
9
Because of the five intemal disulphide bonds in the mature protein, the nicked molecular form
could CO-migrate with the native form on a sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) under no-reducing conditions, but under denatunng/reducing
conditions, this form of PSA separates fiom the native molecular form becoming PSA fragments
with smaller molecular masses (34-36).
Human PSA is also known to be composed of 7 to 12 % carbohydrates (37). Based on
different analytical techniques, the rnolecular mass of this single polypeptide chah could be
different. The molecular weight of the complete protein is around 33 kilodalton (kDa) determined
by gel filtration and gel electrophoresis (38). Based on the component of the PSA protein, i.e. 240
or 237 amino acid residues, the estimated molecular weight is 26.5 or 26.1 ma, respectively (34-
3 6).
Recently, carbohydrate structure analysis of PSA protein revealed that there are 4
carbohydrate side chains conjugated to the PSA polypeptide chain. Of the 4 sugar chains, one is N-
linked to an asparagine at position 45, two O-linked to a senne at positions 69 and 7 1, and one 0-
linked to a threonine at position 70 of the arnino acid sequence (34). However, other studies
reported that there is oniy 1 carbohydrate side chah N-linked to PSA at asparagine. at position 45
(22). The carbohydrate chah consists of hexose, hexosarnine, and sialic acid (20). Approximately
1% of the 7% carbohydrate residues in the PSA molecule is neuraminic acid giving rise to
microheterogeneity when the protein is subjected to isoelectrophoresis. This could explain the
presence of several PS A isofoms with different isoelectnc points (PI) ranging From 6.8 to 7.5. The
major isoform has PI of 6.9 (39). Some features of the human tissue kallikrein gene family are
summarized in Table 2-2- 1 and Figure 2-2- 1 .
TABLE 2-2-1 Numan tissue kollihrein gene fornily
Old
% muence NO. 01 Amino Acids For Mature Proteln:
New lden t i ty in Reglon: M W (kOa) Pr Cene Protein Designation Designation ~ o Q S A pre pro mature obsewed cakulatedb oôsennd calculated hKLK I Pancreaticlrenal kalltkretn hPRK hK I -62 17 7 238 ND 26.4 ND 4.4 hKLK2 Hurnan glandular kallikrein- l hGK- 1 hK2 - 80 17 7 237 ND 26.2 ND 6.9 hKLK3 Prostate-specific antigen PS A hK3 1 O0 17 7 237 28.5 26.1 6.8- 7.2' 7.4
2.3. The PSA Gene
A gene encoding PSA was identified and cloned by several groups in the 1980s (40-42).
The PSA gene is located on the long arm of chromosome 19 and spans a DNA region of DNA about
6 kilobases (kb) in length. It contains 5 exons, 4 introns, 2 promoter sites, and an untranslated
region at 3' end. PSA gene (KLK3 or hKLK3) belongs to a human kallikrein gene family which
contains two other members, the human glandular kallikrein 1 gene (hGK- 1, KLK2 or IiKLKî) and
the tissue (rendpancreatic) kallikrein gene (KLK1 or W U ) . The PSA gene is approximately
80% homologous with human glandular kallikrein (hGK-1) gene and 68% homologous with tissue
kallikrein (hKLK 1) gene (40,43,44). In addition, recent studies of hGK- 1 mRNA suggest that PSA
and hGK-1 genes are very sirnilar as they both seem to be present only in the prostate and are
regulated by androgen via androgen receptors (45.46). In the prostate, the amount of hGK- 1 mRNA
is only 10% to 50% of the arnount of PSA rnRNA (47,48). These two genes, based on their DNA
sequences, are supposed to encode two similar proteins in terms of their polypeptide structure and
enzyrnatic activity (49). Since the protein encoded by hGK-1 gene has not been identified yet,
Peehl suspects that seminal PSA is actually a mixture of two proteins which are separately encoded
by PSA and hGK-1 genes (50). However, no one has been able to prove this hypothesis. Based on
the nucleotide sequence of the hGK-1 gene, the protein is predicted to have trypsin-Iike substrate
activity, in contrast to the chymotrypsin-like activity of PSA (50).
In spite of many similarities between PSA and kallikrein genes, it is stiil believed by other
investigaton that these genes and their related proteins are distinct from each other in many aspects
(45). Fust, these genes are located far apart on the chromosome 19; PSA gene resides 12 kb away
13
from the hGK- 1 gene and at least 15 kb away fkom the tissue kallikrein gene (5 1,52). Secondly, the
promo ter site and other untranslated regions are different between these genes (40,4 1,43.50).
Finally, a crucial difference is found in some arnino acids that determine the binding specificity of
the enzymes to their substrates (35.4 1). PSA is believed to have chyrnotrypsin-like activity since
a serine is present at one of the substrate binding sites (arnino acid 184). whereas hGK-1 is
supposed to have trypsin-like activity as the position is occupied by an aspartic acid.
Several RNA species have been reponed to transcribe frorn the PSA gene (48). A 1.6 kb
transcript is considered to be the major species being translated into the PSA protein. The other two
transcnpts with 1.9 and 0.9 kb in length are thought to be the products of alternative splicing.
Different PSA M A S have been seen in benign prostatic hyperplasia (BPH) and prostate cancer
(48.53). However, no one has been able to identify any different forms of PSA protein translated
from these transcripts as being clinically distinct as well as useful.
2-4. Regulation of PSA Production
2-4- 1. Positive Control Mechanisms
Evidence for androgen regulation of the PSA has been denved pnrnarily from clinical
studies. Goldfarb et a1 (54) studied the age-related changes in PSA expression in human prostate
utilizing irnmunohistochemical techniques. PSA levels in prostatic tissue are altered significantly
d e r birth throughout puberty and these aiterations match well to the changes of androgen level in
serum (5 5). Weber and associates (5 6 ) demonstrated a significant correlation between circulating
androgen levels and semm PSA. At birth, serurn PSA detectable in both genders and no difference
exias between males and femaies. After birth, the levels start to drop to undetectable levels within
14
several months and they begin to rise again in male teenagers at puberty. Considerable evidence
exists in support of clinid studies that therapeutically increasing or decreasing testosterone levels
in the male body are always followed by changes in semm PSA ievels (52,5 8).
Based on these clinicd observations, many investigators suggested a possible role of
androgen in the regulation of PSA gene expression. Since LNCaP cells were used to study the
androgenic effkts on PSA mRNA expression, convincing evidence for androgen regulation of PSA
gene was provided. For example, Northern blot analysis showed that PSA mRNA increases rapidly
(within 2-6 hours) following androgen treatment of LNCaP cells (59). DNA sequence analysis of
the promoter region of the PSA gene revealed a consensus androgen-response element (ARE)
located approximately 160 base pairs upstream of the transcnpt start site (59). Young et al have
demonstrated that in their ce11 culture experiments with the use of the prostate cancer ce11 line
LNCaP, the production of PSA was up-regulated by androgens through the androgen receptor and
that the regulation was accomplished at the level of PSA gene transcription (59). Recently. it has
been demonstrated that vitamin D, as well as severai paracnne factors secreted fiom prostatic
stromal cells can up-regulate PSA protein expression in the absence of androgen (60). An autocrine
factor produced by a subline of LNCaP was found to up-regulate PSA mRNA (6 1 ) .
2 - 4 2 Negatzve Control Mechanisms
There are at least two signalling pathways that exhibit a negative effect on androgen
induction of PSA expression. One is the phorbol ester-activated protein kinase C (PKC) pathway.
In further experiments, a tumor-promoting phorbol ester (TPA) that activates PKC, had profound
effects on blocking androgen induction of PSA rnRNA and protein, whereas forskolin, a protein
15
kinase A activator, had linle or no effect on the expression of PSA (62,63). Therefore, it is
suggested that the PKC activity rnay function to counteract androgen action.
A second pathway that negatively regulates androgen action is mediated by retinoic acid
(RA). Young et al (63) has shown that RA can inhibit ce11 growth as well as repress androgen
induction of PSA &A in LNCaP cells. Kinetic studies have shown that at least 16 hours of RA
treatment were required to effect the repression of PSA gene. This is in marked contrast to the TPA
effect, which occurred fairly rapidly (4-6 hours). Moreover, AR protein levels were also reduced
afier 24 hours of RA treatment. Therefore, it has been postulated that the antagonistic effects of RA
on the proueration and function of prostatic cells may be at least in part via modulatory effects on
the AR, although the acnial mechanism remains to be determined.
2-5. Effects of Growth Factors on the PSA Gene
In addition to steroid hormones, growth factors are thought to also participate in the
regulation of PSA gene expression. Results From ce11 culture studies indicated that epidermal
growth factor (EGF) and transforming growth factor alpha (TGF-a) could down-regulate the level
of PSA mRNA in prostate cancer cells and that TGF-P and bFGF seemed to have little influence
on the regulation of PSA expression (64,13). It was suggested in another study that some
unidentified autocrine regulators may exist modulating the expression of PSA gene in prostate
cancer cells which grew independently fi-om androgen (61). Extracellular matrix also seemed to
affect the expression of PSA gene through its action on the growth of prostate cancer cells (65).
16
2-6. Biologieal Function of PSA
In recent years PSA has been studied extensively and characterized well regarding its
rnolecular and biochemical aspects in biological fluids. Although the physiologicai role and
importance of PSA protein in non-prostatic tissue still needs to be elucidated, our knowledge of its
physiological functions is mostly limited to the findings in the serninal fluid. PSA Functions as a
serine protease and its catalytic triad consists of three amino acids including histidine 4 1, aspartate
96, and serine 189 (34-36). It has been shown that PSA is able to cleave other proteins at certain
leucine and tyrosine residues fiom the protein's carboxy-terminus (66-68). Arginine and methionine
residues at the carboxyl-terminal end have also been mentioned to be the possible cleavage sites
of PSA on protein C inhibitor (PCI) or secretory protease inhibitor, both of which are present in
serninal plasma (69,7O).
PSA is a serine protease, and in al1 tissues and fluids, the predorninant form is the non
complexed, 33 kDa free PSA monomer that is the enzymaticaily active form of this enzyme.
However, complexes with proteinase inhibitors also exist at much lower concentrations. The
sequence of PSA shows extensive hornology with y-nerve growth factor ( 56%), epidermai growth
factor-binding protein (53%), and a-nerve growth factor (5 1%) (35). In spite of having sequence
hornology with these molecules, it is not known that PSA has functions sirnilar to these factors. It
is proposed that PSA may enzymatically act upon one or more substrates and modiQ their action
in a fashion similar to the function of the other proteinases of the kallikrein family. Earlier studies
suggested that PSA had both chymotrypsin-like and trypsin-like enrymatic activities (68,71).
However, it is now believed that PSA has only chymotrypsin-like activity (50.72). The finding of
trypsin-like activity in PSA is suspected to be due to contamination of purified PSA with hGK- 1
17
protein which is supposed to have trypsin-like activity based on the amino acid sequence of kGK- 1
derived from its mRNA (72). However, since no one has been able to isolate the hGK-1 protein,
this view of PSA enzymatic activity is still hypothetical.
Findings by an in-vitro study showed that PSA can hydrolyse a number of substrates
including gelatin, interleukin 2, myoglobin, ovdbumin, fibnnogen, and insulin at both a and P
chains (34). In fact, major physiological function of PSA was initially demonstrated in seminal
plasma. PSA in seminal plasma was found to be able to digest two large seminal vesicle proteins
called seminogelin 1 and II, to smaller molecular weight fragments (66,67). Cleavage by PSA
occurs at carboxy-termini of certain tyrosine and leucine residues in seminogelin (73). Proteins
narned serninal-vesicle-specific antigens were found by another group to be the substrates of PSA
in semen (74) are thought to be identical to the seminogelins (72). The extracelluar matnx
component fibronectin is also a target of PSA's enzymatic activity (73). Fibronectin, iike
seminogelin, is a component of the seminal fluid and was also discovered to be cleaved by PSA in
semen (75,73). Liquefaction of seminal coagulum formed at ejaculation is known to be the
consequence of the cleavage of these proteins by PSA. This PSA function has been linked to
human fertility because, presumably, sperm motility is enhanced after the semen clot is liquefied
(50).
In spite of the PSA function in seminal plasma, the physiological role and importance of
PSA in non-prostatic tissues needs to be elucidated. Recent data on prostatic tissue could be
extrapolated to support the view that PSA may be involved in the growth regulation of mammary
and other tissues. Evidence indicates that PSA may play an imponant role in the regulation of
growth factors. It has been reponed that PSA levels in semm of prostate cancer patients are
18
associated with the semm levels of insulin-Iike growth factor binding proteins (IGFBPs) (76). The
IGFBPs form a large farnily of related proteins with at least six members (77). In one study, PSA
was shown to be able to digest one of the six members, IGFBP-3. This activity is thought to
regulate the insulin-like growth factor-1 (IGF-1) concentration, because digestion of IGFBP-3 by
PSA releases biologically active IGF-1 (78). insulin-like growth factors (IGFs) are known to be
important autocrine, paracrine, and endocrine peptide hormones having a variety of effects on the
proliferation and differentiation of various normal cells as well as on the transformation and
apoptosis of cancer cells (79,80). IGFs must bind to specific cellular membrane receptors to exert
their hnctions. The binding of IGFs to their receptors is modulated by another group of proteins
called IGFBPs (81). It is believed that IGFBPs carry IGFs to their target cells and regulate the
function of IGFs through blocking the binding of IGFs to their receptors. Thus, the digestion of
IGBP-3 by PSA results in the release of IGFs to which IGFBP-3 binds (82). In addition to its
binding to IGFs, IGFBP-3 may have other hnctions independent from IGFs because IGFBP-3
receptor has been found in the membrane of certain type of cells and IGFBP-3 could bind to those
receptors exerting its own hnctions (83). In this case, PSA may regulate the fùnction of IGFBP-3
per se. Ln addition, It has been found that PSA has rnitogenic activity, presumably due to activation
by PSA of latent transforming growth factor+ and through modulation of ceIl adhesion (84).
Al1 these observations have suggested that PSA rnight have a biologic role. This role is
dependent on the enzyrnatic activity of PSA. Proteolytic activity in tissues is usually tightly
regulated. Because of its proteolytic activity, PSA could bind to sorne serine protease inhibitors.
PSA occurs predominantly in complex with a,-antichymotrypsin (PSA-ACT) in semm and several
other serine protease inhibitors (67). Others have shown that in seminal plasma, PSA binds and
19
inactivates another serine protease inhibitor cailed protein-C inhibitor (69.70).
Except serine protease inhibitors, other mechanisms may be involved in the regulatioa of
PSA function. One possible mechanism is that PSA rnay be secreted as a prozymogen and needs
to be processed enzymatically as some other proteolytic enzymes. It has been suggested that the
initial PSA polypeptide chain has 261 arnino acids and it undergoes several proteolytic changes to
become a functionally active mature molecule with 237 amino acids. Three cleavage sites have
been found to exist and cleavage at 2 of the sites may result in release of 2 small peptide chains
fiom the large molecule. One has 17 molecules and the other contains 7 amino acids. These changes
rnay facilitate the activation of PSA fiom zymogen to its active form. This activation process is
thought to take place inside the prostate gland, but the activating enzyme has not been identified.
One study suggested that hGK-1 protein may be the candidate enzyme because the proteolysis is
trypsin-like cleavage and hGK- 1 is supposed to have this activity.
There is a third cleavage site at the lysine-lysine bond between amino acid residues 145 and
146 of PSA aminoacid sequence. Cleavage at this intemal site may produce a two-chain form of
PSA (nicked form of PSA) which is enzymatically inactive. The protease enzyme respoiisible for
this cleavage has not been reponed. The free form of PSA in semm may have this structure, as it
is enzymatically inactive, and is unable to bind to a,-antichymotrypsin. Formation of free PSA is
suspected to occur before it enters the blood circulation because it would be highly unlikely for the
srnall amount of free and active PSA not to react with a,-antichymotrypsin while it is present in
serurn in a large excess arnount over the free PSA. Other cleavage sites on the PSA molecule may
also exist, but the consequence of the cleavages is not clear (66,67). Physiologically, inactivation
of PSA function can also be achieved through the binding of PSA to other proteins. It is believed
20
that once PSA gets into the blood circulation the native form binds quickiy to several senne
protease inhibitors, rnainly a ,-antichymotrypsin, and becomes enzymatically inactive.
2-7. Molecutar Forms of PSA
PSA in semen has two major molecular forms. one of which is the intact polypeptide
chain called the native forrn. n i e other cailed Mcked form. has a cleavage at aminoacids Lys 145
and Lys 146 (85). As mentioned earlier. a small amount of PSA in semen also binds to PCI.
Several molecular forms of PSA exkt in serurn. including free PSA. a 1 -antichymotrypsin-bound
PSA (PSA-ACT complex). and a,-macroglobulin bound PSA (70.85). The PSA-ACT complex
is the major molecular form of PSA in serurn. Al1 these forms are believed to be enzymatically
inactive. Most current PSA assays c m detect free PSA and PSA-ACT. PSA assays which only
detect fiee PSA have been developed and the ratio of free versus total PSA appears to provide
a slightly better sensitivity and specificity for diagnosis of prostate cancer (86). The new
nomenclature of PSA molecular forms and kalliheins is shown in Table 2-7- 1.
2-8. PSA In Non-Prostatic Tissues
2-8- 1. Pen'urethral Glands
The periurethral gland is the first fernale tissue which was suggested to be able to
produce PSA. PSA production by this gland is based on the findings of immunohistochemical
studies which demonstrate positive stainings for PSA in both male and female periurethral glands
(87.88.89.90) and of PSA in male urine of prostate cancer patients who have had their prostate
removed (87). Further histological studies indicate that the tissue structure of female periurethral
Table 2-7-1 Nomencluture of hK proteins Formal Name Common Name Description hK1 PRK Pancreaticl$enal kallikrein h K2 hC K. I hK3 . PSA
Total PSA t-PSA
Free PSA f- PSA
PSA complexes PSA-ACT
PSA-AT
PSA-IT
Clandular kalfikrein Prostate-specific antigen All imrnunodetectable forms in serum. primarily f-PSA and PSA-ACT Noncomplexed B A ; may be proteoiyticaliy active or inactive in seminal muid. oniy inactive in senrm PSA covatently bound to a, -antichymouypsin inhibitor; synonymous with PSA ~0mplex; major immunodetectable form in serum PSA covaiently linked and encapsulated by a,- macroglobulin; not detected in irnrnunoassays; synonymous with occult PSA PSA covaiently bound to protein-C inhibitor: minor component in serninal fluid; not detected in serum PSA covaiently bound to a, .antitrypsin; trace component in serum PSA covalently bound to inter-alpha trypsin inhibitor: trace cornponent in serum
22
gland is similar to that of male prostate before puberty. but the gland remains underdeveloped
throughout the whole life due to the lack of androgenic stimulation (91 .) Therefore. the female
periurethral gland c m be considered the 'fernale prostate' (14,92). An intcresting animal
rxperirnent suggests that endoderm-derived urethral epithelium from newbom female mice would
develop into a prostate-like tissue producing a PSA-like protein if the cells were allowed to grow
in the body of male mice (93).
2-8-2. Breast
2-8-2- 1. Breasr Cancer Tissue
In 1993. PSA imrnunoreactivity in breast cancer cytosolic extracts. prepared for
measurement of steroid hormone receptors. was accidentally discovered in our laboratory while
we were evaluating the analytical specificity of a new ultrasensitive PSA assay developed for
monitoring prostate cancer patients afier radical prostatectomy. PSA irnmunoreactivity in non-
prostatic tissues was thought to be due to the cross reaction of polyclonal antibodies to non-PSA
proteins. However. this possibility was excluded when similar irnmunoreactivity was seen with
double-monoclonal PSA assays (94). Moreover. the possible existence of PSA in breast cancer
was suggested by the molecular weight of PSA immunoreactive species in the cytosols. The
molecular weight determined by gel electrophoresis and gel filtration was identical to that of
PSA in seminal plasma or to the free fom of PSA in male serum. Finally. PSA mRNA was
detected in breast cancer tissues which were positive for PSA protein measured by imrnunoassay
(95). In contrast. tissues negative for PSA protein were also negative for PSA mRNA.
Since PSA concentrations in breast cancer cytosols are relatively low compared to the
levels in male prostate, the number of sarnples which have detectable PSA may Vary depending
upon the sensitivity of the PSA assay used. Using a PSA assay which has a detection limit of
10 n_d . we found about 30% of breast cancer cytosol samples having PSA levels at or higher
than 0.03 ng of PSA per mg of total protein.
2-8-2-2. Breast Cancer Cells
Several breast cancer ce11 lines which do not produce PSA in the absence of steroid
hormones (baseline) are able to produce PSA afier the cells are incubated with androgens.
progestins. glucocorticoids. and mineralocorticoids (96). The stimulation of PSA production by
these steroid hormones is tirne- and dose-dependent. The molecular weight of PSA produced by
these breast cancer cells is identicai to seminal PSA or to PSA from the prostate cancer ce11 line
LNCaP. PSA mRNA is readily identified in these cells lines d e r the stimulation by steroids
(95) . Estrogens do not have the ability to stimulate the cells to produce PSA. but can impair
the stimulating effect of androgens (96). The production of PSA is observed only in breast
cancer ce11 lines which have steroid hormone receptors (MCF-7 or T47-D). but for the cells
which do not possess the receptors (BT-20) no PSA is produced afier similar stimulation (96).
However, for as yet unknown reasons. many ce11 lines which are positive for receptors are not
able to produce PSA after steroid hormone stimulation.
2-8-2-3. Other Breast Tissues
PSA has also k e n found in both normal breast tissues (specimens fiom cosrnetic breast
reduction surgery) and breast tissues from benign breast disease (BBD). The percentage of
24
women who have detectable levels of PSA in their breast tissues is siçnificantly higher in
patients with BBD (60%) than in normal women (30%). but the positivity rates are not different
between breast cancer patients and normal women (97). The molecular weight of PSA in these
specimens is similar to the one found in seminal plasma (33 KDa).
2-8-2-4. Breasr Fluids
PSA is detectable in dl tested breast fluids. including rnilk (9). cyst fluid (98). and nipple
aspirate fluid (99). PSA concentrations in these fluids Vary widely from undetectable to 5.000
rn@. PSA levels in mik decrease post-delivery with t h e but are not affected by mother's age
or baby's gender. Free PSA is the major molecular fonn of PSA in milk. PSA-ACT complex
is also present in milk. but accounts for a5%. In cyst fluid. PSA levels vary between <0.01
and 82 pg/L. and free PSA and PSA-ACT complex are present in roughly equal proportions
(98). Even wider variations of PSA concentrations are seen in nipple aspirate fluid (0-5,000
m a ) : the rnolecular forms of PSA in this fluid are free PSA and PSA-ACT. in roughly equal
proportions.
2-8-2-5. PSA Regulation and Funcrion in Breasr Tissue
Based on the resuits of ce11 culture experiments. we concluded that the PSA gene
expression in breast cancer cells is up-regulated by androgens. glucocorticoids and progestins.
The regulation requires androgen, glucocorticoid and progesterone receptors. respectively
(unpublished data). Estrogen not only does not up-regulate but can interfere with the effect of
androgen. The exact mechanism is unknown. Since al1 these observations are based on in vitro
25
experiments using breast cancer ceil lines, it remains unknown as to which steroid hormones are
involved in the PSA gene expression and PSA protein production in normal breast cells and
during pregnancy. However. there is indirect evidence for PSA production in normal breast tissue
induced by progesterone. We have andyzed normal breast tissues for PSA from I l healthy
women who underwent cosmetic breast reduction surgery. Of these wornen. none but one had
very high levels of PSA in the tissue: that woman was also the only one who was on oral
contraceptives which contained high levels of progestin (8). Clinical studies have s h o w that
the presence of PSA in breast cancer cytosols is associated with the presence of estrogen and
progesterone receptors. Although we did not examine the relationship between androgen
receptors and PSA status in the clinical study. it is known that androgen receptors are present
in breast cancer cytosols (99) and are positively correlated with estrogen and progesterone
receptors ( 100).
Interestingly. PSA is only associated ~4th ER or PR by their dichotomous status. Le.. the
presence or absence of these proteins. No correlation could be seen between the levels of PSA
and ER or PR. This phenornenon may suggest that the associations between PSA and ER or PR
are indirect and the key luikage between PSA and the receptors is the ligands which bind to the
receptors and activate PSA gene transcription. Therefore. the amounts of receptors present in the
cells at and above certain levels are not the key factors for PSA gene transcription. Another
interesting observation is that ER and PR positivity in breast cancer increase with age of the
patients. but PSA positivity decreases slowly with age (6). The opposite direction of correlation
between age and PSA or receptors may indicate that PSA and the receptors are both associated
with the steroid hormones released from the ovary because the production of ovarïan hormones
26
is an age-dependent phenornenon.
The enzyrnatic activity and physiological role of PSA in breast tissue remains
undetermined. In seminal plasma PSA is found to be able to proteolyze IGFBP-3 (78). An
inverse correlation between PSA and IGFBP-3 is also seen in the senun of prostate cancer
patients (76). Based on these information. we have examined PSA levels in breast cancer
cytosols in association with IGFBP-3. However. no correlation or association was seen between
PSA and IGFBP-3 in our preliminary study (101). The relationship between PSA with other
members of IGF farnily. IGF-1. IGF-II. and IGFBP-1. was also analyzed. but no association was
found.
The fact that PSA is present in milk and amniotic fluid (mentioned later) and that PSA
levels in amniotic fluid change with gestational age suggests that PSA may play a role in fetal
andor newbom development.
The relationship between PSA and IGFBP-3 indicates a possible regulatory pathway for
IGFs. In an in vitro study. it was found that IGF-1 could activate androgen receptor (AR)
resulting in AR-mediated gene transcription (102). PSA gene transcription is one of those genes
up-regulated by androgen via AR (59). Taken together. these data suggest that it may be likely
that there is a regulatory loop on IGF-I through PSA and IGF binding proteins. IGF-1 increases
the production of PSA through androgen receptor. and PSA degrades IGFBP-3 causing release
of IGF-1 from its binding protein. It is believed that IGF binding proteins control the
bioavailability of IGFs through regulation of their transportation and binding to IGF receptors.
Whether the regulation results in enhanced or suppressed action of IGF depends on the type of
tissues involved (79). Because of this tissue-specific regulation, it is difficult to speculate
whether the loop is up- or dom-regulating
Other interesting findings about IGF
the action of IGFs.
include estrogen regulated expression of IGFs and
their binding proteins in breast tissue (103) and involvement of the IGF farnily in ce11
transformation and apoptosis (86.1 04). PS A. through IGFBP-3 proteolysis could be indirectly
involved in such phenomena.
2-8-2-6. Clinical Applicatiom
The presence of PSA in breast cancer cells and its association with steroid hormone
recepton indicated potentiai clinical utilities of this protein in breast cancer. Preliminary clinical
studies did suggest the possibility of PSA king a favorable indicator of prognosis. It was found
that PSA presence was associated with early stage of the disease and small size of the tumor.
and that patients with PSA positive breast cancer could have longer survival compared to those
with PSA negative cancer. Furthemore. the reduced risk for relapse by PSA was sustained afier
other major clinical and pathological factors were controlled in the analysis (12). Survival
anaiysis in subgroups of patients indicated that PSA status may help to further identify patients
with favourable prognosis among those who were node positive or estrogen receptor negative.
One recent study on breast nipple aspirate fluid found that PSA levels in this fluid were
significantly lower in women with high risk of breast cancer compared to women with low risk
of the disease (99). However. dl these observations are based on small number of patients.
Large scale and well-designed studies are needed to confirm these findings.
It is still not clear what is the biological rnechanism of PSA being a good prognostic
indicator for breast cancer. One possible interpretation is that PSA can only be produced by well
28
differentiated cancer cells. Being a product of steroid hormone regdation through their receptors.
the presence of PSA indicates the presence of the regulatory system. which is a sign of good
differentiation of cancer cells. Moreover, PSA in breast cancer cells indicates not only the
existence but d so the fûnctional status of the system. Based on the observation of 30% ER
positive patients who do not respond to endocrine treatrnent (105). it is thought that these
receptors are mutant products which are not functional (106). Therefore. the physical existence
of these receptors does not necessarily indicate their functional status. From this point of view.
PSA could be a more direct and useful marker for this system since it is placed downstrearn
from the fünction of the ER and PR.
The other possibility is the regulatory balance between androgens and estrogens in the
target cell. Since PSA is an androgen up-replated protein in the prostate. the impact of androgen
on breast cancer needs to be considered. The androgen receptor is present in breast cancer and
is correlated with ER and PR. Ce11 culture experiments shows that androgen could inhibit the
proliferation of breast cancer cells (107) and counteract the effect of estrogen ( 108). In addition.
androgen has been used effectively in the treatrnent of some breast cancer patients (109). Our
ceII culture study demonstrates that PSA production in breast cancer cells c m be induced by
androgens (96). Based on this evidence. the presence of PSA in breast ceils may suggest an
androgenic suppression of estrogen effect which is believed to play an essential role in the
development and progression of breast cancer ( 1 10.1 1 1.1 12).
2-8-3. Amniotic Fluid
In addition to milk and other breast fluids. amniotic fluid is another female fluid
29
containing detectable levels of PSA ( 1 1). Both free PSA and PSA-ACT complex are found in
the fluid but the former is the major component. The median concentration of PSA in amniotic
fluid increases between gestationai weeks 14 and 21 and then seerns to level off or decrease until
parturition. The presence and change of PSA concentration in arnniotic fluid seem to have an
impact on senun PSA in pregnant women. S e m PSA is significantly higher in pregnant women
than in non-pregnant women. and changes in serum PSA of pregnant wornen match well with
the change of PSA in amniotic fluid (1 13). Extrernely low or high levels of PSA in arnniotic
fluid at a specific gestational age rnay be linked to fetal abnormalities such as trisomy 21 ( 1 l 3 ) .
ïhe source and physiological role of PSA in amniotic fluid remain unknown. but the levels are
not related to fetai gender. The relationship between PSA and IGFBP-3 or other members of IGF
fmily in amniotic fluid has not been examined.
2-8-4. Fernale Semm
Using conventional PSA assays with a detection limit of 0.1 pg/L or higher, it is possible
to find detectable levels of PSA in less than 10% of fernale sera ( 1 14- 1 18). Because of the lack
of interest. the real identity of PSA irnmunoreactivity in female senun has never been studied.
It was assumed that this irnrnunoreactivity was an artifact which was caused either by cross
reaction of polyclonal anti-PSA antibodies to non-specific proteins or by the presence of
heterophilic antibodies or anti-PSA antibodies.
Based on our studies, the percentage of female sera which are positive for PSA increased
when we use an increasingly sensitive PSA method (detection lirnits between 0.01 and 0.001
pg/L). This suggests that trace amounts of PSA are present in the majority of female sera (1 19).
30
The molecular form of PSA in normal fernale senim as determined by gel chromatography is
similar to the one in male s e m i.e. rnainly PSA-ACT complex (1 19).
In the prostate, androgen up-regulates the expression of PSA and the regulation of PSA
production by androgen is also observed in the cultured breast cancer cells. Based on this. it is
interesting to know if PSA is elevated in the s e m of women who have high levels of androgen.
Our recent study shows that serum PSA is increased significantly in hirnite women and that PSA
levels are positively correlated with the levels of testosterone and 3 a -androstanediol glucuronide
(unpublished data). A potential clinical utility of serum PSA in hirsutism is suggested.
The source of PSA in female s e m remains undeterrnined. but several possibilities exist
based on preliminary observations. Serurn PSA in pregnant women could come from amniotic
fluid since PSA levels increases during prepancy and change with the gestational age. Amniotic
fluid has 20-40 times higher PSA than serum of pregnant women. Obviously. this source should
be taken into account only when women are pregnant. It is reasonable to speculate that breast
tissue is an important source of serum PSA in women. However. when we measured PSA in
matched pre- and pst-surgical sera of breast cancer patients. we found no change in s e m PSA
atier surgery and no correlation in PSA levels between breast tissue and serum ( 120). However.
these observations may have the following limitations. First. the PSA assay we used in the study
was still not sensitive enough to detect minute changes of PSA in female senim. Secondly. serum
PSA can come from both normal and cancerous breast tissues. Thus. the presence and removal
of the tumor tissue may not necessarily affect the levels of PSA in s e m . However. our most
recent (unpublished) data does suggest that removal of breast cancer could result in decrease of
semm PSA when a more sensitive PSA assay is used.
3 1
It is possible that PSA in female semm arises from other sources such as ovary.
endometri-. or lung. but the frequency of these tissues h a k g detectable PSA is relatively low.
Although PSA is also produced by the female periurethral gland. the arnount that could enter
blood circulation is thought to be minimal. Thus. PSA from this gland is not expected to be a
major source of PSA in female s e m .
Recently, we observed changes in the molecular t o m of PSA in female serum in
association with disease statu. For hedthy women and post-operative patients with breast cancer.
the major molecular form of PSA in senun is PSA-ACT cornplex. whereas for the pre-operative
women with breast cancer. the major form is free PSA (12 1). However. this observation is
preliminary . The explanation for this phenornenon and its potential clinical utility remain
unknown.
2-8-5. Ovary, Endometrium and Other Tissues
PSA is detectable in some ovarian cancer cytosols. but the frequency of detection in
primary ovarian cancer is much lower (3%) compared to the frequency of detection in breast
cancer (30%) (122). Ovarian cancer metastatic to breast also has detectable PSA provided that
the primary tumor produces it. We have measured such specimens from 3 women. and 2 of them
were found to have detectable PSA. Of the 3 patients. clinical information was available for one
PSA negative patient and one PSA positive patient. Interestingly. the patient with PSA positive
cancer. which was negative for steroid hormone receptors. responded well to the endocrine
treatment and survived many years afier distant metastatsis. However. the patients with PSA
nrgative cancer was positive for the receptors and she died shortly afier the relapse (122).
32
Although this observation is only based on two patients. it is consistent with our îïnding of PSA
being a favourable prognostic indicator in primary breast cancer ( 1 2).
Clements and Mukhtar reported that PSA mRNA is detectable in endometrial tissues
dong with two other members of the Mlikrein gene family (33). Other tissues which have been
reported to have PSA immunoreactivity by imrnunohistochemistry include kidne y. pituitary (44).
normal axillary and perennial apocrine sweat glands. and some apocrine foci in fibrocystic breast
tissues (24). However. most of the immunohistochernical findings are believed to be due to the
cross-reaction of polyclonal antibody to non-specific antigens because the results were not
reproducible when a monoclonal antibody was used (24).
2-8-6. Other Cancers
Some prirnary lung cancer tissues fiom both males and females contain low but detectable
amounts of PSA (123.7). Other cancer tissues which are found to have trace amounts of
detectable PSA in our study include kidney, adrenai. and colon (7). It has also been reported that
serurn PSA levels were increased in a woman with renat ceil carcinoma (124). Van Krieken
found PSA in salivary gland carcinomas (4) and Papoai et al. reported carcinomas in sweat
glands and breast apocrine glands having false-positive staining for PSA (24).
2-9. Steroid Hormone Receptors
2-9- 1. Mechanisms of Gene Replation
It has been known for several years that steroid hormones function ihrough the action of
specific receptor proteins. In target tissues, receptors are activated by hormonal ligands and
33
thereafler modulate the expression of a network of specific target genes. Receptors for steroids,
glucocorticoid. androgen, estrogen, progesterone, and mineraiocorticoid, and the sterol, 1,25-
dihydroxyvitamin D, have been identified and characterized by biochemical and molecular
biologicai methods (125). Other non-steroid, seemingly unrelated molecules such as Thyroxine (T,)
and retinoic acid (RA) have been found to have receptor hormones and appear to act through sirnilar
mechanisms as steroid receptors ( 126).
Even before the steroid receptors were cloned, it was clear that they were organized in
funcrional domains. Proteolytic cleavage analysis first revealed receptor fragments in which DNA-
binding activity was separateci &om steroid binding. Once the receptors were cloned. these domains
were better dehed. Functionaliy, the steroid receptors interact with inhibitory proteins such as heat
shock proteins ( Figure 1-I), bind to ligand, dimenze. bind to specific sequence of DNA with high
affinity, and activate transcription.
A number of structural features are sirnilar in al1 members of the steroid receptor gene
family, suggesting that they are considered rightly as members of a class of regdatory proteins.
These features include a structurally separate hormone binding site that is a fraction of the total
receptor polypeptide chain, the presence of a high-afinity DNA binding site distinct fiom the
hormone site, a tendency to aggregate at low ionic strength to form either dimers or tetramers of
the subunits, and enhanced affinity for the ceil nucleus in the presence of bound hormone. These
four characteristics can be observed in both cmde extracts and punfied preparations and hence are
probably characteristic of the proteins in situ.
On the basis of cDNA cloning and the cornparison of the deduced amino acid sequence of
different hormone receptors, al1 receptors analyzed thus far are structured in a sirnilar way: they
34
exhibit a variable N-terminal region, a short and well conserved cysteine-rich centre domain, and
a relatively well conserved C-terminal half ( Figure 2-9-1). These three main domains are separated
by less well-defined regions, and are probably subdivided into shorter structural motifs. For
instance, the central domain exhibits an array of cysteine residues compatible with the formation
of two- so-called zinc fingers (127), each of them with a zinc atom tetrahedrically coordinated to
four cysteines. This type of structure, as well as in vitro binding studies and functional evidence,
make it highly probable that the central domain is responsible for the DNA-binding activity of the
receptors. The cysteine residues implicated in metal binding have recently been established by
mutational analysis. Interestingly , each one of the hypothetical zinc figures is encoded by a
separate exon of the receptor gene, but one figure alone appears to be unable to bind to DNA or to
activate transcription ( 1 27,128).
Irnmediately adjacent to the C-terminal zinc figure is a short sequence similar to those found
in SV40 T antigen that appears to be in part responsible for intranuclear locaiization of the
glucocorticoid receptors. Another hormone dependent nuclear translocation signai rnay be located
in the carboxy terminal haif overlapping the steroid binding domain. In this region also reside the
amino acid residues thought to be responsible for the interaction with the 90 kDa heat shock
protein. Since binding of the hormone appears to promote dissociation fiom hsp90, the effect of
hormone binding to nuclear translocation rnay be indirect.
The C-terminal; seems to be complex structurally and functionally, since in addition to its
role in hormone binding and nuclear translocation, it may also contain tram-activation and
dimerization tùnctions. Some of the amino acid residues involved in binding of natural or synthetic
ligands have been identified by mutational and chernicd analysis (1 29- 130).
N - Modulator IONAB Ligand ] - c FUNCTldN
- - - iransacirvaiion
CONSENSUS RESPONSlVE ELEMENTS FOR NUCLEAR RECEPTORS
11 13 15 1 2 3 4 5 6 10 1 2 1 4
1. GRE (+ ) GGTACAnnnTGmCT 2. PR€ II
3. ARE 8 @
4. MRE a *
5. ER€ AGGTCAnnnTGACCT 6. EcRE AGGGTTnnnTGCACT
Figure 2-94. General stnictual and Functional organization of nuclear receptors and consensus responsive elements.
36
The N-terminal domain is less well charactenzed and may have a modulatory effect on
tram-activation. Most of the receptor antibodies descnbed thus far are directed against this region
of the protein. in the case of glucocorticoid receptor, the junction between this and the central
domain appears to be very sensitive to proteolytic attack, as a 40 kDa form of the receptor
containing only the central and C-terminal; domains is ofien found in liver cposol as a product of
receptor deregulation (129-13 1). This finding underlines the structural array of the receptor
molecules, with protease-sensitive regions comecting more resistant domains.
The function of the steroid hormone receptors concem the regulation of the expression of
particular categories of genes. this regulation is exerted by interaction with specific DNA sequences
and with other proteins involved in gene expression control. The DNA sequence required to the
specific binding of nuciear receptors (hormone response element; HREs) are generally situated
upstream of the transcription site and present two short (5 to 6 base pairs) segments of a very
similar or identical sequence (in opposite direction for most nuclear receptors), interspersed or not
by a short non-conserved segment. A comparison of the available sequence information yields a
15-mer consensus sequence for the glucocorticoid response element (GRE) and a 13-mer consensus
sequence for the estrogen response element (ERE) (Figure 2-9-1). WhiIe no clear consensus
sequence has yet been defineci for the progesterone and androgen response elements, a GRE 15-mer
is able to mediate induction Dy progesterone, androgen, and mineralocorticoids (1 32).
The specificity of the natural elements, such as that found in the MMTV-LTR. for a
particular hormone receptor is not tightly restricted, and these elements are able to mediate
induction by several hormones, including glucoconicoids, progestins, androgens, and
mineralocorticoids. Detailed studies of the contacts of the glucocorticoid and the progesterone
37
receptors with a particular HRE show very sirnilar patterns for the recepton. These differences are
probably functionally relevant, as mutation of individuai sequences has a differentiai infiuence on
the response to glucocorticoids and progestins. Thus, it seems that although identical sequences can
mediate response to different hormones, the requirements for optimal interaction are not identical
for the different hormone receptors (132).
A consensus sequence has also been postulated for the thyroid hormone and retinoic acid
responsive elements containing half palindromes sirnilar to those of the ERE but with different
spacing (TRE,RRE, Figure 2-9-1). It has been s h o w that the thyroid hormone receptor cm bind
to the ERE, but that this binding does not result in transcriptional activation (126,132).
The ultimate hnction of receptors is to modulate specific effects at the transcriptional level.
This fùnction has been analyzed extensively in vivo using receptor-deficient ce11 lines transfected
with expression vectors contain cDNAs encoding receptors and reporter vector containing specific
response elements linked to a gene whose product is readily assayable such as chiorarnphenicol
acetyltransferase (CAT) or luciferase. Using these analyses, the regions of the receptor responsible
or gene activation can be defined. for exarnple, mutations that affect conserved cysteine in the DNA
binding domain result in loss of glucocorticoid receptor trans-activation fùnction. In the rat
glucocorticoid receptor an 86-amino acid region including the DNA binding region is sufficient
to mediate gene activation in stably and transiently transfected cells. However in some cases the
activation capacity of mutant receptors was low compared with wild-type receptors. A mutant of
the human estrogen receptor that lacked the hormone binding domain exhibited only 5% of activity.
For several receptors, deletion of amino acids in the N-terminus results in reduced activation
capacity. Thus multiple regions of receptors, both N-terminal and C-teminal, are involved in the
38
transcriptional activation process (129).
A series of recent reports suggested that transcription efficiency of steroid hormone
receptors, once positioned on the right promoter, require functional interaction among the receptor
molecules as well as interactions with other essential transcriptional factors. Functional
cooperativity between two binding sites for the glucocorticoid receptor was first demonstrated in
the regdatory region of the tyrosine amino transferase gene. However, in the case of h4MTV.
binding of glucocorticoid and progesterone receptors to linear DNA segments containing severai
receptor binding sites, does not occur in a cooperative fashion. Additional experiments with
artificial combinations of GREs and binding sites for transcriptional factors showed that not only
NF- 1, but a whole battery of other well characterized factors, are able to act in combination with
the hormone receptors for the activation of an adjacent promoter. When the binding site for receptor
is not very strong, the influence of binding sites to other transcription factors is synergetic, whereas
with a strong GRE the effect are additive, in fact another GRE is as effective as a binding site for
NF- l or SP- 1, a CACCC sequence, or an octanucleotide motif (129,134).
Beside transcription factors, a participation of chromatin structural features in the
mechanism of hormone gene regulation has been repeatedly suggested. In particular, the
observation that following hormonal treatment DNAase I hypersensitive regions appear in the
vicinity of the regulated promoters has suggested that the hormones may act by altering the
chromatin structure of target genes. Recently, it has been postulated that phosphorylation of steroid
hormone receptor is another potential pathway for control of hormone action, particularly if
phosp horylation is hormone dependent. For example, hormone treatments increase the
phosphorylation of the progesterone and glucocorticoid receptors and it rnay regulate the DNA-
39
binding and/or transcriptionai activation fùnction of steroid receptors. However, the fùnctional
significance of steroid receptor phosphorylation has yet to be determined (1 3 5).
2-9-2. Clinical Applicafion of Estrogen and Progesterune Recep fors in Breast C'amer
ER and PR belong to a superfamily of nuclear hormone receptors that hnction as
transcription factors when they are bound to their respective ligand. Estrogens and progestins are
considered the pnmary regulators of growth and differentiation of normal mammary tissue.
Moreover, estrogens are thought to play an important role in the development and progression of
breast cancer. This concept is based on severai lines of evidence. First , epidemiologic evidence
indicates a protective effect concomitant with ovariectomy and an increased risk of developing
breast cancer arnong women treated with the estrogen diethylstilbestrol. In addition, the mitogenic
effects of estrogens on various breast cancer ce11 lines grown in culture, and in vivo in
ovariectomized nude mice have been well documented in the literature. Reports have also
demonstrated that the use of oral contraceptives starting at a younger age significantly increase the
risk of breast cancer. This cohort of wornen had a significant relationship between survival and
exposure to oral contraceptives at a young age. These results suggest that estrogen does indeed
participate in tumor growth promotion and, later, in progression of the disease ( 13 6- 1 3 8).
Estrogens and progestins exert their cellular effects through the binding and activation of
specific nuclear receptors, the ER and the progesterone receptor (PR). When evaluating the role of
these receptors in breast cancer, two issues are important. First, the presence of ER and PR predicts
improved disease-free and overall suMval of breast cancer patients. Second, the presence of these
receptors in tumors predicts the likelihood of benefit from endocrine therapy. Since the original
40
publication suggesting the value of ER as a prognostic biomarker of delayed recurrence in primary
breast cancer, ER and PR assays have become standard practice in the management of breast
cancer. As a single factor, PR has been show to be a more powerful independent predictor of
tmor recurrence that ER in certain subset of patients, such as those with stage II breast cancer. It
is now known that the correlation between ER and PR status and patient outcome is a general
reflection of the intrinsic biological behaviour of receptor-positive tumors. ER-and PR-positive
tumors are more likely to be highly differentiated, to be diploid, and to exhibit lower proliferation
rates than are receptor-negative tumors. The value of steroid receptors as single, independent
prognostic biomarkers is limited, however, and they are most effectively used in combination with
their prognostic biomarkers ( 1 39,140).
Potentially of great importance is the ability of ER and PR to predict response to endocrine
therapy. The ER assay is most usefùl if the tumor is ER negative; these patients seldom respond to
endocrine therapy. The knowledge or ER and PR status together improves the ability to predict
endocrine responsiveness. This findings is based on a hypothesis first published by Horwitz and
associates, suggesting that PR rnight be a better marker than ER for an intact endocrine response
pathway because PR is a product of estrogen action. Indeed, this appears to be the case. Response
rates higher than 70% are seen in metastatic ER positive. PR positive tumors. In addition a
quantitative correlation appears to exist between receptor content and response. with some studies
finding a quantitative correlation only with ER levels and other finding correlations with both ER
and PR levels (14 1).
41
2-9-3. Steroid Hormone Receptors and Cancer
The biological effécts of sex steroid hormones on growth and development of reproductive
tissues and on the growth and progression of endocrine-dependent neoplasia ( 142- 149) are rnediated
by intracellular receptors that are members of a gene family of ligand-dependent transcription
factors (149- 152). In addition to sex steroid hormones and glucocorticoids, the gene family also
includes receptors for thyroid hormones (TR), vitamin D, (VDR), retinoic acid (FUR), and a
number of orphan receptors for which a ligand has not yet been identified. Intracellular receptors
are modular proteins composed of separate domains for ligand binding, DNA binding and
transcriptional enhancement. For the steroid hormone group of receptors, the C-terminal ligand
binding domain (LBD) also harbours sequences for a second ligand-dependent transcriptional
activation domain, dimerization, nuclear localization and binding of heat shock proteins (1 49- 142)
There are similarities in the generai rnechanism by which ligands activate steroid hormone
receptors. In the absence of hormone, receptors from an inactive oligometric complex with heat
shock protein 90 (hsp 90), hsp70, immunophilins and possibly other proteins of unknown identity
(1 53- 155). In response to binding hormone, receptors dissociate from the oligornetric complex and
acquire the ability to dimerize (1 56- 159), and to bind to hormone response elernents (HRE) that are
usually located in the regdatory region of steroid responsive genes ( 160). Receptor association with
HREs leads to an increase or decrease in transcnption (161) by mechanisms that are not fully
understood. A central event in the activation process appears to be a ligand induced conformational
change in receptor structure (162).
It has been demonstrated that steroid hormone receptor presence is a necessary condition
for hormone responsiveness in a target tissue. This crucial role has led numerous investigators to
42
examine malignant tissues that are known to respond to hormonal manipulations and to correlate
the clinical responsiveness to the presence and absence of a steroid receptor. Their presence has
been well documented in some human breast cancers (163) and acute leukaernia (164) and
information is accumulating in other hormone dependent cancers, such as prostate (165) and
endometrial cancer ( 166). In addition, steroid hormone receptors have recently been identified in
some rnalignant melanoma sarnples, lung tumors and colon cancer (1 6% 169). The lack of receptor
almost always predicts a lack of response to hormone therapy, while many but not dl tumors that
contain measuriible receptors are responsive to hormonal manipulations at least in the case of breast
cancer and leukaernia.
Function of steroid hormone receptors could be assessed by different biochemical and
molecular biological methodologies. One possible approach is to look at the expression of steroid
hormone-inducible proteins in target tissues using receptor inhibitors. Several synthetic ligands
(both steroid and non-steroidal) for estrogen (ER), progesterone (PR), androgen (AR),
glucocorticoid (GR), rnineralocorticoid (MR), and retinoic acid (RAR) have been developed which
compete for binding with natural hormones and are capable of inhibiting receptor activity The
antagonist forms a complex with the receptor and then interferes with the normal function of a
ligand-bound receptor by not translocating to the nucleus, not binding to the appropriate DNA
sequences with high affinity, or not aflfecting transcription rates. These components have also been
proven to be valuable for dissecting normal receptor activation mechanisms ( 170- 174).
Breast cancer is the second most fiequent cause of cancer death arnong American women,
accounting for 18% of al1 cancer deaths among women and trailing only lung cancer (1 75). AS
mentioned above, PSA is a good prognostic indicator in breast cancer patients and associate with
43
the presence of steroid hormone receptors ( 6 ) and this may be used as a new approach to design
therapeutic agents for treatrnent of endocrine dependent breast and other hormone dependent
cancers. Approximately 30% of aü breast cancer patients respond to endocrine therapy. The overail
response rate to antiestrogens increases to 70% or more in patients whose tumor express receptors
for both estrogen and progesterone (1 75-1 78). To define the mechanism of action of endocrine
therapies and to develop and screen new agents and therapies we require models that exhibit an
endocrine response profile comparable to that found in breast cancers.
In this regard, breast cancer ce11 lines that grow in-vitro represent one of the most widely
used experimental models of breast cancer. For many studies, these models provide the only means
to address a specific hypothesis. Therefore, studying the steroid hormone regdatory pathway of
PSA expression in non-prostatic tumors, particularly breast cancer, should open a new window of
predicting the prognosis of breast cancer patients and possibly the response of breast cancer to
endocrine treatment.
CHAPTER 3. Hypothesis
Prostate specific antigen (PSA) is a protein selectively expressed by prostatic tissue. The
PSA gene contains in its prornoter region an androgen response eiement. PSA gene expression in
the prostate gland as well as in prostatic carcinoma ce11 lines is regulated by the androgen receptor
and androgenic steroids produced by the testes. The epitheiial cells of the prostate, which produce
high arnounts of prostate specific antigen, are rich in androgen receptors. It is now well established
that the PSA gene in prostatic tissue is regulated by the steroid hormone receptor system. PSA was
recently discovered in breast tissue extracts, in breast tumors as well as in al1 breast secretions.
However, the physiological role of PSA in non-prostatic tissues and its mode of regulation are
unknown and not as yet been studied.
In this thesis, we have hypothesized that the expression of PSA in breast tissue is under the
regulation of the steroid hormone receptor system.
We also hypothesize that if the PSA gene is regulated by steroid hormones and their
receptors in non-prostatic tissues, then, PSA would be present and its concentration should be
changing in physiological situations where steroid hormone levels undergo cyclic changes, as it
happens during the menstmal cycle. We have further postulated that apart frorn the breast, which
is known to possess steroid hormone receptors, other tissues which are now known to contain
steroid hormone receptors e.g. lung tissue, may also have the ability to produce PSA.
Consistent with the Hypothesis of this thesis, we have formulated the following research
questions:
1. Could we design highly sensitive reverse transcription-polymerase chah reaction methods
45
to rnonitor the PSA mRNA with extremely high sensitivity and specificity?
2. Is PSA expression in non-prostatic tissues regulated by steroid hormones? 1s this regulation
dose dependent? Are there major differences in the ability of various classes of steroid
hormones to regulate the PSA gene? Are there steroid hormones which act to uprepulate
andor downregulate the PSA gene?
3 . If the PSA gene is indeed regulated by steroid hormones and their receptors in tissue culture
systems, is the same regulation occumng in-vivo? Could we establish cyclic changes of
PSA concentration in semm of women during the menstrual cycle? Couid we irnpiicate
cyclically changing steroid hormones during the menstrud cycle in the regulation of the
PSA gene?
4. 1s breast tissue the only female tissue that produces PSA or are there other tissue that
possess steroid hormone receptors that could also upregulate this gene? For example, is the
PSA gene upregulated in lung tissue, which is known to contain steroid hormone receptors?
Would this gene be upregulated in patients who receive exogenously administered steroid
hormones?
CHAPTER 4. Objectives
The objectives of my investigation are listed below.
1. In order to examine in detail and with high sensitivity, the expression of prostate specific
antigen in non-prostatic tissues under non-stimulatory and stimulatory conditions by various
agonists, I would need to have appropriate tools. A highly sensitive, irnrnuno£luoromet~c
procedure for measuring PSA protein has already been developed in Our laboratory. This
method has a detection limit of 1 ngL and is the rnost sensitive assay presently available
for PSA My first objective was to develop a highly reliable and extremely sensitive reverse
transcription polymerase cham reaction method to monitor PSA mRNA levels. This method
would be invaluable in monitoring PSA expression at the mRNA level in ce11 lines as well
as in clinical material to be studied.
7 -- Among an extensive collection of ce11 lines of which some possess steroid hormone
receptors while others are receptor negative, my objective is to isolate ce11 lines in which
the PSA gene could be reguiated by exogenously administered hormones. This particular
system will be invaluable to study the mechanisms of PSA regulation. Once the ce11 lines
are identified, rny objective is to examine if they could regulate the PSA gene under
stimulation by androgenic, progesterogenic, estrogenic, glucocorticoid and
mineralocorticoid agonists. In parallel experirnents, another objective is to identie which
of the agonist actions could be blocked by molecules that antagonize these particular
steroids. The results fiom these studies will ailow me to delineate which receptors are
involved in PSA gene regdation.
3. Another objective is to examine if the results of the tissue culture system would be
applicable to clinical situations. 1 have thus exarnined if the PSA levels in serum of women
change during the menstrual cycle. Since many aeroid hormones are regulated through the
menstrual cycle and their concentration changes drarnatically during the luteal or follicular
phases. 1 was interested to establish if there is any cyclic change of PSA concentration in
serum of these women.
4. Since tissues other than the prostate and breast possess steroid hormone receptors, another
objective was to establish if such tissues (e-g. lung tissue) are also able to produce PSA
especially after stimulation by exogenously administered hormones.
CHAPTER 5. Materiais and Methods
5-1. Preparation of PSA cDNA
5- f - 1. Transformation Procedure
DHSa E. Coli competent cells were transfected with a recombinant pA75 plasmid (a gifl
from Dr. J. Trapman, University of Texas, M.D. Anderson Cancer, Houston, TX) using the heat-
shoc k transformation method. This plasmid contains the complete sequence of PS A cDNA
(179,180). In brief. DHSa competent cells were thawed on ice and mixed gently. 50pL of
competent cells were aliqoted into chilled microcentrifuge tubes. 1-3 PL plasmid pA75 was added
directiy to a tube containing competent cells, moving the pipette through the cells while dispensing.
Cells were incubated on ice for 30 minutes and then heat-shocked 20 seconds at 37 OC. Following
that, cells were placed on ice for 2 minutes. 0.95 rnL of LB media was added and shaken at 225 rpm
for 1 hour at 37 OC for expression. In pardel, transformation efficiency was controlled by adding
5 p L (0.5 ng) control pUC19 vector to one tube containing 50 pL of competent cells. After
tramfaion, the DH5 a cells were grown on LB-ampicillin media (10 g bacto-tryptone, 5 g bacto-
yeast extract, 10 g NaCl, pH 7.4, per liter, ampicillin 50 mg/mL). Cultures were incubated for 4
hours at 3 7 OC with shaking and then used to inoculate larger volumes of kesh media which were
incubated at 37 OC for 24 hours. Isolation and purification of the pA75 plasrnid was performed using
the Prornega Magic Maxiprep kit (Promega, Madison, WI, 53 1 1-5399). The procedure is described
briefly as follows. About 100-500 mL the cells were pelleted by centrifugation at 14,000 x g for 15
minutes. The ceils pellet was resuspended in 15 mL of ce11 resuspension solution (50 mM Tris-HCI,
pH 7.5, 10 mM EDTq 100 pg/mL RNase A) to disrupt the cells. 15 mL of ce11 lysis solution ( 0.2
49
M N a 0 6 1% SDS) were added and k e d gentiy, but thoroughly, by stimng or inverting. The ce11
suspension should becorne clear and viscous. Following few time stimng or inverting, 15 mL of
neutralization solution (2.55 M potassium acetate, pH 4.8) were added and immediately mixed by
inverting the centrifuge boale several times. Centrifiigation at 14,000 x g for 15 minutes at 4 OC
separates the cleared supernatant from the white precipitate which was removed carefblly. To
extract DNA, 0.6 volumes of isopropanol were used and mixed by inversion. Centrifugation at
14,000 x g for 15 minutes separates the DNA pellet which is resuspended in 2 mL of TE buffer (
50 mh4 Tris-HCI, pH 7.5, 1 rnM EDTA).
Plasmid purification was carried out by adding 10 mL of Magic Maxipreps DNA
purification resin to the DNA solution. The resin/DNA mix was transferred into the Maxicolumn
and washed with 13 rnL of column wash solution (200 rnM NaCL, 20 rnM Tris-HCI, pH 7.5, 5 mM
EDTA diuted 1: 1 with 95% ethanol ). Another 12 mL of column wash solution were added to the
Maxicolurnn and applied a vacuum to draw the solution through the Maxicolumn. To rinse the
resin, 5 mL of 80% ethanol were added to the Maxicolurnn by continuing to draw a vacuum for an
additional 10 minutes. The maxicolurnn was removed from the vacuum manifold and placed in the
provided reservoir (50 rnL screw cap tube). 1.5 mL of preheated (65-70 OC) water was applied to
the Maxicolumn and waited 1 minute. DNA was eluted by centrifugation the
~axko~urnn/Reservoir at 2,500 rpm (1,300 x g ) for 5 minutes. The concentration and purity of the
plasmid DNA was determined spectrophotometrically.
A plasmid containing the complete p-aain cDNA was obtained from Clontech Laboratones
Inc, Palo Alto, CA, 943034407.
50
5-2. Positive and Negative Control Ce11 Lines
The PSA-expressing human prostatic carcinoma ce11 lhe LNCaP and the non-PSA
expressing human breast carcinoma ce11 line BT-20 were used in control experiments. These ce11
lines were obtained fiom the American Type Culture CoIIection, Rockville, MD. Culture of ceII
lines was performed in flasks at 37 'C and 5% COz in RPMI 1640 medium supplemented with
bovine insulin (200 uni t f i ) glutamine (29 g/L) and 10% fetal calf serum. When the cells were
grown to near confiuency, they were washed with isotonic saline, detached by trypsin-EDTA
treatment and counted. 10' cells were kept frozen at -70 OC until RNA extraction was performed.
5-3. Preparation of Tumor Cytosol Extracts
Approximately 0.2 g of tissue £Yom each tumor was quick frozen and pulverized manually
with a hamrner to a fine powder at -80 OC. The cells were lysed for 30 minutes on ice with 1 mL of
lysis buffer (50 mrnoi/L Tris buffer, pH 8.0, containing 150 mmom NaCI, 5 rnmol/L EDTA, IO@
Nonidet NP-40 surfactant and I mmoVL phenylmethylsulfonyl Ruoride). The lysates were
centrifuged at 15,000 g at 4 OC for 30 minutes and the supematants (cytosolic fractions) were
assayed for PSA and total protein.
5-4. PSA Measurements
PSA in the cytosolic extracts andor tissue culture supematants were measured with a highly
sensitive and specific time-resolved immunofluorometric technique previously established and
described in detail elsewhere (1 8 1 ). In bnef, the PS A assay uses a mouse monoclonal anti-PS A
capture antibody coated to polystyrene microtiter wells, a biotinyiated monoclonal anti-PSA
5 1
detection antibody and alkaline phosphatase-labelled streptavidin (SA-ALP). In this imrnunoassay,
100 pL of sample is incubated with the capturing antibody in the presence of 50 pL of assay buffer
containhg the monoclonal biotinylated anti-PSA detection antibody. M e r 1 h incubation followed
by washing x6, the SA-ALP conjugate is added for 15 min followed by another washing x6. The
activity of ALP is then measured by adding the substrate 5'-Buorosalicyl phosphate, incubating for
10 min and then by adding a Tb3* and EDTA-containing developing solution. After 2 min. the
fluorescence is measured in the time-resolved fluorornetric mode with the CyberFluor-615
imrnunoanalyzer (CyberFluor Inc., Toronto, Ontario). This assay has a detection limit of 1 n g L of
PSA and can measure PSA at leveis of I ng/L or higher (up to 10,000 ng/L) with imprecision of
40%. Al1 assays were performed in duplicate. Tissue culture supenatants and/or tumor cytosolic
extracts were measured undiluted using 100 pL aliquots per assay, udess othenvise stated.
5-5. High-Performance Liquid Chromatography (HPLC)
HPLC analysis for molecular weight venfication of immunoreactive substances was
performed with a Hewlett Packard 1050 system. The mobile phase for the gel filtration
chromatography was a 0.1 moVL sodium sulfate and 0.1 moVL sodium dihydrogen phosphate
solution, pH 6.80. The ske exclusion column used was a TSK-GEL G3000SW. 60 cm x 7.5 mm
in combination with a guard column (TosoHaas, Montgomeryville, PA) with exclusion limit of 600
kDa and was calibrated with a molecular mass standard solution fiom Bio-Rad Laboratories,
Hercules, CA, which contains thyroglobulin (670 D a ) , IgG ( 158 kDa), ovalburnin (44 kDa).
myoglobin (1 7 kDa), and cyanocobalamin (1.35 kDa). The fIow rate was 0.5 W m i n and HPLC
was mn isocratically. After injection of 500 pL of centrifùged sample, fractions of 0.5 mL were
collected with a fraction collecter (Mode1 FRAC-100; Pharmacia, Upsala, Sweden).
5-6. Total Protein Determination
The concentration of total protein in ce11 lysates, tissue cytosolic extracts, and biological
fluids were measured in duplicate using a cornrnercially available kit (Pierce Chernical Co.
Rockford IL 61 105) based on the use of the bicinchoninic acid @CA) detection reagent, as
directed by the product literature for perfodng the "microtiter plate protocol". Assayed in parallel
were protein standard solutions, ranging in concentration from 0.05 to 2 g/L, which were made by
serial dilution of a 2 g/L. albumin standard with a buffer containing 50 mmol/L Tris, pH 7.4, and
7.5 mrnol/L NaN,. The same buffer was used to dilute turnor extracts whose initial total protein
concentration exceeded 2 mg/rnL. Ninety-six well, transparent polystyrene microtiter plates
(Dynatech Laboratories Inc., Chantilly VA) served as the reaction vessels. Absorbance at 545 nm
(close to the absorbance maximum of the reaction product, 562 nm) was measured on the ELISA
plate reader (Bio-Tek Instruments Inc., Winooski VT). Calculation of protein concentrations in the
turnor extracts, ce11 lysate, and biologicd fluids by interpolation From a linear calibration curve, was
performed using Deltasofl ELSA Analysis software (BioMetallics Inc., Princeton NJ). PSA
concentration in al1 cytosols is expressed as ng of PSA per g of total protein.
5-7. Isolation of Total RNA
Total RNA isolation from tissues and ce11 lines was performed using the TRIzol method
(GIBCO BRL, Gaithesburg, MD, TRIzol reagent) following the instructions of the manufacturer.
The m o l method is descnbed briefly as follows. About 200-500 mg of fiozen tumor tissue were
53
first pulverized to a fine powder at -80 OC, and homogenized in 1 rnL of TRIzoI reagent per 50 to
100 mg tissue. 10' cells were pelleted and used for total RNA extraction. M e r incubating the
samples and/or ce11 pellets for 5 minutes at room temperature to permit the complete dissociation
of nucleoprotein complexes, 200 pL chloroform per 1 mL of TRIzol reagent were added into the
solution. Following vigorous mixing, the solution was centrifùged at 12,000 x g for 15 minutes at
4 OC. Centrifugation separates the biphase mixture into the lower red phenol-chforoform phase and
upper colorless aqueous phase which was removed carefdly. The RNA was precipitated from the
aqueous phase by rnixing with 508 pL of isopropanol per i mL of initial TRIzol reagent. The
samples were incubated at room temperature for 10 minutes and centrifuged at 12, 000 x g for 10
minutes at 4 OC. The supernatant was removed and the RNA pellet was washed once with 75%
ethanol. M e r centrifugation and removal of the alcohoi, the pellet was air dned and dissolved in
diethyl pyrocarbonate (DEPC) treated water. The integnty of the RNA was checked
electrophoretically on 2% agarose-formaldehyde gels, and the amount and punty by
spectrophotometry at A?, and
5-8. Reverse Transcription
The synthesis of cDNA from the isolated total RNA was carried out with a first-strand
cDNA synthesis kit using Superscript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD).
Briefly, 1-5 pg of RNA and oligo (dT),2.,, primers (500 ng), were first denatured for 10 min at 70
OC, chilleci on ice for 1 min and then incubated for 5 min at 42 OC in a 19 pL reaction mixture which
includes 10 x PCR buffer ( containing 200 mmoi/L of Tris-HCI and 500 rnmoVL of KCl, pH 8.4)
(Boehringer Mannheim), 10 mmoVL of deoxynucleotide triphosphate rnix (Boehringer Mannheim),
54
10 mmoVL of dithiothreitol @TT), and 25 mmoiL of MgCl? Then, 200 units (1 PL) of Superscript
II reverse transcriptase were added to the reaction mixture, incubated for 50 min at 42 OC,
terminated at 70 OC for 15 min and chilled on ice. The mixture was then treated with 1 pL of RNase
H for 20 min at 37 OC before proceeding to amplification of the target cDNA. Negative control
reactions for RT-PCR were performed using al1 reagents but without added Superscript II.
5-9. Oiigonucleotide Primers
Two oligonucleotide primers were used to ampli@ a 754 base pair region of PSA cDNA.
These, originally desctibed by Deguchi et al. (182) have the foliowing sequence: PSA Al : 5'-
TGCGCAAGTTCACCCTC A-3', PS A B I : 5'-CCCTCTCCTTACTTCATCC-3'. We have fiirther
developed another two primers for nested primer PCR as follows: PSA NI: 5'-
CTGTGTGCTGGACGCTGG-3', PS A N3 : 5'-ACCTCAC ACCTAAGGAC A-3'. For actin cDNA
amplification, we used the following primers, previously published (183). ACT 1: 5'-
AC AATGAGCTGCGTGTGGCT-3', ACT 2 : 5'-TCTCCTTAATGTC ACGC ACGA-3'.
PCR with pnmers AIE3 1 yields a 754 bp fragment fiom exons 4, 5 and the 3' untranslated
region, with primers NIN3 for nested primer PCR a 303 bp fragment and with ACTUACTZ a 372
bp fragment.
5-10. PCR Protocol
PCR was performed in 0.2 mL thin-walled MicroArnp reaction tubes on a Perkin-Elmer
Gene Arnp 2400 system. Total volume was 50 PL. The reaction mixture contained PCR buffer (50
mrnol/L KCI, 10 rnrnolR. Tris buffer, pH 8.3, 1.5 mrnoVL MgCl,, I OmgIL gelatin), 200 pmoVL of
55
deoxynucleoside triphosphates, (dNTPs), 1 pmol/L of PCR pnmers, 2.0 units of Taq DNA
polymerase (Boehringer Mannheim) and 5 pL of cDNA target (added last). The PCR was
performed with one cycle at 94 "C for 5 min, 30 cycles with denaturation at 94 "C for 30 S.
annealing at 60 OC for 30 s and extension at 72 "C for 30 s and one cycle at 72 OC for 7 min. Twenty
pL of PCR reactions were electophoresed on 2% agarose gels and visualized with ethidium bromide
staining. Negative controls did not contain template DNA.
5- 1 1. Nested Primer PCR
The procedure was the sarne as for the above PCR protocol except that 25 cycles were used
in the first PCR, pnmers Nl/N3 were used in the nested primer PCR for 20 cycles and 1 pL of the
first PCR product was added as target.
5-12. Labeling of PSA cDNA Probe
PSA cDNA plasmid was linearized with Hind III enzyme digestion and labeled with the
random primer method by incorporation of digoxigenin-labeled deoxyuridine triphosphate (DIG-
dUTP). We used the DIG-DNA labeling kit fiom Boehnnger Mannheim and the protocol
recornmended by the manufacturer. PSA cDNA plasmid was first incubated for 1 hour at 3 7 OC with
150 units of Hind-III restriction enzyme (Boehringer Mannheim) in a 10 rnmol/L of Tris-HCI
buffer, pH 8.0, containing 0.1 mol of NaCI, 5 mrnollL of MgCL, and 1 mmoK of P-2-
rnercaptoethanol per liter. Then, the PSA cDNA plasmid was denatured by heating in a boiling
water bath for 10 minutes and chilled on ice. Thirty pL of, 10 x concentrated dNTP mix, including
I rnmolL of dATP, 1 mmol/L of dCTP, 1 mrnoi/L of dGTP, 0.65 m o V L of dTTP, and 0.35
mrnol/L of DIG-dUTP. The whole mixture was incubated at least 60 minutes at 37 OC.
5- 13. RNA Labeling by In- Vitro Transcription
The PSA PCR product was cloned into the polylinker site of the pCR 2.1 transcription
vector. The recombinant PCR 2.1 transcription vector contains promoters for SP6 and T7 RNA
polymerases. After linearization of the vector with Hind III restriction enzyme, the T7 RNA
polymerase was used to create "mn-off' transcripts using the DIG RNA Labeling Kit (Boehringer
Mannheim, Germany) and the protocol recommended by the manufacturer. DIG-UTP was used as
a substrate and was incorporated into the transcript. The DIG-labelled cRNA was used as a non-
radioactive probe in Southem blots. In brief description, the following reagents were added to a
microfuge tube on ice: 20 pL of, 10 x transcription buffer, 20 PL of, 10 x concentrated NTP
labeling d u r e containing 10 mM ATP, 10 mM CTP, 10 &I GTP, 6.5 mM UTP, 3.5 rnM DIG-
UTP, pH 7.5, 10 pg o f linearized DNA, 400 units of T7 RNA polymerase, and 10 pL of RNase
inhibitor and centrifuged briefly. The reaction mixture was incubated for 2 hours at 37 OC. Longer
incubation does not increase the yield of labelled CRNA. DIG-labelled RNA probes have the
foliowing advantages: 1. they are of defined length; 2. they are single strand specific and therefore
al1 labelled RNA is available for hybridization and does not renature as in the case of DNA. cRNA
which is DIG-labelled according to the above protocol allows the detection of O. 1 pg homologous
DNA or RNA in a dot blot.
5-14. Direct Incorporation of DIGdUTP During PCR
We have used the same PCR protocol to ampli& PSA cDNA but we included a dNTP
57
mixture that contains, in addition :O the four nucleoside triphosphates, DIG-1 1-dUTP. The final
concentration of d N T P s and DIG- 1 1 -dUTP in the PCR reaction were 200 pmoI/L and 0.7 pmoVL,
respectively. PCR products were electrophoresed on 2% agarose gels, transferred and fixed as
descnbed. The DIG- 1 1-dUTP incorporated was detected using cherniluminescence as descnbed
in the previous section.
5-15. Detection Limit of PCR
We have used totai RNA extracteci from 10' LNCaP prostate carcinoma cells as a reference
preparation for determining lirnits of detection. This total RNA was reverse transcribed and then
diluted successively to give cDNA corresponding to a certain number of cells. We have also used
punfied plasrnid pA75 containing full-length of PSA cDNAs to calculate the detection lirnit of the
PCR methods. Plasmid and LNCaP cDNAs were diluted in a 10 rnrnol/L Tris buffer, pH 7.80
containing 1 mg/ml of salmon sperm DNA to avoid losses due to adsorption to tubes. Negative
controls were included as necessary with the diluent used as target.
5-16. Gel Electrophoresis, Southem Blotting and Hybridization
Aliquots of PCR produas (20 pL) were electrophoresed at 100 V for 45 min on 2% agarose
minigels containing ethdium brornide. The gels were then Southem blotted ont0 positiveiy charged
nylon membranes (Boehnnger Mannheim) by overnight alkali capillary blotting with use of 0.4
mol/L NaOH. The membranes were then baked for 15-30 min at 120°C. For hybridization, the
membranes were placed in a roller bottle with 20 rnL of hybndization buffer per 100 cm' of
membrane. We used a commercial hybridization solution @IG Easy Hyb, Boehnnger Mannheim).
58
Prehybridization was at 42 OC for 1 h. The solution was then replaced with 5 mL per 100 cm'
membrane of hybndization buffer containing 50 ng/rnL of fieshly denatured labeled PSA cDNA
andlor 200 ng/rnL DIG-labeled cRNA probe. Hybridization was carried out for 12- 16 h at 42 OC.
Filters were subsequently washed twice with 2 x SSC containing 0.1% SDS at room temperature
(5 min per wash) and twice with O. 1 x SSC containing 0.1% SDS at 68 OC (1 5 min per wash).
5-1 7. Detection Protocol
The detection was performed as follows: The membranes were first washed briefly in a
washing b&er containing 100 mM maleic acid, 150 rnM NaCl, pH 7.5 and 0.3% Tween 20 for 1-5
minutes at room temperature and then were incubated in blocking solution (Boehringer Mannheim)
for 30 minutes. Alkaiine phosphatase-conjugated anti-digoxigenin antibody (Fab Fragment) was
diluted to a final concentration of 35 mU/mL ( 5000-fold) in blocking solution. The membranes
were incubated in diluteci antibody conjugate solution for 30 minutes and then washed twice for 15
minutes each at room temperature in washing buffer. Finally, the membranes were equilibrated in
a buffer containing 0.1 moVL of Tris-HCl , 0.1 moVL of NaCl and 50 rnmoi/L of MgCl,, pH 9.5,
for 5 minutes at room temperature and incubated with 100-fold diluted cherniluminescent substrate
in the bfler stated above, AMPPD (1 0 rng/rnL, Boehringer Mannheim), andior CDP-star (25 mM,
Tropix, Bedford, MA). While CDP-star equilibrating buffer was 0.1 M diethanolamine, 0.1 rnM
MgC12, pH 9.5. The cherniluminescent signal was captured on X-ray film with 15-20 minutes
exposure for AMPPD and 10-60s exposure for CDP-star at room temperature.
Other methods for Southem blotting, probe radiolabeling, hybridization, and
autoradiography were aiso perforrned according to standard techniques (1 84).
59
5-18. Cloning of PCR Products
We have cloned the 754 bp PCR product of PSA cDNA and another five PCR products with
rnolecular weights between 250-500 bp using the TA cloning kit (Invitrogen Corporation, San
Diego, CA). We followed the protocol recomrnended by the manufacturer. Large quantities of the
recombinant plasrnids were prepared by culturing transformed E. coli cells in LB-ampicillin media
and extracting the plasmids with the Qiagen Midi plasmid purification kit (Qiagen Inc, Chatsworth,
CA).
5-19. DNA Sequencing
PCR products were directly sequenced using the Thermo Sequenase kit (Amersham
Internat ional, Buckinghamshire, England). The protocol recomrnended by the manufacturer was
used throughout. Sequencing primers, labelled at the S'-end with Cy5 fluorescent dye? were as
follows: PSA-S 1 5'-AAGGTGACCAAGTTCATG-3' (binds 19 nucleotides intemally from PCR
primer PSA-A 1). PSA-S2: 5'-CCATCCCATGCCAAAGGA-3' (binds 19 nucleotides intemally
6om PCR pimer PSA-A2). Al1 sequencing reactions were loaded on the ALF Express Automatic
Sequencer (Pharmacia Biotech, Uppsala, Sweden). Sequence cornparisons were performed with
BLAST and DNASIS software.
5-20. Tissue Culture System
5-20- l. Conipo~ndr
Al1 steroidal and non-steroidal compounds used in this study were obtained from Sigma
Chernical Co., St. Louis, MO 63 l78., except for the following: ICI 102,780 and Casodex (ICI
60
1 76,334) (Zeneca Pharma Inc., Mississauga, ON, Canada); RU58,668, RU54,876, RU56,187,
Nilutamide (Anandron, RU23,908), and Mifepristone (RU486, RU38,486) (Russel-UCLAF,
Romainville, France), Beclomethasone Dipropionate (Glaxo Wellcome Inc., Mississauga, ON,
Canada), vitamin D analogs ( ' O 8-8892, Ro 2 1-553 5, Ro 23-7553, and Ro 24-553 1) (Hofian-La
Roche Inc, Nutley, New Jersey, USA), EB- 1 O89 (Leo Pharmaceuticai Products Ltd, Allerup.
Denmark), LG100 153, LGlOO272, and Degnelin (LIGAND Pharmaceuticals Inc, San Diego, CA),
Recombinant Human FGF acidic, EGF, IGF-1, and IGF-II (R&D Systems, Minneapolis, MN).
Hydroxyflutarnide was a gifi from Dr. Doma Peehl. Stanford University. Stock solutions ( 10'~ or
lo5 M) were prepared in absolute ethanol. More dilute solutions were also prepared in the sarne
solvent. A total number of 64 substances were tested in tissue culture expenments, which included
androgens, progestins, estrogens, glucocorticoids, mineralocorticoids, vitamin D, retinoic acid,
growth factors, gonadotropin hormones, cyclohexirnide, and steroid hormone antagonists. The list
of al1 compounds along with their biological function are s h o m in Table 5-20-1-1.
5-20-2. Cell lines
In tissue culture expenments various tumor ceil lines were tested for PSA expression. Al1
the ce11 lines used were obtained from the Amencan Type Culture Collection (ATTC), Rockville,
MD 20852., except for the following: SAOS (osteosarcorna; provided by Dr. M. Grynpas, Mount
Sinai Hospital, Toronto), BG-1 (ovanan carcinoma; provided by Dr. H. Rocheford, INSERM,
Montpelier, France), and MFM-223 and MFE-296 (breast and endometrial cancer ce11 lines
provided by DR. R. Hackenberg, Klinikum der Phillips-Universitat, Marburg, Germany). The list
of the ce11 lines tested along with their tissue origin are shown in Table 5-20-2-1.
Table 5-20-1-1. List of Steroid/Cornpounds Tested for PSA Production in Tissue Culture Systern
Biological Action
1 1 1 Testosterone 1 Androgen
--
~ ~ d r o a n d r o s t e r o n e 7 Androgen
2
1 4 1 RI 88 1 (methyltrienolone) 1 Androgen
Androsterone Androgen
6
1 9 1 Estriol 1 Estrogen metabolite
7
8
Dihydrotestosterone Androgen
Dihydroisoandrosteronee Sulfate (DEA-SO,)
Estrone
10
1 13 1 Hydrocortisone 1 Glucocorticoid
Androgen Met abolite
Estrogen
1 1
12
1 7a-Ethynylestradiol Estrogen
P-Estradiol
Corticosterone
14
1 16 1 Prednisone 1 Glucocorticoid
Estrogen
Glucocorticoid
15
Betarnethasone 1 7-valerate Glucocorticoid
Dexamethasone
1 7
Glucocorticoid
1 8
1 21 1 Norethidrone 1 Progestin
Beclomethasone dipropionate
19
20
Glucocorticoid
1 7 a-Hydroxyprogesterone
1 23 1 Depo-Provera 1 Progestin
Progesterone precursor
Progesterone
Norethynodrel
22
Progestin
Progestin
Norgestrel Progestin
Steroid/ Compounds Biological Action
1 25 1 Triarncinolone Acetonide 1 Progestin/ Glucocorticoid 1 24
1 26 1 Aidosterone 1 Mineralocorticoid 1
Norgestimate
( 29 1 Ro23-7553 1 Vitamin D, analog
Progestin
27
28
1 32 1 LG100275 (TTNPB) 1 RAR Agonist
EB1089
Ro 23-553
30
3 1
1 34 1 13-cis Retionoic Acid 1 Retinoic Acic
Vitarnin D, analog
Vitamin D, analog
1 35 1 Al1 Tram Retinoic Acid 1 Retionoic acid
Ro-24-5531
Degnelin
Vitamin D, analog
Rentioc Acid Agonist
1 38 1 ICI 182, 780
36
37
Antiestrogen
Antiestrogen
LG 100153
Tarnoxifen
RXR Agonist
Antiestrogen
1 42 1 Cyproterone Acetate 1 Antiandroged Progestin
40
4 1
1 43 1 Casodex (ICI 176,334) 1 Antiandrogen
1 44 1 RU%, 187 Antiandrogen
RU 54, 876
HydroxJutamide
- .- - - - . - -
I 1 45 1 Nilutamide (Anandron) Antiandrogen
Antiestrogen
Antiandrogen
1 46 1 Mifepristone (RU 4861 RU 38,486) 1 Antiprogestin
1 47 1 ~ortexolone 1 Antiglucocorticoid
1 48 1 Spironolactone 1 htirnineralocorticoid
I i ! Biological Action
51 1 GH 1 Growth Hormone
49
50
Lactogenic
53 1 LH 1 Gonadotropin
Cholesteroi
Cycloheximide
54 1 FSH 1 Gonadotropin
Steroid hormone precusor
Protein Synthsis inhibitor
55 1 P-HCG 1 Chriogonadotropin
56 1 IGF-I 1 Growth Factor
57 1 IGF-II 1 Growth Factor
58 1 a-FGF 1 Growth Factor
59 1 EGF 1 Growth factor
Oral Contraceptive
Oral Contraceptive
Control
64 1 Nothing 1 Control
Table 5-20-2-1. Cell lines Tested for PSA Production in Tissue Culture System
Tissue of Origin 1 Steroid Hormone Receptor Status
1
2
BT-474 1 Brdast 1 ER (+), PR (+), AR (+) '.'
3
4
T-47D
MCF-7
Breast 1 ER (-), PR (-), AR (-)'.'
ZR-75- 1
MDA-M.-453
6
7
9 1 BG- 1
Breast
B reast
10 1 HBT-75 1 0varia.n ( Not Reported
ER (+), PR (+), AR (+)l.'
ER (+), PR (+), AR (+)'.'
Breast
Breast
MFM-23 3
HBL- 1 O0
ER (+), PR (+), AR (+)'y2
ER (-), PR (+), AR (+)L*2
13 ( A-427 1 Lung 1 Not Reported
Breast
B reast
1 1
12
Not Reported
ER (-), PR (-), AR (+)'*'
ER (-), PR (-), AR (-)'s2
NCbH460 1 Lung 1 Not Reported
HBT- 16 1
HBT- 173
16 1 SOAS 1 Bone
- --
Not Reported
Ovarian
Lung
19 1 Co10 320 1 Colon 1 Not Reported
AR (+), ER (+), PR (?)"
Not Reported
prostate 1 ER (-), PR (-), AR (+) '+'
1. J Steroid Biochem Mol Biol. 56: 1 13-1 17, 1996 2. Clarke R Leonessa F, Bmmer N, Thompson EW. ( 1996). Diseases of the breast : In vitro
models of breast cancer.Harris JR, Lippman ME, Morrow M, Hellman S (eds). Lippman- Raven Press: New York, pp.245-26 1.
3. J Clin Endocrino1 Metab 76: 1497- 1502, 1992 4. Cancer Res 4452864290, 1984 5. Biochem Biophys Res Commun, 192:940-947, 1993
5-2 0-3. Ce f f Culture Procedure
fhe ce1 lines were initially grown in plastic culture flasks at 37" with 5% of COZ in RPMI
1640 culture medium (GIBCO BRL, Gaithersburg, MD) supplemented with glutamine (200
mrnollL), bovine insulin ( 10 mg/L), fetai bovine semm ( 1 O%), antibiotics (penicillin, streptomycin)
and antirnycotics (amphitericin B) unless otherwise recornrnended by the providers. The cells were
cultured to near conûuency and then transferred to phenol red-free media containhg 10% charcoal-
stripped fetal bovine serum (Gibco BRL, Gaithesburg, MD)with antibiotics/antimycotics. Phenol
red-free media were used since phenol red was found previously to have weak estrogenic activity
(185) and charcoal-stripped fetal bovine serum is devoid of steroid hormones. The cells were then
detached by trypsin-EDTA treatment, washed with PBS buffer and subcultured as above in 24-well
tissue culture plates (Corning # 25820) until confluence with change in media at 3 days. Each well
contained 2 mL of phenol red-fiee media with 10% charcoai-stripped fetai calf serum and
antibiotics/antimycoticç.
5-20-4. S~imulution w i h Steroid Compozrnak
Stimulation was initiated by adding 2 pL of each steroid and steroid compounds (IO-<- 1
M) dissolved in 100% ethanol and incubating for a cenain penod of time (usually up to 8 days).
Tissue culture supematants (-150 pL ) were removed for PSA anaiysis at days 3,5, and 8 . Slight
modifications of this protocol were introduced as necessaq. Appropriate multiple positive and
negative controls (oniy alcohol added) were included in each expenment. Wells with microbial
contamination were excluded fiom the data anaiysis.
66
5-20-5 Stimulation with S e m Smp[es and Gonadotropin Hormones
Stimulations were camied out with confluent T-47D and BT-474 breast carcinoma ce11 lines
containhg 1 ml of phenol red-ffee media and 10% charcoal-stripped fetal calf serum and
antibiotics/antimycotics. Stimulation was initiated by adding 1 mi of filter-steralized senim sarnple
and incubating for a certain time (24 h). Tissue culture supernatant (- 150 PL) was removed for PSA
protein analysis after 24 h . Slight modifications of this protocol were introduced as necessary.
Appropriate multiple negative controls (no semm added) were included in each experiment .
Positive controls consisted of T-47D cells stimulated with the synthetic progestin Norgestrel which
was found previously (96) to induce PSA mRNA and PSA protein production by T-47D cells.
Additional experiments were performed by using progesterone at final concentrations of 1 O-'- 1 0-l1
M. prolactin (5 ng/mL-5 pg/rnL), growth hormone (10 ng/mL-10 pg/mL), LH (10 ng/mL-10
pg/mL), FSH (1 0 ng/mL- 1 O pg/rnL) and choriogonadotropin (5 IU/mL-0.005 IU/rnL). Al1
concentrations are final, in the microtiter weIls.
5-20-6. Blockuzg Experirnents
Blocking experiments were performed by simultaneously checking for the following
possibilities: (a) Stimulation by the blocker alone at a final concentration of 1 0-8 M. (b) Stimulation
by the stimulating steroid alone at a final concentration of IO-^ MM. (c) Adding the blocker to the
cells at a final concentration of IO-' M, incubating for 1 h and then adding the stimulant at a
concentration of 10 '~ M. (d) Including controls with ethanol only (negative controls). This protocol
allows for a direct cornparison of the stimulating activity of either the blocker or the stimulant and
the effect of the blocker on the ability of the stimulant to induce PSA expression when the blocker
67
is allowed to bind to the receptors at 10-fold higher concentrations for I h before the addition of
the stimulant.
5-20- 7. Kineizc Experiments
The kinetics of PSA production by T-47D and BT-474 cells were studied as follows.
Confluent T-47D and BT-474 cells were stimulated with Norgestrel at a final concentration of 10"
M and the cells were harvested dong with tissue culture supematants at 1, 2, 4, 8, 24 and 48 h.
Control cells were harvested at 48 h without any stimulation (ethanol added only). The tissue
culture supematants and portion of the cells were used for PSA protein analysis; another portion
of the cells was used to extract total RNA for PCR analysis of PSA mRNA.
5-21. Lysis Procedure
AU cell pellets were lysed for 30 minutes on ice with 1 mL of lysis buffer. Lysis buffer was
50 mmol/L Tris, pH 8.0 containing 150 mrnoi/L sodium chioride, 5 mmol/L
ethylenediaminetetraacetic acid (EDTA), 10 g/L Nonidet NP-40 surfactant, 1 mrnoVL
phenylmethylsulphonyi fluoride and 1 m g L each of aprotinin and leupeptin as proteinase
inhibitors. The lysate was centrifugecf at 15,000 g at 4 "C for 30 minutes, the supernatant was
collected and immediately assayed for PSA and total protein.
5-22. Measurement of Steroid Hormone Receptors
The concentration (fhoVrng) of estrogen receptors (ER) and progesterone receptors (PR)
in al1 tumor ceIl lines and breast tumor specirnens were measured with the Abbott enzyme
68
immunoassay kits (Abboa Laboratories, North Chicago, IL) at the Sumybroo k Heaith Science
Centre.
5-23. Measurement of Hormones in Serum
AU serum samples collected dunng the course of the menstrual cycle were analyzed for the
following hormone levels: FSH, LH, estradiol and progesterone. FSH and LH were assayed with
the ~ccess" Immunoassay Analyzer (Sanofi Diagnostics Pasteur, Montreal, Quebec, Canada). The
Iowest detectable level of FSH and LH distinguishable fiom zero with 95% confidence is 0.2 U L
and both assays exhibit total imprecision under 10% across the assay range. Estradiol was measured
by a solid-phase, chemiluminescence enzyme immunoassay system (Immulites, Diagnostic
Products Corporation, Los Angels, CA). Detection limit and coefficient of variation are 0.044
nrn0i.L and less than 10%, respectively. Progesterone was measured with the Ciba-Corning ACS*
progesterone cherniluminescence irnmunoassay (Ciba-Corning Canada, Markharn, ON, Canada).
The ACS" progesterone assay has a minimum detectable concentration of 0.35 nmoVL and
coefficients of variation 4 0 % within the measuring range. The detection limit of the assay is
approximately 0.1 5 pg/rnL and precision < 10%.
5-24. Immunohistochemistry
Immunohistochemical stains for PSA protein were performed on Cpm-thick paraffin
sections of selected blocks of the pnmary lung adenocarcinorna using a streptavidin-biotin
technique and a polyclonal antiserum directed against human seminal plasma PSA protein
(Biomeda, Foster City, CA). Sections were pretreated with microwave heating to facilitate antigen
69
retrieval. Human prostate was used as a positive control while non-immune rabbit serum served
as a negative control for assessrnent of non-specific staining.
5-25. Su bject Selection
5-25 1. Bremi Tisnre Specimens
Thirty breast cancer specimens were obtained from women undergoing surgery for primary
breast cancer. The breast tumor tissues were stored in liquid nitrogen irnrnediately &er surgical
resection, transported to the laboratory and subsequently stored at -70 OC until protein and RNA
extractions were performed. Al1 tumors were primary lesions except for one ovarian carcinoma,
which was metastatic from a primary breast cancer. We also used one lung carcinoma which was
metastatic from a primary prostate cancer, one prirnary liver cancer and one primary prostate
cancer, al1 as control tissues. Al1 tissues were lefkovers from routine pathological exarninations.
Steroid hormone receptor analysis (ER and PR) was also performed on each tumor specimens using
the Abbon enzyme immunoassay kits.
5-25-2. Semm Specimens Obtahed During Menstnial Cycle
Fourteen healthy, reproductive age women with regular owlatory menstrual cycles, as
determined by history and either basal body temperature charting or luteal phase progesterone
measurements, were studied dunng the period of one whole menstrual cycle. Blood was collected
over the one month cycle. Criteria for inclusion in this study were: female between 20-46 years of
age, normal menstrual cycle and not on birth control pills or other medications. Samples were
collected eight times (every 3-4 days) over the course of the cycle, usually three in the phase after
70
menstmation (follicular phase), two near ovulation and three in the luteal phase. Al1 women had
a normal screening medical history; one reported a past history of breast fibroadenoma which was
not treated. Six of the volunteers were nulliparous (no pregnancy) and one had two normal
pregnancies. Race of the volunteers was two Blacks, two Oriental, and ten Caucasians. Menstrual
cycle regularity and ages of the women were between 26-39 days and 29-46 years, respectively.
5-53. Lztng Cancer Patients
5-25-3- I. Case stucs
Patient: The patient was an 87 year old femaie who was initially admitted to the Ernergency
Department of St. Joseph's Health Centre, Toronto, with acute cholecystitis. A chest x-ray at the
time of admission revealed that the patient had fairly severe lung disease with chronic emphysema
but also a nodule in the right lung suspicious for carcinoma. The patient undenvent wedge
resection. On pathological examination, the lung lesion proved to be a moderately differentiated
adenocarcinorna with a maximum dimension of 1.5 cm. The cancer arose in an area of a scar with
extensive deposition of anthracotic pigment. The tumor consisted of sheets and glands of large cells
with eosinophilic cytoplasm and large hyperchromatic nuclei with central nucleoii. The pleural
surface and resection margin were fiee of tumor.
Post-operatively, the patient did well initially, however, she soon developed abdominal pain
and was totally unable to eat. With a diagnosis of cholecystitis, the patient undenvent a video-
assisted cholecystectomy. Post-operatively, she developed complications with abdominal bleeding
and died 32 days d e r admission.
During the course of her disease, she was treated with the following dmgs: Zantac
71
(ranitidine hydrochloride; H, receptor antagonist); Dilitiazem (antianginal agent; calcium channel
blocker); Ventolin (salbutamol; bronchodilator, beta-2 adrenergic stimulant); Lasix (furocexnide,
diuretic), Theo-Dur (theophylline, bronchodilator); Demerol (meperidine, narcotic, analgesic);
Morphine (narcotic, analgesic); and Becloforte (beclomethasone dipropionate, antinflamato~y
corticosteroid).
Tumor tissue was snap-fiozen in liquid nitrogen and subsequently stored at -80 O C . In
parallel, tumor tissue and adjacent normal tissue excised during surgery was fixed in formalin and
embedded in paraffin. Fresh fiozen tissue was used to prepare cytosolic extracts and for RNA
extraction; the parafin-embedded tissue was used for histopathology and immunohistochernistry.
5-25-3-2. Lung Ttmor Tissues
Patients. 52 turnor tissue specimens were obtained h m patients undergoing surgery for primary
lung cancer. Seven specirnens of histologically normal lung tissue were recovered fiom adjacent
tissues of the lung tumors. Diagnosis was histologically confirmed and tumors were staged using
the TNM (T-rimary tumor; N=regional lyrnph nodes; M=distant met astasis) classification system.
Tumor tissues and adjacent normal tissues, excised during surgery, were snap-fiozen in liquid
nitrogen and subsequently stored at -80°C until use.
Detailed clinicopathological features of Our patient population are summarized in Table 5-
25-3-2- 1.
5-26. Ethical Issues
Al1 research projects involving human biological specimens and clinical information were
Table 5-25-3-2-1. Clinicopathological Findings in 52 patients with primary Lung Tumors
Variable* Number of Patients ?40
Sex
Male
Fernale
Stage
Histological Type
Squarnous
Adenocarcinorna
Smail Cell
Carcinoid Tumor
Large Ce11
Nodal Status
Node Negative 38 73
Node Positive 14 27 *Age was between 42 -89 years (median 68). "Grade unknown for 5 patients.
73
approved by the local ethics authorities. Informed consent was signed at the local institution by the
patients who were d i n g to provide their biological specirnens and clinical information for medical
research. Al1 information released or published from the studies referred to code numbers known
only to the principal investigators. Extraordinary care was taken to preserve the confldentiality of
participating patients.
5-27. Statistical Analysis
The distribution of demographic and clinicopathologic variables, including age, stage,
histological grade and type, and nodal status were compared between PSA-positive and PSA-
negative patients with the use of contingency tables analyzed by the chi-square test or Fisher's Exact
test where appropnate. AU analyses were performed using SAS software (SAS Institute, Cary, NC,
USA).
CHAPTER 6, Results
6-1. Optimization of PCR Protocol
The polymerase chain reaction (PCR) is a recently developed procedure for the in-vitro
arnpilification of DNA sequences that has gained widespread acceptance in many areas of
rnolecular biology particularly in tumor biology, clinical medicine and more recently in routine
application for diagnosis. Although the basic principles are simple and straightforward, the reaction
itself involves complex kinetic interactions between template, product, pnmer, nucleotide
triphosphates, and enzymes that change throughoiit the course of the reaction. Despite this
complexity, PCR amplification is remarkably improved with the new thermostable Taq DNA
polymerase. There are, however, adjustments that can be made to some of the reaction parameters
that in some cases will drarnaticaily improve specificity and yield. These adjustments include
alterations of the reaction buffer, particularly MgC12, pnmer concentration, dNTP concentration,
enzyme conccentration, annealing time and temperature, and extension time and temperature. The
efficacy of PCR is measured by its specificity, efficiency (i.e. yield), and fidelity. A highly specific
PCR will generate only one amplification product that is the intended target sequence. More
efficient amplification will generate more product with fewer cycles. A highly accurate (Le. high
fidelity) PCR will contain a negligible amount of DNA polymerase induced errors in its product.
An ideal PCR would be the one with high specificity, yield and fidelity. Therefore, dunng
the initial attempts of developinç a PCR method for PSA gene amplification, we designed specific
primers for PSA and optimized al1 conditions of ampilification including reaction buffer, primer
concentration, annealing tirne and temperature, dNTP concentration, number of cycles, DNA Taq
75
polymerase concentration, and arnount of target used. As mentioned, PCR is a very sensitive
method. It has been s h o w that the technique can arnplfy successfùlly DNA targets that are
presents in complex mixtures as single molecules (186). Such studies are bea done with senal
dilutions of plasmid vector.
6-2. Determining Limits of Detection Methods
6-2- 1. E~hidium Bromide Staining
The detection limit of Our PCR method was first determined by amplification of plasmid
pA75 containing the full-length PSA cDNA. Ethidium bromide staining of the PCR product on a
2% agarose gel revealed a detection limit of - 1,000 copies of PSA cDNA (Figure 6-2-1-1). A
plasmid containing p-actin cDNA was also amplified using p-actin specific primers. The detection
limit of actin cDNA with ethidium bromide staining was -200 copies. In both PCRs for PSA
cDNA and actin cDNA, the PCR products were of the expected length i.e. 754 and 372 bp,
respectively .
Total RNA was extracted from the human prostatic carcinoma cell line LNCaP which is
known to express PSA and from the human breast carcinoma ce11 iine BT-20 which is steroid
hormone receptor negative and does not express PSA protein. The total RNA from both ce11 lines
was reverse transcnbed and the cDNA diluted serially and amplified by PCR. The BT-20 cDNA
did not yield any amplification products at any dilution confirming absence of PSA mRNA in this
ce11 line. The LNCaP cDNA yielded the expected 754 bp amplification product with cDNA
equivalent to 2 LNCaP cells or higher. Actin cDNA was successfully amplified fiom both ce11
lines. These data are presented in Figure 6-2-1-1B.
Figure 6-2-1-2. RT-PCR of PSA &A (A upper panel) and p-actin mRNA (B, lower panel). The PCR product was detected on agarose gels by ethidium bromide staining in both cases. Lane 1. Molecular weight markers in base pain. Lanes 2 - 8. mRNA extracted fkom LNCaP cells corresponding to 20,000 (2). 2,000 (3), 200 (4). 20 (5). 2 (6) , 0.2 (7) and 0.02 ( 8 ) cells. Lane 9. Control PCR with 9,000 molecules of plasmid pA75, containing full-length PSA cDNA as target. Lanes 10-1 1 . mRNA extracted fiom 20,000 (10) or 2,000 (1 1 ) BT-20 cells which are negative for PSA mRNA but positive for p-actin rnRNA. The detection lirnit is PSA mRNA equivalent to 2 LNCaP cells.
6-2-2. Nested PCR
Similar expenments were performed using the nested-primer protocol. With this method,
we could detect less than 10 molecules of plasmid pA75 and cDNA equivdent to 0.02 LNCaP cells
(Figure 6-2-2-1 ). This data suggests that the nested primer PCR method is about 100 times more
sensitive than the regular PCR protocol when using ethidiurn bromide stained gels for detection of
PCR products.
6-2-3. Direct DIGdUTP Incorporation
We have further developed a third highly sensitive PCR procudure for PSA cDNA using
one PCR reaction, in which digoxigenin-labeled Il-dUTP is direaly incorporated into the PCR
product. DIG- 1 1 -dUTP is added in minute amounts in the PCR mix dong with the other dNTPs.
The DIG-1 1-dUTP labeled PCR product is first run on agarose gels, it is then transferred by
Southem blotting to nylon membranes and subsequently detected using anti-digoxigenin antibodies
labeled with aikaiine phosphatase and cherniluminescence. This method gave detection lirnits of
about 10 molecules of pA75 plasrnid and 0.2 LNCaP cells (Figure 6-2-3-1 ).
6 - 2 4 Souihem B Iot Hybridtzation
A fourth detection method of PSA cDNA was based on a single PCR reaction, agarose gel
electrophoresis of the product, Southem transfer to nylon membranes, hybndization with a
digoxigenin-labeled PSA cDNA probe and detection by cherniluminescence. This method gave
detectabilities of about 10 molecules of pA75 plasmid and 0.2 cells of LNCaP. A summary of the
Figure 6-2-2-1. Nested primer PCR protocol for PSA mRNA. A. The target is pA75 plasmid. Number of pA75 m o l d e s per lane (in brackets) as follows: 0(2), 1 (3), 10 (4), 100 ( S ) , 1,000 (6), 10,000 (7). Lane 1 contains molecular wieght markers in base pairs and Lane 8 is negative control. B. The target is LNCaP mRNA equivalent to the following number o f cells: 0.002 (2), 0.02 (3), 0.2 (4), 2 (5), 20 (6). 200 (7). The detection limit in A is 510 pA75 molecuies and in B is 0.02 LNCaP celIs.
Figure 6-2-34. RT-PCR protocol for PSA mRNA with direct incorporation of DIG- 1 1-dUTP and cherniluminescence detection. A The target is pA7S plasmid. Number of pA75 molecules per lane (in brackets) as follows: O ( 2 , 1 ( 3 , O ( 4 100 (5). 1,000 (6), 10,000 (7). Lane 1 contains biotinylated rnolecular weight markers. B. The target is LNCaP rnRNA equivalent to the following number of cefls: O (2), 0.02 (3), 0.2 (4), 2 (9, 20 (6) , 200 (7). The detection lirnit in A is 10 pA75 molecules and in B is 0.2 LNCaP cells.
Table 6-2-4-1. Detection of PSA mRNA with various RT-PCR protocols
PROTOCOL DETECTION METHOD DETECTION LIMIT -
pA75 Plumid LNCaP (No. of copies) (No. of ctlb)
-- - -
1 . PCR with one set of Agarose gel elcctrophorcsis 1000 2 prîmen (AGE); ethidium bromide
staiaing (EBS) - - - -
2. Nestcd primer PCR AGE, EBS 510 0.02 - - - - -- -
3. PCR with one set of Incorporatin of DIG- 1 LdUTP; 1 O 0.2 p rim ers AGE; Southent blotting (SB);
anti-DIG antibodics labeled with alkaline phosphatase (ALP); cherniluminescence. - - -- --
4. PCR with one set of AGE; SB; hybridization with - < I O 0.2 prirners dig-labeled PSA cDNA probe;
anci-DIG antibodies labeled with ALP; cherniluminescence
developed techniques and their detectability is presented in Table 6-2-4-1.
6-3. PSA Gene Expression in Breast Tumors
In order to study PSA mRNA expression in breast tumors, we have initially screened 240
breast tumor cytosolic extracts for PSA protein using a highly sensitive immunological assay (1 74).
We then classified these tumors into three categones based on the arnount of PSA expressed Le.
highly positive tumors (PSA > 100 ng/g of total protein), weakly positive tumors ( 15 ng/g CPSA
< 100 ng/g) and negative tumors (PSA < 1 5 ng/g). We then selected 30 tumors for fùrther study at
the mRNA level using PCR technology. The results are shown in Table 6-11. The nested primer
method was not used for tumor RNA analysis since we found that this assay could not be
completely controlled for PCR contamination.
From the 10 tumors that are highiy positive for PSA protein, 5 were positive with PCR
method 1 (one PCR, ethidium bromide staining), 10 were positive by method 3 (DIG-dUTP
incorporation) and 9 were positive by method 4 (probe hybridization). One sarnple was not
evaluated by method 4. From the 10 tumors which are weakly positive for PSA protein, none was
positive with method 1, 6 were positive by method 3 and 6 were positive by method 4. From the
6 positives there were two discrepant sarnples; sarnples 12 and 1 7. From the 10 tumors which are
negative for PSA protein, none of the methods detected PSA mRNA except for sarnple 24 which
was found weakly positive only by method 4 (Table 6-3-1).
Resuits of PCR analysis of some breast tumors are shown in Figure 6-3-1. In addition to
these tumors, we also tested control tissue fiom prostate (positive with al1 methods), a lung cancer
which was metastatic fiom a prostate primary lesion (positive with al1 rnethods) and one primary
Iiver cancer (negative by dl methods).
Table 11-34 Detection
Spechen #
1. 38101
2. 38 10-5
3. 38 10-7
4. 3810-10
5. 38 14-6
6. 3816-1 1
7. 3828-3
8. 383 1-7
9. 3832-5
10. 3818-1
I I . 3827-5
12. 3833-6
13. 383 1-6
14. 38 13-8
15. 38 14-5
16. 3822-3
17. 3822-1
18. 3818-12
19. 3828-I
20. 3832-7
21. 3827-3
3 3 -- . 3832-2
23. 3832-3
24. 38324
25. 3833-1 1
26. 38 14-3
27. 3822-1 1
28. 3828-10 --
29. 38284
30. 3833-1
1 .Methoci 1 is one PCR, digo'cigenin- 1 I -dUTP
of PSA Protein PSA
( ng/g of total protein)
269
1102
828 1
1 106
3417
2387
IO4
14 15
724 1
18 1
64
8
23
24
12
79
5 2
57
20
86
O
O
O
O
O
O
O
O --
O
O
agarose gel eIectrophoresis incorporation. 3 .Method 4 is
and PSA mRNA in 30 ER/PR
(- of
18 1.'80
2'4
45 1.'661
235,'s 15
71.;16
8i4
143A2 1
16i253
341 19
1901368
0: 5
4: 1
36/73
7321296
3901307
595
21'32
32379
701'1 18
2257
5853
266i5
99; 128
971'85
O/ 0
2781582
01 1
1!0
1891245
6312
and ethidium bromide based on Sûuthern bfot and
Method 4@'
- +
NDW
- +
-
-
.-
based on
Primary Breast
Methd 1'')
-
- -
+
-
stahing. hybridization.
Tumors PSA mRNA
Method 3a)
- - +
- - -
6
- -
A
- -
-
- 2.Method 3 1s 4.ND~iotdone
Figure 11-31. Detection of PSA mRNA by RT-PCR and hybridization (Method 4) in breast tumors weakly positive for PSA protein. Lane 1 . Biotinylated molecular weight markers. Lanes 2- 1 1. Samples 1 1 to 20 of Table 2. Lane 12. Negative control. Actin PCR was positive in al1 samples (data not shown).
6-4. Steroid Hormone Regulation of PSA Gene Expression in Breast Cancer
AI1 ce11 lines used were first measured for the presence of the steroid hormone receptors,
ER and PR. The obtained results were summarized in Table 6-41.
In order to investigate the mechanism of PSA gene regulation in the breast, al1 the breast
cancer ce11 lines tested (Table 6-20-2-1) for PSA production only two steroid hormone receptor
positive ce11 lines, T-47D and BT-474, were able to expressed PSA at the protein and the rnRNA
level in response to stimulatory eRects of steroid hormones. Therefore, we were able to establish
a highly sensitive tissue culture system which reproduces in-vitro the phenornenon of PSA
production by breast cells. These MO steroid hormone receptor-positive breast carcinoma ceil lines,
T-47D and BT-474, do not produce detectable PSA when cultured in media devoid of steroid
hormones. Upon stimulation by steroid hormones , these ce11 lines produce PSA in a dose-response
manner. We have used these system to study the kinetics of PSA production, the dose-response of
PSA production by various steroid hormones and the blocking effect of antihormones.
6-4- 1. Kinetics of PSA Expression
The breast carcinoma ce11 lines, T-47D and BT-474, were cultured in the absence of any
stimulating steroid and in the presence of the stimulating steroid Norgestrel at a concentration of
10' M. The appearance of PSA mRNA was monitored with reverse transcription-polymerase chah
reaction (RT-PCR). The appearance of PSA protein inside and outside of the ce11 (secreted protein)
was monitored by the irnmunoassay procedure. The results are summarized in Figures 6-4-14 and
6-40 1-2.
Table 6-41. Steroid Hormone Receptor Levels in Cell lines Tested for PSA Production
Progesterone Receptor
(PR) fmoümg*
Tissue of Origin
--
Breast 1 1 1 1 1 023
Estrogen Receptor (ER) fmoWmg*
Breast 1 14 1 38
Breast 1 112 1 482
Breast 1 O 1 O
Breast 1 1 I 1
Breast 1 O 1 O
Breast 1 O 1 O
19 1 BG- 1
HBT- 173
Lung I 0 l 1
SOAS
Colo 320 01 Bone 78
Endornetrium 1
Coion I 1 I O
3
1
Prostate I O I O
* hoVrng of total protein
Figure 6-4-1-1. RT-PCR of PSA mRNA extracted from T-47D cells. A. Ethidium bromide-stained agarose gel. B. The gel was Southern-transferred and the PCR product, in which digoxigenin- dUTP was incorporated during PC& was detected with anti-digoxigenin antibodies and chernilurninescence. Lanes 1 and 7. T-47D cells were stimulated with absolute alcohol (solvent) and mRNA extracted at the beginning (O time) or at the end (48 h) of the experiment, respectively. Lanes 2 ,3 ,4,5,6 and 8. The T-47D ceUs were stimulated with IO-' M Norgestrel once and mRNA extracted f i e r 1 h (lane 2), 2 h (lane 3), 4 h (lane 4), 8 h (lane 5), 24 h (lane 6) and 48 h (lane 8). M. W., molecular weight markers. The PCR product is 754 bp in size. Actin RT-PCR was performed in al1 cDNAs Frorn lanes 1-8 and it was positive in al1 cases.
O Hours 1 2 4 8 24 4 8
Figure 6-4-1-2. Appearance of PSA mRNA and PSA protein intracellularly (O) or in the tissue culture supernatant (4) foilowing stimulation of T47D ceils with lod M Norgestrel. The post- stimulation time of first appearance of PSA mRNA is 2 h, of intracellular PSA protein is 4 h and of PSA protein in tissue culture supernatant is 8 h. Cells not stimulated at al1 (data not shown) or stimulated with ethanol did not produce PSA mRNA or protein during the 48 h duration of this experirnent. No PSA mRNA or protein was seen in Norgestrel- stimulated or unstimulated BT-20 breast carcinoma ce11 lines.
88
PSA mRNA is undetectable in either unstimulated cells, cells stimulated with ethanol for
up to 48 h or cells stimulated with Norgestrel after 1 h post-stimulation. PSA mRNA becomes
detectable in the Norgestrel-stimulated cells at 2 h, its concentration increases at 4 h and it persists
for at least 48 h (Figure 6-4-1-1). However, this test is semiquantitative. Quantitative information
was provided by protein data. PSA protein is first detected in the ce11 cytoplasm 4 h post-
stimulation by Norgestrel and accumulates over the 48 h study period. PSA secreted into the
culture medium is first detected at 8 h and its concentration increases rapidly with time (Figure 6-40
1-2).
An identical experiment was perfomed using the steroid hormone receptor-negative and
positive breast carcinoma ce11 lines BT-20, MDA-MB-453, ZR-75- 1, and A-427 lung carcinoma
ce11 line. These ce11 lines did not produce any detectable PSA mRNA or protein after stimulation
with Norgestrel at the indicated time periods.
The identity of the PCR product was verïfied by complete sequencing of both strands.
Partial sequencing data are shown in Figure 6-4-1-3. The entire sequence is shown in Figure 6-4-
1-4. The sequence, spanning 6 16 nucleotides from exons 4, 5 and the 3'-untransiated region is
>99% homologous to the published sequence of PSA cDNA or genomic DNA. We found 1000/0
homology with the sequence published by Lundwall(187), Digby et al (5 1) and Klobeck et al (188).
There is one base difference (A to G) at position 439 (3'-untranslated region) of our
sequence and the sequence published by Lundwall and Lilja (39 , Shultz et al (1 79), Henmi and
Vihko (41), and Riegman et al (180). Also, at position 419 (3'-untranslated region) we and others
have identified G but a few other investigators have identified T. These differences are likely
polymorphisrns.
GCTCGGGTGA
ATCACGTCAT
GTACACCAAG
CWCCCCTG
7TGGAAATGA
GTCCTTAGGT
AGGTGTAGAC
TCCTGGGGAA
GGACACAGAT
AAGAGGGGTG
ACTGTCCATG
TCACAGCAAG
TGGWGCCT
ITCTGGGGGC
GGGGCAGTGA
GTGGTGCATT
AGCACCCCTA
CCAGGCCAAG
GTGAGGTCCA
CAGAGTGrrr
TACTGGCCAT
AGGAT GGGGT
GGATCCACAC
MGCACTGAG
GATGGAGCT G
AGAGAA
CCACftGTCT
ACCAT GTGCC
ACCGGAAGTG
TCAACTCCCT
ACTCAGGCCT
GGGITGCTAG
CTTAMTGGT
GCCTGGAGAC
GTCTGTGTTA
TGAGAGAGTG
CAGAAGCTGG
AAAAC ATAAC
GTAATGGT GT
CTGCCCGAAA
GATCAAGGAC
ARGTAGtAA
CCCCAGiTCT
GMMGAAAT
GTAArnGT
ATATCACT CA
r t f G f GGGGT
GAGAGTGACA
AGGCACAACG
CCACf CTGTC
GGCCTKCCT
ACCATCGTGG
ACITGGAACC
ACTGACCn-r
CAGCAGACAC
CCTCTCTGTG
AmCTCTGA
ACAGAGATGA
TGTGCTGGAC
CACCAGACAC
CTGGAGGCAC
Figure 6-4- 1-4. Complete sequence of the 6 16 nucleotide region of die PSA cDNA sequence from Norgestrel-stimulated T47D cells. For discussion see text. Bold and underhe indicate end of exon 4 and exon 5 , respectively.
9 1
Cornparison of Our 616 nucleotide sequence with the sequence of human glandular
kailikrein gene revealed only about 80% homology. These data confirm that the mRNA isolated
fiom Norgestrel stimulated T-47D and BT-474 cells is indeed the PSA rnRNA.
6-4-2. Induction of PSA Production by Steroid Hormones
In order to establish which of the compounds shown in Table 5-20-1-1 act as PSA gene
regulators, we stimulated T47D and BT-474 breast cancer cells and measured PSA in the tissue
culture supernatant at 3, 5 and 8 days post-single stimulation at a compound concentration of IO-' M.
The compounds were then qualitatively categorized into t hree groups; non-stimulators, weak
stimulators and strong stimulators. For this classification, we arbitrarily selected a scpematant PSA
concentration at 8 days of >ZOO ng/L, 10-200 ng/L or <IOng/L for strong, weak and non-
stimulators, respectively. Some stimulation data are shown graphicaily in Figure 6-4-24. Detailed
data are summarized in Table 6-4-2- 1.
6-4-3. Delermina~ion of Dose- Response Stimulatory Effects of Steroid Hormones
Dose-response experiments were designed to detemine the lowest concentration of
stimulating steroids which could still induce production of PSA. Representative dose-response
experiments are shown in Figure 6-4-3-1. The lowest concentration of stimulating steroids which
could induce PSA production is shown in Table 6-4-2-1. Among the steroids tested, the most
potent were three androgens (dihydroandrosterone, R1881, and dihydrotestosterone) and four
synthetic progestins (Norgestrel, Norethynodrel, R2050, and Depo-provera). These four steroids
couid induce PSA production at final concentrations down to IO-" M. Two other androgens
(testosterone and androsterone) and a synthetic progestin (Norgestimate) were potent at
Stimulation Compound
Figure 64-24. PSA concentration in T-47D ceil line tissue culture supematants d e r a single IO-' M stimulation with various compounds and sarnpling of the supematants at 3(.), 5(.) and 8(A) days post-stimulation. The compounds tested were: 1, testosterone; 2, estrone; 3, vitamin D; 4, corticosterone; 5 , dihydroandrosterone; 6, estriol; 7, 17a-hydroxyprogesterone; 8, androsterone; 9, hydrocortisone; 10, 1 7a-ethynylestradiol; 1 1, norethynodrel; 12, tamoxifen; 13, P-estradiol; 14, betarnethasone- 17-valerate; 15, norethidrone; 16, norgesuel; 1 7, aldosterone; 1 8, dexamethasone; 1 9, cholesterol; 20, Ri88 1; 21, R5020; 22, 23, no stimulation; 24, aicohol. in ali cases the most drarnatic change in PSA concentration occurs in the interval between 3-6 days. The strongest stimulators are androgens (1,5,8,20,21) and progestlis (1 l,l5,l6). More data are given in Table 642-2 .
93 Table 6-4-2-1. PSA concentration in T-47D ce11 Iine tissue culture supernatant after stimulation with various compounds
1
VaHydraxvprogestm Progesteme peairsor Nothing
f+ '%Wtae Rogesîm Weak
8e tamethasone 1 ?-valerat e
Dexarnetha~orie
Prednisone
G l u c a u k n d
Glucocwticoid
Glucocorticoid
- - . - - -
NOrgestrel
DepuPrwera
Norgesh'mate
Aldosteme
RU 58,668 1 Antjeserogen I Nothing I
- - -
s m g
S W
Ming
- -
Progestin Progestin
Triamiandone acetonde
TamOufen
- p. - - -
ND
10.'
Progestin I Strong Mineraloconicoid Weak
1 Antirnineralocorticoid 1 Weak 1 IO-'
- - - -- -
SLrorig
s m l IOW
10"
Progestin/giUOOCOrtjcoid
Anliesîrogen
Casodex (Cl 176.334)
RUS6.187
Nitutmide (Anandron)
Mdepistone (Rü486iRU38.486)
Cuîexdane
1. For definitions. see lexi. 2. ND; not done.
r 10"
rl(rl'
Str0ng
Noihing
Antiandrogen
An fiandmgen
Antiandrogen
Antiprogestin
A n t i g l u m ï d
IDw
Noîhing
Weak
Nothing
Weak
Weak
lo4
l@
10'
Figure 64-11. Dose-response experiments of six representative steroids. For more details and discussion see text.
95
concentrations down to lO-'OM. 17a-Ethinyle~radiol, unlike to al1 other estrogens tested, was able
to positively regulate PS A production but only at relatively high concentrations ( 1 0"- 1 0' M).
Arnong the glucocorticoids, corticosterone was a weak stimulator while betarnethasone and
dexarnethasone were strong stimulators but only at concentrations >IO-' M. Aldosterone was a
weak stimulator but the synthetic compound triamicinolone acetonide (TA) was a strong
stimulator, acting at low concentrations (lO*"' M). None of the antiestrogens exhibited stimulatory
activity. Among the antiandrogens, some did not induce any PSA production, one was a weak
stimulator (RU 56,187) and one (cyproterone acetate) was a strong stimulator, acting at
concentrations as low as 10-'O M. The antiprogestin Mifepristone (RU 38,486; RU 486) as well as
the antiglucocorticoid cortexolone and the antimineralocorticoid spironolactone were weak
stimulators, acting at concentrations 1 O"- 1 0-'M. The androgen rnetabolite dihydroisoandrosterone
sulfate and the progesterone precursor 17a-hydroxyprogesterone were inactive. Dose-response
experiments with the estrogens estrone and estradiol have s h o w that these compounds remain
inactive with respect to PSA gene up-regdation at any concentration between 1 O-'- 1 O-'' M.
Stimulation expenments were funher conducted using the steroid hormone receptor-
negative breast carcinoma ce11 lines BT-20, HBL- 100, and the steroid hormone receptor-positive
breast carcinoma cell iines MCF-7,ZR-75- 1, MDA-MB-453, and MFM-23 3 . The sarne experirnent
was carried out using the steroid hormone receptor positive cell lines SAOS (osteosarcorna), MFE-
296 ( endometnum) and BG-1 (ovarian carcinoma) and the unknown receptor status of ce11 lines
HBT- 16 1 (ovaian), HBT- 16 1 (ovarian), HBT- 173 (ovarian), A-427 (lung), SK-MES- 1 (lung),
NCLH460 (lung), MIA PaCa (pancreas), and Co10 320 (colon). None of the cornpounds listed in
Table 5-20-2-1 was able to induce detectable PSA protein production.
96
6 - 4 4 Blochg Steroid Hormone Receptors
In order to fbnher elucidate the mechanism of regdation of PSA production by steroid
hormones, we have conducted blocking experiments. In these studies, we have first treated the T-
47D and BT-474 cells with a steroid hormone receptor blocker for 1 h followed by the addition of
a 10-fold lower concentration of a stimulating steroid. The data are presented in Table 6-4-44.
Estradiol, as well as the antiandrogens Nilutamide, RU56,187 and hydroxyflutamide, were
able to significantly block the stimulating action of dihydrotestosterone in T-47D but not as strong
as in BT-474 celi line. Mifepristone was also a potent blocker in T-47D and a weak blocker in BT-
474 ; no blocking activity was seen arnong the antiestrogen ICI 182,780 the antimineralocorticoid
spironolactone and the antiglucocorticoid cortexolone in T-47D but only ICI 182, 780 had a weak
blocking effect on dihydrotestosterone in BT-474 ce11 line.
The stimulatory activity of Norgestrel was blocked minimally by estradiol, Nilutamide,
hydroxyflutamide and ICI 182,780 and to a higher degree by RU 56, 187 in T-47D. However,
arnong those blocker only RU 56.187 was able to block a fesser degree in BT-474. The most potent
blocker of Norgestrel's action was the antiprogestin Mifepristone (blocking 90- 100%) in both cell
lines. The stimulatory activity of the highly selective progestin agonist Norgestimate was only
blocked by the antiprogestin Mifepnstone (blocking 100%).
The stimulatory activity of dextarnethasone and aldo sterone were not bloc ked by estradiol
as well as Nilutamide, hydroxyflutarnide, RU56,187, Mifeprktone, ICI 182, 780, spironolactone,
and cortexolone in BT-474 breast carcinoma ceIl Iine.
Table 6 - 4 4 2 . Blocking of PSA Production in T-47D ceiis by Various Compounddl)
Stimularing Compound (1 0%)
Blochg Cornpound ( 10SM)
Estradio1
Nilutamide
RU 56,187
Hyiroyflutamrde
ICI 182,780
Mifepnstone
Spironolactone
Cortexolone
1. For detaiied protocol refer to Methods section.
7 . Range of 3 dfierent experiments.
98
6-5. Variation of PSA Protein Dunng the Menstmai Cycle
We collected approximately 8-10 serum sarnples from each of the 14 normal fernale
volunteers during the foilicular, mid cycle and luteal phase of the menstrual cycle. PSA protein was
measured in these semm sarnpies using a highly sensitive tirne-resolved immunofluorometnc
technique (1 8 1). This assay has within-run coefficients of variation of < 10% at PSA levels around
1 ng/L or higher. AI1 samples from the same patient were measured in duplicate or tnplicate in the
same assay mn to minimize variability. The PSA concentration patterns were verified in at least
3 different assay runs per patient and the results were consistent in al1 cases.
Anaiysis for PSA reveaied that in 11 patients the PSA levels were below 4 ng/L in dl
samples coliected. Since the detection lirnit of the assay used is 1-2 ng/L, we did not use these data
for further analysis. Among the three volunteers whose senim PSA levels were >4 n g L , we
collected additional sera from two. These new sera spanned two consequtive menstruai cycles and
were collected at least two months after the sera f?om initial collection. Thus, in total, we studied
7 informative menstrual cycles From three different patients. The days of the menstrual cycle in
each case was verified by analysis of progesterone, estradiol, LH, and FSH.
In Figure 6-51, we present the semm PSA changes during the menstrual cycle of patient
M.T. (3 cycles) and in Figure 6-5-2 the data for patient C.J. (3 cycles) and L.S. (1 cycle). In the
sarne graph, we present the progesterone values as well. When we plotted the PSA data along with
data for LH, FSH, or estradiol, no recognizable pattern or relationship was seen. The data of
Figures 6-54 and 6-5-2 show a consistent pattern with PSA peaking during the rnid-late follicular
phase and reaching a minimum during the rnid-Iate luteal phase. The difference between PSA and
progesterone peaks is about 10-20 days.
O 1 O 20 30
Days of Menstrual Cycle
O 10 20 30 40 SO 6 0
Days of Menstrual Cycle
Figure 6-51. S e m prostate specific antigen (m) and progesterone (+) Ievels during the menstnial cycle ofvolunteer M.T. A One rnaistnial cyde. B. Sera fiom two additionai consecutive menstrual cycles coilected at least two months after the sera of panel A. Notice the peak of PSA which foiiows the progesterone peak with a delay of 10-20 days.
O 1 O 20 10
Dayr of Mrnstrual Cycle
O t
O 1 O 20 3 O O
Days of Menstrual Cycle
Oays of Menstrual Cycfe
Figure 6-5-2. S e m prostate specific antigen (@) and progesterone (*) levels during the menstrual cycle of volunteers C. J. and L. S. A. One rnenstrual cycle of volunteer C. J. B. One menstruai cycle of volunteer L. S. C .Sera f?om h ~ o additional consecutive menstmal cycles of volunteer C.J. coilected at least two months &er the sera o f panel A Other cornrnents as in Figure 6-54.
101
We have further exarnined if semm obtained during the menstrual cycle has the ability to
stimulate PSA production in the breast carcinoma ce11 line T-47D. This expenment was performed
by measuring either PSA rnRNA or protein. We found that non stimulated T-47D cells do not
produce PSA protein and do not express PSA mRNA. When the cells are stimulated with serum
obtained during the menstnial cycle, the results of Figure 6-5-3 are obtained. The ability of the
semm to induce PSA production parallels the levels of progesterone. Maximal stimulation is
achieved with the semm that contains the maximal progesterone concentration (day 24 of the
menstnial cycle).
PSA mRNA in T-47D cells can be detected in trace amounts if the serum used for
stimulation is collected during the follicular phase of the cycle. When the serum is collected during
the iuteal phase, PSA mRNA expression is dramatically increased (Figure 6-5-4). These changes
in PSA mRNA levels parallel the changes in progesterone levels. PSA mRNA is undetectable in
T-47D cells which are not stimulated by serum.
We have further stirnulated T-47D cells with the glycoprotein hormones LH, FSH, hCG and
with prolactin and growth hormone. Despite the wide range of concentrations used, none was able
to induce PSA production. Progesterone was able to induce T-47D cells for PSA protein production
and PSA mRNA expression at concentrations between 1 O-'- 1 0-l0 M-
6-6. Expression of the PSA Gene in Lung Tissue
The tumor extract from the primary lung adenocarcinorna was measured for PSA protein;
the PSA concentration obtained was 958 ngL and was considered highly positive for PSA
immunoreactivity . In contrast, another 1 0 lung adenocarcinornas from women, when extracted,
O 1 O 20 3 0
Days
Figure 6-5-3. Serum progesterone levels during the rnenstnial cycle of a nomaliy cychg volunteer (m). These sera were used to stimulate PSA production in the breast carcinoma ce11 line T-47D as dûcnbed under methods. The PSA concentration in the asnie culture supematants is shown (+). Notice that sera which contain high levels of progesterone are able to induce PSA production in the c d h e T-47D. Data on PSA rnRNA expression are presented in Figure 6-5-4.
Figure 6-5-4. Reverse transcription polymerase chah reaction of PSA mRNA (A,B) and actin (C) obtahed from T47D celis stimdated with semm of volunteer MM. The serum was drawn at days 5 (lane 2). 7 (lane 3), 10 (lane 4), 14 (lane 9, 16 (lane 6), 21 (lane 7), 24 (lane 8) and 28 (lane 9) of the mensaual cycle. Lane 10 represents a positive PCR control (plasmid containhg full-length cDNA for PSA) and lane 1 1 is the negtive PCR control (no target). Notice the intense PCR bands produced when s e m Eorn the luteal phase of the cycle is used to stimulate T-47D ceils. In panel A the PCR product was detected with cherniluminescence; In panels B and C with ethidium bromide staining of agarose gels.
1 O4
exhibited PSA concentrations <20 n@. Contamination of the tumor extract by blood PSA is
unlikeiy since the PSA concentration in the serum of this woman was 4 ng/L preoperatively and 5
ng/L postoperatively.
In order to estabiish the molecular weight of the immunoreactive species in the tumor
extract, we performed high performance iiquid chromatography with a gel filtration colurnn (Figure
6-44). Virtuaily 100% of the immunoreactive species elutes at fraction 40, corresponding to a
molecular weight of approximately 33 KDa (free PSA). No complex between PSA and a,-
antichymotrypsin (molecular weight - 100 KDa) was seen.
Total RNA exrracted from the lung tissue was reverse-transcnbed to cDNA and then
arnplified by PCR using pnmers specific for the PSA cDNA sequence. Southern blot hybndization
of the PCR product with a PSA DIG-labelled cRNA probe detected the PSA band of the expected
size (754-bp) in the sarnple but not in the negative control (Figure 6-6-2). This band was
reamplified (to obtain enough material) and sequenced. Both strands were sequenced and the data
were compared with the published PSA and glandular kallikrein cDNA sequences.
Partial sequencing data are shown in Figure 6-6-3. The entire sequence obtained is
presented in Figure 6-6-4. The sequence, spanning 549 nucleotides from exons 4, 5 and the 3'-
untramlateci region has >99% sequence similarity to the published sequence of PSA cDNA or genomic
DNA (Table 6-64). However, there is only about 80% similarity with the human glandular kallikrein
gene in accordance with literature reports (22).
We found four positions where there is variation between Our sequence and those reported in
the literahire. These ciifferences, alI present in the 3' untranslated region. may represent polymorphisms.
At two positions (position 427 and 447) the variation appears in the same spot (underlined) of the
Free PSA
2 5 SS 4 S
Fraction Number
Figure 6-64 Molecular weight identification of PSA irnmunoreactivity in the lung tissue exuaa ushg HPLC with a gel filtration column. Mer injecting 500 PL of tumor extract, HPLC &actions (0.5 mL) were analyzed with an assay that recognizes both free PSA (33 KDa) and PSA-ACT complex (100 KDa). Al1 PSA immunoreadvity in the lung tumor extract elutes at a molecular weight of 33 KDa (fraction 40 + 2) and represents f i e PSA PSA-ACT complexes (eluthg at fiaction 3222) were not found.
Figure 6-6-2. Reverse transcription PCR of RNA extracted fiom various tissues. Panel A. The PCR products were run on an agarose gel, stained with ethidium brornide. Lane 1. Molecular weight markers with length in base pairs shown on the left. Lane 2. Digoxigenin-labeled DNA markers (not stained). Lanes 3 and 4. RT-PCR product for the patient described here; no bands are visible with this method. Lane 5. RT-PCR produa for a patient with a PSA-positive breast turnor (positive control). Lane 6 . RT-PCR product for a patient with a PSA-negative breast turnor (negative control). Lane 7. Amplification of 9,000 m o l d e s of a plasrnid containing the f ù U sequence of PSA cDNA (positive control). Lane 8. PCR negative control (no target added). Lane 9. PCR for actin cDNA for the patient described here, venfjmg the success of the reverse transcription. In lanes 5 and 7, the PCR produa is 754 bp and in iane 9 is 372 bp. Panel B. PCR products were run on an agarose gel, Southem-blotted and hybridized with a DIG-labeled RNA probe. Detection of hybrids was achieved with cherniluminescence. Lane 1. DIG labeled molecular weight markers with length in base pairs show on the lefi. Lane 2. RT-PCR produa for the patient described in this paper. The 754 bp PCR product is clearly identified dong with a higher rnolecular weight produa (genomic DNA). Lane 3. PCR negative control.
AAGCACCTGC
TTCAAGGTAT
CCTCCCTGT
CATCGTGGCC
lTGGAACCIT
TGACCllTGT
GCAGACACAG
TCTCTGTGTC
TTCTCTGAGG
AGAGATGAAA
TGCTGGACAC
TCGGGTGATT
CACGTCATGG
ACACCAAGGT
AACCCCTGqG
GGAAATGACC
CCTTAGGTGT
GTGTAGACCA
CTGGGGAATA
ACACAGATAG
GAGGGGTGGG
TGTCCATGAA
CTGGGGGCCC
GGCAGT GAAC
GGTGCAlTAC
CACCCCTATC
AGGCCAAGAC
GAGGTCCAGG
GAGTGrrrCT
CTGGCCATGC
GATGGGGTGT - ATCCACACTG
GCACTGAGCA
ACTTGTCTGT
CATGTGCCCT
CGGAAGTGGA
AACICCCTAT
T CAGGCCTCC
GUGCTAGGA
TAAATGGTGT
CTGGAGACAT
CTGTGlTATT
AGAGAGTGGA
GAAGCTGGAG
AATGGTGTGC
GCCCGAAAGG
TCAAGGACAC
TGTAGTAAAC
CCAGlTCTAC
AAAGAAATCA
A A r n G T C C
ATCACTCAAT
TGTGGGGTAC
GAGTGACATG
GCACAACGC
Figure 6-6-4. Complete sequence of the 549 nucleotide region of the PSA cDNA sequence from the mRNA isolated from the lung tissue. Bold and underline indicate end of exon 4 and exon 5, respectively. Underiined sequences represent a six base repeat in which one nucleotide (double underiined) is fiequently polymorphic (Table 1). Sequence variation was also seen at positions 234 and 184 (underhed).
Table 6-61. C o m p ~ s o n of DNA sequence of PCR product found in this mdy with sequence deposited in GenBank
GenBank Accession Positiodl)
DNA 427 447 234 184
This Study (Lung Tissue) cDNA
cDNA
cDNA
Genomic
cDNA
cDNA
Genomic
cDNA
Genomic
1. Refcn to nucleotide nurnbcr shown in Figure 6-64. Yucleotides not shown are identical in al1 sequencts
Figure 6-64. Immunohistochemical localization of PSA in lung tissue with polyclod antibody. A: The lung tumor does not contain PSA imrnunoreactivity in epithelial neoplastic ceiis or in the stroma1 component. B: A trapped non-neoplastic bronchiole at the edge of the m o r contains PSA imrnunoreactivity in epitheliai lining ceus. The cytoplasmic positivity is arong but restricted to scattered ceus (arrowheads). Tumor ceiis (T) around the bronchiole are negative. (Streptavidin-biotin technique; ~ 2 5 0 ) .
1 1 1
sequence repeat T G G E T (Figure M).
We localized the PSA immunoreactivity with immunohistochemistry (Figure 6-6-5). While
the lung tumor was entirely negative for P S 4 trappeci non-tumorous bronchioles at the edge of the
tumor contained focal PSA immunoreactivity in epithelial lining ceiis.
Since the PSA gene is regulated by steroid hormones (96), we examineci if the only steroid
administered to the patient durùig hospitalization (beclomethasone dipropionate) was able to stimulate
PSA production in a tissue culture system. The breast carcinoma ceii lime BT-474 does not produce
PSA when cultureci in the absence of steroid hormones but it does so when stimuiated by androgens,
progestins and glucocorticoids. Beclomethasone dipropionate was able to stimulate PSA production
in this ce11 line in a dose-dependent manner at levels 10"- 10" moi/L (Figure 6-6-6).
6-7. Frequency of PSA mRNA Expression in Lung Tumon
Due to the marked differences between semm PSA concentrations in males and females
(males have approximately 500-fold higher levels), a11 data were analyzed separately between males
(N=3 5) and females (N=17). PSA levels in tumor cytosols were expressed as ng of PSA per g of
total protein to normaiize for the arnount of tissue extracted. The average total protein
concentration of these extracts was -1 g/L. Any level of PSA 4 1 ng/g or < 1 ng/L was considered
as non-detectable since it is below the detection limit of the PSA assay used (50).
The distribution of lung tumor PSA content between males and females is shown in Table
6-7- 1. In the sarne Table, we summarize data of serum PSA in these male and female patients.
Both pre-operative (-5 days before surgery) and post-operative (-5 days post-surgery), PSA levels
were measured. The following comments apply: (a) Levels of PSA in tissue extracts fkom males
Figure 6-66. Production of PSA by the breast carcinoma ceIl line BT-474 der stimulation by beclomethasone dipropionate and other steroids. PSA was measured in the tissue culture supernatant 5 days post-stimulation of confluent celIs with a simgle dose. 1 . Norgestrel (progestin). 2. Beta- methasone (glucocorticoid). 3. Dexamethasone (glucocorticoid). 4. BecIomethasone (glucocorticoid). 5 . Prednisone (glucocorticoid). 6. Estradio1 (estrogen). Dosedependent PSA production is seen at beclomethasone levels between 1 O-'- 1 0.7 moVL.
Table 6 - 7 4 Distribution of PSA in Lung Tumor Cytosols and in preoprative and Postoperative Sera fiom Male (M) and Fernale (F) patients.
Percentile
Tumor Cytosols (ng/g)
Preoperative Semm (ng/L)
Postoperative Senim (ng/L)
114
are higher than those in females. (b) We found no consistent changes between pre-operative and
post-operative semm PSA in either males or females. (c) Analysis of normal lung tissue extracts
from 3 males and 4 females has revealed that PSA was also detectable at levels 1.9 ng/g (median)
for males and 2.9 ng/g (median) for femaies.
We have examined if the PSA levels in the tumor cytosoIs were associated with the semm
levels in both males and femaies. The Spearman correlation coefficient between tumor cytosol PSA
and preoperative serum PSA in males was 0.80 (p=0.000 1) and in femaies was 0.53 (p=0.05). The
Spearman correlation coefficient between tumor cytosol PSA and postoperative semm PSA in
males was 0.5 1 (p=0.0 1) and in femaies was 0.37 (p=O. 18). These data suggest that there is a
significant correlation between tumor levels of PSA and serum P S 4 at least in male patients.
In Our series, only four patients died during the follow-up of 6-1 2 months. For this reason,
and due to the smdl number of patients, no survival anaiysis in relation to PSA levels in the tumors
was performed.
PSA mRNA was detected in lung tumors with RT-PCR and Southern blot hybridization.
From the 35 lung tumors from males, 24 (69%) were positive for PSA rnRNA. From the 17 lung
tumors from females, 9 (53%) were positive for PSA mRNA Of the 7 adjacent normal lung tissues,
five (71%) were dso positive for PSA mRNA. Examples of PSA mRNA detection in lung tumors
are shown in Figures 6-7-1 and 11-7-2. In addition to the expected 754 bp PCR product, we
occasionally detected bands of molecular weights between 250-500 bp (Figure 6-70 2). The
identity of these bands is discussed below. Sequencing of the 754 bp PCR product from one female
patient with iung cancer confirmed the 100% identity with the published sequence of PSA cDNA
(187).
Figure 6-7-1. A: RT-PCR for PSA mRNA extracted from lung tumors. B: RT-PCR for actin mRNA extracted fiom lung tumors. Lane 1. Molecular weight markers. Sizes in base pairs (bp) are shown in lefi panel. Lanes 2-1 1 . Lung tumors. Lane 12. PCR negative control (no template added). In A, the PCR products were detected by Southem hybridization and cherniluminescence. In B, the PCR products were detected by ethidium bromide staining. The 754 bp PCR product between molecuiar weight markers 900 and 692 bp corresponds to PSA. The 372 bp PCR product (in panel B) corresponds to actin. Tumors in lanes 2, 4, 5, 7, 8, 9 and 10 are positive for PSA rnRNA.
Figure 6-7-2. A: RT-PCR for PSA mRNA extracted from lung tumors. B: RT-PCR for actin mRNA extracted fiom lung tumors. Lane 1 . Molecular weight markers. Sizes in bp are s h o w in Iefl panel. Lanes 2-5. Lung tumors. Other cornments as in Figure 14-7- 1. Aberrant PCR bands appear in lanes 4 and 5 o f panel A Other aberrant bands appeared at molecular weights of 250-500 bp (not shown).
117
The association between PSA protein and mRNA expression in lung tumors and other
clinicopathologicai variables was examined by contingency tables and chi-square statistical
analysis. In males, using as cutoff the median PSA level in lung tumors (expressed as ng/g of total
protein), we found no association between PSA protein and presence or absence of mRNA
(p4 .33) . patient age @=0.88), stage (p=0.24), grade (p=0.36), histological type (p=0.67) or nodal
status (p=0.23). The only statistically significant association was with preoperative (p-0.003) and
postoperative (p4.006) serum PSA, an association also revealed by Spearman correlation analysis
(already described). In females, no significant association was found between tumor PSA levels
and any of the above clinicopathological variables.
We have fùrther examined if the presence of PSA mRNA in lung tumors is associated with
any of the clinicopathologicai variables. In males or females, the presence of PSA M A was not
associated with patient age, stage, grade, pre-surgical or post-surgical serum PSA or nodal status.
We found some evidence that the presence of mRNA in males is associated with squamous ce11
carcinomas. From the I I tumors which were PSA mRNA-negative, four (36%) were squarnous
cell, six (55%) were adenocarcinomas and one (9%) was of other histologic type. From the 24 PS A
mRNA positive tumors, seventeen (7 1 %) were squamous cell, four (1 7%) were adenocarcinomas
and three (13%) were of other histologic type (p=0.07).
In Figure 6-7-2, we have shown that we occasionally detected aberrant PCR products of
rnolecular weights between 250-500 bp. We have isolated and cloned six of these products and
subsequently sequenced al1 of them using vector-specific sequencing primers. Three of these
products proved to be primer-dimer artifacts which hybndized to the probe due to sequence
complementarity between the PCR primers and our riboprobe which contains the primers in its
TGCCAATGGG
T C i G A C A
AACI'TAAAAA
TGGATCTTCA
GGGAGAACTG
CAAGACATGG
T GITCTGCAA
ACTGCAACTA
GGrrrAAGAA
TiATCCAGCA
TGAATGTAGT
TAC CTACAAA
GAATACTGAA
ITCTAAGGTG
AAAAAAACAT
TGCTAGGTGT
GAAITGT GGT
CTAAGCAGTA
AGAAGCAGAT
TGAAATCCAT
ATCCAGGGAG
TCCCAAAm
ACCCAGGAAT
TAAAATGGAT
GCCClTGTAG
CCCTGGTCAT
TGTGTAAGCT
CAGAATATAG GMATATCAC
m A A G T T A GTTAAGGACT
ACTGAClTCA GTAlTïGTCA
CAATACTCGC TTCTCAAACA
GCCAGCCATA CAAATATGCT
TTGCTGCAAC TGATAITCTG
TCCAAGAACT AACACTGCAG
GTCACTTCCA AGAAGTATCA
GAAAAGGGAT TAAGAGTGAG
Figure 6-7-3. Sequence of a 450 bp PCR product detected in addition to the 754 bp PSA PCR product in some lung tumors. This sequence has no homology to any other sequence deposited in GenBank.
118
sequence. The other three aberrant PCR products represented the same sequence consisting of 450
bp (Figure 6-7-3). This PCR produa was created only by primer PSA-A2 since the complete
sequence of this PCR product had in one end the primer PSA-A2 and at the other end of the same
strand a sequence complementary to PSA-A2. The 450 bp sequence, venfied by sequencing of both
strands, had no sequence hornology to PSA cDN4 kallikrein cDNA or any other sequence
deposited in GenBank as revealed by BLAST or DNASIS software analysis.
CHAPTER 7. Discussion
7-1. Detection of PSA Protein and mRNA in Breast Tumors
Prostate specific antiçen (PSA) is a senne protease found at very high concentrations in
seminal plasma. It has been suggested that PSA is involved in semen liquefaction post-ejaculation.
PSA is considered a highiy specific biochemical marker of the prostate gland and is currently used
for diagnosis, prognosis, and management of patients with prostate cancer (1-3).
Our group has recently dernonstrated that approximately 30% of fernale breast tumors
produce a 33 D a protein with stnking sirnilarities to seminal plasma PSA (4-6). This
immunoreactive PSA was shown to be associated with the presence of steroid hormone receptors,
earlier disease stage, and younger patient age (6). Preliminary data suggest that PSA may be a new
favorable pronostic indicator in breast cancer (12).Irnrnunoreactive PSA has been detected in
various tumors including ovarian, colon, h g , and parotid tumors. However, PSA expression in
these tumors is infiequent and occurs at much lower levels than in breast tumors (7). Recently, it
has been dernonstrated that PSA mRNA isolated from a few PSA positive breast tumors had an
identical sequence to the PSA cDNA derived fiorn prostate tissue (95).
Polyrnerase chah reactraction (PCR) is a very sensitive procedure for in-vitro amplification
of DNA sequences. PCR has gained widespread acceptance in many areas of molecular biology
particularly in tumor biology, clinical medicine, and more recently in routine diagnostic
applications. RT-PCR-based methods for analyzing rnRNA expression specific for neoplastic ceils
have been applied for detecting micrometastasis or minimal residual disease (1 82,189).
120
In this study, 1 have developed sensitive RT-PCR methods for detecting PSA rnRNA.
Among the variations tested, the nested primer assay was the most sensitive but this method is
difficult to control for contamination and was not used to assay the breast tumors. The simple PCR
assay in which PCR products are detected by ethidium bromide staining was also found inadequate
since many PSA protein-positive tumors tested negative for PSA mRNA with this method. The
other two modifications in which the PCR products are detected with cherniluminescence with use
of digoxigenin as label were found to be satisfactory. Data generated with these two methods were
in good agreement with data fiom the analysis o f PSA protein with a highly sensitive immunoassay.
The observed few discrepancies are likely due to tumor heterogeneity since in PSA-positive tumors,
not ail tumor cells produce PSA; the immunoreactivity is focal and restricted to clusters of cells as
detected by imrnunohistochernical localization. Tumor heterogeneity is likely the reason why we
did not observe a consistent correlation between levels of PSA protein and intensity of the PCR
bands; two different tumor pieces were used for cytosol extraction and PSA protein analysis and
for rnRNA extraction and PCR.
In the last few years, rnany other groups have developed sensitive PCR procedures for PSA
mRNA. Deguchi et al. (1 82) developed a PCR assay with sensitivity between O. 1- 1 LNCaP ceIl
and used it to detect prostatic carcinoma turnor cells invading the regional lymph nodes. Moreno
et al. (190) developed a PCR assay of unspecified sensitivity and used it to detect prostate cancer
cells in the circulation of patients with advanced cancer. Katz et al. (19 1) developed a PCR assay
for PSA mRNA which incorporates DIG- 1 1 - d W in the reaction products and demonstrated that
they could detect 1 LNCaP ce1 admixed with 100,000 human blood lymphocytes. When they used
their method to assay human blood, they found that 78% of prostate cancer patients with metastatic
121
disease and 39% with localized disease had cancer cells in the circulation. More recently, Jaakkola
et al. (192) have developed a nested primer PCR assay with the ability to detect -2 LNCaP cells
admixed with 106 leukocytes. These authors found that PSA mRNA could not be detected in the
blood of patients with benign prostatic hyperplasia, non-prostatic types of cancer or in patients with
localized prostate cancer; 50% of patients with metastatic disease tested positive.
A universal finding arnong these midies (190- 192) is that none of the numerous bloods From
normal males or females tested positive for PSA mRNA. However, Smith et al. (1 93) reported
100% positivity arnong 7 normal males and 6 normal female blood samples, using a highly sensitive
nested primer protocol. Since nested-primer procedures are prone to contamination, we recommend
caution before concluding that blood samples from any male or female contain traces of PSA
mRNA until the data by Smith et al. are reproduced by others.
The data presented here support the view that assessrnent of PSA production by breast
tumors c m be studied by either highly sensitive immunoassays for PSA protein (1 82) or by highly
sensitive RT-PCR assays, like those described here for PSA mRNA. We anticipate that the RT-
PCR-based assays will be usehl to assess PSA mRNA levels in tumors other than those of the
breast and in non-prostatic tumors and tissues from males. Although the PSA protein assay is
extremely sensitive, it suffers from the major limitation that male blood contains significant
amounts of PSA which may contaminate tumor or tissue extracts from males. Detection of PSA
mRNA would effectively circumvent this potential problem.
7-2. Steroid Hormone Regulation of PSA Gene in Breast Cancer
The PSA gene is known to be regulated by androgens in the male prostate (194,195). The
122
epitheliai cells of the prostate gland are rich in AR, some stromai cells aiso contain AR, as well as
the enzyme 5 a-reductase which reduces testosterone to dihydrotestosterone ( 1 94). The PS A gene
has an HRE to which the activated AR can bind (45,188,195-197). The PSA gene is up-regulated
by androgens and androgen agonists and is down-regulated by antiandrogens.
We have recently shown that female breast tissue and breast secretions contain high levels
of PSA (198). Although the physiological role of this protein in the female breast is still unknown.
we have demonstrated that the presence of PSA is strongly associated with presence of steroid
hormone receptors (6) . I have thus postulated that the PSA gene in the fernale breast is regulated
by steroid hormones. In this study, I have developed a tissue culture syaem to fùrther examine this
regulation and study the involvement of the various steroid hormone receptors.
We have first shown that the steroid hormone receptor-positive breast carcinoma ce11 Iine
T-47D is capable of producing PSA under appropriate stimulation by steroid hormones. T-47D
cells as well as MCF-7 cells do not produce PSA in the absence of steroid hormones (96,193). The
PSA mRNA produced by T47D and BT474 cells is identical to the sequence of PSA mRNA from
prostate celis. In contrast, the breast carcinoma ceIl line BT-20, which is devoid of steroid hormone
receptors did not produce PSA after stimulation by any of the compounds listed in Table 5-20-1-1.
1 have thus postulated that PSA production by breast cells is dependent on the steroid
honnone/steroid hormone receptor system. I fùrther demonstrated that the receptors and hormones
are necessary but not sufficient for PSA production. When I stirnuiated the steroid hormone
receptor-positive ceil Lines SAOS (osteosarcoma) and BG- 1 (ovarian carcinoma), ZR-75- 1, MCF-7,
(breast carcinoma cell lines) with the compounds show in Table 5-20-1-1, none was able to induce
PSA production. The presence of estrogen and progesterone receptors in these ceIl lines was
123
confirmed by aiiysis with estabtished enzyme immunoassay kits (Table 6-4-1). Apparently, either
post-receptor defects are present in these ce11 lines, the receptors are defective or the promoter of
the PSA gene in these ce11 lines is tissue specific. These possibilities were not studied further.
Among al1 androgenic compounds tested, only dihydroisoandrosterone sulfate, an inactive
metabolite, was not able to stimulate PSA production. Al1 other androgens were strong stimulators
(Table 6-4-2-1). The physiologicai androgens testosterone and androsterone and their reduced
forms dihydrotestosterone and dihydroandrosterone were able to induce PSA production at levels
as low as 10-'O and IO-'' M. respectively. The lower active concentration of dihydrotestosterone
(and dihydroandrosterone) is in accord with its higher affinity for the androgen receptor than
testosterone ( 194). Strong stimulation was also observed with the synthetic compounds R 1 88 1 and
RS020. In al1 cases tested, we observed a dose-response relationship. Although there is always a
degree of cross-reaaivity of steroid hormones with receptors other than the cognate receptors. the
activity of androgens at levels around 1 O-'' M (a concentration 10- 100-fold lower than the affinity
constant of the testosterone-AR complex) strongiy suggests that the effect is mediated through high
affinity binding to the androgen receptor and not through low affinity binding to cross-reacting
receptors. Among the four estrogens tested, the three naîural estrogens estradiol, estrone and estnol
did not mediate any PSA production. These data suggest that the estrogen receptor is not involved
in PSA gene up-regdation in the breast carcinoma cell line T-47D and BT-474. 17a-
Ethinylestradiol, a synthetic estrogen, was a weak but consistent stimulator at concentrations 21 0'
M. suggesting that this steroid is not a pure estrogen. Our data suggest that this steroid interacts
with the androgen and/or the progesterone receptor leading to active complexes capable of weakly
up-regulating the PS A gene.
124
Arnong the group of glucocorticoids tested, the physiological glucocorticoid hydrocortisone
and the synthetic glucocorticoid prednisone had no effect. The strong induction of betarnethasone
and dexamethasone and the weak induction by corticosterone, (at concentrations ~ 1 0 . ~ M) dl of
which have higher affinities for the glucocorticoid receptor than hydrocortisone and cortisone and
do not bind to either AR or PR (199,) suggest that the glucocorticoid receptor is capable of
regulating the PSA gene as well. The PSA stimulation at high glucocorticoid concentrations ody,
may reflect the low concentration of this receptor in T47D cells (200,20 1).
Wit h the exception of the inactive progesterone precursor 1 7 a -hydroxyprogesterone, al1
other progesterone agonists tested, were strong stimulators of PSA production. In dose-response
experiments, we have shown that the three tested progestins were active at levels 1 O-"- 1 O-" M. In
particular, Norgestimate, which exhibits highly specific high affinity binding to the progesterone
receptor than other progestin agonists (202-204) and binds to androgen receptor very poorly, was
active at levels down to 10 - '~ M. The data presented for the progestin agonists, in combination with
data of blocking experiments (discussed below) strongly suggest that the progesterone receptor,
activated by progestin, is capable of directly up-regulating the PSA gene.
In males, PSA gene regulation is under the control of testicular androgens through the
androgen receptor. 1 speculate that in fernales, PSA gene regulation in organs like the breast and
the endometnum (33) is mediated by progestins and androgens through the independent action of
the progesterone and the androgen receptor.
Aidosterone, a natural mineralocorticoid, was capable of PSA regulation only weakly and
at concentrations > 1 om8 M.
Triarnicinolone acetonide, a compound known to interact with the PR and GR but not the
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androgen receptor ( 199,205) was found to strongly stimulate PSA production at concenaations as
low as IO-'' M. This finding M e r strengthens our suggestion that the progesterone and
glucocorticoid receptor c m mediate PS A production without involvement of the AR.
Among the four antiestrogens, none was able to mediate PSA production consistent with
the suggestion that the estrogen receptor is not involved in PSA gene up-regdation. Among the
group of antiandrogens, 1 observed some interesting phenornena. Al1 these compounds bind to the
androgen receptor leading to either inactive complexes (pure antiandrogens) or to complexes with
some biologicai stimulatory activity (antiandrogens with weak agonist activity). In our system,
hydroxytlutamide, Casodex and Nilutamide (Anandron) which are known to bind to the androgen
receptor with low atfinity ( ~ 2 % of testosterone affinity) (206) did not mediate any PSA production
suggesting formation of weak and inactive complexes with the androgen receptor. Cyproterone
acetate, which binds to the androgen receptor with affinity -10% of that of testosterone (206), was
found to be a strong stimulator of PSA production, with activity even at concentrations - 10"' M.
This data suggests that cyproterone acetate, a known antiandrogen which also interacts with the
progesterone receptor (194,206) and has biological progestational activity ( 1 94) exerts its action
on PSA replation through binding to the progesterone receptor. Our finding that cyproterone
acetate can up-regulate the PSA gene through the progesterone receptor in parallel to its expected
d o m regulation of the PSA gene through androgen receptor blockade, requires fùrther investigation
since monitoring PSA levels during cyproterone acetate treatment of prostate cancer may not be a
reliable index of clinical response. Up-regulation of the PSA gene by cyproterone acetate through
its progestational activity has not, to Our knowledge, as yet been reported. It is currently unknown
if this indeed happens in prostate cells in addition to breast cancer ce11 lines.
126
The newer antiandrogen RU56,187 has f i n i t y for the androgen receptor similar to
testosterone but no detectable afftnity for progesterone, glucocorticoid, mineralocorticoid or
estrogen receptors (206). In our system, 1 detected weak PSA gene up-regdation at RU56,187
concentration > 1 M. This up-regdation strongly suggests that RU56.187 has weak androgen
agonist activity. In this respect, Our tissue culture system appears to be more sensitive than the in-
vitro systems used by Teutsch et al to evaluate RU56,187, concluding that this compound is totally
devoid of binding to other steroid receptors and of any agonist effect (206). It remains to be
determined if the weak agonist activity of RU56,l87 has any biological significance.
Mifepristone (RU486, RU3 8,486) is a new antiprogestational agent with antiglucocorticoid
and antiandrogenic activity . RU486 has been commercialized as an antiprogestin for first trimester
pregnancy interruption. In Our system, 1 found that Mifepristone has weak agonist activity,
mediating PSA gene up-regulation at concentrations >IO-' M. This agonist activity was not
observed by Philibert et al (207), fbrther suggesting that their biological tests are not as sensitive
as our tissue culture system in detecting such an effect. In support of our data are reports by others
showing weak agonist activity of RU486 in various systems (208,209,99). It remains to be seen if
the weak agonist activity of Mifepristone is mediated by the androgen, glucocorticoid or the
progesterone receptor. Very weak agonist activity was dso observed for the antiglucocorticoid
cortexolone and the antimineralocorticoid spironolactone at concentrations 21 0.' M.
While estradiol has no positive effect on PSA gene regulation, blocking experiments have
revealed that estradiol could block the action of dihydrotestosterone and to a much lesser degree
Norgestrel but not Norgestimate on PSA gene regulation (Table 6-4-2-1). There are two possible
explanations for this phenornenon. First, high doses of estradiol could cause its binding to the
127
androgen receptor thus blocking the action of dihydrotestosterone (1 99,99). Second, estradiol
would bind to the estrogen receptor in T-47D cells and the active cornplex further inhibit the action
of active AR complexes. Active ER, AR and PR complexes compete for the same transcription
factors including c-jun and c-fos as suggested previously (210). The fact that estradiol blocks the
stimulation by dihydrotestosterone but not the stimulation by Norgestimate suggests that estradiol
blockade targets the AR but not the PR. The finding of positive regulation of the PSA gene by
androgen and progestin and the negative regulation by estrogen suggests that PSA is regulated by
a delicate balance between androgens, progestins and estrogens.
Nilutamide and hydroxyflutamide, two antiandrogens that bind with low affinity to the
androgen receptor, had moderate but not complete blocking activity (-30-40% on average) on
dihydrotestosterone and an even lower blocking activity ( 9 0 % ) on Norgestrel and no blocking
activity on Norgestirnate. This data are expected since the stimulating steroids (eg.
dihydrotestosterone), having higlier &nity for the androgen receptor, would displace a fraction
of the blocker after they are added into the tissue culture system. On the other hand, RU56.187
which has an affinity for the androgen receptor similar to testosterone, was able to block 85-9 1%
of the activity of dihydrotestosterone. The lower blockade on Norgestrel action (1 7-40% on
average) and the absence of blockade on Norgestimate action further suggests that a significant
portion of Norgestrel's and 100% of Norgestirnate's stimulation is mediated through the
progesterone receptor to which RU56,187 does not bind and could not block.
The antiestrogen ICI 182,780 had little or no blocking effect on the stimulation of PSA
production by dihydrotestosterone, Norgestrel or Norgestimate, in accordance with our view that
the estrogen receptor does not positively mediate PSA production in Our system.
128
Mifepnstone was an effective blocker of PSA production by dihydrotestosterone (70-80%)
and an almost complete blocker of Norgestrel and Norgestimate (90-1 00%). This is in accord with
Our view that PSA production is mediated independently by the AR and the PR since Mifepnstone
is known to block effectively the progesterone receptor and to a lesser but significant degree the
androgen receptor (207).
As expected. the antiglucocorticoid cortexoione and the antimineralocorticoid
spironolactone had no effect on either dihydrotestosterone, Norgestrel or Norgestimate action since
these two antihormones bind pnmarily to GR and MR and only with Iow afinity to other receptors
which are invoived in PSA production.
Stimulation experiments were also conducted using growth factors, vitamin D, analogs,
dl-tram and cis-retinoids (Table 6-20-1-1) in T-47D and BT-474 breast carcinoma ce11 lines, but
none of thern was able to induce PSA production. However, recently it has been reported that in
addition of steroid hormones, growth factors, IGF-1, KGF, EGF, were thought to participate in the
regulation of PSA gene expression through the androgen receptor activation in prostatic tumour ce11
line (102). In addition to traditional steroid hormones, four growth factors IGF-1, FGF, TGF, and
EGF affect breast cancer and treatment with trans-retinoids (analog of vitamin A) slows tumor
growth and reduces circulating IGF-1, particularly in women under 50. The mechanism by which
retinoids affect IGF-induced growth of breast cancer cells involves modification of local IGFBP
production (2 1 1).
Taken together, our data suggest the following: The breast carcinoma ce11 lines T-47D and
BT-474 have the necessary receptors and other transcriptional machinery to produce PSA. Once
stimulated by a steroid hormone, T-47D and BT-474 cells produce PSA rnRNA within 1-2h,
129
synthesize detectable intracellulas protein within 4 h and secrete detectable protein within 8 h. PSA
gene regulation is under the control of andorgens and progestins through the independent action of
the androgen and progesterone receptors (positive regulation). Weak positive regulation may also
be effected by high concentrations of glucocorticoids and mineralocorticoid. Estrogens do not
positively regulate the PSA gene but they act as blockers of androgen action. The most effective
blockers of PSA gene regulation were found to be the antiandrogen RU56.187 and the
antiprogestin Mifepristone. Our data, showing multihormone regulation of the PSA gene, are
in accord with those of GIover and Darbe (200) who concluded the same using T-47D celis
transfected with the mamrnary turnor virus long terminal repeat sequences.
Our tissue culture system not only reproduces the phenornenon of PSA production by breast
cancers but it also offers a means of testing the biological activity of candidate new hormonal and
antihormonal agents. With this systern, 1 have shown that two new antihormones, the antiandrogen
RU56,187 and the antiprogestin Mifepristone, which were found to be completely devoid of agonist
activity by traditional in-vivo and in-vitro techniques, have Iow but detectable androgen and/or
progestin agonist activity demonstrated by their ability to up-regulate the PSA gene.
Recently, we have obtained evidence that the progestin-mediated up-regulation of the PSA-
gene occurs in-vivo as well. We have reported PSA production by normal breast tissue in a female
patient who was receiving an oral contraceptive containing Norethidrone (8). PSA regulation by
progestins in the prostate has not been reported but it is known that the prostate cells, in addition
to AR, also contain PR (2 12). In another report, we described a patient who was receiving high
doses of glucocorticoids and had a . ovarian tumor producing PSA (27).
This data fùrther supports the view that PSA may be regulated in diverse tissues containing
130
PR. Tissues which contain PR include, the breast (6), the endometrium (33), brain meningiomas
(2 13), blood vesse1 walls (2 14), unnary (2 15) and osteoclasts (2 16). In view of these and other
hdings, 1 believe that it is tirne to snidy in detail the biological role of PSA in non-prostatic tissues.
The presence of a significant prognostic value of this molecule in breast cancer ( 12) suggests that
this eiegantly regulated enzyme rnay play some role in breast cancer initiation and progression.
7-3. PSA Production During the Menstrual Cycle
There is now extensive evidence that the PSA gene is up-regulated by androgens and
progestins but not estrogens. The PSA concentration in female semm is very low and usually not
measurable by commercial PSA assays. Using highiy sensitive PSA procedures that we developed
(1 8 l), we demonstrated that many female sera had measurable PSA concentrations (1 19). We have
further demonstrated that much higher PSA levels can be found in female breast secretions. For
comparative purposes, 1 here report approximate levels of PSA in various human fluids; serninal
plasma, 1,000,000,000 ng/L; male serum, 1,000-2,000 ng/L; normal breast discharge fluid,
5,000,000 ngL; rnilk of lactating women, 100,000 ng/L; female senim, 2-4 n a . There is
approximately a lo4-fold difference between PSA levels in the physiological secretion (e.g. seminal
plasma or breast discharge fluid) and the corresponding serum. The physiological role of PSA in
seminal plasma seerns to be established (66). The role of PSA in the breast and its secretions is still
obscure. We found that in breast cancer, patients with PSA-producing tumors have better prognosis
than patients with tumors which do not express PSA ( 12).
Since PSA expression is under the control of steroid hormones and their receptors, I
speculated that its concentration may change during the menstrual cycle. This was demonstrated
13 1
by assessing both the levels of semm PSA during the menstrual cycle of normal women and by
examining the ability of serum from these women to stimulate PSA production and PSA mRNA
expression in a tissue culture system. 1 demonstrated that semm PSA levels change significantly
during the menstruai cycle, following a specific pattern. This pattern was similar in three patients
and was reproducible on repeated cycles from the same patients (Figure 6-5-1 and 6-5-2). PSA
concentration increase follows the progesterone concentration increase but with a 10-20 day lag
period, suggesting that PSA is kely a product of progesterone action in the target tissue. Based on
many recent indirect findings, we suggest that the target tissue is the female breast but the
contribution of other steroid hormone responsive tissues, like the endornetrium cannot be excluded
(33). Recently, Clements et al. measured PSA mRNA in endometrial tissue of normal cycling
women and found that highest levels occur during the follicular phase and lowest levels during the
late luteal phase in general accordance with the data reported here (2 17). No association was seen
between the changes of PSA and changes in LH, FSH, Free testosterone or estradiol. Our tissue
culture system confirmed the following: (a) That progesterone cm up-regulate PSA production. (b)
that estradiol LH, FSH, prolactin, growth hormone and choriogonadotropin do not rnediate PSA
production in the T-47D and BT-474 ceil lines. (c) That sera collected at the luteai phase of the
cycle (which are high in progesterone) but not sera collected at the follicular phase or rnid-cycle)
have the ability to up-regulate PSA protein production and increase PSA mRNA expression in the
breast carcinoma ce11 line T-47D.
The data presented allow us to speculate that corpus luteum steroids, one of which is
progesterone, stimulate target tissues capable of producing PSA (one of which is the breast) for
PSA production and release into the mamrnary ducts. A minor Fraction of this PSA difises into the
132
general circulation and can be measured in the serum. Peak levels of PSA appear 10-20 days post-
progesterone peak. Once the corpus luteum regesses, PSA concentration decreases with an
apparent half-life of approximately 5-6 days. Based on these findings, we suggest that PSA is a
protein regulated by the corpus luteum and it is a marker of progesterone action in target tissues.
Since this protein has senne protease activity, it will be interesting to find its biological role in the
breast and its secretions and its possible physiological substrates. This issue has recently been
discussed ( 198).
Yu et al. (8) have demonstrated that prostate specific antigen gene can be upregulated by
progestin containing oral contraceptives. Recently, the role of oral contraceptives in the
development of breast cancer has been reviewed in over 150,000 patients. The conclusion fiom this
study is that women who are currently using combined oral contraceptives or have used them in the
past 10 years are at a slightly increased risk of having breast cancer diagnosed, although the
additional cancers diagnosed tend to be localized to the breast. There is no evidence of an increase
in the risk of having breast cancer diagnosed 10 or more years after cessation of use, and the
cancers diagnosed then are less advanced clincally than the cancers diagnosed in never-users (2 18).
7-4. Expression of PSA Gene in Lung Tissue
More recently, others have proposed that PSA may cleave biologically important substrates
like insulin-like growth factor binding protein 3 (IGF'BP-3) (78), parathyroid hormone related
peptide (PTHrP) (219) or unknown substrates which release peptides with smooth muscle
contraction activity (220). The search for new biological functions of PSA has recently been
intensified d e r the realization that PSA is expressed in diverse tissues (198). Striking sirnilarities
133
between PS A expression and expression of the breast cancer susceptibility gene BRC A- l have been
noted (22 1 ). For example, BRC A- 1 and PS A were found to be upregulated dunng pregnancy, are
both secreted into the milk of lactating women and are both regulated by steroid hormones. If
BRCA- 1 is a granin then PSA and BRCA- 1 may be secreted by the breast epithelial cells. Others
have found that PSA may act as a negative growth regulator in hormone-dependent breast cancers
by stimulating the conversion of estradiol to estrone (222). The highest concentration of PSA,
except for seminal plasma, was found in breast discharge fluid (up to 5 mg/L) and in milk of
lactating women (up to 0.3 m a ) (99).
It has been known for years that, in the prostate, the PSA gene is regulated by androgens
through the androgen receptor (22). Using a tissue culture system with breast carcinoma ce11 lines,
we have demonstrated that androgens, progestins and glucocorticoids can independently up-regulate
the PSA gene (96). We have also provided evidence that this regdation occurs irz-vivo by
describing a woman whose breast tissue PSA was very high due to stimulation by a progestin
containing oral contraceptive (8) and another woman whose ovarian tumor was producing PSA,
likely due to stimulation by glucocorticoids (27).
Although two previous studies have suggested that PSA may be produced by lung tissue and
tumon (223,123), their data were based only on protein measurements. Since PSA has extensive
sequence homology with the hGK-1 gene and protein and PSA antibodies cross react with hGK- 1
(224,225), the only certain way to verify the presence of PSA in lung is by either sequencing the
purified protein (which is irnpractical due to the very low levels present) or by molecular analysis
of the expressed PSA rnRNA. 1 describe here a patient whose lung tissue extract was highly positive
for PSA protein in its 33 KDa form. In this patient, 1 was able to arnplify PSA mRNA and sequence
134
PSA cDNA to ven@ its identity as PSA cDNA and not hGK-1 cDNA. Immunohistochernical
localization has shown that the PSA-positive tissue is the normal lung epithelium adjacent to the
tumor. This is in accord with data fiom breast tumors in which we noted more fiequent staùiing
for PSA of normal and hyperplastic breast epithelium and well-differentiated tumors. Poorly
differentiated and steroid hormone receptor-negative breast tumors are usually negative for PSA.
This patient was unique since her lung tissue contained more than 50 times the PSA
immunoreactivity of another 10 lung tumor extracts from women. 1 postulate that in this patient,
the PSA was up-regulated by the steroid beclomethasone dipropionate since this drug was found
to be able to up-regulate PSA production in-vitro, in a dose-dependent manner (Figure 6-6-6).
These data parallei observations in a patient with ovarian carcinoma (27) and a patient receiving
oral contraceptives (8). Recently, in support of our hypothesis, Beattie et al. reported presence of
high affinity receptors for androgen, estrogen and glucocorticoids in lung tissues and carcinomas
(169). Beer and Malkinson reported similar data in mouse lung tumors (226). I conclude that PSA
can be expressed in a variety of tissues bearing steroid homone receptors and that its expression
can be manipulated by exogenously administered hormones. The biological role of PSA in these
tissues is still unknown.
7-5. Frequency of PSA mRNA Expression in Lung tumon
PSA in lung tissue was previously found using irnrnunological assays that measure PSA
protein (123). However, since PSA is also present in the blood, there is always the possibility of
contamination of tissue extracts by residual blood in the tissue. Also, the close sequence homology
berneen PSA and hGK-1 may cause problems of cross-reactivity (22). In this study, 1 have s h o w
135
that contamination of the tumor extracts by blood PSA is likely, since 1 found a signdlcant
correlation between tumor extract PSA and serum P S 4 especially in male patients.
Since PSA mRNA-containing celis do not seem to exist in the circulation of patients without
prostate cancer (208,220,221), 1 have attempted to show evidence for PSA expression in lung tissue
by searching for PSA mRNA presence in lung tissue extracts. 1 found PSA &A in 69% of lung
tumors from males, 53% of lung tumors fiom females and in 71% of normal lung tissues. These
dzta support the view that low levels of PSA mRNA are found in the majority of lung tissue
extracts. These levels are much lower than those found in femde breast since in the latter tissue,
PSA mRNA could be detected without the need of Southem blot hybndization (227) and PSA
protein levels in breast tumor extracts are much higher then those reported in Table 6-7-1 (8). As
in the case of breast, normal lung tissue contains PSA mRNA as well.
1 found no association between PSA mRNA presence and clinicopathoiogical variables
including patient age, stage, grade, histological type or nodal status. Consequently, the prognostic
value of this marker in lung cancer is questionable at present. The regdation of PSA rnRNA
expression in lung tissue and its possible biological role are unknown.
By cloning and sequencing of aberrant PCR products, 1 was able to isolate a 450 bp unique
sequence not previously deposited in GenBank (Figure 6-7-3). Additional work will reveal if this
fragment belongs to a new gene that is overexpressed in lung tumors. Work dong these lines is
currently in progress.
This data support the view that PSA is fiequently expressed at low levels in cancerous and
normal lung tissue. The role of this molecule in lung physiology and pathobiology is currently
unknown.
136
7-6. Conclusion
For studying of PSA gene expression in non-prostatic tissues 1 have first developed reverse
transcription-polymerase chah reaction (RT-PCR) methods for detecting prostate-specific antigen
(PSA) mRNA Using this methods, and a highly sensitive irnmunofluorometnc assay for measuring
PSA proteinJ found good agreement between presence of PSA protein and PSA rnRNA in breast
tumors. 1 thus proposed that in women, detection of PSA protein or PSA mRNA in tissues and
turnors offers equivalent information. Because PSA protein is present in male blood and thus could
contaminate extracts fiom tumors and tissues from men, I propose that the RT-PCR methods 1
describe be used to assess non-prostatic expression of the PSA gene in men.
For investigating the mechanism of PSA gene regulation in the breast, I have developed a
tissue culture system which reproduces in-vitro the phenornenon of PSA production by breast cells.
The tissue culture expenments showed that steroid hormone receptor positive breast cancer cells,
T-47D and BT-474, are capable of producing PSA when stimulated by androgens, progestins and
çlucocorticoidslrnineralocorticoid but not estrogens. However, Our studies demonstrated that those
ce11 lines which were devoid of steroid hormone receptors, PSA production was not observed after
stimulation with steroid hormones. The steroid hormone induced PSA production in breast cancer
cells was time- and dose-dependent. Kinetic studies showed that PSA mRNA expression appears
-2 hours post stimulation; PSA protein appears after 4-8 h. Among sixty four compound tested,
only androgens and progestins were able to stimulate PSA production at concentrations beiow 10"
M. Evidence that the progesterone and androgen receptors cm regulate the PSA gene
independentiy was provided as follows: (a) The progestin Norgestimate which does not bind to the
androgen receptor up-regdates the PSA gene at concentrations as low as 1 O-'' M. (b) Tnamcinolone
137
acetonide which does not bind to the AR but binds to the PR acts sirnilarly to Norgestimate (c) The
antiandrogen cyproterone acetate which blocks the androgen receptor but has progesterone activity,
up-regulates the PSA gene at concentrations as low as 10'" M. (d) the antiprogestin Mifepristone
blocks completely the stimulation of the specific progestin Norgestimate. Our tissue culture system
identified androgedprogestin agonist activities of 17a-ethinylestradiol, the antiandrogen RU56,187
and the antiprogestin Mifepristone. These data suggested that the expression of the PS A gene in the
fernale breast is under the control of androgens and progestins. Our tissue culture system is a highly
sensitive in-vitro method for evaluating the biological activity of candidate compounds having
agonist and antagonist steroid hormone activity.
The regulation of the PSA gene by steroid hormones in the breast led us to speculate that
this protein may be differentially expressed during the menstrual cycle. By examining the PSA
concentration in serum during the menstrual cycle of normal women, 1 found that PSA levels in
serum are highest dunng the mid-late follicular phase, drop continuously with a half-life of 5-6 days
between the late follicular phase and rnid-cycle and reach a minimum dunng the mid-luteal phase.
PSA changes do not correlate with changes in LH, FSH, or estradiol levels. However, PSA peaks
seem to follow the progesterone concentration peaks with a delay of 10-20 days. Using a tissue
culture system based on T-47D breast carcinoma ce11 line, only sera obtained during the rnid-late
luteai phase were able to up-regulate the PSA mRNA and protein. In stimulation experiments in-
vitro, progesterone, but not LEI, FSH, estradiol, hCG, prolactin or growth hormone, was able to up-
regulate PSA mRNA and protein in the T-47D ce11 line. These data suggested that progesterone,
and possibly other corpus luteum steroids, stimulate target tissues for ?SA production in a cyclical
manner during the menstmal cycle. However, the relatively long tirne difference between
138
progesterone and PSA peaks does not support a direct induction-secretion mechanism. This issue
needs clarification with further expenmentation.
Further, the ability of non-prostatic tissues to produce PSA was further reported in a female
patient with lung adenocarcinorna whose tumor extracts was highly positive for PSA
immunoreactivity. The identity of PSA in lung tissue was verified by using RT-PCR, HPLC,
immunohistochemistry, and sequencing of the PCR products. In addition, tissue culture experiments
suggested that beclomethasone, a glucocorticoid used to treat the patient, was able to up-regulate
PSA gene expression. This was the first report that unequivocally demonstrated PSA expression
in lung tissue.
In addition, further studies demonstrated that PSA protein and mRNA was present in
primary lung tumor tissues in both males and fernales. PSA protein was detected more fiequently
and at higher levels in lung tumor extracts fiorn male patients, suggesting a possible contamination
of the tumor extracts with PSA fiom residual blood in the tumor vasculature. Clinicai studies
indicated that the expression of PSA protein did not associate with patient age, stage, grade,
histological type or nodal status. Similarly, PSA mRNA was not associated with any
clinicopathological variables but squarnous ceil carcinomas, especially in males, were more
fiequently positive. Consequently, the prognostic value of this marker in lung cancer is questionable
at present. Our data support the view that PSA is fiequently expressed at low levels in cancerous
and normal lung tissue. The role of this rnolecule in lung physiology and pathobiology is currently
unknown.
More interestingly, by cloning and sequencing of aberrant PCR products, 1 was able to
isolate a 450 bp unique sequence not previously deposited in the GenBank. Additionai work will
139
reveal if this fragment belongs to a new gene that is overexpressed in lung tumors. Work dong
these lines is currently in progress.
CHAPTER 8. Concluding Remarks and Future Directions
In this thesis, 1 have generated the following new information.
1 . 1 have devised an ultrasensitive, highly specific rnethod for monitoring prostate specific
antigen mRNA levels. This method was s h o w to produce results in excellent concordance
with an ultrasensitive imrnunofluorometric procedure for measuring PSA protein.
2. 1 have discovered two breast carcinoma ce11 lines which can upregulate the prostate specific
antigen gene d e r exogenous stimulation by steroid hormones. By using this system, 1 have
s h o w that the PSA gene can be upregulated at steroid hormone concentrations below 10''~
M by progestins and androgens. In addition, higher concentrations of glucocorticoids and
mineralocorticoids can also upregulate the PSA gene. 1 have s h o w that estrogens not only
do not upregulate the PSA gene but they block PSA gene upregulation by progestins and
androgens. 1 have also studied the kinetics of PSA mRNA protein regulation in these ce11
lines. The data generated have shown that the PSA gene, in these ce11 lines is under the
control of steroid hormones.
3 . 1 have shown that the data generated with the VI-vitro system are in accord with the data that
1 have produced Nt-vivo. by studying the PSA gene expression during the menstrual cycle.
1 have shown for the first time that the PSA gene is regulated differentially in a cyclical
manner during the rnenstrual cycle. 1 have also provided evidence that during the menstrual
cycle, the major regulating steroid is progesterone.
By using a combination of protein measurements, high performance liquid chromatography,
RT PCR, Southem blot hybridization, immunohistochernistry and DNA sequencing
141
techniques, 1 have shown unequivocally that the prostate specific antigen gene is expressed
by lung tissue and lung tumors. 1 provided evidence that at least in one patient that the PSA
gene in lung tissue is upregulated by exogenously administered steroids. 1 found no
association between PSA gene expression and lung cancer and clinicopathological variables
of the patients.
It is now very clear that the PSA gene is not expressed specifically in prostatic tissue as it
was believed for many years. However, there are still many questions which have originated fiom
my investigations and those of other members of our research laboratory. For example, 1 tabulate
below a number of unanswered questions.
The physiological role of PSA in breast and other non-prostatic tissues remains obscure.
PSA is a senne protease with chymotrypsin like enzymatic activity and it is likely exerting
its physiological role by cleaving other substrates. It has recently been postulated that PSA
may be a protease for the BRCA-1 gene products (E.P. Diamandis. BRCAl protein
products: Antibody specificity. functional motifs and secreted tumour suppressors. Nature
Genetics, 1 996; 1 3 : 268).
1 postulated that the PSA molecule may be acting through surface receptors in regulating
ce11 proliferation. However, extensive experimentations to identi@ such a receptor have
been unsuccessful. 1 found no evidence of presence of a receptor in these ce11 lines.
It will be interesting to examine and discover why PSA is a favourable prognostic indicator
in breast cancer and a marker of reduced risk for developing breast cancer. It appears that
PSA is a molecule with anticarcinogenic properties in the breast.
I postulated that the tissue culture systems that 1 developed may have potential for screening
142
various candidate compounds with steroid hormone agonist and steroid hormone antagonist
activities. Preliminary results by other members of our research laboratory suppon this
hypothesis.
5 . It will be interesting to examine if the functions of prostate specific antigen are rnediated
through growth factor regulation. It has recently been proposed that PSA is a protease for
IGF binding proteins and this rnay constitute a regulatory loop for growth factor regulation.
6 During my investigations, 1 found two breast carcinoma ce11 lines, which are steroid
hormone receptor positive and can upregulate the PSA gene d e r induction by progestins
and androgens. However, 1 have also identified other ceif lines which contain steroid
hormone receptors (e.g. ZR-75, BG- 1) which do not upregulate the PSA gene after steroid
hormone induction. I have postulated that these ce11 lines have either defective receptors
or are lacking other critical transcription factors that are involved in PSA gene regulation.
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