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Vol. 1, 71-80, Jantiar�’ 1995 Clinical Cancer Research 71
Differential Expression of Multiple MDM2 Messenger RNAs and
Proteins in Normal and Tumorigenic Breast Epithelial Cells
Jean M. Gudas,’ Hoang Nguyen,2
Regina C. Klein, Dai Katayose, Prem Seth,
and Kenneth H. Cowan
Medicine Branch, Division of Cancer Treatment, Medical Breast
Cancer Section, National Cancer Institute, Bethesda, Maryland 20892
ABSTRACT
The MDM2 gene is a nuclear phosphoprotein that is
regulated by p53 and functions, in one capacity, to inhibit
the transcriptional activity of the wild-type p53 protein.
Multiple MDM2 transcripts were detected in human breast
epithelial cells. In estrogen receptor-negative normal, im-
mortal, and tumorigenic breast epithelial cells, we found agood correlation between MDM2 mRNA levels and expres-
sion of wild-type p53. When wild-type p53 was overex-
pressed in estrogen receptor-negative tumor cells containing
a mutant or no endogenous p53, MDM2 mRNA levels in-creased significantly, indicating that wild-type p53 positively
influences MDM2 mRNA levels in these tumor cells. Because
all estrogen receptor-positive breast tumor cells had high
MDM2 mRNA levels regardless of the status of their endo-genous p53 protein, other factors likely influence MDM2
expression in these cells Distinct MDM2 proteins (range, Mr
54,000-68,000 and 90,000-100,000, respectively) were dif-
ferentially expressed in human breast epithelial cells. The
lower molecular weight MDM2 proteins were most abun-
dant in the normal mammary cells but present at varyinglevels in many of the tumor cells examined. MDM2 was a
nuclear protein; however, nuclear staining intensity did notalways correlate with the amount of MDM2-immunoreac-
tive protein as determined by Western blot analysis. This
discrepancy suggests that MDM2 interacts with novel cellu-
lar proteins in different kinds of breast epithelial cells.
INTRODUCTION
The human homologue of the mouse MDM2 gene encodes
a zinc finger-containing protein that is a putative transcription
factor (1, 2). Overexpression of this gene in BALB/c 3T3 cells
increases their tumonigenic potential, suggesting that the MDM2
protein possesses oncogenic activity. Further evidence that
MDM2 plays a role in the process of cell transformation derives
from experiments where this protein was shown to complex
with and inhibit the transcriptional activity of wild-type p53 (3,
Received 8/16/94; accepted 10/20/94.
I To whom requests for reprints should be addressed, at MedicineBranch, Division of Cancer Treatment, National Cancer Institute, Build-ing 10, Room 12N226, Bethesda, MD 20892.
2 Supported by a Cooperative Research Development Agreement pro-vided by the Centocor Corp., Malvern, PA.
4). MDM2 interacts with the amino terminal acidic activation
domain of the p53 protein, raising the possibility that MDM2
inhibits p53 function by disrupting its interaction with the gen-
enal transcription machinery (5).
Recent evidence indicates that MDM2 is one of the down-
stream effector genes in cells that express a wild-type p53
protein (6, 7). The first intron of the MDM2 gene contains a p53
DNA binding site. This element confers wild-type p53 respon-
sivity to the endogenous MDM2 gene and to reporter genes
when placed upstream in a hetenologous promoter (7). More
recent data suggest that this p53 response element may actually
be part of an internal, cryptic p53-dependent promoter (8).
When considered together, these observations suggest that neg-
ulation of p53 and MDM2 are intimately linked and that these
two proteins may act in concert to control cell proliferation.
Our laboratory has been interested in regulatory mecha-
nisms that play a role in the genesis of breast cancer. The
importance of p53 in this process is underscored by the finding
that -40% of primary human breast tumors harbor mutations in
the coding sequences of the p53 gene (9, 10). Moreover, recent
studies indicate that expression of mutant p53 is an important
prognostic marker for node-negative breast cancers (1 1, 12).
Presumably, the remaining tumors utilize other mechanisms to
bypass the negative regulatory effects of the wild-type p.53 gene.
One possibility is that these tumor cells can up-regulate the
expression of MDM2 to a level sufficient for binding and
titrating the effects of wild-type p53. Such a mechanism has
been proposed to explain the ovenexpression of MDM2 in soft
tissue sarcomas, osteosancomas, and a subset of malignant gli-
omas (13-17). Recently, Chen et a!. (18) reported that the
amplification of MDM2 in osteosancoma cells resulted in inhi-
bition of wild-type p53-induced growth arrest following treat-
ment with ionizing radiation. These results have provided im-
portant evidence demonstrating that MDM2 overexpression can
affect p53 function in a known physiological pathway. To date,
amplification and overexpression of MDM2 has not been shown
to play a significant role in the genesis of human epithelial
tumors (19-21).
To begin to understand the complex regulation between
these two cellular proteins, we examined levels of p53 and
MDM2 mRNA and protein in human breast epithelial cells. We
determined the sizes of the mRNAs and proteins for MDM2
present in mammary epithelial cells at different stages of cell
transformation. One goal of oun study was to determine whether
a relationship existed between p53 levels and corresponding
levels of MDM2 in mammary epithelial cells that expressed
wild-type versus mutant p53 proteins. Because MDM2 levels
were high in ER�3 breast cancer cells regardless of their p53
3 The abbreviations used are: ER, estrogen receptor; NMECs, normalhuman mammary epithelial cells.
Research. on May 27, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
72 MDM2 and Breast Cancer
4 D. Katayose, submitted for publication.
status, we examined the possible influence of estrogens on
MDM2 gene expression. Finally, to determine the intracellular
localization of these proteins, we performed immunocytochem-
ical analyses of MDM2 and p53 proteins in representative breast
epithelial cell lines.
MATERIALS AND METHODS
Cells and Cell Culture. NMECs derived from reduction
mammoplasties were purchased fron Clonetics Corp. (San Di-
ego, CA) and the Corniel Institute (Camden, NJ). The immor-
talized breast epithelial cell line MCF-10 was generously pro-
vided by Dr. Sam Brooks, Michigan Cancer Foundation (22).
Immortalized 184B5 and HBL100 cells were obtained from the
ATCC. The ER� cell line MCF-7 was obtained from Dr. R.
Buick, University of Toronto (23) and the ER� BT2OT cells
were provided by Dr. K. Keyomansi, New York State Depart-
ment of Health. Other ER� cells including T47-D, ZR7S-1, and
BT474 and the ER tumor cell lines MDA-MB-157, MDA-
MB-231, MD-MBA- 453, MD-MBA-468, HS578T, and SKBr3
were all obtained from the ATCC.
NMECs and 184B5 cells were cultured in a mammary
epithelial basal medium (Clonetics Corp.) supplemented with
vitamins, 0.5% fetal bovine serum, 20 ng/ml EGF, S jxg/ml
insulin, 0.5 p.g/ml hydnocortisone, and 52 p�g/ml bovine pitu-
itary extract (24). MCF-10 cells were cultured in Dulbecco’s
modified Eagle’s medium/F12 (GIBCO) supplemented with
2.5% horse serum, 10 mM HEPES, 2 msi glutamine, 0.1 m�t
nonessential amino acids, 20 ng/ml EGF, 10 pg/ml insulin, 0.5
�xWml hydnocortisone, and 10 p.g/ml transfennin. The ER� and
ER breast cancer cells were cultured in a-MEM (GIBCO)
supplemented with 10 msi HEPES, 2 msi glutamine, 0.1 mM
nonessential amino acids, 10% fetal bovine serum, 1 ng/ml
EGF, and 2 j.g/ml insulin with the exception of MDA-MB-468,
SKBr3, and BT474 cells. These cells were grown in improved
MEM without EGF and insulin was added to a final concentra-
tion of 5 �i.g/ml. All cells were routinely screened for Myco-
plasma contamination and maintained at 37#{176}Cin an atmosphere
of 6.5% CO2.
Adenovirus Infection of Breast Tumor Cells. An Ad-
enovinus vector containing a eDNA encoding the wild-type p53
gene (AdWTp53) was constructed according to established pro-
cedunes.4 MDA-MB-157 and MDA-MB-231 cells were plated
and allowed to grow for 1 or 2 days before infection with 10
plaque-forming units ofAdWTp53 or the same virus without the
p53 gene (AdCon). Cells were harvested for protein and RNA
analyses 24 h after infection.
RNA Isolation and Northern Blot Analysis. Total
RNA was prepared by Iysing cell monolayers in guanidinium
isothiocyanate and centrifuging over a 5.7 M CsCl cushion as
described previously (25). RNA (20 pig) was electrophoresed on
denaturing formaldehyde gels, transferred to MagnaNT mem-
branes, and cross-linked with UV. The probes used for Northern
blot analyses were obtained as follows. A 2.1-kilobase BamHI-
XhoI fragment, a 750-bp BamHI-Xba I, and an 800-bp HindIll
fragment were excised from different regions of the MDM2
eDNA (a generous gift from Dr. Bert Vogelstein; Ref 13). A
2.0-kilobase BamHI fragment from the human p53 eDNA was
purchased from ATCC and an 800-bp PstI fragment was excised
from p36B4 (26). All probes were labeled with [a-32P]dCTP on
dATP to a specific activity of - 1 X i0� cpm/pg DNA using a
random-primed labeling kit (Boehninger Mannheim).
Immunoprecipitation and Western Blot Analyses. Forimmunoprecipitation studies, cells were washed 3 times in ice-cold
PBS and harvested in lysis buffer (50 m�i Tnis-HC1, pH 8.0, 5 mrvi
EDTA, 150 mM NaC1, 0.75% NP4O, 1 mist phenylmethylsulfonyl
fluoride, S �j.�ml aprotinin, S �ig/ml leupeptin, 50 msi sodium
fluoride, 10 mM sodium o-vanadate). Equal amounts of protein
were subjected to immunoprecipitation by incubating with 1 �xg
IF2 monoclonal antibody (MDM2 antibody 1; Oncogene Science)
or 3 p.1 culture supernatant from 5B10-producing hybnidoma cells
(5). Specific complexes were collected following incubation with
Protein A + G agarose (Oncogene Science). After washing three
times in SNNTE buffer (5% sucrose; 0.5 M NaC1; 1% NP-40; 50
mM Tnis, pH 7.4; 5 mM EDTA) and twice with radioirnmunopre-
cipitation assay buffer, the precipitates were electrophonesed in 8%
SDS-polyacrylamide gels, transferred to nitrocellulose, and probed
for MDM2 as described below.
For Western blot analyses cells were washed three times
with ice-cold PBS, scraped into hot (80-90#{176}C) Laemmli lysis
buffer (62.5 mM Tnis-HC1, pH 6.8, 2 msi EDTA, 15% sucrose,
10% glycerol, 3% SDS, 0.7 M 2-mercaptoethanol) and the
proteins denatured by boiling for 10 mm. After centnifugation,
protein lysate (50 �iWlane) was electrophoresed on 8 or 10%
SDS/polyacnylamide gels and transferred to nitrocellulose mem-
bnanes. The filters were blocked with Tnis-buffered saline con-
taming 8% dried milk and 0.1% Tween 20 and then probed with
the respective antibody. MDM2 was detected using 2.5 p.g/ml
1F2 antibody (Oncogene Science), p53 was detected using 4
p.g/ml PAbs 1801 and DO-i antibodies (Oncogene Science) and
actin was detected with 3 p.g/ml actin antibody-i (Oncogene
Science). After washing in Tnis-buffered saline containing 0.1%
Tween 20, and subsequent incubation with horseradish peroxi-
dase-conjugated goat anti-mouse antibody (Bio-Rad), specific
complexes were detected using a chemiluminescent technique
according to the manufacturer’s recommendations (New En-
gland Nuclear).
Immunocytochemistry and Autoradiography. Cells were
grown in tissue culture chamber slides, washed three times in
ice-cold PBS, and immediately fixed in methanol:acetone (i:i) for
2 mm. After allowing the slides to air dry, they were stained for
MDM2 using the IF2 monoclonal antibody (MDM2 Ab-1; Onco-
gene Science) and PAb 1801 (Ab-2) (Oncogene Science) for de-
tection of p53. The MDM2 and p53 antibodies were diluted to a
final concentration of 4.5 �xg/ml or 1 �xg/ml, respectively. After
washing in Tnis-buffered saline containing 0.05% Tween 20, the
specific complexes were visualized using the avidin-biotin com-
plex immunoperoxidase system (Vector Laboratories, Burlingame,
CA) according to the manufacturer’s recommendations.
RESULTS
MDM2 mRNA Expression in Human Mammary Epi-
thelial Cells. In our initial experiments we sought to deter-
mine whether there were any differences in the expression of
Research. on May 27, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
-� �h��:.:1
II � I
. S � #{216}S#{149}..�#{149}.
Clinical Cancer Research 73
12.5 �
4.5 �
NMEC IMEC ER+ ERU U � II U U U
� �% 4, (�i� � ,-. �C, cJcJ�
MDM-2
ER
p53
36B4
Fig. I Northern blot analysis of MDM2 and p53 RNA in human breast epithelial cells. The p53 status of the cells was obtained from several sources
and, if known, is indicated in parentheses (30, 31, 33, 36). RNA was isolated from three independently derived normal human mammary epithelialcells HMEC 1-3 (Wi’), immortalized, nontumonigenic HBL100, MCF-10 (Wi’) and 184B5 (Wi’) cells, estrogen receptor-positive MCF-7 (WT),ZR7S-1 (WT), T47-D (codon 194 mutation) and BT-474 (codon 285 mutation) tumor cells and estrogen receptor-negative MDA-MB-157 (null),
MDA-MB-231 (codon 280 mutation), MDA-MB-468 (codon 273 mutation), SKBn-3 (codon 175 mutation), HS578T (codon 157 mutation) breast
cancer cells, electrophonesed, transferred, and hybridized with probes for the MDM2, p53. and 36B4 genes, respectively. The lane designated SJSA-1contains RNA from an osteosarcoma line that has an amplification of the MDM2 gene. Arrows to the left of the top panel, sizes of the MDM2transcripts present in these cells. Single panel to the right, a shorter exposure of the lane containing RNA from the SJSA-1 cell line.
MDM2 in normal and immortal versus transformed human
mammary epithelial cells. For these experiments total RNA was
isolated from exponentially growing populations of NMECs
derived from reduction mammoplasties, immortalized but non-
tumonigenic mammary epithelial cells and estrogen receptor-
positive and -negative breast cancer cell lines. Normal and
immortalized, nontumonigenic breast cells grown in culture do
not express the estrogen receptor. This observation likely re-
fleets the biology of mammary epithelial cells as very few cells
are ER� in normal nonlactating mammary glands (27, 28). To
ascertain the estrogen receptor status of the various cells, we
probed the filter for ER mRNA (Fig.i, Panel 2). As expected,
only the ER� breast tumor cells had detectable levels of estro-
gen receptor mRNA.
Relatively high levels of MDM2 mRNA were present in all
three independently derived ER, NMEC strains (Lanes 1-3).
The amount of MDM2 mRNA detected in spontaneously arising
MCF1O (Lane 5) and carcinogen-induced i84B5 (Lane 6) ER,
immortalized, nontumonigenic cells was similar to the amount
present in NMECs. In contrast, HBL100 mammary epithelial
cells, that were immortalized by SV4O large T antigen, ex-
pressed a much lower level of MDM2 mRNA (Lane 4). This is
an important observation as SV4O T antigen binds to and mac-
tivates the function of wild-type p53. The four estrogen receptor
positive breast cancer cell lines MCF-7, ZR7S-1, T47-D and
BT474 (Lanes 7-10) expressed similar MDM2 mRNA levels to
those found in normal mammary epithelial cells. In contrast, the
five estrogen receptor negative breast cancer cell lines exam-
ined, MDA-MB-157, MDA-MB-231, MDA-MB-468, SKBr-3,
and HSS78T, all expressed MDM2 RNA levels that were S to iO
fold lower than in any of the other cells analyzed (Lanes Il-is).
Although all human mammary epithelial cells expressed readily
detectable MDM2 transcripts, the abundance of this mRNA was
well below the amount present in the overexpressing human
osteosarcoma cell line, SJSA-1 (16).
MDM2 mRNAs having several different molecular
weights were detected in our panel of mammary epithelial cells.
To determine the sizes of the various transcripts, identical
Research. on May 27, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
74 MDM2 and Breast Cancer
1F2
1 06
8O-�#{216}�1 �-
5B10
�
�I 06 -
8O-�#{248}� �
Fig. 2 Immunoprecipitation of MDM2 protein from human breast epithelial cells. Protein extracts were prepared from representative breast epithelial
cells and immunoprecipitation was performed by incubating 2 mg protein extract with the lF2 (Oncogene Science) or 5B10 antibody at 4#{176}C.Specific
protein complexes were separated in an 8% SDS-polyacrylamide gel, electrophoretically transferred to a nitrocellulose membrane, and subsequently
probed for MDM2 using the lF2 antibody. Numbers, migration of prestained molecular weight markers that were coelectrophoresed and transferred
with the samples. The lane labeled Control contains 2 mg protein extract from MCF-7 cells precipitated with an isotypic nonspecific antibody. Arrows,
positions and intensities of the MDM2 proteins.
amounts of RNA standards were coeleetnophoresed and their
migration was compared with MDM2 mRNAs from mammary
epithelial and SJSA-l osteosarcoma cells. The major form of
MDM2 mRNA in human mammary epithelial cells migrated as
a 6.7-kilobase mRNA. We often saw higher molecular weight
transcripts of -8.0 and 12.5 kilobase in normal and immortal
mammary epithelial cells hut rarely in tumonigenie mammary
epithelial cells. In the MDM2 ovenexpressing SJSA-l cells,
another band of -4.5 kilobase could be readily detected. This
band was detected occassionally in RNA samples from all
mammary epithelial cells.
Since MDM2 is known to lie downstream of p53 in a
nuclear signal transduction pathway (6, 7), we examined the
relationship between p53 and MDM2 mRNA levels in the same
mammary epithelial cells. When the filter shown in the upper
panel was stripped and hybridized with a probe for p53, most of
the cell lines showed readily detectable levels of p53 mRNA
regardless of their state of transformation. The two exceptions
were ZR75-l cells which expressed significantly lower levels of
p53 mRNA and MDA-MB-157 cells which had no detectable
p53 mRNA at all. The different patterns of p53 and MDM2
mRNA expression observed among the mammary epithelial
cells could not be attributed to unequal loading of the lanes as all
cells expressed similar levels of the control p36B4 mRNA.
MDM2 Protein Levels in Mammary Epithelial Cells.
MDM2 is expressed as a series of phosphoproteins ranging in
molecular weight from 55,000 to 90,000 in munine 3T3DM cells
that contain multiple copies of the MDM2 gene (1, 29). To
determine the molecular weight(s) of the MDM2 proteins cx-
pressed in human mammary epithelial cells, immunoprecipita-
tion studies were performed using the IF2 and 5BlO antibodies
which recognize epitopes in the amino and carboxy terminal
regions of MDM2, respectively (5, 14). The results presented in
Fig. 2 demonstrate that the IF2 antibody recognized at least four
distinct forms of MDM2 in breast epithelial cells. The Mr 90,000
form of MDM2 was the predominant protein present in most of
the cell lines examined. Two additional MDM2 proteins with
molecular weights of around 75,000 and 85,000 were detected at
lower levels from all mammary epithelial and SJSA-i osteosan-
comas cells. In the normal and immortalized 184B5 cells, a
higher Mr 95,00�) MDM2 protein was also detected.
Immunoprecipitations performed with the 5B10 antibody,
which recognizes an epitope in the carboxy terminus of MDM2,
yielded slightly different results. This antibody efficiently pre-
cipitated the Mr 90,000 and 95,000, and, to a lesser extent, the
Mr 75,000 forms of MDM2. The relative differences in inten-
sities of the various bands likely reflect bonafide differences in
protein levels as equivalent amounts of actin were detected from
each sample (data not presented). As a control for these exper-
iments we demonstrate that numerous protein bands with mo-
leculan weights ranging from 75,000 to 90,000 are also precip-
itated from SJSA-1 cells that contain an amplified MDM2 gene.
The level of many cell cycle-regulated proteins does not
always correlate with the abundance of their mRNAs. To deter-
mine quantitative differences in the levels of MDM2 protein in
our panel of mammary epithelial cells, we prepared total cell
protein extracts and performed Western blot analyses. The pre-
dominant form of MDM2 detectable by Western blot was the Mr
90,000 protein (Fig. 3). We found a good correlation between
the level of MDM2 mRNA and the amount of the Mr 90,000
MDM2 protein in the different cells. The normal (Fig. 3, Lanes
I and 2), immortal, nontumonigenic (Fig. 3, Lanes 3 and 4) and
the ER� breast cancer cells (Fig. 3, Lanes 5-8) all had higher
levels of the Mr 90,000 MDM2 protein than the ER� breast
cancer cells (Fig. 3, Lanes 9-13). An additional MDM2 protein
having a Mr 57,000 was present in the SJSA-i osteosarcoma
cells and many of the breast epithelial cells examined. This band
was not detectable in the immunoblotting experiments because
it comignates on gels with the mouse monoclonal antibodies
used for immunoprecipitation. The bands corresponding to the
major MDM2 proteins detected in the SJSA-l osteosancoma line
are indicated (Fig. 3, arrows on the right). To ensure that the
differences in protein levels detected in the various mammary
epithelial cells were not due to unequal loading and transfer of
proteins, the lower portion of the same membrane was subse-
quently probed for actin (Fig. 3, bottom).
Several groups have described a binding site for wild-type
p53 within the first intron of the MDM2 gene (7, 8). This DNA
sequence is apparently involved in the regulation of MDM2
gene transcription by wild-type p53. Thus, in the presence of
wild-type p53, MDM2 mRNA and protein levels were concom-
itantly increased presumably due to the influence of wild-type
Research. on May 27, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
ER� ERTumor
�w � MDM-2
�4- p53
Clinical Cancer Research 75
5 F. Diella, personal communication.
Norm 1mm TumorI 11 1 1 II I
I- C�1 I_tnc�w,- .� I.- CO
� � �. �F,� “��‘ 1’ . � r;; � ‘� �� � � (a 0’rJ� � I- I-. � U) 0 0 0
z z,- � N � C/) �
106-� -
80-
49.5 - .
49.5- � .t�-..p.--�R
49.5 -‘4- Actin
Fig. 3 Western blot analysis of MDM2 and p53 proteins in human breast epithelial cells. Total protein extracts from exponentially growing normal
mammary epithelial cells (Lanes 1 and 2), 184B5 cells (Lane 3), immortalized MCF-l0 (Lane 4), estrogen receptor-positive MCF-7 (Lane 5), T47-D(Lane 6), ZR7S-l (Lane 7), and BT-474 (Lane 8) tumor cells, and estrogen receptor-negative SKBr3 (Lane 9), HS578T (Lane 10), MDA-MB-157(Lane 11), MDA-MB-231 (Lane 12), and MDA-MB-468 (Lane 13) tumor cells were prepared in Laemmli lysis buffer. Protein (50 �xWlane) was
separated in an 8% SDS polyacylamide gel, electroblotted onto nitrocellulose, and the membranes were reacted with antibodies corresponding to
MDM2, PS3, or actin. Protein molecular weight markers that were coelectrophoresed and transferred are indicated on the left of each panel. Lanedesignated SJSA-l contains protein extract from osteosarcoma cells that ovenexpress the MDM2 protein.
p53 in the cells (6, 7). To determine whether there was a
correlation between MDM2 and p53 protein levels in different
mammary epithelial cells, we performed p53 Western blot anal-
yses on the same extracts. These results showed no apparent
correlation between the absolute levels of p53 and MDM2
proteins in these cells.
A more critical consideration in these comparisons is
whether the p53 expressed in the various cells is mutant or wild
type. Normal and immortalized, nontumonigenic breast cells
grown in culture do not express the estrogen receptor (Fig. 1).
This finding likely reflects the biology of mammary epithelial
cells since very few cells are ER� in normal nonlactating
mammary glands (27, 28). The normal and immortal 184B5
mammary epithelial cells used in these studies express a wild-
type p53 protein that has increased stability (30-32). The p.53
gene in the immortalized MCF1O cells has been sequenced
along its entirety and shown to be wild type.5 All of these cells
expressed relatively high levels of MDM2 protein. In contrast,
the ER tumor cells (Lanes 10-14) expressed either a mutant on
no p53 and had relatively low levels of MDM2 mRNA and
protein (33). Thus, when examining ER mammary epithelial
cells, we found a good correlation between wild-type p53 ex-
pression and corresponding levels of MDM2 mRNA and the Mr
90,000 MDM2 protein.
The same positive correlation between wild-type p53 and
MDM2 protein expression did not hold true for the ER� breast
cancer cells. All of the ER� breast cancer cells expressed
relatively high levels of full length MDM2 protein when com-
pared to the ER cells. MCF-7 and ZR75-l cells express a
wild-type p53 protein (34, 35); however, T47-D and BT-474
cells both express mutant p53 (33, 36). Thus, factors other than
p53 are likely responsible for the high levels of MDM2 mRNA
and protein in ER� breast tumor cells. Among the various cell
types examined, MDM2 mRNA levels showed proportionally
greater variations than did MDM2 protein levels. These findings
suggest additional regulation of MDM2 protein accumulation at
some posttnanscniptional level in human breast epithelial cells.
Wild-Type p53 Induces MDM2 Expression in ER Tu-
mor Cells. To obtain independent evidence that wild-type p53
influences MDM2 expression in human breast tumor cells, two
ER tumor cell lines, MDA-MB-157 and MDA-231, were
infected with an adenovirus vector carrying a wild-type p.53
gene at 10 plaque-forming units/cell. The relative levels of
wild-type p53 protein present in the cells 24 h after infection are
shown in Fig. 4A. We observed no difference in p513 expression
between uninfected cells (Fig. 4A, Lanes 1 and 4) and cells
Research. on May 27, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
MDA-1 57
123
MDA-231
456
A
B
p
I I
ences MDM2 mRNA expression in ER breast tumor cells that. express mutant on no endogenous p53 protein.
Effect of Estrogen on MDM2 Expression. Because all
of the ER� tumor lines contained high levels of MDM2 protein,
regardless of the status of the p.53 gene, we examined whether
estrogen could influence MDM2 expression in these cells.
- 49 5 MCF-7 and ZR75-1 cells were grown in phenol red-free me-
U dium containing charcoal-stripped serum for 5 days. These
conditions have been shown previously to slow the growth of
the cells and down-modulate the expression of estrogen-depen-
dent genes (37, 38). After scanning the autoradiograms and
correcting for slight loading differences, a 2-fold increase in
MDM2 mRNA levels was observed in MCF-7 cells after refeed-
ing the cells with estrogen-containing medium for 4 to 8 h (Fig.
5A). The slight increase in MDM2 mRNA detected in ZR75-l
cells at 4 and 8 h after estrogen repletion could be attributed to
minor loading differences as the level of the control mRNA,
36B4, was also increased slightly in these samples (bottom, Fig.
SB). We examined MDM2 protein levels in the same samples
and found that they were not affected by estrogen levels in the
M DM2 culture medium!’ In contrast to the minor changes observed in
MDM2 mRNA levels, the estrogen inducible gene, pS2, was
down-regulated in estrogen-depleted cells and increased -20-
fold 4 to 8 h after the addition of �3-estradiol to the culture
medium (middle). When considered together these data suggest
that estrogen levels may not directly influence MDM2 mRNA
expression in cultured breast epithelial cells.
Immunocytochemical Localization of MDM2 and p53
in Human Mammary Epithelial Cells. To determine the
intracellular location of the MDM2 protein, we stained repre-
sentative breast cell lines using the IF2 antibody. After evalu-
ating different fixatives, methanol:acetone (1:1) was found to
yield the most reproducible staining results. All of the mammary
epithelial cells studied contained cleanly detectable MDM2 pro-
tein using Western blot analysis (Fig. 3); however, considerable
variation in immunocytochemical staining patterns was ob-
served. The NMECs and immortalized l84B5 mammary epi-
thelial cells showed distinct nuclear staining for MDM2 with
some variation in intensity from one cell to another (Fig. 6A).
When the same cells were stained for p53 using the PAb 1801
antibody, a similar pattern of heterogenous nuclear staining was
observed. In contrast another immortalized, mammary epithelial
cell line MCF-10 showed less intense and more variable staining
for both MDM2 and p53 proteins. The decreased staining in-
tensity observed in these cells may be due to the slightly lower
levels of protein accumulation as determined by Western blot-
ting (Fig. 3).
The estrogen receptor-positive breast epithelial lines all
contained relatively high levels of MDM2 protein when exam-
med using Western blots (Fig. 3). However, when analyzed
immunocytochemically, the ER� tumor cells stained poorly, if
at all, for MDM2. In MCF-7 cells the small amount of peninu-
clear staining obtained with the MDM2 antibody is likely non-
specific because we see similar results using isotypic monoclo-
nal antibodies for other cellular proteins that are not expressed
36B4
Fig. 4 A, infection of ER� tumor cells with an adenovirus vector
containing a wild-type gene. Protein lysate prepared from exponentially
growing MDA-MB-157 and MDA-MB-231 ER tumor cells (Lanes 1
and 4), cells infected with a control adenovirus vector (Lanes 2 and 5),
and cells infected with AdWTpS3 virus (Lanes 3 and 6) was examined
for p53 expression using Western blot analysis. B. RNA was isolatedfrom cells treated as described in the legend to A and examined forMDM2 expression using Northern blot analysis. Lower panel, 36B4
expression from the same filter.
infected with a control virus lacking the p53 gene (Fig. 4A,
Lanes 2 and 5). In contrast, cells infected with a virus carrying
the wild-type p53 gene expressed significantly increased levels
of p53 protein (Fig. 4A, Lanes 3 and 6).
MDM2 mRNA levels were examined from cells indepen-
dently infected with the same viral constructs (Fig. 4B). MDM2
mRNA levels were relatively low in the uninfected cells (Fig.
4B, Lanes I and 4) and in the cells infected with a control virus
(Fig. 4B, Lanes 2 and 5). MDA-MB-157 and MDA-MB-231
cells expressing a wild-type p53 protein showed 6- and 10-fold
respective increases in MDM2 mRNA levels (Fig. 4B, Lanes 3
and 6). These increases in MDM2 mRNA levels are in accon-
dance with the finding of slightly higher levels of wild-type p53
protein in the MDA-MB-231 cells. When considered together
these data support the idea that wild-type p53 positively influ-
76 MDM2 and Breast Cancer
6 J� M. Gudas, unpublished data.
MDA-157 MDA-231
1 2 34 5 6
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Clinical Cancer Research 77
A MCF-7
� 1 2 4 8 12 24
‘�.
w�
B ZR75i
0 1 2 4 8 12240
MDM2
pS2
36B4
Fig. 5 Northern blot analysis of MDM2 mRNA in ER� cells following estrogen stimulation. MCF-7 and ZR75-l cells were depleted of estrogen
for S days and subsequently refed with medium containing charcoal-stripped serum and 10 nM �3-estradiol. At the times indicated above each lanetotal RNA was prepared, electrophoretically separated, transferred to filters, and probed for expression of MDM2 (upper panels), p52 (middle panels),
and 36B4 (lower panels). Large and small arrows, migration of the 6.7-kilobase and 4.5-kilobase MDM2 transcripts. respectively.
in these cells.6 Very few nuclei from ER� ZR7S-1 cells stained
for MDM2 protein and some cells may show some cytoplasmic
localization. Interestingly, staining of the ZR75-i cells for p53
revealed a distinct cytoplasmic staining that was highly local-
ized within discrete foci. To our knowledge, a similar staining
pattern for p53 has not been described in other cell types.
The ER, MDA-MB-231 breast cancer cells had very low
levels of MDM2 when examined usingWestern blot analysis
(Fig. 3) and did not stain at all for MDM2. Two other ER
breast cancer cell lines that expressed very low levels of MDM2
protein by immunoprecipitation and Western blotting, MDA-
MB-157 and MDA-MB-453, were also negative for MDM2
when examined immunocytochemically (data not shown). We
believe that the level of MDM2 protein may be below the
detection limit by immunocytochemistry in the ER breast
cancer cells. As expected for cells that express a mutant p53, all
nuclei in MDA-MB-231 cells stained intensely for p53. As
controls for the immunocytochemistry, we show that SJSA-1
cells, which overexpress MDM2, stained intensely for this an-
tigen while very few nuclei from these cells were positive for
p53 (Fig. 6).
DISCUSSION
In this study we report two independent observations con-
cerning MDM2 expression in human breast epithelial cells. The
first finding is that a good correlation exists between expression
of wild-type p53 in normal and immortalized mammary epithe-
hal cells with accumulation of relatively high levels of MDM2
mRNA and protein in the same cells. Because the magnitude of
the difference in relative MDM2 mRNA levels was greaten than
that observed for corresponding MDM2 protein levels, other
factors, including protein stability and translational efficiency,
likely play a role in determining the total amount of MDM2
protein in a cell. The normal and immortalized human mammary
epithelial cells used for these studies are estrogen receptor
negative and express a wild-type p53 protein. The wild-type p53
is unusual in these cells because it has a half-life of -3 h (30r
compared to a half-life of -20 mm in many other cell types (30,
39, 40).
In contrast, all of the estrogen receptor-negative breast
tumor cell lines studied had either a mutant on no p53 protein
and expressed significantly less MDM2 mRNA and protein.
Because the ER cell lines we examined all had mutations in the
conserved DNA binding domain of the protein (41), they are all
likely defective in their ability to transactivate cellular target
genes. Previous studies from other laboratories have demon-
strated a consensus p53 DNA binding site in the MDM2 gene
which is necessary for regulation by wild-type p53 (6, 7). To
determine whether wild-type p53 could, indeed, affect MDM2
expression, we introduced a wild-type p53 gene into ER breast
tumor cells that contained a mutant on no p53 protein. In both
cell types wild-type p53 expression resulted in an increase in
MDM2 mRNA levels. These data suggest that wild-type p53
functions to increase the expression of MDM2 mRNA and
protein in estrogen receptor-negative breast epithelial cells and
this regulatory circuit becomes disrupted when the cells
progress and gain mutations in the p.53 gene. ER� breast tumors
are usually more differentiated than ER tumor cells (42, 43).
Because MDM2 mRNA and protein levels are lower in ER
breast cancer cells, our data suggest a diminution rather than an
increase in MDM2 expression during the progression of mam-
many tumors.
The second major finding from this study is that MDM2
mRNA and protein levels are significantly higher in ER� breast
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Fig. 6 A, immunocytochemical localization of MDM2 and p53 proteins in normal and immortal human breast epithelial cells. Cells were grown on
glass tissue culture chamber slides, fixed in methanol:acetone (1:1), and subsequently stained for MDM2 and p53 using the 1F2 or PAb 1801
antibodies, respectively. After washing, specific complexes were detected using the avidin-biotin complex immunoperoxidase detection system as
recommended by the manufacturer (Vectastain). B, MDM2 and p53 immunostaining in tumorigenic breast epithelial cells.
78 MDM2 and Breast Cancer
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cancer cells independent of their p53 status. Only MCF-7 and
ZR75-1 cells have a wild-type p53 gene (35). The other estro-
gen ER� breast cell lines examined, T47-D and BT474, both
expressed mutant p53 proteins (33, 36). Despite the presence of
a mutant p53 gene, these cells had relatively high levels of
MDM2 protein. Thus, other cellular factors, apart from p53,
must account for the high levels of MDM2 mRNA and protein
in these cells.
Because this phenotype was only observed in the ER�
breast cancer cells, we asked whether some function or genes
regulated by the estrogen receptor could be involved in the
increased expression of MDM2 in these cells. When ER� cells
were cultured in estrogen-depleted medium, the mRNA for the
estrogen-regulated gene, pS2, was decreased and increased sig-
nificantly on nefeeding the cells with medium containing �3-es-
tradiol. Under the same conditions only a 2-fold change in
MDM2 mRNA levels in MCF-7 cells and no change in ZR75-l
cells was detected. Therefore, the elevated levels of MDM2
mRNA and protein in these cells may not involve a direct effect
of the estrogen receptor itself. Many other genes including
glutathione-S-transferase in and gluathione penoxidase are dif-
ferentially expressed in ER� versus ER breast cancer cells
(44-46). Presumably, differences in the posttranscniptional pro-
cessing of these mRNAs differences and/on differences in their
chnomatin structure accounts for preferential mRNA expression
in one cell type versus another. These results are similar but not
identical to those reported by Saeed et al. (47) who found that
estrogen increased MDM2 expression in ER cells transfected
with a gene encoding the estrogen receptor. Because the estro-
gen receptor does not always induce estrogen-dependent pro-
moters when introduced into ER cells (48), the two results are
not directly comparable.
Research. on May 27, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 79
Several different MDM2 transcripts, ranging in size from
4.5 to 12.5 kilobases, were detected in mammary epithelial cells.
At present we do not know if these various mRNA species have
functional significance or if some of the higher molecular
weight transcripts represent partially on alternatively processed
MDM2 mRNAs. Interestingly, evidence from DNA sequencing
studies indicates that both the human and mouse MDM2
mRNAs can be alternatively spliced (1, 49). Because all mRNA
bands hybridized with probes obtained from both the 5’ and 3’
end of the coding region of the human MDM2 eDNA, we
believe that they are all bona fide MDM2 transcripts.
Results from immunoprecipitation and Western blot anal-
yses showed a complex pattern of many different MDM2 pro-
teins in human breast epithelial cells. The predominant protein
detected from all mammary epithelial cells migrated as a Mr
90,0000 band. This form likely represents the full-length
MDM2 protein while the smaller, -57,000 form, may be an
alternatively spliced variant as described in mouse and rat cells
(I, 29). Haines et al. (29) have necently reported that the
different molecular weights of the MDM2 proteins in mouse
cells represent alternatively spliced variants. Moreover, they
have demonstrated that these proteins differ in their ability to
physically associate with, and thus inhibit, the function of wild-
type p53 (29). When considered along with the finding of
multiple mRNA species, we believe that the different forms
of MDM2 present in breast epithelial cells likely represent
distinct MDM2 proteins that may have both overlapping and
unique biological functions.
Immunocytochemical staining for MDM2 showed that the
protein was nuclear in breast epithelial cells; however, the
intensity and number of positive cells did not always correlate
with the level of protein detectable by biochemical methods.
Some of the variation in nuclear MDM2 staining seen in the
NMECs and l84B5 cells may reflect differences in cell cycle
distribution as we have previously shown that these cells stain
variably for p53, depending upon their position in the cell cycle
(32). ER� MCF-7, ZR75-1, and BT474 (data not shown) cells
showed little or no staining for MDM2 although they had
comparable amounts of the Mr 90,000 MDM2 when assayed
using Western blot analysis (Fig. 3). One possibile explanation
for the anomolous immunocytochemical results in ER� cells is
that MDM2 is somehow sequestered in a larger complex that is
only dissociated on lysis of the cells. We are presently perform-
ing more detailed experiments to test this hypothesis. With the
exception of a unique cytoplasmic focal staining pattern for p53
observed in ZR7S-1 cells, our results for p53 staining in breast
epithelial cells are similar to those reported by other investiga-
tons (30, 33, 50).
Increasing evidence suggests that p53 and its downstream
effector proteins MDM2 and CIP/Waf-1 play a role in deter-
mining cellular responses to endogenous and exogenous signals.
The differential expression of multiple MDM2 mRNAs and
proteins raises the possibility that these products may perform
distinct functions in mammary epithelial cells at different stages
of tumor development. We believe that further study of p53 and
its downstream effector proteins MDM2 and CIP-1 (51, 52)
should enhance our understanding of the processes of cell trans-
formation and the means by which cells modulate their response
to DNA damage and environmental stress. A better understand-
ing of these cellular processes should in turn translate into more
specific, and, hopefully, more effective therapeutic approaches
for the treatment of breast cancers.
ACKNOWLEDGMENTS
We thank Drs. Arnold Levine and Vincent Marachel for providing
us with the 5BlO monoclonal antibody. We are also grateful to Dr. Bert
Vogelstein for providing the human MDM2 eDNA clone, Dr. Thomas
Look for the SJSA-1 osteosarcoma cells, and Dr. Erasmus Schneider for
helpful comments on the manuscript.
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1995;1:71-80. Clin Cancer Res J M Gudas, H Nguyen, R C Klein, et al. proteins in normal and tumorigenic breast epithelial cells.Differential expression of multiple MDM2 messenger RNAs and
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