Transforming growth factor beta I expression in benign and malignant prostatic tumors

The Prostate 25:2 10-2 I8 (I 994)

Transforming Growth Factor Beta I Expression in Benign and Malignant Prostatic Tumors

E. Glynne-Jones, M.E. Harper, L. Goddard, C.L. Eaton, P.N. Matthews, and K. Griffiths

Tenovus Cancer Research Centre (€.G.-J, M.E.H., L.G., C.L.E., K.G.), and Department of Surgery (P.N.M.), University of Wales College of Medicine, Heath Park, Cardiff,

United Kingdom

ABSTRACT: The expression of transforming growth factor beta 1 (TGF-P1) in prostate specimens obtained from patients with benign prostatic hyperplasia (BPH, n = 32) and prostate carcinoma (n = 66) was investigated using Northern blot analysis and immunohistochemistry. Northern blot analysis revealed TGF-Pl message (2.5 kb) in virtually all of the samples examined, reflecting the ubiquitous nature of this growth factor. No statistical difference was found between the levels of mRNA detected in benign and malignant tissues due, in part, to the inherent heterogeneity of prostate tissue. Immunohistochemical methods using an antibody to native TGF-P1 revealed a novel pattern of immunoreactivity. Staining observed only in certain epithelial cells of benign glands was associated with areas of infection rather than tumorigenesis. Interestingly, intense staining was also seen in polymorphonuclear leukocytes. No correlation was found with the mRNA results, suggesting that this antibody is binding to TGF-P1 activated in response to infection rather than de- tecting sites of synthesis of latent TGF-P1. 0 1994 Wiley-Liss, Inc.

KEY WORDS: BPH, prostate cancer, polymorphonuclear leucocytes, infection

INTRODUCTION

The transforming growth factor Ps are a family of dimeric polypeptides of which five have been identi- fied to date, either by isolation of the proteins or by cDNA cloning. Transforming growth factor (31 (TGF- P l ) is a 25 kilodalton pluripotent regulator of cell growth and function [1,2]. Although originally described as an activity produced by retrovirally transformed cells, both TGF-P1 and its receptors are expressed in many normal tissues and cell types. The widespread distribution suggests that the activation process may play an important part in the control of TGF-P1. This view is supported by the finding in a number of investigations that TGF-P1 mRNA levels remain constant, while protein secretion increases IW.

Important functions of TGF-Pl include the promo- tion of matrix formation and tissue repair [5,6], immunoregulation [7], mediation of the inflammatory response [6], and regulation of epithelial cell growth [S-lo], all of which are important in the normal and

abnormal growth of the prostate. Although widely reported as an epithelial cell inhibitor, the role of TGF-Pl in carcinogenesis is complex. Primary cul- tures of benign, hyperplastic, and malignant prostate tissues are very responsive to the growth inhibitory effects of TGF-P1, whereas the established, androgen-independent prostatic cell lines PC3 and DU145, although initially inhibited, return to their normal growth pattern despite retreatment [9,10]. Stimula- tion of the growth of transformed rat prostate cells by TGF-Pl has been reported [ll], and enhanced tumor growth demonstrated in an in vivo model using MATLyLu rat prostate carcinoma cells transfected with a vector producing overexpression of TGF-Pl

Received for publication November 24, 1993; accepted February 15, 1994. Address reprint requests to Dr. M.E. Harper, Tenovus Cancer Re- search Centre, College of Medicine, Heath Park, Cardiff CF4 4XX, U.K.

0 1994 Wiley-Liss, Inc.

TGF-P Expression in Human Prostatic Tumors 21 I

[12]. The potential tumor growth-promoting proper- ties include the stimulation of angiogenesis [5], sup- pression of the immune system [13], and stimulation of invasion and metastatic potential [14].

The control of TGF-(3 expression in the prostate is poorly understood. Investigations, carried out by both in vitro and in vivo studies, have implicated TGF-p1 in the progression to malignancy [15-181. Im- munoreactive TGF-p has been detected using a number of antibodies with very different primary epitope specificity [19-211. The object of this study was to investigate the expression of TGF-p1 in a large series of prostate biopsy specimens using a Northern blot analysis to detect mRNA, in conjunction with immunohistochemistry using an antibody to native TGF-P1 to detect the peptide.

MATERIALS AND METHODS

Tissue Samples

Prostate tissue was obtained from 66 patients with histologically diagnosed prostatic carcinoma, and 32 patients with benign prostatic hyperlasia (BPH). All of the patients with carcinoma and 27 of the patients with BPH underwent transurethral resection (TUR) of their tumors, while the remaining 5 patients with BPH had open prostatectomy operations. A representative sample of the curettings from the operations was fixed in Bouins fluid, followed by paraffin wax embedding for subsequent histopathological examination and for the TGF-p1 immunostaining assay. The remainder was snap-frozen in liquid N, and stored at -70°C. Of the frozen tissue, 50 carcinoma samples and 19 BPH samples were used for mRNA analysis. A representative piece of each of the curettings selected for RNA extraction was embedded in OCT mountant to allow histological comparison with the wax embedded tissue.

A range of human and murine tissues known to express TGF-P1 [20,22-241 were examined for immunoreactivity using Bouins fixed archival material. The human tissues included colon carcinoma, breast carcinoma, adrenal gland, testis, normal prostate, normal kidney, infected kidney, and small bowel. Mouse tissues included normal prostate, spleen, salivary gland, kidney, testis, vas deferens, pancreas, and bladder.

Cell Lines

DU145 and PC3 prostatic carcinoma cell lines were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 i.u./ml), streptomycin (100 i.u./ml), and fungizone (2.5 pg/ml). Cells were grown in Nunc

T75 flasks (Gibco, Paisley, Scotland) for RNA extraction and in chamber slides (Gibco) for immunohistochemical analysis.

lmmunohistochemical Assay

Wax-embedded, Bouins-fixed tissue sections 5 pm thick were mounted on chrome aludgelatin-coated slides and dried at 37°C overnight. Sections were de- waxed and taken through graded alcohols to phos- phate buffered saline (PBS, 0.01 M, pH 7.4). Sections were washed in PBS followed by incubation for 1 hr with a polyclonal rabbit antibody to TGF-P1 (TGF-p1 detection antibody, British Biotechnology Ltd., Ox- ford, UK) at a dilution of 1:40 (1 pg/ml in 0.05 M Tris-HC1 prepared in 0.01 M PBS), which was found to be optimal for this assay system. Antigen binding was detected by the streptavidin-biotin immunoper- oxidase method, using the streptavidin-biotin univer- sal kit (Diagnostic Products Corporation, Oxford, UK) as per protocol with the chromogen 3 amino-9- ethyl carbazole. Sections were lightly counterstained with 10% hematoxylin and mounted in Aquamount (BDH Chemical Co., Poole, UK). Negative controls consisted of consecutive sections in which rabbit IgG at the appropriate dilution (10 pg/ml) replaced the primary antibody. Specificity of the staining reaction was tested by preabsorption of the primary antibody with two different preparations of the TGF-P1 peptide (British Biotechnology Ltd., Sigma, Poole, Dor- set, UK) at a concentration of 100 pg/ml, overnight, at 4°C. A number of methods of denaturing the tissue sections prior to incubation with the primary antibody were investigated. These included saponin (0.05% in water, 20 min at room temperature), pronase (0.01%, 0.001%, in PBS, 15 min at 37"C), hyaluronidase (1 mg/ml in 0.1 M NaAc, 0.15 M NaCl, pH 5.5, 30 min at 37"C), acid urea (6 M urea in 0.1 M glycine HCl buffer, pH 3.5, 1 hr at 4"C), and microwave treatment (4 x 5 min, high power, 850 W, in citrate buffer, pH 6, at 100°C). Cells cultured in chamber slides were rinsed in PBS, fixed in Bouins fluid (8 min), and rinsed in 70% ethanol prior to TGF-P1 detection as previously described.

RNA Extraction and Northern Blot Analysis

Total RNA was extracted using the guanidine isothiocyanate/cesium chloride method 1251. Briefly, frozen tissue (0.5 g) was pulverized in liquid N, and homogenized at 4°C in guanidine isothiocyanate buffer (4 M containing 0.25 M sodium acetate, pH 6, and 0.1 M 2-mercaptoethanol). After centrifugation at 25,000 rpm (lOO,OOOg, 4°C) to remove cell debris, a CsCl gradient was set up by the addition of 0.4 g/ml CsCl to the lysate, which was then layered onto a

212 Glvnne-lones et al.

Fig. 1.

TGF-P Expression in Human Prostatic Tumors 213

cushion of 5.7 M CsCl (containing 0.1 M EDTA). Cen- trifugation was at 41,000 rpm (288,00Og, Beckman SWTi41) at 20°C for 5 hr. RNA pellets were purified by phenolichloroform extraction and ethanol/sodium acetate precipitation. Precipitates were resuspended in autoclaved analar H,O prior to spectrophotometric RNA estimation. RNA samples (20 pg) were electrophoresed on denaturing formaldehyde/agarose (1 %) gels and transferred to Hybond N (Amersham Int., plc, Amersham, UK) nylon filters. RNA from the prostatic carcinoma cell lines, DU145 and PC3, were included on every blot to act as positive controls and as a measure of interblot variation.

Blots were prehybridized (4 hr) and then hybrid- ized (overnight at 55°C) with a 1 kb human TGF-p1 cDNA ECORl fragment [26]. The probe was labeled with [a3’P]dCTP using the random prime labeling system (Prime-a-gene labeling kit, Promega Corp, Southampton, UK), and 1 x lo6 dpm labeled probe/mI was added to the hybridization solution.

Prehybridization and hybridization solutions contained 50% deionized formamide, 5 x SSPE (900 mM NaCl, 5 mM EDTA, 50 mM Na, PO4 pH 7.4), 5 x Denhardt’s solution (0.1% Ficoll 400, 0.1% polyvinyl pyrrolidone, 0.1 % bovine serum albumin), 6% poly- ethylene glycol 6000, 0.5% sodium dodecyl sulphate (SDS), and salmon sperm DNA (100 pg/ml). After hybridization, the filters were washed twice in 2 x SSC containing 0.1% SDS at 45°C for 30 min, and once in 0.1 x SSC containing 0.1% SDS at 60°C for 30 min (1 x SSC consists of 150 mM NaC1, 15 mM tri- sodium citrate, pH 7), followed by autoradiography using Fuji RX film with intensifying screens. Filters were washed in 1% glycerol at 80°C for 2 min to remove the probe prior to rehybridization (overnight at 45”C, 5 x lo5 dpm labeled probe/ml) with a 3.5 kb p-actin cDNA ECORUHind I11 fragment of PBR 322.

Fig. I . lmmunohistochemical distribution of TGF-P I (a-e) and prostate specific antigen (PSA, f) in human prostatic tumor sections. All sections were lightly counterstained with hematoxylin to clarify the morphology. a: Heterogeneous TGF-P I immunostaining is seen in the secretory epithelium of benign glands. Staining is predominantly cytoplasmic. x 450. b: Anaplastic carcinoma cells (open arrow) are negative, whereas the adjacent benign gland (solid arrow) has positive TGF-PI immunostaining. ~ 4 5 0 . c: In- tense staining in a dysplastic gland in an area of lymphocytic infiltration. No immunostaining was seen in the lymphocytes. x 450. d: Several PMNL within a blood vessel (arrow) exhibit immunoreactivity for TGF-P I . X 670. e,f: Consecutive sections demonstrating an inverse correlation between the presence of TGF-P I and PSA in epithelial cells. Positive staining for TGF-P I is seen in the benign dysplastic gland and negative reaction in the adjacent area of well- differentiated carcinoma (e), whilst the latter exhibits intense PSA immunostaining which is not seen in the dysplastic area (f). X 450.

Quantitation of mRNA

Sizes of detectable transcripts were determined with reference to RNA molecular weight markers (Promega Corp.). Densitometry was performed on X-Ray films with a Bio-Rad scanning densitometer . Density values were adjusted to allow for nonlinear- ity of dpm vs. optical density (OD) using an empiri- cally derived correction table (Eaton et al., unpub- lished data) prior to normalization to the values from the p-actin probe to allow for variability in the loading and transfer of RNA samples. Results were expressed as a percentage of the DU145 control mRNA which was included on every blot. The signal from the cell line DU145 was chosen, since density levels were within the same range as those obtained from the prostate tissue mRNA, whereas those of PC3 were much higher.

Statistical Analysis

The Kruskal-Wallis test and the Mann-Whitney test were used to test for significance among the three histological groups: BPH, carcinoma seen in conjunction with BPH, and carcinoma alone.

RESULTS

TGF-f3 I lmmunohistochemistry

Focal areas of immunoreactive TGF-Pl were located in the secretory epithelium of benign ducts and glands in BPH specimens. Staining was localized to areas of infection and strongly associated with foci of lymphocytic infiltration and the presence of polymorphonuclear leukocytes (PMNL). Such areas were evident to some degree (varying from <1% to 60% tissue involvement) in 92% of the tissues studied. Staining was observed in columnar secretory epithelial cells (Fig. la,b), but was most intense and widespread (up to 90% of cells within a focal area of staining) where there had been a response to the infection, i.e., lymphoid invasion, dysplasia, atrophy, and in- volution of glandular epithelium (Fig. k ) . Immuno- reactivity was predominantly in the cytoplasm but, within an affected acinus, staining was also seen in a proportion of the nuclei of these cells (0-60%). Car- cinoma cells were consistently negative (Fig. lb) irre- spective of lymphocytic infiltration or histological grade of the tumor. The occurrence of TGF-P immunostaining in the various prostate specimens is sum- marized in Table I.

The presence of PMNL was a common featui-e OC-

curring in 75% of the tissues. Numbers varied from a few PMNL infiltrating the stromal matrix to large numbers seen within blood capillaries and acinar lumen. This cellular population also exhibited a strong

214 Glynne-Jones et al.

TABLE I. lmmunohistochemical Staining for TGF-6 I

BPH with BPH carcinomaa Carcinoma

Total no. 32 42 24

Negative 6 (19%) 12 (29%) 24 (100%) Positive 26 (81%) 30 (71%) 0

"Specimens in which BPH was seen in conjunction with carcinoma.

staining reaction with this TGF-P1 antibody (Fig. Id) whereas lymphocytes, monocytes, and macrophages were negative. There was a wide variation in staining intensity with generally stronger staining where PMNL were located in blood vessels than within the stromal compartment or the lumen of affected acini. Positively stained PMNL were sometimes seen in capillaries adjacent to morphologically typical BPH tissue with no apparent infection in the immediate location.

In tissues which contained both BPH and carcinoma, the epithelial cells in benign glands displayed the same range of positivity and association with infection as tissues with exclusively BPH morphology. PMNL, occasionally seen infiltrating areas of carcinoma, displayed the same range of positivity as those within benign tissue specimens. Monolayers of DU145 and PC3 cells grown in chamber slides were negative for TGF-P1 when tested in this assay system.

A number of other human and mouse tissues were examined for TGF-P1 immunoreactivity using the same assay protocol. The human tissues included colon carcinoma, breast carcinoma, adrenal gland, testis, normal prostate, normal kidney, infected kidney, and small bowel. Mouse tissues included normal prostate, spleen, salivary gland, kidney, testis, vas deferens, pancreas, and bladder. The only positivity detected in these tissues was in the tubules of the infected kidney and PMNL present in a specimen of human colon. Specificity of the staining reaction was demonstrated by preabsorption of the TGF-PI antibody with two different preparations of TGF-P1 peptide (R&D systems, Sigma) which eliminated immunoreactivity in both the prostatic epithelial cells and the PMNL.

Northern Blot Analysis

TGF-P1 message was detected in 97% of the samples. Northern blots with the TGF-P1 probe showed a 2.5 kb transcript and, with the actin probe, showed a 2.1 kb transcript in all cases (Fig. 2).

Of the 50 carcinoma samples selected for study,

examination of hematoxylin and eosin-stained frozen sections from OCT blocks made prior to pulverization of the tissue for RNA extraction revealed that 11 consisted only of BPH tissue, and these have been recat- egorized accordingly. Figure 3 shows the relative abundance of TGF-P1 mRNA in samples with only BPH, samples in which both BPH and carcinoma were evident, and those in which only carcinoma was seen. There is no significant difference among these groups as tested by the Kruskal-Wallis test (P =

0.607) and the Mann-Whitney test (BPH vs. carcinoma + BPH, P = 0.726; BPH vs. carcinoma, P = 0.313; carcinoma + BPH vs. carcinoma, P = 0.691).

DISCUSSION

These studies provide further evidence of the mul- tifunctional nature of TGF-6. TGF-Pl mRNA was detected in virtually all of the prostate samples studied, reflecting the widespread distribution of TGF-P1 which is released in its latent form by most cells [1,2]. TGF-P1 mRNA has been reported in 12 BPH and 3 normal prostate specimens by Mori and et al. [22] who, using a synthetic oligonucleotide probe, found TGF-P1 message in all of their tissue samples with no significant difference in expression between the two groups, although TGF-P2 expression was elevated in BPH compared to normal prostate. Many tumor cells express higher levels of TGF-P1 than their normal counterparts and have lost their ability to respond to the peptide leading to the suggestion that TGF-P1 may affect carcinogenesis by a paracrine effect on stromal elements of the tumor [l]. Elevated mRNA levels have been found in rat prostate carcinoma [16] and, using a transgenic mouse prostate reconstitution model system, Thompson et al. [27] found that increased TGF-P1 mRNA was involved with the progression to malignancy.

In our study, no significant difference was found among the three specimen groups, BPH, BPH associated with carcinoma, and carcinoma alone. This may be an indication of the importance of the activation of the latent form in the mechanism of action of TGF-P1 but to some degree can be accounted for by the inherent heterogeneity of prostate tissue. Variables include the percentage of stromal compared to epithelial tissue, the degree of involvement of the tissue with carcinoma, the percentage of carcinoma compared to BPH, the presence and extent of inflammation and infection, and the extent of damage by TUR, all of which may influence the level of detection of TGF-P1 mRNA. Further insight could be gained by in situ hybridization which would elucidate further the to- pographical distribution of the sites of synthesis of TGF-P1 mRNA. Detection of TGF-P1 peptide has been

TGF-f3 Expression in Human Prostatic Tumors 2 I 5

I n

m 0 c 0

E .- L

8 I Q m m 7

E"

8 0 c .- 2

(D 7

I n m b 7

7

3 n 0" R

TGFPI 2.5kb

Actin 2.1 kb

Fig. 2. Northern blot analysis of TGF-PI and actin expression in a group of prostate tumor specimens. RNA from the prostatic cell lines DU 145 and PC3 were included on every blot as positive controls. RNA was electrophoresed and transferred to nylon filters followed by sequential hybridization with each probe. Exposure times for TGF-P I and actin were 45 hr and 20 hr, respectively.

achieved by immunohistochemical studies using a number of antibodies with very different primary epitope specificity. Flanders et al. [ZO] developed two antibodies raised against synthetic preparations of a peptide, corresponding to the amino terminal 30 amino acids of mature TGF-P1. Although raised to the same peptide sequence, due presumably to exposure of different epitopes in the various forms of the peptide, two distinct staining patterns were observed in a range of human and murine tissues [20,23]. Staining was located in the extracellular space with the antibody LC(1-30) and in the intracellular space with antibody CC(1-30). It was postulated that the LC antibody binds to sites of synthesis since the same pattern of intracellular localization of immunoreactive TGF-P1 was also seen with an antibody to the mature peptide, anti P(50-75), and two antibodies raised to peptide sequences only seen in the precursor form of TGF-P1

Using the transgenic mouse prostate reconstitution model, Thompson et al. [27] found elevated expression of extracellular TGF-P1 with the CC(1-30) antibody which, in agreement with their mRNA results, was associated with the progression from the benign state to malignancy. Truong et al. [18] reported extracellular staining in prostatic specimens using the CC(1-30) antibody with more extensive immunoreactivity associated with prostate cancer com-

P O I .

pared with BPH specimens. Intracellular staining with the LC(1-30) antibody was found in both stro- ma1 and epithelial cells with the former being more pronounced in the stromal cells of BPH specimens, whereas epithelial staining was more intense and more widespread in prostate carcinoma specimens. An antibody to residues 4-19 was used to demonstrate the association of TGF-P1 with the rate of disease progression in breast cancer [24].

A number of antibodies have been raised to purified native TGF-P1 extracted from porcine platelets (R&D Systems). This is the large latent form of TGF-P1 in which mature TGF-P1 is noncovalently associated with sequences from the remainder of the precursor, also known as the latency-associated peptide (TGF- PlLAP), and a third high molecular weight binding protein found only in platelets [28,29]. In this biolog- ically latent form, TGF-P1 cannot bind to its cellular receptor nor can it be immunoprecipitated by polyclonal antisera to TGF-Pl suggesting that the receptor binding site and other epitopes may be masked. Char- acterization of a neutralizing antibody to TGF-PI [30] using immunoblotting showed binding to mature TGF-P1 and TGF-plLAP with weak association to the binding protein. This antibody was used to investigate the role of TGF-P1 in normal and diseased human kidneys [30]. Immunoreactive TGF-Pl was found to be localized in association with matrix components of

216 Glynne-Jones et at.

-

-,

-

4

3 *d

ti 4)

d s 2 E

1

0

a

a a 0 a a 0

08 I

A

.. a 0 a a 4 i 0 a a

8 j a

- - - -

a

0

i w a 0

Specimen Histology

Fig. 3. Relative abundance of TGF-6 I mRNA in the three specimen groups: BPH specimens, samples in which both BPH and carcinoma were present, and tissue containing only carcinoma.

glomerular basement membrane or mesangium and with immune deposits in the glomeruli.

The results of our study indicate the immunohistochemical localization of a different epitope of TGF- P l to those described above. The antibody used (R&D Systems, detection antibody), also raised to native TGF-PI extracted from porcine platelets, does not neutralize the physiological activity of TGF-P1.

Immunoreactivity was detected in epithelial cells in benign prostatic glands in association with areas of inflammation and infection. TGF-P1 plays an important role in immunoregulation, inflammation, and tissue repair by both paracrine and autocrine mecha- nisms [6,7,10]. The activation of TGF-P1 in the prostate cells in response to infectious agents would aug- ment the inflammatory reaction by priming these tissue sites for enhanced PMNL recruitment [31]. Di- rected migration of PMNL caused by femtomolar concentrations of TGF-P1 has shown that this peptide is the most potent PMNL chemotactic factor yet identi- fied [32]. TGF-P1 is also a powerful chemoattractant of monocytes, which when recruited, are further stimulated by locally increased concentrations of TGF-P1 to synthesize and secrete other growth factors and cytokines such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF), and interleukin 1 (IL-l), thus promoting tissue repair by regulating fibrosis and angiogenesis.

mRNA is expressed in arbitrary units represented as a percentage of the mRNA level detected for DU I45 which is included on every blot as a control.

Morphological changes resulting from infection in the prostate include dysplasia, metaplasia, and invo- lution of glandular epithelium and initial changes along these pathways can be seen in glands where TGF-P1 immunostaining was most intense. It is of interest to note that, in general, there was a negative correlation between the location of TGF-P1 and prostate-specific antigen (Fig. le,f) and between TGF-P1 and prostatic acid phosphatase in the glands (unpub- lished data), which may indicate that a reduction or loss of secretory activity is one of the early events in this process. Damage to the glandular tree due to trauma and infection must result in cellular remodel- ing in order to seal off the damaged area, which of necessity will also induce death of certain cellular populations at these points. This process may also involve TGF-P1 which has been shown to be an important factor in apoptosis [33-351.

Carcinoma cells did not exhibit any binding with the TGF-P1 antibody used in these studies suggesting that the epitope of interest is either not present or is masked. Activation of the latent form of TGF-Pl can be achieved in vitro by heat, proteolytic enzymes, low or high pH, denaturing with SDS, or a high concentration of urea [l]. Acid urea and hyaluronidase have been used to demonstrate TGF-P1 staining in immunohistochemistry [18,30]. In order to assess the possibility of hidden epitopes in our studies, a number of methods of denaturing the prostate tissues

TGF-P Expression in Human Prostatic Tumors 2 I7

were investigated, prior to the addition of the primary antibody, including acid urea, pronase, saponin, hyaluronidase, and microwave treatment. None of these treatments altered the staining pattern and in some instances the intensity was reduced. The two prostatic cancer cell lines PC3 and DU145 grown as monolayers, like the carcinoma cells of the prostatic biopsy specimens, showed no immunoreactivity with this antibody despite the high levels of TGF-P1 mRNA detected after Northern blotting, indicating that this antibody does not detect sites of synthesis.

Interestingly, a feature of this immunocytochemical study was the strong staining seen in PMNL (Fig. Id). This observation suggests, for the form of TGF-p1 in which this epitope is exposed, a role in inflammation and infection rather than tumorigenesis. Using the LC(1-30) antibody, immunoreactivity for TGF-P1 has been detected in PMNL in the initial inflammatory response to the implantation of demineralized rat bone matrix in postnatal rats in an in vivo model of endochondral bone formation [36]. It has been reported that cultured human peripheral blood monocytes and PMNL secrete TGF-P1 which was deduced to be in the active form, since acid treatment of the conditioned medium did not increase the amount of TGF-Pl activity [37]. TGF-Pl transcripts were detected at similar levels in monocytes and in PMNL. Although TGF-Pl is a potent chemoattractant for both these cell types and both express high affinity type I receptors, there are fundamental differences in their response to, and regulation by, the peptide. In contrast to the receptors on monocytes, neutrophil TGF-p1 receptors are not down-regulated by exposure to inflammatory mediators [32]. In addition, exposure to the peptide does not enhance specific functions of PMNL as occurs with monocytes. Neutrophil production of reactive oxygen intermediates is not induced by TGF-P1 nor does it suppress the production induced by other stim- uli [32]. The fact that, in our assay system, monocytes did not show immunoreactivity for TGF-p1 supports the view that staining in the PMNL may be due to a scavenging mechanism either for excess TGF-P1 in order to restrict the local action of the activated peptide or for the latency-associated peptide after cleavage from the mature peptide.

It is not known to which part of the large latent form of TGF-P1, extracted from platelets, this antibody is raised. Binding may be to an epitope, or epitopes, in the mature peptide, the latency-associated peptide, and/or the binding protein. In view of the fact that the results of the immunocytochemical study do not correlate with the mRNA studies, it is unlikely that we are seeing sites of synthesis. The occurrence of different forms and complexes of TGF- p l , depending on the physiological conditions, may

account for the specificity of the staining reactions seen with the various antibodies which have been produced. A panel of well characterized TGF-Pl antibodies is needed to find out which forms of this peptide correlate with the many different functions of TGF-P1.

ACKNOWLEDGMENTS

The authors thank the Tenovus organization for their generous financial support.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

REFERENCES

Roberts AB, Sporn MB: The transforming growth factors-p. In Sporn MB, Roberts AB (eds): ”Handbook of Experimental Pharmacology Peptide Growth Factors and Their Receptors.” Heidelberg: 1990, pp 419-472. Masague J: The transforming growth factor-p family. Annu Rev Cell Biol 6:597-641, 1990. Kerhl JH, Wakefield LM, Roberts AB, Jakowlew S, Al- varezmon M, Derynck R, Sporn MB, Fauci AS: Produc- tion of transforming growth factor-p by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 163:1037-1050, 1986. Assoian RK, Fleurdelys BK, Stevenson HC, Miller PJ, Madtes DK, Raines EW, Ross R, Sporn MB: Expression and secretion of type p transforming growth factor by activated macrophages. Prof Natl Acad Sci USA 84:

Roberts AB, Sporn MB, Assoian RK. Transforming growth factor type beta: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 83:4167-4174, 1986. Wahl SM, Wong H, McCartney-Francis N: Role of growth factors in inflammation and repair. J Cell Bio- chem 40:193-199, 1989. Kehrl JH: Transforming growth factor+: An important mediator of immunoregulation. Int J Cell Cloning

Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB: Type beta transforming growth factor: A bifunctional regulator of cellular growth. Proc Natl Acad Sci USA 82119-123, 1985. Wilding G, Knabbe C, Zugmaier G, Flanders K, Gelp- mann El’: Differential effects of TGFP on human prostate cancer cells in vitro. Mol Cell Endocrinol62:79-87, 1989. Wilding G: Response of prostate cells to peptide growth factors: Transforming growth factor p. Cancer Surv 11:147-161, 1991. Shain S, Lin A, Koger J, Karaganis A: Rat prostate cancer cells contain functional receptors for transforming growth factor p. Endocrinology 126:818-825, 1990. Steiner M, Barrack E: Transforming growth factor p l overproduction in prostate cancer: Effects on growth in vivo and in vitro. Mol Endocrinol 6:15-25, 1992. Torre-Amione G, Beauchamp RD, Koepper H, Park BH, Schreiber H, Moses HC, Rowley DA: A highly im- munogenic tumour transfected with murine transforming growth factor p l cDNA escapes immune surveil- lance. Proc Natl Acad Sci USa 87:1486-1490, 1990. Welch DR, Fabra A, Nakajima M: Transforming growth

6020 -6024, 1987.

9~439-450, 1991.

218 Glynne-Jones et al.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

factor p stimulates mammary adenocarcinoma cell invasion and metastatic potential. Proc Natl Acad Sci USA 87:7678-7682, 1990. Thompson TC: Growth factors and oncogenes in prostate cancer. Cancer Cells 2345-354, 1990. Steiner MS, Barrack ER Expression of transforming growth factors and epidermal growth factor in normal and malignant rat prostate. J Urol 143:240A, 1990. Merz VW, Miller GJ, Krebbs T, Timme TL, Kadmon D, Park SH, Egawa S, Scardino PT, Thompson TC: Ele- vated TGF p l mRNA levels are associated with ras plus myc induced carcinomas in reconstituted mouse prostate: Evidence for a paracrine role during progression. Mol Endocrinol 5:503-513, 1991. Truong LD, Kadmon D, McCune B, Flanders KC, Scar- din0 A, Thompson TC: Association of transforming growth factor-pl with prostate cancer. An immunohistochemical study. Hum Pathol 24:4-9, 1993. Flanders KC, Roberts AB, Lung N, Fleurdelys E, Sporn MB: Antibodies to peptide determinants of TGFP and their application. Biochemistry 27:739-746, 1988. Flanders KC, Thompson NL, Cissel DS, Van Ob- berghen-Schilling E, Baker C, Kass ME, Ellingsworth LR, Roberts A, Sporn MB: Transforming growth factor p l . Histochemical localisation with antibodies to different epitopes. J Cell Biol 108:653-660, 1989. Ellingsworth LR, Brennan JE, Fok K, Rosen DM, Bentz H, Piez KA, Seyedin SM: Antibodies to the N-terminal portion of cartilage-inducing factor A and transforming growth factor p. J Biol Chem 261:12362-12367, 1986. Mori H, Maki M, Oishi K, Jayne M, Igarashi K, Yoshida 0, Hakanaka M: Increased expression of genes for basic fibroblast growth factor and transforming growth factor p2 in human benign prostatic hyperplasia. Pros- tate 16:71-80, 1990. Thompson NL, Flanders KL, Smith J, Ellingsworth LR, Roberts AB, Sporn MB: Expression of transforming growth factor p l in specific cells and tissues of adult and neonatal mice. J Cell Biol 108:661-668, 1989. Gorsh SM, Memoli VA, Stukei TA, Gold LI, Arrick BA: Immunohistochemical staining for transforming growth factor 01 associates with disease progression in human breast cancer. Cancer Res 52:6949-6952, 1992. Davies LG, Dibner MD, Battey JF: ”Basic Methods in Molecular biology.” New York: Elsevier, 1986, pp 130- 135. Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR,

Assoin RK, Roberts AB, Sporn MB, Goeddell DV: Hu- man transforming growth factor beta. Complementary DNA sequence and expression in normal and transformed cells. Nature 316:701-705, 1985.

27. Thompson TC, Truong LD, Timme TL, Kadmon D, MC- Cune B, Flanders K, Scardino P, Park SH: Transgenic models for the study of prostate cancer. Cancer Suppl 71:1165-1171, 1992.

28. Wakefield LM, Smith DM, Flanders KC, Sporn MB: Latent transforming growth factor p from human platelets. J Biol Chem 263:7646-7654, 1988.

29. Miyazono K, Olofsson A, Colosetti P, Heldin CH: A role of the latent TGF-pl binding protein in the assem- bly and secretion of TGF-p1. EMBO 10:1091-1101,1991.

30. Yoshioka K, Takemura T, Murakami K, Okada M, Hino S, Miyamoto H, Maki S: Transforming growth factor p protein and mRNA in glomeruli in normal and diseased human kidneys. Lab Invest 68:154-163, 1993.

31. Drake WT, Issekutz AC: Transforming growth factor-pl enhances polymorphonuclear leucocyte accumulation in dermal inflammation and transendothelial migration by a priming action. Immunology 78:197-204, 1993.

32. Brandes ME, Mai VEH, Ohura K, Wahl S: Type I transforming growth factor-p receptors on neutrophils me- diate chemotaxis to transforming growth factor-p. J Im- munol 147:1600-1606, 1991.

33. Martikainen P, Kyprianou N, Isaacs JT: Effect of transforming factor-pl on proliferation and death of rat prostatic cells. Endocrinology 127:2963-2968, 1990.

34. Kyprianou N, English HF, Isaacs JT: Programmed cell death during regression of PC-82 human prostatic cancer following androgen ablation. Cancer Res 50:3748- 3753, 1990.

35. Bursch N, Oberhammer F, Jirtle RL, Askari M, Sedivy R, Grasl-Kraupp 8, Purchino AF, Schulte-Hermann R: Transforming growth factor-pl as a signal for induction of cell death by apoptosis. Br J Cancer 67531-537, 1993.

36. Carrington JL, Roberts AB, Flanders KC, Roche NS, Reddi AH: Accumulation, localisation and compart- mentation of transforming growth factor p during endochondral bone development. J Cell Biol 107:1969- 1975, 1988.

37. Grotendorst GR, Smale G, Pencev D: Production of transforming growth factor beta by human peripheral blood monocytes and neutrophils. J Cell Physiol 140: 396-402, 1989.

Documents

Transforming growth factor beta I expression in benign and malignant prostatic tumors