J. Steroid Biochem. Molec. Biol. Vol. 41, No. 3-8, pp. 659-663, 1992

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REGULATION PROTEIN(S)

OF IN

FIBROBLAST

THE

PROSTATE

GROWTH

ANDROGEN-RESPONSIVE

CARCINOMA

CELL

LINE

FACTOR-LIKE HUMAN LNCaP

B. ZtJCK,~C. GOEPFERT, 2 A. NEDLIN-CHITTKA,l K. SOHRT,~K.-D. VOIGT1'2 and C. KNABBE2. llnstitute of Hormone and Fertility Research, Grandweg 64, 2000 Hamburg 54 and 2Department of Clinical Chemistry, Medical Clinic, University Hospital Eppendorf, Martinistr. 52, 2000 Hamburg 20, Fed. Rep. Germany Summary--Growth of the normal and malignant prostate is known to be regulated by androgens. Part of their effect has been suggested to be mediated through coordinated regulation of secreted growth factors with autocrine function. We now examine the biological role of preferentially paracrine acting factors in growth control of prostate cancer, i.e. fibroblast growth factor(s) (FGF). Coculture experiments using the androgen-responsive human prostate carcinoma cell line LNCaP as feeder cells and the FGF-dependent human adrenal carcinoma SW-13 cell line as target cells show that (i) LNCaP cells induce growth of SW-13 cells, (ii) even higher stimulation of SW-13 cells is seen in the presence of androgen treated LNCaP cells and (iii) a specific anti-bFGF antibody inhibits growth of SW-13 cells induced by androgen treated LNCaP cells; no proliferation of SW-13 cells occurs in the absence of LNCaP cells. Partial purification of the secretory products of LNCaP cells was performed by affinity chromatography using a heparin sepharose column. Fractions were tested for biological activity in a soft agar assay with SW-13 cells. Several activities could be detected, the main activity was eluted with about 1.5 M NaCl. These data suggest that androgen treatment of LNCaP cells leads to enhanced secretion of proteins which belong to the FGF-family.

INTRODUCTION The prostate is an androgen-dependent gland [1] which often develops to benign hyperplasia or malignant cancer and in about 20% causes death of older men in the Western world (for a review see [2]). One theory for this development is the hypothesis of autocrine growth regulation of cancer cells. Malignant transformation is associated with enhanced secretion of growth stimulating factors or decreased secretion of growth inhibitory factors which bind to specific receptors of the secreting cell and modulate their own proliferation[3,4]. For estrogenresponsive human breast cancer cells it could be demonstrated that estrogens act as mitogens through expression of a variety of stimulatory and/or inhibitory growth factors [5-7]. Recently a similar mechanism for androgen action on SC-3 cells, a newly established cloned cell line derived from the mouse m a m m a r y tumor Shionogi carcinoma 115 (SCl15) cell line Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.

*To whom correspondence should be addressed.

was proposed. Evidence that fibroblast growth factors (FGF) could be involved was provided by the observation that a b F G F - a n t i b o d y was able to partially inhibit androgen induced growth of SC-3 cells [8]. The established human prostate cancer cell line L N C a P is a useful tool for in vitro studies because of its ability to be stimulated by androgens [9]. This androgen stimulation is accompanied by enhanced growth factor secretion which can eventually stimulate growth in an autocrine fashion [10]. To investigate the role of the preferentially paracrine acting b F G F we examined the proliferation of the F G F dependent human adrenal carcinoma cell line SW-13 [11, 12] as target cells in coculture with L N C a P cells. Other hormones like hydrocortisone, dexamethasone, the synthetic androgen R1881 or growth factors like E G F or insulin do not show any stimulatory effects on the growth of SW-13 cells. Our coculture experiments using L N C a P cells as feeder cells in soft agar result in enhanced cloning tendency of SW-13 target cells. This could be due to growth factors belonging to the FGF-family secreted by L N C a P cells. In order to confirm this hypothesis soft agar was complemented with a neutralizing

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anti-basic FGF antibody to inhibit the FGFinduced growth of SW-13 cells. Before its final characterization FGF was termed heparin binding growth factor. This property had been extensively utilized by many investigators for isolation and purification of FGF from tissues or media by use of a heparin affinity chromatography [13-15]. We made use of this technique for fractionating conditioned media obtained from LNCaP cells with or without androgen treatment. Fractions were tested for biological activity in a soft agar assay with SW- 13 cells. EXPERIMENTAL

insulin (Gibco/BRL) for LNCaP cells, respectively. Charcoal treatment was performed to remove endogenous steroids from serum [16]. Colonies larger than 50/~m were counted after 2 weeks of incubation.

MTT-assay The MTT-assay is based on the mitochondrial reduction of a yellow tetrazolium salt (Sigma) to a purple-colored formazan product in living metabolically active cells. This reaction was measured colorimetrically at a wavelength of 492 nm using a multiwell plate reader (SLTLabinstruments, Groedig/Salzburg, Austria) and carried out as described previously [17].

Chemicals

Coculture experiments

Recombinant human TGF~ was obtained from Bissendorf (Hannover, F.R.G.), mouse EGF (receptor grade) from Gibco/BRL (Berlin, F.R.G.) and R1881 (methyltrienolone) from NEN/Du Pont (Bad Homburg, F.R.G.). Recombinant bFGF was generously provided by Progen (Heidelberg, F.R.G.). Heparin Sepharose CL-6B was obtained from Pharmacia LKB (Freiburg, F.R.G.) and 1-125 labeled bFGF was purchased from Amersham (Braunschweig, F.R.G.)

Coculture experiments were run in 24 well dishes (Nunc) (for details see[18]). After the LNCaP cells had grown to near confluence, the media was removed and changed to soft agar. After gelatinizing the target cells were plated in another soft agar layer. SW-13 colonies larger than 50/~m were counted after 2 weeks of incubation.

Antibody IgG against bFGF Neutralizing antibody against human bFGF was purchased from R&D Systems (Minneapolis, MN, U.S.A.). Unspecific control rabbit IgG was purchased from Sigma (Deisenhofen, F.R.G.). Anti-FGF antibody was used in a concentration of 350 #g/ml, control IgG in a concentration of 250 #g/ml in the feeder layer assay as described below.

Cell culture and cloning LNCaP cells were kindly provided by Dr Horoszewicz (Buffalo, NY, U.S.A.). SW-13 cells were from American Type Culture Collection (Rockville, MD, U.S.A.). Cells were routinely cultured in Dulbecco's modified Eagle medium (DMEM, Gibco/BRL) supplemented with 10% (v/v) fetal calf serum (FCS, Gibco/BRL) and 50 mg/1 Gentamycin (Gibco/BRL) in an atmosphere of 5% CO2 in air. Soft agar assays in 24 well dishes (Nunc, Denmark) were performed in media supplemented with 0.4-0.8% agar (Difco, Detroit, MI, U.S.A.) and with 10% (v/v) FCS for SW-13 cells and 5% (v/v) charcoal- and sulfatasetreated FCS (CCS), enriched with 50ng/ml

Conditioned media and affinity chromatography Conditioned medium was prepared as described earlier [6] with the modification that serum free medium was used without any supplementation of protein. After 120-fold concentration by ultrafiltration (Amicon, F.R.G.; cutoff = 5000 Da) fractionation was performed over a heparin sepharose affinity column (HR 10/10, Pharmacia) with 50raM T r i s ~ l (pH = 7.0) and a linear NaCl gradient from 0.0 to 3.0 M (Fig. 3) until a final elution volume of 200 ml was reached. Fractions of 8 ml each were collected at a rate of 1 ml/min, lyophilized, resuspended in and consequently dialyzed against phosphate buffered saline (PBS) (Gibco/BRL). After sterile filtration (0.22/~m, Millipore, F.R.G.) 160/~1 of each fraction were tested in triplicate in a soft agar-assay [final concentration was 20% fraction tested (v/v) of the total volume] for stimulating the growth of SW- 13 cells. RESULTS

Effects of androgens or various growth factors on growth of LNCaP and SW-13 cells in M T T - and soft agar-assay The proliferation of LNCaP and SW-13 cells was tested under treatment with different

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Fig. la. Effects of 0.1-100ng/ml EGF or TGF and 10-t4-10-s M R1881 on proliferation of LNCaP cells. Proliferation was determined by MTT-assays as described in Experimental. Means are givenas percentageof 00492of the untreated control (n = 4, SD < 8%). iooo

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Fig. 2. Effect of antibodies on SW-13 growth stimulated by androgen treated LNCaP cells in coculture. Soft agar-assay was performed with and w/o 10-1° M R1881 under simultaneous addition of a specific anti-bFGF antibody, or a nonspecific IgG, or w/o any further supplementation (control). Number of SW-13 colonies was counted after 14d incubation (n = 3, mean + SD).

t ~ c ~ ceUm , 100

[ng/ml]

bFGF-concentration

Fig. lb. Effects of 0.1-100 ng/ml bFGF on proliferation of LNCaP or SW-13 cells. Proliferation was determined in soft agar-assays, counting the number of colonies after 14d incubation (n = 3, mean + SD). concentrations of the synthetic androgen R 1881 or the growth factors E G F and TGF~t in a MTT-assay (Fig. l a). The influence of b F G F on both cell lines was investigated under anchorage-independent conditions in a soft agar-assay (Fig. lb). L N C a P cells were stimulated by R1881 (maximum stimulation at a concentration of 10-1°M), by E G F (maximum stimulation at about 10 ng/ml) and by T G F a (stimulation occurred at a concentration more than 2 ng/ml) (Fig. la). Furthermore b F G F has a stimulating influence upon the proliferation of L N C a P cells (maximum stimulation at 1 ng/ml) (Fig. l b). Notice that b F G F on L N C a P cells shows a biphasic dose response. In contrast SW-13 showed no response to R1881 or growth factors given in Fig. l b except b F G F . 7-Fold stimulation was obtained at a concentration between 10 and 30 ng b F G F / m l (Fig. lb).

Inhibitory effects of anti-bFGF antibody on androgen induced growth of SW-13 cells in the presence of LNCaP cells L N C a P cells were used as feeder cells in the bottom layer and SW-13 cells as target cells in

the upper layer of a soft agar-assay. L N C a P cells were able to stimulate the growth of SW- 13 cells in these coculture experiments. Treatment with 10 -'° M R1881 resulted in a >2-fold stimulation of SW-13 cells in the presence of L N C a P cells (Fig. 2). No stimulation occurred when SW-13 cells were treated with R1881 in the absence of L N C a P cells (data not shown). An a n t i - b F G F antibody had an inhibitory effect on the stimulation of SW-13 cells by L N C a P cells. The growth of SW- 13 could not be significantly diminished through unspecific IgG (Fig. 2). These data suggest that androgen induced growth of SW-13 cells is mediated through secretion of FGF-like peptides by L N C a P cells. Since the starting cell density corresponds to near confluence, a further stimulation of SW-13 cells does not correspond to higher densities of L N C a P cells but androgen induced secretion of F G F by L N C a P cells. Furthermore the SW-13 cell density was not very high (8000cells/well) therefore autocrine growth by reported endogenously produced b F G F [12] should be excluded and consequently no significant cloning is seen in the absence of L N C a P cells.

Effects of heparin affinity chromatographic fractionated conditioned media onto SW- 13 cells in a soft agar-assay 120-Fold concentrated conditioned medium obtained from L N C a P cells under androgen treatment was fractionated by heparin sepharose affinity chromatography by running a linear NaC1 gradient from 0 to 3.0 M solution and the lyophilized and dialyzed fractions were tested for biological activity in a soft agar-assay

662

B. ZUCKet al.

and SW-13 target cells show that LNCaP cells are able to stimulate SW-13 proliferation in an U androgen regulated fashion. Since b F G F is a 2 Z© potent mitogen for SW-13 cells we suggest that j~ 200" growth stimulation is due to growth factors belonging to the FGF-family. Because of this tOO paracrine action these growth factors have to be secreted in biologically active form. Further260 ~ 10 16 20 more androgen treatment of LNCaP cells fraction n u m b e r Fig. 3. Elution profileof conditionedmedia of LNCaP cells, increased significantly SW-13 colony formation. supplemented with 10-~°M R1881 (0 0) or 0.1% SW-13 cells could not be stimulated by androethanol ( O - - - O ) for control, which was applied to a gens only, so induced proliferation has to be heparin sepharose column as described in Experimental. Fractions were tested in triplicate for mitogenicactivity on attributed to a rising secretion of FGF-like SW-13 cells in a soft agar-assay. peptides. This hypothesis is further supported by the finding that a specific antibody against with SW-13 cells (Fig. 3). The number of SW-13 b F G F reduces growth of SW-13 cells in colonies without the addition of any growth coculture. Finally, affinity chromatography demonfactor or fraction was usually < 40 (basal). The strated that LNCaP cells produce and secrete main activity was obtained from a fraction heparin binding growth factor(s) into the eluted at a NaCI concentration of about 1.5 M. medium. The main biological activity of fracThis is in good agreement with elution profiles tionated conditioned medium was eluted at a obtained by other investigators for b F G F from NaC1 concentration of about 1.5 M. It is known different sources [13-15]. Furthermore chromafrom previous reports[13-15] that bFGF-like tography with 1-125 labeled b F G F under proteins elute at this concentration of NaCI and analogous elution conditions resulted in a simicalibration runs with 1-125 labeled b F G F under lar elution behavior (data not shown). This analogous conditions showed a similar elution stimulation did not appear with fractions which profile (data not shown). This data can be were produced without androgen treatment, interpreted as an additional important hint to suggesting that FGF-like molecules are only FGF-like growth factors. However, other secreted under androgen stimulation. At present the other heparin binding activities cannot be growth stimulating factors were eluted from heparin sepharose affinity column. With all the ascribed to known biological compounds. data taken together it cannot be excluded that LNCaP cells also secrete other heparin binding DISCUSSION mitogens than b F G F after stimulation with The proliferation of the human prostate carci- androgen. We conclude that members of the FGFnoma cell line LNCaP is stimulated by androgens. Postulating an autocrine mechanism for family are androgen regulated and secreted cancer cells this stimulation should be mediated growth factors in human prostate cancer cells by secreted growth factors. In fact it was found which represent regulatory molecules in the that TGF~ and E G F effect prostate cancer cells paracrine communication but might also act as in vitro and have autocrine potential[19, 20]. androgen regulated autocrine growth factors. Little is known about the role of F G F in prostate cancer. Recently Lawson et al.[21] Acknowledgement--This work was supported by a Grant found an enhanced expression of b F G F in from the Deutsche Forschungsgemeinschaft(Ho 388/6-1/6). benign prostate hyperplasia. And although it was assumed that acidic or basic F G F were REFERENCES hardly released into conditioned medium be1. Sandberg A. A.: Endocrine control and physiologyof cause of the absence of a typical amino-terminal the prostate. Prostate 1 (1980) 169-184. signal peptide [22], there are now hints, that 2. Dhom G.: Epidemiology of hormone-depending tuF G F could be secreted by tumor cells [8]. mors. In Comprehensive Endocrinology; Endocrine Dependent Tumors (Edited by K.-D. Voigt and C. We investigated the possible role of secreted Knabbe). Raven Press, New York (1991) p. 1. FGF-like growth factors acting in paracrine 3. Sporn M. B. and Todaro G. J.: Autocrinesecretionand growth control of human prostate carcinoma. malignant transformation of cells. N. Engl. J. Med. 303 (1980) 878-880. Coculture experiments with LNCaP feeder cells "d

.~ 400 -

5

Regulation of FGF-like protein(s) in the LNCaP cell line 4. Sporn M. B. and Roberts A. B.: Autocrine growth factors and cancer. Nature 313 (1985) 745-747. 5. Lippman M. E., Dickson R. B., Bates S., Knabbe C., HuffK., Swain S., McManaway M., Bronzert D., Kasid A. and Gelmann E. P.: Autocrine and paracrine growth regulation of human breast cancer. Breast Cancer Res. Treat. 7 (1986) 59-70. 6. Knabbe C., Lippman M. E., Wakefield L. M., Flanders K. C., Kasid A., Derynck R. and Dickson R. B.: Evidence that transforming growth factor-/3 is a hormortally regulated negative growth factor in human breast cancer cells. Cell 48 (1987) 417-428. 7. Takahashi K., Suzuki K., Kawahara S. and Ono T.: Growth stimulation of human breast epithelial cells by basic fibroblast growth factor in serum-free medium. Int. J. Cancer 43 (1989) 870-874. 8. Tanaka A., Matsumoto K., Nishizawa Y., Lu J., Yamanishi H., Maeyama M., Nonoumra N., Uchida N. and Sato B.: Growth stimulation by androgens, glucocorticoids or fibroblast growth factors and the blocking of the stimulated growth by antibody against basic fibroblast growth factor in protein-free culture of Shionogi carcinoma 115 cells. J. Steroid Biochem. Molec. Biol. 37 (1990) 23-29. 9. Horoszewicz J. S., Leong S. S., Kawinski E., Karr J. P., Rosenthal H., Ming Chu T., Mirand E. A. and Murphy G. P.: LNCaP model of human prostatic carcinoma. Cancer Res. 43 (1983) 1809-1818. 10. Knabbe C., Kellner U., Schmahl M. and Voigt K.-D.: Growth factors in human prostate cancer cells: implications for an improved treatment of prostate cancer. J. Steroid Biochem. Molec. Biol. Submitted. 11. Halper J. and Moses H. L.: Epithelial tissue-derivedgrowth factor-like polypeptides. Cancer Res. 43 (1983) 1972-1979. 12. Corin S. J., Chen L. C. and Hamburger A. W.: Enhancement of anchorage-independent growth of a human adrenal carcinoma cell line by endogenously produced basic fibroblast growth factor. Int. J. Cancer 46 (1990) 516-521. 13. Story M. T., Livingston B., Baeten L., Swartz S. J.,

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Jacobs S. C,, Begun F. P. and Lawson R. K.: Cultured human prostate-derived fibroblasts produce a factor that stimulates their growth with properties indistinguishable from basic fibroblast growth factor. Prostate 15 (1989),355-365. Gospodarowicz D., Abraham J. A. and Schilling J.: Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc. Natn. Acad. Sci. U.S.A. 86 (1989) 7311-7315. Mydlo J. H., Bulbul M. A., Richon V. M., Heston W. D. W. and Fair W. R.: Heparin-binding growth factor isolated from human prostatic extracts. Prostate 12 (1988) 343-355. Darbre P., Yates J., Curtis J. and King R. J. B.: Effects of estradiol on human breast cancer cells in culture. Cancer Res. 43 (1983) 349-354. Romijn J. C., Verkoelen C. F. and Schroeder F. H.: Application of the MTT assay to human prostate cancer cell lines in vitro: establishment of test conditions and assessment of hormone-stimulated growth and druginduced cytostatic and cytotoxic effects. Prostate 12 (1988) 99-110. Kasid A., Knabbe C. and Lippman M. E.: Effect of v-ras oncogene transfection on estrogen independent tumorigenicity of estrogen dependent human breastcancer ceils. Cancer Res. 47 (1987) 5733-5738. Wilding G., Valverius E., Knabbe C. and Gelmann E. P.: Role of transforming growth factor-or in human prostate cancer cell growth. Prostate 15 (1989) 1-12. Schuurmans A. L. G., Bolt J. and Mulder E.: Androgens stimulate both growth rate and epidermal growth factor receptor activity of the human prostate tumor cell line LNCaP. Prostate 12 (1988) 55-63. Lawson R. K.: Benign prostatic hyperplasia and growth factors. Urologe 29 (1990) 5-7. Schweigerer L., Neufeld G., Friedman J., Abraham J. A., Fiddes J. C. and Gospodarowicz D.: Basic fibroblast growth factor: production and growth stimulation in cultured adrenal cortex cells. Endocrinology 120 (1987) 796-800.

Regulation of fibroblast growth factor-like protein(s) in the androgen-responsive human prostate carcinoma cell line LNCaP.

Growth of the normal and malignant prostate is known to be regulated by androgens. Part of their effect has been suggested to be mediated through coor...
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