JOURNAL OF CELLULAR PHYSIOLOGY 148:260-266 (1991)

Heparin Inhibits Autocrine Stimulation But Not Fibroblast Growth Factor Stimulation of CelI Pro1iferation of Androgen- Responsive Shionogi Carcinoma 1 1 5 SOJIKASAYAMA,* SATORU SUMITANI, AKIRA TANAKA, HlROSHl YAMANISHI, NOBUYOSHI NAKAMURA, KElSHl MATSUMOTO, A N D B U N Z O S A T 0 Third Department of Internal Medicine (S.K.,S.S.,B.S.), and Second Department of Pathology (A.T.,H.Y.,N.N.,K.M.), Osaka University Hospital, Osaka 553, lapan An androgen-responsive cloned cell line (SC-3) derived from Shionogi carcinoma 1 15 (SC115) has been shown to secrete fibroblast growth factor (FCF)-like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC-3 cells in an autocrine mechanism. Since the androgeninduced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC-3 cells. Heparin was found to exhibit significant inhibition of testosterone-induced growth in a concentration-dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 pgiml. D N A synthesis of SC-3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC-3 cells induced by acidic (a) or basic (b) FCF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen-induced growth factor in the conditioned medium from testosteronetreated cells. These results indicate that heparin inhibits autocrine loop in SC-3 cells induced by androgen. Thus, the autocrine growth factor possesses a different characteristic from aFGF and bFCF in that its bioactivities are negatively modulated by the glycosaminoglycan.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been shown to be mitogenic for a wide variety of cell types (Gospodarowicz et al., 1986). These bioactivities of both aFGF and bFGF have been demonstrated to be mediated through the same cell surface receptor (Neufeld and Gospodarowicz, 1986; Olwin and Hauschka, 1986). Their mitogenic activity can be modulated by many compounds such as heparin (Schreiber et al., 1985; Gospodarowicz and Cheng, 19861, transforming growth factor-@(TGF-P) (Baird and Durkin, 1986) and protamine (Neufeld and Gospodarowicz, 1987; Dauchel et al., 19891, although their molecular mechanism remains largely unknown. Particularly, many investigators have paid attention to the effect of heparin on the bioactivity of FGFs. Heparin, a sulfated glycosaminoglycan, is able to bind aFGF and bFGF (Gospodarowicz et al., 1986; Burgess and Maciag, 1989). Heparin has been shown to potentiate aFGFinduced proliferation of human endothelial cells (Thornton et al., 1983; Schreiber et al., 1985; Mueller et al., 1989) and rat pheochromocytoma cells (Damon et al., 1989). In contrast, it exerts positive or negative effects on the bioactivity of basic FGF, depending on cell types and on experimental conditions (Neufeld et al., 1987; Miller-Davis et al., 1988; Bgrzu et al., 1989; Shipley et al., 1989; Gospodarowicz e t al., 1990). Therefore, these observations raise the possibility that hep-

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1991 WILEY-LISS, INC

arin can be used as a probe to differentiate the growth factor associable with FGF receptor. The molecular mechanism of androgen-induced cell proliferation remains largely unknown. Prostate cells have been reported to be growth-stimulated by the heparin-binding growth factor, so-called prostatropin (Crabb et al., 1986). Androgen-dependent rat prostate cancer cells (Dunning tumor) have been found to contain the heparin-binding growth factor (Matuo et al., 1987). Androgen-responsive mouse mammary tumor, Shionogi carcinoma 115 (SC1151, which was established by Mineshita and Yamaguchi (19641, has been shown to grow in the presence of androgen both in vivo (Mineshita and Yamaguchi, 1965; Bruchovsky and Meakin, 1973; Suzuki e t al., 1983) and in cell culture (Desmond et al., 1976; King et al., 1976; Jung-Testas et al., 1976). The growth of SC115 cells has been demonstrated also to be stimulated by FGFs, but not by other growth factors, such a s epidermal growth factor, insulin, and platelet-derived growth factor (Nakamura et al., 1989). In addition, we have shown that SC115 cells produce heparin-binding growth factor in response to androgen stimuli (Nonomura et al., 1988) and that Received January 3, 1991; accepted April 5, 1991.

"To whom reprint requestsicorrespondence should be addressed.

GROWTH INHIBITION OF SC115 CELLS BY HEPARIN

neutralizing antibody against bFGF inhibits androgeninduced a s well as bFGF-induced proliferation of the cells (Lu et al., 1989), suggesting th at the androgendependent growth of SC115 cells is mediated through a FGF-like peptide in a n autocrine fashion. Combined with these previous observations, therefore, it seems to be quite important to clarify the action mechanism of the heparin-binding growth factor in androgen-sensitive cells. I n this regard, the growth-modulating ability of glycosminoglycans should be carefully examined to obtain the clue for the action mechanism of heparinbinding growth factors in androgen-sensitive cells. In the present study, we examined the effect of heparin on androgen- or FGFs-induced proliferation of SC115 cells. Our results indicate that heparin dramatically inhibits the androgen-induced autocrine loop, whereas it does not block FGF-stimulated cell growth.

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onto a 96-well plate ( 5lo3 ~ cellsiwell) in 0.15 ml MEM supplemented with 2% DCC-treated FCS. On the following day, the cells were washed once with the serum-free medium and cultures were continued in the same medium for 24 h. The quiescent cells were treated with or without testosterone, aFGF, or bFGF in the presence or absence of glycosamino lycans. After 24 h, the cells were pulse-labeled with [8Hlthymidine (0.30 pCi/well) for 2 h . The radioactivity incorporated into the cells, which were collected by Cell Harvester (LKB Wallac, Turk, Finland) after trypsinization, was determined by Betaplate (LKB Wallac).

Autocrine growth-promotingactivities in conditioned medium Serum-free conditioned medium (2 liters) obtained from the cultures of SC-3 cells in the presence of testosterone (10p8M) was collected, filtered, and conMATERIALS AND METHODS centrated (up to 25-fold) as described previously (NonoMaterials mura et al., 1988). The conditioned medium was apBovine brain-derived aFGF and bovine brain-derived plied onto a heparin-Sepharose column (gel bed bFGF were purchased from R & D systems, Inc. (Min- volume, 2 ml) which had been equilibrated with 10 mM neapolis, MN). Glycosaminoglycans (heparin, low mo- Tris-HC1 (pH 7.0)-0.6 M NaCl-0.1% (wiv) 3-[(3-cholecular weight-heparin, heparan sulfate, dermatan sul- lamidopropyl) -dimethylammonio] - 1-propanesulfonate fate, chondroitin sulfate) were obtained from Sigma (CHAPS) buffer. After being washed with 100 ml of 10 Chemical Co. (St. Louis, MO). Heparin-Sepharose was mM Tris-HC1 (pH 7.01-0.6 M NaC1-0.1% ( w h ) CHAPS from Pharmacia (Piscataway, NJ). [Meth~l-~HIthymi-buffer, materials bound to the column were eluted with dine (70-90 Ciimmol), Ampure'" SA, and Ampure'" DT 20 ml of 10 mM Tris-HC1 (pH 7.0)-2 M NaCl-O.l% (w/v) were obtained from Amersham Japan (Tokyo, Japan). CHAPS buffer to concentrate the growth-promoting All other reagents were of analytical grade. activity by 100-fold. Our recent studies revealed that the eluate shows growth-stimulatory activity for SC-3 Cell culture cells (Nonomura et al., 1989, 1990). CHAPS was exSC-3 cells, a cloned cell line obtained from SC115 cluded from the eluate by means of a n Ampure'" DT tumor (Noguchi et al., 19851, were maintained in a column. Subsequently the eluate was desalted by medium composed of Eagle's minimum essential me- means of a n Ampure'" SA column to serum-free medium (MEM) containing 2% fetal calf serum (FCS) and dium. The eluate from the heparin-Sepharose column lO-'M testosterone. Mouse NIH 3T3 cells, obtained was diluted finally into 0.25-4% (viv) and processed for from RIKEN (Saitama, Japan), were cultured in Dul- DNA synthesis assay. becco's modified Eagle's medium (DMEM) suppleRESULTS mented with 10% calf serum. Cells were grown a t 37" C Heparin inhibits the androgen-induced in a n atmosphere of 5% C02/95%air. proliferation of SC-3 cells Cell growth experiment The SC-3 cells were cultured with or without 1O-'M SC-3 cells were plated onto a 24-well plate (5x lo3 testosterone in the presence or absence of 10 pgiml cells/well) in 1ml MEM containing 2% dextran coated heparin in the serum-free medium for various periods charcoal (DCC)-treated FCS. On the following day (day of time and the cell number was counted. As described O), the medium was replaced with 1 ml serum-free earlier (Nakamura et al., 1989; Lu et al., 19891, the medium [Ham's F-12: MEM ( l : l , v/v) containing 0.1% growth of SC-3 cells was markedly stimulated with bovine serum albumin (BSA)] in the presence or testosterone, whereas no significant increase in cell absence of testosterone ( 10psM) or heparin (0.01-10 number was observed in the culture without testosterpg/ml). The serum-free medium was changed every one (Fig. 1).The addition of heparin (10 pg/ml) resulted other day until the cells were harvested by trypsiniza- in a marked inhibition (approximately 60-70%) of tion and counted in an electronic particle counter testosterone-stimulated cell proliferation, while it did (Coulter Electronics, Ltd., Luton, UK). In some exper- not alter the cell growth in the absence of testosterone iments to monitor the cell growth induced by FGFs, a (Fig. 1). Next, the effect of the increasing concentramedium composed of MEM supplemented with 1% tions of heparin on the cell number on day 6 of DCC-treated FCS was substituted for the serum-free testosterone-stimulated SC-3 cells was examined. As shown in Figure 2, heparin exhibited the inhibition of medium. androgen-induced growth in a concentration-depenDNA synthesis dent manner. Fifty-percent inhibition was observed at [3H]Thymidine incorporation into DNA of SC-3 cells a concentration of approximately 0.1 pg/ml. and NIH 3T3 cells in the serum-free medium was When SC-3 cells were cultured in the serum-free measured by the method as described (Hiraoka et al., medium for 24 h, DNA synthesis determined by 1987) with slight modifications. The cells were plated [3H]thymidine incorporation into the cells was stimu-

KASAYAMA ET AL.

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Days Fig. 1. Inhibitory effects of heparin on androgen-induced growth of SC-3 cells. SC-3 cells were plated onto a 24-well plate (5 X lo3 cellsiwell) containing MEM supplemented with 2% DCC-treated FCS. On the following day (0)the medium was changed to the serum-free medium with or without 10-8M testosterone in the presence or absence of 10 pgiml heparin, The medium was replaced every other day and the cell number was counted on the indicated day. 0 , control: 0 , testosterone; A , heparin; A , testosterone plus heparin. Values represent means obtained in triplicate in one of three separate experiments. Bars show SD.

lated with 10paM testosterone to about 40-fold. Heparin inhibited the DNA synthesis concentration-dependently (Fig. 3), which occurred almost in parallel with the inhibition of the cell growth (Fig. 2). The maximum inhibition was obtained above concentrations of 1 pg/ ml heparin. Low molecular weight-heparin (molecular weight, -3,000) also inhibited testosterone-induced DNA synthesis to the same extent. Heparan sulfate and dermatan sulfate inhibited androgen-induced DNA synthesis, but about 100-fold higher concentrations were necessary to obtain the levels of inhibition equivalent to heparin. In contrast, chondroitin sulfate had little effect on the DNA synthesis a t concentrations up to 10 pg/ml (Fig. 3).

Heparin does not inhibit the ability of FGF to stimulate the proliferation of SC-3 cells Since the proliferation of SC-3 cells has been reported to be stimulated also with aFGF or bFGF (Nakamura et al., 19891, we tested the effect of heparin on FGFsinduced DNA synthesis of SC-3 cells. Incubation with

Fig. 2. Effect of increasing concentrations of heparin on androgeninduced growth of SC-3 cells. SC-3 cells were seeded as described in Figure 1. The cells were cultured with 10-'M testosterone in the presence or absence of 0.01-10 pgiml heparin in the serum-free medium. On day 6 the cells were harvested and the cell number was counted. The control value (100%) represents the cell number produced by the addition of 10-8M testosterone alone. The data represent means i SD in triplicate assay. Results are typical of three separate experiments.

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Fig. 3. Effects of various glycosaminoglycans on t3Hlthymidine incorporation into SC-3 cells stimulated with androgen. SC-3 cells were plated at 5 x lo3 cellsiwell in 96-well plates containing MEM with 2% DCC-treated FCS. The next day, the medium was replaced with the serum-free medium and the cultures were continued for 24 h. The cells were treated with lo-' M testosterone in the presence of increasing concentrations of heparin (01, low molecular weight-heparin (01, heparan sulfate (m), dermatan sulfate (o), or chondroitin sulfate (A).After 24 h the cells were pulsed with [3H]thymidinefor 2 h, and the radioactivity incorporated into the cells was determined. In this experiment, the value of C3Hlthymidine incorporation into the cells not treated with testosterone was 250 i7 20 cpndwell. Values represent means in triplicate in one of four separate experiments. SD values were consistently less than 10%of the mean values.

263

GROWTH INHIBITION OF SC115 CELLS BY HEPARIN

aFGF or bFGF for 24 h dramatically increased l3HIthymidine incorporated into the cells in a concentration-dependent way (Fig. 4). The maximum increase, about 50-fold, was reached at a concentration of 1ng/ml aFGF and bFGF. Heparin, at concentrations up to 10 pg/ml, appeared to have no effect on the saturating concentrations of aFGF- or bFGF-induced stimulation of DNA synthesis. When the cells were stimulated with submaximal concentrations (viz., 0.01 or 0.1 ng/ml) of aFGF or bFGF, heparin showed slight but significant potentiation of the growth factor activity (Fig. 4).Neither heparan sulfate, chondroitin sulfate, nor dermatan sulfate appeared to alter the i3H1thymidine incorporation stimulated by aFGF or bFGF (data not shown). Heparin alone induced no significant changes in the levels of [3H]thymidine incorporation (Fig. 4). During the present work, we have found that in serum-free cultures aFGF or bFGF showed a less prominent increase in the number of SC-3 cells than testosterone, while the addition of low concentrations of DCC-treated FCS to the cultures enabled FGFs to stimulate the cell growth to levels equivalent to those of testosterone. Thus, in the next experiment 1%DCCtreated FCS, which alone had no apparent effect on the cell number, was included in the cultures to examine the effect of FGFs on the cell yield. As demonstrated in Figure 5, 10 pg/ml heparin inhibited testosteroneinduced cell growth by approximately 80%.In contrast, the cell growth elicited by aFGF or bFGF (1ngiml each) was not significantly inhibited by heparin. Again, the addition of heparin alone to the cultures without the growth stimulators showed no significant effect.

Heparin inhibits the ability of androgen-induced autocrine factor to stimulate the proliferation of SC-3 Cells Growth-promoting activities in the conditioned medium from the cultures of testosterone-treated SC-3 cells were partially purified by means of a heparinSepharose column as described in Materials and Methods. The active fractions were found to contain no androgen activities, since cyproterone acetate, a n antiandrogen, did not inhibit the growth-promoting activities (data not shown). From the eluate from heparin-Sepharose column, CHAPS and 2 M NaCl were excluded by using Ampure'" DT and Ampure'" SA, respectively. The fractions containing growthpromoting activities were diluted in the serum-free medium to 0.254% (v/v). The active fractions elicited a significant stimulation of [3H]thymidine incorporation into SC-3 cells in a concentration-dependent fashion: 4% (v/v)-diluted fractions showed about 80-fold stimulation (Fig. 6). Heparin efficiently decreased the r3H1thymidine incorporation concentration-dependently: 50% decrease was observed a t a concentration of 0.1 p,g/ml heparin (Fig. 6). Heparin inhibits the ability of androgen-induced autocrine factor but not FGF to stimulate the proliferations of NIH 3T3 cells Next we examined the effects of bFGF and androgeninduced growth factor secreted from SC-3 cells upon DNA synthesis in mouse NIH 3T3 cells under the

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Fig. 4. Effects of increasing concentrations of heparin on r3H1thymidine incorporation in SC-3 cells stimulated with aFGF or bFGF. The cells were plated as shown in Figure 3. Increasing concentrations (M, 0.01 ngiml; A , 0.1 ngiml; 0 , 1 ngiml; 0 , 10 ngiml) of aFGF (left)or bFGF (right)were added to the quiescent cells with or without various concentrations of heparin, and r3H1thymidine incorporated into the cells was determined. A , no FGF added. Values are means 2 SD of triplicate incubations.

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Fig. 5. Differential effects of heparin on androgen-, aFGF-, or bFGF-induced growth of SC-3 cells. SC-3 cells (5 x lo3 cellsiwell) were plated as shown in Figure 1.The next day (0)the medium was changed to MEM supplemented with 1%DCC-treated FCS in the presence of testosterone (lo-* M; T), aFGF (1 ngiml), or bFGF (1 ngiml) with (dotted bars) or without (open bars) of 10 pgiml heparin. The medium was changed every other day and the cell number was counted on day 6. Values represent means -t SD.

serum-free condition. bFGF, a t a concentration of 10 ng/ml, stimulated [3H]thymidine incorporation to about 15-fold. Heparin did not alter the bFGF-induced stimulation of DNA synthesis (Fig. 7). Growth-pro-

264

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Heparin (pg/rnl) Fig, 6. Effects of increasing concentrations of heparin on [3H]thymidine incorporation in SC-3 cells stimulated with growthpromoting activities from conditioned medium of androgen-treated SC-3 cell cultures. The growth-promoting activities were partially purified by heparin-Sepharose as described in Materials and Methods. SC-3 cells were plated and treated with various dilutions (0.254% [viv]) of the active fractions in the presence of increasing concentrations of heparin. [3H]Thymidine incorporation into the cells was measured. Values are means in triplicate assay. Results are typical of three separate experiments.

Heparin (pg/rnl) Fig. 7. Effects of increasing concentrations of heparin on ['Hlthymidine incorporation into NIH 3T3 cells stimulated with bFGF (1ngiml; A ) or growth-promoting activities from conditioned medium of androgen-treated SC-3 cell cultures (2% diluted samples; 0 ) . The growth-promoting fractions used were the same as described in Figure 6. NIH 3T3 cells were plated in 96-well plates (5 x lo3 cellsiwell) containing MEM with 2% DCC-treated FCS and the medium was replaced with the serum-free medium. On the following day, the cultures were treated for 24 h with the growth factors in the presence of increasing concentrations of heparin. 0,no growth factor added. Values are means ? SD in triplicate assay.

sistent with our proposal that the androgen-induced cell proliferation is mediated through the FGF-like autocrine factor. The molecular mechanism of heparin-induced inhibition of androgen-dependent growth is worth discussing. The possibility that heparin exerts the inhibitory activity via the androgen-induced growth factor-independent pathway seems to be very unlikely, since it specifically inhibited the bioactivity of the partially purified autocrine growth factor. On the other hand, DISCUSSION heparin appeared to have no effect on aFGF- or bFGFThe present study clearly shows t h a t heparin and its induced stimulation of DNA synthesis when saturating related glycosaminoglycans (heparan sulfate and der- concentrations (more than 1 ngiml) of FGFs were used. matan sulfate) exerted negative effects on the prolifer- In contrast, heparin enhanced the bioactivity of low ation of SC-3 cells when the cells were stimulated with concentrations of aFGF and bFGF. These phenomena testosterone. On the other hand, heparin had no inhib- may be due to heparin-mediated stabilization of FGFs. itory effect on the growth of SC-3 cells stimulated with (Gospodarowicz and Cheng, 1986; Rosengart et al., aFGF or bFGF. These results suggest that the inhibi- 1988; Sommer and Rifkin, 1989). One interpretation is tory activity of heparin on androgen-induced prolifer- that the association of the androgen-induced autocrine ation of SC-3 cells is not caused by nonspecific cytotox- factor, but not FGFs, with heparin masks the binding icity. This is also supported by the observations that sites for FGF receptor. Heparin also inhibited DNA treatment with heparin alone showed no negative synthesis of NIH 3T3 cells stimulated with the androeffect on the cell growth. gen-induced growth factor derived from SC-3 cells, but Previous studies have revealed t h a t the growth of not with FGF. Thus, the inhibitory effect of heparin on SC-3 cells is promoted by a n FGF-like peptide, which is the androgen-induced growth factor is elicited in a secreted into conditioned medium in response to andro- factor-specific, but not a cell-type-specific manner. gen (Nonomura et al., 1988; Lu et al., 1989). The These results may support the masking theory. Alterpresent study showed that heparin efficiently de- natively, it is also possible that the difference in the creased r3H1thymidine incorporation induced by the heparin effects reflects the difference in relative affingrowth-promoting activities in the conditioned medium ities of the growth factors for heparin compared to from testosterone-treated cells. The heparin-induced receptors. aFGF and bFGF have higher affinities for decrease in DNA synthesis stimulated with the andro- FGF receptors than for heparin, favoring binding to gen-induced growth factor almost paralleled the de- receptors. By contrast, the androgen-induced growth crease in DNA synthesis induced by testosterone. Thus, factor derived from SC-3 cells may have a lower affinity this heparin-induced growth inhibition would be con- for the receptor, and thus binding to heparin may be moting activities in the conditioned medium from testosterone-treated SC-3 cell cultures, which were partially purified by heparin-Sepharose column, also had the ability to increase the DNA synthesis of NIH 3T3 cells. The addition of heparin significantly decreased the DNA synthesis stimulated with the SC3-derived growth factor as a function of concentration. About 0.1 pg/ml of heparin exhibited a 50% inhibition (Fig. 7).

GROWTH INHIBITION OF SC115 CELLS BY HEPARIN

favored over binding to receptors. In addition, it also cannot be ruled out that heparin activates some latent inhibitor of the androgen-induced growth factor, which may be present in the partially purified preparation. A final conclusion cannot be drawn until the amino acid sequence of SC-3 derived autocrine growth factor is determined and the respective domains responsible for binding to receptor and heparin are identified. It has been shown that FGFs exist in the subendothelial extracellular matrix (Baird and Ling, 1987; Vlodavsky et al., 1987) and are able to be released by heparitinase and heparin-like molecules (Bashkin et al., 1989). Recently we observed that the growthpromoting activities are also present in the extracellular matrix produced by SC-3 cells (unpublished data). The association with the extracellular matrix is thought to be via binding to heparan sulfate proteoglycans present in the matrix. The growth-promoting activities would be inactive under these forms. Thus extracellular matrix may function as a modulator of the autocrine cell growth mechanism of SC-3 cells. Conversely, the modification of the extracellular glycosaminoglycan environment possibly alters the proliferation rate. In conclusion, the autocrine growth factor produced by androgen-treated SC-3 cells possesses a different characteristic from aFGF as well as bFGF in that its bioactivity is negatively modulated by heparin and its related glycosaminoglycans. In this regard, it would be of interest to examine the effect of the glycosaminoglycans on the biological activities of other FGF families such as Int-2, HstiKS3, FGF-5, and KGF.

ACKNOWLEDGMENTS The authors would like to thank Kaori Shiwa for help in preparing the manuscript. This work was supported in part by grants-in-aid for Cancer Research from the Ministry of Education, Science, and Culture and from the Cancer Research Promotion Fund, and by grants from the Foundation €or the Growth Science in Japan and from the Hirai Cancer Research Fund. LITERATURE CITED Baird, A,, and Durkin, T. (1986) Inhibition of endothelial cell proliferation by type P-transforming growth factor: interactions with acidic and basic fibroblast growth factors. Biochem. Biophys. Res. Commun., 138t476482. Baird, A,, and Ling, N. (1987)Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in uitro: implications for a role of heparinase-like enzymes in the neovascular response. Biochem. Biophys. Res. Commun., 142t428-435. BBrzu, T., Lormeau, J.-C., Petitou, M., Michelson, S., and Choay, J . (1989) Heparin-derived oligosaccharides: Affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells. J . Cell. Physiol., 140t538-548. Bashkin, P., Doctrow, S., Klagsbrun, M., Svahn, C.M., Folkman, J., and Vlodavsky, I. (1989) Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry, 28:1737-1743. Bruchovsky, N., and Meakin, J.W. (1973)The metabolism and binding of testosterone in androgen-dependent and autonomous transplantable mouse mammary tumors. Cancer Res., 33t1689-1695. Burgess, W.H., and Maciag, T. (1989)The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem., 58575-606. Crabb, J.W., Armes, L.G., Carr, S.A., Johnson, C.M., Roberts, G.D., Bordoli, R.S., and Mckeehan, W.L. (1986) Complete primary structure of prostatropin, a prostate epithelial cell growth factor. Biochemistry, 25r49884993.

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Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115.

An androgen-responsive cloned cell line (SC-3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)-li...
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