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[CANCER RESEARCH 49, 3474-3481. July 1, 1989] Negative Controls of Cell Proliferation: Human Prostate Cancer Cells and Androgens1 C. Sonnenschein,2 N. Olea, M. E. Fasanen, and A. M. Soto Department of Anatomy anil Cellular Biology, Tufts University Health Science Centers, Boston, Massachusetts 02111 ABSTRACT LNCaP cells represent a useful tool to explore the mechanism of sex hormone action on cell proliferation in an "in culture-in animal" model. Results indicated that: (a) these cells were inhibited from proliferating for extended periods (up to 30 days) when placed in charcoal-dextranstripped sera; they remained, however, viable because they proliferated when sex hormones were added to this medium; (b) the inhibitory effect of sera was reversed by the addition of Sa-dihydrotestosterone at 3 x I0~"' M, 17/3-estradiol at 3 x 10~" M and higher concentrations, and progesterone at 3 x Kl'" M and higher concentrations; (c) while the dose response to androgens was biphasic (i.e., 5a-dihydrotestosterone at con centrations higher than 3 x 10~'°M resulted in progressively lower cell yields), estrogens and progestagens exhibited a monophasic pattern; (d) these cells were exceedingly sensitive to the nutritional environment in which they grew; (<•) while these cells have androgen receptors (68 fmol/ mg protein; A,, = 2 x 10 ' M), estrogen and progestagen receptors could not be detected by biochemical and ¡mmunocytochemicaltechniques; (/) tumors grew at the site of inoculation in castrated nude mice carrying 170-estradiol and progesterone pellets and in intact male nude mice implanted with placebo pellets, while tumors did not grow in castrated nude mice implanted with a 5a-dihydrotestosterone pellet. Taken together the data collected are compatible with the following conclusions: (a) the proliferatiti- response in LNCaP cells seems not to be directly mediated by their intracellular androgen receptors; (b) plasma-borne trypsin-sensitive inhibitors of the proliferation of these cells (androcolyone I) appear to play a significant role in the proliferative event; (c) natural and synthetic androgens, estrogens, and progestagens cancelled the inhibition by duiri-oal-di-Mrail-stripped human sera; (¡I)only androgens were able to trigger an inhibition of cell proliferation (shutoff effect) at concentra tions higher than those that affected maximal cell yields (direct negative hypothesis); and (<•) a faulty shutoff response is probably a crucial event for the tumorigenesis of these human prostate cells. INTRODUCTION Our knowledge of the mechanisms whereby cells proliferate is sketchy and full of controversy. Several hypotheses have been proposed and they vary significantly depending on the premises on which they are based. For example, while hypotheses on positively regulated mechanisms rely on the notion that cells are waiting for a signal to trigger their entering the cell cycle (1, 2), hypotheses on negatively controlled mechanisms are based on the premise that cells are constitutively poised to proliferate and will do so when all inhibitory signals are can celled or no longer recognized (3). The most frequently explored hypotheses have been those dealing with the positive options (direct and indirect) (1, 2, 4-6). Alternatively, using a variety of vertebrate cell types, we have proposed and presented evi dence compatible with the notion that only negative controls are prevalent on the regulation of cell proliferation (3). Still others have proposed that a combination of growth factors and growth inhibitors is responsible for an integrated regulation of Received 9/13/88: revised 3/15/89; accepted 3/28/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by Grants USPHS NIH CAI3410 and NSF DCB8711746. 2To whom requests for reprints should be addressed. the proliferation of cells in metazoans (7-10). Sex hormone-sensitive mammalian cell lines represent useful experimental models to study the control of cell proliferation in animals because sex steroids can be easily manipulated to regulate the proliferation rate of these cells. In addition, sex hormone-sensitive tumors represent a sizable proportion of tumors that increase the morbidity and mortality of human populations (11). After testing several putative androgen-sensitive models for cell proliferation (12),' we focused on the human prostate cancer cell line LNCaP described by Horoszewicz et al. (13). LNCaP cells provide a useful human prostate "in culture-in animai" model to assess proximate and ultimate elements of causality in this mechanism. MATERIALS AND METHODS Cell Line. LNCaP-FGC cells were graciously made available to us by Dr. Julius Horoszewicz from the State University of New York, at Buffalo. They were established from a metastatic supraclavicular lymph node removed from a 50-year-old patient with a prostate adenocarcinoma (13). When we obtained them these cells were on their 60th passage after their establishment. Culture Conditions. Cells were maintained routinely in DIME4 pur chased from GIBCO, Grand Island, NY, and supplemented with 5% FBS from Sterile Systems (Lot 1115715, Logan, UT). Cells were routinely grown in 75- and 250-cm2 plastic flasks (Corning Plastics, Corning, NY). To test their proliferative rate and proliferation yields cells were seeded in 12-well plates (Costar, Cambridge, MA) in 5% FBS-supplemented DME for 48-72 h so that cells would attach to the plastic surface. Then, seeding media were quickly changed to the experimental media; steroid hormones were added according to the experimental design (see below). Because LNCaP cells attach loosely to the plastic surface every effort was taken not to disrupt them; for quantitation purposes, the cell-lysing solution was added to the exper imental media in each well so that nuclei from attached and floating cells would be counted. For cell yield experiments, cultures were kept untouched for 7-10 days after the experimental media were added; for proliferation rate experiments, cells were harvested every 1-2 days and experiments were stopped after the slopes of the curve reached a plateau. Steroids and Other Substances Tested. Estrone, 17/J-estradiol, and estriol were from Calbiochem, Richmond, CA. DHT, 5/3-dihydrotestosterone, testosterone, 19-nortestosterone, progesterone, pregnenolone, androstenedione, 5n-androstane-3n,17/i-diol, 5«-androstane, 3/3,17/3-diol,hydrocortisone, ethynyl estradici, and DES were obtained from SUTalo ids, Keene, NH. Moxestrol and R1881 were graciously provided to us by Roussell-UCLAF, Romainville, France: R5020 was purchased from Du Pont-New England Nuclear, Boston, MA. Org4333 (11/3-chloromethyl estradiol) was made available to us by Organon, Oss, The Netherlands. Mibolerone (7a,17a-dimethyl-l 9-nortestoster one) was a gift by Dr. A. Traish, Boston University School of Medicine. Epidermal growth factor was purchased from Collaborative Research, Lexington, MA (Lot 88-1341) and graciously supplied by Creative Biomolecules, Inc., Holliston, MA (Lot 055-A); insulin (Lot 615-0753C. Sonnenschein and A. M. Soto, unpublished data. 4 The abbreviations used are: DME. Dulbecco's modification of Eagle's me dium; FBS, fetal bovine serum; DHT. dihydrotestosterone; DES. diethylstilbestrol; IET. 100 ng/ml insulin. 100 ng/ml epidermal growth factor, and 2 ng/ml transferrin; CD. charcoal-dextran-stripped; RBA, relative binding affinity; CDHuS. charcoal-dextran-stripped human serum. 3474 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research. HUMAN ANDROGEN-SENSITIVE from 5 x 10 '°to 5 x 10~6M. The assay was carried out as described 256) was from Eli Lilly Co., Indianapolis, IN; and human transform) (Lot 100F-9002) was from Sigma. This combination, called IET, was added to DME when cells were grown in "serumless" media. Experimental Design. Because LNCaP cells are of human origin, we tested their proliferation properties using homologous sera. Blood was drawn from male and female healthy volunteers. Sera were obtained from clotted blood, centrifuged to eliminate cells, heat inactivated for 30 min at 56°C,and stored frozen at — 20°C.When needed, these sera were thawed and charcoal-dextran stripped according to a protocol described elsewhere (14). CD sera were used either immediately after stripping or were kept frozen at —20°C. Proliferation yields of LNCaP cells in heat-inactivated human male and female sera were measured to establish whether or not the sex origin of the sera mattered. Next, similar experiments were run using heat-inactivated alone and heat-inactivated plus charcoal-dextranstripped sera. In these experiments, the different sera were tested at various concentrations between 0.5 and 50% to determine whether the addition of various fixed sex hormone concentrations would elicit comparable responses. Toward the end of their exponentially growing period, cells were lysed with a detergent and nuclei were counted in duplicate in a Coulter Counter Zf¡Model Apparatus. These data were statistically analyzed and represented graphically using the LOTUS 123 and the Sigma plot packages in an IBM XT. A similar procedure was followed for proliferation rate curves (3, 14). Tumorigenesis in Nude Mice. Groups of 5 nude mice (nu/nu CD1 from Charles River Research Laboratories, Wilmington, MA) were castrated and immediately implanted with pellets containing 15 mg of DHT, 0.72 mg of 170-estradiol or 20 mg of progesterone. A group of 5 intact males and females were implanted with a placebo pellet and a group of 5 castrated male mice were implanted with placebo pellets. Pellets were purchased from Innovative Research of America, Toledo, OH. One day after these procedures took place each nude mouse was inoculated s.c. with approximately IO7cells suspended in 0.25 ml DME in the interscapular area. Animals were observed twice weekly and tumor development was recorded. Tumors were excised to measure levels and localization of estrogen receptor. This experiment ended 75 days after cell inoculation. Sex hormone receptors were measured by biochemical procedures described below; estrogen and progesterone receptors were characterized also by a commercially available kit (Ab bott Laboratories, Chicago, IL). Binding Studies. Labeled steroids |ïI (testosterone (specific activity, 3.1 TBq/mmol), [3H]l7ß-estradio\ (specific activity, 3.8 TBq/mmol) and [3H]R5020 (specific activity, 3.2 TBq/mmol) were purchased from Du Pont-New England Nuclear. Intracellular sex steroid-binding pro teins were investigated in whole cell extracts as well as in intact cells. Cytosolic and nuclear extracts were obtained after centrifugation (105,000 x g, 45 min) of a sonicated cellular suspension in 10 mM Tris-500 mivi KC1-0.5 mM EDTA buffer, pH 7.4. Aliquots of this extract were incubated for 18 h at 4°Cwith increasing concentrations (2 x 10~'-2 x 10~8M) of labeled steroids, with and without a 200-fold excess of the corresponding unlabeled steroid. Bound and free fractions were separated by dextran-coated charcoal adsorption (14). Intact cells grown in monolayer culture were assayed for specific steroid binding (15). Briefly, the cells were incubated in 12-well plates during 50 min at 37°Cin DME containing the labeled steroid with and without excess of cold competitor. The range of concentrations used was the same as that in the cytosolic-nuclear extract assay. Previously, it was determined that specific uptake of labeled steroids in these cells reaches plateau during the first 25 min of incubation.5 Intracellular radioactivity was extracted with ethanol after ice-cold phosphate-buffered saline washing. In both types of experiments, Scatchard analysis of saturation data was used to quantify maximal binding capacity (/f,,,.,J and affinity parame ters (dissociation constant, Ka) by the SCAFIT program (16). Cytosol-nuclear extracts were used to study competition for androgen receptors. Aliquots were incubated with 6 x 10~9 M [3H]testosterone and variable concentrations of cold competitors (testosterone, DHT, 5«-androstane-3a, 17/3-diol, 5a-androstane-3/3,17/3-diol, mibolerone R1881, 17/3-estradiol, estrone, estriol, ethynyl estradici, DES, Org4333, moxestrol, progesterone, pregnenolone, and R5020) ranging 5 N. Olea, unpublished results. TUMOR CELLS above for the receptor assay. The RBA was calculated from the equation RBA = Testosterone (I50) x 100 Test competitor (I50) i.e., the ratio between the concentration of unlabeled testosterone and that of the competitor that inhibits 50% (I50) of the [3H]testosterone bound to androgen receptors. Partial Purification and Trypsin Treatment of the Plasma-borne In hibitors. Human plasma was treated with 80 mM BaCI2, 15 mM sodium citrate, and 1 mM benzamidine for l h at 4°C.After centrifugation at 2000 rpm for 20 min, the supernatant depleted of coagulation factors was dialyzed against 25 mM Tris-HCl, pH 7.4. Plasma protease inhib itors (a,-antitrypsin, a2-macroglobulin, etc.) were removed from 10 ml of BaCl2-treated plasma by chromatography through a 25-ml Cibacronblue agarose column (Pierce Chemical Co., Rockford, IL); the retained fraction was eluted with 2 M NaCl-25 mM Tris, pH 7.4. Aliquots of this fraction were treated as follows: (a) chromatography through a 5ml benzamidine-Sepharose column (Pharmacia-LKB, Piscataway, NJ) as a control; and (¿>) incubation with 0.5 mg/ml of trypsin for l h at 37°Cfollowed by chromatography in benzamidine-Sepharose to remove trypsin from the androcolyone preparation. Both benzamidine-Sepha rose eluates were concentrated by ultrafiltration, dialyzed against 100 mM NaCl-25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4, and rendered sterile by filtration before adding to IET-supplemented DME. Proliferation yield experiments to test the effect of these plasma fraction-supplemented media were done as described above and elsewhere (14). RESULTS Proliferative Properties of LNCaP Cells Effects of Heat-inactivated Human Male and Female Sera. Human female serum was more inhibitory than male serum when heat inactivated (Fig. I A). DHT increased cell yields in 10% female serum; in contrast, DHT added to male serumsupplemented medium did not significantly increase cell yields. However, 3 x 10~7 M DHT consistently inhibited cell prolifer ation (Fig. IA); this was also seen when 10% female serum was used. a 2 UJ IO 1 1CT12 IO"10 10"e DHT CONCENTRATION 1CT (M) 10-12 ESTRADIOL ,0-10 ,0-e CONCENTRATION ,0-6 (M) Fig. 1. Cell proliferation yields obtained when established human prostate tumor cells (LNCaP) were grown in media supplemented with 10% human male (O) and female (•)sera that were heat-inactivated (A and C) or heat-inactivated and charcoal-dextran-stripped sera (B and D). These sera were supplemented with either 3 x KT12-3 x IO'" M DHT (A and A) or 3 x l(T"-3 x irr7 M 17/3estradiol (C and D). Values are mean ±SD (bars) of 4 experimental measure ments; when SD was less than or equal in magnitude to the size of the plotted data point bars were not plotted. Cells were grown undisturbed in experimental media for 7 days. 3475 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research. HUMAN ANDROGEN-SENSITIVE TUMOR CELLS Effect of DHT on Cell Proliferation Yields. Charcoal-dextranstripped male and female human sera showed a comparable inhibitory effect on the proliferation yield of LNCaP cells (Fig. \B). Addition of DHT to either female or male CD serumsupplemented media generated a biphasic proliferative response (Fig. \B); the peak was reached at 3 x 10~'" M. Concentrations above 3 x 10"'" M DHT resulted in progressively lower cell yields. As a result of this type of experiment, heretofore, we used sera from either sex when heat-inactivated CD-stripped human sera was required. Effect of Other Sex Hormones on Cell Proliferation. Fig. 1, C and D, shows the effect of 17/3-estradiol at concentrations between 3 x 10~" and 3 x 10~7 M when supplemented to (a) intact heat-inactivated and (b) heat-inactivated and charcoaldextran-stripped 10% human male and female sera. Cell yields were approximately 2-fold higher in plates treated with 3 x NT* M 17/3-estradiol than with 3 x 1(T10M DHT. Testosterone showed a proliferative pattern similar to that obtained with DHT (not shown). 3«-Androstanediol was 103fold and 3/3-androstanediol was 10-fold less potent than DHT (Fig. 2A). However, 3/3-androstanediol increased the cell yield to values approximately 2-fold higher than those obtained with DHT; this effect was comparable to that seen with 17/3-estradiol. In addition, 30-androstanediol concentrations above those required for maximal cell yield resulted in a shutoff effect similar to that observed with DHT (Fig. 2A). 19-Nortestosterone was 10-fold more potent than DHT (not shown). On the other hand, 5/3-DHT and androstenedione were 100-fold less potent than DHT (not shown). Estrone and estriol did not affect the proliferation yields of LNCaP cells. 17a-Estradiol was 100 times less effective than 17/3-estradiol (Fig. 2B). Pro gesterone (Fig. 2C) and pregnenolone (not shown) elicited a monophasic proliferative pattern. Cholesterol and hydrocortisone did not induce LNCaP cell proliferation over control values (not shown). Effect of Synthetic Androgens, Estrogens, and Progestagens on Cell Proliferation. The synthetic androgens R1881 (Fig. 2A) and mibolerone (not shown) were 100 times more potent than DHT. Both androgens triggered the shutoff response as DHT did. Among synthetic estrogens neither DES nor moxestrol was active; Org4333 increased the proliferation yield of these cells at concentrations of 3 x 10~9 M and higher (Fig. 2B). The proliferative pattern of Org4333 was similar to that shown by 17/3-estradiol, i.e., no shutoff effect. Ethynyl estradici behaved similarly to 17/3-estradiol (Fig. 2B). Of synthetic progestagens, R5020 was approximately 2 or ders of magnitude less potent than progesterone (Fig. 2C); higher concentrations of either progesterone or R5020 did not result in lower cell yields. Proliferation Rate of LNCaP Cells. The population-doubling time of LNCaP cells was 40 h when 3 x 10-"' M DHT was added to 10% charcoal-dextran-stripped human sera-supple mented medium (Fig. 3). The inhibitory effect of CD human sera was not instantaneous; at the end of 3-4 days the popula tion increase was halted and remained stable for up to 30 days without media changes. That these cells were then still viable is suggested by increased cell proliferation rates when 3 x IO'"' M DHT was added (not shown). The proliferation rate of LNCaP cells growing in the presence of DHT was significantly faster than that of the control (minus DHT). 170-Estradiol showed a slightly shorter population-doubling time. It appears as if 17/3-estradiol allowed for an additional population dou bling over what DHT influenced (Fig. 3). -10 1CT1U 10~8 STEROID CONCENTRATION 10r« (M) Fig. 2. Dose-response cunes in medium supplemented with 10% CDHuS and with different hormones. A, DHT (O). 5«-androstane-3n,17^-diol (•),5ir-androstane-.V.17rf-diol (A). R1881 (A). B, DHT (O), 17«-estradiol(A). 17f(-estradiol (•),cthynyl estradiol (A). Org4333 (D). C. DHT (O). progesterone (•).R5020 (A). Value* are mean ±SD (bars) of 4 experimental measurements. Cells were kept in experimental media without change for 7 days. This is 1 of 20 represent ative experiments. Serum Concentration Effects on Proliferation Yields. Absolute yields varied according to the serum and sex hormone concen trations used (Fig. 4). The proliferative effect of DHT appeared to be influenced by serum concentration in the media; lower DHT concentrations (3 x 10~" M) were necessary to cancel the inhibitory effect of low CD human serum-supplemented media (1-5%). Higher DHT concentrations (3 x 10"'°M) were needed to cancel the inhibitory effect of high CD human serum-supple mented media (10-50%). In addition, lower serum concentra tions generated lower yields when compared with higher serum concentrations in experiments seeded and harvested simulta neously. A comparable pattern was obtained with 17/3-estradiol (Fig. 4B). Effect of Putative Growth Factors and Growth Inhibitors. Testing of proliferation yields under putative serumless condi tions revealed information worth reporting. As described above (see "Experimental Design"), cells were kept for 48 h in 5% FBS so that a sufficient number of cells would become attached 3476 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research. HUMAN ANDROGEN-SENSITIVE TUMOR CELLS the proliferative potency of some of the compounds tested. [3H]17/3-Estradiol binding was displaced by androgens and pro gestagens. DES and moxestrol, which bind to estrogen receptor present in estrogen-sensitive cells, did not compete with [3H]17/3-estradiol binding. These observations, as well as the ab sence of immunoreaction with monoclonal antibodies against human estrogen receptor (not shown), indicate that no estrogen receptor is present in these cells; this confirms data of others (17). R5020 binding was also displaced by androgens and estrogens with RBAs comparable to those observed when com petition between ['Hjtestosterone and progestagens was meas ured. These observations indicate that steroid-binding activity seems to represent androgen receptor. While a good correlation between relative binding affinities and proliferative potency was established for many compounds tested, R1881 and mibolerone were more potent to induce cell proliferation than predicted by their RBA. In addition, both progesterone and pregnenolone were more potent inducers of cell proliferation than predicted by their RBAs; pregnenolone displayed the most extreme lack of correlation between its RBA (<0.01%) and its proliferative potency (1%) when compared with DHT values. LU ce m m 5 D Z LU ü 10 5_ Tumorigenesis in Nude Mice Tumors developed in castrated mice implanted with 17/3estradiol and progesterone and in intact male animals (Table 3). Tumors did not develop in castrated mice implanted with placebo pellets, in mice implanted with DHT pellets, and in female nude mice. Plasma hormone levels were not determined in these mice. 4x10 3 5 time (days) Fig. 3. Proliferation curve of the LNCaP cells. Similar inocula were grown in 10% CDHuS with 3 X IIT10 M DHT (•),with 3 X KT" M 17/3-estradiol (D), or without hormone (O). Media were not changed for 7 days when cells were harvested. Each point represents three wells. Values are mean ±SD (bars). to the plastic surface. Cells seeded and kept for 48 h in media supplemented with increasing concentrations of FBS (from 1 to 40%) showed a significantly different cell yield when chal lenged to proliferate in IET-supplemented DME; a direct pos itive correlation could be established between the serum con centration in the seeding media and the cell yield achieved in DME plus IET (Fig. 5). When cells were seeded in 1% FBS the difference between cell yields of controls and sex hormonesupplemented cultures were less significant than those recorded when cells were seeded in 5% or higher FBS concentrations. Effect of Trypsin Treatment on Partially Purified Plasmaborne Inhibitors. Table 1 shows that the partially purified frac tion inhibited the proliferation of LNCaP cells when compared to IET medium. In addition, DHT added to the purified fraction significantly increased cell yield, whereas IET was ineffectual. Trypsinization of the plasma fraction resulted in a significant cell yield increase in the absence of androgen; moreover, DHT did not increase the cell yield when added to the trypsin-treated purified fraction. Remarkably, adding 10% CDHuS to the trypsin-treated fraction restored both inhibition and hormone responsiveness. 3-2-jIti¡S** T' Ei 1-^^to ^Iss ^^Blr*i/^f|in/ f\ fey//vl fsvs^sssssN _! r1ifi* o's«"(flm3Z 3-immtLUa sssssS NSSsNs 2-1-n-fr _Ls\s\s\utl. ///tf/ Intracellular Sex Steroid-binding Proteins LNCaP cells have been reported to contain androgen recep tors (13). We confirmed this. The KA of the receptor-DHT complex was 3 x 10~9 M; the concentration of binding sites was 68 fmol/mg of protein in the 100,000 x g extract (Fig. 6). The whole cell assay indicated about 40,000 binding sites/cell. Testosterone binding was displaced by other androgens, some estrogens, and progestagens. Table 2 compares the RBA and _ riJ| r1fEs^s ÕA[ SÃŽ t/ /// / 1% 5% 10% 20% 30% 40% 50% Fig. 4. Serum concentration effects on proliferation when LNCaP cells were grown in media supplemented with 1-50% CDHuS P, A and B); plus DHT (A: m. 3 x 10~" M; P, 3 x 10"'°M; ®,3 x 10"' M) or plus 17i)-estradiol (B: •.3 x 10"' M; M, 3 x I0~8 M). Cells were harvested at the end of 7 days of culture without media change. Values are mean ±SD (bars) of 4 experimental measure ments. 3477 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research. HUMAN ANDROGEN-SENSmVE 25201510Ltl 5 5- o C o1% ce. £ 25- 5% 10% 20% 40% B Z 20- 105- 1% 5% 10% 20% 40% Fig. 5. Serum concentration effects on LNCaP cell proliferation. Cells were seeded in 1-40% FBS and 48 h later were changed to IET-supplemented DME alone (O, A and A) or plus DHT (A: •,3 x 10~" M;^, 3 x \0~">M;M, 3 X 10~" M) or plus 17/i-estradiol (B: •,3 X 10"* M; Ãœ,3 X 10"' M). Cells were harvested after 7 days of culture without media change. Values are mean ±SD (bars) of 4 experimental measurements. Table 1 Trypsin treatment of partially purified androcolyone I Results are expressed as cell number/well (mean ±SD) after 5 days of exposure to experimental media. TreatmentAndrocolyone frac ratio151,050± +DHT TUMOR CELLS that androgens directly stimulate cell proliferation; we will expand below on why we consider this latter option unlikely. On the other hand, androgen-mediated inhibition of LNCaP cell proliferation appears to be a consequence of the androgenspecific induction of a gene product the function of which is shutting off the entry of these cells in the cell cycle. These conclusions are not significantly different from those drawn more than a decade ago by Bruchovsky et al. (18). These interpretations will be confirmed when the following evidence will become available: (a) the purification of the plasma-borne specific inhibitor of the proliferation of androgen-sensitive cells (androcolyone I); (b) the characterization of specific receptors for these inhibitors; and (c) the characterization and purification of the intracellular androgen-induced proliferation inhibitor (androcolyone II). While the task of generating evidence to verify these inter pretations is completed using the LNCaP model, it is worth while attempting the reconstruction of probable regulatory mechanisms of the proliferation of these human androgensensitive cells. Tumor Nature of LNCaP Cells. LNCaP cells were established from a supraclavicular lymph node metastasis of a malignant prostatic adenocarcinoma in a patient who died within 2 years of the apparent onset of the disease (13). Figs. 1 to 5 indicate, however, that these cells can be effectively arrested by sex steroid-depleted sera. Both male and female human sera were effective and to the same extent as far as we could measure it. This inhibition, however, was cancelled by physiological con centrations of natural androgens (Figs. 1 and 2A). That this is not just a fortuitous, idiosyncratic "in culture" event is indicated by the lack of tumor formation by these cells in castrated nude mice (Table 3). Comparable data have been generated when another human prostate carcinoma was challenged to prolifer ate in nude mice (19). These data are, therefore, compatible with the notion that human and mouse sera contain an inhibitor of the proliferation of human prostate cells. Bovine, horse, pig, ±5,184 18,835 tion" Trypsin-treated an 145.540 ±10,440 153,720 ±5,399 0.92.35 drocolyone* 10% CDHuS-DHT98,93084.260 ±11,527+DHT/-DHT 198,1 20 ±25,8801.5 Trypsin-treated an- 101,290 ±10,603 224,610 ±10,503 2.22 drocolyone + 10% CDHuS IET 143,020 ±6,747 148,430 ±31,954 1.04 °Androcolyone fraction was obtained by Cibacron blue and benzamidine- Table 2 Comparison between RBAs to the androgen receptor and proliferation potencies of diverse steroidal and nonsteroidal compounds on LNCaP cells HormoneDHT5a-Androstane-3a, proliferative potency*1000.11010,00010,0001— 7/3-diol5«-Androstane-3/3,l 1 7/3-diolR1881Mibolerone1 Sepharose chromatography and added to IET to a final concentration of 3 mg/ ml. * Androcolyone fraction was incubated with 0.5 mg/ml trypsin for l h at 37"C; trypsin was removed by benzamidine-Sepharose chromatography; 3 mg/ml were added to IET. Cells were harvested after 5 days of culture without changing media. 7/3-EstradiolEstroneEstriolEthynyl '1__1—10011 estradiciR2858Org4333DESProgesteronePregnenoloneR5020RBA°1001.214200150.90.1<0.011. DISCUSSION The human prostate tumor LNCaP cell line has proved to be a reliable tool to explore competing hypotheses on the mecha nism of androgen regulation of cell proliferation. Results sug gest a complex interaction among the protagonists of such regulation. Thus far, our data point to the participation of distinctive pathways favoring the proliferation and the inhibi tion of this proliferation occurring in a successive fashion. On the one hand, data are compatible with the notion that prolif eration occurs when some natural and synthetic sex hormones cancel the inhibition generated by human serum depleted of sex hormones by charcoal-dextran stripping. While we favor this interpretation we cannot yet completely rule out the alternative " Calculated as indicated in "Materials and Methods." *The relative proliferative potency was the ratio (xlOO) between the concen tration of testosterone and agonist necessary to elicit maximal cell yield for a 104 cell inoculum grown in CDHuS for 8 days. c—, no proliferative potency at the range of concentrations tested. Table 3 Tumorigenesis when iff1 LNCaP cells were inoculated in nude mice Intact female + placebo Intact male + placebo Castrate male + placebo Castrate male + DHT pellet Castrate male + 17/3-estradiol pellet Castrate male -I-progesterone pellet 0/5 4/5 0/5 0/5 3/5 3/5 3478 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research. HUMAN ANDROGEN-SENSITIVE dog, and chicken sera also inhibit the proliferation LNCaP cells.6 of these Proliferative and Inhibitory Effects of Androgens. Androgen administration generated a biphasic proliferative pattern whereby higher concentrations than those required for maximal cell yield resulted in a dose-dependent inhibitory' effect (Figs. 1 and 2A). This phenomenon resembles the time-dependent shutoff effect of androgens on the proliferation of prostate epithelium in castrated rats (18). Proliferative Effect of Estrogens and Progestagens. Addition of 17/3-estradiol, 17a-estradiol, ethynyl estradici, and Org4333 to 10% charcoal-dextran-stripped human serum-supplemented medium resulted in increased cell yields. Both estrogens and progestagens showed a monophasic dose-response curve. Un like androgens, estrogens and progestagens failed to trigger the inhibitory shutoff response. Estrogens and progestagens also induced proliferation of LNCaP cells inoculated into castrated male nude mice (Table 3). Thus, good correlation between in culture and in animal studies has been established in this model. Correlation between Intracellular Receptor Binding and Pro liferative Potency of Sex Steroids and Synthetic Agonists. LNCaP cells contain an intracellular androgen-binding moiety which binds DHT (Ka 3 x 10~9 M) and is present at a concen tration of 68 fmol/mg protein (Fig. 6). These cells lack estrogen receptors because (a) no immunoreactive estrogen receptor was detected; (¿>) the Kd for [3H]17/3-estradiol was lower than ex pected for human estrogen receptor (20); and (c) [3H]17/3estradiol binding was not displaced by DES and moxestrol while it was displaced by androgens and progestagens. That pH]17|0-estradiol binding by LNCaP cytosol represents androgen receptor is suggested by the fact that DES does not displace [3H]DHT from the rat ventral prostate androgen receptor (21), while 17/3-estradiol is known to reduce ['HJDHT binding to androgen receptors; this evidence was collected by sucrose gradient analysis (22) and by measuring 17ß-estradioleffects on the nuclear retention of [3H]DHT in prostate slices (23). When comparing the proliferative potency of diverse hor mones with their relative binding affinities a good correlation was found for 3/3-androstanediol, DHT, testosterone, and 6 of the 7 estrogens shown in Table 2. On the other hand, proges terone, pregnenolone, mibolerone, and R1881 were more po tent to induce cell proliferation than predicted by their relative binding affinities for the androgen receptor. For example, the proliferative potency of pregnenolone was 1% of the DHT potency, while its RBA was less than 0.01% when compared to DHT. In addition, 3a-androstanediol, estrone, and R5020 dis played lower proliferative potency than predicted by their re ceptor-binding affinities. Lack of correlation between affinity for the androgen receptor and activity was reported for both synthetic agonists and antagonists using the rat ventral prostate model (21). These data are interpreted as evidence for (a) metabolic conversion of the hormone into a more potent me tabolite when hormonal action exceeds binding activity or (b) fast metabolic clearance when hormonal activity is lower than anticipated by the receptor binding data. The proliferative effect of estrogens and progestagens could not be explained by meta bolic conversion to androgens since (a) estrogens and proges tagens displayed a monophasic proliferative response while all androgens show a biphasic response and (b) addition of estro gens and progestagens consistently resulted in a 2-fold higher cell yield than that generated by the androgens tested. Because progestagens showed a proliferative effect comparable in po 6Unpublished data. TUMOR CELLS LU O rr LU « 0.2CO I 0.1 Ul LU ce o D Å“ 0 25 50 75 BOUND [3H] -TESTOSTERONE (fmol/mg protein) Fig. 6. Scatchard plot of the saturation data obtained when LNCaP cell cytosol was incubated during 18 h at 4°Cwith [3H]testosterone with and without cold competitor. The maximal binding capacity and the value of the dissociation constant were 68.73 fmol/mg of cytosol protein and 3.2 x 10"' M, respectively. Correlation coefficient of point fit 0.977. tency to DHT while displaying cell yields consistently higher than those evoked by androgens, a critical evaluation on the role of the androgen receptors is in order. The direct positive hypothesis for androgen action on cell proliferation proposes that hormone action is directly mediated by steroid binding to the intracellular receptor. Hormone recep tor complexes would interact with regulatory sequences of genes under androgen control and elicit their expression. However, there is no compelling evidence supporting this pathway for the control of cell proliferation. In fact, if one assumes that cell proliferation was directly mediated through receptor binding a strong correlation between receptor binding affinity and prolif erative potency should be apparent. Data on LNCaP cells do not provide evidence for this mode of action because: (a) while estrogens and progestagens induce cell proliferation, these cells do not have estrogen or progesterone receptors; (b) if the action of estrogen and progesterone were mediated through interaction with the androgen receptor, a good correlation between binding affinity and proliferative potency should have been apparent (this is not the case at least for estrone and the progestagens tested); and (c) if the proliferative action of estrogens and progestagens were mediated through the androgen receptor, a similar response pattern should have been obtained with all these sex steroids. Instead, all androgens displayed both a proliferative and an inhibitory response (shutoff) whereas estro gens and progestagens only displayed the proliferative response and consistently induced higher cell yields than androgens (Table 2). The data discussed above are incompatible with, but do not rule out, the direct and indirect positive hypotheses. To the contrary, these data are compatible with the direct and indirect negative hypotheses; according to the indirect negative hypoth esis androgens, estrogens, and progestagens induce cell prolif eration by neutralizing a serum-borne specific inhibitor of androgen-sensitive cell proliferation (androcolyone I). On the other hand, the inhibitory effect of androgens seems to be mediated through the androgen receptor since (a) there is a good correlation between the potency of these hormones to induce the shutoff effect and their relative binding affinities to the androgen receptor and (b) estrogen and progestagens are unable to evoke the shutoff response. Proliferative Response of LNCaP Cells to Human Sera. Our previous work on estrogen-sensitive cell proliferation revealed 3479 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research. HUMAN ANDROGEN-SENSITIVE that (a) charcoal-dextran-stripped serum inhibited the prolif eration of estrogen-sensitive cells in a dose-dependent manner and (b) estrogen sensitivity was not apparent in serumless medium where cells proliferated maximally regardless of the presence of estrogens (3, 14, 20). Similar results were expected with LNCaP cells. The proliferation yields of LNCaP cells in 1-50% CD human serum were comparable while the yields obtained with DHT or 17/3-estradiol increased with serum concentration reaching a maximum at 20% CDHuS (Fig. 4). The hormone concentration needed to obtain maximal cell yields also increased as the serum concentration increased (Fig. 4). These results suggest that serum plays a prominent nutritive role which overlaps either with the inhibitory role of the androcolyone I it carries (indirect negative hypothesis) or with the stimulatory role of sex hormones (direct positive hypothesis). Fig. 5 shows that the cell yield of LNCaP cells in serumless media (DME plus IET) was also dependent on the fetal bovine serum concentration in the seeding medium, i.e., prior to the cells being exposed to serumless media. The more serum in the seeding medium, the higher was the cell yield in both the control and sex hormone-treated wells; however, the proliferative yield generated by DHT and 17/3-estradiol-treated cells was consid erably smaller in comparison to that of controls when cells were seeded in 1% fetal bovine serum. These data are compatible with the notion that LNCaP cells are exceedingly sensitive to nutritional availability and that sex hormones do not compen sate for lack of nutrients. Also, these data suggest that heatinactivated fetal bovine serum contains a surplus of active androcolyone I that is cancelled by DHT and 17/3-estradiol. These data by itself, however, do not allow us to discriminate between sex hormones inducing cell proliferation directly or indirectly (by cancelling the inhibitory effect of the serum-borne androcolyone I). Table 1 further substantiates the presence of a plasma/serumborne androcolyone I; trypsin treatment of a partially purified androcolyone I preparation resulted in the loss of both inhibi tory and hormone effects. That trypsinization releases a growth factor is unlikely because adding 10% CDHuS to this prepara tion restored inhibition and hormone responsiveness. Role of Putative Growth Factors on the Proliferation Yield of LNCaP Cells. The indirect positive hypothesis proposes that sex steroids induce the synthesis of growth factors that in turn trigger the entry of their target cell into the cell cycle (1, 2, 4, 19,24). We tested the effect of insulin, epidermal growth factor, and transferrin when added to phenol red-free DME plus 1% and 10% CDHuS. The results shown in Fig. 7 indicate that the cell yield of LNCaP cells was similar regardless of whether or not these growth factors were added to the serum-containing media. DHT added to serumless, growth factor-supplemented media resulted in a marginal increase of the cell yield; however, the effect of DHT was proportionally higher as the CD serum concentration in the media increased. The growth factors tested appear to play no significant role in these sex steroid-specific proliferative events. Results described in this paper provide interesting clues on the hormonal control of cell proliferation. They can be sum marized as follows, (a) The proliferative response obtained with androgens, estrogens, and progestagens challenges the notion that this event is mediated through the androgen receptor pathway and suggests an alternative hypothesis for hormone action on cell proliferation, i.e., the involvement of plasmaborne inhibitors of androgen-sensitive cell proliferation (andro colyone I), (b) The potent inhibitory effect of androgens on cell proliferation seems to be mediated through the androgen recep- TUMOR CELLS 3 - 2 - o i H "^ ce LU m C D 111 U B 3 - 2 - 1 - 10% IET 1% Fig. 7. Cell proliferation yields obtained when LNCaP cells were grown in IET or 1 and 10% CDHuS-supplemented DME medium. A and B: D, control wells (no hormone added); A: DHT supplemented at 3 x 10"'°M (•),3 x 10"' M (^), and 3 x 10~s M (^), respectively. B: 17/3-estradiol supplemented at 3 x 10~' M(•),3 x IO'8 Mp), and 3 x 10~7M (M), respectively. Cells were harvested after 7 days of culture in media kept undisturbed. Values are mean -I-SE (bars) of 4 experimental measurements. tor pathway (direct negative hypothesis); because the prolifer ative response seems to be triggered by all three sex steroid groups and the inhibitory response is only triggered by andro gens, these results suggest that the ultimate proliferative control in normal prostate cells is the shutoff mechanism triggered by high androgen plasma levels, (c) The development of an androgen-dependent tumor is probably due to a defective shutoff mechanism; this will allow proliferation to occur in the presence of androgen concentrations otherwise sufficient to trigger the shutoff mechanism in normal cells. In addition, these results provide leads to gain a mechanistic understanding of the role of sex steroid on the control of cell proliferation by searching for: (a) a serum-borne inhibitor of androgen-sensitive cell pro liferation (androcolyone I); (b) androcolyone I receptors on androgen sensitive cells; (c) sex hormone-androcolyone I inter actions; (d) the gene product(s) responsible for the androgentriggered shutoff effect (androcolyone II); and (e) target genes for the action of androgens on the induction of androcolyone II (colyogenes) (3). ACKNOWLEDGMENTS The authors would like to acknowledge the technical support of RenéeSilvia and the secretarial help of Virginia Olson and Mary Currier. REFERENCES 1. Erikson, R. L. Regulation of cell proliferation by oncogene products and growth factors. Somatic Cell Mol. Genet., 13: 459-461, 1987. 2. Heldin, C. H., Betsholtz, C., Claesson-Welsh, L., and Westermark, B. Sub version of regulatory pathways in malignant transformation. Biochim. 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