Neither fibroblast growth factor-1 nor fibroblast growth factor-2 is an androgen receptor coactivator in androgen-resistant prostate cancer.

We used rat prostate cancer cell stable transfectants that lacked either endogenous fibroblast growth factor (FGF)-1 secondary to constitutive expression of FGF-1 antisense RNA (aFa2-transfectants) or endogenous FGF-2 isoforms secondary to constitutive expression of FGF-2 antisense RNA (bFa9-transfectants) to examine the potential synergistic effects of mitogen and androgen as modulators of proliferation. During culture on 5% charcoal-stripped fetal bovine serum (CS-FBS), FGF-1 caused a 2- to 2.5-fold increase in the proliferation of aFa2-transfectants that lacked endogenous FGF-1 and retained full expression of FGF-2 isoforms. In marked constrast, bFa9-transfectants that lacked FGF-2 isoforms and retained full expression of FGF-1 died with exponential kinetics when cultured on either 5% CS-FBS or 5% FBS in the absence of FGF-2. However, FGF-2 promoted bFa9-transfectant survival and exponential proliferation during culture on either 5% CS-FBS or 5% FBS. The nonmetabolizable androgen R1881 did not affect proliferation of either the aFa2- transfectants, the bFa9-transfectants, or the parental prostate cancer cells used to generate these transfectants. Additionally, neither of the androgen receptor antagonists RU23908 or bicalutamide affected either FGF-1-mediated aFa2-transfectant proliferation or FGF-2-mediated bFa9-transfectant proliferation during culture on 5% CS-FBS. Notably, transient transfection analyses established R1881 concentration-dependent induction of chloramphenicol acetyltransferase activity in both aFa2-transfectants and bFa9-transfectants. Thus, the failure of either androgen or antiandrogen to affect either FGF-mediated or FGF-independent antisense-transfectant proliferation is not attributable to absence of functional androgen receptors. The results indicate that FGF effects in these androgen-resistant antisense transfectants do not involve either androgen-dependent or androgen-independent, mitogen-mediated androgen receptor activation. Our studies show that these rat prostate cancer cells are characterized by both retention of functional androgen receptors during development of androgen resistance and mitogen-mediated, autocrine or paracrine (or both) modulated proliferation. These are two prominent properties characteristic of advanced human prostate cancer.

Androgen regulation of prostate cancer cell FGF-1, FGF-2, and FGF-8: preferential down-regulation of FGF-2 transcripts.

Using quantitative RT-PCR, we found that T1 rat prostate cancer cell relative FGF-1 transcript content was about 180-fold greater than that of FGF-2. This difference in transcript content was not representative of T1 cell relative FGF-1 and FGF-2 protein content which showed, at most, only a 4- to 5-fold greater FGF-1 content. Testosterone caused time-dependent down-regulation of prostate cancer cell FGF-2 transcript content without influencing either FGF-1 or FGF-8 transcript content or T1 cell proliferation. Moreover, testosterone-mediated down-regulation of prostate cancer cell FGF-2 transcripts did not result in a statistically significant change in 21.5 or 17.0 kD FGF-2 isoform content. By contrast, an approximately 20% statistically significant decrement in 19.5 kD FGF-2 isoform content was demonstrable following 24 h testosterone treatment. However, following 72 h testosterone treatment, T1 cell 19.5 kD FGF-2 isoform content was not statistically significantly different from that of control. It is probable that the modest and variable decrement in 19.5 kD isoform content is not physiologically significant and is attributable to artifact resulting from difficulty quantifying this minor component of the FGF-2 isoforms. Transient transfection analysis showed that androgen caused concentration-dependent increases in MMTV-LTR regulated expression of chloramphenicol acetyl transferase activity. Consequently, the failure of androgen to affect either T1 cell FGF-1 and FGF-8 transcript content or T1 cell proliferation could not be attributed to defective androgen receptor function. Moreover, the absence of a close relationship between T1 cell FGF-2 transcript and FGF-2 protein content implies that FGF-2 transcript content is not the dominant determinant of prostate cancer cell FGF-2 protein content. Testosterone-mediated down-regulation of prostate-cancer-cell gene expression may have significance for clinical management of human disease that is treated by androgen ablation. The possibility that such ablation may enhance aggressiveness of "androgen-independent" cells by selective upregulation of gene expression merits further consideration.

Fibroblast growth factor-2 and TPA enhance prostate-cancer-cell proliferation and activate members of the Ras and PKC signal transduction pathways.

Rat prostate-cancer-cell stable-transfectants expressing either antisense-fibroblast growth factor (FGF-1) or antisense-FGF-2 transcripts that respectively have either undetectable FGF-1 or profoundly diminished FGF-2 protein content, were used for analyses of FGF-2 and/or 12-O-tetradecanoylphorbol 12-acetate (TPA) modulation of cell proliferation. Antisense-FGF-2 transfectant doubling-time was 2.6-fold greater than that of vector-control transfectants. FGF-2 and TPA respectively caused 2.5- and 3.0-fold reductions in antisense-FGF-2 transfectant doubling-time. Culture of antisense-FGF-2 transfectants in medium containing both FGF-2 and TPA further reduced their doubling time; however, this effect was not statistically different from that achieved by TPA treatment alone. Antisense-FGF-1 transfectant doubling-time was 2.2-fold greater than that of vector-control transfectants and was reduced 2.0- or 2.3-fold, respectively, when these cells were cultured in medium containing FGF-2 or TPA. In contrast to the results for antisense-FGF-2 transfectants, culture of antisense-FGF-1 transfectants in medium containing both FGF-2 and TPA caused a 2.6-fold reduction in transfectant doubling-time that was significantly greater than that caused by independent treatment with either FGF-2 or TPA. FGF-2 promoted rapid activation of rat prostate-cancer-cell PKCalpha and PKCepsilon, as assessed by isozyme translocation from the soluble to particulate cell fraction, and only moderately altered PKCdelta distribution. By contrast, TPA promoted rapid activation of all three PKC isozymes. Both the TPA- and FGF-2-mediated PKC activation were prolonged and possibly involved cyclic redistribution of isozymes between soluble and particulate cell fractions. FGF-2 also caused rapid phosphorylation of prostate-cancer-cell Shc, the adapter protein that mediates FGF-receptor-modulated ras signaling. The results of these studies indicate that FGF-2 and TPA independently and conjointly modulate rat prostate-cancer-cell antisense-transfectant doubling time and suggest that effector modulation of rat prostate-cancer-cell proliferation is achieved by processes involving PKC and/or ras mediated signaling.

Endogenous fibroblast growth factor-1 or fibroblast growth factor-2 modulate prostate cancer cell proliferation.

We constructed expression vectors containing either rat fibroblast growth factor (FGF)-1 for FGF-2 cDNA cloned in either the sense orientation or antisense orientation relative to the metallothionein promoter of plasmid pMTneo.1. Stable AXC/SSh rat prostate cancer cell transfectants expressing either chimeric FGF-1-sense, chimeric FGF-1-antisense, or chimeric FGF-2-antisense transcripts were obtained. Stable transfectants expressing chimeric FGF-2-sense transcripts were not obtained. Control, sense, and antisense transfectants expressed endogenous FGF-1 and endogenous FGF-2 transcripts, implying that transfection did not eliminate endogenous FGF transcripts. Control transfectants and sense transfectants contained FGF-1 isoforms having a mass of 16.4 or 17.3 kDa and FGF-2 isoforms having a mass of 17, 19.5, or 21.5 kDa. Significantly, adult AXC/SSh rat prostate contained only the 17.3 kDa FGF-1 isoform and the 17 kDa FGF-2 isoform, indicating that neoplastic transformation was associated with elaboration of novel, prostate epithelial cell-derived FGF-2 isoforms. FGF-1 antisense RNA expression eliminated transfectant FGF-1 isoforms without affecting FGF-2 isoform content. Similarly, FGF-2-antisense RNA expression eliminated the transfectant 21.5 kDa FGF-2 isoform, diminished the 19.5 kDa FGF-2 isoform content, and reduced the 17 kDa FGF-2 isoform content to barely detectable levels without affecting the FGF-1 isoform content. This established that FGF-antisense RNAs specifically inhibited translation of cognate, endogenous FGF transcripts. Doubling times of control transfectants and sense transfectants were indistinguishable and were not affected by including FGF-1 or FGF-2 in the culture medium. Doubling times of FGF-1-antisense or FGF-2-antisense transfectants were 1.3- to 1.4-fold greater than those of control transfectants or sense transfectants, and either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling times to values indistinguishable from those of control transfectants or sense transfectants. This established that with regard to prostate cancer cell proliferation: (a) endogenous FGF-1 cannot substitute for endogenous FGF-2 eliminated by FGF-2-antisense RNA expression; and (b) endogenous FGF-2 cannot substitute for endogenous FGF-1 eliminated by FGF-1-antisense RNA expression. In contrast, either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling time. The results of these studies establish that endogenous FGF-1 and endogenous FGF-2 modulate prostate cancer cell proliferation and imply that FGF-1 and FGF-2 of endogenous and exogenous origin conjointly control aspects of prostate cancer cell homeostasis. Our findings suggest complex interaction between components of prostate cancer cell regulatory processes and endogenously produced and exogenously accessible FGF-1 and FGF-2.

Protein kinase C-dependent and -independent pathways of signal transduction in prostate cancer cells: fibroblast growth factor utilization of a protein kinase C-independent pathway.

To examine the possibility that differences in protein tyrosine phosphorylation contributed to differences in fibroblast growth factor (FGF) responsiveness of clonally derived C3 (modestly responsive) and T5 (highly responsive) rat prostate cancer cells, we evaluated the ability of orthovanadate to affect prostate cancer cell thymidine incorporation. These analyses showed that C3 cell FGF insensitivity was not attributable to enhanced protein phosphotyrosine phosphatase activity. Analyses of acidic FGF (aFGF)-mediated protein phosphorylation showed mitogen-caused, time-dependent tyrosine phosphorylation of C3 and T5 cell FGF receptors (FGFRs) and other proteins having a mass of 190, 150, 120, 100, 90, 80, 74, 60/62, 50, 42, or 28 kilodaltons. Although marked differences characterized aFGF mediated intensity of tyrosine phosphorylation, the notable commonality of tyrosine phosphorylation and the mass of the phosphorylated proteins suggested that C3 and T5 cells may use the ras and/or protein kinase C (PKC) pathways for FGF-mediated signal transduction. The PKC agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused concentration-dependent increases in T5 cell thymidine incorporation. In contrast, TPA did not enhance thymidine incorporation of C3 cells or mitogen-sensitive NRK cells included as a nonneoplastic control. TPA also significantly enhanced T5 cell proliferation, whereas identical treatment did not affect proliferation of either C3 or NRK cells. Either 12 or 24 h treatment with 200 or 2000 ng/ml TPA caused complete PKC alpha and partial PKC delta down-regulation in C3, T5, and NRK cells. Consequently, the failure of TPA to affect C3 or NRK cell thymidine incorporation or proliferation was not attributable to potential TPA ineffectiveness in these cells. Survey immunological analyses showed that all three cell lines lacked PKC beta, PKC eta, and PKC theta. In contrast, T5 cells contained abundant amounts of PKC epsilon, whereas the PKC epsilon content of C3 and NRK cells was near the limit of detection. TPA treatment of T5 cells evoked only partial PKC epsilon down-regulation. Both aFGF and basic FGF (bFGF) promoted concentration-dependent enhancement of TPA-pretreated T5 cell thymidine incorporation, and the effects of combined TPA and either aFGF or bFGF treatment were additive. Neither aFGF nor bFGF was able to enhance thymidine incorporation of TPA-pretreated C3 cells beyond the modest effects elicited by FGF treatment of C3 controls.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid, two-step method for high-yield purification of recombinant rat acidic and basic fibroblast growth factors.

We describe the preparation of vectors for T7 polymerase-driven, high-level expression of rat acidic (aFGF) or basic (bFGF) fibroblast growth factors in Escherichia coli. Following isopropyl-beta-D-thiogalactoside induction of T7 polymerase, rat aFGF or bFGF represented a major portion of the proteins synthesized by vector-transformed E. coli. Passage of cell extracts through an Amicon YM-100 membrane provided ultrafiltrates containing either aFGF or bFGF as the principal component. A single heparin-Sepharose chromatography of the ultrafiltrates yielded essentially homogenous, biologically active, recombinant rat aFGF or bFGF. By silanizing vessels and using buffers containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, we could store homogenous aFGF or bFGF preparations without significant loss during repeated freezing and thawing. Previous methods for purifying aFGF or bFGF utilized salt fractionation followed by sequential ion exchange and heparin-Sepharose chromatography. These purified aFGF or bFGF preparations routinely were stored in buffered carrier protein to minimize mitogen loss resulting from adsorption to glass or plastic surfaces. In contrast, the methods that we detail are rapid, require minimal manipulation of preparations, and permit storage of carrier-free, homogenous preparations without loss resulting from surface adsorption. The protocols can be used for preparation of homogenous aFGF or bFGF of other species and may be readily applied to the isolation and characterization of FGF-like mitogens present in cultured cell extracts, conditioned medium, or tissue preparations.

Signal transduction defect appears to be the cause of rat prostate cancer cell fibroblast growth factor insensitivity.

Using monolayer cultures of clonally isolated C3 and T5 rat prostate cancer cells, we determined that acidic (aFGF) and basic (bFGF) fibroblast growth factors profoundly enhanced T5 cell thymidine incorporation with half-maximum stimulation at 0.53 and 0.35 ng/ml, respectively. In contrast, aFGF or bFGF enhancement of C3 cell thymidine incorporation was about 5% of that of T5 cells, and effects were principally mitogen concentration independent. Saturation analyses and cross-linking studies established that both C3 and T5 cells contained high-affinity FGF receptors of 120 and 145 kilodaltons and that receptor content and Kd of C3 and T5 cells were comparable. aFGF or bFGF stimulation of T5 cell thymidine incorporation profoundly decreased as cell plating density was reduced from 1.5 x 10(5) to 1.0 x 10(4) cells/well. The modest response of C3 cells to either aFGF or bFGF also decreased as cell plating density was reduced. Because heparin preserves FGF biological activity and enhances bFGF binding to high-affinity FGF receptors, we examined the effect of heparin on FGF stimulation of C3 cell thymidine incorporation. We found that changes in cell plating density and/or medium heparin concentration had variable, inconsistent effects. These were C3 cell plating density associated and included inhibition or modest enhancement of FGF effects. Binding analyses established that high-affinity bFGF binding of C3 and T5 cells immediately prior to assessing FGF-stimulated thymidine incorporation was comparable and independent of cell plating density, implying that C3 cell FGF insensitivity was not attributable to differences in C3 and T5 cell FGF receptor content at the time of mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat prostate cancer cell line-specific production and apparent secretion of heparin-binding growth factors.

Cultured C3 and T5 AXC/SSh rat prostate cancer cells and their conditioned media contained 1000-fold more heparin-binding mitogens than did normal AXC/SSh rat prostates. Immunological analyses confirmed that rat ventral prostate contained only a 17.5 kilodalton (kDa) basic fibroblast growth factor (bFGF)-like mitogen. In contrast, combined immunological and metabolic radiolabeling analyses showed that C3 cells contained 23/24 and 14 kDa bFGF-like polypeptides, whereas the principal bFGF-like polypeptides of C3 cell conditioned medium were proteins having masses of 17.5 and 14 kDa. Identical analyses showed that T5 cells contained 17.5 and 14 kDa bFGF-like polypeptides, whereas the principal bFGF-like polypeptides of T5 cell conditioned medium were proteins having masses of 17.5, 16, and 14 kDa. We found that bFGF-like proteins of mass less than 17.5 kDa, which were present in conditioned medium, were not derived by proteolysis of higher molecular weight bFGF-like mitogens after these were processed into conditioned medium. Northern analyses showed that normal prostate and prostate cancer cells contained acidic fibroblast growth factor transcripts of 6.5 and 3.4 kilobases (kb) and bFGF transcripts of 6.0 and 2.5 kb. Prostate cancer cells also contained a 12-kb bFGF transcript that was not present in normal prostate. Southern analysis of restriction endonuclease-digested normal prostate or prostate cancer cell genomic DNA showed that the 12-kb bFGF transcript was not the product of a rearranged bFGF gene. Our data show that rat prostate cancer cells contain bFGF-like polypeptides of mass 14 to 23/24 kDa and suggest that these cells secrete bFGF-like polypeptides.

Sexual dimorphism characterizes baboon myocardial androgen receptors but not myocardial estrogen and progesterone receptors.

Using biochemical methods we established that estrogen receptor content and distribution and progesterone receptor content in female and male baboon myocardium did not differ between sexes. In contrast, myocardial androgen receptor distribution between cytosolic and nuclear compartments was sexually dimorphic. Female baboon myocardial androgen receptors were restricted to the cytosolic compartment, whereas male myocardial androgen receptors were distributed between the cytosolic and nuclear compartments. Using human estrogen receptor cDNA we showed that baboon aorta, myocardium and uterus contain a 6.3 kb estrogen receptor transcript. Analyses performed with human progesterone receptor cDNA established that baboon aorta and uterus contain an 8 kb progesterone receptor transcript; however, progesterone receptor transcripts were not demonstrable in baboon myocardial RNA preparations. Because relative hybridization signal intensity reflected known uterine and aortic progesterone receptor content, failure to detect progesterone receptor transcripts in myocardial preparations may reflect sensitivity limitations and the fact that aortic progesterone receptor content is 5-fold greater than that of myocardium. Immunocytochemical analyses demonstrated that baboon myocardial progesterone receptors were present in greater than 25% of myocytes and generally absent from other myocardial cells. Our studies establish that: (1) gonadal steroid hormone receptor gene transcription occurs in cells of the baboon cardiovasculature, (2) these steroid hormone receptors may be physiologically functional, and (3) gonadal steroid hormone receptors may be restricted to specialized cells of the cardiovasculature.

Rat prostate cancer cells contain functional receptors for transforming growth factor-beta.

Clonally derived C-, D-, and T-family AXC/SSh rat prostate cancer cell lines contain transforming growth factor-beta (TGF beta) receptors. The content in C3, D1, T1, and T5 cells, respectively, was 8,560 +/- 1,450, 13,160 +/- 1,240, 2,425 +/- 490, and 10,540 +/- 1,025 sites/cell (mean +/- SEM). Respective Kd values were 160 +/- 48, 200 +/- 53, 24 +/- 3, and 115 +/- 15 pM (mean +/- SEM). T1 cell TGF beta receptor site content and Kd differed significantly from those of other prostate cancer cell lines (P less than 0.05). TGF beta is a bifunctional concentration-dependent modulator of T1 and T5 cell thymidine incorporation. At low concentrations, thymidine incorporation was inhibited, whereas as the medium TGF beta content was increased, T1 and T5 cell thymidine incorporation was stimulated. The concentrations of TGF beta causing half-maximum inhibition of T1 or T5 cell thymidine incorporation, respectively, were 0.11 and 0.24 pM, whereas the respective TGF beta concentrations causing half-maximum stimulation of thymidine incorporation were 14.4 and 134 pM. These findings establish that rat prostate cancer cell sensitivity to TGF beta inhibition of function is at least 2 orders of magnitude greater than that of most other mammalian cells. In contrast, the sensitivity of rat prostate cancer cells to TGF beta enhancement of function is comparable to that of other mammalian cells. TGF beta inhibited basic fibroblast growth factor (bFGF) stimulation of T1 and T5 cell thymidine incorporation. Because the concentration of bFGF required for half-maximum increase of T5 cell thymidine incorporation was independent of medium TGF beta content, the effect of TGF beta is distal to the T5 cell bFGF receptor. In contrast, the concentration of bFGF required for half-maximum increase in T1 cell thymidine incorporation increased 5-fold as the medium TGF beta content was increased; suggesting that the effect of TGF beta in T1 cells is proximal to the T1 cell bFGF receptor. Our studies establish that rat prostate cancer cells contain functional TGF beta receptors, imply the presence of functional bFGF receptors, and demonstrate that mitogen modulation of prostate cancer cell function is multifactorial. The finding that TGF beta is a bifunctional effector of prostate cancer cell DNA synthesis provides some insight into the potential complexity of mitogen modulation of prostate cancer cell proliferation. The mechanism by which these mitogens interact is unknown; however, our studies suggest that some interactive effects may be cell line specific.

Differences in responsiveness of clonally derived AXC/SSh rat prostate cancer cells to secreted or prototypic mitogens.

AXC/SSh rat prostate cancer cells elaborate heat-sensitive, heparin-binding mitogens which include members of the fibroblast growth factor (FGF)-like family of growth factors. The quantity of FGF-like growth factors, relative to total growth factor production, was cell line specific as was prostate cancer cell response to secreted or the prototypic mitogens basic FGF (bFGF), acidic FGF (aFGF), or epidermal growth factor (EGF). C3 cell proliferation, assayed either by cell counting or thymidine incorporation, was not affected by mitogens secreted by C3, D1, T1, or T5 prostate cancer cells or by bFGF, aFGF, or EGF. In contrast, C3, D1, T1, or T5 cell-secreted mitogens enhanced proliferation of T1 and T5 cells, and proliferation of D1, T1, and T5 cells was enhanced by bFGF, aFGF, and EGF. D1 cell response to prototypic mitogens was 3- to 12-fold less than that of T1 or T5 cells. By comparison, the NRKF cell response to prototypic mitogens was qualitatively comparable but quantitatively greater than that of rat prostate cancer cells. The relative and absolute bFGF or aFGF concentrations necessary for half-maximum stimulation of prostate cancer or normal rat kidney fibroblast cell thymidine incorporation were comparable to that known to effect half-maximum increase in proliferation of mesoderm-derived cells. Similarly, the EGF concentration required for half-maximum prostate cancer or normal rat kidney fibroblast cell thymidine incorporation was comparable to that known to effect half-maximum fibroblast thymidine incorporation or granulosa cell proliferation. Our data establish that prostate cancer cell response to prototypic mitogens is representative of that of nonneoplastic cells and imply that C3 cell insensitivity to prostate cancer cell or prototypic mitogens represents defects in cellular response mechanisms. The basis for C3 cell unresponsiveness or D1 cell-diminished responsiveness remains to be elaborated.

Androgen modulation of prostate cancer cell androgen receptor content is cell line specific.

Using methods for cell lysis and fractionation which yield essentially quantitative recovery of rat prostate cancer cell cytosolic and nuclear androgen receptors, we examined androgen modulation of androgen receptor content of clonally derived prostate cancer cell lines. We showed that testosterone elicited a concentration-dependent 2.3-fold increase in T5 cell androgen receptor content which was maximum after 48 h and was maintained through at least 72 h of culture. Testosterone caused only a 1.4-fold elevation in D2 cell androgen receptor content which was maximum between 6 and 12 h of culture and was maintained through at least 72 h culture. In contrast, testosterone did not cause a change in C3 cell androgen receptor content. Cycloheximide inhibition showed that both the testosterone-mediated increase in and maintenance of basal prostate cancer cell androgen receptor content required protein synthesis. Because testosterone and the nonmetabolizable androgen R1881 were essentially equipotent as effectors of the increase in T5 cell androgen receptor content, findings using testosterone appear to represent maximum effects. RU 23908 antagonized both R1881 and testosterone promoted elevations of prostate cancer cell androgen receptor content. Effectiveness of RU 23908 was comparable to the relative binding affinity of R1881, testosterone and RU 23908 for androgen receptors. This implies that at least part of the androgen-promoted increase in prostate cancer cell androgen receptor content is mediated through the action of androgen receptors and suggests that androgen receptors may act as both cis and trans regulatory elements. The mechanisms which determine basal or androgen-modulated prostate cancer cell androgen receptor content remain to be elucidated.

Antiandrogen effects in models of androgen responsive cancer.

The ability of antiandrogens to antagonize androgen effects in androgen responsive tissues is well established. Antiandrogens may diminish in vivo or in vitro proliferation of some androgen responsive cancer cells without causing cessation of multiplication. These model studies are representative of clinical experience in treatment of human prostate cancer with antiandrogen therapy. Recent studies in the AXC/SSh rat prostate cancer model show that these cancer cells elaborate polypeptide growth factors which stimulate their proliferation. If growth factor production by these cells is androgen independent, this may provide an explanation for failure of androgen ablation or antiandrogen treatment to effectively halt prostate cancer cell proliferation.

Differential metabolism of dehydroepiandrosterone sulfate and estrogen conjugates by normal or malignant AXC/SSh rat prostate cells and effects of these steroid conjugates on cancer cell proliferation in vitro.

Normal AXC/SSh rat ventral prostate and clonally derived AXC/SSh rat prostate cancer cells were evaluated for ability to metabolize estrone sulfate (E1S), estrone glucuronide (E1G), or dehydroepiandrosterone sulfate (DHEAS). Both normal and malignant prostate cells converted E1S to estrone. Neither normal nor malignant prostate cells had significant ability to metabolize DHEAS to DHEA, indicating differential specificity of prostate sulfatases(s) for estrogen and androgen sulfates. Both normal and neoplastic prostate cells possess beta-glucuronidase which hydrolyzed E1G to estrone. To assess potential physiologic consequences of these enzymatic activities, we determined the effect of steroid conjugates on in vitro proliferation of selected clonal lines of AXC/SSh rat prostate cancer cells. DHEAS, 10(-6) to 10(-9) M in decade intervals, did not affect in vitro proliferation of AXC/SSh prostate cancer cells; however, 10(-5) M DHEAS decreased in vitro proliferation of these cells. Neither E1S nor E1G, 10(-5) to 10(-9) M in decade intervals, affected in vitro proliferation of AXC/SSh prostate cancer cells. These findings suggest that low residual levels of steroid conjugates, which are not removed by charcoal stripping of serum, do not affect demonstrated in vitro androgen modulation of AXC/SSh rat prostate cancer cell proliferation (Cancer Res. 46, 3775-3781, 1986).

Effect of aging on AXC/SSh rat ventral and dorsolateral prostate S-adenosyl-L-methionine decarboxylase and L-ornithine decarboxylase messenger ribonucleic acid content.

Ventral prostate S-adenosyl-L-methionine decarboxylase (AMDC) and L-ornithine decarboxylase (ODC) transcript content decreased 5-fold as males aged from 6 to 26 months. In contrast, dorsolateral prostate AMDC and ODC transcript content in these individuals was age invariant. The differential effect of aging on tissue transcript levels reflected respective 3.5- and 5-fold decreases in ventral prostate total and poly(A)+ RNA content in aging males, whereas dorsolateral prostate RNA levels essentially were age invariant. Testosterone injection of 26-month-old males increased ventral and dorsolateral prostate content of AMDC and ODC transcripts 4- and 2.5-fold, respectively. Changes in ventral prostate ODC transcript levels correlated well with previously reported age- and testosterone-mediated changes in prostate ODC protein content; however, the relation between ventral or dorsolateral prostate AMDC mRNA levels and previously determined AMDC protein content was less stringent. Our observation that ventral prostate ODC transcript content was 7-fold greater than that of dorsolateral prostate was insufficient to account for the established 200-fold difference in ODC activity in these tissues. Our data imply that transcript content is a principal determinant of ventral prostate ODC activity in aging AXC/SSh rats. However, this relationship does not appear to characterize either ventral or dorsolateral prostate AMDC activity or relative ventral and dorsolateral prostate ODC activity and transcript content. The cause of the age-related preferential loss of ventral prostate RNA remains to be evaluated.

Androgen receptors in biopsy specimens of prostate adenocarcinoma. Heterogeneity of distribution and relation to prognostic significance of receptor measurements for survival of advanced cancer patients.

Needle biopsy specimens of primary adenocarcinoma and surgical specimens of carcinomatous nodal tissue were obtained from previously untreated clinical D stage prostatic adenocarcinoma patients. Assessment of the relation between specimen androgen receptor site content and survival using either scatterplots or Kaplan-Meier analyses showed specimen receptor content was a poor prognostic P greater than 0.1, of survival subsequent to orchiectomy or diethylstilbestrol (DES) therapy. The possibility that heterogeneity of specimen androgen receptor site content contributed to this finding was evaluated by comparing receptor content of multiple small or large tissue specimens from the same prostate gland of patients with benign prostatic hyperplasia or nonmetastatic prostatic cancer. This evaluation showed significant microheterogeneity of human prostate androgen receptor site content which was substantially masked in large tissue specimens. We conclude that microheterogeneity of human prostate androgen receptor site content compromises the use of biopsy specimen androgen receptor measurements as a prognostic of patient survival subsequent to initiation of hormonal therapy.

Gender and baboon aortic steroid hormone receptors.

To examine the potential of steroid hormones to serve as putative regulators of aortic cell function, we defined hormone receptor content and distribution in intact baboons. Total androgen receptor content in baboon aortic arch, thoracic arch, and abdominal aorta of young mature males was indistinguishable from that of proestrus females. However, 30% to 40% of male aortic androgen receptors were in the nuclear fraction, whereas all aortic androgen receptors of proestrus females were in the cytoplasmic fraction. Cytoplasmic fraction estrogen receptor content of aortic arch and thoracic aorta of intact males was indistinguishable from that of proestrus females. However, cytoplasmic fraction estrogen receptor content of abdominal aorta of proestrus females was significantly greater than that of males. Nuclear fraction estrogen receptors were not detectable in either male or proestrus female baboon aortas. To assess effects of endogenous estrogen on aortic progesterone receptor content, we quantified cytoplasmic fraction progesterone receptors and found that content of proestrus female aortic arch was not significantly different from that of males. However, cytoplasmic fraction progesterone receptor content of thoracic and abdominal aorta of proestrus females was significantly higher than that of males. To determine whether differences in aortic receptor content or distribution were associated with changes in aortic cell function, we quantified the activity of two enzymes of glycosaminoglycan metabolism. Aortic beta-glucuronidase activity was not different in male or proestrus female baboons.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation of AXC/SSh rat prostate cancer cells in vitro is androgen modulated.

We used heterogeneous parental cultures of AXC/SSh rat prostate cancer cells to isolate clonally derived prostate cancer cell lines. Light and electron microscopic analyses established that parental and clonally isolated cells possess features characteristic of secretory epithelium. Biochemical analyses showed that these cells contained androgen receptors and acid phosphatase and 5 alpha-reductase activity; phenotypic markers characteristic of differentiated prostate epithelium. Content of these prostate epithelial cell markers was variable and cell line specific. We used selected cell lines to examine androgen modulation of AXC/SSh rat prostate cancer cell proliferation in vitro. We found that proliferation of C-family or D-family cells, those respectively maintained on medium without additions or medium containing 10(-7) M 5 alpha-dihydrotestosterone, was not affected by changes in medium testosterone concentration through the range 10(-6)-10(-9) M. In contrast, testosterone modified proliferation of T-family cells, those maintained on medium containing 10(-7) M testosterone, and effects were antagonized by the anti-androgen RU 23908. Preliminary studies established that AXC/SSh rat prostate cancer cells elaborate polypeptide components which stimulate in vitro cell proliferation. Both the ability to elaborate these components and their effects on in vitro cell proliferation appeared to be cell line specific.