Fibroblast growth factor-2 mediates transforming growth factor-beta action in prostate cancer reactive stroma.

Transforming growth factor-beta (TGF-beta) is overexpressed at sites of wound repair and in most adenocarcinomas including prostate cancer. In stromal tissues, TGF-beta regulates cell proliferation, phenotype and matrix synthesis. To address mechanisms of TGF-beta action in cancer-associated reactive stroma, we developed prostate stromal cells null for TGF-beta receptor II (TbetaRII) or engineered to express a dominant-negative Smad3 to attenuate TGF-beta signaling. The differential reactive stroma (DRS) xenograft model was used to evaluate altered stromal TGF-beta signaling on LNCaP tumor progression. LNCaP xenograft tumors constructed with TbetaRII null or dominant-negative Smad3 stromal cells exhibited a significant reduction in mass and microvessel density relative to controls. Additionally, decreased cellular fibroblast growth factor-2 (FGF-2) immunostaining was associated with attenuated TGF-beta signaling in stroma. In vitro, TGF-beta stimulated stromal FGF-2 expression and release. However, stromal cells with attenuated TGF-beta signaling were refractory to TGF-beta-stimulated FGF-2 expression and release. Re-expression of FGF-2 in these stromal cells in DRS xenografts resulted in restored tumor mass and microvessel density. In summary, these data show that TGF-beta signaling in reactive stroma is angiogenic and tumor promoting and that this effect is mediated in part through a TbetaRII/Smad3-dependent upregulation of FGF-2 expression and release.

Reactive stroma in prostate cancer progression.

PURPOSE: The development of an altered stromal microenvironment in response to carcinoma is a common feature of many tumors. We reviewed the literature describing characteristics of reactive stroma, how reactive stroma affects cancer progression and how carcinoma regulates reactive stroma. Moreover, we present a hypothesis of reactive stroma in prostate cancer and discuss how the biology of reactive stroma may be used in novel diagnostic and therapeutic approaches. MATERIALS AND METHODS: An extensive literature search was performed to review reports of the general features of wound repair stroma, general stromal responses to carcinoma, and stromal biology of normal and prostate cancer tissues. These studies were analyzed and a reactive stroma hypothesis in prostate cancer was developed. RESULTS: Modifications to the stroma of breast, colon and prostate tumors parallel the generation of granulation tissue in wound repair. These changes include stromal cell phenotypic switching, extracellular matrix remodeling and angiogenesis induction. Therefore, it is predicted that a modified wound healing response induces the formation of reactive stroma in cancer to create a tumor promoting environment. Based on its role in wound repair and its over expression in prostate cancer, transforming growth factor-beta stands out as a potential regulator of reactive stroma. CONCLUSIONS: Reactive stroma in prostate cancer and granulation tissue in wound repair show similar biological responses and processes that are predicted to promote cancer progression. Further identification of specific functional and regulatory mechanisms in prostate cancer reactive stroma may aid in the use of reactive stroma for novel diagnostic and therapeutic approaches.

Adenovirus-mediated suicide gene therapy for bladder cancer: comparison of the cytomegalovirus- and Rous sarcoma virus-promoter.

BACKGROUND: To compare efficacy and toxicity of the human cytomegalovirus-immediate-early (CMV) promoter and the Rous-sarcoma-virus (RSV) promoter to express thymidine kinase (tk) for adenovirus-mediated suicide gene therapy of experimental bladder cancer in vivo and in vitro. MATERIALS AND METHODS: In vitro: 3 human (5637, RT-4 and TCC-SUP) and one murine (MBT-2) bladder cancer cell line were exposed to ADV/RSV-tk or ADV/CMV-tk vectors and cell survival was determined. In vivo: Subcutaneous tumors were established and adenovirus vectors were injected 10 days later. RESULTS: In vitro: ADV/CMV-tk was up to 4 times more potent in terms of cell killing than ADV/RSV-tk. In vivo: ADV/CMV-tk had a three-fold higher antitumor potency per viral particle as compared to ADV/RSV-tk. Higher doses of ADV/CMV-tk caused treatment-associated hepatotoxicity. CONCLUSIONS: Our results confirm the efficacy of adenovirus-mediated tk suicide gene therapy in the treatment of experimental bladder cancer. Dose-related toxicity was greater with the use of ADV/CMV-tk, but lower doses achieved the same efficacy as ADV/RSV-tk.

In vivo adenovirus-mediated suicide gene therapy of orthotopic bladder cancer.

These studies were undertaken to determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase (ADV/RSV-tk) and the prodrug ganciclovir (GCV) in an orthotopic murine bladder cancer model. We utilized a replication-defective adenoviral construct containing the beta-galactosidase gene as a control and the herpes simplex virus thymidine kinase gene as the therapeutic vector under the transcription control of the Rous sarcoma virus long terminal repeat promoter. Intravesically created, orthotopic bladder tumors were established in syngeneic C3H/He female mice. India ink injection and beta-galactosidase studies were performed to determine if transurethral administration, direct tumor injection, or the combination was the most efficient route of virus administration. Optimal dosing of ADV/RSV-tk was determined by direct tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate but similar controlled experiments. Growth curve studies demonstrated reliable tumor formation by 14 days. Direct transvesical tumor injection resulted in the best distribution and intratumor gene expression as measured by X-gal staining. Dose-ranging experiments demonstrated an optimal viral dose of 5 x 10(8) plaque-forming units and a greater than twofold reduction in tumor growth for the animals treated with ADV/RSV-tk compared to controls. Efficacy studies demonstrated a greater than threefold reduction in tumor growth. No clinical or gross pathologic toxicity was detected. Long-term survival results suggested a survival benefit for the treatment animals compared to controls. We conclude that ADV/RSV-tk in combination with GCV provides effective therapy for orthotopic murine bladder cancer by significantly inhibiting tumor growth with limited toxicity to the host. These data provide further support for testing this suicide gene therapy strategy in human Phase I trials.

The WFDC1 gene encoding ps20 localizes to 16q24, a region of LOH in multiple cancers.

We previously identified ps20 protein as a secreted growth inhibitor and purified the protein from fetal rat prostate urogenital sinus mesenchymal cell conditioned medium. The rat cDNA was subsequently cloned, and ps20 was found to contain a WAP-type four-disulfide core motif, indicating it may function as a protease inhibitor. We now report cloning and characterization of the mouse ps20 gene (designated Wfdc1), the human homolog cDNA, and the human gene (designated WFDC1). Both the mouse and human WFDC1 genes consist of seven exons and encode respective ps20 proteins sharing 79.1% identity and nearly identical WAP motifs in exon 2. The WFDC1 gene was mapped by FISH analysis to human Chromosome (Chr) 16q24, an area of frequent loss of heterozygosity (LOH) previously identified in multiple cancers including prostate, breast, hepatocellular, and Wilms' tumor. Identification and characterization of the WFDC1 gene may aid in better understanding the potential role of this gene and ps20 in prostate biology and carcinogenesis.

Transforming growth factor-beta1 induces nuclear to cytoplasmic distribution of androgen receptor and inhibits androgen response in prostate smooth muscle cells.

Stromal-epithelial interactions in the prostate gland are dependent on androgen regulation of prostate stromal cells, yet little is known about androgen action in these cell types. Recent reports have demonstrated that androgen-regulated gene transcription can be stimulated or inhibited by certain growth factors, indicating cross-talk mechanisms. To address potential cross-talk in signaling pathways between androgen and transforming growth factor-beta1 (TGFbeta1) in prostate stromal cells, the PS-1 prostate smooth muscle cell line was examined. In the presence of physiological concentrations of androgen, PS-1 cell proliferation was stimulated, and androgen receptor (AR) exhibited a nuclear localization pattern. The addition of TGFbeta1 (25 pM) was capable of blocking androgen-induced proliferation, but had no direct effect in cultures without androgen. Immunocytochemistry to localize AR subcellular distribution showed that TGFbeta1 (5-100 pM) altered the distribution of AR from the nucleus to the cytoplasm. Other growth factors, including fibroblast growth factor-2, epidermal growth factor, and TGFbeta2 had no effect on AR distribution. The TGFbeta1-induced nuclear to cytoplasmic change in receptor localization was rapid (initiated within 30 min), was neutralized by TGFbeta1 antibodies, did not require new protein synthesis, and was complete by 6 h. Removal of TGFbeta1 from the culture medium resulted in a rapid redistribution of AR to the nucleus, indicating reversible mechanisms. Northern analysis of the ddp17 marker transcript for androgen action in PS-1 cells showed that androgen-stimulated ddp17 expression was inhibited in the presence of TGFbeta1 (25 pM). TGFbeta1 induced a similar nuclear to cytoplasmic distribution of AR in primary cultures of rat prostate stromal cells. TGFbeta1, however, had no effect on AR distribution in either the LNCaP prostatic carcinoma cell line or the DDT1MF-2 leiomyosarcoma cell line. Specific cross-talk between TGFbeta1 and AR signaling pathways in prostate stromal cells may play a significant role in prostate development and stromal cell response in carcinoma progression.

Localization of transforming growth factor-beta1 and type II receptor in developing normal human prostate and carcinoma tissues.

Transforming growth factor-beta1 (TGF-beta1) is implicated in prostate development, and elevated expression of TGF-beta1 has been correlated with prostate carcinogenesis. In this study, cell type specificity of TGF-beta1 and TGF-beta receptor Type II (RcII) protein expression was determined by immunocytochemistry in human normal prostate and compared to prostate carcinoma tissues. Heterogeneous localization patterns of LAP-TGF-beta1 (TGF-beta1 precursor) and RcII were observed in both epithelial and mesenchymal cells in fetal prostate, with LAP-TGF-beta1 localizing to more basal epithelial cells. Homogeneity of LAP-TGF-beta1 staining was increased in neonatal, prepubertal, and adult prostate, with elevated immunoreactivity noted in epithelial acini relative to stromal tissue for both LAP-TGF-beta1 and RcII proteins. In stromal tissues, RcII cell localization exhibited staining patterns nearly identical to smooth muscle alpha-actin. In prostate carcinoma, LAP-TGF-beta1 localized to carcinoma cells with an increased staining heterogeneity relative to normal prostate. In contrast to normal epithelial cells, carcinoma epithelial cells exhibited low to nondetectable RcII staining. Stromal cell staining patterns for LAP-TGF-beta1 and RcII in carcinoma, however, were identical to those of normal prostate stromal cells. These studies implicate both epithelial and stromal cells as sites of TGF-beta1 synthesis and RcII localization in the developing and adult normal human prostate. In addition, these data indicate a loss of epithelial expression of RcII concurrent with altered LAP-TGF-beta1 expression in human prostate carcinoma cells.

Molecular cloning and expression of ps20 growth inhibitor. A novel WAP-type "four-disulfide core" domain protein expressed in smooth muscle.

We previously reported the purification of ps20 (Rowley, D. R., Dang, T. D., Larsen, M., Gerdes, M. J., McBride, L., and Lu, B. (1995) J. Biol. Chem. 270, 22058-22065), a urogenital sinus mesenchymal cell secreted protein having growth-inhibitory properties. We report here cloning of the 1.03-kilobase rat ps20 cDNA clone from the PS-1 (adult rat prostate smooth muscle) cDNA library. Partial clones were obtained by nested polymerase chain reaction with degenerate primers, and full-length ps20 cDNA clones were isolated by plaque hybridization. Sequence analysis revealed that ps20 protein contains a WAP-type "four-disulfide core" motif and is a novel member of the WAP signature protein family composed primarily of secreted serine protease inhibitors. Native ps20 immunoprecipitated from smooth muscle cells and recombinant ps20 both resolved on SDS-polyacrylamide gel electrophoresis with apparent molecular mass of 27-29 kDa under reducing conditions and 21-23 kDa under non-reducing conditions, respectively. Stable ps20-transfectant COS-7 cell lines secreted ps20 and were growth-inhibited relative to mock transfectants. In addition, COS-7 and prostate carcinoma PC-3 cells were growth-inhibited by bacterially expressed ps20. Northern analysis indicated differential expression by tissue with highest expression in the heart. Immunohistochemical localization of ps20 protein showed cell-specific expression by both visceral and vascular smooth muscle in all tissues, including the prostate gland. These results indicate ps20 is a novel growth-regulatory member of the WAP signature family expressed by smooth muscle cells.

What might a stromal response mean to prostate cancer progression?

Many similarities exist between the stroma at sites of wound repair and reactive stroma in cancer. Common features include an elevated stromal cell proliferation, altered expression of matrix components, elevated expression of TGF beta-1, neovascularization, and expression of several common stromal markers. In addition, proliferative stromal cells at these sites generally express myodifferentiation markers. A comparison between the many common features and the biologically active molecules observed in reactive stroma in carcinoma and reactive stroma in wound repair is discussed in this review. An extended analysis of the literature suggests a functional link between mechanisms in wound repair response and the stromal reaction in many cancers including prostate cancer. We propose in this review, that the fundamental mechanisms of stroma in providing a rapid response to altered homeostasis in wounding, also provides for a tumor-regulating stromal microenvironment in cancer. The functional consequences of this stromal response to carcinoma progression and how the stromal response might be used in extended diagnosis and in therapeutic approaches are discussed.

Adenovirus-mediated suicide gene therapy for experimental bladder cancer.

OBJECTIVES: To determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase gene (HSV-tk) and the prodrug ganciclovir (GCV) in a murine model of human transitional cell carcinoma. METHODS: We used a replication-defective adenoviral construct containing the beta-galactosidase gene (ADV/Rous sarcoma virus [RSV]-beta-gal) as a control or ADV/RSV-tk as the therapeutic vector under the transcriptional control of the RSV long-terminal repeat promoter. Transduction efficiency was assessed in vitro by infection of MBT-2 cells with ADV/RSV-beta-gal at various multiplicities of infection (MOI) utilizing 5-bromo-4-chlor-3-indolyl-beta-D-galactoside (X-gal) staining. Sensitivity of MBT-2 cells to the therapeutic vector was determined after infection with ADV/RSV-tk with or without GCV. Subcutaneous tumors were established in syngeneic C3H/He female mice with 5 x 10(5) MBT-2 cells. Optimal dosing of ADV/RSV-tk was determined by direct percutaneous tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate, similar, controlled experiments. RESULTS: In vitro studies indicated greater than 95% transduction 96 hours after inoculation at an MOI of 3000 and a greater than 95% cell death rate with RSV-tk + GCV at an MOI of 61 or greater. In vivo experiments demonstrated an optimal viral dose of 3 x 10(8) plaque-forming units (pfu) and a greater than fourfold reduction in tumor growth for the animals treated with ADV/RSV-tk compared with control animals (P = 0.0013). Toxicity was limited to histologic evidence of hepatitis with ADV/RSV-tk doses greater than 3 x 10(8) pfu + GCV. Long-term survival of treatment animals was significantly increased over that of control animals (59%, P = 0.0001). CONCLUSIONS: ADV/RSV-tk with GCV treatment results in efficient gene transfer in vitro and provides effective therapy in experimental murine bladder cancer by significantly inhibiting tumor growth and improving host survival.

Androgen-regulated proliferation and gene transcription in a prostate smooth muscle cell line (PS-1).

Androgens play a key role in directing stromal-epithelial interactions in prostate gland development, in maintenance of adult phenotype, and in disease progression. To address the molecular mechanisms of androgen action in prostate stromal cells and the genes regulated by androgens in this cell type, a stromal cell line (PS-1) was developed from rat ventral prostate. The PS-1 cell line was adapted to chemically defined media and characterized as smooth muscle based on expression of desmin, smooth muscle alpha-actin, and nuclear androgen receptor, markers for prostate smooth muscle. To examine steroid hormone regulation, PS-1 cells were analyzed for response to a panel of steroid hormones in serum-free, chemically defined media. PS-1 cells were significantly growth stimulated by physiological concentrations of androgens (10nM) relative to other steroids tested. To ascertain whether this cell line could be used to examine androgen-regulated gene expression, differential display PCR was used to demonstrate the presence of androgen-regulated transcripts and provide the initial steps in cloning these genes. Replicate differential display PCR analyses showed consistent androgen stimulation of 16 messenger RNA species. Northern analysis confirmed androgen regulation of 6 species. Sequence analysis of each indicated no regions of significant homology or direct matches to existing sequences. Further study of the PS-1 stromal cell line and androgen-regulated genes identified here will provide a novel in vitro system for defining molecular mechanisms of androgen action in prostate stromal cells and the significance of stromal androgen-regulated genes to stromal-epithelial interactions.

Purification of a novel protein (ps20) from urogenital sinus mesenchymal cells with growth inhibitory properties in vitro.

Our previous studies have characterized mesenchyme-derived proteins to identify biologically active proteins and novel markers for stromal cell paracrine action relative to stromal-epithelial interactions. Previous reports have characterized properties of a growth inhibitory activity (to bladder and prostatic epithelial cells), secreted by U4F fetal rat urogenital sinus mesenchymal cells, not cross-reactive with antibodies to known cytokines, and provisionally termed UGIF. The present study reports the characterization, purification, and biological properties of a 20-21-kDa protein responsible for UGIF activity. The 20-21-kDa protein (termed ps20) was purified to near homogeneity, the amino-terminal sequence was determined, and biological properties were characterized in vitro. Amino-terminal sequence analysis indicated no direct matches or regions of homology with known proteins. Purified ps20 induced a linear and saturable inhibition of [3H]thymidine incorporation in PC-3 prostatic carcinoma cells (half-maximal activity at 2.6 nM), inhibited cell proliferation (increased population doubling time from 19.8 to 25.8 h), and induced a 210% stimulation in the synthesis of secreted proteins. These data suggest that ps20 may be a candidate paracrine effector protein and may play a role in stromal-epithelial cell interaction in the prostate gland.

Beta-2 microglobulin is mitogenic to PC-3 prostatic carcinoma cells and antagonistic to transforming growth factor beta 1 action.

Previous studies have identified a M(r) 12,000 protein in rat prostatic stromal cell-conditioned medium with growth stimulatory activity to human prostatic carcinoma cells as a direct match with beta 2-microglobulin (beta 2-m). The present study was conducted to characterize the activities of human beta 2-m directly, using commercially available, purified human beta 2-m. Beta 2-m was assayed for growth stimulatory activity to human PC-3 prostatic carcinoma cells and rat PS-1 prostatic stromal cells and for antagonistic activity to transforming growth factor beta 1 (TGF-beta 1)-induced growth inhibitory actions. Beta 2-m acted to stimulate [3H]thymidine incorporation in PC-3 cells in a linear, concentration-dependent and saturable manner in serum-free medium. Beta 2-m stimulated cell proliferation and significantly decreased population doubling times in both PC-3 and PS-1 cell lines. At half-maximal concentrations of TGF-beta 1 and lower, beta 2-m acted in a concentration-dependent, antagonistic manner, acting to stimulate growth-inhibited PC-3 cells to fully neutralize TGF-B1 activity. In contrast, cells exposed to maximum activity TGF-beta 1 concentrations were refractory to beta 2-m action, regardless of the concentration tested. This represents the first report to demonstrate a growth-stimulatory activity of B2-m with carcinoma/epithelial cells and to show beta 2-m antagonistic activity to TGF-B1 growth-induced inhibition. Beta 2-m has been shown previously to associate with hormone/growth factor receptors. Together, these data suggest that beta 2-m may play a role in modulating cell proliferation, possibly through modification of ligand/receptor kinetics. Owing to the elevation of both beta 2-m and TGF-beta 1 in many dysplastic-neoplastic conditions, beta 2-m may be relevant to mechanisms of abnormal proliferation disorders and in modulating TGF-beta 1 mechanisms of actions.

Glucocorticoid regulation of transforming growth factor-beta activation in urogenital sinus mesenchymal cells.

The transforming growth factors-beta (TGF-beta s) regulate many aspects of cell proliferation and differentiation. The TGF-beta isoforms are produced by most cell types in the biologically inactive, latent form. The physiological relevance of latent TGF-beta and the regulation of activation to the biologically active form are not well understood. Although expression of TGF-beta messenger RNA is regulated by the steroid hormone family, the mechanisms of hormonal regulation of TGF-beta activation have not been well studied. Fetal rat urogenital sinus organ cultures and derived mesenchymal cell lines (U4F, U4F1) have been established in order to analyze the expression of growth and differentiation regulatory factors which may function in mesenchymal induction of epithelial differentiation. The U4F1 cell line in chemically defined medium upon supplementation with dexamethasone (10 nM-1.4 microM), produced an activity which was growth inhibitory to PC-3 prostatic carcinoma epithelial cells. Analysis of physicochemical properties and purification of activity demonstrated a 25-kDa protein was responsible for activity. The activity cross-reacted to antisera specific for TGF-beta 1, beta 2 and for TGF-beta 2 exclusively, but not with antisera to rat interferon (alpha-beta) or rat interleukin 6. Acid treatment of control (unsupplemented) conditioned medium and cultures supplemented with other steroid hormones produced identical levels of activated TGF-beta as nonacid-treated conditioned medium from dexamethasone supplemented cultures which did not increase levels of activity upon acid activation. Activity from the acid-treated control conditioned medium was neutralized by TGF-beta antibodies. These data suggest latent TGF-beta is produced constitutively by U4F1 mesenchymal cultures in steroid unsupplemented medium and these cultures are induced by dexamethasone to activate identical levels of TGF-beta. These observations may be relevant to understanding diverse aspects of glucocorticoid regulation of tissue function and suggests that TGF-beta may be relevant to paracrine and autocrine growth regulation in the developing urogenital sinus.

Teicoplanin--home therapy for prosthetic joint infections.

Infection with methicillin-resistant organisms is increasingly common among implanted orthopaedic devices. The organisms involved are often multiresistant to other commonly used antibiotics. Rifampicin resistance is less common at isolation but may develop during treatment unless combination therapy with another drug is employed. However, tolerance of combinations is poor, particularly among elderly patients. These patients may require at least 6 weeks of antibiotic therapy, and where there is no suitable oral therapy, this has meant prolonged hospitalization solely for administration of parenteral antibiotics. Recently we have started treating such patients with teicoplanin, 6 mg/kg once daily, by intravenous bolus injection. The injections are administered by relatives at home and the patients attend the ward twice weekly for inspection and changing of the intravenous cannula. The potential for home administration of parenteral teicoplanin gives this drug a major advantage over other parenteral drugs for the treatment of prosthetic joint infections.

Characterization of a fetal urogenital sinus mesenchymal cell line U4F: secretion of a negative growth regulatory activity.

Mesenchymal cell lines derived from fetal rat urogenital sinus organ cultures have been characterized to establish an in vitro system for addressing growth and differentiation regulatory factors involved in mesenchymal-epithelial interactions during prostate morphogenesis. A continuous cell line was developed and designated U4F. Immunocytochemical analysis showed vimentin intermediate filament content confirming a mesenchymal origin. Previous studies with urogenital sinus organ cultures have reported the expression of a negative growth activity, which is stimulatory to protein synthesis and secretion and alters phenotypic morphology of NBT-II bladder epithelial cells. Subconfluent and confluent U4F monolayers did not produce this growth inhibitory activity. Foci of stacked cells were observed 3 wk postconfluency, which evolved into multicellular spheroids. The negative growth activity was expressed in the conditioned medium coordinate with spheroid formation. Transplanted spheroids continued to express the growth inhibitory activity. Morphologic analysis of spheroids showed a cellular capsule and a core of extracellular matrix. A continuous cell strain (U4F1) with altered phenotypic properties, arose spontaneously from long-term U4F cultures. The U4F1 cell strain did not form spheroids, yet expressed the negative growth activity constitutively in monolayer culture. Analyses of physicochemical, immunological, and biological properties showed the activity is identical in conditioned media from urogenital sinus organ cultures, U4F spheroids, and U4F1 monolayers. Based on the combined properties, this activity cannot be ascribed to previously characterized negative growth factors. The establishment of this mesenchymal cell culture system will aid in the further identification of paracrine-acting growth and differentiation regulatory factors secreted by fetal mesenchyme.

Monoclonal antibodies against the androgen receptor: recognition of human and other mammalian androgen receptors.

Monoclonal antibodies against the androgen receptor (AR) will provide useful probes for elucidating both the structure and function of this important regulatory protein. Recently, human autoimmune anti-AR sera have been described. The purpose of the current work was to immortalize lymphocytes from the blood of patients with high titer anti-AR antibodies and to produce monoclonal antibodies against the receptor in vitro. Human serum samples (10 microliters) were incubated in high ionic strength buffer (400 mM KCl) for 16 h at 0 C with [3H]Mibolerone-labeled cytosol (100-200 fmol AR) from Dunning tumors. Receptor-antibody complexes were precipitated with goat antihuman immunoglobulin (Ig) antibody. From our 1005 serum samples examined, 5 specimens were detected which precipitated greater than 40% of the AR. These antibodies recognized the AR from human, rat, mouse, dog, steer, chicken, and hamster, but did not recognize estrogen, progesterone, or glucocorticoid receptors. By sucrose gradient analysis in high salt (0.4 M KCl) 1 of the antisera shifted the 4.4S monomeric receptor to 8S, and the others shifted the receptor to 18S. However, all of the antibodies were shown to be IgG class by immunoprecipitation with class-specific second antibodies. Peripheral blood lymphocytes donated by these patients were isolated by histopaque density gradient sedimentation, activated in vitro, transformed with Epstein-Barr virus, and seeded into 96-well plates. From 263 million human lymphocytes plated in 96-well dishes, 1215 wells gave rise to Epstein-Barr virus-transformed lymphoblastoid cells, and 8 of these wells were determined to be anti-AR positive. Cells from 2 of the positive wells were cloned and designated CB54 and UA67, both of which secreted IgG class antibodies against the AR. These 2 monoclonal antibodies have been shown to be highly specific for the AR and to cross-react with the AR from human, rat, and hamster. Studies with the monomeric form of the AR and its proteolytic fragment using sucrose density gradients have suggested that the 2 antibodies recognize different epitopes on the monomeric AR molecule. Furthermore, by Western blot analysis the antibodies have identified the AR as an 118K protein on a sodium dodecyl sulfate gel, which is consistent with our previous findings of the mol wt of the AR.

Responses of NBT-II bladder carcinoma cells to conditioned medium from normal fetal urogenital sinus.

In vitro studies were conducted to determine whether conditioned medium from rat fetal urogenital sinus explants would affect phenotypic characteristics of NBT-II urinary bladder carcinoma cells in culture. NBT-II cells were exposed to medium (30%, v/v) conditioned for 48 h by intact urogenital sinus explants derived from 18-day fetal rats. Upon exposure for 23 h the [3H]thymidine incorporation by NBT-II cells was decreased by 40.3% relative to control cultures. This effect was paralleled by a similar decrease in proliferation. NBT-II cultures decreased in cell number by 32.1 and 45.8% on days 2 and 4, respectively, after exposure to conditioned medium. Although cell proliferation was inhibited, conditioned medium acted to induce an increase in protein secretion. An increase of 18.6% was observed in the incorporation of [35S]methionine into newly synthesized, secreted proteins by NBT-II cells exposed to conditioned medium for 23 h. Morphologically the NBT-II cells exposed to conditioned medium were larger, more spread out, and exhibited a greater array of lamellipodia and filopodia, although [35S]methionine incorporation into cellular proteins was decreased by 11.1%. These results suggest that diffusable factors produced by fetal urogenital sinus explants can induce changes in proliferation, protein synthesis, protein secretion, and phenotypic morphology of NBT-II carcinoma cells in culture.

A common molecular weight of the androgen receptor monomer in different target tissues.

Previously reported molecular weights for the monomeric steroid binding subunit of the androgen receptor protein have ranged from 25,000 to 167,000. The molecular weight appeared to vary among different species and target organs, as well as between different investigators. This study has examined androgen receptors from a diverse group of organs and species to determine whether these tissues share a common monomeric form. Gel filtration revealed peaks of specific [3H]dihydrotestosterone binding activity corresponding to Stokes radii of 54, 33, and 20 A in cytosols from several tissues. Phosphocellulose chromatography diminished the appearance of the smaller androgen receptor forms and facilitated the appearance of the larger 54-A form. Mixing experiments suggested that phosphocellulose was stabilizing the 54-A form by binding putative proteases which cleave this larger form. Methods were developed to generate homogenous preparations of a given androgen receptor size for comparative study. Sucrose density gradient analysis showed sedimentation coefficients of 4.5-5.0, 3.5-4.0, and 2.5-3.0 S, respectively. The corresponding calculated molecular weights were 109,000-121,000, 52,000-59,000, and 22,000-27,000. Scatchard analysis of each of these androgen receptor forms demonstrated very similar affinity for [3H]dihydrotestosterone (Kd approximately 1 nM), and each form possessed the ability to bind to DNA-cellulose. Extensively purified preparations of androgen receptor from R3327 tumor contained varying amounts of the three receptor forms even though molybdate and phosphocellulose were used to stabilize the androgen receptor protein during purification.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of an intermediate-sized androgen receptor: association with RNA.

This study identifies an intermediate-sized androgen receptor and characterizes its relationship with the 9.1S and 4.4S receptor forms. Under low ionic conditions, at 2-4 degrees C, there exists a 9.1S (+/- 0.17) (n = 30) oligomeric form which does not bind to DNA. Under high ionic conditions, this form dissociates to a 4.4S (+/- 0.08) (n = 18) monomeric form. When the salt concentration is lowered, the 4.4S monomer converts to a species with an intermediate sedimentation coefficient of 7.7S (+/- 0.15) (n = 17) which binds to DNA. Unlike the 9.1S oligomer the 7.7S form is not maintained by sodium molybdate under high ionic conditions but rather dissociates to the 4.4S monomer. To determine whether these forms were associated with RNA, the 7.7S form was incubated with RNase A and analyzed by density gradient centrifugation. The 7.7S form was digested fully by RNase to the 4.4S monomer. The 7.7S form demonstrated a buoyant density of 1.2459 +/- 0.014 g/cm3 (n = 6) in metrizamide gradients, suggesting a ribonucleoprotein component. The sedimentation coefficient of the 9.1S form was unaffected by RNase. These data suggest that the intermediate 7.7S receptor form is composed of 4.4S monomer associated with a ribonucleoprotein molecule(s).