NRP-152 Cells Research Articles

17-Beta-estradiol induces neoplastic transformation in prostatic epithelial cells

Although estrogens have been long implicated in the prostate carcinogenesis, direct evidence showing their carcinogenicity on prostatic epithelial cells has not yet been clearly demonstrated. In this study, we treated an immortalized, non-transformed and androgen-responsive rat prostatic epithelial cell line NRP-152 with 17β-estradiol (E 2) at concentrations 1–3 μM for period 2–6 weeks. After in vitro treatment, we evaluated the anchorage-independent growth of E 2-treated NRP-152 cells by soft agar assay and isolated the colonies formed by the transformed E 2-NRP-152 cells in soft agar for further growth phenotype characterization. Our results showed that the isolated E 2-NRP-152 clones displayed neoplastic transformation phenotype, as demonstrated by their capacity of forming colonies in soft agar and tumors in immunodeficient nude mice, while losing their spheroid formation capacity in Matrigel 3D-culture. Western blot and RT-PCR analyses showed that the transformed E 2-NRP-152 cells expressed increased levels of ERα and several putative prostate cancer stem cell markers (integrins α2β1, CD44, CD133, ABCG2 and CXCR4) but decreased levels of ERβ and AR. Comet assay revealed that E 2-treatment also induced formation of comet cells, indicating that E 2 caused DNA damage to the NRP-152 cells. Our present findings demonstrated that in vitro E 2 exposure could neoplastically transform the rat prostatic epithelial cells, indicating that E 2 is carcinogenic to the prostatic epithelial cells.

Critical Role of Smad2 in Tumor Suppression and Transforming Growth Factor-β–Induced Apoptosis of Prostate Epithelial Cells

Transforming growth factor-beta (TGF-beta) functions as a tumor suppressor of the prostate through mechanisms that remain unresolved. Although TGF-beta receptors directly activate both Smads 2 and 3, to date, Smad3 has been shown to be the essential mediator of most Smad-dependent TGF-beta responses, including control of gene expression, cell growth, apoptosis, and tumor suppression. Using a robust lentiviral short hairpin RNA system to silence Smads 2 and/or 3 in the NRP-152 nontumorigenic rat prostate basal epithelial cell line, we provide the first evidence for Smad2 as a critical mediator of TGF-beta-induced apoptosis and gene expression. Parallel analyses revealed that Smad3 is the major mediator of TGF-beta-induced transcriptional and apoptotic responses in the NRP-154 rat prostate carcinoma cell line. Remarkably, silencing Smad2 alone caused malignant transformation of NRP-152 cells, as assayed by s.c. tumor growth in athymic mice, whereas silencing Smad3 alone did not induce tumors. Nevertheless, tumors induced by silencing both Smads 2 and 3 were larger than those from silencing Smad2 alone. Given previous reports that NRP-152 cells have a stem cell phenotype, we speculate a critical role for Smad2 as a tumor suppressor in the basal epithelial or stem cell compartment of the prostate.

The amino-terminal peptide of Bax perturbs intracellular Ca2+ homeostasis to enhance apoptosis in prostate cancer cells

During apoptosis, proteolytic cleavage of Bax at the amino terminus generates a truncated Bax of approximately 18 kDa (p18Bax) and an amino-terminal peptide of approximately 3 kDa (p3Bax). Whereas extensive studies have shown that p18Bax behaves like a BH3 protein with enhanced pro-apoptotic function over that of the full-length Bax (p21Bax), little is known about the function of p3Bax in apoptosis. We have previously shown that Bax and Ca2+ play a synergistic role in amplifying apoptosis signaling and that store-operated Ca2+ entry (SOCE) contributes to Bax-mediated apoptosis in prostate cancer cells. Here we test whether p3Bax can contribute to regulation of Ca2+ signaling during apoptosis through use of a membrane-penetrating peptide to facilitate delivery of recombinant p3Bax into NRP-154 cells, a prostate epithelial cell line with tumorigenic capacity. We find that human immunodefficiency virus transactivator of transcription protein (TAT)-p3Bax fusion peptide can enhance thapsigargin-induced apoptosis in NRP-154 cells, elevate SOCE activity, and increase inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores. Our data indicates that p3Bax can modulate the entry of extracellular Ca2+ and thus regulate the amplification of apoptosis in prostate cancer cells.

Overexpression of bax induces down‐regulation of store‐operated calcium entry in prostate cancer cells

Store-operated Ca2+ channels control homeostasis between extracellular Ca2+ reservoir and intracellular Ca2+ storage and play important roles in apoptosis in a wide variety of cells, including prostate epithelia. Recent studies have shown that the acquired apoptosis-resistant nature of androgen-independent prostate cancer is associated with reduced function of store-operated Ca2+ entry (SOCE). This study investigates the functional interaction between Bax and SOCE in the apoptosis signaling cascade in prostate cancer. Our previous findings show that NRP-154, an androgen-independent prostate cancer cell line, could sustain overexpression of exogenous Bax without undergoing apoptosis. Here we show that sustained overexpression of Bax in NRP-154 cells leads to down-regulation of SOCE and reduced Ca2+ storage inside the endoplasmic reticulum. While reduced SOCE may represent an adaptive mechanism for cell survival, increased levels of Bax in the latent state enhances the sensitivity of NRP-154 cells to TGF-beta and thapsigargin-induced apoptosis. This enhanced apoptosis can be reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of SOCE, or reversed under conditions where SOCE is only partially activated. Our results demonstrate a functional interaction between Bax and SOCE in apoptosis of prostate cancer, and support the concept that improving this interaction has therapeutic implications for prostate cancer.

FLICE-Like Inhibitory Protein Blocks Transforming Growth Factor β1–Induced Caspase Activation and Apoptosis in Prostate Epithelial Cells

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal-induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor beta1 (TGFbeta1), mimicking androgen withdrawal-induced apoptosis. FLIP levels decline with TGFbeta1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFbeta1-induced apoptosis. Small interfering RNA-mediated knockdown of FLIP recapitulates and enhances TGFbeta1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFbeta1-induced apoptosis. TGFbeta1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFbeta-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFbeta1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFbeta stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis.

Epigallocatechin‐3‐gallate and bicalutamide cause growth arrest and apoptosis in NRP‐152 and NRP‐154 prostate epithelial cells

A number of epidemiological studies have suggested that consumption of green tea reduces the risk of prostate cancer. The aim of this study was to elucidate the effects of epigallocatechin-3-gallate (EGCG), one of the major constituents of green tea, on growth inhibition and apoptosis in prostate epithelial cell lines with and without bicalutamide. The effects of EGCG and bicalutamide alone and in combination were examined on NRP-152 and NRP-154 cells derived from the dorso-lateral prostate of the Lobund-Wistar rat. Following treatments, cell number and levels of apoptosis were assessed. After treatment with EGCG, both cell lines displayed a dose-dependent decrease in cell number; this effect was more pronounced in NRP-154 cells. This decrease in cell number was caused by growth arrest in NRP-152 cells and apoptosis in NRP-154 cells. The apoptotic events in the NRP-154 cells were concurrent with a loss of manganese superoxide dismutase expression. Androgen ablation was achieved by androgen withdrawal using charcoal stripped serum or treatment with bicalutamide. Bicalutamide decreased cell number and induced apoptosis in a dose-dependent manner in both cell lines; however, androgen withdrawal did not. There was a loss of androgen receptor expression in NRP-152 cells with bicalutamide treatment. However, as the NRP-154 cells are androgen receptor negative, the loss in cell number and increased apoptotic events in these cells cannot be attributed to the anti-androgenic activity of bicalutamide. Cells treated with a combination of bicalutamide and EGCG also demonstrated a dose-dependent decrease in cell number that was significantly greater than bicalutamide alone. This study demonstrates the potential use of EGCG and other antioxidants as therapeutic candidates for prostate cancer.

Tumor Evasion of the Immune System by Converting CD4+CD25− T Cells into CD4+CD25+ T Regulatory Cells: Role of Tumor-Derived TGF-β

CD4+CD25+ T regulatory (T(reg)) cells were initially described for their ability to suppress autoimmune diseases in animal models. An emerging interest is the potential role of T(reg) cells in cancer development and progression because they have been shown to suppress antitumor immunity. In this study, CD4+CD25- T cells cultured in conditioned medium (CM) derived from tumor cells, RENCA or TRAMP-C2, possess similar characteristics as those of naturally occurring T(reg) cells, including expression of Foxp3, a crucial transcription factor of T(reg) cells, production of low levels of IL-2, high levels of IL-10 and TGF-beta, and the ability to suppress CD4+CD25- T cell proliferation. Further investigation revealed a critical role of tumor-derived TGF-beta in converting CD4+CD25- T cells into T(reg) cells because a neutralizing Ab against TGF-beta, 1D11, completely abrogated the induction of T(reg) cells. CM from a nontumorigenic cell line, NRP-152, or irradiated tumor cells did not convert CD4+CD25- T cells to T(reg) cells because they produce low levels of TGF-beta in CM. Finally, we observed a reduced tumor burden in animals receiving 1D11. The reduction in tumor burden correlated with a decrease in tumor-derived TGF-beta. Treatment of 1D11 also reduced the conversion of CD4+ T cells into T(reg) cells and subsequent T(reg) cell-mediated suppression of antitumor immunity. In summary, we have demonstrated that tumor cells directly convert CD4+CD25- T cells to T(reg) cells through production of high levels of TGF-beta, suggesting a possible mechanism through which tumor cells evade the immune system.

The Presenilin-2 Loop Peptide Perturbs Intracellular Ca2+ Homeostasis and Accelerates Apoptosis

In cells undergoing apoptosis, a 22-amino-acid presenilin-2-loop peptide (PS2-LP, amino acids 308-329 in presenilin-2) is generated through cleavage of the carboxyl-terminal fragment of presenilin-2 by caspase-3. The impact of PS2-LP on the progression of apoptosis, however, is not known. Here we show that PS2-LP is a potent inducer of the mitochondrial-dependent cell death pathway when transduced as a fusion protein with HIV-TAT. Biochemical and functional studies demonstrate that TAT-PS2-LP can interact with the inositol 1,4,5-trisphosphate receptor and activate Ca(2+) release from the endoplasmic reticulum. These results indicate that PS2-LP-mediated alteration of intracellular Ca(2+) homeostasis may be linked to the acceleration of apoptosis. Therefore, targeting the function of PS2-LP could provide a useful therapeutic tool for the treatment of cancer and degenerative diseases.

Apoptosis Induction and Clusterin Expression of NRP-152 Cells by Tamsulosin

Apoptosis Induction and Clusterin Expression of NRP-152 Cells by Tamsulosin

Overexpression of Bax sensitizes prostate cancer cells to TGF-β induced apoptosis

NRP-154 is a tumorigenic epithelial cell line derived from the preneoplastic dorsal-lateral prostate of rats. These cells are exquisitely sensitive to TGF-beta induced apoptosis. In contrast, we find that NRP-154 cells can sustain overexpression of exogenous Bax protein, which is different from non-tumor cells where Bax functions as a ubiquitous stimulator of apoptosis. NRP-154 cells stably overexpressing Bax show increased sensitivity to TGF-beta induced apoptosis. The degree of TGF-beta induced apoptosis displays high correlation with cleavage of Bax at the amino-terminus. Our data indicate that prostate cancer cells can host high levels of latent Bax which can be activated through post-translational modification.

Signal transducer and activator of transcription 3 (STAT3) activation in prostate cancer: Direct STAT3 inhibition induces apoptosis in prostate cancer lines

Signal transducers and activators of transcription (STAT) were originally discovered as components of cytokine signal transduction pathways. Persistent activation of one STAT, STAT3, is a common feature of prostate cancer. Activated STAT3 was found in pathology specimens obtained from prostatectomy in the cancerous areas but not in the normal margins. Because the activation of STAT3 is mediated by the action of an upstream Janus kinase (JAK) kinase, usually JAK1 or JAK2, the activation step for STAT3 might itself be a target for therapy in prostate cancer. However, the redundancy of upstream kinases may make this strategy unreliable for therapy. To develop molecular targets for prostate cancer treatment, JAK kinase and STAT3 inhibition of two prostate cancer lines were compared. DU145 and NRP-154 cells were treated with JAK kinase inhibitors, analyzed for onset of apoptosis, and measured by annexin V binding and propidium iodide uptake. Activation of caspases in the cells was determined by measuring cleaved caspase-3 following treatment. For determining the effect on mitochondrial membrane depolarization that accompanies apoptosis, the fluorescent dye JC-1 was used. STAT3 was specifically inhibited by transfecting either a dominant-negative (DN) STAT3 plasmid or antisense STAT3 oligonucleotides into the cells. To look for reduction in STAT3 levels within cells, fixed and permeabilized prostate cancer cells were stained with antibody to STAT3. We found that more than one JAK kinase is involved in STAT3 activation in prostate cancer lines. AG490 (JAK2 specific) induced apoptosis in DU145 but not in NRP-154 prostate cancer lines, whereas piceatannol (JAK1 specific) induced apoptosis in NRP-154 but not in DU145 cells. Next, we demonstrated efficacy of specific STAT3 inhibitors in prostate cancer lines. Both induction of apoptosis and reduction in intracellular STAT3 protein were observed following treatment with antisense STAT3 oligonucleotides, while transfection of a DN-STAT3 plasmid into both prostate cancer cell lines resulted in loss of viability and onset of apoptosis. We conclude that STAT3-specific inhibitors, rather than JAK kinase-specific inhibitors, should be more useful therapeutically in treating androgen-resistant prostate cancer and that STAT3 is an appropriate target in the treatment of prostate cancer.

Regulation of trespin expression by modulators of cell growth, differentiation, and apoptosis in prostatic epithelial cells

We recently identified a novel rat ov-serpin, Trespin, which inhibits the trypsin-like serine proteinase plasmin and is expressed in several tissues, including prostate. In this report Trespin expression was studied in prostatic cell lines, NRP-152, NRP-154, and DP-153, derived from the Lobund-Wistar rat. Northern blots revealed Trespin mRNA is expressed in NRP-152 and DP-153 basal epithelial cell lines but not in the luminal line, NRP-154. Similarly, Trespin levels drop >30-fold following transdifferentiation of NRP-152 cells toward a luminal variant, further suggesting Trespin expression is specific for basal prostatic epithelial cells. Trespin expression in NRP-152 cells is up-regulated by dexamethasone (Dex) and insulin-like growth factor-I (IGF-I), each of which stimulate growth and prevent differentiation and apoptosis. However, Dex (alone) facilitates loss of Trespin by TGF-β, yet enhances the ability of LR 3-IGF-I to reverse such loss, similar to the pattern of apoptosis induced by TGF-β. Likewise, several apoptosis inducers markedly decrease Trespin mRNA levels. HEK293 cells stably overexpressing Trespin display increased cell proliferation and partial resistance to growth inhibition and phosphorylation of c-Jun induced by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA). Together these data strongly suggest that Trespin has critical functions tied to the regulation of growth, differentiation, and apoptosis of prostatic epithelial cells.

IL-6 signaling by STAT3 participates in the change from hyperplasia to neoplasia in NRP-152 and NRP-154 rat prostatic epithelial cells.

BackgroundSTAT3 phosphorylation is associated with the neoplastic state in many types of cancer, including prostate cancer. We investigated the role of IL-6 signaling and phosphorylation of STAT3 in 2 rat prostatic epithelial lines. NRP-152 and NRP-154 cells were derived from the same rat prostate, yet the NRP-152 cells are not tumorigenic while the NRP-154 cells are tumorigenic. These lines are believed to represent 2 of the stages in the development of prostate cancer, hyperplasia and neoplasia. Differences in signaling pathways should play a role in the 2 phenotypes, hyperplastic and neoplastic.MethodsWe looked at the phosphorylation state of STAT3 by intracellular flow cytometry, using phospho-specific antibodies to STAT3. We used the same method to examine IL-6 production by the cell lines. We also measured apoptosis by binding of fluorescent annexin V to the cells.ResultsAlthough both cells lines made IL-6 constitutively, phosphorylated-STAT3 was present in untreated NRP-154 cells, but not in NRP-152 cells. Treatment with dexamethasone inhibited the IL-6 production of NRP-152 cells, but enhanced that of NRP-154 cells. Treatment with the JAK2 inhibitor AG490 induced apoptosis in NRP-152, but not NRP-154 cells.ConclusionsWe conclude from these experiments that STAT3 activity plays a role in the phenotype of NRP-154 cell, but not NRP-152 cells. The significance of alternative IL-6 signaling pathways in the different phenotypes of the 2 cell lines is discussed.

Bcl-xL Blocks Transforming Growth Factor-β1-induced Apoptosis by Inhibiting Cytochrome c Release and Not by Directly Antagonizing Apaf-1-dependent Caspase Activation in Prostate Epithelial Cells

The mechanism by which transforming growth factor-beta1 (TGF-beta1) induces apoptosis of prostate epithelial cells was studied in the NRP-154 rat prostate epithelial cell line. TGF-beta 1 down-regulates expression of Bcl-xL and poly(ADP-ribosyl)polymerase (PARP), promotes cytochrome c release, up-regulates expression of latent caspase-3, and activates caspases 3 and 9. We tested the role of Bcl-xL in this cascade by stably overexpressing Bcl-xL to prevent loss by TGF-beta 1. Clones overexpressing Bcl-xL are resistant to TGF-beta 1 with respect to induction of apoptosis, cytochrome c release, activation of caspases 9 and 3, and cleavage of PARP; yet they remain sensitive to TGF-beta 1 by cell cycle arrest, induction of both fibronectin and latent caspase-3 expression, and loss of PARP expression. We show that Bcl-xL associates with Apaf-1 in NRP-154 cells; but this association does not inhibit the activation of caspases 9 and 3 by cytochrome c. Together, our data suggest that TGF-beta1 induces apoptosis through loss of Bcl-xL, leading to cytochrome c release and the subsequent activation of caspases 9 and 3. Moreover, our data demonstrate that the antiapoptotic effect of Bcl-xL occurs by inhibition of mitochondrial cytochrome c release and not through antagonizing Apaf-1-dependent processing of caspases 9 and 3.

Retinoid and androgen regulation of cell growth, epidermal growth factor and retinoic acid receptors in normal and carcinoma rat prostate cells

Recent in vivo and in vitro studies suggest that retinoic acid receptor (RAR)-mediated processes may be involved in androgen regulation of prostate cells in a manner that may be altered during prostatic carcinogenesis. We tested this hypothesis in the newly established carcinoma and non-carcinoma rat prostate epithelial cell lines, NRP-154 and NRP-152, respectively. In DMEM/F-12 medium supplemented with 10% charcoal stripped fetal calf serum (cFCS), the number of both NRP-152 and NRP-154 cells were stimulated by testosterone (T), with a 4-fold greater effect in NRP-152 than in NRP-154 cells. Retinoic acid (RA) alone also stimulated the growth of NRP-152 cells, but failed to induce cell growth of NRP-154 cells. Importantly, the level of RAR α mRNA was elevated whereas the levels of RAR γ and androgen receptor (AR) mRNA were lower in NRP-154 cells compared to those in NRP-152 cells. Treatment of NRP-152 cells with increasing doses of T resulted in a dose-dependent decrease and rebound of the level of RAR α and γ mRNA in NRP-152 cells; these effects were not apparent, if not reversed, in NRP-154 cells. Both ligand binding and Western blot analyses revealed that epidermal growth factor receptor (EGF-R) was stimulated by 20 nM T but was suppressed by 0.1 μM RA, which also attenuated the stimulating effects of T on EGF-R in NRP-152 and to a lesser extent in NRP-154 cells. The differences in the level and androgen regulation of RAR mRNAs and reciprocal regulation of EGF-R expression by T and RA between NRP-154 and NRP-152 cells suggest that variations in the EGF-R and RAR signal events may contribute to differences in growth rate between these two cell lines.

The rat prostatic epithelial cell line NRP‐152 can differentiate in vivo in response to its stromal environment

BACKGROUND The clonally derived rat prostatic epithelial cell line NRP-152 was examined to determine its ability to differentiate in a tissue recombination model. METHODS NRP-152 cells alone, or combined with urogenital mesenchyme (UGM) or 10T1/2 fibroblasts, were grafted beneath the renal capsule of athymic rodent hosts. After 1 and 3 months, grafts were examined grossly and immunohistochemically. RESULTS NRP-152 cells grafted alone formed small (10–25 mg) grafts without recognizable architecture. NRP-152 cells recombined with UGM formed larger grafts (50–100 mg after 28 days) containing glandular epithelium. Columnar luminal cells expressed cytokeratins 8 and 18 and rat prostatic secretory markers (DP-1 and DP-2). The epithelial ducts were surrounded by well-differentiated smooth muscle. The glandular epithelial cells were shown to be of rat origin. NRP-152 + 10T1/2 tissue recombinants formed small grafts (10–40 mg wet weight) after 1 month. The epithelial component of these grafts formed solid unbranched cords expressing cytokeratins 5 and 14; no glandular epithelial structures were observed. The stromal matrix was densely packed with a few cells expressing α-actin. CONCLUSIONS A clonally derived prostatic epithelial cell line can form structurally and functionally normal prostatic tissue. This suggests that prostatic basal and luminal epithelial cells can be derived from a common progenitor. Prostate 39:205–212, 1999. © 1999 Wiley-Liss, Inc.

The rat prostatic epithelial cell line NRP-152 can differentiate in vivo in response to its stromal environment.

The clonally derived rat prostatic epithelial cell line NRP-152 was examined to determine its ability to differentiate in a tissue recombination model. NRP-152 cells alone, or combined with urogenital mesenchyme (UGM) or 10T1/2 fibroblasts, were grafted beneath the renal capsule of athymic rodent hosts. After 1 and 3 months, grafts were examined grossly and immunohistochemically. NRP-152 cells grafted alone formed small (10-25 mg) grafts without recognizable architecture. NRP-152 cells recombined with UGM formed larger grafts (50-100 mg after 28 days) containing glandular epithelium. Columnar luminal cells expressed cytokeratins 8 and 18 and rat prostatic secretory markers (DP-1 and DP-2). The epithelial ducts were surrounded by well-differentiated smooth muscle. The glandular epithelial cells were shown to be of rat origin. NRP-152 + 10T1/2 tissue recombinants formed small grafts (10-40 mg wet weight) after 1 month. The epithelial component of these grafts formed solid unbranched cords expressing cytokeratins 5 and 14; no glandular epithelial structures were observed. The stromal matrix was densely packed with a few cells expressing alpha-actin. A clonally derived prostatic epithelial cell line can form structurally and functionally normal prostatic tissue. This suggests that prostatic basal and luminal epithelial cells can be derived from a common progenitor.

Transdifferentiation of NRP-152 rat prostatic basal epithelial cells toward a luminal phenotype: regulation by glucocorticoid, insulin-like growth factor-I and transforming growth factor-beta.

The role of basal epithelial cells in prostatic function, development and carcinogenesis is unknown. The ability of basal prostatic epithelial cells to acquire a luminal phenotype was explored in vitro using the NRP-152 rat dorsal-lateral prostate epithelial cell line as a model system. NRP-152, which was spontaneously immortalized and clonally derived, is an androgen-responsive and nontumorigenic cell line that has a basal cell phenotype under normal growth conditions. However, when placed in mitogen-deficient media, these cells undergo a dramatic morphological change to a luminal phenotype. Under these growth-restrictive conditions, immunocytochemical analysis shows that NRP-152 cells acquire the luminal markers Z0-1 (a tight-junction associated protein), occludin (integral tight-junction protein), and cytokeratin 18, and lose the basal markers cytokeratins 5 and 14. Total protein and mRNA levels of cytokeratins 8, 18, c-CAM 105 (the calcium-independent cell adhesion molecule) and Z0-1, as detected by western and/or northern blot analyses, respectively, are induced, while cytokeratin 5 and 15 are lost, and occludin is unchanged. Concomitant with this differentiation, expression of transforming growth factor-beta2 (TGF-beta2), TGF-beta3, and TGF-beta receptor type II (TbetaRII) is induced, while those of TGF-beta1 and TbetaRI remain essentially unchanged. Mitogens, such as insulin-like growth factor-I and dexamethasone inhibit luminal differentiation, while exogenous TGF-beta induces such differentiation. These data together with TGF-beta neutralization experiments using pan-specific antibody implicate an important role for autocrine TGF-beta in the induction of the luminal differentiation.

Identification of the start sites for the 1.9- and 1.4-kb rat transforming growth factor-beta1 transcripts and their effect on translational efficiency.

Three distinct transforming growth factor-beta1 (TGF-beta1) transcripts of 2.5, 1.9 and 1.4kb have been described, but the start sites and functional significance of the shorter transcripts are unknown. Here, we have cloned and sequenced a rat genomic fragment encoding approximately 1250 bases upstream of the start of the TGF-beta1 open reading frame. Using a combination of ribonuclease protection and 5' RACE-PCR analysis, we have mapped the start sites for the two shorter TGF-beta1 transcripts in NRP152 cells, a rat prostatic epithelial cell line that expresses all three transcripts at high levels. The 1.4-kb mRNA starts 25 bases upstream of the initiator AUG, whereas the 1.9-kb mRNA has two start sites 366 and 401 bases upstream of the AUG. Polysome analysis of the NRP152 cells indicates that the 1.9-kb transcript is very efficiently translated, whereas the 2.5- and 1.4-kb transcripts appear to be poorly translated. Differential regulation of TGF-beta1 transcript size may therefore represent an important mechanism for regulating TGF-beta1 protein levels.

The role of transforming growth factor-beta1, -beta2, and -beta3 in androgen-responsive growth of NRP-152 rat prostatic epithelial cells.

We have investigated the role of autocrine/paracrine TGF-beta secretion in the regulation of cell growth by androgens as demonstrated by its inhibition by two androgen response modifiers; the nonsteroidal antiandrogen hydroxyflutamide (OHF), believed to act by inhibiting androgen binding to androgen receptors, or finasteride, an inhibitor of 5alpha-reductase, the enzyme necessary for the conversion of testosterone to 5alpha-dihydrotestosterone (DHT), using the nontumorigenic rat prostatic epithelial cell line NRP-152. Growth of these cells was stimulated three- to sixfold over control by either testosterone or DHT under serum-free culture conditions. This was accompanied by a two- to threefold decrease in the secretion rate of TGF-beta1, -beta2, and -beta3. Finasteride reversed the ability of testosterone but not DHT to stimulate growth and downregulate expression of TGF-beta1, -beta2, and -beta3 in a dose-dependent fashion, suggesting that this activity of testosterone required its conversion to DHT. OHF antagonized the stimulatory effects of DHT on NRP-152 cell growth but could reverse the inhibitory effects of DHT only on TGF-beta2 and TGF-beta3 and not TGF-beta1 secretion. This suggests that either TGF-beta1 regulation by DHT or the androgen antagonism of OHF occurs independent of androgen receptor binding. Neutralizing antibodies to TGF-beta (pantropic and isoform-specific) were able to block the ability of finasteride to antagonize the effects of testosterone nearly completely while only partially inhibiting the antiandrogenic effects of OHF. Thus, the ability of androgens to stimulate growth of NRP-152 cells involves the downregulation of the production of TGF-beta1, -beta2, and -beta3 in addition to other growth-stimulatory mechanisms.