RESUMEN
OBJECTIVES: To elucidate the mechanism of transforming growth factor (TGF)-ß1 overexpression in prostate cancer cells. METHODS: Malignant (PC3, DU145) and benign (RWPE1, BPH1) prostate epithelial cells were used. Phosphatase activity was measured using a commercial kit. Recruitment of the regulatory subunit, Bα, of protein phosphatase 2A (PP2A-Bα) by TGF-ß type I receptor (TßRI) was monitored by coimmunoprecipitation. Blockade of TGF-ß1 signaling in cells was accomplished either by using TGF-ß-neutralizing monoclonal antibody or by transduction of a dominant negative TGF-ß type II receptor retroviral vector. RESULTS: Basal levels of TGF-ß1 in malignant cells were significantly higher than those in benign cells. Blockade of TGF-ß signaling resulted in a significant decrease in TGF-ß1 expression in malignant cells, but not in benign cells. Upon TGF-ß1 treatment (10 ng/mL), TGF-ß1 expression was increased in malignant cells, but not in benign cells. This differential TGF-ß1 auto-induction between benign and malignant cells correlated with differential activation of extracellular signal-regulated kinase (ERK). Following TGF-ß1 treatment, the activity of serine/threonine phosphatase and recruitment of PP2A-Bα by TßRI increased in benign cells, but not in malignant cells. Inhibition of PP2A in benign cells resulted in an increase in ERK activation and in TGF-ß1 auto-induction after TGF-ß1 (10 ng/mL) treatment. CONCLUSIONS: These results suggest that TGF-ß1 overexpression in malignant cells is caused, at least in part, by a runaway of TGF-ß1 auto-induction through ERK activation because of a defective recruitment of PP2A-Bα by TßRI.
Asunto(s)
Adenocarcinoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Adenocarcinoma/patología , Comunicación Autocrina , Línea Celular Tumoral/metabolismo , Activación Enzimática , Humanos , Masculino , Fosforilación , Próstata/metabolismo , Neoplasias de la Próstata/patología , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
BACKGROUND: Stromal smooth muscle cells (SMCs) play an important role in the pathogenesis and clinical symptom of benign prostatic hyperplasia. We had reported that estrogen enhances the phenotype of SMC in cultured prostate stromal cells (PRSCs). Here we further investigate the mechanism by which estrogen affects the differentiation of PRSCs. METHODS: Primary cultured PRSCs were stimulated with E2 or BSA-E2. The mRNA level of SMC-specific genes, smoothelin, and SM-MHC were measured by qRT-PCR. The SM-MHC protein was measured by Western blot. The mRNA and protein levels of TGF-beta1 were measured by qRT-PCR and ELISA. The MAPK inhibitor PD98059, the estrogen receptor antagonist ICI182,780 and neutralizing antibody to TGF-beta1 were used to reveal the mechanism of estrogen effect. RESULTS: E2 and BSA-E2 significantly up-regulate the expression of SMC-specific genes in PRSCs. Both forms of estrogen could increase the expression of TGF-beta1, which can be blocked by pre-treating with PD98059. Moreover, PD98059 and TGF-beta1 neutralizing antibody could abrogate the effect of BSA-E2 on cell differentiation. However, they could only inhibit part of E2-induced SMC phenotype enhancement. ICI182,780 could partially suppress the pro-differentiation effect of E2 but had no influence on the effect of BSA-E2. Combined treatment with ICI182,780 and PD98059 can completely abrogate the effect of E2. CONCLUSIONS: Estrogen could promote the expression of TGF-beta1 in PRSCs through nongenomic activation of MAPK pathway, and in turn enhance the SMC phenotype. Besides for this nongenomic effect, estrogen can also enhance the SMC phenotype through classical genomic action.
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Estradiol/farmacología , Genoma Humano/efectos de los fármacos , Miocitos del Músculo Liso/citología , Próstata/citología , Células del Estroma/citología , Anticuerpos Neutralizantes/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Sinergismo Farmacológico , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Flavonoides/farmacología , Fulvestrant , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Miocitos del Músculo Liso/metabolismo , Fenotipo , Albúmina Sérica Bovina/farmacología , Factor de Crecimiento Transformador beta1/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The aim of this study was to identify differential responses to low concentrations of 17beta-estradiol (E2) in primary stromal cell cultures derived from either normal organ donors or benign prostatic hyperplasia or hypertrophy (BPH) specimens. Furthermore, we sought to identify the potential mechanism of E2 action in these cell types, through either a genomic or nongenomic mechanism. We initially treated stromal cells derived from five normal prostates or five BPH specimens with low concentrations of E2 (0.001-1.0 nM) and analyzed their growth response. To determine whether genomic or nongenomic pathways were involved, we performed studies using specific estrogen receptor antagonists to confirm transcriptional activity or MAPK inhibitors to confirm the involvement of rapid signaling. Results of these studies revealed a fundamental difference in the mechanism of the response to E2. In normal cells, we found that a nongenomic, rapid E2 signaling pathway is predominantly involved, mediated by G protein-coupled receptor-30 and the subsequent activation of ERK1/2. In BPH-derived prostate stromal cells, a genomic pathway is predominantly involved because the addition of ICI 182780 was sufficient to abrogate any estrogenic effects. In conclusion, prostate stromal cells respond to far lower concentrations of E2 than previously recognized or examined, and this response is mediated through two distinct mechanisms, depending on its origin. This may provide the basis for new insights into the causes of, and possible treatments for, BPH.
Asunto(s)
Estradiol/metabolismo , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Adolescente , Adulto , Anciano , Células Cultivadas , Receptor alfa de Estrógeno/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Próstata/citología , Receptores de Estrógenos , Receptores Acoplados a Proteínas G , Transducción de Señal , Células del Estroma/metabolismoRESUMEN
It is known that human benign prostatic hyperplasia might arise from an estrogen/androgen (E/T) imbalance. We studied the response of castrated rat prostate to different ratios of circulating E/T. The castrated male Wistar rats were randomly injected with E/T at different ratios for 4 weeks. The prostates of E/T (1:100) group showed a distinct prostatic hyperplasia response by prostatic index, hematoxylin and eosin staining, and quantitative immunohistochemical analysis of alpha-smooth muscle actin (SMA). In this group, cells positive for Vimentin, non-muscle myosin heavy chain (NMMHC) and proliferating cell nuclear antigen (PCNA) increased in the stroma and epithelium. Furthermore, the mRNA levels of smooth muscle myosin heavy chain (SMMHC) and NMMHC increased. So E/T at a ratio of 1:100 can induce a stromal hyperplastic response in the prostate of castrated rats. The main change observed was an increase of smooth muscle cells, whereas some epithelial changes were also seen in the rat prostates.
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Proliferación Celular , Estradiol/sangre , Orquiectomía , Células del Estroma/citología , Testosterona/sangre , Animales , Inmunohistoquímica , Masculino , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ratas , Ratas WistarRESUMEN
OBJECTIVES: The mouse renal cell carcinoma line, Renca, is insensitive to transforming growth factor-beta (TGF-beta) in vitro. The present study was conducted to determine whether removal of TGF-beta from these tumor cells would inhibit tumor progression in vivo. METHODS: TGF-beta elimination was accomplished either by administration of neutralizing TGF-beta antibody into mice receiving intravenous injection of Renca tumor cells or infection of TGF-beta antisense expression vector into these tumor cells before subcutaneous injection into recipient mice. RESULTS: Although a low dose of TGF-beta antibody (5 mg/kg every 3 days) was without any effect, a high dose of TGF-beta antibody (50 mg/kg every 3 days), administered to recipient mice, resulted in a significant reduction in lung metastasis and was accompanied by increased apoptosis in the tumor cells. When the tumor cells were transfected with a TGF-beta1 antisense expressing vector, a significant reduction occurred in the tumor incidence, as well as the tumor burden. However, in nude mice, cells with reduced TGF-beta1 production grew almost as well as did the unmodified Renca cells, suggesting that the host's immune system might play an antitumor role. CONCLUSIONS: These results indicate that progression of Renca tumor can be inhibited by eliminating TGF-beta from the tumor cells. Our results also suggest that, although insensitive to TGF-beta under in vitro conditions, Renca tumors could be inhibited by TGF-beta removal through the systemic host environment.
Asunto(s)
Carcinoma de Células Renales/terapia , Neoplasias Renales/terapia , Factor de Crecimiento Transformador beta/fisiología , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/secundario , Línea Celular Tumoral , Proliferación Celular , Técnicas In Vitro , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Transfección , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
BACKGROUND: Estrogen is involved in the development and progression of benign prostatic hyperplasia (BPH). It can stimulate proliferation of prostate stromal cells (PrSCs). However, the exact mechanism remains unclear. METHODS: We used the primary cultured human PrSCs and a prostate stromal cell line, WPMY-1, to examine the signaling pathways involved in estrogen-mediated proliferation of PrSCs. Cells were treated with 17beta-estradiol (E(2)) or BSA-E(2). Cell proliferation was assessed by the MTT assay and by cell counting. Western blot analysis was used to determine the status of activation of ERK1/2. RESULTS: Results indicated that both E(2) and BSA-E(2) stimulated proliferation of primary PrSCs and WPMY-1 cells. ERK was rapidly activated by E(2) and BSA-E(2). PD98059, which is a selective ERK inhibitor, significantly inhibited estrogen-induced cell proliferation. PrSCs expressed estrogen receptor alpha (ERalpha) and GPR30 but not ERbeta. Small hairpin RNA (shRNA) to ERalpha, but not to GPR30, blocked estrogen-mediated ERK activation and cell proliferation. CONCLUSIONS: The results indicated that estrogen could activate ERK pathway through the non-genomic ERalpha pathway, leading to proliferation of PrSCs.
Asunto(s)
Proliferación Celular , Estradiol/fisiología , Estrógenos/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Próstata/citología , Próstata/metabolismo , Células Cultivadas , Receptor alfa de Estrógeno/antagonistas & inhibidores , Flavonoides/farmacología , Péptido Liberador de Gastrina/antagonistas & inhibidores , Humanos , Masculino , ARN/farmacología , Receptores de Estrógenos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
OBJECTIVE: To characterize a paracrine effect of prostatic epithelial cells in the presence or absence of oestradiol on the differentiation and proliferation of prostatic stromal cells. MATERIALS AND METHODS: Conditioned media (CM) collected from a prostatic epithelial cell line (BPH-1), which was pre-treated with different concentration of oestradiol, were added to cultures of primary prostatic stromal cells. The proliferation rates of stromal cells were determined using a tetrazolium assay. The mRNA level was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR), and the protein level of smooth muscle myosin heavy chain (SM-MHC), fibronectin and collagen IV were determined with Western blotting, enzyme- linked immunosorbent assay and radioimmunoassay, respectively. The expression of transforming growth factor beta1 (TGF beta 1) in the BPH-1 cell line was analysed. RESULTS: The rate of proliferation of stromal cells increased when they were cultured with CM harvested from oestradiol-treated BPH-1 cells, but there was no remarkable change when they were cultured with CM from untreated cells. The level of smoothelin mRNA and SM-MHC protein increased after treatment with CM from BPH-1. The CM from BPH-1 with oestradiol stimulation was more effective in stimulating smoothelin mRNA and SM-MHC protein level. The protein level of collagen type IV, but not fibronectin, was up-regulated in the supernatants and cell extracts of CM-treated stromal cells. Oestradiol enhanced the expression and secretion of TGF beta 1 in BPH-1 cells. TGF beta 1-neutralizing antibody abrogated the effect of BPH-1 CM on the synthesis of collagen IV and SM-MHC in stromal cells. CONCLUSION: These results suggest that oestradiol-stimulated proliferation and differentiation of prostatic stromal cells could be regulated by factors secreted from prostatic epithelial cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Estradiol/farmacología , Próstata/patología , Hiperplasia Prostática/patología , Factor de Crecimiento Transformador beta1/metabolismo , Western Blotting , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Matriz Extracelular/efectos de los fármacos , Humanos , Masculino , Prostatectomía , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Regulación hacia ArribaRESUMEN
Estradiol (E2) level in stroma of benign prostatic hyperplasia (BPH) increases with age, and this increase was associated with an elevated expression of aromatase in prostatic stromal cells (PrSCs). Here, we showed that conditioned medium (CM) of BPH-1 (a benign hyperplastic prostatic epithelial cell line), but not of prostate cancer cell lines (LNCaP, DU-145, and PC-3), stimulates aromatase expression in PrSCs. Cyclooxygenase-2 (COX-2) mRNA level in BPH-1, as well as prostaglandin E2 (PGE2) concentration in BPH-1 CM, was significantly higher than that of prostate cancer cell lines. CM of BPH-1 treated with NS-398 (a specific inhibitor of COX-2) failed to stimulate aromatase expression in PrSCs. And PGE2 can stimulate aromatase expression in PrSCs. Our data suggested that BPH-1 induced aromatase expression in PrSCs through the production of PGE2 in a paracrine mechanism.
Asunto(s)
Aromatasa/metabolismo , Dinoprostona/metabolismo , Comunicación Paracrina/fisiología , Próstata/enzimología , Hiperplasia Prostática/metabolismo , Células del Estroma/enzimología , Aromatasa/análisis , Aromatasa/genética , Western Blotting/métodos , Línea Celular Tumoral , Células Cultivadas , Medios de Cultivo Condicionados , Células Epiteliales , Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
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.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-2/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/inmunología , Escape del Tumor , Animales , Línea Celular Tumoral , Medios de Cultivo Condicionados , Citocinas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Linfocitos T Reguladores/patologíaRESUMEN
OBJECTIVE: To separate smooth muscle cells (SMCs) from fibroblasts in cultured human prostatic stromal cells (PrSCs) by characterizing the SM22 promoter as a prostatic SMC-specific gene promoter, and to investigate its use for a promoter-based cell-sorting method, as SMCs are critical for stromal function and the pathological changes in the development of benign prostatic hyperplasia. MATERIALS AND METHODS: Human PrSCs were cultured in SMC-selective medium or standard medium, respectively, to obtain typical cultures of SMCs and fibroblasts. SM22 promoter activity and specificity were analysed by luciferase reporter-gene assay. A dual-colour vector was constructed with the expression of the red fluorescent protein (RFP) under the control of the 1.4 kb SMC-specific SM22 promoter, and the expression of the green fluorescent protein (GFP) under cytomegalovirus promoter. Fluorescence-activated cell sorting (FACS) was used to isolate and enrich GFP+/RFP+ and GFP+/RFP- cells. Cell phenotype was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence. RESULTS: The 1.4 kb SM22 promoter activity was much higher in PrSCs cultured in SMC-selective medium. Immunofluorescence staining and merged fluorescence microscopy ensured that SM22 promoter-driven GFP positive cells were SMCs. After transfection of the dual-colour vector into PrSCs, GFP+/RFP+ cells (SMCs) and GFP+/RFP- cells (fibroblasts) were isolated by FACS. The phenotype of FACS-enriched SMCs and fibroblasts was confirmed. CONCLUSION: These results indicate that the 1.4 kb SM22 promoter is specific for prostatic SMCs. This dual-colour vector could be a useful tool for separating living SMCs from fibroblasts using FACS.
Asunto(s)
Separación Celular/métodos , Fibroblastos/citología , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas , Próstata/citología , Hiperplasia Prostática/patología , Western Blotting , Células Cultivadas , Clonación Molecular/métodos , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: TGF-beta is a potent immunosuppressant. High levels of TGF-beta produced by cancer cells have a negative inhibition effect on surrounding host immune cells and leads to evasion of the host immune surveillance and tumor progression. In the present study, we report a distinct ability of tumor reactive, TGF-beta-insensitive CD8+ T cells to infiltrate into established tumors, secrete relevant cytokines, and induce apoptosis of tumor cells. METHODS: CD8+ T cells were isolated from the spleens of C57BL/6 mice, which were primed with irradiated mouse prostate cancer cells, the TRAMP-C2 cells. After ex vivo expansion, these tumor reactive CD8+ cells were rendered TGF-beta-insensitive by infection with a retroviral (MSCV)-mediated dominant negative TGF-beta type II receptor (TbetaRIIDN). Control CD8+ cells consist of those transfected with the GFP-only empty vector and naïve CD8+ T cells. Recipient mice were challenged with a single injection of TRAMP-C2 cells 21 days before adoptive transfer of CD8+ T cells was performed. Forty days after the adoptive transfer, all animals were sacrificed. The presence of pulmonary metastases was evaluated pathologically. Serial slides of malignant tissues were used for immunofluorescent staining for different kinds of immune cell infiltration, cytokines, and apoptosis analysis. RESULTS: Pulmonary metastases were either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells (3 out of 12) when compared to GFP controls (9 out of 12), and naïve CD8+ T cells (12 out of 12). Results of immunofluorescent studies demonstrated that only tumor-reactive TGF-beta-insensitive CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis when compared to CD4+ T cells, NK cells, and B cells. A large amount of cytokines such as perforin, nitric oxide, IFN-gamma, IL-2, TNF-alpha were secreted in tumor tissue treated with tumor-reactive TGF-beta-insensitive CD8+ T cells. No immune cells infiltration and cytokine secretion were detected in tumor tissues treated with naïve T cells and GFP controls. CONCLUSIONS: Our results demonstrate the mechanism of anti-tumor effect of tumor-reactive TGF-beta-insensitive CD8+ T cells that adoptive transfer of these CD8+ T cells resulted in infiltration of these immune cells into the tumor parenchyma, secretion of relevant cytokines, and induction of apoptosis in tumor cells. These results support the concept that tumor-reactive TGF-beta-insensitive CD8+ T cells may prove beneficial in the treatment of advanced cancer patients.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias de la Próstata/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Línea Celular Tumoral , Citocinas/inmunología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias de la Próstata/patología , Organismos Libres de Patógenos EspecíficosRESUMEN
Prostate cancers often develop insensitivity to TGF-beta to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-beta receptors (TbetaRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-beta. Sensitivity to TGF-beta was restored in cells treated with 5-Aza-2'-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TbetaRI), and type II (TbetaRII) TGF-beta receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-beta. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TbetaRI and 33 sites in TbetaRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TbetaRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TbetaRII promoter and the -140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TbetaRI promoter and of Sp1 in the TbetaRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TbetaRI or TbetaRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TbetaRI (six sites) and TbetaRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TbetaRI and/or TbetaRII, especially those with high Gleason's scores. In those specimens showing a loss of TbetaR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-beta in some prostate cancer cells is due to promoter methylation in TbetaRs.
Asunto(s)
Metilación de ADN , Neoplasias de la Próstata/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacología , Inmunoprecipitación de Cromatina , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Decitabina , Humanos , Masculino , Fosforilación , Regiones Promotoras Genéticas , Neoplasias de la Próstata/metabolismo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The murine renal cell carcinoma (Renca) cells are insensitive to TGF-beta due to a lack of TGF-beta type II receptor (TbetaR-II). The objective of the present study is to determine the mechanism of this loss of sensitivity to TGF-beta in Renca cells. Renca cells were cultured and treated with 5-Aza-2'-Deoxycytidine (5-Aza), a specific inhibitor of methylation. Expression of TGF-beta type I receptor (TbetaRI) and TbetaRII was determined by RT-PCR and Western blot analysis before and after the treatment of Renca cells with 5-Aza. The expression of phosphorylated Smad2 (P-Smad2) was determined by Western blot analysis. TGF-beta levels in the conditioned medium were measured by ELISA. Renca cells did not express TbetaR-II prior to 5-Aza treatment. After 5-Aza treatment, these cells expressed TbetaR-II at both mRNA and protein levels, which corresponded to the restoration of sensitivity to TGF-beta by an increase in P-Smad2. Levels of TGF-beta1 were similar before and after 5-Aza treatment. Results of the present study indicated that, in Renca cells, the loss of sensitivity to TGF-beta is likely due to a promoter hypermethylation in the TbetaR-II gene.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Animales , Carcinoma de Células Renales/tratamiento farmacológico , Línea Celular Tumoral , Metilación de ADN , Decitabina , Neoplasias Renales/tratamiento farmacológico , Ratones , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas , ARN Mensajero/análisis , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/análisis , Factor de Crecimiento Transformador beta/fisiología , Factor de Crecimiento Transformador beta1RESUMEN
In a preliminary study, we observed that TGF-beta1 induced both proliferation and growth arrest in prostatic stromal cells, depending on the concentration of TGF-beta1 used in the culture medium. In this study, we explored possible mechanisms of this dual effect of TGF-beta. Primary cultures of prostatic stromal cells, established from clinical surgical specimens and treated with low doses of TGF-beta1 (0.001-0.01 ng/ml), resulted in an increase in cell proliferation. The addition of neutralizing antibody against platelet-derived growth factor (PDGF)-BB, but not anti-PDGF-AA, abrogated this stimulatory effect of TGF-beta1. TGF-beta1 treatment resulted in a dose-related increase in PDGF-BB production as measured by ELISA. Cells underwent growth arrest at high concentrations of TGF-beta1 (1.0 and 10 ng/ml). An inhibitor of cyclin-dependent kinase (cdk), p15INK4b, was up-regulated at both transcript and protein levels in these cultures by TGF-beta1 in a dose-related manner as determined by RT-PCR and Western blot analysis. The transcript, but not the protein, for another cdk inhibitor, p21Cip1, was up-regulated with treatment of TGF-beta1 to these cells. Levels of other cdk inhibitors, such as p16INK4a and p27Kip1, were constitutively expressed in prostatic stromal cells and were not significantly affected by TGF-beta1 treatment. Finally, the growth arrest effect of TGF-beta1 was abrogated when antisense oligonucleotides to p15INH4b, but not p21Cip1, were added to the culture medium. These data indicate that the dual effect of TGF-beta1 is mediated, at least, by up-regulation of PDGF-BB and p15INK4b, respectively.
Asunto(s)
Próstata/citología , Células del Estroma/citología , Factor de Crecimiento Transformador beta/farmacología , Proteínas Supresoras de Tumor , Anticuerpos/farmacología , Becaplermina , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Oligonucleótidos Antisentido/administración & dosificación , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/inmunología , Próstata/metabolismo , Proteínas Proto-Oncogénicas c-sis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismo , Timidina/metabolismo , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta1 , Regulación hacia ArribaRESUMEN
BACKGROUND: Clusterin is a ubiquitous, secretory glycoprotein with a wide array of functions. Recent studies have implicated that clusterin functioned as heat shock response proteins. The objective of the present study was to determine the fate of clusterin expression in cancer cells, which were subjected to a lethal dose of heat shock, in an attempt to shed light on mechanisms of action of clusterin. METHODS: A prostate cancer line, PC-3, and a bladder cancer line, TSU-Pr1, were selected owing to their aggressive growth behaviors. Apoptosis was assessed by enzymatic activities of terminal deoxynucleotidyl transferase as well as by activities of caspase-3. Cells were exposed to 45 degrees C for a period of 60 min. RESULTS: Both cell lines underwent a transient up-regulation of clusterin expression followed by down-regulation and apoptosis. TSU-Prl cells produced higher levels of clusterin and displayed a greater resistance to apoptosis than did PC-3 cells. The addition of exogenous clusterin to the cultures of PC-3 cells protected apoptosis. Treatment of oligonucleotide antisense to clusterin to the cultures of TSU-Pr1 cells enhanced apoptosis mediated by heat shock. CONCLUSION: Clusterin offers a protection to PC-3 and TSU-Prl cells against heat shock and plays an important role in the cascade of events initiated by heat shock. Prostate 53: 277-285, 2002.
Asunto(s)
Glicoproteínas/biosíntesis , Calor , Chaperonas Moleculares/biosíntesis , Neoplasias de la Próstata/patología , Neoplasias de la Vejiga Urinaria/patología , Apoptosis , Clusterina , Fragmentación del ADN , Glicoproteínas/farmacología , Humanos , Masculino , Chaperonas Moleculares/farmacología , Neoplasias de la Próstata/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Clusterin is a ubiquitous secretory glycoprotein that is known to suppress certain forms of apoptosis. Since apoptosis and proliferation are two opposing cellular events, it remains unclear if clusterin has any effect on cellular proliferation. The objective of the present study was to examine the effects of clusterin on proliferation in a prostate cancer cell line, LNCaP. We found that clusterin inhibited EGF-mediated proliferation in these cells, as measured by (3)H-thymidine incorporation and by cell counting. Clusterin did not bind with EGF nor did it block phosphorylation of the EGF receptor. Treatment of LNCaP cells with EGF resulted in a transient increase in the expression of both c-Fos and c-Jun. Addition of clusterin to these cultures significantly down-regulated the protein level of c-Fos, but not c-Jun. These results demonstrated a novel biological role for clusterin. Clusterin is not only anti-apoptotic but also anti-proliferative. The anti-proliferative event maybe associated with a down-regulation of c-Fos.