Inhibition of telomerase is related to the life span and tumorigenicity of human prostate cancer cells.

PURPOSE: Telomerase, the enzyme that catalyzes the elongation of telomeres, is illegitimately activated in the majority of cancers, including that of the prostate, where it may greatly extend the life span of malignant cells. The inhibition of telomerase by molecular intervention has been shown to lead eventually to cell death in several tumor or in vitro immortalized cell lines and in 1 case prevent tumor growth in vivo. Therefore, we tested whether a similar strategy may be used to limit the tumorigenic potential of late stage prostate cancer cells. MATERIALS AND METHODS: PC-3, LNCaP and DU-145 human prostate cancer cells were infected with a retrovirus encoding a dominant-negative version of the catalytic subunit of telomerase (DN-hTERT). Subclones or polyclonal populations were assayed for DN-hTERT expression, telomerase activity, telomere length, cell life span and in most cases tumorigenicity in nude mice. RESULTS: DN-hTERT expression levels directly correlated with cell life span and tumorigenic growth. PC-3 cells expressing high levels of DN-hTERT died rapidly and failed to form tumors in nude mice, whereas cells expressing the lowest levels proliferated the longest and generated tumors that later spontaneously regressed. Similarly the inhibition of telomerase activity in LNCaP cells was greater than in DU-145 cells and correspondingly LNCaP cells had a shorter life span. CONCLUSIONS: DN-hTERT expression limits the life span and tumorigenic potential of human prostate cancer cells, although the onset of these effects appears to be dictated by the expression level of DN-hTERT. Therefore, telomerase represents an attractive target for potentially managing prostate cancer. Nevertheless, effective means of inhibiting the enzyme may be required for a therapeutically useful outcome.     

前列腺癌细胞中表皮生长因子受体的表达和激活

目的：探讨前列腺癌（ＰＣａ）细胞中表皮生长因子受体（ＥＧＦＲ）表达和激活及其与雄激之间的相互作用。方法：采用免疫沉淀和Ｗｅｓｔｅｒｎ ｂｌｏｔ方法,检测雄激素依赖人ＰＣａ细胞株ＬＮＣａＰ及雄激素非依赖人ＰＣａ细胞株ＤＵ－１４５、ＰＣ－３中ＥＧＦＲ的表达和磷酸化激活,测定雄激素对ＬＮＣａＰ ＥＧＦＲ表达和激活的影响。结果：在ＤＵ－１４５和ＰＣ－３中,ＥＧＦＲ表达和激活的基础与表皮生长因子（ＥＧＦ）处     

Effect of the neuropeptides bombesin and calcitonin on the growth of prostate cell lines, PC-3, DU 145, and LNCaP

Neuroendocrine cells are present in normal and tumoral prostate tissue, the neuropeptides secreted by this cells have a biological functions that have not been fully elucidated. The presence of neuroendocrine cells in prostatic carcinoma have been shown to increase tumor progression. We characterized the in vitro proliferative influence of bombesin and calcitonin in androgen-insensitive, PC-3 and DU-145, and androgen-sensitive, LNCaP, cell lines of human prostate cancers. The influence of these neuropeptides on proliferation were assessed using the colorimetric XTT assay and by cells counts with a hemocytometer. The growth of PC-3 and DU-145 cell lines is stimulated by bombesin and calcitonin but exerted any stimulatory effect on the proliferation of the LNCaP cell line. This indicate that bombesin and calcitonin can modulate proliferation of androgen-insensitive human prostate cell lines "in vitro" and may be potential paracrine growth promoters in stablished androgen irresponsive human prostatic carcinoma cells.     

Prostasin基因在前列腺细胞株中表达情况研究

目的 探讨Prostasin在前列腺癌中的功能。方法 运用RT-PCR方法检测人前列腺增生细胞株BPH-1，无转移能力前列腺癌细胞株LNCaP，及有转移能力的前列腺癌细胞株PC-3、DU-145中Prostasin基闪表达情况。结果 Prostasin基斟在BPH-l和LNCaP中正常表达，在PC-3、DU-145中低表达。BPH-l中Prostasin的表达与DU-145和PC-3比较差异有显著性，同样LNCaP中Prostasin的表达与DU-145和PC-3比较差异亦有显著性（P〈0．01）。BPH-1与LNCaP之间表达差异无显著性，DU-145和PC-3之间表达差异亦无显著性，（P〉0.05）。结论 ProStasin可能是前列腺癌的转移抑制剂。     

Secretion of prostate-specific antigen-suppressing activity by two human prostate carcinoma cell lines

The well-differentiated human prostatic carcinoma cell line LNCaP has often been used as a model to study the modulation of prostate-specific antigen (PSA) expression. In this study, we examined the effect of conditioned medium from two androgen-unresponsive, PSA-negative human prostatic carcinoma cell lines, PC-3 and DU-145, on PSA expression by the LNCaP cells. Our results show, for the first time, that the undifferentiated PC-3 and DU-145 cells secrete a factor that lowers PSA mRNA and protein levels in LNCaP cells. Further characterization of the PSA-suppressing activity indicated that it was trypsin as well as acid sensitive, heat-stable, and ammonium sulfate precipitable. The activity was present in the 30-100-kd fraction of PC-3 and DU-145 conditioned media. This PSA-suppression factor could act in an autocrine manner to render undifferentiated prostatic carcinoma cells PSA negative. It may also be responsible for the lack of increase in serum PSA levels observed in a subset of patients with hormone-resistant prostatic carcinoma.     

Androgen-independent growth is induced by neuropeptides in human prostate cancer cell lines

BACKGROUND: Androgen-independent growth leads to progressive prostate cancer after androgen-ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand-independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells. METHODS: We established and characterized the androgen-independent FGC-DCC from the androgen-dependent LNCaP fast growing colony (FGC) cell line. The androgen-independent DU-145, FGC-DCC, and PC-3, and the androgen-dependent LNCaP and PC-346C cell lines were used to study growth modulation of gastrin-releasing peptide (GRP), calcitonin (CT), serotonin (5-HT), and vasoactive intestinal peptide (VIP) by (3)H-thymidine incorporation. Specificity of the growth-modulating effects was tested with the anti-GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides. RESULTS: Androgen-independent growth stimulation by neuropeptides was shown in DU-145 and PC-346C. 2A11 inhibited GRP-induced (3)H-thymidine incorporation in DU-145 and PC-346C and inhibited proliferation of the FGC-DCC and PC-3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP-induced growth effect in DU-145 and PC-346C, whereas oxadiazoloquinoxaline-1-one (ODQ) had no effect on (3)H-thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC-DCC, or PC-3. CONCLUSIONS: GRP-induced growth of DU-145 and PC-346C was specific and cAMP-mediated. Androgen-independent growth of FGC-DCC cells was mainly due to an induction of Bcl-2 expression and possibly through the activation of an autocrine and NE-like pathway, as has been shown also for the PC-3 cell line. Growth induction of non-NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer.     

凋亡抑制基因XIAP在前列腺癌细胞中的表达及与临床病理特征的关系

目的 :研究XIAP基因在前列腺癌细胞系和前列腺癌组织的表达情况 ,及其与前列腺癌临床病理特征的关系。　方法 :应用RT PCR检测前列腺癌组织、正常前列腺组织和前列腺癌细胞株PC 3,DU 14 5 ,LNCaP细胞XIAP基因的表达 ,并通过免疫组化SP法检测 5 6例前列腺癌组织标本XIAP蛋白的表达情况。　结果 :XIAP基因在前列腺癌组织和前列腺癌细胞株PC 3,DU 14 5 ,LNCaP细胞高表达 ,正常前列腺组织无表达。在前列腺癌组织和癌旁组织中 ,XIAP蛋白阳性检出率分别为 5 3.6 % (30 / 5 6 )和 2 1.5 % (12 / 5 6 ) (P <0 .0 1) ;不同分期、分级组XIAP阳性检出率相比差异无显著性 (P >0 .0 5 )。　结论 :凋亡抑制基因XIAP与前列腺癌相关 ,在前列腺癌发生过程中具有重要的作用 ,有可能成为前列腺癌治疗的靶标。     

Antitumor activity of the histone deacetylase inhibitor MS-275 in prostate cancer models

BACKGROUND: Histone deacetylase (HDAC) inhibitors represent a novel class of therapeutic agents with antitumor activity currently in clinical development. In this study, we tested the biological effects of the HDAC inhibitor MS-275 in various pre-clinical prostate cancer models both in'vitro and in vivo. METHODS: In vitro cell proliferation XTT assay and protein expression analysis by Western blot were performed. In vivo tumor growth assessment in subcutaneous, orthotopic, and transgenic mouse models were conducted. RESULTS: MS-275 significantly upregulated histone H3 acetylation and p21 gene expression in human prostate cancer cell lines. MS-275 exerted growth arrest in PC-3 and LNCaP cells, and induced cell death in DU-145 cells. Prostate specific antigen protein levels were increased by MS-275 in LAPC4 cell line. In vivo, MS-275 inhibited the growth of DU-145, LNCaP, and PC-3 in subcutaneous xenografts. MS-275 had also a significant inhibition of PC-3 cells growth in a mouse intratibial model. Molecular analysis showed increased histone acetylation and p21 expression in tumor samples from MS-275-treated mice. In transgenic adenocarcinoma of mouse prostate (TRAMP) mice, long-term treatment of MS-275 slowed the progression of prostate carcinomas with significant reduction in cell proliferation. CONCLUSIONS: Taken together, these data support the clinical testing of MS-275 for the treatment of prostate cancer.     

米非司酮诱导雄激素非依赖性前列腺癌细胞凋亡的实验研究

目的探讨米非司酮在体外诱导雄激素非依赖性前列腺癌DU-145、PC-3细胞凋亡的作用。方法采用四甲基偶氮唑蓝法检测1,10,50和100μmol／L米非司酮作用于前列腺癌DU-145、PC-3细胞24～120h的吸光度（A）值,用流式细胞仪检测10μmol／L米非司酮作用24和48h后DU．145、PC-3细胞凋亡率的变化;采用免疫组化法检测米非司酮作用DU-145、PC．3细胞后bax、bcl-2、血管内皮生长因子（VEGF）蛋白表达的变化。结果1μmol／L米非司酮组的A值与对照组相比,差异无统计学意义（P〉0．05）;10,50和100μmol／L米非司酮组的A值与对照组比较,差异有统计学意义（P〈0．01）;米非司酮对前列腺癌DU-145、PC-3细胞的抑制作用呈时间-剂量依赖性。10μmol／L米非司酮作用前列腺癌DU-145细胞24和48h的凋亡率分别为15．3％和30．4％,PC-3细胞的凋亡率分别为22．2％和32．0％。经10μmol／L米非司酮作用后,DU-145、PC-3细胞中VEGF和bcl-2蛋白的表达明显减少,而bax的表达显著增加（P〈0．05）。结论米非司酮以时间．剂量依赖性方式抑制激素非依赖性前列腺癌DU-145和Pc-3细胞的增殖,其作用可能是通过降低VEGF蛋白的表达。从而下调bcl-2、激活bax蛋白的表达来实现。     

Effects of interferon beta ser and transforming growth factor beta on prostatic cell lines

The effect of interferon beta ser (IFN beta ser) on the growth of three prostatic cancer cell lines DU-145, PC-3 and LNCaP was studied. IFN beta ser inhibited growth of anchorage dependent semiconfluent monolayers and anchorage dependent colony formation of both DU-145 and PC-3 in a dose dependent manner but had no effect on LNCaP. Transforming growth factor beta (TGF beta 1) inhibited proliferation of DU-145 and PC-3 cells in 1% but not 8% fetal calf serum. The combination of TGF beta 1 and IFN beta ser was additive in its effects on growth. Neither epidermal growth factor (EGF) nor transforming growth factor alpha (TGF alpha) reduced the antiproliferative effect of IFN beta ser on these cells. These antiproliferative effects were reproduced in studies on primary epithelial cell cultures derived from prostate specimens with various pathologies. The potential use of IFN beta ser in combination with hormonal therapy to delay the development of hormone refractory tumors is discussed.     

Expression of the matrix metalloproteinase promatrilysin in coculture of prostate carcinoma cell lines

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in tumor progression. Matrilysin (MMP-7) has been shown to be upregulated in prostatic carcinomas and can increase the invasive capacity of DU-145 cells. Because of the heterogenous nature of prostatic tumors, we examined promatrilysin expression in cocultures containing two different prostatic carcinoma cell lines, DU-145 and LNCaP. METHODS: Using enzyme linked immunosorbent assay (ELISA) analyses, promatrilysin expression was measured in DU-145/LNCaP cocultures and conditioned media cross-cultures. The effects of blocking IL-6 on promatrilysin expression were examined by pretreating conditioned media with IL-6 neutralizing antibody. RESULTS: A significant induction of promatrilysin expression was observed in DU-145/LNCaP cocultures compared to LNCaP cells alone. In addition, DU-145 conditioned medium induced the same fold induction of promatrilysin as was observed in the cocultures. LNCaP cell conditioned medium did not induce promatrilysin expression in DU-145 cells. Neutralization of IL-6 with neutralizing antibody abrogated DU-145 conditioned media induced promatrilysin expression to baseline levels. CONCLUSIONS: IL-6 secreted by DU-145 cells can induce promatrilysin expression in LNCaP cells. IL-6, in vivo, may act as a paracrine signaling factor that regulates matrix metalloproteinase expression. Therefore, IL-6 may play a role in invasive metastatic processes of a prostate carcinoma.     

Phenotypic and genotypic characterization of commonly used human prostatic cell lines

OBJECTIVE: To investigate and catalogue systematically the phenotypic and genotypic characteristics of the commonly used prostatic cell lines using immunocytochemistry and polymerase chain reaction (PCR) of hypervariable sequences within the genome to provide a 'fingerprint' characteristic of each cell line. Materials and methods Malignant (LNCaP, LNCaP-r, PC-3, DU-145) and benign immortalized prostatic cell lines (PNT-1A, PNT-1B, BPH-1) were grown on four-well slides, fixed and subjected to indirect streptavidin-biotin immunocytochemistry. Twenty-three antibodies were used in the following groups: cytoskeletal elements: cytokeratins (CK)-5, -7, -8, -14 (two), -16, -18, -19 (three), -20, vimentin and desmin; MUC1 (three); cell adhesion molecules (E-cadherin, alpha-beta-and gamma-catenin); and prostatic associated proteins: prostate specific antigen (PSA), prostatic acid phosphatase (PAP) and androgen receptor (AR). For the PCR, genomic DNA was extracted from the cell lines and from SKOV3 and MCF7 (positive controls). PCR was performed on three variable regions which were then sequenced: AR exon 1 (CAG repeat polymorphism), and two areas of microsatellite instability (MSI): AR exon 8 and hypoxanthine-guanine phosphoribosyl transferase (HPRT) exon 3. RESULTS: All cell lines were CK-8/18 positive and most also expressed CK-7 and -19. Heterogeneous CK-20 expression was detected for the first time in prostatic cell lines. All lines were positive for vimentin and negative for desmin. MUC1 was expressed in one malignant (DU-145) and all immortalized cell lines. E-cadherin expression was low or absent in three lines: PNT1A, 1B and PC-3. Only PC-3 failed to express alpha-catenin; beta- and gamma-catenin were expressed by all lines. PSA, PAP and AR were only expressed by LNCaP and LNCaP-r. On PCR, the CAG repeat lengths in exon 1 of the AR ranged from 19 to 27. Three pairs of cell lines had the same exon 1 CAG repeat length: LNCaP/PC-3 (26 repeats), BPH-1/DU-145 (19 repeats) and PNT1 A/1B (20 repeats). Exon 8 sequences were identical except for LNCaP, which showed a single base mutation, and HPRT exon 3 sequences were all identical. There was no evidence of generalized MSI in any of the cell lines examined. CONCLUSIONS: The cell lines studied fell into three broad groups according to their phenotypic characteristics: (i) prostatic marker positive (LNCaP and LNCaP-r); (ii) high expression of most antigens (DU-145, PC-3 and BPH-1); and (iii) low or absent expression of most antigens (PNT1 A and 1B). Each of the cell lines derived from PC could be identified on the basis of exon 1 and 8 AR sequence variability. DU145 and BPH-1 had identical profiles of the three areas studied, but these cell lines are easily distinguished by their different phenotypic characteristics. PNT1A and 1B had identical genetic and similar phenotypic profiles, which is unsurprising given that they are subclones derived from the same parental line. Even so, these were separable on the basis of CK-19 immunostaining. Using a combination of geno- and phenotypic markers it was possible to derive a 'fingerprint' for each of the cell lines assessed, which will allow meaningful comparison between similar cell lines held in other laboratories.     

Heterogeneity in plasminogen activator (PA) levels in human prostate cancer cell lines: increased PA activity correlates with biologically aggressive behavior

Plasminogen activators (PA), particularly the lower Mr urokinase (u-PA) type, have been associated with tumor cell invasion and metastasis. We have examined the expression of PA by two human prostate cancer cell lines (PC-3 and DU-145) using functional and immunologic techniques. The culture media and cell extracts of the more aggressive PC-3 cell line contained more than two-fold greater PA activity than the relatively indolent DU-145 cell line. Zymographic studies identified the PA expressed as u-PA. PC-3 cells expressed an additional lower molecular weight form of u-PA not noted in DU-145 cells. Heterogeneity in u-PA expression was shown by the fibrin lysis assay, immunohistochemistry, and dual parameter flow cytometry indicating the presence of phenotypically divergent cell populations. Increased u-PA expression may identify those tumor cells that possess aggressive biological potential.     

In vitro proton magnetic resonance spectroscopy of four human prostate cancer cell lines

There is accumulating evidence that some biochemical pathways observable by magnetic resonance spectroscopy, e.g., citrate acid and phospholipid metabolism, are altered in human prostate cancer. Four well-established human prostate cancer cell lines were therefore studied with magnetic resonance spectroscopy to compare differences in metabolic content with tumor biological behavior. Herein we demonstrate that, although each cell line has its own metabolic profile, relative creatine and citrate levels can be used to discriminate the androgen-dependent LNCaP cell line from the androgen-independent DU-145, TSU, and PC-3 cell lines.     

Expression of gonadotropin-releasing hormone (GnRH) and GnRH receptor mRNA in prostate cancer cells and effect of GnRH on the proliferation of prostate cancer cells

The purpose of this study was to determine the production of gonadotropin-releasing hormone (GnRH), the co-occurrence of GnRH receptors in prostate cancer cells, and the effect of GnRH on prostate cancer cell proliferation. Four human prostate cancer cell lines were studied. LNCaP is an androgen sensitive prostate cancer cell line, DU-145 and PC-3 are androgen resistant, and TSU-Pr1 is uncharacterized. The expression of GnRH and GnRH receptor mRNAs were assessed by in situ hybridization and the effect of exogenous GnRH on proliferation of prostate cancer cells was measured by thymidine incorporation assay. GnRH mRNA expression, determined by in situ hybridization, was found in 83.48% of the LNCaP, 89.7% of the TSU-Pr1, 86.2% of the PC-3 and 95.3% of the DU-145. Signals of GnRH receptor mRNA were detected in more than 95% of the cells of all four cell lines. The proliferation of the prostate cancer cells grown in media supplemented with peptide hormone lacking charcoal-stripped serum was significantly (P < 0.05) suppressed. No significant effect of GnRH on the proliferation of all four prostate cancer cells was observed. In summary, prostate cancer cells produced GnRH and its receptors, and exogenous GnRH treatment did not affect the prostate cancer cell proliferation. The existence of GnRH and GnRH receptor mRNA in the same cell suggests that the role of GnRH produced by prostate cancer cells would be autocrine. Received: 21 August 1997 / Accepted 15 April 1998     

Down-regulation of prostasin serine protease: a potential invasion suppressor in prostate cancer.

BACKGROUND: Prostasin is a serine protease predominantly expressed in normal prostate epithelial cells. The biological function of prostasin has not been determined. METHODS: Western blot and RT-PCR analyses were used to examine the expression of prostasin in prostate cancer cell lines. Immunohistochemistry was used to evaluate prostasin protein expression in human prostate cancer. An in vitro Matrigel invasion assay was used to test the invasiveness of prostate cancer cell lines forced to express recombinant prostasin. RESULTS: Both prostasin protein and mRNA were found to be expressed in normal human prostate epithelial cells and a non-invasive human prostate cancer cell line, the LNCaP, but neither was found in invasive human prostate cancer cell lines DU-145 and PC-3. Prostasin mRNA expression was absent in invasive prostate cancer cell lines of a transgenic mouse model. Immunohistochemistry analysis showed that prostasin protein expression is down-regulated in high-grade prostate cancer. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68 and 42%, respectively. CONCLUSIONS: Our data indicate that prostasin may be implicated in normal prostate biology and is able to suppress prostate cancer invasion in vitro.     

Inhibitory effect of IL-6-induced neuroendocrine cells on prostate cancer cell proliferation

BACKGROUND: The role of increased neuroendocrine (NE) differentiation in prostate cancer (PCA) is not well understood. Long-term exposure of the prostate cancer cell line LNCaP to high concentrations of the cytokine interleukin-6 (IL-6) results in permanent transdifferentiation of these cells into a NE phenotype. In this study, we evaluated the effect of IL-6-induced NE cells on the growth of the PCA cell lines LNCaP, PC-3, and DU-145 in vitro. RESULTS: Co-culture of NE cells with PCA cells significantly inhibited DNA synthesis in all three PCA cell lines by 30-90% compared to controls. NE cell co-culture resulted in an increased percentage of PCA cells arrested in the S-phase of cell cycle and PCA cell apoptosis. CONCLUSIONS: Our results imply that NE cells suppress the proliferation of surrounding PCA cells by release of inhibitory factors.