Lineage relationship between LNCaP and LNCaP-derived prostate cancer cell lines

BACKGROUND: LNCaP and its derivative cell lines, which include C4-2 (and the related C4-2B) and CL1, are used as models of prostate cancer. Unlike LNCaP, the other cell lines show features of progressed disease such as metastatic capability and hormone independence. Analyses were done to determine if C4-2 or CL1 cells were selected from pre-existent subpopulations in LNCaP. METHODS: Prostate cancer cells were characterized by cluster designation (CD) phenotyping. Specific cell populations were sorted by flow cytometry. DNA array analysis was used to probe differential gene expression. RESULTS: CD phenotyping showed that CL1 and C4-2 (and C4-2B) were very dissimilar, and C4-2 was more similar to LNCaP. One common difference between LNCaP and its derivatives was CD26, in which virtually all C4-2 or CL1 cells were CD26(+) but only approximately 10% of LNCaP cells were CD26(+). The CD26(+) subpopulation of LNCaP was isolated and cultured in vitro. After culture, a high percentage of the cells (descended from the sorted cells) were CD26(+), in contrast to those sorted by CD13 or CD44. The cultured CD13 and CD44 populations did not show a high percentage of CD13(+) and CD44(+) cells, respectively. CD13 and CD44 are markers, in addition to CD26, for CL1 but not for C4-2. CONCLUSIONS: C4-2 arose probably from CD26(+) LNCaP cells, while CL1 arose de novo.     

Relaxin becomes upregulated during prostate cancer progression to androgen independence and is negatively regulated by androgens

BACKGROUND: Relaxin is a potent peptide hormone normally secreted by the prostate. This study characterized relaxin expression during prostate cancer progression to androgen independence (AI), and in response to androgens. METHODS: The prostate cancer cell line, LNCaP, was assayed by microarrays and confirmatory Northern analysis to assess changes in relaxin levels due to androgen treatment and in LNCaP xenografts following castration. Relaxin protein levels were examined by immunohistochemistry (IHC) in tissue microarrays of human prostate cancer samples following androgen ablation. RESULTS: Relaxin levels decreased in a time and concentration-dependent manner due to androgens in vitro, and increased in xenografts post-castration. Relaxin increased in radical prostatectomy specimens after 6 months of androgen ablation and in AI tumors, was highest in bone metastases. CONCLUSIONS: Relaxin is negatively regulated by androgens in vitro and in vivo, which correlates to clinical prostate cancer specimens following androgen ablation. The role of relaxin in angiogenesis and tissue remodeling suggests it may contribute to prostate cancer progression.     

Increased expression of the metastasis-associated gene Ehm2 in prostate cancer

BACKGROUND: Alterations of fibroblast growth factors and their receptors contribute to prostate cancer progression by enhancing cell survival, motility, and proliferation. The expression of the FGFR-4 Arg(388) variant is correlated with the occurrence of pelvic lymph node metastasis and biochemical (PSA) recurrence in men undergoing radical prostatectomy. Ehm2 is an androgen-regulated gene that has been associated with metastasis in other systems, so we sought to determine if it is expressed in prostate cancer and if the FGFR-4 Arg(388) variant can increase its expression. METHODS: Expression of Ehm2 was examined by quantitative RT-PCR and Western blotting in prostate cell lines and by quantitative RT-PCR, in situ hybridization, and immunohistochemistry in prostate tissues. The effect of Ehm2 expression on collagen IV adhesion was tested by transient overexpression and RNA interference. RESULTS: Ehm2 expression is upregulated in prostate cancer cell lines and prostate cancer tissues. Expression of the FGFR-4 Arg(388) variant results in increased expression of Ehm2. Increased expression of Ehm2 leads to decreased adhesion to collagen IV, which has been associated with metastasis in cancers. Analysis of tissue microarrays revealed that increased Ehm2 expression is associated with biochemical recurrence after radical prostatectomy, which is indicative of more aggressive disease. CONCLUSIONS: Ehm2 is overexpressed in prostate cancer and may enhance disease progression and metastasis.     

Androgen receptor-dependent regulation of Bcl-xL expression: Implication in prostate cancer progression

BACKGROUND: Recently we reported that silencing the androgen receptor (AR) gene reduced Bcl-xL expression that was associated with a profound apoptotic cell death in prostate cancer cells. In this study we further investigated AR-regulated Bcl-xL expression. METHODS: Prostate cancer cell line LNCaP and its sublines, LNCaP/PURO and LNCaP/Bclxl, were used for cell proliferation assay and xenograft experiments in nude mice. Luciferase gene reporters driven by mouse or human bcl-x gene promoter were used to determine androgen regulation of Bcl-xL expression. RT-PCR and Western blot assays were conducted to assess Bcl-xL gene expression. Chromatin immunoprecipitation assay was performed to determine AR interaction with Bcl-xL promoter. Bcl-xL-induced alteration of gene expression was examined using cDNA microarray assay. RESULTS: In cultured prostate cancer LNCaP cells, androgen treatment significantly increased Bcl-xL expression at mRNA and protein levels via an AR-dependent mechanism. Promoter analyses demonstrated that the AR mediated androgen-stimulated bcl-x promoter activation and that the AR interacted with bcl-x promoter. Enforced expression of Bcl-xL gene dramatically increased cell proliferation in vitro and promoted xenograft tumor growth in vivo. Genome-wide gene profiling analysis revealed that Bcl-xL expression was significantly higher in metastatic and castration-resistant diseases compared to normal prostate tissues or primary cancers. Bcl-xL overexpression significantly increased the expression of cyclin D2, which might be responsible for Bcl-xL-induced cell proliferation and tumor growth. CONCLUSIONS: Taken together, our data strongly suggest that androgen stimulates Bcl-xL expression via the AR and that increased Bcl-xL expression plays a versatile role in castration-resistant progression of prostate cancer.     

CL1-GFP: an androgen independent metastatic tumor model for prostate cancer

PURPOSE: The mechanisms responsible for tumor progression to androgen independence in prostate cancer (CaP) remain unknown. To characterize these changes and provide a basis for rational therapeutic strategies for advanced CaP, an in vivo model from a highly aggressive androgen independent CaP cell line with distinct cellular and molecular properties was developed. MATERIALS AND METHODS: An aggressive androgen-independent cell line designated CL1 was derived from a slow-growing, and androgen-dependent, parental LNCaP cell line through in-vitro androgen-deprivation and selection. CL1 was stably transfected with a green fluorescence protein gene (CL1-GFP) and orthotopically injected into SCID mice. The pathologic behavior, histology, and molecular determinants of CL1 tumor and metastases were determined and characterized by standard light and fluorescent microscopy, and quantitative RT-PCR analysis. RESULTS: CL1 is an anaplastic prostate cancer cell line which demonstrates extensive local invasion and metastases to various organs that can be visualized via GFP expression. When compared with parental LNCaP cells, RT-PCR analysis of the tumor revealed an over-expression of EGFR, b-FGF, VEGF, TGF-beta, IL-8, IL-6, and bcl-2 and a down regulated expression of the p53, E-cadherin and PTEN. In contrast to LNCaP cells, CL1 tumors express lower levels of androgen receptor and barely detectable PSA mRNA. CONCLUSIONS: CL1-GFP represents an aggressive androgen-independent CaP tumor model derived through androgen deprivation whose pathologic development and molecular properties in animals resembles the clinical characteristics of hormone refractory prostate cancer (HRPC). Metastatic sites of CL1-GFP can be visualized with fluorescence microscopy offering a unique therapeutic model for the evaluation of drug sensitivity and other therapeutic modalities.     

Overexpression of protease-activated receptors-1,-2, and-4 (PAR-1, -2, and -4) in prostate cancer

BACKGROUND: Although protease-activated receptors (PARs) have been described to play a role in different malignancies, their expression and biological activity in prostate cancer are mostly unknown. METHODS: PAR expression in radical prostatectomy specimens was investigated by immunohistochemistry (IHC, 40 patients) and RT-PCR. Their role in LNCaP prostate cancer cell migration and Rac1/Cdc42 signaling was assessed with Boyden chamber analysis and Western blot, respectively. RESULTS: PAR mRNA expression was higher in cancer, and protein expression was increased in PAR-1 (45%), PAR-2 (42%), and PAR-4 (68%), compared to normal glands. Increased PAR-1 (periglandular stroma) was associated with higher rates of biochemical recurrence (median follow-up, 5 years; P = 0.006). LNCaP migration was enhanced twofold and Rac1/Cdc42 signaling was activated by stimulation of PAR-1 and PAR-2. CONCLUSIONS: PARs are overexpressed in prostate cancer and may serve as potential predictors of recurrence. The data suggest potential role of PARs in autocrine and paracrine mechanisms of prostate cancer.     

Androgens induce CD-9 in human prostate tissue

Based on microarray analyses of LNCaP and LNCaP-r prostatic cell-lines we tentatively identified CD-9 as an androgen sensitive protein. This prompted us to characterize the androgen sensitivity and the correlation to malignancy of CD-9 at the protein level. Using Western blot, RT-PCR and immunohistochemistry the expression of CD-9 was analysed in LNCaP cells stimulated during increasing time by the synthetic androgen R1881 and also in 88 specimens of human prostate cancer tissues. Expression of CD-9 was induced by R1881 in LNCaP. CD-9 was immunolocalized in human prostate tissue sections representing non-malignant tissue as well as tumour areas. In non-malignant glands CD-9 immunoreactivity was observed at the apical and lateral cell borders of luminal epithelial cells. Basal epithelial cells were largely unstained. In tumour areas CD-9 staining intensity was variable and apparently not related to primary Gleason grade. In prostate tissue from a patient under androgen ablation therapy no staining was observed in luminal epithelial cells or in the tumour areas, but some staining was observed in basal epithelial cells. CD-9 is regulated by androgens in LNCaP and present in human prostate specimens. The expression is variable and changes in expression levels. These and earlier studies using other tissues indicate that CD-9 and its cellular localization could have an important role in prostate cancer cell development.     

Alterations in beta-catenin expression and localization in prostate cancer

BACKGROUND: Wnt signaling is thought to be important in prostate cancer, in part because proteins such as beta-catenin can also affect androgen receptor signaling. beta-Catenin forms a cell adhesion complex with E-cadherin raising the possibility that loss of expression or a change in beta-catenin distribution in the cell could also alter downstream signaling, decreased inter-cellular adhesion and the promotion of metastasis. A number of studies have reported the altered expression and/or localization of beta-catenin as a biomarker in prostate cancer. METHODS: Tissue microarrays comprised of BPH and low, moderate and high-grade prostate cancer (n=77) were assessed for beta-catenin expression and distribution using immunohistochemistry. Staining was also performed on a tissue microarray containing tissue from patients before and after hormone manipulation. The effects of fixation and different antibodies was assessed on fixed LNCaP cell pellets and small prostate tissue microarrays. RESULTS: We have observed increased beta-catenin expression in only high Gleason score (>7) prostate cancer. A nuclear re-distribution of beta-catenin has previously been reported. We noted nuclear beta-catenin in benign prostatic hyperplasia and a gradual loss in nuclear distribution with increasing Gleason grade. We found no evidence for an alteration in beta-catenin expression or re-distribution with hormone ablation. Altered fixation, antibodies and antibody concentration did affect the intensity and specificity of staining. CONCLUSIONS: A loss of nuclear beta-catenin is the most consistent feature in prostate cancer rather than absolute levels of expression. We also suggest that variation in immunohistochemical protocols may explain variations in the reported literature.     

The significance of the expression of dihydropyrimidine dehydrogenase in prostate cancer

OBJECTIVE: To measure dihydropyrimidine dehydrogenase (DPD), an enzyme involved in the metabolism of 5-fluorouracil (5-FU), expression in prostate cancer and determine whether 5-chloro-2,4-dihydroxypyridine (CDHP), a potent inhibitor of DPD, enhances the antitumoral activity of 5-FU against prostate cancer. PATIENTS, MATERIALS AND METHODS: In all, 44 prostate tissue specimens were obtained from men who had a radical prostatectomy alone for prostate cancer, and 38 specimens from men who had had neoadjuvant hormonal therapy. We analysed the cancerous tissue and normal prostate tissue for DPD expression using immunohistochemistry, and determined its prognostic significance. In cultured human prostate cancer lines (DU145 and LNCaP), we compared the cytotoxicity of 5-FU/CDHP with that of 5-FU alone. Finally, in experiments on immunodeficient mice, we studied the effect of oral administration of tegafur, a pro-drug for 5-FU, with or without CDHP on the growth of tumours introduced by injection of DU145 cells. RESULTS: The expression of DPD was significantly higher in cancerous than normal prostate tissue; 36 of 44 (82%) specimens of prostate cancer expressed DPD, whereas only 25 of 44 (57%) specimens of normal prostate tissue expressed DPD. For men with prostate cancer who had radical prostatectomy alone, men with negative DPD expression tended to have a longer recurrence-free survival than those with positive expression; there were no recurrences in men with prostate cancer and negative DPD expression in the 5-year follow-up. DPD expression was significantly lower in men with prostate cancer who received neoadjuvant hormonal therapy. In vitro treatment of human prostate cancer cell lines with 5-FU/CDHP showed more cytotoxicity than with 5-FU treatment alone. Finally, DU145 tumours in mice treated with tegafur and CDHP were significantly smaller than in mice given tegafur alone. CONCLUSION: The present study showed that DPD expression is elevated in prostate cancer, and indicate that DPD inhibitors might enhance the antitumour activity of 5-FU against prostate cancer.     

Expression of prothymosin alpha is correlated with development and progression in human prostate cancers

BACKGROUND: Our previous study clearly demonstrated that decreased expression of prothymosin alpha (PTMA) was associated with inhibition of rat prostate carcinogenesis by isoflavones. The purpose of the present investigation was to provide a better understanding of the role of PTMA in human prostate cancers. METHODS AND RESULTS: PTMA expression in 68 prostate cancer cases and in prostate cancer cell lines was examined by immunohistochemistry and immunoblotting, and its levels were increased with progression from normal epithelium, through prostatic intraepithelial neoplasia (PIN) to carcinomas, correlating with the Gleason's pattern. All cell lines studied (LNCaP, 22Rv1, DU145, and PC3) showed high PTMA expression compared with prostate epithelial cells (PrEC). Knockdown of PTMA expression in PC3 cells by RNAi resulted in the inhibition of both cell growth and invasion in vitro. CONCLUSIONS: The present study clearly demonstrated that PTMA expression is intimately involved in the differentiation and progression of human prostate cancers, and could be a target for therapy and diagnostic purposes.     

90K/Mac-2 binding protein is expressed in prostate cancer and induces promatrilysin expression

BACKGROUND: 90K/Mac-2 binding protein is a cell adhesive protein whose level of expression has been correlated with metastatic potential in many different tumor types. The purpose of this investigation was to examine 90K expression in prostate cancer and to determine a possible role for 90K in cancer progression. METHODS: 90K expression in prostate cell lines and tissue samples was evaluated by immunohistochemistry. Expression in cell lines was also evaluated by Western blot analysis and real-time RT-PCR. Induction of promatrilysin by 90K was evaluated by ELISA. RESULTS: Some of the human prostate cell lines studied expressed 90K. 90K was over-expressed in 38.8% of prostate cancer tumor samples, 7.14% of PIN lesions, and 18.6% of normal tissue. 90K was also shown to induce promatrilysin expression in the prostate cell line, LNCaP. CONCLUSIONS: These data demonstrate that 90K is over-expressed in a large fraction of malignant tumors. The fact that 90K can induce expression of promatrilysin indicates a possible role for 90K in cancer progression and metastasis. This suggests that 90K over-expression may be a useful marker for examining prostate cancer progression.     

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。     

趋化因子CXC受体4在前列腺癌细胞中的表达

目的:对趋化因子CXC受体4(CXCR4)在前列腺癌(PCa)组织以及PCa细胞系PC-3M中的表达进行研究。方法:提取正常前列腺组织、PCa组织和PC-3M细胞的总RNA,采用RT-PCR方法,于mRNA水平检测其CXCR4的表达情况;制备PC-3M细胞爬片和MCF-7细胞爬片(人乳腺癌细胞系,作为阳性对照),采用SABC免疫组织化学方法,于蛋白水平测定CXCR4在PC-3M细胞中的表达。结果:RT-PCR方法显示PC-3M细胞和PCa组织CXCR4 mRNA有较高水平的表达,正常前列腺组织未见CXCR4 mRNA的表达;SABC显示PC-3M细胞CXCR4蛋白呈强表达。结论:CXCR4在PCa组织和PC-3M细胞中有明显表达,在正常前列腺组织中无明显表达,CXCR4可能在PCa的发生发展中发挥重要作用。     

Immunohistochemistry and biochemistry in detection of androgen, progesterone, and estrogen receptors in benign and malignant human prostatic tissue.

The relative distribution of androgen (AR), progesterone (PR), and estrogen receptors (ER) was localized and estimated in human prostate tissue by immunohistochemistry in five normal tissue samples, in eight benign hyperplastic (BPH) samples, in nine primary cancers, and in seven prostate cancer metastases. Moreover, three prostatic cancer cell lines (LNCaP, DU 145, and PC 3) were analyzed. A comparison between the results obtained by radioligand binding assays and immunohistochemistry was performed for the AR and PR. Using immunohistochemistry, the AR was exclusively detected in the nuclei of both benign and malignant prostatic epithelial cells. The highest proportion of AR-positive cells was found in BPH and in prostate cancer metastases as compared with normal prostatic tissue. In a majority of the cases, the PR was only present in the nuclei of stromal cells. Benign hyperplastic prostates contained higher proportions of PR-positive cells as compared with primary carcinoma. PR was sparse in epithelial cells. ER-positive stromal cell nuclei were only detected in carcinomatous prostates. A few ER-positive epithelial cell nuclei were found in one sample each of a BPH and normal prostate. All cells from the androgen-dependent, LNCaP, cell line and a majority of the cells from the androgen-independent, DU 145, cell line were AR-positive. In contrast, the cells from the androgen-independent, PC 3, cell line were all AR-negative. All three cell lines were PR- and ER-negative. The radioligand binding technique detected the AR in extracts from both the cytosol and the nucleus. Again BPH contained higher amounts of AR as compared with normal prostatic tissue. The LNCaP cells contained high amounts of cytosolic AR while cells from the DU 145 and PC 3 cell lines lacked detectable AR as estimated by biochemical techniques. There seemed to be a discrepancy between biochemically measured and immunohistochemically estimated receptor content.     

Expression profile of an androgen regulated prostate specific homeobox gene NKX3.1 in primary prostate cancer

PURPOSE: NKX3.1, a member of the family of homeobox genes, exhibits prostate tissue specific expression and appears to play a role in mouse prostate development. Rapid induction of NKX3.1 gene expression in response to androgens has also been described. On the basis of the established role of androgens in prostatic growth and differentiation and studies showing an association of aberrant homeobox gene expression with the neoplastic process, we hypothesize that alterations of NKX3.1 gene expression play a role in prostate tumorigenesis. MATERIALS AND METHODS: NKX3.1 expression was analyzed in matched, microdissected normal and tumor tissues from 52 primary prostate cancer specimens from radical prostatectomy by semiquantitative RT-PCR and in situ hybridization and correlated with the clinicopathologic features. NKX3.1 expression was quantified as differential expression between matched tumor and normal tissues and was grouped as overexpression in tumor tissue, reduced expression in tumor tissue and no change between tumor and normal tissues. Androgen regulation of NKX3.1 expression was also studied in LNCaP cells. Androgen receptor (AR) expression in prostate tumor and normal tissue was correlated with NKX3.1 expression. RESULTS: Comparison of NKX3.1 expression between normal and tumor tissues revealed overexpression in 31% tumor specimens (16 of 52), decreased expression in 21% tumor specimens (11 of 52) and no change in 48% specimens (25 of 52). When these expression patterns were stratified by organ confined and non-organ-confined tumor, a higher percentage of patients exhibited NKX3.1 overexpression in non-organ confined tumor (40%) versus organ confined tumor (22%). Elevated NKX3.1 expression significantly correlated with tumor volume and serum prostate specific antigen (PSA) level in the NKX3.1 overexpression group (p<0.05). Metastatic prostate cancer cell lines did not exhibit mutations in the protein coding sequence of NKX3.1. Additionally, the NKX3.1 expression correlated with AR expression (p<0.01) in vivo in human prostate tissues. Comparison of PSA and NKX3.1 expression in response to androgen revealed a rapid androgen mediated induction of NKX3.1 expression in LNCaP cells. In situ hybridization analysis of representative specimens confirmed RT-PCR observations. CONCLUSIONS: These results suggest an association of NKX3.1 with a more aggressive phenotype of carcinoma of the prostate. Correlation of AR expression with NKX3.1 in human prostate tissues underscores the androgen regulation of NKX3.1 in the physiologic context of human prostate tissues.