Expression of a human cell adhesion molecule, MUC18, in prostate cancer cell lines and tissues

BACKGROUND: Over expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. METHODS: RT-PCR and Western blot analyses were used to analyze the expression of MUC18 mRNA and protein in four human prostate cancer cell lines, cultured primary normal prostate epithelial cells, normal prostate and malignant prostate tissues. Immunohistochemistry was used to determine the expression of MUC18 antigen in prostatic tissues at different stages of malignancy. RESULTS: Human MUC18 mRNA and protein was expressed in three different prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one prostate cancer cell line (LNCaP.FGC). HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from either cultured primary normal prostatic epithelial cells or the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas and in cells of a lymph node metastasis compared to that in normal or benign hyperplastic epithelium (BPH). CONCLUSIONS: We therefore conclude that MUC18 is expressed at higher levels in pre-malignant and malignant prostatic epithelium, including metastasis. We suggest that over-expression of MUC18 may be a new marker of human prostate cancer and also implicates its possible role in development and progression of prostate cancer.     

KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that is expressed at lower levels in prostate cancer cells than in normal prostate cells

BACKGROUND: Fifteen human tissue kallikrein (KLK) genes have been identified as a cluster on chromosome 19. KLK expression is associated with various human diseases including cancers. Noncoding RNAs such as PCA3/DD3 and PCGEM1 have been identified in prostate cancer cells. METHODS: Using massively parallel signature sequencing (MPSS) technology, RT-PCR, and 5' rapid amplification of cDNA ends (RACE), we identified and cloned a novel gene that maps to the KLK locus. RESULTS: We have characterized this gene, named as KLK31P by the HUGO Gene Nomenclature Committee, as an unprocessed KLK pseudogene. It contains five exons, two of which are KLK-derived while the rest are "exonized" interspersed repeats. KLK31P is expressed abundantly in prostate tissues and is androgen regulated. KLK31P is expressed at lower levels in localized and metastatic prostate cancer cells than in normal prostate cells. CONCLUSIONS: KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that may play a role in prostate carcinogenesis or maintenance.     

Identification of a novel gene on chromosome 13 between BRCA-2 and RB-1

METHODS AND RESULTS: By differential display we isolated a new cDNA-fragment, named C13, that is downregulated in malignant prostate tissues. Northern hybridization revealed the fragment to be part of 3.0 and 4.4 kb mRNAs. Fluorescence in situ hybridization, Southern blotting and radiation hybrid mapping demonstrated a chromosomal localization of C13 on 13q12-14 closest to the SHGC-34125 marker. In the 5% chromosomal environment of C13 we detected changes of the allelic status in 13 of 21 prostate cancers. A downregulation was detected at the mRNA level in patients with advanced carcinoma. The 3.0 kb full length cDNA clone encodes a protein with an open reading frame of 2,202 bp or 733 amino acids. The corresponding protein contains a putative nuclear localization signal, several glutamine clusters and an alpha-helix-rich domain. By in situ RNA hybridization we could demonstrate the mainly epithelial expression of the C13 mRNA in prostatic tissue. CONCLUSIONS: The localization of C13 between the tumor suppressor genes BRCA-2 and RB-1, the detected allelic imbalances, the downregulation of its mRNA in some prostatic cancer tissues, the epithelial expression and the described protein structure suggest that this gene encodes a protein that may have tumor or metastasis suppressing function in prostate tissue.     

人前列腺干细胞抗原在前列腺组织中的表达

目的：克隆人前列腺干细胞抗原(PSCA)基因，并利用半定量逆转录聚合酶链反应(RT—PCR)对其组织表达谱进行分析。方法：从人前列腺癌组织提取总RNA，利用RT—PCR技术扩增出人PSCA基因编码区序列，并通过半定量RT—PCR方法检测6例人新鲜正常前列腺组织、16例前列腺增生组织及11例前列腺肿瘤组织中PSCA基因表达水平。结果：序列测定表明，克隆获得的369bp片段与文献报道的人PSCA基因编码区cDNA序列一致，PSCA为前列腺组织特异表达，在前列腺癌组织中的表达明显高于前列腺增生及前列腺正常组织。结论：PSCA是一种新的前列腺肿瘤标记物。     

Activation of epithelial sodium channels by prostasin in Xenopus oocytes

Prostasin, a novel serine protease, was purified from seminal fluid, and its cDNA sequence was determined. Expression of prostasin was detected in human tissues, including prostate, kidney, and lung, as well as bodily fluids, including seminal fluid and urine. However, its physiologic role in the human body is not known. Recently, a novel regulatory mechanism by which serine proteases activate epithelial sodium channel in the Xenopus oocyte was identified. Therefore, it was hypothesized that prostasin could activate sodium currents, and a rat prostasin cDNA clone was isolated to investigate its physiologic function. Rat prostasin mRNA is expressed predominantly in kidney, and lower levels of expression were detected in prostate, lung, colon, stomach, and skin. These all are epithelial tissues in which the epithelial sodium channel (ENaC) is expressed. Coexpression of rat prostasin and rat ENaC in Xenopus oocytes increased the amiloride-sensitive sodium current by twofold. Preincubation of oocytes that expressed prostasin with aprotinin did not result in an increase in sodium current, compared with the control. The removal of aprotinin from the bath solution resulted in a twofold increase of the current only in oocytes that expressed prostasin, which indicates that protease activity of prostasin is required for the ENaC activation. Expression of rat prostasin had no effect on the potassium current when expressed with rat renal outer medulla K channel, which shows specificity of prostasin action for ENAC: These results indicate that prostasin acts as an extracellular regulator of ENAC:     

Caspase 3 expression in benign prostatic hyperplasia and prostate carcinoma

BACKGROUND: Apoptotic resistance to androgen ablation represents a significant problem in the treatment of prostate cancer. Over expression of antiapoptotic proteins such as Bcl-2 and mutations in p53 contribute to this resistance. The caspase family of proteases are central executioners of the cell death pathway. They are expressed in normal prostate secretory epithelial cells. Altered expression may represent an additional component leading to cell resistance. The aim of this study was to determine by immunohistochemistry caspase 3 expression in benign prostatic hyperplasia and prostate cancers. METHODS: Twenty-two patients with histologically determined prostate cancer and benign prostatic hyperplasia (BPH) were investigated. All specimens were obtained from patients undergoing surgical resection of the prostate. Immunohistochemical analysis was performed on formalin fixed paraffin embedded sections to assess caspase 3 expression. RESULTS: Caspase 3 was expressed in 18/22 (81.1%) samples, with high expression in BPH which demonstrated staining in both basal and secretory epithelial cells. Increasing grades of prostatic cancer showed a significant loss of expression in secretory epithelial layers and little staining in epithelial cells in high-grade prostatic carcinoma. CONCLUSIONS: Altered caspase 3 expression may represent an additional mechanism of apoptotic resistance to androgen ablation. Prostate 47:183-188, 2001.     

Radiation and androgen withdrawal alter expression of apoptosis pathway genes of prostate cancer cells

Aim: To investigate the altered expression of apoptosis pathway genes of prostate cancer cells treated by radiation and androgen withdrawal and whether the combined treatment may induce additive apoptosis. Methods: Androgen sensitive prostate cancer cell line LNCaP was cultured and treated by radiation, androgen withdrawal and combination of the two. Apoptosis was determined using apoptotic cells staining and mononuclear cell direct cytotox-icity assay. The total RNA were extracted and harvested. cDNA probes were prepared and labeled with biotin-16-dUTP and then hybridized to commercially available cDNA arrays, including apoptosis pathway-specific genes. The expression of important gene was further determined using RT-PCR. Results: Radiation induced additive apoptosis of prostate cancer cells; androgen withdrawal exhibited synergetic action. TNFRSF8 variant 2, DFFA, LTbR, mdm2, Myd88, TNFRSF14 and TNFSF4 mRNA were up regulated by radiation, while Survivin and Bar mRNA were down regulated. Mcl-1, TNFRSF14     

Isolation and characterization of mouse probasin: An androgen-regulated protein specifically expressed in the differentiated prostate

BACKGROUND: The development and growth of the prostate gland is regulated, in part, by a variety of steroid and polypeptide growth-factor hormones. As a consequence of hormone action, the prostate gland will produce a number of tissue-restricted gene products. Characterization of the regulation, expression, and function of genes encoding prostate-specific proteins is critical to our understanding of prostate biology. Probasin is a prostate-specific gene originally isolated from the rat and has been exploited as a marker of prostate differentiation and to elucidate androgen action. Furthermore, a number of transgenic mouse models of prostate cancer have been established based on the regulatory elements derived from the rat probasin gene. In this report, we describe the isolation and characterization of the mouse probasin ortholog to further facilitate studies related to hormone action in the prostate and the generation and characterization of novel autochthonous models of prostate cancer. METHODS: Mouse probasin cDNA was isolated from a phage library, and the DNA sequence was determined. The predicted protein sequence was used to generate specific oligonucleotide primers and antibodies. Probasin protein and RNA expression were examined by immunobloting, immunohistochemistry, and RT-PCR, in normal mouse prostate tissue and tumor tissues derived from the autochthonous "transgenic adenocarcinoma of the mouse prostate" (TRAMP) model. Regulation of probasin expression in response to surgical castration and hormone supplementation was also characterized. RESULTS: Several points of evolutionary sequence conservation were identified between mouse and rat probasin, especially in the 3' untranslated region. Specific polyclonal antibodies were generated to peptide fragments, and the temporal and spatial pattern of probasin expression was examined. The expression of probasin was primarily localized to the apical membrane of differentiated secretory epithelium. Probasin mRNA and protein were absent from the poorly differentiated tissue of TRAMP tumors. Probasin was found to be androgen-regulated. In contrast to data from studies on rat probasin, no postcastration rebound of mouse probasin mRNA was observed. CONCLUSIONS: Probasin is a marker of differentiation and androgen action in the mouse prostate, and strong sequence conservation between mouse and rat probasin supports an essential role for this gene in the biology of the prostate gland. Isolation and characterization of mouse probasin will facilitate further development and analysis of autochthonous mouse models of prostate cancer.     

Androgen regulation of spermidine synthase expression in the rat prostate

BACKGROUND: Spermidine synthase, an essential enzyme in the polyamine synthesis pathway, was identified as one of the androgen-response genes in the rat ventral prostate. Characterization of androgen regulation of spermidine synthase is important to the understanding of androgenic regulation of polyamine synthesis. METHODS: Full-length cDNA encoding rat spermidine synthase was isolated from a lambdaZAP cDNA phage library. Young male adult Sprague-Dawley rats were used for castration and androgen replacement. Northern blot and in situ hybridization were used to characterize gene expression. RESULTS: The amino acid sequence of rat spermidine synthase shares 99% and 94% identity with that of mouse and human spermidine synthase, respectively. Spermidine synthase gene is abundantly expressed and regulated by androgens in the ventral, dorsal, and lateral lobes of the rat prostate, and its expression is localized to the epithelial cells. Spermidine synthase also is regulated by androgens in the seminal vesicles but not in the muscle, brain, kidney, thymus, heart, or liver, suggesting that this enzyme is responsive to androgen in the male sex accessory organs only. The expression of spermidine synthase and two other enzymes involved in polyamine synthesis, S-adenosylmethionine decarboxylase and ornithine decarboxylase, are regulated by androgens coordinately. CONCLUSIONS: Spermidine synthase is most abundantly expressed and regulated by androgens in the prostatic epithelial cells, suggesting that regulation of spermidine synthase is likely a key step in coordinated androgen regulation of polyamine synthesis in the prostate.