Developmental expression of Magmas in murine tissues and its co-expression with the GM-CSF receptor.

Magmas is a protein that is involved in GM-CSF signaling in a myeloid cell line. Its precise role in the signal transduction process is unclear. To accurately characterize Magmas expression in a variety of cells, mouse embryos and adult murine tissues were analyzed for both mRNA and protein content. Magmas expression was detected as early as the day 6.5 embryo. The level of expression was developmentally regulated. During embryogenesis, elevated Magmas was observed in several structures, including heart, liver, notochord, choroid plexus, cervical ganglion, and nasal mucosa. Muscle, pancreas, intestinal mucosa, and testes were among the adult tissues with high Magmas expression. Most cell types, including hepatocytes and skeletal, smooth, and cardiac myocytes, also expressed the GM-CSF receptor (GMR) but the relative tissue levels of GMR were not always proportional to Magmas. The expression patterns suggest that Magmas has a role in both developing and mature tissues.     

Reduced expression of NGEP is associated with high-grade prostate cancers: a tissue microarray analysis

New gene expressed in prostate (NGEP) is a newly diagnosed prostate-specific gene that is expressed only in normal prostate and prostate cancer cells. Discovery of tissue-specific markers may promote the development of novel targets for immunotherapy of prostate cancer. In the present study, the staining pattern and clinical significance of NGEP were evaluated in a series of prostate tissues composed of 123 prostate cancer, 19 high-grade prostatic intraepithelial neoplasia and 44 samples of benign prostate tissue included in tissue microarrays using immunohistochemistry. Our study demonstrated that NGEP localized mainly in the apical and lateral membranes and was also partially distributed in the cytoplasm of epithelial cells of normal prostate tissue. All of the examined prostate tissues expressed NGEP with a variety of intensities; the level of expression was significantly more in the benign prostate tissues compared to malignant prostate samples (P value <0.001). Among prostate adenocarcinoma samples, a significant and inverse correlation was observed between the intensity of NGEP expression and increased Gleason score (P = 0.007). Taken together, we found that NGEP protein is widely expressed in low-grade to high-grade prostate adenocarcinomas as well as benign prostate tissues, and the intensity of expression is inversely proportional to the level of malignancy. NGEP could be an attractive target for immune-based therapy of prostate cancer patients as an alternative to the conventional therapies particularly in indolent patients.     

前列腺组织中神经生长因子(NGF)表达的研究

研究前列腺组织中神经生长因子（ＮＧＦ） 的生理学意义。采用原位杂交和免疫组化法, 检测４３ 例前列腺增生组织, ８ 例腺癌组织和８ 例正常组织中β－ＮＧＦｍ ＲＮＡ及其蛋白的表达及分布。结果显示β－ＮＧＦｍ ＲＮＡ 在正常组织及增生组织中定位于间质细胞, 偶见于上皮细胞中; 而在癌组织中, 上皮细胞和间质细胞有同样强度的β－ＮＧＦｍ ＲＮＡ染色。其蛋白在良性组织中表达主要着色在间质细胞中,上皮细胞呈弱表达,而癌组织中上皮细胞见着色明显增强（Ｐ＜ ０．０５）。ＮＧＦ的自分泌异常可见是前列腺组织由良性向恶性转变的原因之一。     

Differences in steroid 5alpha-reductase iso-enzymes expression between normal and pathological human prostate tissue.

We studied the expression level and cell-specific expression patterns of 5alpha-reductase (5alpha-R) types 1 and 2 iso-enzymes in human hyperplastic and malignant prostate tissue by semi-quantitative RT-PCR and in situ hybridisation analyses. In situ hybridisation established that 5alpha-R1 mRNA is preferentially expressed by epithelial cells and little expressed by stromal cells whereas 5alpha-R2 mRNA is expressed by both epithelium and stroma. Semi-quantitative RT-PCR has been performed on total RNA from different zones of normal prostate, BPH tissues and liver. We found that 5alpha-R1 and 5alpha-R2 mRNAs expression was near the same in all zones of normal prostate. In BPH tissue, 5alpha-R1 and 5alpha-R2 mRNAs expression was slightly but significantly increased, when it was compared to the levels recorded for normal prostate. In cancer samples, 5alpha-R1 mRNA expression was higher than in normal and hyperplastic prostate but the level of 5alpha-R2 mRNA was not statistically different from that observed in the different zones of normal prostate. In liver, 5alpha-R2 mRNA level was similar to that measured in BPH but 5alpha-R1 mRNA expression was ten times higher. The increase observed in 5alpha-R isoenzymes expression in BPH tissue could play an important role in the pathogenesis and/or maintenance of the disease.     

Peroxisome proliferator-activated receptor- gamma expression in human malignant and normal brain, breast and prostate-derived cells.

The constitutive and gamma -linolenic acid (GLA)-induced expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) immunoreactive protein in a panel of human malignant brain (U87MG, T98G); breast (MCF-7, MB MDA-231, MB MDA 435) and prostate (ALVA, DU-145, LNCaP, PC3) cell lines have been compared with those for their normal cell counterparts, the human normal astrocyte (NHA), mammary epithelial (HMEC) and prostate epithelial (PrEC) cells, respectively. Constitutive levels of expression for PPAR gamma protein were significantly higher in the malignant cell lines relative to their normal cells. GLA supplementation did not affect the protein expression in malignant cells but caused 6- and 3-fold increases in normal breast and prostate cells, respectively. Since activation of PPAR gamma protein in some human malignant cell lines has been demonstrated to induce tumour cell death, these findings signal the need to exploit the significantly elevated expression of this protein in the therapy of human cancer.     

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical 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 development and 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. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. 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 cultured primary normal prostatic epithelial cells and 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 perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.     

Zinc transporter mRNA expression in the RWPE-1 human prostate epithelial cell line

The human prostate gland undergoes a prominent alteration in Zn+2 homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU-145 and PC-3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 microM Zn+2 for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn+2 with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn+2 had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.     

N-cadherin mediates angiogenesis by regulating monocyte chemoattractant protein-1 expression via PI3K/Akt signaling in prostate cancer cells

Over the past decade, evidence continues to mount showing that N-cadherin is a critical protein in cancer progression and metastasis. In the present study, we evaluated the expression of N-cadherin in human prostate cancer tissue specimens and cell lines. Enhanced expression of N-cadherin was observed in both the malignant and bone-metastasized prostate tissue specimens compared to the healthy prostate tissues. Consistent with the tissue array data, N-cadherin was highly expressed in PC3, but not in Du145 and LNCaP human prostate cell lines. Based on cell to cell binding assay, we found that N-cadherin expression facilitates homotypic interaction between human prostate cancer cells and human microvascular endothelial cells (HMEC). Human angiogenesis antibody array and in vitro angiogenesis assay showed that siRNA-mediated knockdown of N-cadherin reduced the secretion of monocyte chemoattractant protein-1 (MCP-1), which played a potential role in stimulating capillary network formation of HMEC. Additionally, culture supernatant of Du145 cells transfected with full-length N-cadherin expressing plasmid showed increased MCP-1 expression and chemoattractant ability compared to normal Du145 cells. Further, we noticed that blocking PI3K activity inhibited N-cadherin mediated MCP-1 expression. Our data demonstrated that N-cadherin in prostate cancer cell mediates cell–cell adhesion and regulates MCP-1 expression via the PI3K/Akt signaling pathway.     

Expression of the human copper influx transporter 1 in normal and malignant human tissues.

The major copper influx transporter, copper transporter 1 (hCTR1), controls the cellular accumulation of cisplatin in mammalian cells. The goal of this study was to determine the pattern of hCTR1 expression in normal and malignant human tissues. Tissue arrays were stained with an antibody specific for hCTR1 using standard immunohistochemical techniques. Particularly strong staining was noted in the alpha cells of the pancreatic islets, enteroendocrine cells of the gastric mucosa and bronchioles, C cells of the thyroid, and a subset of cells in the anterior pituitary. Frequency and intensity of hCTR1 staining in malignant tissues reflected the levels found in their normal tissue counterparts. For example, neither normal prostate nor prostate cancers expressed hCTR1, whereas it was commonly expressed in both normal colonic epithelium and in colon carcinomas. Strong staining was observed in a limited number of cases of carcinoid tumors, Ewing's sarcoma, and undifferentiated carcinomas. Although all tissues require copper, expression of hCTR1 was highly variable among normal tissues and among the major human malignancies, with the highest levels found in enteroendocrine cells. No hCTR1 expression was found in several common types of cancer, suggesting that hCTR1 expression is not commonly enhanced by transformation.     

Akt Mediates Metastasis-Associated Gene 1 (MTA1) Regulating the Expression of E-cadherin and Promoting the Invasiveness of Prostate Cancer Cells

Human metastasis-associated gene 1 (MTA1) is highly associated with the metastasis of prostate cancer; however, the molecular functions of MTA1 that facilitate metastasis remain unclear. In this study, we demonstrate that the silencing of MTA1 by siRNA treatment results in the upregulation of E-cadherin expression by the phosphorylation of AKT (p-AKT) and decreases the invasiveness of prostate cancer cells. We show that MTA1 is expressed in over 90% of prostate cancer tissues, especially metastatic prostate cancer tissue, comparing to non-expression in normal prostate tissue. RT-PCR analysis and Western blot assay showed that MTA1 expression is significantly higher in highly metastatic prostate cancer PC-3M-1E8 cells (1E8) than in poorly metastatic prostate cancer PC-3M-2B4 cells (2B4). Silencing MTA1 expression by siRNA treatment in 1E8 cells increased the cellular malignant characters, including the cellular adhesive ability, decreased the cellular invasive ability and changed the polarity of cellular cytoskeleton. 1E8 cells over-expressing MTA1 had a reduced expression of E-cadherin, while 1E8 cells treated with MTA1 siRNA had a higher expression of E-cadherin. The expression of phosphorylated AKT (p-AKT) or the inhibition of p-AKT by wortmannin treatment (100 nM) significantly altered the function of MTA1 in the regulation of E-cadherin expression. Alterations in E-cadherin expression changed the role of p-AKT in cellular malignant characters. All of these results demonstrate that MTA1 plays an important role in controlling the malignant transformation of prostate cancer cells through the p-AKT/E-cadherin pathway. This study also provides a new mechanistic role for MTA1 in the regulation of prostate cancer metastasis.     

Magmas gene structure and evolution

Magmas is a nuclear encoded protein found in the mitochondria of mammalian cells. It participates in granulocyte-macrophage-colony stimulating factor (GM-CSF) signaling in hematopoietic cells and has an essential role in invertebrate development. In order to characterize the protein structural features and gene evolution of Magmas, a dataset containing 61 Magmas homologs from 52 species distributed among animals, plants and fungi was analyzed. All Magmas members were found to possess three novel sequence motifs in addition to a conserved leader peptide. Phylogenetic tree and dN/dS rate ratios showed that Magmas was evolutionarily conserved. Analysis of Magmas gene organization demonstrated incremental intron acquisition in plants and vertebrates. Significant genetic diversity in Magmas was observed from kingdom specific amino acid signatures, the presence of predicted signal peptides that target the protein to other intracellular locations besides the mitochondria, and the detection of multiple isoforms in higher animals. These studies demonstrate that Magmas members constitute an important family of conserved proteins having multifunctional activities, and provide a basis for future experiments.     

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post-messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock-induced gene expression, leading to abundant HSP induction in vitro or in vivo.     

Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer

The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.     

Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression

High levels of peripheral-type benzodiazepine receptor (PBR), the alternative-binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non-tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon-rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.     

Immunoexpression Analysis and Prognostic Value of BLCAP in Breast Cancer

Bladder Cancer Associated Protein (BLCAP, formerly Bc10), was identified by our laboratory as being down-regulated in bladder cancer with progression. BLCAP is ubiquitously expressed in different tissues, and several studies have found differential expression of BLCAP in various cancer types, such as cervical and renal cancer, as well as human tongue carcinoma and osteosarcoma. Here we report the first study of the expression patterns of BLCAP in breast tissue. We analyzed by immunohistochemistry tissue sections of normal and malignant specimens collected from 123 clinical high-risk breast cancer patients within the Danish Center for Translational Breast Cancer Research (DCTB) prospective study dataset. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. We observed weak immunoreactivity for BLCAP in mammary epithelial cells, almost exclusively localizing to the cytoplasm and found that levels of expression of BLCAP were generally higher in malignant cells as compared to normal cells. Quantitative IHC analysis of BLCAP expression in breast tissues confirmed this differential BLCAP expression in tumor cells, and we could establish, in a 62-patient sample matched cohort, that immunostaining intensity for BLCAP was increased in tumors relative to normal tissue, in more than 45% of the cases examined, indicating that BLCAP may be of value as a marker for breast cancer. We also analyzed BLCAP expression and prognostic value using a set of tissue microarrays comprising an independent cohort of 2,197 breast cancer patients for which we had follow-up clinical information.