Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

Expression analysis of thrombospondin 2 in prostate cancer and benign prostatic hyperplasia.

Thrombospondin 2 (TSP2) is a protein with important roles in different tumor types, mainly related to tumor inhibition. However, there are limiting data regarding TSP2 in prostate cancer (PCa) and benign prostatic hyperplasia (BPH). We aimed to investigate TSP2 transcript and protein expression in tumoral and non-tumoral prostate tissues and cell lines, and its implications for PCa diagnosis and progression. TSP2 transcript expression was evaluated by real time PCR in PCa and BPH tissue samples and in tumoral and non-tumoral cell lines. TSP2 protein expression analysis was conducted by immunohistochemistry in a tissue microarray (TMA) containing PCa and BPH tissue samples. TSP2 transcript was down-regulated in PCa tissue samples and cell lines, when compared to BPH and non-tumoral samples (P<0.01). Receiver Operating Curve (ROC) analysis demonstrated that TSP2 transcript levels can better distinguish PCa from BPH tissue samples (P<0.01) than serum PSA levels (P=0.299). TSP2 protein expression has been observed in the cytoplasm of both PCa and BPH epithelial and stromal compartments. TSP2 stromal staining scores were significantly lower in PCa than in BPH tissues (P<0.01), while similar TSP2 epithelial staining patterns were observed in both diseases. Notably, the TSP2 epithelial staining score was significantly correlated to vascular invasion and biochemical recurrence in PCa tissue samples (P<0.05). Our data indicate that TSP2 is down-regulated at PCa tissues and cell lines, especially at stroma compartment, which could be related to PCa progression. TSP2 levels could potentially be applied for differential PCa and BPH diagnosis.

Expression analysis of osteopontin mRNA splice variants in prostate cancer and benign prostatic hyperplasia.

Osteopontin splicing isoforms (OPN-SI) present differential expression patterns and specific tumor roles. Our aims were to characterize OPN-SI expression in prostate cancer (PCa) and benign prostate hyperplasia (BPH) tissues, besides evaluating their potential as biomarkers for PCa diagnosis and prognostic implications. Prostatic tissue specimens were obtained from 40 PCa and 30 benign prostate hyperplasia (BPH) patients. Quantitative real time PCR (qRT-PCR) was used to measure OPN-SI mRNA expression. Immunohistochemical analysis was performed using an anti-OPNc polyclonal antibody. Biostatistical analyses evaluated the association of OPN-SI and total Prostate Specific Antigen (PSA) serum levels with clinical and pathological data. PCa tissue samples presented significantly higher levels of OPNa, OPNb and OPNc transcripts (p<0.01) than in BPH specimens. OPN-SI mRNA expression were positively correlated with Gleason Score (p<0.01). ROC curves and logistic regression analyses demonstrated that OPN-SI and PSA were able to distinguish PCa from BPH patients (p<0.01). The OPNc isoform was the most upregulated variant and the best marker to distinguish patients' groups, presenting sensitivity and specificity of 90% and 100%, respectively. Immunohistochemistry analysis also demonstrated OPNc upregulation in PCa samples as compared to BPH tissues. OPNcprotein was also strongly stained PCa tissues presenting High Gleason Score. Multivariate analysis indicated that OPNc expression levels above the cut-off value presented a chance 4-fold higher for PCa occurrence. We conclude that OPN-SI were overexpressed in PCa tissues, strongly associated with PCa occurrence and with tumor cell differentiation. Our results suggest OPNc splicing isoform as an important biomarker contributing to improve PCa diagnosis and prognosis, besides providing insights into early steps of PCa carcinogenesis.

Analysis of inherited genetic variants in ret proto-oncogene of Brazilian patients with apparently sporadic medullary thyroid carcinoma.

Mutations in different exons of ret proto-oncogene are responsible for the development of medullary thyroid carcinoma (MTC). The mutations can occur as sporadic or as part of multiple endocrine neoplasia (MEN) type 2 hereditary syndromes. Here we report the first focused study of sporadic MTC in Brazilian patients regarding clinical and molecular analysis of ret proto-oncogene. Our study seeks to estimate the risk of hereditary MTC cases among apparently sporadic cases in a Brazilian population and describe ret genetic variants in their germinative lineage. Germinative sequence variants were screened by DNA sequencing and denaturing gradient gel electrophoresis (DGGE) analysis of exons 10, 11, 13, 14, 15, and 16 of 24 Brazilian patients with apparently sporadic MTC. We identified 1 inherited case of 24 (4%) patients with apparently sporadic MTC. Polymorphisms for the ret proto-oncogene coding region were identified in codon 769 of exon 13 (LeuCTT--> LeuCTG) at a frequency of 13% (3/24) and in codon 904 of exon 15 (SerTCC--> SerTCG) at a frequency of 16.6% (4/24). The observed frequency (4%) of inherited disease among apparent sporadic MTC strengthens routine application of ret proto-oncogene germinative DNA screening in all cases of apparently sporadic MTC ascertained at Brazilian cancer hospitals.

Dolichol phosphate mannose synthase is differentially expressed in male and female worms of Schistosoma mansoni.

The cDNA encoding the Schistosoma mansoni dolichol phosphate mannose synthase was completely sequenced, displaying the highest homology with Cricetulus griseus and Saccharomyces pombe genes. The Schistosome enzyme had a K(m) of 0.127 microM, a value that is within the range of those reported for several other species. Thin-layer chromatography of the radiolabelled schistosome lipid intermediate showed it was identical to dolichol-phosphate (C80-C105). Expression of dolichol phosphate mannose synthase of S. mansoni (SmDPMS) was analysed by Northern blot and quantified by semi-quantitative RT-PCR with cDNA from mature and immature male and female worms. Northern blot analysis revealed a single 1-kb band. Both approaches confirmed a higher level of expression in mature female worms, as compared to immature and male worms.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line.

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.

Human osteopontin splicing isoforms: known roles, potential clinical applications and activated signaling pathways.

Human osteopontin is subject to alternative splicing, which generates three isoforms, termed OPNa, OPNb and OPNc. These variants show specific expression and roles in different cell contexts. We present an overview of current knowledge of the expression profile of human OPN splicing isoforms (OPN-SIs), their tissue-specific roles, and the pathways mediating their functional properties in different pathophysiological conditions. We also describe their putative application as biomarkers, and their potential use as therapeutic targets by using antibodies, oligonucleotides or siRNA molecules. This synthesis provides new clues for a better understanding of human OPN splice variants, their roles in normal and pathological conditions, and their possible clinical applications.

Auto-antibodies in prostate cancer: humoral immune response to antigenic determinants coded by the differentially expressed transcripts FLJ23438 and VAMP3.

BACKGROUND: Here we evaluate auto-antibody response against two potential antigenic determinants of genes highly expressed in low Gleason Score prostate cancer (PC) tumor samples, namely FLJ23438 and VAMP3. METHODS: RT-PCR assays were used to analyze mRNA expression profiles of FLJ23438 and VAMP3 transcripts. The auto-antibody response against FLJ23438 and VAMP3 recombinant proteins was tested by immunoblot assays using PC, benign prostate hyperplasia (BPH), healthy donors (HD), and other human cancers plasma samples. RESULTS: Our data showed that 37% (10/27) and 7.4% (2/27) of PC plasma samples presented auto-antibodies against FLJ23438 and VAMP3, respectively. Only 8.3% (1/12) of BPH plasma samples were reactive for both auto-antibodies, while none (0/12) of HD plasma samples tested were reactive. CONCLUSIONS: The prevalence of 37% of positive PC plasma samples for anti-FLJ23438 antibodies suggests that humoral immune response against this antigenic determinant could be a potential serum marker for this cancer.

Lack of DNA methylation in Schistosoma mansoni.

Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines