Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis

To better understand the carcinogenesis of bladder cancer in Taiwan, we utilized the proteomic approach to search for potential biomarkers of transitional cell carcinoma (TCC). Analysis by 2-DE and MS/MS indicated that seven proteins are down-regulated and three proteins up-regulated in grade III samples as compared with those of grade II. Of these deregulated proteins, fatty acid binding proteins, annexin V, heat-shock protein 27, and lactate dehydrogenase have been shown to be associated with bladder cancer. Our studies also found altered expression of a group of proteins that have not been documented previously in bladder cancer, including annexin I, 15-hydroxyprostaglandin dehydrogenase, galectin-1, lysophospholipase and mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor. These results illustrate a pattern of differential protein expression between low- and high-grade tumors and it may be utilized as the molecular fingerprinting of a subset of bladder cancers. In addition, the present study provides a valuable resource in the study of pathological mechanisms in cancers of urothelial origin. The immunohistochemical staining of grade II and III TCC samples with antiserum to annexin I protein was utilized to confirm that the annexin I protein is up-regulated in grade III TCC.     

Cloning,differential tissue expression of a novel hcApo gene,and its correlation with total carotenoid content in purple and white inner-shell color pearl mussel Hyriopsis cumingii

As a molecular carrier and storage protein, apolipoprotein (Apo) mediates the intracellular uptake of lipids, proteins, vitamins and carotenoids. In this study, we identified a novel Apo gene, designated hcApo, from the freshwater pearl mussel Hyriopsis cumingii. The complete hcApo cDNA consists of 4104 nucleotides with an open reading frame encoding 1155 amino acid residues. The hcApo protein contains a conserved lipoprotein N-terminal domain (LPD-N) that is a characteristic of the large lipid transfer protein (LLTP) superfamily. The hcApo mRNA is constitutively expressed in a wide range of tissues with the highest expression level in the liver. Moreover, differential expression analysis revealed that the hcApo gene is more highly expressed in the liver, kidney, mantle and gill of purple line mussels compared to white line mussels. In situ hybridization investigations of the precise expression site of hcApo mRNA in the mantle showed that hcApo mRNA is specifically expressed in the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold of the mantle, as well as throughout the outer epithelium of the outer fold and ventral mantle. Another very important finding is that significantly positive correlation existed between the hcApo gene expression level and the total carotenoid content in purple line mussels. These findings may provide a better understanding of the roles of hcApo in the molecular mechanisms of shell formation and coloring of H. cumingii.     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

Co-localization of GSTP1 and JNK in transitional cell carcinoma of urinary bladder

Transitional cell carcinoma (TCC) of urinary bladder belongs to glutathione S-transferase P1 (GSTP1) overexpressing tumors. Upregulated GSTP1 in TCC is related to apoptosis inhibition. This antiapoptotic effects of GSTP1 might be mediated through protein:protein interaction with c-Jun NH(2) -terminal kinase (JNK). Herein, we analyzed whether a direct link between GSTP1 and JNK exists in TCC. The presence of GSTP1/JNK complexes was analyzed by immunoprecipitation and Western blotting in 20 TCC specimens, obtained after surgery. Co-localization of GSTP1 and JNK was also investigated in the 5637 TCC cell line by immunofluorescence confocal microscopy. By means of immunoprecipitation we show for the first time the presence of GSTP1/JNK complexes in all TCC samples studied. A co-localization of GSTP1 and JNK was also demonstrated in the 5637 TCC cell line by means of confocal microscopy. Protein-protein interactions, together with co-localization between GSTP1 and JNK provide evidence that GSTP1 most probably inhibits apoptosis in TCC cells by non-covalent binding to JNK.     

Flow Cytometric analysis of Th1 and Th2 cytokines in PBMCs as a parameter of immunological dysfunction in patients of Superficial Transitional cell carcinoma of bladder

Transitional cell carcinoma (TCC) is the commonest cancer of the bladder. Although majority of TCC can be diagnosed at an early stage and removed easily by transurethral resection of tumor (TURT), the management of this carcinoma is complicated due to frequent recurrences usually within 6 months to one-year period. An imbalance between the Th1 and Th2 immune responses has been attributed to immune dysregulation in various malignancies. The present study aims to evaluate the Th1 and Th2 balance in Peripheral Blood Mononuclear Cells of 41 TCC patients (20 recurrent and 21 non-recurrent) using flow cytometry. It also further assesses immunological and cellular factors influencing the anti-neoplastic activity of the TCC patients and in 21 normal healthy subjects in terms of their cytokine expression and various cell surface markers. The findings of the study revealed that the cell surface markers CD3+, CD4+ and CD8+ along with NK cells were found to be significantly lower in patients than healthy controls (p<0.01). The mean percent expression of CD4+ was significantly lower in patients showing recurrence (23.9±9.84) as compared to patients with non-recurrence (31.1±12.27). The percentage of CD4+T-cells (mean±SD) producing IFN-γ, IL-2 and TNF-α were statistically significantly reduced in patients (19.1±4.94, 52.3±20.86 and 12.8±4.49) as compared to healthy controls (23.3±3.67, 67.5±12.0 and 17.6±5.96 respectively), (p<0.01, 0.018, 0.001). On the contrary, the mean levels of IL-4, IL-6 and IL-10 in patients (63.8±17.01, 60.4±14.79 and 65.7±14.84 respectively) were significantly higher as compared to healthy controls (24.4±8.77, 26.5±5.28 and 20.6±3.81 respectively), (p<0.001). No statistically significant difference was observed in the cytokine expression between patients showing recurrence and non-recurrence. Patients with bladder cancer seem to develop a Th2 dominant status with a deficient type1 immune response. The lymphocyte evaluation along with cytokine measurement can provide a sensitive and valuable tool for evaluating the function of cell-mediated immunity in these patients and can also find application in therapeutic monitoring of bladder cancer patients as new targets for immunotherapy.     

The role of chemokine and chemokine receptor gene variants on the susceptibility and clinicopathological characteristics of bladder cancer

The gene variants of the chemokine and chemokine receptor genes associated with inflammation may be involved in cancer initiation and progression. The aim of this study was to explore the possible association of monocyte chemoattractant protein-1 (MCP-1) A2518G, stromal cell derived factor 1 (SDF-1) 3′A and chemokine receptors CCR2A V64I, CCR5 Δ32, CCR5 59029 and CXCR4 gene polymorphisms with the risk and clinicopathological characteristics of bladder cancer (BC) in a Turkish population.     

A mutation in the heparin-binding site of noggin as a novel mechanism of proximal symphalangism and conductive hearing loss

The access of bone morphogenetic protein (BMP) to the BMP receptors on the cell surface is regulated by its antagonist noggin, which binds to heparan-sulfate proteoglycans on the cell surface. Noggin is encoded by NOG and mutations in the gene are associated with aberrant skeletal formation, such as in the autosomal dominant disorders proximal symphalangism (SYM1), multiple synostoses syndrome, Teunissen–Cremers syndrome, and tarsal–carpal coalition syndrome. NOG mutations affecting a specific function may produce a distinct phenotype. In this study, we investigated a Japanese pedigree with SYM1 and conductive hearing loss and found that it carried a novel heterozygous missense mutation of NOG (c.406C > T; p.R136C) affecting the heparin-binding site of noggin. As no mutations of the heparin-binding site of noggin have previously been reported, we investigated the crystal structure of wild-type noggin to investigate molecular mechanism of the p.R136C mutation. We found that the positively charged arginine at position 136 was predicted to be important for binding to the negatively charged heparan-sulfate proteoglycan (HSPG). An in silico docking analysis showed that one of the salt bridges between noggin and heparin disappeared following the replacement of the arginine with a non-charged cysteine. We propose that the decreased binding affinity of NOG with the p.R136C mutation to HSPG leads to an excess of BMP signaling and underlies the SYM1 and conductive hearing loss phenotype of carriers.     

UCA1, a non-protein-coding RNA up-regulated in bladder carcinoma and embryo, influencing cell growth and promoting invasion

A non-protein-coding RNA, UCA1, has been cloned from human bladder TCC cell line BLZ-211 by using 5' and 3' RACE. The UCA1 full-length cDNA was 1442 bp. RT-PCR analysis indicated that UCA1 is an embryonic development and bladder cancer-associated RNA. The proliferative, migrative, invasive, and drug resistance behaviors of human bladder TCC cell line BLS-211 were enhanced by exogenous UCA1 expression in vitro. Several potential target genes of UCA1 were identified through microarray analysis. Moreover, the expression of UCA1 also increased tumorigenic potential of BLS-211 cells in nude mice. Results from the present study suggested that UCA1 might play a pivotal role in bladder cancer progression and embryonic development.