To modify the splicing pattern of Bcl-x and compare the effect of this approach with that of the antisense gene therapy in BIU-87 cell line of bladder cancer, by using 5'-Bcl-x AS to target downstream alternative 5'-Bcl-x splice site to shift splicing from Bcl-xL to Bcl-xS and 3'-Bcl-x AS antisense to the 3'-splice site of exon III in Bcl-x pre-mRNA to down regulation of Bcl-xL expression, the inhibitory effects on cancer cells by modification of alternative splicing and antisense gene therapy were observed and compared by microscopy, MTT Assay, RT-PCR, FACS, Westhern bloting and clone formation. The growth of cells BIU-87 was inhibited in a dose- and time-dependent manner. Its inhibitory effect began 12 h after the exposure, reaching a maximum value after 72h. The number of cells decreased in S phase and the number increased in G1 phase. The ability to form foci was reduced and the antisense gene therapy was approximately half as efficient as modification of alternative splicing in inducing apoptosis. It is concluded that modification of splicing pattern of Bcl-x pre-mRNA in bladder cancer cell BIU-87 is better than antisense gene therapy in terms of tumor inhibition.

The expression of survivin, a member of inhibitor of apoptosis (IAP) family, was examined in bladder transitional cell cancer (BTCC) tissue and adjacent normal tissues to examine its clinical implication in the development of BTCC. Thirty specimens of bladder cancer were detected for the expression of survivin by using immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction (RT-QPCR) in BTCC tissue and adjacent normal tissues. Our results showed that the positive rate of survivin immunostaining specimen were 0 and 60% (18/30) in the adjacent normal tissues, bladder cancer, respectively. The-DeltaDeltaCT value of survivin in bladder cancer tissue was 10.2829 (9.0034-11.5624) times that in the adjacent normal tissues. The expressions of survivin were correlated with the pathological grades of tumor and clinical stages. It is concluded that there was only weak expression of survivin mRNA in the adjacent normal tissues, but the expression of survivin mRNA in bladder cancer tissue was much higher than that in the adjacent normal tissues and the expression of survivin was correlated with pathological grades and clinical stages of tumor.
*Carcinoma, Transitional Cell/metabolism ; *Carcinoma, Transitional Cell/pathology ; Microtubule-Associated Proteins/genetics ; Microtubule-Associated Proteins/*metabolism ; Prognosis ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Tumor Markers, Biological/genetics ; Tumor Markers, Biological/*metabolism ; Urinary Bladder Neoplasms/*metabolism ; Urinary Bladder Neoplasms/*pathology

To evaluate the therapeutic efficiency of combined use of p16-expressing adenovirus and chemotherapeutic agents CDDP or As2O3 on human bladder cancer cell line EJ, the human bladder cancer cell line EJ were transfected with adenovirus-mediated p16 gene (Ad-p16), with administration of cisplatin (CDDP) or arsenic trioxide (As2O3). The cell growth, morphological changes, cell cycle, apoptosis and molecular changes were measured using cell counting, reverse microscopy, flow cytometry, cloning formation, immunocytochemical assays and in vivo therapy experiments to evaluate the therapeutic efficacy of such combined regimen. Ad-p16 transfer and CDDP or As2O3 administration to EJ cells could exert substantially stronger therapeutic effects than the single agent treatment. Especially in in vivo experiments, combined administration of p16 and CDDP or As2O3 induced almost tumor diminish compared to the partial tumor diminish induced by single agent. Moreover, delivery of Ad-p16, or administration of minimal-dose CDDP or As2O3 or combined regimen could induce massive apoptosis of EJ cell. Cell cycle analysis demonstrated that administration of CDDP or As2O3 remarkably arrested EJ cell in G1 prior to apoptotic cell death. When treated with combined regimen, cells were arrested in G1 to a greater extent prior to apoptotic cell death. It is concluded that after introduction into EJ cell, Ad-p16 shows enhanced therapeutic efficacy for EJ cell when used in combination with CDDP or As2O3.
Adenoviridae ; genetics ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; Cisplatin ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Oxides ; pharmacology ; Transfection ; methods ; Tumor Cells, Cultured ; Urinary Bladder Neoplasms ; metabolism ; pathology

To modify the splicing pattern of Bcl-x and compare the effect of this approach with that of the antisense gene therapy in BIU-87 cell line of bladder cancer, by using 5'-Bcl-x AS to target downstream alternative 5'-Bcl-x splice site to shift splicing from Bcl-xL to Bcl-xS and 3'-Bcl-x AS antisense to the 3'-splice site of exon Ⅲ in Bcl-x pre- mRNA to down regulation of Bcl-xL expression,the inhibitory effects on cancer cells by modification of alternative splicing and antisense gene therapy were observed and compared by microscopy, MTT Assay, RT-PCR, FACS, Westhern bloting and clone formation. The growth of cells BIU-87 was inhibited in a dose- and time-dependent manner. Its inhibitory effect began 12 h after the exposure, reaching a maximum value after 72h. The number of cells decreased in S phase and the number increased in G1 phase. The ability to form foci was reduced and the antisense gene therapy was approximately half as efficient as modification of alternative splicing in inducing apoptosis. It is concluded that modification of splicing pattern of Bcl-x pre-mRNA in bladder cancer cell BIU-87 is better than antisense gene therapy in terms of tumor inhibition.

The expression of survivin, a member of inhibitor of apoptosis (IAP) family, was examined in bladder transitional cell cancer (BTCC) tissue and adjacent normal tissues to examine its clinical implication in the development of BTCC. Thirty specimens of bladder cancer were detected for the expression of survivin by using immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction (RT-QPCR) in BTCC tissue and adjacent normal tissues. Our results showed that the positive rate of survivin immunostaining specimen were 0 and 60% (18/30) in the adjacent normal tissues, bladder cancer, respectively. The - △△ CT value of survivin in bladder cancer tissue was 10.2829 (9.0034-11.5624) times that in the adjacent normal tissues. The expressions of survivin were correlated with the pathological grades of tumor and clinical stages. It is concluded that there was only weak expression of survivin mRNA in the adjacent normal tissues, but the expression of survivin mRNA in bladder cancer tissue was much higher than that in the adjacent normal tissues and the expression of survivin was correlated with pathological grades and clinical stages of tumor.