| Bcl10 is one of the apoptosis regulatory protein. It is located at 1p22,one site harbor tumor suppressor tumor gene.We screen Bcl10 expression in different tumor tissue by immunohistochemistry (IHC), and three tumor cell lines by western blot(WB). The results showed Bcl10 protein expression was found in Hela cell line, CA46 cell line and no expression in 293 cell line in western blot assay. In immunohistochemistry, we found the Bcl10 protein has positive finding selectively in Colon cancer , Cervical cancer ,Breast cancer , But increased in MALT lymphoma. On contrary, there are almost tumor tissues with positive finding of p53 protein including Colon cancer , Cervical cancer ,Breast cancer , MALT lymphoma, gastric cardiac carcinoma and Hepatocellular cancer.The Bcl10 gene, unlike other tumor suppressor genes such as p53, may be selectively targeted by different human tumors, specially increased in MALT lymphoma. In our study, Bcl10 play a role at MALT lymphoma tumorgenesis.The IHC results might be explained for one possibility: Bcl10 promoter might controll gene expression in a tissue- specific manner. To investigate the mechanism of regulation of human Bcl10 gene expression, at first, we extracted total RNA from Hela cells, and the 5'-RACE was performed to determine the transcription initiation site of Bcl10 gene after nest PCR. The sequence of 5'-flank regulation region of human Bcl10 gene obtained from GenBank was scanned for predicting transcription factors binding sites in www.genomatix.de. Secondly, A 5.7kB 5'-flanking region of Bcl10 was amplifed by PCR using primers based on the genomic DNA sequence of human Bcl10 gene. a series of the Bcl10 promoter deletion fragments were also generated by PCR,and were constructed and ligated into the pEGFPD vector and the pGL3 luciferase reporter vector respectively. After these vectors were transfected into Hela cells 48h, EGFP fluorescence expression was observed and the luciferase activity was analyzed to determine the promoter sites.The results showed the transcription start site was determined at 189bp upstream of ATG. There were no TATA box, seven GC-Box ,one CAAT box and some putative transcription factors binding sites such as AP-1, in the upstream regulation region in the bioinformatics analysis. Among the six deletion fragments, P4,which containing 1.9kB regulation region, was shown to have relative lower basic transcription activity, while one fragments P2,had higher promoter activity, which located at–477bp～+1bp.A 440bp CpG island of Bcl10 promoter ,which located at–495bp～-56bp, was found by methylation scan software. We detected the status of methylation in 12 MALT lymphoma tissue by means of methyltion specific polymerase chain reaction (MSP). The results showed methylation of Bcl10 promoter was detected in one MALT lymphoma tissue (8%). We suggest that the methylation of Bcl10 CpG island can result in its transcriptional inactivation in some extend, which may be close related to the development of MALT lymphoma.At last, we wish to find a antitumor agent for cancer therapy which related with Bcl10+.Hela cell and 293 cell were used as material for testing Complexes with three copper ratios to chitosan. The results showed that all of the copper complexes of chitosan inhibited the proliferation of HeLa and 293 cells. CTS–Cu(3 ) exhibited a higher antitumor activity than other copper-chitosan complexes tested. Compared with Hela cell lines, the copper complexes of chitosan were found to be more selective to 293 cell lines in this experiment.we might assume that Bcl10 lost the ability to inhibite the proliferation of cancer, moreover, the trunct of Bcl10 can promote the proliferation of cancer. In addition, this study showed that copper-chitosan complex inhibited tumor cell proliferation by arresting the cell cycle progression at the S-phase in 293 cells.Anyway, the precise knowledge of Bcl10 gene need further study, That will be important for the truth under Bcl10 which give us the vision for cancer therapy. |
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