The Inhibition Of Retroviral Vector Containing DPC4/Smad4 Gene On Human Colon Carcinoma Cells

Colorectal cancer are the most common malignant tumors in humanbeing, which account for 40% of humanbeing tumors.There is a trend of rise in some hig cities in our coumtry, meaningwhile, The trend towards Younger and proximal Colon, in recent year,there is a obvious rise in diagnosis and treatment of colon Cancer, but the prognosis is still poor, It is a urgent question to solve.With development of molecular-biology,The tumors are kmown is a more-steps process, involving a lot of genes There is evidence to suggest an association between colon cancer and diets containing a high proportion of fat and meat.The process of colon cancer include three parts(1)oncogenes and Suppressed genes, (2)actives of MMR, (3)Methylation of DNA.TGF-β(Transforming growth factor-β) plays an important role in modulating immune response, regulating cell differentiation and growth, stimulating extracellular matrix formation, and so on. However, epithelial derived tumor cells has often lost their sensitivity to the growth inhibition of TGF-β, and lost their growth inhibition. DPC4 was thought as an important tumor suppressor gene.Despite the extensive research of DPC4, we little know of whether the loss of DPC4 function contributes to the invasion and metastasis of colorectal carcinoma cells. The experiment were devoted to research the expression of DPC4 on the cells of colorectal carcinoma, construction of The Retroviral Vectors Containing DPC4. and Inhibition of DPC4/Smad4 on colorectal carcinoma cells. Attempts to study the effect of transfected DPC4 gene on biological behavior of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis.1,Expression of DPC4 on the cells of colorectal carcinomaTotal 80 patients pathological diagnosed with colorectal Cancer surgery in The China Japan union Hospital of jilin university from January 2006 to of January 2007 were collected . Methods including nomal Expression of DPC4 was detected in 108 samples mucosa membrane and carcinoma tissue by immunohistochemistry ABC method. DPC4 positive signal was localized in cytoplasm and nucleus. The positive rate of DPC4 protein expression in the tumor tissues and normal mucosa were 75 % (60/80)and 100 %(28/28 ) respectively and their difference is statistically (P<0.05).The immunostaining of DPC4 was strong positive in normal colon mucosa. The positive cells distributed in the whole colon mucosa, and weakly positive or negative in poorly differentiated adenocarcinoma and metastatic focus. The high level expression of DPC4 protein in tumor tissue was relative to the earlier tumor stage (P<0.05 );.Thereis no relationship between DPC4 protein and gender, age,tumor location (P>0.05). The expression of DPC4 was associated with tumor stage, tumor differentiation degree and metastasis.2,Construction of The Retroviral Vectors Containing DPC4/Smad4.In recent years the retroviral vector and packaging cells continuously updated stability in the carrier, such as effication and safety, made it to be an important means of gene therapy tools.The human DPC4/Smad4 complementary DNA was amplified fromSmad4/ DPC4-pBluescript plasmid by PCR and subcloned to the retroviral vector pLXSN to obtain pLXSN/ DPC4/Smad4+ recombinant with direct inserting, and then packaged with PA317 amphotropic packaging cells. AntiG418 clones were acquired and named as PA317/ pLXSN DPC4+ cells. As a control, pLXSN also was packaged with PA317 cells and the antiG418 clones were named as PA317/pLXSN. The virus titer was reached 6.0×105 pfu/L. The PCR assay confirmed there was DPC4/Smad4 gene integration in PA317/pLXSN DPC4+ cells, but PA317/pLXSN. This suggested that introduction of DPC4 gene to retroviral vectors was an effective way, which maybe can provide theoretical basis for the gene therapy of colorectal cancer andother malignancy tumor.3,Inhibition of DPC4/Smad4 on The colorectal carcinoma cellsThe retroviral vector pLXSN containing DPC4 which could be used to transfect the colorectal carcinoma cells SW480 was constructed, with the empty vector pLXSN as a positive control. AntiG418 clones were acquired and the daughter cells were named as SW480/DPC4 and SW480/pLXSN respectively. As a negative control, the parent SW480 cells were named as SW480/-. All the cells were identified by PCR and Western assay. The cell in vitro proliferation was assessed by MTT assay. Our study suggested that the retroviral vector pLXSN contaiming DPC4 could inhibit the proliferation of the colorectal carcinoma cells, which indicated that DPC4 could mediate tumor suppression through suppression of angiogenesis.