Protein Expression And Proliferation Ability In Pancreatic Cancer Cells Transfected With KAI1 Gene

Objective Metastasis is main reason of the death for the cancer, especially in pancreatic cancer. This cancer is the fifth leading of cancer death in the United States, with an overall 5-year survival rate of less than 5%, although radiotherapy and chemotherapy, particularly gemcitabine, have produced modest benefits in some patients, no currently available treatment affects 2-year survival for patients with locally advanced and metastatic disease. Similarly, for patients with localized disase, current adjuvant radiation and chemotherapy have shown only modest benefits after surgical resection. One promising alternative is gene therapy, so to seak right genes and find out the mechanism is the important work.KAI1(kang ai 1), called CD82, is a metastasis suppressor gene located on human chromosome 11p11.2. It is a member of the structurally distinct family of cell surface glycoprotein, which is a member of the transmembrance 4 superfamily (TM4SF). TM4SF members are characterized by four highly conserved transmembrane domains, which include two relatively divergent extracellular domains, the larger of which contains at the NH2 and COOH termini. The precise biochemical function of the TM4SF is not clear yet. However, the current data suggest a role for this superfamily largely in the regulation of cell proliferation, activation, and motility. KAI1 expression was reported to correlated with the progression of a variety of human cancers such as non-small cell lung cancer, pancreatic cancer, bladder caner, gastric cancer, and breast cancer, as well as colon cancer cells and melanoma cells.Methods To determine whether KAI1 expression is responsible for the metastasis suppression and the proliferation ability in pancreatic cancer, the human KAI1 gene was transfected into human pancreatic cell lines MiaPacaII, which has low levels of endogenous KAI1 expression to study the effects of the gene in the celllines. One microgram of colum purified pCMV-KAI1 and pCMV-neo DNA were used for subsequent transfection of the Miapacall using lipofectamine. The positive clones were selected by the antibiotics G418 400 g/ml for two weeks. The expression of the KAIl protein in the cells of the.KAI1 transfectant clones, vector-only transfectants and the MiaPacaII were detected by immunocytochemistry. Parental, the proliferation ability of the three kinds of the MiaPacall colnes was assayed by MTT [3- (4, 5-dimethylthiazol-2-yl) -2,5-dipenyl terazolium bromide]) and clonegenicity test.Results Transfected cells were first grown in DMEM medium for 48h, then subjected to be selected in the medium containing G418 for 35 days. Some of the MiaPacall and the MiaPacall transfected with pCMV-KAIl and pCMV-neo were died and suspending after three days. The MiaPacall all died whitin 14 days, but small parts of the other two kinds of cells were still alive after 14 days, and small and scattering cell clones could be observed after 28 days, and the clones could be cultured expansively and grow well. The cells transfected with pCMV-KAIl looks like willow leaves, and more loosing than the MiaPacall. The KAI1/CD82 protein was detected by immunocytochemical staining using monoclonal antibody CD82. Cells were grown and transfected as above. More transfected with pCMV-KAIl plasmid DNA showed positive CD82 staining compared with its corresponding transfectant cells with the empty vector and the cells without transfection. MTT test showed that proliferation ability of the cells transfected with pCMV-KAI1 was much lower compared with the control parental vector pCMV-neo and the Miapacall cells(P<0.05 and P<0.01) ,and the there was not difference between the later two kinds of the cells (t=0.731,P>0.05). Clonegenicity assays showed that the clones could be observed two weeks after the three kinds of the cells plated in the dishes, after stained and calculated the clone numbers of them, the clonegenicity efficiency were 14.00%, 49.56% and 49.23% respectively. The ability of the cells transfeted with KAIl were much lower than that two kinds of cells(P<0...