Effects Of Adenovirus-Mediated PTEN Gene On Biological Behaviour Of Human Colon Cancer Cells

Background: Colon caner is one of the most common malignancies whose incidence is increasing more rapidly than that of rectal cancer in China. Surgery is still the most important and effective way. Despite introduction of new chemotherapeutic agents into treatment protocols, to data no overall improvement has been achieved in the 5-year survival rate. So, new therapeutic regimens for colon cancer are therefore urgently needed. With the quick development of molecular biology, many advancements have been made in gene therapy. To data, there have been about one thousand clinical trials of gene therapy approved in the world. The tumor suppressor genes play a critical role in tumorigenesis and tumor development that they are often used as targets in gene therapy. PTEN is a new tumor suppressor gene on chromosome 10q23, encodes a phosphatidylinositol phosphatase that antagonizes activation of the phatidylinositol 3' -kinase-mediated pathway involved in cell growth. PTEN gene is frequently mutated or lost in colon cancer, especially in advanced stages. Therefore, introduction PTEN gene into PTEN-null colon cancer cell might be an effective strategy for colon cancer. To test the the potential for this type of gene therapy, we constructed a recombinant adenovirus encoding wild-type PTEN and examined its effects on the PTEN-null colon cell line LoVo. Methods: PTEN cDNA was was subcloned into shuttle plasmid pDC315 to construct recombinant shuttle plasmid pDC315.PTEN. Then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHGlox A El,3Cre were cotransfected into 293 cells to construct recombinant adenovirus Ad.PTEN by site special recombination. The viral stock reproduced in 293 cells and was concentrated by cesium chloride ultracentrifugation. The PCR technique was used to detect target gene fragment and adenovirus genomic characteristic fragment. B -galactosidase transduction assay was used to assess efficiency of the adenovirus-mediated gene transfer. After Ad.PTEN infected LoVo cells, the PTEN mRNA, PTEN and nm23-Hl were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. The effects of Ad.PTEN on proliferation andcell cycle progress of LoVo cells were analysed respectively by MTT test, clone forming test and flow cytometry. The apoptosis of LoVo cells was assayed by Annexin-V-PI. The Millicell chamber were coated with a barrier of reconstituted basement membrane proteins to detect the migration of LoVo cells. The levels of nm23-Hl were examined by Western blot technology. Results: (1)A replication-deficient adenovirus type 5 carrying PTEN gene was constructed via site special recombination in 293 cells. Plaque titration on 293 cells showed titers of 5*1010 pfu/ml. The optimal MOI was 50. (2)PTEN mRNA and PTEN was not found in LoVo cells of Ad.LacZ and PBS group, but was very obvious in Ad.PTEN group. The level of PTEN mRNA and PTEN increased significantly with duration of infection in Ad.PTEN infected cells. (3)Ad.PTEN transfection significantly inhibited the proliferation of LoVo cells. Flow cytometric analysis displayed an accumulation of cell in the Gl phase of cell cycle and a decreased percentage of cell in S and G2 phase and proliferative index in Ad.PTEN group, but the apoptosis rate increased. Cloning efficiency of Ad.PTEN group in soft agar was markedly inhibited..(4)Ad.PTEN significantly inhibited the migration of LoVo cells and increased nm23-Hl protein expression in LoVo cells compared with treatment withAd.LacZorPBS. Conclusions: (1)Ad.PTEN can be efficiently expressed in LoVo cells. (2)Ad.PTEN can significantly inhibit the growth and proliferation of LoVo cells and migration as well, moreover, upgrade the nm23-Hl level.(3) Our results lay a basis for further study on colon cancer gene therapy.