레트로바이러스와 p53항암유전자가 암세포의 유전자치료에 미치는 영향

Abstract

Cancer is considered to occur through abnormal growth and differentiation processes, in which oncogenes and tumor suppressor genes are deeply related. Cellular responses to DNA-damaging agents are believed to be critical determinants of human tumorigenesis. Cell cycle arrests and DNA repair following DNA damage require the coordination of multiple gene products that, as a whole, serve to maintain the integrity of the genome. Within the cell cycle, both Gl-S and G2-M phase transitions are under constant surveillance by checkpoint genes for the protection of cells from either exogenous or endogenous DNA-damaging agents. p53 tumor suppressor gene mediates cell cycle perturbations in response to DNA damage, and play a role in cell death, genetic stability, and cancer susceptibility. Recently, gene therapy with p53 tumor suppressor gene is expected as a new effective therapeutic strategy in many kinds of cancer. By using retroviral vector system, we transduced p53 tumor suppressor gene into human osteosarcoma cells, and analysed its growth suppression and apoptosis inducing effects. Combined effects of p53WT gene therapy with chemotherapeutic agent or radiation were also analysed. Titer of ecotrophic p53wT retrovirus was 5.0xl05/ml，and that of amphotrophic p53wT retrovirus was 2.0xl0s/ml when N1H3T3 cells were used as target cells. Human osteosarcoma cells infected with amphotrophic p53wT retroviruses showed increased p21WAF1 gene expression, which acts as a major cyclin-dependent kinase inhibitor in DNA damage responses. In normal DMEM media, human skin fibroblasts infected with amphotrophic retroviruses showed very slow growing (1.7 fold increase in doubling time) and very low saturation density (50% decrease in cell density). In media containing chemotherapeutic agent, human osteosarcoma cells infected with p53wT retroviruses died rapidly; 75% of them died within 4 days and all of them died within 10 days of incubation with chemotherapeutic agent. Their DNAs were extracted and electrophoresed in agarose gel, and we identified DNA ladders characteristic of apoptotic cell death. When human osteosarcoma cells infected with p53wT retroviruses were irradiated with ultraviolet light, more than 95% of cancer cells died within 1 day; whereas mock infected cells showed only less than 5% of cell death. These findings suggest that retroviral vector mediated p53 tumor suppressor gene transfer into cancer cells can suppress tumor cell growth and decrease tumor cell density effectively. These findings also suggest that effective induction of tumor cell apoptosis can be obtained when p53wT gene therapy is used in combination with chemotherapy or radiotherapy. Key Words: p53 gene therapy, Retroviral vector, Human osteosarcoma, Apoptos