Effects Of Inhibitor Of Differentiation 3 On Apoptosis And Sensitivity To Cisplatin Of Cisplatinresistant Human Lung Adenocarcinoma Cells A549/DDP

Inhibitor of differentiation or DNA binding(Id) proteins,which do not contain basic DNA-binding domain and function as dominant negative regulator of basic helix-loop-helix(bHLH) proteins through the formation of inactive heterodimers with bHLH transcription factors, result in the inhibition of lineage-specific gene expression.The Id family of proteins comprises of 4 members which designated Id1～Id4 in mammalian cells.Human Id3 gene was located at lp36.1 Evidences to date indicate its involvements in many diverse developmental, physiological and pathophysiological processes including T and B cells development,skeletal muscle differentiation,vascular smooth muscle cell proliferation,embryonic neurogenesis,osteogenesis and tumor-induced angiogenesis.Objective:To investigate the effect of Id3 on apoptosis and sensitivity to cisplatin of cisplatin-resistant human lung adenocarcinoma cells A549/DDP in vitro.Method:A549/DDP cells were cultured routinely in RPMI-1640 medium.The recombinant eukaryotic expression vector containing the fusion gene of human Id3 and enhanced green fluorescent protein(EGFP) Id3-pEGFP was transfected into A549/DDP cells by liposome-mediated method.After transfection,EGFP expression was observed and photographed by fluorescent microscopy.The alteration of Id3 expression was identified by semi-quantitative RT-PCR and Westernblotting. Cell apoptosis rates were detected by Annexin V/7-ADD staining followed by flow cytometry(FCM),Hoechst33528 staining was used to observe the nuclear morphologyical changes.MTT assay was performed to survey the proliferation rate of the transfected cells and the cells treated with different concentrations of anti-cancer durg DDP.Thereby we calculated the half-maximum inhibitory concentration (IC50).Result:RT-PCR and Western-blotting results showed that Id3 mRNA and protein were up-regulated in Id3/pEGFP-transfected A549/DDP cells.Annexin V/7-AAD and Hoechst33258 staining showed that the apoptotic rate of Id3/pEGFP group was markedly higher than that of control group and pEGFP transfected group.The cells growth rate in the cells transfected with Id3/pEGFP was signifycently inhibited.The inhibitory rates of DDP on cell growth of A549/DDP transfected with Id3/pEGFP were significantly higher than that of control and pEGFP group.Conclusion:Exogenous Id3 gene was able to suppress proliferation or induce apoptosis of A549/DDP cells.The transfection of Id3/pEGFP to A549/DDP cells could increase the sensitivity to cisplatin.