Radiobiological Effects Of A549 Cells And Radio-induced Differential Expressions Of Mitochondrial DNA Genes

Objective Radiotherapy is one of the major means for lung adenocarcinoma treatment and DNA is the target of irradiation. As the only extrachromosomal DNA, mitochondrial DNA (mtDNA) has been shown to play a central role in oxidative phosphorylation (OXPHOS) and adenosine triphosphate synthesis, which decide the activities of cells. For this reason, it is of great importance to investigate the correlation between the effects of radiation on A549 cells and radio-induced differential expressions of mtDNA genes. Methods Besides control group (synchronous cultivated A549 cells without irradiation), two test group were designed: (1)Four bottles of exponential growth A549 cells were irradiated with 8 MV X-ray at the absorbed dose of 2,4,6,8Gy respectively, and the cells were cultured 24h after irradiation.(2) Four bottles of exponential growth A549 cells were all irradiated with 8 MV X-ray at the absorbed dose of 4Gy, and after that, the cells were cultured 12h,24h,48h,72h respectively. Cell proliferation was evaluated by MTT assay. Cell cycle distribution and apoptotic rates were determined by flow cytometry (FCM). The apoptotic morphology was confirmed by transmission electron microscope (TEM). The differently expressed mtDNA genes were examined with the mtDNA oligonucleotide microarray. Results X-radiation inhibited the proliferation of A549 cells, especially at the dose of 4Gy. The morphological characters of apoptosis such as chromatin broken and cytoplasm vacuolation were observed after radiation. Radiation (6 and 8Gy) caused arrest at the G2/M phase of the cell cycle and dose-dependant radio-induced apoptosis was observed. G2/M phase arrest was manifested 12h after exposure to 4Gy dose of radiation and collapsed after 24h incubation. The apoptotic rates increased in a time-dependant way after 4Gy radiation and reached the climax at 48h incubation. X-radiation induced differently expressed mtDNA genes. Genes of tRNA-ASP,tRNA-HIS and tRNA-GLU, genes of tRNA-HIS,tRNA-GLU,ND1,ND2,ND3 and ATPase6, genes of tRNA-GLU,tRNA-HIS,tRNA-ASN,tRNA-CYS,tRNA-ALA,tRNA-SER and 16SrRNA, genes of tRNA-HIS ,tRNA-GLU encoded by mtDNA showed remarkable decrease expression 24h after 2,4,6 ,8Gy radiation, respectively. After exposure to 4Gy radiation, the expressions of mtDNA genes were different at different incubation time. Genes of tRNA-HIS,ND1,ND2,ND4L and ND5, genes of 16SrRNA,ND1,ND2,ND4,ND4L,COX Ⅰ,COXⅡ,COXⅢ,ATPase6 and ATPase8, the 13 rRNA genes and 2 rRNA genes encoded by mtDNA,genes of tRNA-ALA,tRNA-ASP,tRNA-ARG,tRNA-HIS, the 11 mRNA genes (except for genes COXⅠand Cyt b) and 2 rRNA genes encoded by mtDNA showed remarkable decrease expression at 12,24,48,72h, respectively. Conclusions G2/M phase arrest of A549 cells was manifested after irradiation and the most dramatic apoptotic response was observed at the dose of 4Gy. Also, expressions of mtDNA genes were decreased widespreadly and permanently, especially the OXPHOS correlated genes encoded by the mtDNA at 48h～72h incubation after 4Gy irradiation in A549 cells. These results suggested that remarkable decrease expression of mtDNA genes is one mechanism of radio-related apoptosis.