ABSTRACT
<p><b>OBJECTIVE</b>To study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion.</p><p><b>METHODS</b>HepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay.</p><p><b>RESULTS</b>HepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness.</p><p><b>CONCLUSION</b>HepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.</p>
Subject(s)
Humans , Apoptosis , Culture Media , Chemistry , DNA, Mitochondrial , Genetics , Ethidium , Chemistry , Hep G2 Cells , Radiation Effects , Radiation Tolerance , Genetics , Sequence Deletion , X-RaysABSTRACT
<p><b>BACKGROUND</b>It has been proven that mitochondrial DNA (mtDNA) noncoding region plays a very important role in process of transcription. The mutation in mtDNA noncoding region can influence the transcription and translation process of mtDNA. The aim of this study is to investigate the variation status in noncoding region of mtDNA in lung cancer cell lines, and discuss its meaning in carcinogenesis.</p><p><b>METHODS</b>The 7 lung cancer cell lines, A549, SPC-A-1, Calu-3, LTEP-a-2, QG-56, 95-D and NCI-H446, were cultured. Then the mtDNA of them was extracted with one-step method and variation status of noncoding region was analyzed, compared to the Cambridge sequence of mtDNA.</p><p><b>RESULTS</b>Different variations existed in the mtDNA noncoding regions of 7 lung cancer cell lines. The variation rate of LTEP-a-2 (1.82%) was the highest one, and A549 (0.21%) was the lowest one.</p><p><b>CONCLUSIONS</b>The mtDNA noncoding region sequencing is accomplished firstly in 7 lung cancer cell lines. It may provide a reference and background for related researches.</p>
ABSTRACT
Objective To investigate the differential expression of mitochondrial genome and apoptosis-related genes in human lung adenocarcinoma A549 cell line induced by cisplatin. Methods A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone) in 150-mm dishes, and the test cells were exposed to 0.5?g/ml cisplatin continuously for 20h. Same volume PBS solution was added to culture of control cells instead of cisplatin. Human mitochondrial genome oligonucletide micoarray was used to detect the differential expression of 26 target genes. The apoptosis status was analyzed by the flow cytometry technique. Results Compared with the control cells, the expression levels of tRNA-Cys, tRNA-Asn, 12S rRNA and cytochrome oxidase subunit I were remarkably elevated in test cells. The expression levels of bax, NADH dehydrogenase subunit I, NADH dehydrogenase subunit Ⅱ and tRNA-Ser, tRNA-Asp, tRNA-Arg and tRNA-Ala also were elevated to some extent. Apoptosis rate of test cells was 8.5%?0.78%(n=5), which was remarkably higher than that of control cells 1.3%?0.56%(n=5,P