The Comparison Of DNA Double Strand Breaks Between Nuclear DNA And Mitochondrial DNA In Human Liver Cancer Cell Line SMMC-7721 Exposed To Low Liner Energy Transmission Ray

Now a days,, tumor is still the major reason which causes death of people, and the attack rate is increasing every year. As one of the three main treatments of cancer (chemiotherapy, radiotherapy and surgery), Radiotherapy is too important to be substituted. People find that the radioactive ray can break the DNA molecular of cancer cell. Then the damaged cell loses vigor and chromosome undergoes aberration. At last, apoptosis is irreversible. There have been many studies about the relationship between DNA change and apoptosis in malignant cells. Thus we have obtained a vast number of information about nuclear DNA (nDNA). Compared with nDNA, there are less knowledge about mitochondrial DNA(mtDNA)in the cancer cells. However, there are many evidences indicating mtDNA is tightly connected with the happen,development and death of cancer cell.Compared with our limited knowledge about damage of mtDNA after radiation, previous literature mainly focused on the damage of nDNA. Some documents have reported that high Liner energy transmission(LET) can cause nonrandom break of the double DNA strand, and low LET cause random break. Now, we hope to get some information about the damage of mtDNA and nDNA of cancer cell after radiation.We want to know whether there exist radioactive sensitive sites in the mtDNA and nDNA,whether these sites are distributed randomly and what the damage state of mtDNA and nDNA. The results of the work may provide some guidance for the clinical therapy of cancer, and offer a new reference index for the study of radiation sensitiveness of cell.In the present, the most frequently used ray is low LET, namely in the aspect ofγand X treatment. In the present study, through exposure to different dose ofγand X ray, we detected the distribution of sensitive site of mtDNA and nDNA; And we compare the degree of damage between mtDNA and nDNA, coding region and D-loop region,heavy strand and light strand, the two strands of nDNA.Radiation can cause direct and indirect damage of DNA. The former is free radical produced due to the direct damage of polar molecule by the radiation; The latter is mainly the radiation leads to ionization of H20 in the cells, and produces free radicals(most of them are·OH ).There is interaction of free radicals and biomacromolecule, and results in irreversible damage. The two ways are tantamount important. In this study, SMMC-7721 cell are radiated by different dose, namely 10Gy,20Gy,30Gy and 50Gy, and the survival rate of cell, the break of mtDNA and nDNA are detected respectively. The mtDNA is multi-copy molecular, one mitochondria contains 2-10 mtDNA,thus one cell contains hundreds to thousands of mtDNA. To compare the damage degree of mtDNA and nDNA, we established methods based on PCR through which the copy number of mtDNA can be determined. Immediately after radiation, the genome DNA of cells was extracted from experimental and control group. Ligation mediated PCR (LM-PCR) and alkaline gel electrophoresis were used to detect the break site and observe the damage degree. At last, the concrete damage site and frequency can be analyzed by the result of gene-scan technique. We found that there exists sensitive break site of mtDNA and nDNA, moreover,the degree of damage showed an increasing tendency with the increase of irradiation doses, and new break points appeared with the increase of irradiation doses. The sensitive sites ofγray and X ray are identical; The damage degree of mtDNA is more serious than nDNA in SMMC-77221 cell after radiation. We observed that the damage degree of heavy strands(H-strands) is higher than that of light strands(L-stRanDs), coding region(cytochrome b gene)higher than D-loop region ,and the damage degree of the template strands of nDNA(18SrRNA gene) is higher than coding region. From the point of composition of base, damaged thymine(T)and guanine(G)accounts for 81% of the whole damaged base. The reason may be this two types of bases are major attacking target of·OH, this also indicates that radiation mainly cause indirect damage to DNA. We think that protein has definite function of protecting DNA from the damage of radioactive ray, this can explain why the damage degree of mtDNA is higher than that of nDNA and the damage degree of coding region is higher than that of D-loop region. The damage degree of template strand is higher than the coding strand for nDNA and the damage degree of the heavy strand is greater than light strand for mtDNA. We proposed that template strand and heavy strand are exposed more frequently during transcription and thus are more susceptible to radioactive damage.