Study Of Sensitive Breakpoints Within The D-loop Region Of MtDNA

Radiotherapy is one of three main treatments of cancers-chemiotherapy,radiotherapy and surgery.Over the last decades there has been many studies about the relationship between DNA changes in cells and apoptosis of malignant diseases.Thus we obtaining a vast number of information about nuclear DNA(nDNA ) of cells in malignant diseases. Compared with nDNA ,there are less knowledge about mtiochondrial DNA (mtDNA ) in the cancer cells.However, there are many evidences regarding mitochondrion is involved in apoptosis in the carcinoma cells..mtDNA is more prone to be damaged for lack of protection from histones and effective repair system. So we suspect that mtDNA is more sensitive for ionizing radiation. A cell contains about 10,000 mitochondrion,every mitochondrion contains about 10 copies mtDNA , which provide convenience for our study.Mitochondrial DNA(mtDNA) is the only extrachromosomal DNA in human cells.The mitochondrial DNA is a double-stranded,circular molecule of 16569 bp .Because of a bias in base composition between the two helical strands,it isolated a heavy strand (or H-strand)and a light strand (or L-strand).mtDNA contain coding region and non-coding region .The coding region encode two rRNAs,22tRNAs and 13 polypeptides.The mtDNA-encoded polypeptides are all subunits ofenzyme complexes of the oxidative phosphorylation system. rRNAs and tRNAs are involved in the synthesis of polypeptides.The noncoding region includes D-loop and the origin of L-strand replication.The D-loop region is involved in and control the replication and transcription of mtDNA,and have important function in the expression of mtDNA.Thus, any changes in the D-loop region could affect the function of mtDNA even whole cell.We wished to know more information on mtDNA in cancer cells by this study ,and provide a valuable genetic dosimeter.On the other hand ,these studies can provide a new research strategy for detection of sensitivity of cells to radiation.mtDNA could be a particularly sensitive target of radiation.Our study mainly was distributions of the sensitive breakpoints within the non-coding region of mitochondrial DNA in human liver cancer cell line SMMC-7721 irradiated by 60Co Y -ray.At the same time ,observe the differences between non-coding region and coding region in the sensitivity to radiation.Meanwhile we carried out elementary study about the gene expression of mtDNA in SMMC-7721 cells. 1.Study of the sensitive breakpoints within the D—loop region of mitochondrial DNA in human liver cancer cell line SMMC-7721 radiated by 60Co γ-rayThere are two ways of DNA strands break by the radiation,directdamage and indirect damage.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 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. Compared with nDNA , mtDNA is more prone to be damaged by ionizing radiation and free radical for lack of protection from histones.These damages may be involved in apoptosis of cancer cells.SMMC-7721 are subjected to irradiation from 60Co radiation source, dose range of which from low to high. We observed the relation between dose of radiation and the breakspots of mtDNA ,and wish finding some regularity.In this study, SMMC-7721 cells radiated by 10Gy,20Gy,30Gy dose,and their mtDNA were extracted,and detected the damage by the methods of LM-PCR and genescan.The consequence exhibited sensitive breakspots in the D-loop region,and showed incrementing tendency and new breakspots with the dose .At the same time , we found there were different damage degree between H-strands and L-strands,and same condition appeared between coding region and non-coding region .These hinted there are different sensitivity for radiation in the two strands and different region of mtDNA .We thought that protection of protein and formation of mtDNA could involved in . H. Gene expression of mtDNA in the SMMC-7721 cells...