Study On The Variations Of Mitochondrial DNA In Nasopharyngeal Carcinoma

Purpose Mitochondiral (MT) plays an important role in cell apoptosis and the progress of cell aging. Previous investigation revealed that there are some variations of mtDNA in degenerative disease such as mitochondrial mypathics and malignancies such as uterine serous carcinoma, hepatocellular carcinoma and breast carcinoma and so on. But knowledge about the relevance between the variation of mtDNA and nasopharyngeal carcinoma (NPC) are rare. The mtDNA aberration includes point mutation and large-scale deletion. The control region (D-loop region) of mtDNA is a polymorphism region in which is easily to take point mutation. But in the coding region, the most frequently reported mutation is the 4977bp deletion, called the common deletion (CD). In this investigation, we try to detect the variations and mutation in D-loop and it's neighboring region of mtDNA in NPC tissues. Mutation of mtDNA in NPC cell line CNE-2 after irradiation was furtherly studied for to find the correlation between mtDNA mutation and the cell apoptosis. Materials and methods Part 1: Total DNA was extracted from tumor tissues and paired periphery blood monocyte (PBMC) cellss derived from 23 patients with NPC. D-loop and the neighboring region of mtDNA were directly sequenced by PCR methods. Polymorphisms of mtDNA were determined when the sequences of PBMC are not same as Cambridge sequences. Mutations of mtDNA were determined when the sequence of tumor tissues are not same as the PBMC's. Part 2: CNE-2 cells were exposed to X-ray of 0Gy, 2.0Gy, 4.0Gy, 6.0Gy, 8.0Gy and were harvested for 24hr, 48hr, 72hr. Total DNA was extracted for mutation detection in D-loop and the neighboring region of mtDNA by PCR sequence analysis. Mutation difference of mtDNA was compared in the cells before and after irradiation to Cambridge sequences. At the same time total WT-mtDNA and CD-mtDNA were also quantitatived analyzed by using Taqman PCR. The ratio of CD/WT was used to evaluate the CD mutation level of mtDNA after irradiation. Part 3: CNE-2 was exposed to X-ray of 0Gy, 0.5Gy, 2.0Gy, 4.0Gy, 6.0Gy and 8.0Gy. The clonogenic assay was performed to determine the survival fraction. The parametersα,β,α/βand SF2 for the linear-quadratic model were calculated. Furthermore CNE-2 was exposed to X-ray of 0Gy, 2.0Gy, 4.0Gy, 6.0Gy, 8.0Gy and harvested for 24hr, 48hr, 72hr. DAPI and HE staining was used to observe the morphological changes of apoptotic cells. TUNEL labeling method was used for detection of DNA fragments in the apoptotic cells. Cell cycle progression and the apoptosis index were assessed by flow cytometer analysis. The correlation analysis between the apoptosis index and the ratio of CD/WT was evaluated by using soft SPSS11.0.Results 1. MtDNA variation and mutation in NPC tissue Totally 248 polymorphisms were revealed in mtDNA in the 23NPC patients. In D-loop, the polymorphisms are in 63 nucleotide positions and the frequency is 6.38% (63/988) including polymorphisms in 59 nucleotide positions recorded by Mitomap (www.mitomap.org) and 3 are the newly reported (nt360A →AT, nt517G-del, nt16038A→G) in this investigation.There is 1 (nt513 G-del) is not same as the record in Mitomap. In the D-loop neighbor region, the polymorphisms are in 6 positions and the frequency of base polymorphisms is 0.75% (6/795) and 1 polymorphism (nt15787T→C) is the newly reported in this investigation. But this polymorphism can't change the coding amino acid. The number of variants of mtDNA in this group population ranges from 6 to 17 in each person, but in different positions and types of the polymorphisms. In D-loop, 7of 23 (34.8%) patients presented 34 mutations in NPC tissue including 17 in hypervariable segment1, 12 in hypervariabe segment2, one in the termination-associated sequence, and 4 unknown. The hypervariabe segment2 presented highst frequency of point mutation. There are 7 mutations in the H-strand origin region and 4 in the mitochondrial transcription factor bindng site. In the neighboring peptide-coding regions, 6 of 23 (26.1%) patients take 9 mutations in NPC tissues. The function of the mutations in the gene of 12sRNA is unclear but the inserting G at nt15673 and nt15675 can make abnormal expression of cytB, while A→G substitution at nt15769 in cytB gene is a synonymy mutation. T→C substitution at nt15970 in tRNApro gene can make the transport of proline out of way. 88.4% (38/43) mutations are homoplasmic while 11.6% (5/43) are heteroplasmic in this group of NPC patients. 2. Variations in mtDNA of CNE-2 after irradiation By contrasting with the Cambridge Sequence, 11 variations were revealed in mtDNA of CNE-2 before irradiation including 10 in D-loop and 1 in 12sRNA. Butthere was no difference in the sequences of mtDNA between irradiation and un-irradiation. By Taqman PCR, the ratios of CD/WT were 9.26E-06, 1.76E-05, 2.18E-0, 2.77E-05 and 1.03E-04, respectively; in cells exposed to X-ray of 0Gy, 2.0Gy, 4.0Gy, 6.0Gy, 8.0Gy and were harvested for 24hr, 48hr, 72hr. The ratios of CD/WT were 2.01E-05, 3.19E-05, 1.03E-04, respectively in cells exposed to X-ray of 8.0Gy and were harvested for 24hr, 48hr, 72hr. These results indicated that the irradiation could induce the ratio of CD/WT of CNE-2 cells in a dose and time dependent manner. 3. The survival and apoptosis of CNE-2 after irradiation By the clonogenic assay the survival fraction of CNE-2 after irradiation is decreasing as the dose is increasing. Using the linear-quadratic model to fit the survival fraction after irradiation, the parameter αis 0.030Gy-1, βis 0.12Gy-2, α/βis 0.24Gy, and SF2 is 0.58. DAPI staining of irradiation-treated CNE-2 cells revealed extensive nuclear condensation and fragmentation, as well as the appearance of apoptotic bodies. In contrast, control cells and nuclei remained morphologically unchanged. By DNA labeling method (TUNEL), the positive cells showed the indigo spots in the nuclear of the apoptotic cells. When the CNE-2 cells were exposed to the X-ray 0Gy, 2.0Gy, 4.0Gy, 6.0Gy, 8.0Gy for 72 hr, the apoptosis indexes (AI) were 2.8%, 12.4%, 41.3%, 47.9%, 70.8%, respectively. A dose-dependent accumulation of apoptotic cells was found. After CNE-2 cells were treated with X-ray dose of 8.0Gy for 24hr, 48hr, 72hr, the AI were 7.8%,36.3%,70.8%, respectively. This indicated that the irradiation could induce CNE-2 cells apoptosis in a dose and time dependent manner. By the cell cycle analysis, an accumulation of cells in S or G2/M phase was found as the dose was increasing. Significant correlation between the ratio of common deletion and AI induced by irradiation was observed in this study (r = 0.853, P<0.0001). Conclusions 1. MtDNA variations were frequent in NPC patients from Southern China population. In D-loop region, the rate of base polymorphisms was 6.38% (63/988), and 3 variations (360A→AT, 517A-del, 16038A→G) are newly reported and 1 is not same as the mitomap's. In the non-D-loop region the variation rate was 0.75% including 1 new reported (15787T→C) in this investigation. 2. About 30.4% (7/23) NPC patients presented mutations in D-loop of mtDNA, and these mutations may cause the abnormal replication and transcription of mtDNA. 26.1% (6/23) NPC patients presented somatic mutations in the neighboring region of mtDNA in NPC tissue, and these may lead to abnormal oxidative phosphorylation. 3. There was no any difference of variation of mtDNA sequence in CNE-2 cell line when cells treated with different dose of X-ray. 4. Taqman PCR analysis revealed that the ratio of common deletion was increasing by a time and dose dependent manner. There is significant correlation between the ratio of common deletion and apoptosis index induced by irradiation (r = 0.853, P<0.0001), Thus the common deletion of mtDNA was involved the pathway of the apoptosis inducing by irradiation.