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Mitochondrial DNA alterations of peripheral lymphocytes in acute lymphoblastic leukemia patients undergoing total body irradiation therapy

Mitochondrial DNA alterations of peripheral lymphocytes in acute lymphoblastic leukemia patients... Background: Mitochondrial DNA (mtDNA) alterations, including mtDNA copy number and mtDNA 4977 bp common deletion (CD), are key indicators of irradiation-induced damage. The relationship between total body irradiation (TBI) treatment and mtDNA alterations in vivo, however, has not been postulated yet. The aim of this study is to analyze mtDNA alterations in irradiated human peripheral lymphocytes from acute lymphoblastic leukemia (ALL) patients as well as to take them as predictors for radiation toxicity. Methods: Peripheral blood lymphocytes were isolated from 26 ALL patients 24 hours after TBI preconditioning (4.5 and 9 Gy, respectively). Extracted DNA was analyzed by real-time PCR method. Results: Average 2.31 times mtDNA and 0.53 fold CD levels were observed after 4.5 Gy exposure compared to their basal levels. 9 Gy TBI produced a greater response of both mtDNA and CD levels than 4.5 Gy. Significant inverse correlation was found between mtDNA content and CD level at 4.5 and 9 Gy (P = 0.037 and 0.048). Moreover, mtDNA content of lymphocytes without irradiation was found to be correlated to age. Conclusions: mtDNA and CD content may be considered as predictive factors to radiation toxicity. Keywords: mtDNA, 4977-bp Common deletion, Total body irradiation, Real-time-PCR, Acute lymphoblastic leukemia Background possible radio-protective mechanism is that enhanced Breakage of cellular DNA following radiation is a dose replication of mtDNA reduces the mutation frequency of dependent phenomenon and occurs in both the nuclear total mtDNA and delays the onset of lethal radiation and extra-nuclear DNA. Thus, besides nuclear nDNA, damage to the mitochondria [5,6]. This hypothesis has mitochondrial DNA (mtDNA) is equally affected as an been recently supported by Zhang et al with exhibiting only extra-nuclear genome [1,2]. Numerous investigations increased mtDNA copy number in gut and bone marrow showed that mtDNA can be an easily available target for of total body irradiated rats [7]. On the other hand, IR endogenous reactive oxygen species (ROS) and free radi- usually prompts cell apoptosis by displaying an accumula- cals caused by ionizing radiation (IR), which resulted in tion of large scale mtDNA deletions, especially the specific mtDNA copy number alteration and mtDNA damage 4977 bp deletion, referred to as the “common deletion (such as mutation and depletion) [3,4]. (CD)" [8]. The site of CD is flanked by two13 bp direct The mechanisms of cellular response to radiation with repeats (ACCTCCCTCACCA) at mtDNA nucleotide site 8470 and 13447 respectively, and easy to make deletion regard to mtDNA alterations were mainly involved in the following two ways. On one hand, mtDNA has few repair for its unique formation mechanism [9]. Studies have mechanisms and continued mitochondrial function is pre- shown that CD can be as a sensitive marker of oxidative served primarily due to its high copy number. One of damage to mtDNA [10-12]. Unfortunately, only few experiments have evaluated the association between CD and IR till now. For example, accumulation of CD * Correspondence: huyide_mit@yahoo.com.cn 1 has been identified by qualitative PCR method on several Third Department of Oncology, The second affiliated hospital, Third Military Medical University, Chongqing 400037, China irradiated cell lines (such as human skin fibroblasts, Full list of author information is available at the end of the article © 2011 Wen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wen et al. Radiation Oncology 2011, 6:133 Page 2 of 7 http://www.ro-journal.com/content/6/1/133 glioblastoma and colon carcinoma lines) and primary lym- Co., Stockholm, Sweden) set to deliver a dose rate of phocytes [13-15]. Furthermore, CD was induced by IR in 4.5-4.9 cGy/min over two successive days. None of the human hepatoblastoma cell line performing on real-time patients had prior exposure to any cytotoxic treatment PCR with nonspecific dsDNA-binding dye SYBR Green. for at least 2 weeks before the start of radiotherapy. All However, their conclusions were largely controversial. The patients had 7 ml of PB collected prior to and 24 h fol- inconsistency may be due, in part, to the use of non-quan- lowing exposure for each radiation treatment. Besides, titative PCR strategies. Additionally, none of these studies 39 healthy donors had the same volume of PB collected have assayed mtDNA or CD level in peripheral blood lym- without ionizing radiation. Preparation of PBLs followed phocytes (PBLs) after in vivo irradiation exposure for lack standard methods, using human lymphocyte isolation of appropriate human beings radiation model. reagent (TBD Biological Technology Co., Tianjin, In this study, we performed real-time PCR technique China) for separation of mononuclear cells. with a specific fluorogenic TaqMan probe conjugated with minor groove binder (MGB) groups, which is more sensi- DNA extraction tive and appropriate than nonspecific dsDNA-binding dye DNA from lymphocytes in vivo and the skeletal muscle PCR methods previously used [16]. Besides, we taken the was obtained with the TIANamp Genomic DNA Kit acute lymphoblastic leukemia (ALL) patients undergoging (Tiangen Ltd, Beijing, China), and stored at -70°C until total body irradiation (TBI) precondionting as human further study. beings in vivo irradiation model. The advantage of using this model lies in full view of in vivo microenvironment, Analysis of amount of mtDNA and CD by real-time PCR and without need for irradiating healthy individuals. We TaqMan probes with conjugated MGB groups were per- attempted to address the mtDNA status in irradiated formed to ensure maximal specificity in real-time PCR human peripheral blood lymphocytes in vivo to elucidate reaction. Nuclear DNA content was estimated by mea- whether alterations in mtDNA can be linked to exposure suring the human ß-actin gene. The hypervariable region to total body irradiation. 2 (HVR2) in the mitochondrial D-Loop was used to represent the total amount of mtDNA since this region is Materials and methods relatively conserved in Han Chinese [19]. The forward Study participants primer (ß-actin: 5’-AGGACCCTGGATGTGACAGC-3’; This study comprised peripheral blood (PB) samples from HVR2: 5’-GCTTTCCACACAGACATCATAACAA-3’; 26 high risked ALL patients undergoing TBI as pre-trans- CD: 5’-CTTACACTATTCCTCATCACCCAACTAA plantation treatment in their first complete remission AAA-3’), reverse primer (ß-actin: 5’-TGGCATTGCCGA- (CR1) at hematology department of our institution. The CAGGAT-3’;HVR2:5’-GTTTAAGTGCTGTGGCCA- diagnoses were. according to world healthy organization GAAG-3’;CD:5’-GGAGTAGAAACCTGTGAGGAA (WHO) classification and high risk factors were measured AGG-3’) and TaqMan MGB hybridization probes on Ribeca’s report [17]. The patients age from 19 to 56 (ß-actin: 5’-AAAGACACCCACCTTGAT-3’;HVR2:5’- years with a mean of 39.4 ± 10.5. Of these, 10 are females AATTTCCACCAAACCCC-3; CD: 5’-CATTGGCAGCC and 16 males. Besides, a total of 39 healthy volunteer indi- TAGCATT-3’) were synthesized by GeneCore Bio Tech- viduals without IR were included in this study for compar- nologies Co. Ltd., Shanghai, China. Dose-dependent plas- ing the difference of basal mtDNA and CD levels between mid-constructed ß-actin, HVR2 and CD standards were ALL patients and normal donors before IR. The donors used in each run of real-time PCR. Of these, both plas- age from 18 to 55 years with a mean of 37.2 ± 9.4. 19 are mids containing the CD breakpoint and the HVR2 region females and 20 males. All tested subjects signed an were kindly provided by Professor E. Kirches [20]. All informed consent to the use of blood samples in accor- TaqMan reactions were carried out in 96-well plates on dance with the Declaration of Helsinki and with the an ABI 7500 Real-Time PCR instrument (Applied Biosys- approval from our Institutional Review Board. The tems, Foster City, CA, USA) using the Real-Time PCR amount of CD in skeletal muscle under physiological con- Master Mix kit from Toyobo Co. (Osaka, Japan). Each ditions is relatively high (up to 1-2% from total mtDNA reaction was carried out in total volume of 25 μlwith content) [18]. Therefore, DNA isolated from skeletal mus- 50 ng total DNA template, 300 nM each primer, and cle of a 75-year-old male at autopsy was used as positive 100 nM TaqMan-MGB probe. After an initial denatura- control in the present study. tion step at 94°C for three minutes, 40-45 PCR cycles of 15 s at 94°C, 20 s at 60°C, and 30 s at 72°C were per- In vivo irradiation and peripheral blood lymphocyte formed. Real-time PCR of all samples and standards were isolation carried out in quadruplicate. The data from a PCR All patients were treated with two 4.5 Gy TBI sessions run were rejected if the correlation coefficient was less than 0.98. daily using an Elekta SLi 8 MV linear accelerator (Elekta Wen et al. Radiation Oncology 2011, 6:133 Page 3 of 7 http://www.ro-journal.com/content/6/1/133 Statistical analysis MGB PCR approach produces high sensitivity, and could All statistical computations were done using the SPSS give reliable and corroborating data in our study. v15.0 (SPSS, Chicago, IL). Logarithmic transformation of data was essential for further parameter statistical analysis Basal level of mtDNA content and CD ratio from healthy since the original values of the mtDNA and CD copy donors and ALL patients number in lymphocytes showed a nonnormal distribution. We first quantified the mtDNA content (median = 197, Univariate analysis of variance and Student-Newman- minimum = 65, maximum = 1124 in ALL; median = 398, Keuls post hoc tests were used to analyze the difference in minimum = 39, maximum = 1283 in healthy donors) and mtDNA and CD level with IR exposure. The relative CD ratio (median = 0.0116%, minimum = 0.0019%, maxi- change of mtDNA and CD levels after different dosage mum = 0.085% in ALL; median = 0.0193%, minimum = exposure were tested by nonparametric Friedman test. 0.0027%, maximum = 0.121%) per cell in PBLs from ALL The Pearson’s correlation test was used to explore associa- patients and healthy donors before irradiation to deter- tion between mtDNA and CD levels. The correlation mine the distribution pattern. Since both variables did not between mtDNA, CD level and gender, age was analyzed show normal distribution (P < 0.01, Kolmogorov-Smirnov by the nonparametric Spearman’s rho correlation test and test), a logarithm of the mtDNA content and CD ratio was the Pearson’s correlation test individually. P values < 0.05 made for normal distributions (see details in additional file are considered statistically significant. All reported 1, Figure S1). Data of mtDNA content and CD ratio after P values are two sided. logarithm in the three study groups (0, 4.5 and 9 Gy TBI respectively) were given in Table 1 as mean ± SD, median Results and range. Mean ± SD values of initial mtDNA and CD Reliability and reproducibility of the TaqMan-MGB PCR level in healthy donors cohort were at 2.507 ± 0.281 and assay -3.683 ± 0.414. No statistically significant difference was The level of mtDNA and CD from lymphocytes was deter- found for logarithm of basal mtDNA and CD level mined in a set of independent experiments. First, a Taq- between healthy donors and patients with ALL. Man reaction targeting the house keeping gene ß-actin was used to measure the amount of genomic DNA present Changes of mtDNA content and CD ratio after TBI in in cells. A second TaqMan assay was designed to the patients HVR2 region to quantitate the total amount of mitochon- Next, we investigated whether the irradiation dose has an drial DNA. The mtDNA content was normalized to the effect on mtDNA and CD level with lymphocytes. Signifi- amount of genomic DNA in a lymphocyte and expressed cant differences were found between IR status and as a ratio of mtDNA molecules relative to total genomic mtDNA alteration among lymphocytes 24 h after the irra- DNA molecules per cell. A third TaqMan assay targeted diation (P = 0.038 for mtDNA content, 0.027 for CD ratio, the CD breakpoint and measured the abundance of the Univariate analysis of variance). Furthermore, Student CD in the samples. The level of CD was normalized using Newman-Keuls post-hoc tests were used to compare the mtDNA amount and was expressed as a ratio to the mito- difference among the three groups. mtDNA content was chondrial DNA amounts. In other words, the CD ratio significantly increased in 4.5 and 9 Gy irradiation groups was expressed as a percentage of deleted mtDNA mole- compared with 0 Gy group (mean value of mtDNA con- cules relative to total mtDNA molecules in per genomic tent 2.526 and 2.711 compared with 2.360), as well as CD DNA molecules. These primer sets have been used exten- ratio reduced in 4.5 and 9 Gy irradiation groups compared sively for measuring the CD and mtDNA in tissues con- with 0 Gy group (mean value of CD ratio -4.148 and taining low CD and give reliable results [20,21]. Figure 1 -4.233 compared with -3.935). showed the standard curve for the mtDNA common dele- tion and the CD amplification plots for the samples exam- Relative change of mtDNA and CD in lymphocytes from ined. It demonstrated that employed TaqMan assay was each patient after TBI sensitive enough to detect single molecule of CD and high The results above obtained from in vivo lymphocytes iso- -12e-0.6358x linearity was found (y =3E ) in the range of stan- lated from patients suggest a correlation of increased dard samples. CD levels in most of the samples were mtDNA and decreased CD level with dosage (4.5, 9 Gy) detected between Ct 35 and 39. In all samples examined, irradiation in cohort study. To better examine the associa- PCR products were amplified within the linear range of tion between mtDNA alterations and IR in individuals, assays (r > 0.98). Positive control DNA from a 75 year relative changes of mtDNA and CD levels after different old male skeletal muscle contained about 0.729% CD ratio dose TBI were compared for each patient. As shown in and most of the lymphocytes samples contained from Figure 2, the increase in mtNDA content was average 1.87 0.003% to 0.04% CD ratio, consistent with other measure- and 2.13 times individually after 4.5 and 9 Gy TBI (P < ments [18,22]. These results suggest that the TaqMan- 0.001, Friedman test). Meanwhile, decrease in CD was Wen et al. Radiation Oncology 2011, 6:133 Page 4 of 7 http://www.ro-journal.com/content/6/1/133 Figure 1 TaqMan PCR assay for measuring the common mitochondrial deletion in DNA extracted from lymphocytes.The top panel shows the amplification plot for the standard curve whereas the bottom panel shows the amplification plot for the lymphocyte samples. The level of the common mitochondrial deletion in the lymphocyte samples is within the linear range of the standard curve. 0.78 and 0.61 when 4.5, 9 vs. 0 Gy cohorts respectively (P TBI. Similar trends occurred after 9 Gy exposure, where < 0.001, Friedman test). Moreover, significant difference 84.6% of increased mtDNA content (22/26) and 88.5% of was observed in mtDNA copy (P = 0.041) and CD ratio (P decreased CD ratio (23/26) observed. < 0.001) in each patient when comparing 9 Gy vs. 4.5 Gy exposure. Besides, proportions of increased mtDNA con- Relation between mtDNA and CD level after irradiation tent in lymphocytes was found to be 80.8% (21/26) and No relation was found between the level of mtDNA and decreased CD ratio to be 84.6% (22/26) after 4.5 Gy of CD at 0, 4.5 and 9 Gy, when they were analyzed as Wen et al. Radiation Oncology 2011, 6:133 Page 5 of 7 http://www.ro-journal.com/content/6/1/133 Table 1 Logarithm of mtDNA and CD levels in peripheral blood lymphocytes from patients before and after irradiation Group Log (mtDNA content) Log (CD ratio) Median (range) Mean ± SD Median (range) Mean ± SD 0 Gy 2.294 (1.811~ 3.051) 2.360 ± 0. 320 -3.934 (-4.730 ~ -3.071) -3.935 ± 0.459 4.5 Gy 2.566 (1.950 ~ 3.069) 2.526 ± 0. 384 -4.069 (-4.857 ~ -3.063) -4.148 ± 0. 531 9 Gy 2.715 (1.956 ~ 3.186) 2.711 ± 0. 363 -4.437 (-4.952 ~ -3.255) -4.233 ± 0.527 P 0.038 0.027 Abbreviations: SD, standard devitation; CD, common deletion; P value was demonstrated by univariate analysis of variance. continuous variables (Pearson test used in all correla- thirds of the distribution), a modest inverse correlation tions). However, when CD values were segregated in was found reaching significant level for mtDNA content two populations (the lower third against the two upper at different dosage (P = 0.037 for 4.5 Gy, 0.048 for 9 Gy, shown in Figure 3). Besides, significant elevated mtDNA content was observed not in high but in low CD popula- tion (P = 0.021) after 4.5 Gy TBI exposure. Effect of age and gender Finally, the correlations between age, gender, mtDNA and CD level were analyzed individually. No relationship was found between mtDNA, CD level and gender. How- ever, a significant positive effect of age was found for basal logarithm mtDNA content in PBLs. A regression analysis allowed quantification of the effect of age on basal mtDNA content (regression coefficient = 0.0085 y- 1; r2 = 0.251; P = 0.011). The corresponding graphs are presented in Figure 4. These results suggest that older people contained higher mtDNA content in general in the age range of 19-56. Discussion In this paper, we described a sensitive and reliable real- time PCR assay of identifying the mtDNA and common deletion levels. As expected, employed TaqMan-MGB probe was sensitive enough to detect single molecule of CD in our experiment. The sensitivity increased at least 5 fold compared with non specific SYBR Green dye real- time PCR experiment [23]. Besides the improvement of PCR method, we used human tissues and in vivo irradia- tion model, whereas the cell strains and ex vivo irradia- tion model was exclusively used in other studies. As we known, the ex vivo cultured cells is unlikely to reflect full view of in vivo microenvironment. What is more, a lots of apoptotic cell occurs after IR, which is hardly to isolate from the whole cell population of strain, and will extre- Figure 2 Relative change of mtDNA content (A) and CD ratio mely affect the accurate quantification of mtDNA and (B) from patients’ PBLs (n = 26) after different dose of total CD level for cell heterogeneity [23]. In contract, lympho- body irradiation therapy. Significant difference was observed in cytes in vivo mostly consist of survival cells (> 95%) and relative mtDNA (*P = 0.041) and CD (*P < 0.001) change of every could avert the effect of apoptosis [24]. No doubt, it had patient when comparing 9 Gy vs. 4.5 Gy exposure. A circle integrity advantage and is a big step up compared to ex represents mean value of relative change level from each patient undergoing irradiation compared to their basal levels. The lines vivo model. Based on these evidences above mentioned, connect the mean values of relative change level from all cases. we can declare that direct analysis of lymphocytes Wen et al. Radiation Oncology 2011, 6:133 Page 6 of 7 http://www.ro-journal.com/content/6/1/133 content of irradiated PBLs elevated consisting with a dose response. This phenomenon has been explained as a com- pensatory replication of mtDNA to replace damaged mtDNA . Our statistical analysis showed induced levels of CD after TBI in PBLs, compared to some other reports that IR-induced oxidative stress may cause increase of CD ratio [13,14]. Considering that high deletion level of mtDNA increases the susceptibility of human cell to apoptosis[25], the difference is most likely due to the fact that IR exposure causes lymphocytes differentiate into two major populations immediately: apoptotic population usually containing relative high CD level and thus being sensitive to apoptosis, while surviving population con- taining relatively low CD level and more resistant to IR. The cell source in other studies is likely mixed with many apoptotic cells, which may resulted in relatively high CD level detection. Here, we report a statistical inversely association between these two predictive values (mtDNA content and CD ratio) for radiation toxicity. That is to say, lowest Figure 3 Box plot shows an association between CD ratio and values of CD ratio were related to higher values of mtDNA mtDNA content. The lines connect the medians, the boxes cover content, at the same radiation dose in our experiment. Cell th th the 25 to 75 percentiles, and the minimal and maximal values response to IR is individual, and the amount of initial are shown by the ends of the bars. Patients with lower amount of mtDNA and CD levels depend on each patient. The CD ratio suffered higher levels of mtDNA. mechanism behind the relationship remains unclear. One possible reason is that lymphocytes containing lower level isolated from human bodies who received TBI would of deleted mtDNA have stronger ability to replicate wild greatly improve specificity and reliability. These techni- mtDNA than cells with high CD level, in order to resist the que refinements take us closer to a methodology that is irradiation induced mitochondrial damage [26]. Besides, likely to produce reliable and quantitative results. abundant mtDNA replication only occurred in low CD The role of mtDNA content has been investigated in population after moderate dosage treatment, which sug- relation to TBI therapy for the first time in ALL patients. gests the stronger replication ability of low CD population The number of mtDNA copies was elevated in lympho- and a mass of mtDNA copy number production. However, cytes from above 80% of cases after TBI. Besides, mtDNA the strong replication ability was not shown in high CD population after modest dosage treatment. Cellular oxidative stress is thought to play a role in the aging process and may affect mtDNA replication. In the present study, we found that mtDNA copy number is increased with age by lineal regression in our limited cohorts. Similar results has been described that individuals after middle age may be attributed to the enhanced oxida- tive stress than young adults [27], suggesting age factor should be considered when measuring mtDNA content from both nonirradiated and irradiated lymphocytes. Conclusion This study describes the development of a rapid, sensitive, and practical real-time PCR method to quantify the mtDNA copy number and common deletion in PBL sam- ples. Our results suggest that radiation increased mtDNA content and declined common deletion ratio in peripheral Figure 4 Regression analysis of the relationship between age lymphocytes of ALL patients, and an inverse association and basal mtDNA content from patients’ lymphocytes (n = 26). was observed between both parameters after irradiation, Wen et al. Radiation Oncology 2011, 6:133 Page 7 of 7 http://www.ro-journal.com/content/6/1/133 9. Krishnan KJ, Reeve AK, Samuels DC, Chinnery PF, Blackwood JK, Taylor RW, which may be considered as predictive factors to radiation Wanrooij S, Spelbrink JN, Lightowlers RN, Turnbull DM: What causes toxicity. mitochondrial DNA deletions in human cells? Nat Genet 2008, 40:275-279. 10. Gerhard GS, Benko FA, Allen RG, Tresini M, Kalbach A, Cristofalo VJ, Gocke CD: Mitochondrial DNA mutation analysis in human skin fibroblasts from Additional material fetal, young, and old donors. Mech Ageing Dev 2002, 123:155-166. 11. Chabi B, Mousson de Camaret B, Chevrollier A, Boisgard S, Stepien G: Additional file 1: Figure S1. The histograms show the frequency Random mtDNA deletions and functional consequence in aged human distribution of logarithm of both mtDNA content (A) and CD ratio (B) skeletal muscle. Biochem Biophys Res Commun 2005, 332:542-549. from patients (n = 26) after different dose of irradiation. Both population 12. Pavicic WH, Richard SM: Correlation analysis between mtDNA 4977-bp showed normal distributions (P = 0.488 and P = 0.753 respectively, deletion and ageing. Mutat Res 2009, 670:99-102. Kolmogorov-Smirnov test). 13. Prithivirajsingh S, Story MD, Bergh SA, Geara FB, Ang KK, Ismail SM, Stevens CW, Buchholz TA, Brock WA: Accumulation of the common mitochondrial DNA deletion induced by ionizing radiation. Febs Letters 2004, 571:227-232. 14. Kubota N, Hayashi J, Inada T, Iwamura Y: Induction of a particular deletion List of abbreviations in mitochondrial DNA by × rays depends on the inherent mtDNA: mitochondrial DNA; CD: common deletion; PB: peripheral blood; PBLs: radiosensitivity of the cells. Radiat Res 1997, 148:395-398. peripheral blood lymphocytes; TBI: total body irradiation; IR: ionizing radiation; 15. Wardell TM, Ferguson E, Chinnery PF, Borthwick GM, Taylor RW, Jackson G, HVR2: hypervariable region 2; nDNA: nuclear DNA; ALL: acute lymphoblastic Craft A, Lightowlers RN, Howell N, Turnbull DM: Changes in the human leukemia; MGB: minor groove binder; ROS: reactive oxygen species. mitochondrial genome after treatment of malignant disease. Mutat Res 2003, 525:19-27. Acknowledgements 16. Willoughby K, Valdazo-Gonzalez B, Maley M, Gilray J, Nettleton PF: We would like to acknowledge Professor E. Kirches for his assistance with Development of a real time RT-PCR to detect and type ovine plasmids donation. We are indebted to associate Professor Jieqiong Lei pestiviruses. J Virol Methods 2006, 132:187-194. (Mathematics department, College of biotechnology, TMMU University) for 17. Ribera JM, Oriol A, Bethencourt C, Parody R, Hernandez-Rivas JM, statistical assistance, to the patients and donors who donated blood for this Besalduch J, Sanz MA, Arias J, Fernandez-Calvo J, Moraleda JM, et al: study. This study was supported by grants from the Keystone Project of the Comparison of intensive chemotherapy, allogeneic or autologous stem “Eleventh Five-year Plan” for Medical Science Development of PLA (No.06G068) cell transplantation as post-remission treatment for adult patients with and the National Natural Science Foundation of China (No.30772144). high-risk acute lymphoblastic leukemia. Final results of the PETHEMA ALL-93 Trial. Bone Marrow Transplantation 2005, 35:S14-S14. Author details 1 18. Moraes CT, Sciacco M, Ricci E, Tengan CH, Hao H, Bonilla E, Schon EA, Third Department of Oncology, The second affiliated hospital, Third Military DiMauro S: Phenotype-genotype correlations in skeletal muscle of Medical University, Chongqing 400037, China. Institute of Human patients with mtDNA deletions. Muscle Nerve 1995, 3:S150-153. Respiratory Disease, The second affiliated hospital, Third Military Medical 19. Yao Y, Kong Q, Bandelt H, Kivisild T, Zhang Y: Phylogeographic University, Chongqing 400037, China. differentiation of mitochondrial DNA in Han Chinese. The American Journal of Human Genetics 2002, 70:635-651. Authors’ contributions 20. Sabunciyan S, Kirches E, Krause G, Bogerts B, Mawrin C, Llenos IC, Weis S: QW and YH designed the study, FJ provided real-time PCR assay, QW Quantification of total mitochondrial DNA and mitochondrial common analyzed the data and written the paper, GQ contributed to revising the deletion in the frontal cortex of patients with schizophrenia and bipolar paper. All authors read and approved the final manuscript. disorder. Journal of Neural Transmission 2007, 114:665-674. 21. Pogozelski WK, Hamel CJ, Woeller CF, Jackson WE, Zullo SJ, Fischel- Competing interests Ghodsian N, Blakely WF: Quantification of total mitochondrial DNA and The authors declare that they have no competing interests. the 4977-bp common deletion in Pearson’s syndrome lymphoblasts using a fluorogenic 5’-nuclease (TaqMan) real-time polymerase chain Received: 26 May 2011 Accepted: 6 October 2011 reaction assay and plasmid external calibration standards. Mitochondrion Published: 6 October 2011 2003, 2:415-427. 22. Mohamed SA, Wesch D, Blumenthal A, Bruse P, Windler K, Ernst M, Kabelitz D, References Oehmichen M, Meissner C: Detection of the 4977 bp deletion of 1. Iliakis G, Wang H, Perrault AR, Boecker W, Rosidi B, Windhofer F, Wu W, mitochondrial DNA in different human blood cells. Experimental Gerontology Guan J, Terzoudi G, Pantelias G: Mechanisms of DNA double strand break 2004, 39:181-188. repair and chromosome aberration formation. Cytogenet Genome Res 23. Wang L, Kuwahara Y, Li L, Baba T, Shin RW, Ohkubo Y, Ono K, Fukumoto M: 2004, 104:14-20. Analysis of Common Deletion (CD) and a novel deletion of 2. Purkayastha S, Milligan JR, Bernhard WA: On the chemical yield of base mitochondrial DNA induced by ionizing radiation. International Journal of lesions, strand breaks, and clustered damage generated in plasmid DNA Radiation Biology 2007, 83:433-442. by the direct effect of × rays. Radiat Res 2007, 168:357-366. 24. Schmitz A, Bayer J, Dechamps N, Thomas G: Intrinsic susceptibility to 3. Yakes FM, Van Houten B: Mitochondrial DNA damage is more extensive radiation-induced apoptosis of human lymphocyte subpopulations. and persists longer than nuclear DNA damage in human cells following International Journal of Radiation Oncology Biology Physics 2003, 57:769-778. oxidative stress. Proc Natl Acad Sci USA 1997, 94:514-519. 25. Liu CY, Lee CF, Hong CH, Wei YH: Mitochondrial DNA mutation and 4. Rodemann HP, Blaese MA: Responses of normal cells to ionizing depletion increase the susceptibility of human cells to apoptosis. Ann N radiation. Elsevier 2007, 81-88. Y Acad Sci 2004, 1011:133-145. 5. Larsen NB, Rasmussen M, Rasmussen LJ: Nuclear and mitochondrial DNA 26. Wai T, Teoli D, Shoubridge EA: The mitochondrial DNA genetic bottleneck repair: similar pathways? Mitochondrion 2005, 5:89-108. results from replication of a subpopulation of genomes. Nature Genetics 6. Yang JL, Weissman L, Bohr VA, Mattson MP: Mitochondrial DNA damage 2008, 40:1484-1488. and repair in neurodegenerative disorders. DNA repair 2008, 27. Liu CS, Tsai CS, Kuo CL, Chen HW, Lii CK, Ma YS, Wei YH: Oxidative stress- 7:1110-1120. related alteration of the copy number of mitochondrial DNA in human 7. Zhang H, Maguire D, Swarts S, Sun W, Yang S, Wang W, Liu C, Zhang M, leukocytes. Free Radic Res 2003, 37:1307-1317. Zhang D, Zhang L, et al: Replication of murine mitochondrial DNA following irradiation. Adv Exp Med Biol 2009, 645:43-48. doi:10.1186/1748-717X-6-133 8. Cortopassi GA, Arnheim N: Detection of a specific mitochondrial DNA Cite this article as: Wen et al.: Mitochondrial DNA alterations of deletion in tissues of older humans. Nucleic Acids Res 1990, 18:6927-6933. peripheral lymphocytes in acute lymphoblastic leukemia patients undergoing total body irradiation therapy. Radiation Oncology 2011 6:133. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

Mitochondrial DNA alterations of peripheral lymphocytes in acute lymphoblastic leukemia patients undergoing total body irradiation therapy

Wen, Quan; Hu, Yide; Ji, Fuyun; Qian, Guisheng
Radiation Oncology , Volume 6 (1) – Oct 6, 2011
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Springer Journals
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Copyright © 2011 by Wen et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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Abstract

Background: Mitochondrial DNA (mtDNA) alterations, including mtDNA copy number and mtDNA 4977 bp common deletion (CD), are key indicators of irradiation-induced damage. The relationship between total body irradiation (TBI) treatment and mtDNA alterations in vivo, however, has not been postulated yet. The aim of this study is to analyze mtDNA alterations in irradiated human peripheral lymphocytes from acute lymphoblastic leukemia (ALL) patients as well as to take them as predictors for radiation toxicity. Methods: Peripheral blood lymphocytes were isolated from 26 ALL patients 24 hours after TBI preconditioning (4.5 and 9 Gy, respectively). Extracted DNA was analyzed by real-time PCR method. Results: Average 2.31 times mtDNA and 0.53 fold CD levels were observed after 4.5 Gy exposure compared to their basal levels. 9 Gy TBI produced a greater response of both mtDNA and CD levels than 4.5 Gy. Significant inverse correlation was found between mtDNA content and CD level at 4.5 and 9 Gy (P = 0.037 and 0.048). Moreover, mtDNA content of lymphocytes without irradiation was found to be correlated to age. Conclusions: mtDNA and CD content may be considered as predictive factors to radiation toxicity. Keywords: mtDNA, 4977-bp Common deletion, Total body irradiation, Real-time-PCR, Acute lymphoblastic leukemia Background possible radio-protective mechanism is that enhanced Breakage of cellular DNA following radiation is a dose replication of mtDNA reduces the mutation frequency of dependent phenomenon and occurs in both the nuclear total mtDNA and delays the onset of lethal radiation and extra-nuclear DNA. Thus, besides nuclear nDNA, damage to the mitochondria [5,6]. This hypothesis has mitochondrial DNA (mtDNA) is equally affected as an been recently supported by Zhang et al with exhibiting only extra-nuclear genome [1,2]. Numerous investigations increased mtDNA copy number in gut and bone marrow showed that mtDNA can be an easily available target for of total body irradiated rats [7]. On the other hand, IR endogenous reactive oxygen species (ROS) and free radi- usually prompts cell apoptosis by displaying an accumula- cals caused by ionizing radiation (IR), which resulted in tion of large scale mtDNA deletions, especially the specific mtDNA copy number alteration and mtDNA damage 4977 bp deletion, referred to as the “common deletion (such as mutation and depletion) [3,4]. (CD)" [8]. The site of CD is flanked by two13 bp direct The mechanisms of cellular response to radiation with repeats (ACCTCCCTCACCA) at mtDNA nucleotide site 8470 and 13447 respectively, and easy to make deletion regard to mtDNA alterations were mainly involved in the following two ways. On one hand, mtDNA has few repair for its unique formation mechanism [9]. Studies have mechanisms and continued mitochondrial function is pre- shown that CD can be as a sensitive marker of oxidative served primarily due to its high copy number. One of damage to mtDNA [10-12]. Unfortunately, only few experiments have evaluated the association between CD and IR till now. For example, accumulation of CD * Correspondence: huyide_mit@yahoo.com.cn 1 has been identified by qualitative PCR method on several Third Department of Oncology, The second affiliated hospital, Third Military Medical University, Chongqing 400037, China irradiated cell lines (such as human skin fibroblasts, Full list of author information is available at the end of the article © 2011 Wen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wen et al. Radiation Oncology 2011, 6:133 Page 2 of 7 http://www.ro-journal.com/content/6/1/133 glioblastoma and colon carcinoma lines) and primary lym- Co., Stockholm, Sweden) set to deliver a dose rate of phocytes [13-15]. Furthermore, CD was induced by IR in 4.5-4.9 cGy/min over two successive days. None of the human hepatoblastoma cell line performing on real-time patients had prior exposure to any cytotoxic treatment PCR with nonspecific dsDNA-binding dye SYBR Green. for at least 2 weeks before the start of radiotherapy. All However, their conclusions were largely controversial. The patients had 7 ml of PB collected prior to and 24 h fol- inconsistency may be due, in part, to the use of non-quan- lowing exposure for each radiation treatment. Besides, titative PCR strategies. Additionally, none of these studies 39 healthy donors had the same volume of PB collected have assayed mtDNA or CD level in peripheral blood lym- without ionizing radiation. Preparation of PBLs followed phocytes (PBLs) after in vivo irradiation exposure for lack standard methods, using human lymphocyte isolation of appropriate human beings radiation model. reagent (TBD Biological Technology Co., Tianjin, In this study, we performed real-time PCR technique China) for separation of mononuclear cells. with a specific fluorogenic TaqMan probe conjugated with minor groove binder (MGB) groups, which is more sensi- DNA extraction tive and appropriate than nonspecific dsDNA-binding dye DNA from lymphocytes in vivo and the skeletal muscle PCR methods previously used [16]. Besides, we taken the was obtained with the TIANamp Genomic DNA Kit acute lymphoblastic leukemia (ALL) patients undergoging (Tiangen Ltd, Beijing, China), and stored at -70°C until total body irradiation (TBI) precondionting as human further study. beings in vivo irradiation model. The advantage of using this model lies in full view of in vivo microenvironment, Analysis of amount of mtDNA and CD by real-time PCR and without need for irradiating healthy individuals. We TaqMan probes with conjugated MGB groups were per- attempted to address the mtDNA status in irradiated formed to ensure maximal specificity in real-time PCR human peripheral blood lymphocytes in vivo to elucidate reaction. Nuclear DNA content was estimated by mea- whether alterations in mtDNA can be linked to exposure suring the human ß-actin gene. The hypervariable region to total body irradiation. 2 (HVR2) in the mitochondrial D-Loop was used to represent the total amount of mtDNA since this region is Materials and methods relatively conserved in Han Chinese [19]. The forward Study participants primer (ß-actin: 5’-AGGACCCTGGATGTGACAGC-3’; This study comprised peripheral blood (PB) samples from HVR2: 5’-GCTTTCCACACAGACATCATAACAA-3’; 26 high risked ALL patients undergoing TBI as pre-trans- CD: 5’-CTTACACTATTCCTCATCACCCAACTAA plantation treatment in their first complete remission AAA-3’), reverse primer (ß-actin: 5’-TGGCATTGCCGA- (CR1) at hematology department of our institution. The CAGGAT-3’;HVR2:5’-GTTTAAGTGCTGTGGCCA- diagnoses were. according to world healthy organization GAAG-3’;CD:5’-GGAGTAGAAACCTGTGAGGAA (WHO) classification and high risk factors were measured AGG-3’) and TaqMan MGB hybridization probes on Ribeca’s report [17]. The patients age from 19 to 56 (ß-actin: 5’-AAAGACACCCACCTTGAT-3’;HVR2:5’- years with a mean of 39.4 ± 10.5. Of these, 10 are females AATTTCCACCAAACCCC-3; CD: 5’-CATTGGCAGCC and 16 males. Besides, a total of 39 healthy volunteer indi- TAGCATT-3’) were synthesized by GeneCore Bio Tech- viduals without IR were included in this study for compar- nologies Co. Ltd., Shanghai, China. Dose-dependent plas- ing the difference of basal mtDNA and CD levels between mid-constructed ß-actin, HVR2 and CD standards were ALL patients and normal donors before IR. The donors used in each run of real-time PCR. Of these, both plas- age from 18 to 55 years with a mean of 37.2 ± 9.4. 19 are mids containing the CD breakpoint and the HVR2 region females and 20 males. All tested subjects signed an were kindly provided by Professor E. Kirches [20]. All informed consent to the use of blood samples in accor- TaqMan reactions were carried out in 96-well plates on dance with the Declaration of Helsinki and with the an ABI 7500 Real-Time PCR instrument (Applied Biosys- approval from our Institutional Review Board. The tems, Foster City, CA, USA) using the Real-Time PCR amount of CD in skeletal muscle under physiological con- Master Mix kit from Toyobo Co. (Osaka, Japan). Each ditions is relatively high (up to 1-2% from total mtDNA reaction was carried out in total volume of 25 μlwith content) [18]. Therefore, DNA isolated from skeletal mus- 50 ng total DNA template, 300 nM each primer, and cle of a 75-year-old male at autopsy was used as positive 100 nM TaqMan-MGB probe. After an initial denatura- control in the present study. tion step at 94°C for three minutes, 40-45 PCR cycles of 15 s at 94°C, 20 s at 60°C, and 30 s at 72°C were per- In vivo irradiation and peripheral blood lymphocyte formed. Real-time PCR of all samples and standards were isolation carried out in quadruplicate. The data from a PCR All patients were treated with two 4.5 Gy TBI sessions run were rejected if the correlation coefficient was less than 0.98. daily using an Elekta SLi 8 MV linear accelerator (Elekta Wen et al. Radiation Oncology 2011, 6:133 Page 3 of 7 http://www.ro-journal.com/content/6/1/133 Statistical analysis MGB PCR approach produces high sensitivity, and could All statistical computations were done using the SPSS give reliable and corroborating data in our study. v15.0 (SPSS, Chicago, IL). Logarithmic transformation of data was essential for further parameter statistical analysis Basal level of mtDNA content and CD ratio from healthy since the original values of the mtDNA and CD copy donors and ALL patients number in lymphocytes showed a nonnormal distribution. We first quantified the mtDNA content (median = 197, Univariate analysis of variance and Student-Newman- minimum = 65, maximum = 1124 in ALL; median = 398, Keuls post hoc tests were used to analyze the difference in minimum = 39, maximum = 1283 in healthy donors) and mtDNA and CD level with IR exposure. The relative CD ratio (median = 0.0116%, minimum = 0.0019%, maxi- change of mtDNA and CD levels after different dosage mum = 0.085% in ALL; median = 0.0193%, minimum = exposure were tested by nonparametric Friedman test. 0.0027%, maximum = 0.121%) per cell in PBLs from ALL The Pearson’s correlation test was used to explore associa- patients and healthy donors before irradiation to deter- tion between mtDNA and CD levels. The correlation mine the distribution pattern. Since both variables did not between mtDNA, CD level and gender, age was analyzed show normal distribution (P < 0.01, Kolmogorov-Smirnov by the nonparametric Spearman’s rho correlation test and test), a logarithm of the mtDNA content and CD ratio was the Pearson’s correlation test individually. P values < 0.05 made for normal distributions (see details in additional file are considered statistically significant. All reported 1, Figure S1). Data of mtDNA content and CD ratio after P values are two sided. logarithm in the three study groups (0, 4.5 and 9 Gy TBI respectively) were given in Table 1 as mean ± SD, median Results and range. Mean ± SD values of initial mtDNA and CD Reliability and reproducibility of the TaqMan-MGB PCR level in healthy donors cohort were at 2.507 ± 0.281 and assay -3.683 ± 0.414. No statistically significant difference was The level of mtDNA and CD from lymphocytes was deter- found for logarithm of basal mtDNA and CD level mined in a set of independent experiments. First, a Taq- between healthy donors and patients with ALL. Man reaction targeting the house keeping gene ß-actin was used to measure the amount of genomic DNA present Changes of mtDNA content and CD ratio after TBI in in cells. A second TaqMan assay was designed to the patients HVR2 region to quantitate the total amount of mitochon- Next, we investigated whether the irradiation dose has an drial DNA. The mtDNA content was normalized to the effect on mtDNA and CD level with lymphocytes. Signifi- amount of genomic DNA in a lymphocyte and expressed cant differences were found between IR status and as a ratio of mtDNA molecules relative to total genomic mtDNA alteration among lymphocytes 24 h after the irra- DNA molecules per cell. A third TaqMan assay targeted diation (P = 0.038 for mtDNA content, 0.027 for CD ratio, the CD breakpoint and measured the abundance of the Univariate analysis of variance). Furthermore, Student CD in the samples. The level of CD was normalized using Newman-Keuls post-hoc tests were used to compare the mtDNA amount and was expressed as a ratio to the mito- difference among the three groups. mtDNA content was chondrial DNA amounts. In other words, the CD ratio significantly increased in 4.5 and 9 Gy irradiation groups was expressed as a percentage of deleted mtDNA mole- compared with 0 Gy group (mean value of mtDNA con- cules relative to total mtDNA molecules in per genomic tent 2.526 and 2.711 compared with 2.360), as well as CD DNA molecules. These primer sets have been used exten- ratio reduced in 4.5 and 9 Gy irradiation groups compared sively for measuring the CD and mtDNA in tissues con- with 0 Gy group (mean value of CD ratio -4.148 and taining low CD and give reliable results [20,21]. Figure 1 -4.233 compared with -3.935). showed the standard curve for the mtDNA common dele- tion and the CD amplification plots for the samples exam- Relative change of mtDNA and CD in lymphocytes from ined. It demonstrated that employed TaqMan assay was each patient after TBI sensitive enough to detect single molecule of CD and high The results above obtained from in vivo lymphocytes iso- -12e-0.6358x linearity was found (y =3E ) in the range of stan- lated from patients suggest a correlation of increased dard samples. CD levels in most of the samples were mtDNA and decreased CD level with dosage (4.5, 9 Gy) detected between Ct 35 and 39. In all samples examined, irradiation in cohort study. To better examine the associa- PCR products were amplified within the linear range of tion between mtDNA alterations and IR in individuals, assays (r > 0.98). Positive control DNA from a 75 year relative changes of mtDNA and CD levels after different old male skeletal muscle contained about 0.729% CD ratio dose TBI were compared for each patient. As shown in and most of the lymphocytes samples contained from Figure 2, the increase in mtNDA content was average 1.87 0.003% to 0.04% CD ratio, consistent with other measure- and 2.13 times individually after 4.5 and 9 Gy TBI (P < ments [18,22]. These results suggest that the TaqMan- 0.001, Friedman test). Meanwhile, decrease in CD was Wen et al. Radiation Oncology 2011, 6:133 Page 4 of 7 http://www.ro-journal.com/content/6/1/133 Figure 1 TaqMan PCR assay for measuring the common mitochondrial deletion in DNA extracted from lymphocytes.The top panel shows the amplification plot for the standard curve whereas the bottom panel shows the amplification plot for the lymphocyte samples. The level of the common mitochondrial deletion in the lymphocyte samples is within the linear range of the standard curve. 0.78 and 0.61 when 4.5, 9 vs. 0 Gy cohorts respectively (P TBI. Similar trends occurred after 9 Gy exposure, where < 0.001, Friedman test). Moreover, significant difference 84.6% of increased mtDNA content (22/26) and 88.5% of was observed in mtDNA copy (P = 0.041) and CD ratio (P decreased CD ratio (23/26) observed. < 0.001) in each patient when comparing 9 Gy vs. 4.5 Gy exposure. Besides, proportions of increased mtDNA con- Relation between mtDNA and CD level after irradiation tent in lymphocytes was found to be 80.8% (21/26) and No relation was found between the level of mtDNA and decreased CD ratio to be 84.6% (22/26) after 4.5 Gy of CD at 0, 4.5 and 9 Gy, when they were analyzed as Wen et al. Radiation Oncology 2011, 6:133 Page 5 of 7 http://www.ro-journal.com/content/6/1/133 Table 1 Logarithm of mtDNA and CD levels in peripheral blood lymphocytes from patients before and after irradiation Group Log (mtDNA content) Log (CD ratio) Median (range) Mean ± SD Median (range) Mean ± SD 0 Gy 2.294 (1.811~ 3.051) 2.360 ± 0. 320 -3.934 (-4.730 ~ -3.071) -3.935 ± 0.459 4.5 Gy 2.566 (1.950 ~ 3.069) 2.526 ± 0. 384 -4.069 (-4.857 ~ -3.063) -4.148 ± 0. 531 9 Gy 2.715 (1.956 ~ 3.186) 2.711 ± 0. 363 -4.437 (-4.952 ~ -3.255) -4.233 ± 0.527 P 0.038 0.027 Abbreviations: SD, standard devitation; CD, common deletion; P value was demonstrated by univariate analysis of variance. continuous variables (Pearson test used in all correla- thirds of the distribution), a modest inverse correlation tions). However, when CD values were segregated in was found reaching significant level for mtDNA content two populations (the lower third against the two upper at different dosage (P = 0.037 for 4.5 Gy, 0.048 for 9 Gy, shown in Figure 3). Besides, significant elevated mtDNA content was observed not in high but in low CD popula- tion (P = 0.021) after 4.5 Gy TBI exposure. Effect of age and gender Finally, the correlations between age, gender, mtDNA and CD level were analyzed individually. No relationship was found between mtDNA, CD level and gender. How- ever, a significant positive effect of age was found for basal logarithm mtDNA content in PBLs. A regression analysis allowed quantification of the effect of age on basal mtDNA content (regression coefficient = 0.0085 y- 1; r2 = 0.251; P = 0.011). The corresponding graphs are presented in Figure 4. These results suggest that older people contained higher mtDNA content in general in the age range of 19-56. Discussion In this paper, we described a sensitive and reliable real- time PCR assay of identifying the mtDNA and common deletion levels. As expected, employed TaqMan-MGB probe was sensitive enough to detect single molecule of CD in our experiment. The sensitivity increased at least 5 fold compared with non specific SYBR Green dye real- time PCR experiment [23]. Besides the improvement of PCR method, we used human tissues and in vivo irradia- tion model, whereas the cell strains and ex vivo irradia- tion model was exclusively used in other studies. As we known, the ex vivo cultured cells is unlikely to reflect full view of in vivo microenvironment. What is more, a lots of apoptotic cell occurs after IR, which is hardly to isolate from the whole cell population of strain, and will extre- Figure 2 Relative change of mtDNA content (A) and CD ratio mely affect the accurate quantification of mtDNA and (B) from patients’ PBLs (n = 26) after different dose of total CD level for cell heterogeneity [23]. In contract, lympho- body irradiation therapy. Significant difference was observed in cytes in vivo mostly consist of survival cells (> 95%) and relative mtDNA (*P = 0.041) and CD (*P < 0.001) change of every could avert the effect of apoptosis [24]. No doubt, it had patient when comparing 9 Gy vs. 4.5 Gy exposure. A circle integrity advantage and is a big step up compared to ex represents mean value of relative change level from each patient undergoing irradiation compared to their basal levels. The lines vivo model. Based on these evidences above mentioned, connect the mean values of relative change level from all cases. we can declare that direct analysis of lymphocytes Wen et al. Radiation Oncology 2011, 6:133 Page 6 of 7 http://www.ro-journal.com/content/6/1/133 content of irradiated PBLs elevated consisting with a dose response. This phenomenon has been explained as a com- pensatory replication of mtDNA to replace damaged mtDNA . Our statistical analysis showed induced levels of CD after TBI in PBLs, compared to some other reports that IR-induced oxidative stress may cause increase of CD ratio [13,14]. Considering that high deletion level of mtDNA increases the susceptibility of human cell to apoptosis[25], the difference is most likely due to the fact that IR exposure causes lymphocytes differentiate into two major populations immediately: apoptotic population usually containing relative high CD level and thus being sensitive to apoptosis, while surviving population con- taining relatively low CD level and more resistant to IR. The cell source in other studies is likely mixed with many apoptotic cells, which may resulted in relatively high CD level detection. Here, we report a statistical inversely association between these two predictive values (mtDNA content and CD ratio) for radiation toxicity. That is to say, lowest Figure 3 Box plot shows an association between CD ratio and values of CD ratio were related to higher values of mtDNA mtDNA content. The lines connect the medians, the boxes cover content, at the same radiation dose in our experiment. Cell th th the 25 to 75 percentiles, and the minimal and maximal values response to IR is individual, and the amount of initial are shown by the ends of the bars. Patients with lower amount of mtDNA and CD levels depend on each patient. The CD ratio suffered higher levels of mtDNA. mechanism behind the relationship remains unclear. One possible reason is that lymphocytes containing lower level isolated from human bodies who received TBI would of deleted mtDNA have stronger ability to replicate wild greatly improve specificity and reliability. These techni- mtDNA than cells with high CD level, in order to resist the que refinements take us closer to a methodology that is irradiation induced mitochondrial damage [26]. Besides, likely to produce reliable and quantitative results. abundant mtDNA replication only occurred in low CD The role of mtDNA content has been investigated in population after moderate dosage treatment, which sug- relation to TBI therapy for the first time in ALL patients. gests the stronger replication ability of low CD population The number of mtDNA copies was elevated in lympho- and a mass of mtDNA copy number production. However, cytes from above 80% of cases after TBI. Besides, mtDNA the strong replication ability was not shown in high CD population after modest dosage treatment. Cellular oxidative stress is thought to play a role in the aging process and may affect mtDNA replication. In the present study, we found that mtDNA copy number is increased with age by lineal regression in our limited cohorts. Similar results has been described that individuals after middle age may be attributed to the enhanced oxida- tive stress than young adults [27], suggesting age factor should be considered when measuring mtDNA content from both nonirradiated and irradiated lymphocytes. Conclusion This study describes the development of a rapid, sensitive, and practical real-time PCR method to quantify the mtDNA copy number and common deletion in PBL sam- ples. Our results suggest that radiation increased mtDNA content and declined common deletion ratio in peripheral Figure 4 Regression analysis of the relationship between age lymphocytes of ALL patients, and an inverse association and basal mtDNA content from patients’ lymphocytes (n = 26). was observed between both parameters after irradiation, Wen et al. Radiation Oncology 2011, 6:133 Page 7 of 7 http://www.ro-journal.com/content/6/1/133 9. Krishnan KJ, Reeve AK, Samuels DC, Chinnery PF, Blackwood JK, Taylor RW, which may be considered as predictive factors to radiation Wanrooij S, Spelbrink JN, Lightowlers RN, Turnbull DM: What causes toxicity. mitochondrial DNA deletions in human cells? Nat Genet 2008, 40:275-279. 10. Gerhard GS, Benko FA, Allen RG, Tresini M, Kalbach A, Cristofalo VJ, Gocke CD: Mitochondrial DNA mutation analysis in human skin fibroblasts from Additional material fetal, young, and old donors. Mech Ageing Dev 2002, 123:155-166. 11. Chabi B, Mousson de Camaret B, Chevrollier A, Boisgard S, Stepien G: Additional file 1: Figure S1. The histograms show the frequency Random mtDNA deletions and functional consequence in aged human distribution of logarithm of both mtDNA content (A) and CD ratio (B) skeletal muscle. Biochem Biophys Res Commun 2005, 332:542-549. from patients (n = 26) after different dose of irradiation. Both population 12. Pavicic WH, Richard SM: Correlation analysis between mtDNA 4977-bp showed normal distributions (P = 0.488 and P = 0.753 respectively, deletion and ageing. Mutat Res 2009, 670:99-102. Kolmogorov-Smirnov test). 13. Prithivirajsingh S, Story MD, Bergh SA, Geara FB, Ang KK, Ismail SM, Stevens CW, Buchholz TA, Brock WA: Accumulation of the common mitochondrial DNA deletion induced by ionizing radiation. Febs Letters 2004, 571:227-232. 14. Kubota N, Hayashi J, Inada T, Iwamura Y: Induction of a particular deletion List of abbreviations in mitochondrial DNA by × rays depends on the inherent mtDNA: mitochondrial DNA; CD: common deletion; PB: peripheral blood; PBLs: radiosensitivity of the cells. Radiat Res 1997, 148:395-398. peripheral blood lymphocytes; TBI: total body irradiation; IR: ionizing radiation; 15. Wardell TM, Ferguson E, Chinnery PF, Borthwick GM, Taylor RW, Jackson G, HVR2: hypervariable region 2; nDNA: nuclear DNA; ALL: acute lymphoblastic Craft A, Lightowlers RN, Howell N, Turnbull DM: Changes in the human leukemia; MGB: minor groove binder; ROS: reactive oxygen species. mitochondrial genome after treatment of malignant disease. Mutat Res 2003, 525:19-27. Acknowledgements 16. Willoughby K, Valdazo-Gonzalez B, Maley M, Gilray J, Nettleton PF: We would like to acknowledge Professor E. Kirches for his assistance with Development of a real time RT-PCR to detect and type ovine plasmids donation. We are indebted to associate Professor Jieqiong Lei pestiviruses. J Virol Methods 2006, 132:187-194. (Mathematics department, College of biotechnology, TMMU University) for 17. Ribera JM, Oriol A, Bethencourt C, Parody R, Hernandez-Rivas JM, statistical assistance, to the patients and donors who donated blood for this Besalduch J, Sanz MA, Arias J, Fernandez-Calvo J, Moraleda JM, et al: study. This study was supported by grants from the Keystone Project of the Comparison of intensive chemotherapy, allogeneic or autologous stem “Eleventh Five-year Plan” for Medical Science Development of PLA (No.06G068) cell transplantation as post-remission treatment for adult patients with and the National Natural Science Foundation of China (No.30772144). high-risk acute lymphoblastic leukemia. Final results of the PETHEMA ALL-93 Trial. Bone Marrow Transplantation 2005, 35:S14-S14. Author details 1 18. Moraes CT, Sciacco M, Ricci E, Tengan CH, Hao H, Bonilla E, Schon EA, Third Department of Oncology, The second affiliated hospital, Third Military DiMauro S: Phenotype-genotype correlations in skeletal muscle of Medical University, Chongqing 400037, China. Institute of Human patients with mtDNA deletions. Muscle Nerve 1995, 3:S150-153. Respiratory Disease, The second affiliated hospital, Third Military Medical 19. Yao Y, Kong Q, Bandelt H, Kivisild T, Zhang Y: Phylogeographic University, Chongqing 400037, China. differentiation of mitochondrial DNA in Han Chinese. The American Journal of Human Genetics 2002, 70:635-651. Authors’ contributions 20. Sabunciyan S, Kirches E, Krause G, Bogerts B, Mawrin C, Llenos IC, Weis S: QW and YH designed the study, FJ provided real-time PCR assay, QW Quantification of total mitochondrial DNA and mitochondrial common analyzed the data and written the paper, GQ contributed to revising the deletion in the frontal cortex of patients with schizophrenia and bipolar paper. All authors read and approved the final manuscript. disorder. Journal of Neural Transmission 2007, 114:665-674. 21. Pogozelski WK, Hamel CJ, Woeller CF, Jackson WE, Zullo SJ, Fischel- Competing interests Ghodsian N, Blakely WF: Quantification of total mitochondrial DNA and The authors declare that they have no competing interests. the 4977-bp common deletion in Pearson’s syndrome lymphoblasts using a fluorogenic 5’-nuclease (TaqMan) real-time polymerase chain Received: 26 May 2011 Accepted: 6 October 2011 reaction assay and plasmid external calibration standards. Mitochondrion Published: 6 October 2011 2003, 2:415-427. 22. Mohamed SA, Wesch D, Blumenthal A, Bruse P, Windler K, Ernst M, Kabelitz D, References Oehmichen M, Meissner C: Detection of the 4977 bp deletion of 1. Iliakis G, Wang H, Perrault AR, Boecker W, Rosidi B, Windhofer F, Wu W, mitochondrial DNA in different human blood cells. Experimental Gerontology Guan J, Terzoudi G, Pantelias G: Mechanisms of DNA double strand break 2004, 39:181-188. repair and chromosome aberration formation. Cytogenet Genome Res 23. 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Rodemann HP, Blaese MA: Responses of normal cells to ionizing depletion increase the susceptibility of human cells to apoptosis. Ann N radiation. Elsevier 2007, 81-88. Y Acad Sci 2004, 1011:133-145. 5. Larsen NB, Rasmussen M, Rasmussen LJ: Nuclear and mitochondrial DNA 26. Wai T, Teoli D, Shoubridge EA: The mitochondrial DNA genetic bottleneck repair: similar pathways? Mitochondrion 2005, 5:89-108. results from replication of a subpopulation of genomes. Nature Genetics 6. Yang JL, Weissman L, Bohr VA, Mattson MP: Mitochondrial DNA damage 2008, 40:1484-1488. and repair in neurodegenerative disorders. DNA repair 2008, 27. Liu CS, Tsai CS, Kuo CL, Chen HW, Lii CK, Ma YS, Wei YH: Oxidative stress- 7:1110-1120. related alteration of the copy number of mitochondrial DNA in human 7. Zhang H, Maguire D, Swarts S, Sun W, Yang S, Wang W, Liu C, Zhang M, leukocytes. Free Radic Res 2003, 37:1307-1317. Zhang D, Zhang L, et al: Replication of murine mitochondrial DNA following irradiation. Adv Exp Med Biol 2009, 645:43-48. doi:10.1186/1748-717X-6-133 8. Cortopassi GA, Arnheim N: Detection of a specific mitochondrial DNA Cite this article as: Wen et al.: Mitochondrial DNA alterations of deletion in tissues of older humans. Nucleic Acids Res 1990, 18:6927-6933. peripheral lymphocytes in acute lymphoblastic leukemia patients undergoing total body irradiation therapy. Radiation Oncology 2011 6:133.

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Published: Oct 6, 2011

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