Abstract
The 4977-base pair common deletion DmtDNA4977 is the most frequently observed mitochondrial DNA mutation in human tissues. Because mitochondrial DNA mutations are mainly caused by reactive oxygen species (ROS), and given that oxidative stress plays an important role in melanoma carcinogenesis, the investigation of DmtDNA4977 may be particularly relevant to the development of melanoma. In the current study, we compared DmtDNA4977 levels in blood leucocytes from 206 melanoma patients and 219 healthy controls. Overall, melanoma cases had significantly higher levels of DmtDNA4977 than healthy controls (median: 0.60 vs 0.20, P=0.008). The difference was evident among individuals who were older than 47 years old, women, and had pigmentation risk factors (e.g. blond or red hair, blue eye, fair skin, light or none tanning ability after prolonged sun exposure, and freckling in the sun as a child). The difference was also evident among those who had at least one lifetime sunburn with blistering and had no reported use of a sunlamp. Interestingly, among controls, DmtDNA4977 levels differed by phenotypic index and reported use of a sunlamp. In the risk assessment, increased levels of DmtDNA4977 were associated with a 1.23-fold increased risk of melanoma (Odds ratio (OR): 1.23, 95% Confidence Interval (90% CI): 1.01, 1.50). A significant dose-response relationship was observed in quartile analysis (P=0.001). In summary, our study suggests that high levels of DmtDNA4977 in blood leucocytes are associated with increased risk of melanoma, and that association is affected by both pigmentation and personal history of sun exposure.
Introduction
Somatic mutations in mitochondrial DNA (mtDNA) have been shown to accumulate with age in a variety of tissues in various organisms, including humans (1–3). These mutations seem to be primarily caused by reactive oxygen species (ROS) that arise as a by-product of oxidative phosphorylation in mitochondria (4). Mutated and wildtype mtDNA molecules often coexist in the heteroplasmic state within the same cell, tissue or organ (5, 6). Different types of deletions have been identified in the mtDNA of various tissues of humans (2, 7). Among them, the 4977-base pair ‘common deletion’, or DmtDNA4977, which is located between nt 8,470 and nt 13,447, is the most commonly identified (8, 9). DmtDNA4977 results in complete or partial truncation of subunits of F0–F1-ATPase, cytochrome c oxidase, and nicotinamide adenine dinucleotide-coenzyme Q (NADH-CoQ) oxidoreductase, and subsequently alters mitochondrial capacity to modulate the apoptotic process. Although the DmtDNA4977 occurs frequently in tissues of high oxygen demand and low mitotic activity, e.g. brain, heart, skeletal muscle, or skin, it can also be detected – albeit in much lower amounts – in rapidly replicating cells such as blood leukocytes (10). In many of these tissues, DmtDNA4977 demonstrates an age-related increase. However, other factors ranging from lifestyle to environmental to genetic factors have been shown to affect the levels of DmtDNA4977 significantly (11–14).
Although DmtDNA4977 exists in low amount in various types of cells, an accumulation of DmtDNA4977 in cells may lead to mitochondrial dysfunction and further increased levels of ROS; this accumulation has been implicated in the carcinogenesis of various cancers, including melanoma. In a study of 67 melanoma patients, Hubbard et al. found that levels of DmtDNA4977 in melanoma tissues were increased with age and differed by degree of overall pigmentation and response to sunlight (15). In non-melanoma skin cancer, Durham et al. found that levels of DmtDNA4977 were significantly higher in tumor than adjacent normal tissues from non-melanoma skin cancer patients (16). In addition, in a recent analysis of ultraviolet-radiation-independent melanoma carcinogenesis, Mitra et al. showed that mechanisms of oxidative damage, including mtDNA mutations were active contributors (17). Importantly, these studies were conducted using combinations of melanoma tumor tissues and adjacent normal tissues. So far, no study has been carried out to assess the levels of DmtDNA4977 in blood leukocytes (surrogate tissues) from melanoma patients and healthy controls, and explored their relationship with melanoma risk.
Therefore, in the current study, we analyzed levels of DmtDNA4977 in blood leukocytes from melanoma patients and healthy controls using semi-quantitative real-time PCR. We further explored the relationship between DmtDNA4977 levels and known melanoma risk factors, including pigmentation and history of sunlight exposure.
Materials and Methods
Study Population
The study protocol was approved by the Institutional Review Board at MD Anderson. Detailed information on study participants has been described previously (18). In brief, patients and controls were recruited at The University of Texas MD Anderson Cancer Center. All patients with either newly diagnosed or surgically treated, histopathologically confirmed melanoma who were Texas residents and registered at MD Anderson between April 1994 and July 2013 were eligible for inclusion. Healthy controls were recruited from unrelated clinic visitors (82.7%) and patient spouses (17.3%). Exclusion criteria for patients were prior chemotherapy or radiation therapy, presence of metastasis, prior cancer diagnosis, and any blood transfusion in the 6 months prior to recruitment. After obtaining written informed consent, each participant was given a self-administered questionnaire to collect demographic data and information on risk factors, including natural hair color, skin color, eye color, history of sunlight exposure (e.g. freckling in the sun as a child, tanning ability, and number of sunburns), presence of moles and dysplastic nevi, number of first-degree relatives with any cancer. In the current study, 206 melanoma cases and 219 controls were included. All study participants were Caucasian Americans. The selection was mainly based on the completion of questionnaire data on demographic data and melanoma risk factors, and leukocyte DNAs availability. In addition, the cases and controls were frequency matched on the dates of blood drawn and DNA extraction.
Determination of DmtDNA4977 levels
Analysis of DmtDNA4977 levels was conducted using the CFX96 real time system from Bio-Rad, and the method has been detailed by Pogozelski et al (19). The specific primers and probe used to detect DmtDNA4977 are located at nt 8,416 to 8,437, nt 13,519 to 13,498, and nt 13,461–13,480. The amplicon spans the DmtDNA4977 region. The specific primers and probe used to detect the total mtDNA are located at nt 1,307–1,328, nt 1,433–1,414, and nt 1,340–1,359. Each plate included 2 wells containing control DNA (Promega). Briefly, equal concentration of sample DNA (2ng/ul) was prepared based on picogreen assay. The reaction mixture included 1xBiorad iTaq SMX (Biorad iTaq UniveralProbes SMX 200), 250nM each forward and reverse primer, 200nM probe, 100ng genomic DNA in a total volume of 50ul. Real-Time PCR was performed under multiplex conditions; that is, primers and probes for both mtDNA total (labelled with TET) and mtDNA deletion (labelled with FAM) were present in each reaction. Amplification conditions were 50°C 2min, 95°C for 10 min, followed by 40 cycles of 95°C for 15sec, and 60°C for 1 min. The threshold was manually chosen either based on 10 times the standard deviation of the baseline or default as determined by the program. The relative mitochondrial deletion copy number was calculated by the equation: 2−((sample del Cq-sample total Cq)-(control del Cq-control total Cq)). A dose response was found in the DNA range of 0.2–125ng in both deletion and total mtDNAs respectively, (Supplemental Figure 1).
Statistical analysis
Statistical analyses were performed using the STATA statistical package (version 10, STATA Inc., College Station, TX). Differences in the distributions of demographic variables and known risk factors for melanoma obtained from the self-administered questionnaire between cases and controls were evaluated with multivariate logistic regression analysis. Adjusted ORs and 95% CIs for each of the known risk factors were obtained from unconditional multivariate logistic regression analysis to evaluate their associations with melanoma risk (Table 1). To examine differences between cases and controls for the median DmtDNA4977 levels associated with selected categorical characteristics and differences between selected categorical characteristics for the median DmtDNA4977 levels within the case or control group, the Wilcoxon rank-sum test was used (Table 2). Phenotypic index (PI) was calculated by combining hair color, eye color, and tanning ability to burn characteristics as indicators of cutaneous phenotype as previously described (20). During the analysis, the phenotypic index was further grouped into three categories: low (0), intermediate (1–2), and high (3–5). Multivariate logistic regression analysis was used to assess the relationship between phenotypic index and DmtDNA4977 levels among control study subjects (Table 3). For the main effect of DmtDNA4977 levels on melanoma risk, ORs and 95% CIs were estimated with unconditional multivariate logistic regression (Table 4). Potential confounders were adjusted in the analysis. DmtDNA4977 levels were examined in several ways, including as a continuous variable, as a categorical variable divided by the median value in controls, and as a categorical variable based on quartile distributions in controls. Cutoff points for all constructed categorical variables were based on the distribution within the control population. The dose response was tested for the quartile distribution of DmtDNA4977 levels by inserting the mean value of each quartile and then treating the variable as a continuous variable in the logistic regression model. Multivariate logistic regression analysis was used to assess the joint effects of phenotypic index, use of sunlamp, lifetime sunburn, and DmtDNA4977 levels on melanoma risk (Table 5).
Table 1.
Multivariate analysis of selected known risk factors between melanoma cases and healthy controls
| variables | Controls (n = 219) N (%) |
melanoma cases (n = 206) N (%) |
OR (95% CI)* |
|---|---|---|---|
| Age (y) | |||
| ≤47 | 113 (51.6) | 87 (42.2) | 1.00 |
| >47 | 106 (48.4) | 119 (57.8) | 1.65 (1.06, 2.58) |
| Gender | |||
| Male | 81 ((37.0) | 114 (55.3) | 1.00 |
| Female | 138 (63.0) | 92 (44.7) | 0.33 (0.20, 0.52) |
| Hair color | |||
| Black or brown | 169 (77.2) | 136 (66.0) | 1.00 |
| Blond or red | 50 (22.8) | 70 (34.0) | 1.62 (1.00, 2.64) |
| Eye color | |||
| Not blue | 158 (72.1) | 126 (61.2) | 1.00 |
| Blue | 61 (27.9) | 80 (38.8) | 1.37 (0.87, 2.16) |
| Skin color | |||
| Dark brown | 114 (52.1) | 85 (41.3) | 1.00 |
| Fair | 105 (47.9) | 121 (58.7) | 0.97 (0.93, 1.02) |
| Tanning ability after prolonged sun exposure | |||
| Average or deep | 165 (75.7) | 150 (73.9) | 1.00 |
| Light or none | 53 (24.3) | 53 (26.1) | 0.81 (0.48, 1.39) |
| Lifetime sunburns with blistering | |||
| 0 | 93 (42.7) | 46 (22.4) | 1.00 |
| ≥1 | 125 (57.3) | 159 (77.6) | 2.27 (1.23, 2.10) |
| Freckling in the sun as a child | |||
| No | 108 (49.5) | 88 (42.7) | 1.00 |
| Yes | 110 (50.5) | 118 (57.3) | 1.17 (0.75, 1.82) |
| Reported use of sunlamp | |||
| No | 149 (68.0) | 128 (62.1) | 1.00 |
| Yes | 70 (32.0) | 77 (37.9) | 2.68 (1.59, 4.51) |
Multivariate logistic analysis including age, gender, hair color, skin color, eye color, tanning ability, lifetime sunburns, freckling in the sun as a child, and use of sunlamp.
Table 2.
Median DmtDNA4977 levels by selected known risk factors between melanoma cases and healthy controls
| Cases (n=206) | Controls (n=219) | P value* | |
|---|---|---|---|
| Overall (median) | 0.38 | 0.20 | 0.008 |
| Age (y) | |||
| ≤47 | 0.36 | 0.19 | 0.350 |
| >47 | 0.42 | 0.22 | 0.010 |
| P value# | 0.617 | 0.261 | |
| Gender | |||
| Male | 0.40 | 0.23 | 0.061 |
| Female | 0.35 | 0.13 | 0.027 |
| P value# | 0.670 | 0.871 | |
| Hair color | |||
| Black or brown | 0.37 | 0.21 | 0.100 |
| Blond or red | 0.38 | 0.19 | 0.018 |
| P value# | 0.613 | 0.607 | |
| Eye color | |||
| Not blue | 0.25 | 0.21 | 0.287 |
| Blue | 0.42 | 0.18 | 0.003 |
| P value# | 0.051 | 0.662 | |
| Skin color | |||
| Dark brown | 0.29 | 0.21 | 0.104 |
| Fair | 0.49 | 0.19 | 0.007 |
| P value# | 0.046 | 0.562 | |
| Tanning ability after prolonged sun exposure | |||
| Average or deep | 0.49 | 0.32 | 0.313 |
| Light or none | 0.34 | 0.16 | 0.005 |
| P value# | 0.595 | 0.198 | |
| Lifetime sunburns with blistering | |||
| 0 | 0.27 | 0.22 | 0.468 |
| ≥1 | 0.37 | 0.17 | 0.005 |
| P value# | 0.538 | 0.283 | |
| Freckling in the sun as a child | |||
| No | 0.33 | 0.27 | 0.168 |
| Yes | 0.41 | 0.20 | 0.010 |
| P value# | 0.843 | 0.826 | |
| Reported use of sunlamp | |||
| No | 0.35 | 0.17 | 0.017 |
| Yes | 0.39 | 0.24 | 0.123 |
| P value# | 0.512 | 0.565 | |
The P-value comparing median DmtDNA4977 levels between cases and controls.
The P-value comparing median DmtDNA4977 levels between groups defined by selected characteristics.
Table 3.
Association between phenotypic index and DmtDNA4977 levels among controls
| Phenotypic index | Low (<0.20) | High (≥0.20) | |
| low | 60 (55.0%) | 47 (43.1%) | |
| Intermediate and high | 49 (45.0%) | 62 (56.9%) | 1.34 (0.70, 2.57)* |
| Reported no use of sunlamp | |||
| Low (<0.20) | High (≥0.20) | ||
| low | 42 (53.2%) | 25 (35.7%) | |
| Intermediate and high | 37 (46.8%) | 45 (64.3%) | 2.17 (1.10, 4.28)* |
| Reported use of sunlamp | |||
| Low (<0.20) | High (≥0.20) | ||
| low | 18 (60.0%) | 22 (56.4%) | |
| Intermediate and high | 12 (40.0%) | 17 (43.6%) | 0.61 (0.17, 2.15)* |
ORs were adjusted by age and gender.
Table 4.
Risk of melanoma as estimated by DmtDNA4977 levels.
| DmtDNA4977 levels | Number of cases (%) | Number of controls (%) | OR (95% CI)* |
|---|---|---|---|
| Continuous variable | 219 (100) | 206 (100) | 1.23 (1.01, 1.50) |
| Categorical variable | |||
| By mean in controls | |||
| <0.20 | 81 (39.3) | 110 (50.2) | 1.00 |
| ≥0.20 | 125 (60.7) | 109 (49.8) | 1.67 (1.09, 2.54) |
| By quartile in controls | |||
| 1st | 36 (17.5) | 55 (25.1) | 1.00 |
| 2nd | 45 (21.8) | 55 (25.1) | 1.25 (0.65, 2.37) |
| 3rd | 58 (28.2) | 56 (25.6) | 1.60 (0.87, 2.92) |
| 4th | 67 (32.5) | 53 (24.2) | 2.16 (1.18, 3.97) |
| P for trend = 0.001 |
ORs were adjusted by age, gender, hair color, skin color, eye color, tanning ability, lifetime sunburns, freckling in the sun, and use of sunlamp
Table 5.
Joint effects among DmtDNA4977 levels, phenotypic index, and history of sun exposure on melanoma risk*
| DmtDNA4977 levels | Low (<0.20) | High (≥0.20) | P for interaction |
|---|---|---|---|
| Phenotypic index | |||
| low | 1 | 2.08 (1.10, 3.90) | |
| intermediate | 1.47 (0.69, 3.15) | 2.22 (1.12, 4.39) | |
| high | 2.09 (0.95, 4.60) | 2.46 (1.15, 5.27) | 0.284 |
| Reported use of Sunlamp | |||
| No | 1 | 1.84 (1.09, 3.09) | |
| Yes | 3.11 (1.48, 6.51) | 4.45 (2.24, 8.82) | 0.673 |
| Lifetime sunburn | |||
| No | 1 | 1.22 (0.58, 2.60) | |
| Yes | 1.83 (0.93, 3.58) | 3.56 (1.84, 6.88) | 0.314 |
ORs were adjusted by age and gender, skin color, lifetime sunburns, freckling in the sun, and use of sunlamp as appropriate.
Results
First, we performed the multivariate analysis to assess the relationship between age, gender, pigmentation, history of sunlight exposure, and melanoma risk in our study population. The results were shown in Table 1. We found that melanoma cases were older (OR=1.65, 95% CI: 1.06, 2.58), more likely to have blond or red hair (OR=1.62, 95% CI: 1.00, 2.64), and were more likely to report a history of blistering sunburn (OR=2.27, 95% CI: 1.23, 2.10) and sunlamp use (OR=2.68, 95% CI: 1.59, 4.51) than controls. Women had a lower risk of melanoma than men (OR=0.33, 95% CI: 0.20, 0.52). Skin color, eye color, tanning ability, and freckling in the sun as a child were not associated with melanoma risk in our population.
The comparison of DmtDNA4977 levels between melanoma cases and healthy controls is summarized in Table 2. Overall, melanoma cases had significantly higher levels of DmtDNA4977 than controls (median: 0.60 vs 0.20, P=0.008). When we compared median DmtDNA4977 levels between the case and control group according to age group, gender, pigmentation, and history of sunlight exposure, significant difference was observed among older study subjects (>47 years old) (P=0.010), women (P=0.027), and those with blond or red hair (P=0.018), blue eyes (P=0.003), fair skin (P=0.007), light/none tanning ability (P=0.005), at least one lifetime sunburn with blistering (P=0.005), freckling in the sun as a child (P=0.010), and no reported sunlamp use (P=0.017). When we compared DmtDNA4977 levels within the case or control group by age, gender, pigmentation, and history of sunlight exposure, borderline differences were observed according to eye and skin colors in the melanoma case group. Study subjects who had blue eyes had higher levels of DmtDNA4977 than those who did not have blue eyes (P=0.051), and those who had fair skin had higher levels of DmtDNA4977 than those who had dark skin (P=0.046).
We then investigated the relationship between phenotypic index and DmtDNA4977 levels among control subjects. Those in the intermediate or high risk group tended to have higher DmtDNA4977 levels, although the association did not reach statistical significance (OR=1.34, 95%CI: 0.70, 2.57) (Table 3). However, when the study subjects were stratified by reported sunlamp use, significant association was observed among those who had no reported sunlamp use (OR=2.17, 95%CI: 1.10, 4.28), but not among those who reported to use sunlamp (OR=0.61, 95%CI: 0.17, 2.15).
The relationship between DmtDNA4977 levels and risk of melanoma is summarized in Table 4. In the multivariate linear regression analysis as a continuous variable, increased DmtDNA4977 levels were associated with a 1.23-fold increased risk of melanoma after adjusting for age, gender, hair color, skin color, eye color, tanning ability, lifetime sunburns, freckling in the sun as a child, and reported use of a sunlamp (adjusted OR = 1.23, 95% CI = 1.01, 1.50). When we dichotomized DmtDNA4977 levels into two groups (high or low) using the median levels of DmtDNA4977 in the control group (0.20), we found that high levels of DmtDNA4977 were associated with a 1.67-fold increased risk of melanoma after adjusting co-variates (adjusted OR = 1.67, 95% CI = 1.09, 2.54). In further quartile analysis using 25%, 50%, and 75% values of DmtDNA4977 levels among the control group as cutoff points, we found that those in the 4th (highest) quartile of DmtDNA4977 levels had a 2.16-fold increased risk of melanoma (adjusted ORs = 2.16, 95% CI = 1.18, 3.97) when compared to those with the lowest quartile. A statistically significant dose–response trend was observed (P = 0.001).
We next examined the joint effects of phenotypic index, reported use of sunlamp and lifetime sunburn, and DmtDNA4977 levels on melanoma risk (Table 5). Compared to study subjects in the low risk group who had low DmtDNA4977 levels, those in the high risk group with high DmtDNA4977 levels had a 2.46-fold increased risk of melanoma (OR=2.46, 95%CI: 1.15, 5.27). Similarly, compared to study subjects who had no reported sunlamp use and had low DmtDNA4977 levels, those who had reported sunlamp use and had high DmtDNA4977 levels had a 4.45-fold increased risk of melanoma (OR=4.45, 95%CI: 2.24, 8.82). Finally, compared to study subjects who never had a sunburn and had low DmtDNA4977 levels, those who had at least one sunburn and had high DmtDNA4977 levels had a 3.56-fold increased risk of melanoma (OR=3.56, 95%CI: 1.84, 6.68). No interaction was observed in the analysis.
Discussion
To our knowledge this study represents the first investigation of the relationship between DmtDNA4977 levels measured in blood leukocytes and melanoma risk. Overall, we found that DmtDNA4977 levels in blood leukocytes were higher in melanoma cases than healthy controls. The difference was evident among individuals greater than 47 years old, women, and have blond or red hair, blue eyes, fair skin, light or no tanning ability after prolonged sun exposure, at least 1 lifetime sunburn with blistering, freckling in the sun as a child, and no reported use of a sunlamp. In further analysis, we found increased levels of DmtDNA4977 were associated with increased risk of melanoma.
Our findings support a role for ROS in melanoma carcinogenesis (21–24). Mitochondria are the major source and target of the intracellular ROS. It has been reported that the presence of melanin in cultured primary human melanocytes is associated with a higher degree of ROS accumulation and a simultaneous reduction in the cellular antioxidant glutathione (25, 26). In a recent study of the relationship between pigment production, ROS, and mitochondrial function, Boulton et al. found a direct relationship between pigment production and the production of ROS by mitochondria, indicating an intimate relationship among pigment production, ROS, and mitochondria (27). It has also been suggested that increased ROS production in melanocytes may not only allow the accumulation of potentially oncogenic mutations in genomic DNAs but also cause significant damage on mtDNAs, such as DmtDNA4977 (28).
Levels of DmtDNA4977 have been reported to increase with increasing age (2, 29). In apparently normal skin obtained from wide excision in melanoma, Hubbard et al. observed a significant increase of DmtDNA4977 levels with patient age (15). However, in the current study, we were not able to document an association with age. While DmtDNA4977 levels in our study trended higher in older versus younger subjects among both cases and controls, these differences did not reach statistical significance. The discrepancy between our study and others may be due to the difference in biospecimens. DmtDNA4977 levels in blood leukocytes tend to be much lower than many other tissues with high oxygen demand and low mitotic activity (30). Thus, the effect size of the relationship may be too small to be detected given the modest sample size of our study. It could also be related to difference in lifestyle, environmental, or genetic factors between our study population and those reported by others. Interestingly, significant difference in DmtDNA4977 between cases and controls were only observed among older study subjects (>47 years old) (P=0.010), but not among younger ones (≤47 years old). This is consistent with the role of ROS in ageing and melanocytic carcinogenesis.
One interesting observation is that the significant difference in DmtDNA4977 levels between the case and control groups was only observed among study subjects already at higher risk of developing melanoma based on their pigmentation characteristics. This suggests that mitochondrial DNA deletion may be particularly relevant to the ultraviolent dependent pathway in melanocytic carcinogenesis. Solar exposure is a major inducer of ROS formation in the melanocytes and a major contributor to melanoma (31). Irradiation of the skin by UVA and/or UVB impairs natural antioxidant defenses and induces high levels of ROS (32). Genetically, the ultraviolent dependent pathway is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene, whose protein product is a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Loss-of-function alleles of the MC1R that are associated with increased melanoma risk cause sustained oxidative stress in human melanocytes owing to the inability to respond to α-MSH (33). Thus, it would be interesting to investigate the relationship between MC1R gene polymorphisms and DmtDNA4977 levels in melanoma risk.
Unlike pigmentation characteristics, sunlamp use is a modifiable risk factor for melanoma. In the current study, we found that the difference of DmtDNA4977 levels between melanoma cases and controls was significant among those who had no reported sunlamp use, but not those who had reported sunlamp use. Intriguingly, in further analysis, we observed that the relationship between DmtDNA4977 levels and pigmentation characteristics was significantly affected by the reported sunlamp use. The significant association was only observed among controls who had no reported sunlamp use, but not among controls who had reported sunlamp use. Our findings, although still preliminary, highlight the importance of healthy behaviors (e.g. no use of sunlamp) on reducing melanoma risk.
There are limitations in this study. Due to the nature of retrospective study design, we cannot infer a causal relationship between DmtDNA4977 levels in blood leucocytes and melanoma risk. Furthermore, any correlation between DmtDNA4977 levels in blood leucocytes and melanocyte tissue has not yet been evaluated. Thus, the biological relevance of increased DmtDNA4977 levels in blood leucocytes to melanocytic carcinogenesis remains to be further determined. Nevertheless, our study provides evidence linking mitochondrial DNA deletions in blood leucocytes and melanoma risk, and further suggests that mitochondrial DNA deletions may mediate the association between solar exposure and melanoma risk. Additional studies are warranted to confirm the results identified in this investigation and to improve understanding the contribution of ROS and mitochondrial DNA to melanoma carcinogenesis.
Supplementary Material
Acknowledgments
This work was supported by 5R01CA195614 and The University of Texas MD Anderson Cancer Center Various Donors Melanoma and Skin Cancers Priority Program Fund; the Miriam and Jim Mulva Research Fund; the McCarthy Skin Cancer Research Fund and the Marit Peterson Fund for Melanoma Research.
Grant: P30 CA016672, R01 CA195614.
References
- 1.Kazachkova N, Ramos A, Santos C, Lima M. Mitochondrial DNA damage patterns and aging: revising the evidences for humans and mice. Aging Dis. 2013;4(6):337–50. doi: 10.14336/AD.2013.0400337. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Greaves LC, Reeve AK, Taylor RW, Turnbull DM. Mitochondrial DNA and disease. J Pathol. 2012;226(2):274–86. doi: 10.1002/path.3028. [DOI] [PubMed] [Google Scholar]
- 3.Greaves LC, Turnbull DM. Mitochondrial DNA mutations and ageing. Biochim Biophys Acta. 2009;1790(10):1015–20. doi: 10.1016/j.bbagen.2009.04.018. [DOI] [PubMed] [Google Scholar]
- 4.Wei YH, Lee HC. Oxidative stress, mitochondrial DNA mutation, and impairment of antioxidant enzymes in aging. Exp Biol Med (Maywood) 2002;227(9):671–82. doi: 10.1177/153537020222700901. [DOI] [PubMed] [Google Scholar]
- 5.Li H, Liu D, Lu J, Bai Y. Physiology and pathophysiology of mitochondrial DNA. Adv Exp Med Biol. 2012;942:39–51. doi: 10.1007/978-94-007-2869-1_2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Barritt JA, Brenner CA, Willadsen S, Cohen J. Spontaneous and artificial changes in human ooplasmic mitochondria. Hum Reprod. 2000;15(Suppl 2):207–17. doi: 10.1093/humrep/15.suppl_2.207. [DOI] [PubMed] [Google Scholar]
- 7.Damas J, Samuels DC, Carneiro J, Amorim A, Pereira F. Mitochondrial DNA rearrangements in health and disease–a comprehensive study. Hum Mutat. 2014;35(1):1–14. doi: 10.1002/humu.22452. [DOI] [PubMed] [Google Scholar]
- 8.Arnheim N, Cortopassi G. Deleterious mitochondrial DNA mutations accumulate in aging human tissues. Mutat Res. 1992;275(3–6):157–67. doi: 10.1016/0921-8734(92)90020-p. [DOI] [PubMed] [Google Scholar]
- 9.Wei YH. Mitochondrial DNA alterations as ageing-associated molecular events. Mutat Res. 1992;275(3–6):145–55. doi: 10.1016/0921-8734(92)90019-l. [DOI] [PubMed] [Google Scholar]
- 10.Meissner C, Ritz-Timme S. Molecular pathology and age estimation. Forensic Sci Int. 2010;203(1–3):34–43. doi: 10.1016/j.forsciint.2010.07.010. [DOI] [PubMed] [Google Scholar]
- 11.Iwai K, Iwamura Y, Yamashita S, Wadano Y, Mesaki N. Effect of tea catechins on mitochondrial DNA 4977-bp deletions in human leucocytes. Mutat Res. 2006;595(1–2):191–5. doi: 10.1016/j.mrfmmm.2005.10.001. [DOI] [PubMed] [Google Scholar]
- 12.Iwai K, Miyao M, Wadano Y, Iwamura Y. Dynamic changes of deleted mitochondrial DNA in human leucocytes after endurance exercise. Eur J Appl Physiol. 2003;88(6):515–9. doi: 10.1007/s00421-002-0745-7. [DOI] [PubMed] [Google Scholar]
- 13.von Wurmb-Schwark N, Schwark T, Caliebe A, Drenske C, Nikolaus S, Schreiber S, et al. Low level of the mtDNA(4977) deletion in blood of exceptionally old individuals. Mech Ageing Dev. 2010;131(3):179–84. doi: 10.1016/j.mad.2010.01.005. [DOI] [PubMed] [Google Scholar]
- 14.von Wurmb-Schwark N, Ringleb A, Schwark T, Broese T, Weirich S, Schlaefke D, et al. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood. Mutat Res. 2008;637(1–2):73–9. doi: 10.1016/j.mrfmmm.2007.07.003. [DOI] [PubMed] [Google Scholar]
- 15.Hubbard K, Steinberg ML, Hill H, Orlow I. Mitochondrial DNA deletions in skin from melanoma patients. Ethn Dis. 2008;18(2 Suppl 2):S2, 38–43. [PubMed] [Google Scholar]
- 16.Durham SE, Krishnan KJ, Betts J, Birch-Machin MA. Mitochondrial DNA damage in non-melanoma skin cancer. Br J Cancer. 2003;88(1):90–5. doi: 10.1038/sj.bjc.6600773. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Mitra D, Luo X, Morgan A, Wang J, Hoang MP, Lo J, et al. An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background. Nature. 2012;491(7424):449–53. doi: 10.1038/nature11624. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Shen J, Gopalakrishnan V, Lee JE, Fang S, Zhao H. Mitochondrial DNA copy number in peripheral blood and melanoma risk. PLoS One. 2015;10(6):e0131649. doi: 10.1371/journal.pone.0131649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Pogozelski WK, Hamel CJ, Woeller CF, Jackson WE, Zullo SJ, Fischel-Ghodsian N, et al. Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson’s syndrome lymphoblasts using a fluorogenic 5′-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards. Mitochondrion. 2003;2(6):415–27. doi: 10.1016/S1567-7249(03)00033-3. [DOI] [PubMed] [Google Scholar]
- 20.Kanetsky PA, Rebbeck TR, Hummer AJ, Panossian S, Armstrong BK, Kricker A, et al. Population-based study of natural variation in the melanocortin-1 receptor gene and melanoma. Cancer Res. 2006;66(18):9330–7. doi: 10.1158/0008-5472.CAN-06-1634. [DOI] [PubMed] [Google Scholar]
- 21.Jiao Q, Zou L, Liu P, Xu Q, Zhang Y, Yu Y, et al. Xanthoceraside Induces Apoptosis in Melanoma Cells Through the Activation of Caspases and the Suppression of the IGF-1R/Raf/MEK/ERK Signaling Pathway. Journal of medicinal food. 2014;17(10):1070–8. doi: 10.1089/jmf.2013.3035. [DOI] [PubMed] [Google Scholar]
- 22.Kim SH, Yoo YH, Lee JH, Park JW. Mitochondrial NADP(+)-dependent isocitrate dehydrogenase knockdown inhibits tumorigenicity of melanoma cells. Biochemical and biophysical research communications. 2014;451(2):246–51. doi: 10.1016/j.bbrc.2014.07.105. [DOI] [PubMed] [Google Scholar]
- 23.Ardestani S, Deskins DL, Young PP. Membrane TNF-alpha-activated programmed necrosis is mediated by Ceramide-induced reactive oxygen species. Journal of molecular signaling. 2013;8(1):12. doi: 10.1186/1750-2187-8-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Burgeiro A, Bento AC, Gajate C, Oliveira PJ, Mollinedo F. Rapid human melanoma cell death induced by sanguinarine through oxidative stress. European journal of pharmacology. 2013;705(1–3):109–18. doi: 10.1016/j.ejphar.2013.02.035. [DOI] [PubMed] [Google Scholar]
- 25.Wittgen HG, van Kempen LC. Reactive oxygen species in melanoma and its therapeutic implications. Melanoma Res. 2007;17(6):400–9. doi: 10.1097/CMR.0b013e3282f1d312. [DOI] [PubMed] [Google Scholar]
- 26.Godic A, Poljsak B, Adamic M, Dahmane R. The role of antioxidants in skin cancer prevention and treatment. Oxid Med Cell Longev. 2014;2014:860479. doi: 10.1155/2014/860479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Boulton SJ, Birch-Machin MA. Impact of hyperpigmentation on superoxide flux and melanoma cell metabolism at mitochondrial complex II. FASEB J. 2015;29(1):346–53. doi: 10.1096/fj.14-261982. [DOI] [PubMed] [Google Scholar]
- 28.Kim ES, Park SJ, Goh MJ, Na YJ, Jo DS, Jo YK, et al. Mitochondrial dynamics regulate melanogenesis through proteasomal degradation of MITF via ROS-ERK activation. Pigment Cell Melanoma Res. 2014;27(6):1051–62. doi: 10.1111/pcmr.12298. [DOI] [PubMed] [Google Scholar]
- 29.Prigione A, Cortopassi G. Mitochondrial DNA deletions induce the adenosine monophosphate-activated protein kinase energy stress pathway and result in decreased secretion of some proteins. Aging Cell. 2007;6(5):619–30. doi: 10.1111/j.1474-9726.2007.00323.x. [DOI] [PubMed] [Google Scholar]
- 30.Gattermann N, Berneburg M, Heinisch J, Aul C, Schneider W. Detection of the ageing-associated 5-Kb common deletion of mitochondrial DNA in blood and bone marrow of hematologically normal adults. Absence of the deletion in clonal bone marrow disorders. Leukemia. 1995;9(10):1704–10. [PubMed] [Google Scholar]
- 31.Jenkins NC, Grossman D. Role of melanin in melanocyte dysregulation of reactive oxygen species. Biomed Res Int. 2013;2013:908797. doi: 10.1155/2013/908797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Xiao L, Matsubayashi K, Miwa N. Inhibitory effect of the water-soluble polymer-wrapped derivative of fullerene on UVA-induced melanogenesis via downregulation of tyrosinase expression in human melanocytes and skin tissues. Arch Dermatol Res. 2007;299(5–6):245–57. doi: 10.1007/s00403-007-0740-2. [DOI] [PubMed] [Google Scholar]
- 33.Kadekaro AL, Leachman S, Kavanagh RJ, Swope V, Cassidy P, Supp D, et al. Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation. FASEB J. 2010;24(10):3850–60. doi: 10.1096/fj.10-158485. [DOI] [PMC free article] [PubMed] [Google Scholar]
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