The Mitochondrial DNA Common Deletion Is Present in Most Basal and Squamous Cell Carcinoma Samples Isolated by Laser Capture Microdissection but Generally at Reduced Rather than Increased Levels

Abstract

basal cell carcinoma mitochondrial DNA squamous cell carcinoma TO THE EDITOR Mitochondrial (mt) contribution to carcinogenesis has been critically discussed with circumstantial evidence against as well as for a causal role. Proliferating cancer cells are dependent on sufficient energy supply that would argue against defective mitochondria in tumor cells. On the other hand, mitochondria are the main endogenous source of reactive oxygen species as highly mutagenic side products of oxidative phosphorylation (Fridovich, 2004Fridovich I. Mitochondria: are they the seat of senescence?.Aging Cell. 2004; 3: 13-16Crossref PubMed Scopus (165) Google Scholar) and increased amounts of mtDNA mutations have been detected in a variety of tumors (reviewed by Berneburg et al., 2006Berneburg M. Kamenisch Y. Krutmann J. Repair of mitochondrial DNA in aging and carcinogenesis.Photochem Photobiol Sci. 2006; 5: 190-198Crossref PubMed Scopus (32) Google Scholar). One of the best described mt deletions is the 4977 bp common deletion (Cortopassi and Arnheim, 1990Cortopassi G.A. Arnheim N. Detection of a specific mitochondrial DNA deletion in tissues of older humans.Nucleic Acids Res. 1990; 18: 6927-6933Crossref PubMed Scopus (637) Google Scholar). The common deletion affects several transfer RNA and respiratory chain genes. It is known to be involved in myopathies (Holt et al., 1989Holt I.J. Harding A.E. Morgan-Hughes J.A. Deletions of muscle mitochondrial DNA in mitochondrial myopathies: sequence analysis and possible mechanisms.Nucleic Acids Res. 1989; 17: 4465-4469Crossref PubMed Scopus (84) Google Scholar), Alzheimer's disease (Corral-Debrinski et al., 1994Corral-Debrinski M. Horton T. Lott M.T. Shoffner J.M. McKee A.C. Beal M.F. et al.Marked changes in mitochondrial DNA deletion levels in Alzheimer brains.Genomics. 1994; 23: 471-476Crossref PubMed Scopus (241) Google Scholar), as well as chronological aging, and photoaging of the skin (Cortopassi and Arnheim, 1990Cortopassi G.A. Arnheim N. Detection of a specific mitochondrial DNA deletion in tissues of older humans.Nucleic Acids Res. 1990; 18: 6927-6933Crossref PubMed Scopus (637) Google Scholar; Berneburg et al., 1997Berneburg M. Gattermann N. Stege H. Grewe M. Vogelsang K. Ruzicka T. et al.Chronically ultraviolet-exposed human skin shows a higher mutation frequency of mitochondrial DNA as compared to unexposed skin and the hematopoietic system.Photochem Photobiol. 1997; 66: 271-275Crossref PubMed Scopus (112) Google Scholar; Berneburg et al., 1999Berneburg M. Grether-Beck S. Kurten V. Ruzicka T. Briviba K. Sies H. et al.Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion.J Biol Chem. 1999; 274: 15345-15349Crossref PubMed Scopus (307) Google Scholar). Generally, chronological aged skin has an elevated level of the common deletion (Zhang et al., 1992Zhang C. Baumer A. Maxwell R.J. Linnane A.W. Nagley P. Multiple mitochondrial DNA deletions in an elderly human individual.FEBS Lett. 1992; 297: 34-38Abstract Full Text PDF PubMed Scopus (188) Google Scholar; Yang et al., 1994Yang J.H. Lee H.C. Lin K.J. Wei Y.H. A specific 4977-bp deletion of mitochondrial DNA in human ageing skin.Arch Dermatol Res. 1994; 286: 386-390Crossref PubMed Scopus (65) Google Scholar; Liu et al., 1998Liu V.W. Zhang C. Pang C.Y. Lee H.C. Lu C.Y. Wei Y.H. et al.Independent occurrence of somatic mutations in mitochondrial DNA of human skin from subjects of various ages.Hum Mutat. 1998; 11: 191-196Crossref PubMed Scopus (25) Google Scholar). Besides this, it has been shown that the common deletion can be induced by UV irradiation in human skin (Berneburg et al., 2004Berneburg M. Plettenberg H. Medve-König K. Pfahlberg A. Gers-Barlag H. Gefeller H. et al.Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin.J Invest Dermatol. 2004; 122: 1277-1283Crossref PubMed Scopus (131) Google Scholar). Furthermore, skin cancer has been linked to chronic UV exposure (Pang et al., 1994Pang C.Y. Lee H.C. Yang J.H. Wei Y.H. Human Skin Mitochondrial DNA Deletions Associated with Light Exposure.Arch Biochem Biophys. 1994; 312: 534-538Crossref PubMed Scopus (78) Google Scholar). This link has been investigated by Eshaghian et al., 2006Eshaghian A. Vleugels R.A. Canter J.A. McDonald M.A. Stasko T. Sligh J.E. Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers.J Invest Dermatol. 2006; 126: 336-344Crossref PubMed Scopus (74) Google Scholar who published data on mt deletions in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). They showed that most of these tumors had lower or undetectable amounts of mt deletions compared to marginal non-tumor tissue. However, there are methological restrictions to this study. Although the number of BCC was 25, the number of investigated SCC tumors is considerably low (four SCC investigated). Furthermore, the extraction method of tumor and non-tumor tissue does not exclude contamination by other tissues. Tumors and margins were excised surgically and examined histologically. As also mentioned by the authors there is a possibility, that extracted tumor samples contain a portion of non-tumor tissue. This contamination makes the distinction of mt deletions in tumor tissue and non-tumor tissue difficult, as unknown levels of non-tumor tissue may also be present in tumor samples. Furthermore, epidermal tissue was analyzed in a low portion of non-tumor samples whereas the other samples contained a mixture of epidermal and dermal tissue whereas investigation of only epidermal tissue would be desirable. Therefore, we initiated a study approved by the Department of Dermatology at the Eberhard Karls University, Tübingen, Germany and the local ethics committee, similar to Eshaghian et al., carrying out laser capture microdissection to extract tumor cells and epidermal cells from paraffin-embedded histological samples. This allows accurate isolation of tumor cells avoiding contamination from tumor surrounding stromal tissue. In addition, tumor and epidermal cells from samples of 21 BCC and 20 SCC of patients with different age and at different body sites were isolated after histological examination by laser capture microdissection. As the level of the common deletion can also be influenced by chronological aging and UV exposure, to exclude a bias of this non-tumor specific influence on the common deletion, levels of the common deletion from tumor samples were normalized to appropriate epidermal non-tumor tissue of the same patient. Detection of the common deletion was carried out as described previously. Briefly, after DNA extraction, levels of the common deletion and undeleted mtDNA sequences were detected with real-time PCR as published (Koch et al., 2001Koch H. Wittern K.P. Bergemann J. In human keratinocytes the Common Deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation.J Invest Dermatol. 2001; 117: 892-897Crossref PubMed Google Scholar; Pfaffl, 2001Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: 2003-2007Crossref PubMed Scopus (22910) Google Scholar). Levels of the common deletion in relation to surrounding tissue are given as percentage of total mtDNA. The common deletion could be detected in 85.7% (18/21) BCC and 95% (19/20) SCC and in all but one of the non-tumor samples (Tables 1 and 2). This is in accordance to the work by Eshaghian et al. where in some samples the common deletion was undetectable. It has been shown that the total common deletion level present in most tumor samples is low. However, it has also been shown that the common deletion accumulates during normal aging. As skin tumors occur at higher age, from our point of view, it is not surprising that the common deletion can be detected in most investigated samples.Table 1Age and localization of 20 patients with SCC (sun exposed: head and face and upper extremities; non-sun exposed: trunk and lower extremities)Patient (number)Age of patients (years)LocalizationLevel of the common deletion (in tumor) as percentage of total mtDNALevel of the common deletion (in epidermis) as percentage of total mtDNA 166Head0.060.62 268Nose0.170.28 368Forehead0.360.60 471Retro auricular0.000.03 574Lower lip0.010.25 678Nose0.151.09 782Nose0.843.15 889Temple0.980.92 991Ear0.030.171091Ear0.090.221191Nose0.160.281291Ear0.050.081391Temple0.080.301491Temple0.020.151597Nose0.310.541697Vertex0.023.931797Head0.090.121856Shoulder0.010.011973Shoulder0.090.132075Hip1.150.60mtDNA, mitochondrial DNA; SCC, squamous cell carcinoma.There is no evidence for difference in relative levels of common deletion between age and gender of the patients. Open table in a new tab Table 2Age and localization of 21 patients with BCC (sun exposed: head and face and upper extremities; non-sun exposed: trunk and lower extremities)Patient (number)Age of patients (years)LocalizationLevel of the common deletion (in tumor) as percentage of total mtDNALevel of the common deletion (in epidermis) as percentage of total mtDNA 149Nose0.061.87 251Retro auricular0.100.37 363Forehead0.000.00 465Temple0.042.04 570Nose0.040.31 670Nose0.442.86 770Nose1.465.00 871Nose0.170.07 973Corner of the eye1.081.181073Nose0.031.101174Pre auricular0.020.041274Retro auricular0.110.351375Temple0.053.921479Cheek0.280.561581Cheek0.240.161681Forehead0.921.081782Nose0.000.061883Lower palpebra0.090.371987Temple0.000.042091Pre auricular0.110.142164Breast0.010.06BCC, basal cell carcinoma; mtDNA, mitochondrial DNA.There is no evidence for difference in relative levels of the common deletion between age, gender of the patients. Open table in a new tab mtDNA, mitochondrial DNA; SCC, squamous cell carcinoma. There is no evidence for difference in relative levels of common deletion between age and gender of the patients. BCC, basal cell carcinoma; mtDNA, mitochondrial DNA. There is no evidence for difference in relative levels of the common deletion between age, gender of the patients. In our study, the common deletion levels of SCC and BCC show similar patterns as determined by real-time PCR (Figures 1a and 2a) and confirmed by agarose gel analysis of real-time PCR results revealing only a single band for the common deletion or the fragment representing undeleted mtDNA (housekeeping sequence) (Figures 1b and (2b). The amount of the common deletion in tumor cells taken together is significantly lower than the common deletion levels of epidermal cells (SCC: P=0.0005; BCC: P=0.0005). In the majority of SCC and BCC tumors, the level of the common deletion is lower than in corresponding non-tumor epidermal tissue. Earlier findings have shown that the level of the common deletion can be induced by UV exposure and chronological aging. These effects must be considered when examining the level of the common deletion in tumors of the patients and comparing it to control tissue. Thus, it is necessary to extract perilesional control tissue near the tumor location to make sure that the control tissue was exposed to the same UV radiation and has the same chronological age as the corresponding tumor tissue in each patient. Owing to our experimental design it is not possible to differentiate between aging or UV effects on the level of the common deletion. Superimposing effects of age or UV irradiation on the common deletion in control tissue can only be ruled out if samples are taken from the same location or patients have the same age. Owing to the existing cohort of patients and the fact that patients with SCC or BCC tumors are usually of relatively high age our data did not reveal age-related changes of the common deletion. This is in line with results of other groups which also failed to establish an age-related change of the common deletion (Cortopassi et al., 1992Cortopassi G.A. Shibata D. Soong N.W. Arnheim N. A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues.Proc Natl Acad Sci. 1992; 89: 7370-7374Crossref PubMed Scopus (534) Google Scholar; Koch et al., 2001Koch H. Wittern K.P. Bergemann J. In human keratinocytes the Common Deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation.J Invest Dermatol. 2001; 117: 892-897Crossref PubMed Google Scholar). Koch et al., 2001Koch H. Wittern K.P. Bergemann J. In human keratinocytes the Common Deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation.J Invest Dermatol. 2001; 117: 892-897Crossref PubMed Google Scholar suggested that mainly donor variabilities are responsible for different levels of the common deletion in keratinocytes. Neither in SCC nor in BCC an association between tumor size and relative level of the common deletion could be detected (data not shown). Two BCC tumors and two SCC tumors showed slightly increased levels of the common deletion compared to the corresponding non-tumor tissue. But in these samples epidermal and tumor tissue revealed low levels of the common deletion (0.17 and 0.24% common deletion in tumor probes and 0.07 and 0.16% in their corresponding epidermal tissue) (Table 2) and the difference between tumor and non-tumor tissue in these samples is not very high. In SCC two tumors showed a slightly higher amount of the common deletion (0.98% and 1.15% in tumor probe) compared to non-tumor epidermal tissue (0.92 and 0.60%) (Table 1). Thus, in our study the overall level of the common deletion is reduced rather than increased although higher or similar amounts of the common deletion can also be observed in single cases.Figure 2The levels of mitochondrial DNA common deletion are generally reduced in basal and squamous cell carcinoma samples isolated by laser capture microdissection. (a) Levels of mtDNA deletions in BCC and in surrounding epidermal non-tumor tissue. The level of the common deletion in epidermal (black bars) and tumor tissue (gray bars) of different patients (1–21) with BCC is shown as percentage of total mtDNA. In comparison to epidermal cells all BCC tumors together show significantly lower levels of the common deletion (Wilcoxon test P-value=0.0005). (b) Amplified DNA fragments from patient 14 separated by agarose gel electrophoresis taken during log phase of the real-time PCR. Top panel: PCR fragment for the common deletion. Bottom panel: PCR fragment for non-mutated mtDNA region serving as internal control (housekeeping sequence).View Large Image Figure ViewerDownload (PPT) In aggregate, our data support the results by Eshaghian et al. in skin tumors as well as the findings of other groups in different tumor entities. In an earlier study, BCC and SCC tumors with higher levels of the common deletion as well tumors with low levels of the common deletion were observed (Durham et al., 2003Durham S.E. Krishnan K.J. Betts J. Birch-Machin M.A. Mitochondrial DNA damage in non-melanoma skin cancer.Br J Cancer. 2003; 88: 90-95Crossref PubMed Scopus (74) Google Scholar). Dormant status or low metabolism rates of these BCC or SCC cells is also a possible explanation why these cells can afford higher levels of the common deletion. Generally increased levels of the common deletion seem to be an exception. In many other carcinomas (colorectal, breast, gastric, and head and neck cancers) the common deletion was reduced or almost absent when compared to normal tissue (Heerdt and Augenlicht, 1990Heerdt B.G. Augenlicht L.H. Absence of detectable deletions in the mitochondrial genome of human colon tumors.Cancer Commun. 1990; 3: 109-111Google Scholar; Fukushima et al., 1995Fukushima S. Honda K. Awane M. Yamamoto E. Takeda R. Kaneko I. et al.The frequency of 4977 base pair deletion of mitochondrial DNA in various types of liver disease and in normal liver.Hepatology. 1995; 6: 1547-1551Google Scholar; Habano et al., 1998Habano W. Nakamura S. Sugai T. Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.Oncogene. 1998; 17: 1931-1937Crossref PubMed Scopus (139) Google Scholar, Habano et al., 1999Habano W. Sugai T. Yoshida T. Nakamura S. Mitochondrial gene mutation, but not large-scale deletion, is a feature of colorectal carcinomas with mitochondrial microsatellite instability.Int J Cancer. 1999; 83: 625-629Crossref PubMed Scopus (103) Google Scholar; Kotake et al., 1999Kotake K. Nonami T. Kurokawa T. Nakao A. Murakami T. Shimomura Y. Human livers with cirrhosis and hepatocellular carcinoma have less mitochondrial DNA deletion than normal human livers.Life Sci. 1999; 64: 1785-1791Crossref PubMed Scopus (25) Google Scholar; Angela et al., 2004Angela M. Dani C. Dani S.U. Lima S.P.G. Martinez A. Rossi B.M. Less ΔmtDNA4977 than normal in various types of tumors suggests that cancer cells are essentially free of this mutation.Genet Mol Res. 2004; 3: 395-409PubMed Google Scholar; Shieh et al., 2004Shieh D.B. Chou W.P. Wei Y.H. Wong T.Y. Jin Y.T. Mitochondrial DNA 4,977-bp deletion in paired oral cancer and precancerous lesions revealed by laser microdissection and real-time quantitative PCR.Ann NY Acad Sci. 2004; 1011: 154-167Crossref PubMed Scopus (29) Google Scholar). This appears reasonable as dividing tumor cells depend on abundant energy supply during proliferation. High levels of the common deletion, implying that approximately one-third of the mt genome is missing, may lead to insufficient expression of mt proteins, thus leading to an impaired respiratory chain. Thus, defective energy production and high levels of the common deletion would preclude proliferation in tumor cells. Therefore, it is tempting to speculate that a possible increase of the common deletion, if it exists at all, is generally a transient phenomenon during early stages of carcinogenesis which disappears at later stages of the tumor owing to selection mechanisms for depletion of the common deletion below the level of progenitor cells. This could at least explain the significantly lower level of the common deletion in the majority of tumor cells compared to epidermal cells. Further work is needed to undermine a potential role of the common deletion as initial cancer promoting factor as well as the exact mechanisms responsible for possible selection against high levels of large mt deletions.