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Methylation of Mitochondrial DNA Is Not a Useful Marker for Cancer Detection

Posted on: Friday, 6 August 2004, 06:00 CDT

To the Editor:

The human mitochondrial (mt) DNA is small (16.5 kb) and encodes 13 respiratory chain subunits, 22 transfer RNAs, and 2 ribosomal RNAs. mtDNA is present at 1000-10000 copies/cell, and the vast majority of these copies are identical (homoplasmic) at birth. A large number and wide variety of mtDNA mutations have been identified, and >200 disorders are associated with specific point mutations, single deletions, multiple deletions, or depletion of mtDNA (1 ).

Several mtDNA mutations were recently found in human colorectal cancers and bladder, head and neck, and lung tumors (2). Mutated mtDNA was detected in body fluids from patient with each type of the above cancers and was much more abundant than was mutated nuclear p53 DNA (2). Aberrant DNA methylation has been identified as an important mechanism for inactivation of carcinogenesis-related genes in neoplasias (3). By virtue of the clonal nature and high copy number of mtDNA, we hypothesized that methylation and mutations of mtDNA could be detected in body fluids and be useful as molecular markers for detection of cancer. In the present study, we focused on the aberrant methylation of mtDNA in cancer cell lines and tissue specimens from patients with gastrointestinal cancers.

The present study included 15 cancer cell lines and tissues from 31 patients with gastric cancer and 25 patients with colorectal cancer. The patients were similar to those who participated in our previous studies (4). For the patients, both malignant and nonmalignant tissues were examined. Each patient consented to the experimental use and pathology examination of the specimens.

To examine methylation, we performed bisulfite-PCR-single- stranded DNA conformation polymorphism (SSCP) analysis as described previously (4). Three pairs of primers based on GenBank accession no. NC_ 001807 were used for amplification of mtDNA. Primer pairs were as follows:

Sense, 5'-TAAGTTATTTTAGGGATAAT-3' (nucleotides 29202941); antisense, 5'-ATCTTAACAAACCCTATTCTTA-3' (31953216)

Sense, 5 '-TTG AGTTATG ATATTAATTGGT-3' (6725-6746); antisense, 5'- CATAATAAAAATAAACTACAAC-3' (6999-7020)

Sense, 5 '-TTGGTTATTAATGGTATTGAAT-3' (7884-7905); antisense, 5'- AAAAACAACTCATAAATACAAA-3' (8041-8062).

In the 16.5-kb human mitochondrial genome there are 435 CpG sites and 4747 cytosines at non-CpG sites. The primers were selected to amplify the maximum number of CpG sites. PCR products were expected to contain 318 bp and 13 CpG sites, 317 bp and 13 CpG sites, and 200 bp and 11 CpG sites, respectively. PCR products were subjected to SSCP analysis and sequenced directly with a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and a PRISM 310 Genetic Analyzer (Applied Biosystems).

Bisulfite-PCR-SSCP analysis revealed only unmethylated bands for all analyzed samples. Several SSCP bands were analyzed by direct sequencing, and the lack of methylated DNA was confirmed. Therefore, we believe that methylation of mtDNA is a rare event in the regions we analyzed in cancer cell lines and tissues from patients with gastric and colorectal cancer.

CpG dinucleotides are pervasively underrepresented in all animal mitochondria but vary in frequency in fungal, protist, and plant mitochondrial genomes (5). The methylationdeamination-mutation scenario may not apply to mtDNA genomes because the necessary methylase is not produced by most invertebrates or the methylase does not or can not access mitochondria in vertebrates. In contrast, endogenous methylation of 5-methylcytosine has been reported in mtDNA of rodents and human fibroblasts in culture (6). In that study, ~2-5% of CCGG sites were fully methylated, suggesting that nuclear methylases enter a subset of the mitochondria. Whether mtDNA can be methylated remains controversial. In the present study we examined selective regions containing more CpG sites. For an exhaustive study targeting whole mtDNA sequences, procedures more effective than bisulfite modification should be developed.

In conclusion, hypermethylation of mtDNA occurs at a very low frequency and does not appear to be a sensitive marker for detection of cancer.

This research was supported in part by a Labour Sciences Research grant (H15-Cancer Prevention-9) and by a Grant-in-Aid for Scientific Research (14657625) from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

References

1. Marcelino LA1 Thilly WG. Mitochondrial mutagenesis in human cells and tissues. Mutat Res 1999;434:177-203.

2. Baylin SB, Herman JG, Graft JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 1998; 72:141-96.

3. Fliss MS, Usadel H, Caballero OL, Wu L, Buta MR, Eleff SM, et al. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science 2000;287:2017-9.

4. Maekawa M, Sugano K, Ushiama M, Fukayama N, Nomoto K, Kashiwabara H, et al. Heterogeneity of DNA methylation status analyzed by bisulfite-PCR-SSCP and correlation with clinico- pathological characteristics in colorectal cancer. Clin Chem Lab Med 2001;39:121-8.

5. Cardon LR, Burge C, Clayton DA, Karlin S. Pervasive CpG suppression in animal mitochondrial genomes. Proc Natl Acad Sci U S A 1994;91: 3799-803.

6. Shmookler Reis RJ, Goldstein S. Mitochondrial DNA in mortal and immortal human cells. Genome number, integrity, and methylation. J Biol Chem 1983;258:9078-85.

Masato Maekawa1

Terumi Taniguchi1

Hitomi Higashi1

Haruhiko Sugimura2

Kokichi Sugano3

Takashi Kanno1

1 Department of Laboratory Medicine

and

2 first Department of Pathology

Hamamatsu University

School of Medicine

Hamamatsu, Japan

3 Oncogene Research Unit

Cancer Prevention Unit

Tochigi Cancer Center

Research Institute

Utsunomiya, Japan

* Address correspondence to this author at: Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan. Fax 81-53-435-2794; e-mail mmaekawa@ hama- med.ac.jp.

DOI: 10.1373/clinchem.2004.035139

Copyright American Association for Clinical Chemistry Aug 2004

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