Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium AACR Conference on Molecular Diagnostics - 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishikawa, M.
Right arrow Articles by Inoue, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishikawa, M.
Right arrow Articles by Inoue, M.
[Cancer Research 61, 1843-1845, March 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Somatic Mutation of Mitochondrial DNA in Cancerous and Noncancerous Liver Tissue in Individuals with Hepatocellular Carcinoma

Manabu Nishikawa1, Shuhei Nishiguchi, Susumu Shiomi, Akihiro Tamori, Noritoshi Koh, Tadashi Takeda, Shoji Kubo, Kazuhiro Hirohashi, Hiroaki Kinoshita, Eisuke Sato and Masayasu Inoue

Departments of Biochemistry [M. N., E. S., M. I.], Internal Medicine [M. N., S. N., S. S., A. T., N. K., T. T.], and Surgery [S. K., K. H., H. K.], Osaka City University Medical School, Osaka 545-8585, Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Unlike other types of cancer, hepatocellular carcinoma (HCC) is usually preceded by chronic inflammation caused by viral infection. The mutation of mitochondrial DNA (mtDNA) in hepatocarcinogenesis associated with viral infection was investigated. Compared with control liver tissue, the frequency of mtDNA mutations was markedly increased in both noncancerous and cancerous liver specimens from individuals with HCC. The accumulation of mtDNA mutations in HCC tissue reflected the degree of malignancy. The frequency of mtDNA mutations in HCC tissue was also greater than that described previously for other types of tumors. These observations suggest that the repeated destruction and regeneration of liver tissue associated with chronic viral hepatitis lead to the accumulation of mtDNA mutations. The genetic instability that results in the high rate of mtDNA mutation in cancerous liver tissue is also consistent with the multicentric hepatocarcinogenesis detected clinically.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Most cases of chronic hepatitis are caused by hepatitis viruses. Persistent infection with hepatitis B virus or hepatitis C virus also ultimately results in the development of HCC.2 Thus, unlike other types of cancer, HCC is usually preceded by chronic infection for 20–40 years. The development of HCC is rapid, however, in individuals with viral hepatitis and high activities of alanine aminotransferase in plasma (1) . Treatment of hepatitis patients with IFN reduces the incidence of HCC (2 , 3) . Normalization of the plasma activity of alanine amino transferase by IFN treatment reduces the rate of hepatocarcinogenesis, even if the hepatitis virus is not eliminated (4) . Chronic inflammation induced by hepatitis viruses thus plays an important role in hepatocarcinogenesis.

The extent of oxidative stress is increased in the liver of individuals infected with hepatitis viruses (5 , 6) , and reactive oxygen species are important inducers of DNA mutations (7) . Mutations accumulate to a greater extent in mtDNA than in nuclear DNA, in part because mitochondria lack histones and DNA repair systems (8) . The human mitochondrial genome comprises a 16.5-kb circular double-stranded DNA molecule that encodes 13 polypeptides of the respiratory chain, 22 transfer RNAs, and 2 rRNAs required for protein synthesis. Expression of the entire mitochondrial genome is necessary for the maintenance of mitochondrial functions, suggesting that small changes in the sequence of mtDNA might result in profound impairment of such functions. Mutations in mtDNA are associated with mitochondrial myopathy, encephalopathy, and diabetes mellitus (9) . Although it is unclear whether mutations in mtDNA underlie carcinogenesis, several such mutations have been detected in human colorectal (10) , neck, urinary bladder (11) , and thyroid (12) cancers.

We hypothesized that the increase in oxidative stress associated with carcinogenesis in individuals with chronic hepatitis might result in mutation of mtDNA. We therefore sequenced the mitochondrial genome of cancerous and noncancerous regions of the liver of individuals with HCC and compared the sites and frequencies of mutations with those detected in normal liver.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Tissue Specimens.
We obtained 149 liver specimens with informed consent from 78 consecutive Japanese individuals who underwent surgery at Osaka City University Medical School. These specimens included 59 HCC samples and 59 corresponding samples of noncancerous regions of the liver of individuals with chronic hepatitis C virus infection; 12 HCC samples and 12 corresponding samples of noncancerous regions of the liver of individuals with chronic hepatitis B virus infection; and 7 control liver samples from individuals without viral infection but with liver disease other than HCC (including hepatolithiasis and colorectal carcinoma with liver metastasis). HCC was diagnosed histologically as well differentiated, moderately differentiated, or poorly differentiated in 7, 32, and 32 individuals, respectively. This study was performed in accordance with the Helsinki Declaration of 1975 (1983 revision) and was approved by the ethics committee of Osaka City University Medical School.

PCR Amplification.
Liver specimens were frozen, microdissected with a cryostat, and digested with proteinase K (0.1 mg/ml) in the presence of 1% SDS. DNA was extracted with the use of phenol-chloroform and ethanol precipitation as described previously (13) . Each DNA sample (50 ng) was subjected to amplification by PCR with the use of overlapping sets of primers to screen the entire mitochondrial genome. PCR (an initial incubation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min) was performed in a final volume of 50 µl with a GeneAmp PCR system 9600 (Perkin-Elmer).

The generation of large PCR products excluded the possibility of nuclear pseudogenes complicating the analysis (14) . In addition, the primers were selected to avoid such coamplification by analysis with cell lines devoid of mtDNA.

Sequence Analysis.
Aberrant PCR products were purified with a QIAquick PCR purification kit (Qiagen) and sequenced with an Applied Biosystems DNA sequencer (Perkin-Elmer) and a Dye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems). The entire sequence of mtDNA was examined for 1 control subject and 2 individuals with HCC, and the sequence of the displacement (D)-loop (nucleotides 100–600) was examined for the remaining 6 control subjects and 69 individuals with HCC. All mutations were confirmed by repeated analysis of DNA extracted from the specimens.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
The entire mitochondrial genomes of two poorly differentiated HCC specimens, the corresponding noncancerous tissue, and one control liver specimen were amplified by PCR and sequenced manually. The mtDNA mutations detected are indicated in Fig. 1Citation . Compared with a mtDNA sequence deposited in GenBank (accession no. J01415), the mtDNA sequence obtained from the liver specimen of our control subject contained three single-base mutations, all of which were located in the D-loop. The mtDNA sequences obtained from the two cancerous tissue specimens contained 67 and 77 mutations, half or more of which were also apparent in the mtDNA obtained from the paired noncancerous tissue specimens. Consistent with previous observations (10, 11, 12) , most of the mutations detected in the present study were homoplasmic.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1. Location of mtDNA mutations in specimens of control liver and paired cancerous and noncancerous tissue from individuals with HCC. Arrows, positions of mutations in mtDNA obtained from a liver specimen of a control subject as well as from paired noncancerous and cancerous liver tissue from two individuals with poorly differentiated HCC. The three common mutations (#) as well as identical mutations in the cancerous and noncancerous specimens of the two HCC patients (*, patient 1; §, patient 2) are indicated. ND1, NADH dehydrogenase 1; COI, cytochrome c oxidase I; ATPase 8, ATP synthase 8; Cyt b, cytochrome b. One-letter codes are transfer RNAs.

 
The greatest frequency of mtDNA mutations was apparent in the D-loop, especially in the region between nucleotides 100 and 600. All mtDNA samples contained a G->A transition at nucleotide 263, a T->C transition at nucleotide 489, and C insertion between nucleotides 311 and 312. We then compared the number of mutations in the D-loop region (between nucleotides 100 and 600), excluding the three mutations common to all samples, among specimens of control liver, noncancerous tissue from individuals with HCC, and well-differentiated, moderately differentiated, and poorly differentiated HCC (Fig. 2)Citation . For the 7 control specimens, the median number of mutations in the D-loop was 0 (25th and 75th percentiles: 0 and 1, respectively). The corresponding values were 2 (0 and 2), 3 (2 and 4.5), and 4 (2.5 and 6.5) for specimens of well-differentiated, moderately differentiated, and poorly differentiated HCC (n = 7, 32, and 32), respectively. Most of these mutations were also apparent in the paired noncancerous tissue specimens. Only 28 (39%) of the 71 individuals with HCC exhibited a greater number of mtDNA mutations in this region in cancerous tissue than in noncancerous tissue. The median difference was 0 (-1 and 1) in individuals with well-differentiated HCC, 1 (0 and 2) in those with moderately differentiated HCC, and 0 (0 and 2) in those with poorly differentiated HCC.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2. Frequency distributions for the number of mutations in the D-loop of mtDNA of control liver and paired noncancerous and cancerous liver specimens from individuals with HCC. The number of mutations in the D-loop (nucleotides 100–600), with the exception of the three mutations common to all subjects, was compared among control liver (n = 7) as well as noncancerous and cancerous liver tissue from individuals with well-differentiated (n = 7), moderately differentiated (n = 32), or poorly differentiated (n = 32) HCC.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Our data show that the number of mtDNA mutations in HCC tissue (~70 sites/tumor) is substantially greater than that detected previously for other types of cancer (10, 11, 12) . Furthermore, the number of mtDNA mutations in noncancerous liver tissue from individuals with HCC (~40 sites) was shown to be markedly greater than that in control liver specimens.

Most of the identified mutations in mtDNA were homoplasmic, although hepatocytes contain hundreds of mitochondria and each mitochondrion contains 1–10 genomes (15) . Mitochondria of tumor cells proliferate selectively when such cells are fused with normal cells (10 , 16) , possibly because certain mutant mtDNA molecules exhibit a replicative advantage. The D-loop region of mtDNA is important for both replication and expression of the mitochondrial genome because it contains the leading-strand origin of replication and the promoters of transcription (17) . Thus, mutations in this region might affect the rate of DNA replication by modifying the binding affinity of important trans-acting factors. Mitochondria that undergo rapid replication appear to acquire DNA damage more readily, resulting in an accumulation of mutations, than do those maintained under resting conditions. Our data now suggest that mtDNA mutations accumulate during the neoplastic transformation of hepatocytes. Mitochondrial DNA harboring certain mutations might generate abnormal RNAs or proteins, the latter of which may promote leakage of electrons from the mitochondrial electron transport chain. The amounts of endogenously produced reactive oxygen species, such as superoxide and related free radicals, might thus be increased in cells with mutant mtDNA. The resulting oxidative modification of DNA therefore may contribute to the early stages of hepatocarcinogenesis.

Although inflammatory cells may infiltrate HCC tissue, the number of such cells is small compared with the number of cancer cells. Moreover, the density of mitochondria in hepatocytes and HCC cells is much greater than that in inflammatory cells. Thus, the possible contribution of contaminating inflammatory cells to the extent of mtDNA mutation observed in HCC tissue is negligible. Furthermore, for some individuals with HCC, mtDNA preparations from liver tissue were compared with those from paired blood samples; almost no mutations were found in blood samples excluding the three common mutations. In contrast to the single-nucleotide mutations observed in the present study, large deletions have been described previously in mtDNA from certain types of tumors (18, 19, 20) . Despite attempts to detect deletions in mtDNA with the use of multiple primers for PCR analysis, none was apparent in either cancerous or noncancerous tissue from individuals with HCC. Our results thus suggest that hepatocytes and HCC cells with large deletions in their mtDNA might be eliminated, and that cells without such deletions undergo continuous proliferation.

The high frequency of mtDNA mutations in noncancerous liver tissue of individuals with HCC suggests that hepatocytes in such tissue may already have undergone the initial stage of malignant transformation in chronic inflammation induced by virus infection. The observation that most of the mutations detected were homoplasmic indicates that the mutated mtDNA had become dominant in both HCC tissue and hepatocytes in noncancerous regions of the liver. Given the clonal nature and large number of copies of mtDNA, mutation of the mitochondrial genome in noncancerous regions of the liver of individuals with HCC is indicative of genomic instability and likely contributes to hepatocarcinogenesis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Biochemistry, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan. Phone: 81-6-6645-3722; Fax: 81-6-6645-3721; E-mail: nishikawa{at}med.osaka-cu.ac.jp Back

2 The abbreviations used are: HCC, hepatocellular carcinoma; mtDNA, mitochondrial DNA. Back

Received 12/15/00. Accepted 1/10/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Tarao K., Rino Y., Ohkawa S., Shimizu A., Tamai S., Miyakawa K., Aoki H., Imada T., Shindo K., Okamoto N., Totsuka S. Association between high serum alanine aminotransferase levels and more rapid development and higher rate of incidence of hepatocellular carcinoma in patients with hepatitis C virus-associated cirrhosis. Cancer (Phila.), 86: 589-595, 1999.[Medline]
  2. Nishiguchi S., Kuroki T., Nakatani S., Morimoto H., Takeda T., Nakajima S., Shiomi S., Seki S., Kobayashi K., Otani S. Randomised trial of effects of interferon-{alpha} on incidence of hepatocellular carcinoma in chronic active hepatitis C with cirrhosis. Lancet, 346: 1051-1055, 1995.[Medline]
  3. Imai Y., Kawata S., Tamura S., Yabuuchi I., Noda S., Inada M., Maeda Y., Shirai Y., Fukuzaki T., Kaji I., Ishikawa H., Matsuda Y., Nishikawa M., Seki K., Matsuzawa Y. Relation of interferon therapy and hepatocellular carcinoma in patients with chronic hepatitis C. Osaka Hepatocellular Carcinoma Prevention Study Group. Ann. Intern. Med., 129: 94-99, 1998.[Abstract/Free Full Text]
  4. Yoshida H., Shiratori Y., Moriyama M., Arakawa Y., Ide T., Sata M., Inoue O., Yano M., Tanaka M., Fujiyama S., Nishiguchi S., Kuroki T., Imazeki F., Yokosuka O., Kinoyama S., Yamada G., Omata M. Interferon therapy reduces the risk for hepatocellular carcinoma: national surveillance program of cirrhotic and noncirrhotic patients with chronic hepatitis C in Japan. IHIT Study Group. Inhibition of Hepatocarcinogenesis by Interferon Therapy. Ann. Intern. Med., 131: 174-181, 1999.[Abstract/Free Full Text]
  5. Kane J. M., III, Shears L. L., II, Hierholzer C., Ambs S., Billiar T. R., Posner M. C. Chronic hepatitis C virus infection in humans: induction of hepatic nitric oxide synthase and proposed mechanisms for carcinogenesis. J Surg. Res., 69: 321-324, 1997.[Medline]
  6. Schwarz K. B. Oxidative stress during viral infection: a review. Free Radical Biol. Med., 21: 641-649, 1996.[Medline]
  7. Beckman K. B., Ames B. N. Oxidative decay of DNA. J. Biol. Chem., 272: 19633-19636, 1997.[Free Full Text]
  8. Croteau D. L., Bohr V. A. Repair of oxidative damage to nuclear and mitochondrial DNA in mammalian cells. J. Biol. Chem., 272: 25409-25412, 1997.[Free Full Text]
  9. Johns D. R. Mitochondrial DNA and disease. N. Engl. J. Med., 333: 638 1995.[Free Full Text]
  10. Polyak K., Li Y., Zhu H., Lengauer C., Willson J. K., Markowitz S. D., Trush M. A., Kinzler K. W., Vogelstein B. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nat. Genet., 20: 291-293, 1998.[Medline]
  11. Fliss M. S., Usadel H., Caballero O. L., Wu L., Buta M. R., Eleff S. M., Jen J., Sidransky D. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science (Washington DC), 287: 2017-2019, 2000.[Abstract/Free Full Text]
  12. Yeh J. J., Lunetta K. L., van Orsouw N. J., Moore F. D., Jr., Mutter G. L., Vijg J., Dahia P. L., Eng C. Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours. Oncogene, 13: 2060-2066, 2000.
  13. Jen J., Kim H., Piantadosi S., Liu Z. F., Levitt R. C., Sistonen P., Kinzler K. W., Vogelstein B., Hamilton S. R. Allelic loss of chromosome 18q and prognosis in colorectal cancer. N. Engl. J. Med., 331: 213-221, 1994.[Abstract/Free Full Text]
  14. Parfait B., Rustin P., Munnich A., Rotig A. Co-amplification of nuclear pseudogenes and assessment of heteroplasmy of mitochondrial DNA mutations. Biochem. Biophys. Res. Commun., 247: 57-59, 1998.[Medline]
  15. Wallace D. C. Diseases of the mitochondrial DNA. Annu. Rev. Biochem., 61: 1175-1212, 1992.[Medline]
  16. Shay J. W., Ishii S. Unexpected nonrandom mitochondrial DNA segregation in human cell hybrids. Anticancer Res., 10: 279-284, 1990.[Medline]
  17. Taanman J. W. The mitochondrial genome: structure, transcription, translation and replication. Biochim. Biophys. Acta, 1410: 103-123, 1999.[Medline]
  18. Yamamoto H., Tanaka M., Katayama M., Obayashi T., Nimura Y., Ozawa T. Significant existence of deleted mitochondrial DNA in cirrhotic liver surrounding hepatic tumor. Biochem. Biophys. Res. Commun., 182: 913-920, 1992.[Medline]
  19. Burgart L. J., Zheng J., Shu Q., Strickler J. G., Shibata D. Somatic mitochondrial mutation in gastric cancer. Am. J. Pathol., 147: 1105-1111, 1995.[Abstract]
  20. Heerdt B. G., Chen J., Stewart L. R., Augenlicht L. H. Polymorphisms, but lack of mutations or instability, in the promotor region of the mitochondrial genome in 6human colonic tumors. Cancer Res., 54: 3912-3915, 1994.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
A. Tamori, Y. Yamanishi, S. Kawashima, M. Kanehisa, M. Enomoto, H. Tanaka, S. Kubo, S. Shiomi, and S. Nishiguchi
Alteration of Gene Expression in Human Hepatocellular Carcinoma with Integrated Hepatitis B Virus DNA
Clin. Cancer Res., August 15, 2005; 11(16): 5821 - 5826.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
A. Lievre, C. Chapusot, A.-M. Bouvier, F. Zinzindohoue, F. Piard, P. Roignot, L. Arnould, P. Beaune, J. Faivre, and P. Laurent-Puig
Clinical Value of Mitochondrial Mutations in Colorectal Cancer
J. Clin. Oncol., May 20, 2005; 23(15): 3517 - 3525.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Shidara, K. Yamagata, T. Kanamori, K. Nakano, J. Q. Kwong, G. Manfredi, H. Oda, and S. Ohta
Positive Contribution of Pathogenic Mutations in the Mitochondrial Genome to the Promotion of Cancer by Prevention from Apoptosis
Cancer Res., March 1, 2005; 65(5): 1655 - 1663.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
L-J C Wong, D-J Tan, R-K Bai, K-T Yeh, and J Chang
Molecular alterations in mitochondrial DNA of hepatocellular carcinomas: is there a correlation with clinicopathological profile?
J. Med. Genet., May 1, 2004; 41(5): e65 - e65.
[Full Text] [PDF]

Home page
 CarcinogenesisHome page
J. Z. Chen, N. Gokden, G. F. Greene, B. Green, and F. F. Kadlubar
Simultaneous generation of multiple mitochondrial DNA mutations in human prostate tumors suggests mitochondrial hyper-mutagenesis
Carcinogenesis, September 1, 2003; 24(9): 1481 - 1487.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pathol.Home page
E Kirches, G Krause, S Weis, C Mawrin, and K Dietzmann
Comparison between mitochondrial DNA sequences in low grade astrocytomas and corresponding blood samples
Mol. Pathol., June 1, 2002; 55(3): 204 - 206.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
S. Nomoto, K. Yamashita, K. Koshikawa, A. Nakao, and D. Sidransky
Mitochondrial D-loop Mutations As Clonal Markers in Multicentric Hepatocellular Carcinoma and Plasma
Clin. Cancer Res., February 1, 2002; 8(2): 481 - 487.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
V. W. S. Liu, H. H. Shi, A. N. Y. Cheung, P. M. Chiu, T. W. Leung, P. Nagley, L. C. Wong, and H. Y. S. Ngan
High Incidence of Somatic Mitochondrial DNA Mutations in Human Ovarian Carcinomas
Cancer Res., August 1, 2001; 61(16): 5998 - 6001.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishikawa, M.
Right arrow Articles by Inoue, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishikawa, M.
Right arrow Articles by Inoue, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online