Oncogene (1998) 17, 1931 ± 1937  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc instability in the mitochondrial DNA of colorectal carcinomas: Evidence for mismatch repair systems in mitochondrial

Wataru Habano, Shin-ichi Nakamura and Tamotsu Sugai

Division of Pathology, Central Clinical Laboratory, School of Medicine, Iwate Medical University, 19-1 Uchimaru, Morioka 020, Japan

The role, if any, that mitochondrial (mt) DNA Mitochondria contain their own genetic systems for alterations play in the carcinogenic process remains replication, transcription and translation (Wallace, unclear. To determine whether mtDNA instability occurs 1992a). Mitochondrial DNA (mtDNA) is a 16 569- in cancers, nine microsatellite sequences in the mtDNA base pair (bp) double-stranded, closed circular were examined in 45 sporadic colorectal carcinomas. molecule, which codes for both a small (12S) and a Alteration in a polycytidine (C)n tract within a non- large (16S) ribosomal RNA gene, 22 transfer RNAs coding displacement-loop (D-loop) region was detected in and 13 protein-coding genes. All of the mitochondrial 20 carcinomas (44%), three of which also exhibited genes encode subunits of the oxidative phosphorylation frameshift mutations in a polyadenosine (A)8 or (OXPHOS) enzymes that are responsible for the polycytidine (C)6 tract within NADH dehydrogenase energy-generating pathway. MtDNA is thought to be (ND) genes. Interestingly, all three mutant genes were more susceptible to damage by mutagens than nuclear predicted to encode truncated ND proteins, which lacked DNA for the following reasons: (a) mtDNA polymer- a large portion of the C-terminus. These results ase g replicates the DNA with poor ®delity (Kunkel suggested that certain repair systems, like the mismatch and Loeb, 1981), (b) mtDNA is a naked molecule to repair systems in the nuclear genome, are required for which chemical carcinogens can easily bind, while mtDNA maintenance and that defects in these systems nuclear DNA is protected by histones (Backer and can lead to target mitochondrial gene mutations in Weinstein, 1980; Allen and Coombs, 1980) and (c) a colorectal carcinomas. high concentration of reactive oxygen species in mitochondria can induce DNA damage (Oberley and Keywords: genomic instability; mitochondrial DNA; Buettner, 1979). Indeed, structural changes in mtDNA colorectal carcinoma are common events in various degenerative mitochon- drial diseases (Wallace, 1992a,b). However, only a few studies have demonstrated structural changes in mtDNA in human cancers, including large-scale Introduction deletions (450 bp) in gastric (Burgart et al., 1995) and renal cell (Horton et al., 1996) carcinomas. Almost Carcinogenesis is a multistep process involving the all other studies of cancer have described changes in accumulation of genetic changes (Bishop, 1993). Much mitochondrial gene expression (Heerdt et al., 1990; attention has been directed to genetic events, such as Sharp et al., 1992). This observation prompted us to activation of oncogenes, inactivation of tumor speculate that some DNA repair mechanisms are suppressor genes and defects of mismatch repair genes potentially involved in maintenance of the normal (Karp and Broder, 1995). However, there are still many mitochondrial genome. aspects that cannot be explained by these genetic Hypermutable status (instability) in the nuclear changes. Somatic cell hybrid studies have demonstrated genome has been observed in certain types of that tumor-suppressive activity can be involved in the cancers, in which microsatellite sequences are prefer- extra-chromosomal fractions (Jonasson and Harris, entially a€ected due to slippage during the replication 1977; Sager, 1985). This implies that both nuclear process (Ionov et al., 1993; Aaltonen et al., 1993; and cytoplasmic elements are responsible for the steps Thibodeau et al., 1993). These replication error of carcinogenesis. Indeed, mitochondrial abnormalities phenotypes are related to functional loss of mismatch have been observed in many human cancers, including repair (MMR) genes, including the hMSH2, hMLH1, changes in structure, number, respiratory enzyme hPMS1 and hPMS2 genes (Parsons et al., 1993; Boyer components and transport systems (Pedersen, 1978; et al., 1995). On the other hand, in the mitochondrial Wilkie et al., 1983). However, the vast majority of genome, the MMR system has been found only in proteins in mitochondria are encoded by the nuclear yeast strains, in which MSH1 and MSH2 are separately genome. Therefore, it is not clear whether these involved in mitochondrial and nuclear DNA repair mitochondrial abnormalities result from defects in the systems, respectively (Reenan and Kolodner, 1992). No mitochondrial genome. MSH1 homologue has been found in mammalian cells and therefore, it remains uncertain whether an MMR system plays a role in the maintenance of mammalian mitochondrial genome. Correspondence: S-I Nakamura Received 29 December 1997; revised 6 May 1998; accepted 6 May The aim of the present study was to determine 1998 whether certain MMR systems play a role in mtDNA mtDNA instability in colorectal carcinoma W Habano et al 1932 maintenance and whether impairment of this system gene mutations. Because the mitochondrial genome is can lead to a hypermutable status of mtDNA in cancer highly heterogeneous (heteroplasmy), analysis needs to cells. Thus, we evaluated mitochondrial microsatellite be performed using pure normal or carcinoma cells for instability (MSI) in the non-coding D-loop region accurate estimation. Therefore, in the present study, we because any alterations in the coding region can a€ect applied a crypt isolation technique (Habano et al., in mitochondrial function and as a result lead to press; Habano et al., 1996; Nakamura et al., 1993, exclusion by negative selection after mitochondrial 1994; Yoshida et al., 1996) that allows the complete segregation. Two simple repeat sequences in the D- separation of non-neoplastic and neoplastic crypts loop region were then examined in 45 colorectal from stromal cells. carcinomas. We also examined mutations in seven independent repeat sequences in the mitochondrial coding region to determine whether mitochondrial Results MSI can be associated with speci®c mitochondrial Alterations in mtDNA in the D-loop region Two distinct microsatellite sequences within the mitochondrial D-loop region were examined for mutations in 45 sporadic colorectal carcinomas. A 157-bp portion including a polycytidine (C)n tract (Burgart et al., 1995) was examined using the polymerase chain reaction-single strand conformation polymorphism (PCR ± SSCP) method. Twenty carcino- mas (44%) exhibited abnormal bands with di€erent mobilities (Figure 1). Direct DNA sequencing revealed that most alterations consisted of deletion or insertion of cytidine residues in the (C)n tract (Figure 2, case 59 and 57). Two cases revealed more obvious changes in this region (Figure 2, case 501). None of the individuals examined had the same repeat number in germline status (normal mucosa) (7 4n 49), suggest- ing that this variation re¯ected a neutral polymorphism with respect to mitochondrial function. Some normal and carcinoma tissue contained two distinct mtDNA subpopulations with respect to (C)n status (hetero- Figure 1 PCR ± SSCP analysis in the mitochondrial D-loop plasmy). In some cases, a mtDNA subpopulation that region. Cases 57 and 59 exhibited di€erent mobilities in appeared to be speci®c to carcinoma tissue in the SSCP carcinoma tissue (C), compared to each normal counterpart analysis was also detectable in the normal counterpart (N), while cases 84 and 506 exhibited no mobility shift. Case 501 showed more obvious changes in this region. All alterations were using direct sequencing (Figure 2, case 59). Such cases detectable in both single-strand (ss) and double-strand (ds) DNA revealed a di€erent proportion of mtDNA subpopula- patterns tions between normal and carcinoma tissue. Whether

Figure 2 Direct sequencing of the mitochondrial D-loop region. In case 59C, the (C)n status of the major population changed from (C)8 to (C)7, but normal tissue (59N) contained a minor (C)7 subpopulation. Arrowheads `T (thymidine)' and `C (cytidine)' indicate (C)7 and (C)8 subpopulations, respectively. In case 57, the (C)8 population was completely exchanged to (C)9. Case 501 showed more extensive change, but the altered sequence could not be determined mtDNA instability in colorectal carcinoma WHabanoet al 1933 the alterations resulted from sequence changes (genuine hibited alterations in this (CA)n tract in comparison mutations) or simply re¯ected changes in the propor- with each normal counterpart. Thus, microsatellite tion of mtDNA subpopulations during mitochondrial alteration in the mitochondrial D-loop region (MSI) segregation could not be determined. Therefore, these occurred commonly in the (C)n tract, but not in the alterations were comprehensively characterized as (CA)n tract, in colorectal carcinomas. changes in mitochondrial heteroplasmy (CMH) and distinguished from genuine mutations that were never Mutations in the coding region of mtDNA detected in normal counterparts (Figure 3). If the cases with CMH were excluded from the positive group with In order to determine whether microsatellite alteration mitochondrial instability, mtDNA alterations in this occurred in the mitochondrial coding regions, seven region were found in nine cases (20%). independent repeat sequences (Table 1) were examined The other D-loop portion including a dinucleotide using an automated DNA sequencer. As shown in (CA)n repeat sequence was examined by electrophor- Table 2, three cases (7%) revealed mutations in one of esis under denaturing conditions using an automated these sequences. Case 505C exhibited a cytidine DNA sequencer. A polymorphism with respect to the deletion in the (C)6 tract within the NADH repeat number was observed among normal tissues dehydrogenase 5 (ND5) gene (Figure 4). This frame- (4 4n 47). However, none of the carcinomas ex- shift mutation created a stop codon (TAA) down-

Figure 3 Patterns of mtDNA alteration. For the purpose of simpli®cation, distribution of mtDNA in normal or carcinoma tissue is schematically displayed in a single cell. MtDNA population is homoplasmic (a) or heteroplasmic (b),(c) with respect to (C)n status in normal cells. If a certain population with di€erent (C)n status (open-triangle) is detected in carcinoma cells, the alteration is de®ned as a genuine mutation that indicates sequence change in this (C)n tract ((a) and (b)). If only a proportion of distinct mtDNA subpopulations is changed in carcinoma cells without any di€erent subpopulations (c), this alteration is de®ned as a change in mitochondrial heteroplasmy (CMH) and is distinguished from a genuine mutation mtDNA instability in colorectal carcinoma W Habano et al 1934 Table 1 List of primers for ampli®cation of each repeat sequence Repeat mtDNA region sequence ( pairs)a mtRNA Primer sequences (CA)n 467 ± 556 ±b 5'-CCCATACTACTAATCTCATCAA-3' 5'-TTTGGTTGGTTCGGGGTATG-3' (C)6 3529 ± 3617 ND1 5'-CCGACCTTAGCTCTCACCAT-3' 5'-AATAGGAGGCCTAGGTTGAG-3' (A)7 4555 ± 4644 ND2 5'-CCTGAGTAGGCCTAGAAATAAA-3' 5'-ACTTGATGGCAGCTTCTGTG-3' (A)7 6644 ± 6733 COI 5'-CCTACCAGGCTTCGGAATAA-3' 5'-ATAGCTCAGACCATACCTATG-3' (T)7 9431 ± 9526 COIII 5'-CCAAAAAGGCCTTCGATACG-3' 5'-GCTAGGCTGGAGTGGTAAAA-3' (C)6 and (A)8 12360 ± 12465 ND5 5'-CACCCTAACCCTGACTTCC-3' 5'-GGTGGATGCGACAATGGATT-3' (CCT)3-(AGC)3 12940 ± 13032 ND5 5'-GCCCTTCTAAACGCTAATCC-3' 5'-TCAGGGGTGGAGACCTAATT-3' aNucleotide pairs as numbered by Anderson et al. (1981). bA (CA)n is located in the non-coding D-loop region. For each primer pair, the ®rst oligonucleotide represents the forward primer, and the second corresponds to the reverse primer

Table 2 Mitochondrial gene mutations and predicted protein alterations Mutant mtDNA Created Changes in the (C)n instability Cases Mutations status stop codon no. of amino acids in the D-loop 505 ND5 (C)6?5 homoplasmy TAA 603?35 CMHa 124 ND5 (A)8?9 homoplasmy AGA 603?57 CMH 20 ND1 (C)6?7 heteroplasmy AGG 318?100 CMH aChanges in mitochondrial heteroplasmy

stream and was predicted to encode a truncated ND5 protein (a change from 603 to 35 amino acids). The mutant mtDNA was homoplasmic throughout carci- noma tissue. Case 124 and 20 (Figure 4) also exhibited frameshift mutations in the (A)8 and (C)6 tracts and created mitochondria-speci®c stop codons AGA and AGG, respectively. The three carcinomas also showed alterations in the (C)n sequences within the D-loop region. None except the above three cases exhibited alterations in the coding repeat sequences examined. Thus, microsatellite alteration in the mitochondrial coding region was observed in some colorectal carcinomas, and appears to be associated with alteration in the D-loop region.

Discussion

We assessed mtDNA instability in the non-coding D- loop region in 45 colorectal carcinomas. Burgart et al. Figure 4 Direct sequencing of the mitochondrial ND5 and ND1 (1995) demonstrated that mtDNA alterations in the D- genes. In case 505, a cytidine deletion in the (C)6 tract in the ND5 loop region are found in gastric cancers (12.5%); all of gene resulted in the (C)5 in carcinomas, with the status of their series had deletions in the 50 bp portion including homoplasmy. In case 20, a cytidine insertion in the (C)6 tract in the ND1 gene resulted in the (C)7 in carcinomas, with the status the (C)n tract. Heerdt et al. (1994) examined colorectal of heteroplasmy. Arrowheads `T (thymidine)' and `C (cytidine)' carcinomas in the promoter region adjacent to the (C)n indicate (C)6 and (C)7 subpopulations, respectively tract and found neither mutation nor instability. In the present study, mtDNA alterations within the D-loop region were frequently detected in colorectal carcino- mas (44%), most of which involved insertion or heteroplasmy in the normal counterparts retained deletion of a cytidine in the (C)n sequence. None of exactly the same proportion of mtDNA subpopula- the cases revealed such large-scale deletions as Burgart tions. We believe that the real status of mitochondrial et al. demonstrated in gastric carcinomas. Some heteroplasmy was able to be estimated in this study, positive cases exhibited genuine mutations, but the because mtDNA was obtained from pure normal or other positive cases exhibited changes in mitochondrial neoplastic cells using the crypt isolation technique. heteroplasmy (CMH) with respect to (C)n status The (C)n tract is located in a conserved sequence (Figure 3). We assume that the CMH in the block 2 (CSB2) that is an essential element for mtDNA mitochondrial genome plays a role in colorectal replication (Chang et al., 1985). We have not found carcinogenesis, because many carcinomas that showed any evidence for a change in replication or transcrip- mtDNA instability in colorectal carcinoma WHabanoet al 1935 tion eciency among these positive cases. However, chondrial . Therefore, the truncated ND5 these mutations were most likely neutral with respect proteins probably conferred some speci®c cell growth to mitochondrial function and could not in¯uence advantage. This also indicates that the ND5 mutations tumorigenesis since these sequence variations were did not result from non-random targeting of mitochon- primarily found in individual normal tissue. This can drial MSI. The other case 20 was a frameshift mutation be supported by a recent study of Parsons et al. (1997) in a polycytidine tract (C)6 in the ND1 gene and which proves a high substitution rate in this sequence. predictably led to a truncated protein. This ND1 Because of aspects similar to nuclear genomic mutation may also be important in colorectal instability characterized as (CA)n alteration (Aaltonen carcinogenesis because a large amount of the mutant et al., 1993; Thibodeau et al., 1993), the (C)n (C)7 population had accumulated in the carcinoma alterations probably re¯ect microsatellite instability tissue. (MSI) in the mitochondrial genome. On the other Interestingly, the three cases also exhibited altera- hand, because we used total cellular DNA including tions in the (C)n tract in the D-loop region. Therefore, both mitochondrial and nuclear genome in the present the ND1 or ND5 gene may be one of the target genes study, the possibility that we examined mtDNA-like for mitochondrial MSI in colorectal carcinomas. The sequences in the nuclear genome cannot be excluded situation is similar to that in the nuclear genome, in (Fukuda et al., 1985). However, the ampli®ed sequence which mutations in the mononucleotide repeat around the (C)n tract was completely consistent with sequence in the transforming growth factor btype II the mtDNA sequence previously reported (Anderson et receptor (Markowitz et al., 1995), insulin-like growth al., 1981). In addition, there is no evidence that factor II receptor (Souza et al., 1996) and BAX mtDNA sequences are stably integrated into the (Rampino et al., 1997) genes are preferentially nuclear genome in human cells. Therefore, we believe observed in colorectal carcinomas with MSI. We that genetic instability detected in the present study found that the mitochondrial genome has no repeat occurred in the mitochondrial genome, but not in the sequence that consists of more than eight mononucleo- nuclear genome. The results also indicate that certain tides. Therefore, mtDNA instability in the coding repair systems, like mismatch repair (MMR) systems in region may not be frequently detected in colorectal the nuclear genome, play a role in the maintenance of carcinomas. However, other mitochondrial repeat the mitochondrial genome in human cells. Conse- sequences that we had not examined in this study quently, repair de®ciencies may lead to mtDNA may have been a€ected in these carcinomas. instability in some colorectal carcinomas. In contrast, Both ND5 and ND1 gene products are subunits of none of the cases exhibited instability in the (CA)n the NADH dehydrogenase complex. Horton et al., tract, although the alteration in this tract is also (1996) detected mtDNA deletions in the ND1 region in thought to be neutral with respect to mitochondrial renal cell carcinomas, suggesting that functional function. Heerdt et al. (1994) also found no alteration changes in a NADH dehydrogenase component may in the same (CA)n tract in colorectal carcinomas. We signi®cantly in¯uence carcinogenesis, although these believe that relatively small repeat numbers (4 4n 47) alterations most likely do not accelerate tumor cell in the normal status prevent the creation of slippage growth directly. One attractive hypothesis (Bandy and mutations in this tract. Parsons et al. (1995) also Davison, 1990) is that damage to the mitochondrial showed that the probability of maintaining a nuclear gene may result in impaired function of normal microsatellite sequence in MMR-de®cient cells is respiration and release abnormally high levels of inversely proportional to the length of the micro- reactive oxygen species, which amplify damage to the satellite tract. The similarity in the target speci®city nuclear genome (such as oncogenes and suppressor suggests that both mitochondrial and nuclear MSI may genes). Ozawa (1995) also suggested that production of result from the same mechanism, by which MMR reactive oxygen species can be synergistically enhanced de®ciencies lead to slippage mutation during the by the malignant cycle and results in mtDNA replication process. mutations and deletions. These hypotheses provide a On the other hand, mtDNA mutations in the coding possible mechanism by which speci®c mitochondrial region were detected in three cases (505, 124 and 20). mutations promote carcinogenesis. In contrast, abnor- Because each mutant mtDNA was not detected in the mal mitochondrial gene products may also lead to normal counterpart (Figure 4), these mutations lethal changes in mitochondrial respiratory functions. re¯ected the generation of new mutant mtDNA during However, structural and functional changes in cancer development. The cases 505 and 124 showed mitochondria are found in various human cancer cells frameshift mutations in the ND5 gene and predictably (Wilkie et al., 1983) and some of these cancers reveal led to truncated proteins that lacked a large portion of remarkable defects in respiratory metabolism. There- the C-terminus. Interestingly, these mtDNA mutations fore, certain cancer cells, even if they are rapidly were homoplasmic throughout the carcinoma tissue. It growing, appear to adapt to life under anaerobic seems unlikely that only a minor component of mutant conditions. mtDNA has accumulated in each carcinoma tissue Also interesting is that, in the present study mtDNA without any selection because the number of mitochon- instability in the D-loop region correlated with nuclear drial genomes per cell is quite large (Shay and Werbin, genomic instability (data not shown). The results 1987). In a yeast model (Bernardi, 1979), a truncated suggest that nuclear and mitochondrial genomes mitochondrial genome can gain a selective advantage partly share the same DNA repair system. In our over normal mitochondrial genomes due to faster previous study (Habano et al., 1998), loss of the replication. However, an insertion or deletion of only hMSH2 and hMLH1 genes was observed in 21% and one nucleotide in the ND5 sequence most likely did not 20% of the same series of colorectal carcinomas, result in a replicative advantage over normal mito- respectively. However, mtDNA instability was not mtDNA instability in colorectal carcinoma W Habano et al 1936 associated with these gene defects, suggesting that a carcinomas. Corresponding tissue specimens were ob- certain MMR enzyme other than hMSH2 or hMLH1 tained from normal mucosa located distal to the tumor. may be responsible for maintenance in both nuclear All specimens were surgically resected at Iwate Medical and mitochondrial genomes. In contrast, some University Hospital, Morioka, Japan, between 1993 and carcinomas with mtDNA instability, including cases 1995, as described previously (Habano et al., 1998). Crypt isolation and total cellular DNA extraction were 505, 124 and 20 in the present study that showed ND performed as previously described (Habano et al., 1996). gene mutations, do not exhibit any nuclear MSI. Therefore, it is more likely that there is another MMR system speci®c to mitochondrial genome maintenance. PCR ± SSCP analysis The situation is similar to MMR systems in yeast To screen for mutations in a 157-bp portion (Burgart et al., strains, in which maintenance of the mitochondrial and 1995) of the mitochondrial D-loop region, nonRI PCR ± nuclear genomes is separately regulated by MSH1 and SSCP analysis was performed as described previously MSH2, respectively. We suggest that an MSH1-like (Habano et al., 1996). PCR products were electrophoresed enzyme plays a role in mtDNA maintenance in at 300 V at 238C for 2 h through a 7.5% polyacrylamide mammalian cells. gel (138613061 mm) containing 50 mM Tris-base, 50 mM boric acid and 1 mM EDTA. After silver staining, DNA To our knowledge, this is the ®rst study to identify fragments that reproducibly showed mobility shifts microsatellite instability in the mitochondrial genome according to independent two-repeated SSCP analyses of carcinomas. Certain mitochondrial gene mutations were subjected to direct sequencing using dye-labeled (ND1 and ND5) were associated with the mtDNA dideoxynucleoside triphosphates as terminators and ana- instability, suggesting that unknown mismatch repair lysed using an automated DNA sequencer (Smith et al., systems play a role in mtDNA maintenance and that 1986). repair defects lead to speci®c mitochondrial gene mutations in carcinomas. Therefore, alterations in the Microsatellite analysis mitochondrial genome, in combination with alterations in nuclear oncogenes and tumor suppressor genes, may Mutations in eight independent microsatellite sequences were examined using a model 373A automated ¯uorescent be important in the process of a certain type of DNA sequencer and GENESCANTM 672 (Version 1.2.2-1, colorectal carcinoma. Hence, more attention must be Perkin-Elmer Cetus, Norwalk, Connecticut). The primers focused on the role of the mitochondrial genome in for ampli®cation of each microsatellite sequence are listed carcinogenesis. in Table 1. Each antisense primer was labeled with 6-FAM, TET or HEX ¯uorescent dye. PCR was performed as previously described (Habano et al., 1996), although the annealing temperature was 578C for all reactions. Materials and methods Carcinoma samples reproducibly showing extra DNA signals according to two-repeated GENESCAN analyses Crypt isolation and DNA extraction were subjected to direct sequencing. Forty-®ve tissue specimens were obtained from 44 patients (31 male and 14 female) with primary colorectal

References

Aaltonen LA, PeltomaÈ ki P, Leach FS, Sistonen P, PylkkaÈ nen Heerdt BG, Chen J, Stewart LR and Augenlicht LH. (1994). L, Mecklin J-P, JaÈ rvinen L, Powell SM, Jen J, Hamilton Cancer Res., 54, 3912 ± 3915. SR, Petersen GM, Kinzler KW, Vogelstein B and de la Heerdt BG, Halsey HK, Lipkin M and Augenlicht LH. Chapelle A. (1993). Science, 260, 812 ± 816. (1990). Cancer Res., 50, 1596 ± 1600. Allen JA and Coombs MM. (1980). Nature, 287, 244 ± 245. Horton TM, Petros JA, Heddi A, Sho€ner J, Kaufman AE, Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Graham Jr SD, Gramlich T and Wallace DC. (1996). Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Genes Chromosom. Cancer, 15, 95 ± 101. Sanger F, Schreier PH, Smith AJH, Staden R and Young Ionov Y, Peinado MA, Malkhosyan S, Shibata D and IG. (1981). Nature, 290, 457 ± 465. Perucho M. (1993). Nature, 363, 558 ± 561. Backer JM and Weinstein IB. (1980). Science, 209, 297 ± 299. Jonasson J and Harris H. (1977). J. Cell Sci., 24, 255 ± 263. Bandy B and Davison AJ. (1990). Free Radicals Biol. Med., Karp JE and Broder S. (1995). Nature Med., 1, 309 ± 320. 8, 523 ± 539. Kunkel TA and Loeb LA. (1981). Science, 213, 765 ± 767. Bernardi G. (1979). TIBS, 4, 197 ± 201. Markowitz S, Wang J, Myero€ L, Parsons R, Sun L, Bishop JM. (1993). Cell, 75, 235 ± 248. Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, BoyerJC,UmarA,RisingerJI,LipfordJR,KaneM,YinS, Vogelstein B, Brattain M and Willson JKV. (1995). Barrett JC, Kolodner RD and Kunkel TA. (1995). Cancer Science, 268, 1336 ± 1338. Res., 55, 6063 ± 6070. Nakamura S, Goto J, Kitayama M and Kino I. (1994). Burgart LJ, Zheng J, Shu Q, Strickler JG and Shibata D. Gastroenterology, 106, 100 ± 107. (1995). Am.J.Pathol.,147, 1105 ± 1111. Nakamura S, Kino I and Baba S. (1993). Gut, 34, 1240 ± Chang DD and Clayton DA. (1985). Proc. Natl. Acad. Sci. 1244. USA, 82, 351 ± 355. Oberley LW and Buettner GR. (1979). Cancer Res., 39, Fukuda M, Wakasugi S, Tsuzuki T, Nomiyama H and 1141 ± 1149. Shimada K. (1985). J. Mol. Biol., 186, 257 ± 266. Ozawa T. (1995). Biochim. Biophys. Acta, 1271, 177 ± 189. Habano W, Sugai T and Nakamura S. (1998). Oncogene, 16, Parsons R, Myero€ LL, Liu B, Willson JKV, Markowitz SD, 1259 ± 1265. Kinzler KW and Vogelstein B. (1995). Cancer Res., 55, Habano W, Sugai T, Nakamura S and Yoshida T. (1996). 5548 ± 5550. Lab. Invest., 74, 933 ± 940. mtDNA instability in colorectal carcinoma WHabanoet al 1937 Parsons R, Li G-M, Longley MJ, Fang W, Papadopoulos N, Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Jen J, de la Chapelle A, Kinzler KW, Vogelstein B and Connell CR, Heiner C, Kent SBH and Hood LE. (1986). Modrich P. (1993). Cell, 75, 1227 ± 1236. Nature, 321, 674 ± 679. Parsons TJ, Muniec DS, Sullivan K, Woodyatt N, Alliston- Souza RF, Appel R, Yin J, Wang S, Smolinski KN, Greiner R, Wilson MR, Berry DL, Holland KA, Weedn AbrahamJM,ZouT-T,ShiY-Q,LeiJ,CottrellJ,Cymes VW, Gill P and Holland MM. (1997). Nature Genet., 15, K,BidenK,SimmsL,LeggettB,LynchPM,FrazierM, 363 ± 367. Powell SM, Harpaz N, Sugimura H, Young J and Meltzer Pedersen PL. (1978). Prog. Exp. Tumor Res., 22, 190 ± 274. SJ. (1996). Nature Genet., 14, 255 ± 257. Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed Thibodeau SN, Bren G and Schaid D. (1993). Science, 260, JC and Perucho M. (1997). Science, 275, 967 ± 969. 816 ± 819. Reenan RAG and Kolodner RD. (1992). Genetics, 132, 975 ± Wallace DC. (1992a). Ann. Rev. Biochem., 61, 1175 ± 1212. 985. Wallace DC. (1992b). Science, 256, 628 ± 632. Sager R. (1985). Adv. Cancer Res., 44, 43 ± 68. Wilkie D, Evans IH, Egilsson V, Diala ES and Collier D. Sharp MGF, Adams SM, Walker RA, Brammar WJ and (1983). Int. Rev. Cytol., 15, 157 ± 189. Varley JM. (1992). J. Pathol., 168, 163 ± 168. Yoshida T, Nakamura S and Sugai T. (1996). J. Surg. Oncol., Shay JW and Werbin H. (1987). Mutation Res., 186, 149 ± 63, 9 ± 16. 160.