Mechanisms for the Involvement of DNA Methylation in Colon Carcinogenesis1

[CANCER RESEARCH 56. 2375-2381, May 15. 1996] Mechanisms for the Involvement of DNA Methylation in Colon Carcinogenesis1

Christoph Schmutte, Allen S. Yang, TuDung T. Nguyen, Robert W. Beart, and Peter A. Jones2

Departments of Biochemistry and Molecular Biology Â¡C.S.. A. S. Y.. T. T. N.. P. A. J.I and Colorectal Surgery Â¡K.W. B.Â¡,University of Southern California, School of Medicine, Los Angeles. California 90033-0800

ABSTRACT mutations at these sites in colon tumors are C â€”¿Â»Tor G â€”¿>A transitions (10); and all of these hot spots in p53 have been found to C â€”¿>Ttransitions at CpG sites are the most prevalent mutations found be methylated (9, 11, 12), which is consistent with a methylation- in the p53 tumor suppressor gene in human colon tumors and in the induced mutational mechanism. Other genes, such as the factor IX germline (Li-Fraumeni syndrome). All of the mutational hot spots are methylated to 5-methylcytosine, and it has been hypothesized that the gene, show a similar accumulation of transition mutations at CpG sites majority of these mutations are caused by spontaneous hydrolytic deami- (13). In addition, CpG sites are underrepresented in human DNA by nation of this base to thymine. We have previously reported that bacterial a factor of 5 (14), and it is believed that transition mutations from C methyltransferases induce transition mutations at CpG sites by increasing â€”¿>Tor G â€”¿Â»Aon the opposite strand, respectively, caused the loss the deamination rate of C â€”¿Â»Uwhen the concentration of the methyl of CpGs during evolution (15, 16). We have argued that these muta group donor S-adenosylmethionine (AdoMet) drops below its A1M,suggest tions are caused by an endogenous mechanism, because epidemiolog- ing an alternative mechanism to create these mutations. Unrepaired uracil ical studies have not revealed any regional differences in the patterns pairs with adenine during replication, completing the ( ' â€”¿â€¢Ttransition of germline mutations (17), and no strand bias has been found in the mutation. To determine whether this mechanism could contribute to the repair process (10). development of human colon cancer, we examined the level of DNA Two mechanisms have been proposed to explain why these CpG (cytosine-S)-methyltransferase (MTase) expression, the concentration of sites are mutational hot spots in human tumors (9, 18). Amino groups AdoMet, and the activity of uracil-DNA glycosylase in human colon of cytosine and 5mC3 are less stable in DNA than the amino groups tissues, and searched for the presence of mutations in the MTase gene. Using reverse transcription-PCR methods, we found that average MTase of guanine and adenine and hydrolyze spontaneously, forming uracil niKNA expression levels were only 3.7-fold elevated in tumor tissues and thymine, respectively (19, 20; for review, see Ref. 21). Differ compared with surrounding normal mucosa from the same patient. Also, ences in the repair efficiencies of the U:G versus T:G mismatches no mutations were found in conserved regions of the gene in 10 tumors created by this process are also likely to contribute substantially to the sequenced. High-performance liquid Chromatographie analysis of extracts formation of mutational hot spots at CpG sites (22, 23). On the other from the same tissues showed that AdoMet concentrations were not hand, the methylation process itself could increase the deamination reduced below the A,,, value for the mammalian enzyme, and the concen rate at the target cytosine. During the normal transfer reaction of the tration ratio of AdoMet:5-adenosylhomocysteine, the breakdown product methyl group, the MTase flips its target cytosine out of the DNA helix of AdoMet and the competitive MTase inhibitor, did not differ signifi into the catalytic pocket of the enzyme, where a cysteine residue of the cantly. Finally, extracts from the tumor tissue efficiently removed uracil enzyme forms a covalent intermediate with the C-6 of the cytosine from DNA. Therefore, biochemical conditions favoring a mutagenic path way of C â€”¿>Uâ€”¿>Twere not found in a target tissue known to undergo a ring, destroying the aromatic character of the base and activating the high rate of C â€”¿Â»Ttransitions at CpG sites. C-5 position to accept the methyl group from the cofactor AdoMet (24-26). ÃŸelimination of the cysteine moieties of the MTase restores INTRODUCTION the aromatic character of the base and completes the methylation process (Fig. 1, lower pathway). When AdoMet is present in concen The dinucleotide CpG has been found to have a special role in the trations below the Km value for the enzyme, the half-life of the genome and in the development of human cancers. Most of the CpGs activated intermediate is increased, destabilizing the amino group in in human DNA are methylated at the 5-position of cytosine, which is the 4-position of the cytosine ring. We have shown previously (27) the only known covalent modification in DNA. Areas of the genome that the bacterial MTase M.Hpall increases the hydrolytic deamina without methylation are clustered as "CpG islands," which are often tion rate of C â€”¿>Uin vitro by a factor of IO4 over background under associated with promoter regions of genes (1,2) and are generally these conditions. Because uracil codes for thymine during replication, methylated on the inactive X chromosome (3) and on imprinted genes unrepaired mismatches would be expected to result in C â€”¿Â»Ttransi (4). Changes in global genomic methylation and methylation patterns tion mutations (Fig. 1, upper pathway). Subsequently, we (16) and are among the most consistent findings in the development of human others (28, 29) have demonstrated the same mutational mechanism for cancers (5-7). This has led to the hypothesis of an epigenetic mech the MTases M.Hhal, M.Sssl, and M.EcoRII in vitro and in vivo, anism for cancer development, in which changes in methylation suggesting that the ability to deaminate the target cytosine is a interfere with expression of proto-oncogenes and tumor suppressor common feature of all MTases, thus offering an alternative pathway genes (8). In addition, involvement of methylation is also the most for creating the high rate of C â€”¿Â»Ttransition mutations at methylation plausible explanation for the high rate of C â€”¿Â»Ttransition mutations sites found in human tumors. at CpG sites, which are frequently found in several human cancers in Low AdoMet concentrations might not only lead to an increase in key genes such as the p53 tumor suppressor gene, especially in colon the numbers of mutations via this pathway but also reduce the overall tumors and in the germline (Li-Fraumeni syndrome; Ref. 9). Five of DNA methylation level, because fewer methyl groups would be six mutational hot spots in the p53 gene are CpG sites; 47% of all available for transmethylation reactions. Interestingly, as mentioned before, the content of 5mC in DNA isolated from various colon Received 11/7/95; accepted 3/14/96. tumors is significantly reduced compared with that in normal tissue (5, The costs of publication of this article were defrayed in pan 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. ' The abbreviations used are: 5mC, 5-methylcytosine; AdoMet. S-adenosylmethionine; 1This work was supported by National Cancer Institute Grant R35 CA49758. AdoHcy. S-adenosylhomocysteine; AdoEt. 5-adenosylethionine; MTase. cytosine (DNA- " To whom requests for reprints should be addressed, at University of Southern 5)-methyltransferase; UDG. uracil-DNA glycosylase; GAPDH. glyceraldehyde-3-phos- California/Morris Cancer Center, 1441 Easttake Avenue. Los Angeles. CA 90033. Phone: phate dehydrogenase; SSCP. single-stranded conformational polymorphism; RT. reverse (213) 764-0816; Fax: (213) 764-0102. transcription; HPLC, high-performance liquid chromatography. 2375

Normal Reactive Premntagenlc MÂ«**Â» B-.P"Â«* Intermediate Lesion

Uracll-DNA Glycosylaae

Fig. I. Proposed mechanisms for the formation of C â€”¿Â»Ttransition mutations at CpG sites induced by methyltransferase and by spontaneous deamination of 5mC and its repair.

Methyltransferase 5mC:G

r.-G mismatch repair

30-32),4 although regions of hypermethylation of CpG islands have methylation in colon cancer specimens and the adjacent normal co- been found (33, 34). In contrast, the overall decrease in 5mC content Ionic epithelium. cannot be explained by reduced MTase activity; in fact, levels of MTase expression are increased in this tissue type (35, 36; see below), MATERIALS AND METHODS and high levels of MTase activity have also been found in various cell lines (37). Further evidence supporting the hypothesis that alterations Colon Specimen. Human colon cancer tissue and surrounding normal in DNA methylation are causally related to colon cancer development mucosa were obtained from colon cancer patients undergoing colectomy. have been provided by several studies on the MTase and AdoMet for Normal mucosa was stripped from fat and muscle tissues. Samples were frozen in liquid nitrogen or ethanol and dry ice within 30 min after colectomy and the development of cancer in rodents. Laird et al. (38) found a kept at -70Â°C until use. Colon carcinomas were at least 1 cm in diameter and reduction in tumor incidence in min~ mice with reduced activity of of high grade and stage. MTase, due to heterozygosity of the MTase gene. In addition, treat Analysis of the Methylation Status ofp53 Codons 248,282, and 283. A ment of rats with the drug 5-azacytidine, which reduces MTase previously described PCR-based methylation assay was modified to determine activity, further reduced the number of polyps detected in these mice. the methylation status at mutational hot spots of the p5J gene (11). Genomic Moreover, overexpression of MTase leads to transformation of NIH/ DNA ( 100 ng) was digested for l h at 37Â°Cwith 10 units of C/oI, Hpall, Rsal, 3T3 cells (39). In another approach, feeding rats folate- or methi- or Mspl. Hpa\\ was used as a methylation-sensitive enzyme for the analysis of onine-deficient diets, precursors in the biosynthesis of AdoMet, led to the methylation status of codons 248 (CGC) and 282 (CGC), and C/01 was used to determine whether codon 283 (CGC) was methylated. The digested lower concentrations of AdoMet in the liver, and the DNA was DNA (10 ng) was PCR amplified using primers that flanked the codons to be hypomethylated within 1 week of treatment (40-42). In addition, the studied. The primers for codon 248 were 5'-GTTGCCTCTGACTGTACCAC- rate of liver cancer has been found to be elevated under these condi CATC-3' and 5'-CAAGTGGCTCCTGAC-3'. The primers for codon 282 and tions. Smith et al. (43) sequenced relevant exons in the p53 gene of 283 were 5'-CCTATCCTGAGTAGTGGT-3' and 5'-TCGCTTAGTGCTC- hepatomas induced by feeding Fischer 344 rats with a choline-defi- CCTGG-3'. The PCR was performed at 94Â°Cfor 75 s, 62Â°Cfor 75 s, and 72Â°C cient diet and found two C â€”¿Â»Ttransitions at CpG sites of nine for 90 s for 26 cycles, which was in the linear range of the assay. The PCR mutations detected. It has been shown that diets low in methionine and products were run on 1.2% agarose gels, transferred to a Zeta probe membrane, folate also increase the risk of colon cancer in humans (44), and and probed with the p53 cDNA (46). Quantitative RT-PCR Analysis of MTase Expression. A quantitative interestingly, many transformed cells are methionine dependent (45). RT-PCR assay was used to measure the level of MTase mRNA. as described These findings are consistent with a model that MTase increases the previously (47). Total mRNA was extracted from normal and cancerous colon rate of transition mutations at CpG sites in genes such as p53, which mucosa. mRNA was reverse transcribed using random hexamers and Molony are critical for cancer development (Fig. 1, upper pathway), and low murine leukemia virus reverse transcriptase (Boehringer Mannheim). The AdoMet concentrations could play a role in inducing a mutator cDNA was then PCR amplified using primers for DNA MTase (5'-GTGC- phenotype. Interestingly, these findings, except for the accumulation GAGACACGATGTC-3' and 5'-TTGTCCAGGATGTTGCCG-.V) and GAPDH (S'-TGAGGCTGTTGTCATACTTCTC-S' and 5'-CAGCCGAGC- of CpG transition mutations, can also be explained by the epigenetic CACATCG-3'). The PCR conditions were 94Â°Cfor 45 s, 55Â°Cfor 30 s, and model of carcinogenesis, and both models are not exclusive. 72Â°for 90 s for 22 and 27 cycles for MTase and GAPDH, respectively. The The above studies all point to alterations in DNA methylation patterns and control of the DNA methylation process as having PCR conditions were optimized to assure that the reaction was in the linear phase of amplification. To assure consistency between experiments, a stock of important potential roles in colon cancer development. Alterations in HeLa cell cDNA was serially diluted and run for each experiment. MTase expression levels, mutations in the MTase gene, and variations Quantitative Analysis of AdoMet and AdoHcy Concentrations in Colon of AdoMet concentrations may contribute not only to changes in DNA Tissue. Colon tissue was homogenized in 2 ml 0.2 N perchloric acid, centri- methylation levels and, therefore, to alterations in gene expression, fuged for 30 min at full speed in an Eppendorf centrifuge, and subsequently but also to enzymatic deamination of cytosine residues at mutational filtered through a 0.2-^.m syringe filter. All operations were performed on ice hot spots (Fig. 1). Therefore, we examined these aspects of DNA or at 4Â°C.AdoEt was added before tissue homogenization as an internal standard. The crude extract was analyzed by ion-pair HPLC using a slightly 4 P. Cheng. C. Schmulte. K. F. Cofer, F. Felix. M. L. Yu. and L. Dubeau. Differential modified method of Wagner et al. (48). A Waters HPLC apparatus equipped with a 440 absorbance detector and a 10-cm Brownlee RP-18 column guarded role of DNA methylation in the development of benign and malignant ovarian tumors, submitted for publication. by a Brownlee Aquaphore ODS precolumn was used. The columns were 2376

Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 1996 American Association for Cancer Research. DNA METHYLATION AND CpG TRANSITION MUTATIONS adjusted to 40Â°C.AdoMet, AdoHcy, and AdoEt were separated by a linear A. gradient, starting with 100% of eluent A [0.1 MNaH2PO4, 2% acetonitrile, and Normal Adenoma Carcinoma 8 mM octadecanoylsulfonic acid (pH 2.65)] and leading in 40 min to eluent B [0.2 M NaH2PO4, 26% acetonitrile, and 8 mM octadecanoylsulfonic acid (pH o re CL. n ro Â£ O. (o Â£ o. 3.25)]. The flow rate was set at 1 ml/min. Absorbance was monitored at 254 O I 2 O I nm. Under these conditions, the retention times of the standard substances u were 18.1 min (AdoMet), 19.6 min (AdoHcy), and 21.5 min (AdoEt). AdoMet and AdoHcy were identified in tissue extracts on the basis of their retention times and cochromatography with reference substances. The purity of the peaks was verified by variation of the mobile phase pH and the concentration of acetonitrile. Standard substances were obtained from Sigma Chemical Co. Mutational Analysis of the Human Methyltransferase cDNA from Hu man Colon Tissues. cDNA from total RNA was prepared as described above. PCR was performed using primers for regions I-III (ml, 5'-CAAGCCTGT- GAGCCGAGCGAGC-3'; m2, 5'-CCAGCCATGACCAGCTTCAGCAG- GATGTT-3'), regions IV-VI (m3, 5'-AAGGGAGACGTGGAGATGCTGT- B. GCG-3'; m4, 5'-GGAGCGCTTGAAGGAGACAAAGTTCCTGA-3'), and Normal Adenoma Carcinoma regions IX and X (m5, 5'-CAGCACAACCGTCACCAACCC-3'; m6, 5'- CAACATACAAAGCTTGATCTCCAAGCCAATG-3'; see Fig. 5). PCR products were analyzed for mutations using a SSCP technique and by direct sequencing. Determination of UDG Activity. We used a reporter plasmid (pSV2neoJ) containing an ampicillin- and a neomycin-resistance gene. An inactivating point mutation in the neomycin-resistance gene was introduced by site-directed Fig. 2. Methylation analysis of codons 248. 282, and 283 of the p53 tumor suppressor mutagenesis, changing GTGC to GCGC, as described previously (16). A U:G gene in human colon tissues. DNA was isolated and incubaled with restriction enzymes mismatch at this site (GUGC) was created by incubating the plasmid with following PCR. using primers that flank codons 248 or 282/283. PCR products were M.Hhal MTase (a generous gift of Dr. Sha Mi, Cold Spring Harbor Labora separated by agarose gel electrophoresis. A, codon 248 (CGG): Cfol cuts outside of the sequence analyzed: Hpall cuts only unmethylated CCGG; and Mspl is insensitive for tory) for 16 h without AdoMet, which leads to deamination of cytosine to methylation at the CpG site. B, codons 282 (CGG) and 283 (CGC): Rsal cuts outside of uracil at the recognition site (16). The plasmid was purified by phenol- the sequence analyzed: and Hpall and Cfol cut only if their recognition sites are chloroform extraction and ethanol precipitated, and 100 ng were incubated unmethylated. with various concentrations of extract from human normal and cancerous colon tissue (23) in 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 2 /xg BSA, and 5 Â¿IM DTT in a total volume of 10 /il. Purified UDG (Boehringer Mannheim) was examined were methylated in the target colon tissue, showing that also serially diluted and added to the experiment as a control. Subsequently, these sites are targeted by MTase. the plasmid was isolated by phenol-chloroform extraction and ethanol precip Analysis of Methyltransferase Expression Levels. We showed itation and electrotransformed into Escherichia coli NR8052 (^[pro-lac], thi, earlier that the levels of active MTase enzyme are often markedly ara, trpE9777, ungi), which are devoid of UDG activity. After incubation on elevated in transformed cell lines relative to their nononcogenic SOB plates containing either ampicillin to score the transformation efficiency counterparts (37). Also, El-Deiry et al. (35) found considerable over- or kanamycin, the reversion frequency was determined by dividing the number expression of the MTase mRNA in colorectal tumors, a situation that of kanamycin-resistant colonies by the number of colonies on the ampicillin might lead to an enhanced rate of MTase-induced cytosine deamina plates. The efficiency of uracil repair was scored by comparing the reversion tion. According to our findings in the in vitro studies with the bacterial frequencies of bacteria transformed with the plasmid, which was incubated MTases M.Hpall and M.Hhal (16, 27), deamination of cytosine at the with colon tissue extracts, with the reversion frequencies obtained from bac teria transformed with plasmids incubated without extracts. target site is dependent on the concentration of MTase and the cofactor AdoMet. Deamination was only measurable when AdoMet concentrations dropped below its Km. To test whether this mechanism RESULTS might contribute to mutations at methylation sites in the human p53 gene, we first measured MTase mRNA expression and AdoMet con Analysis of the Methylation Status of Mutational Hot Spots in centrations in colon carcinoma tissues and normal mucosa. RT-PCR the p53 Gene. Previous studies in this (9, 11) and other laboratories analysis using primers specific for region VIII of MTase (see Fig. 5 (12) have shown that all CpG sites in the p53 gene are methylated in for diagram) and primers specific for GAPDH as a control revealed human WBC, kidney, muscle, bladder, and sperm DNA. We first that MTase expression was increased in six of seven tumors compared confirmed that the CpG dinucleotides at codons 248, 282, and 283, with the normal epithelium from the same patient (Fig. 3). However, which are mutational hot spots in colorectal cancer (49), were meth increases were generally modest and far less than the 60-200-fold ylated in normal colonie mucosa, adenomas, and carcinomas. Fig. 2 differences reported by El-Deiry et al. (35) but were similar to shows a typical analysis with primers flanking codons 248 (Fig. 2A) increases of MTase activity levels reported by Issa et al. (36). Because and 283 (Fig. 25). Methylation analysis of codon 248 showed strong tumor tissue presumably contains a greater percentage of dividing signals in template DNA cut with Cfol, which cuts outside of the cells than normal tissue, it remains to be seen whether increases in sequence analyzed. No signals were generated when the template MTase expression are due to an increase in the proportion of dividing DNA was precut with Mspl, which is insensitive to methylation of the cells or due to an acute increase in MTase expression per individual internal cytosine of the CCGG recognition sequence. On the other cell. hand, the CCGG sequences were methylated in all tissues, because Analysis of AdoMet Concentrations. Ion-pair HPLC was used to strong signals were generated after digestion with Hpall. Similar measure concentrations of AdoMet and the breakdown product results were obtained for codons 282 and 283, which are substrates for AdoHcy in extracts of mucosal and carcinoma tissue samples. To the methylation-sensitive enzymes Hpall and Cfol, respectively (Fig. prevent breakdown of AdoMet, an acidic total cell extract was pre 25). In this case, Rsal, which cuts outside of the sequence analyzed, pared, filtered, and injected directly into the HPLC. AdoEt, which has was used as a control. Therefore, all of the mutational hot spots chemical stability similar to AdoMet under these conditions, was used 2377

Patient from these data that AdoMet is not limiting for methylating DNA by

HeLa cDNA BC MTase in this late stage of tumor development. oÂ°g88NTNTNTNT Mutational Analysis of Human Methyltransferase cDNA. Be cause we found adequate concentrations of AdoMet for DNA methylation in the tumor tissue, we hypothesized that a mutation in the AdoMet-binding pocket of MTase might create an enzyme that po tentially inhibits efficient AdoMet binding. This could prevent effi cient cytosine methylation without reducing the DNA-binding ability and thus increasing the frequency of C â€”¿Â»Ttransitions at the target site. Indeed, we have recently shown that point mutations in the highly conserved domain I of the bacterial MTase M.H/xdl reduce the ability of the enzymes to methylate DNA and also increase the deamination rate at the target C dramatically in vitro and in vivo (50). Therefore, we analyzed these highly conserved regions of the MTase cDNA to test whether a similar mutation in domains coding for the AdoMet-binding pocket of the human MTase, which might lead to a mutator phenotype. could be involved in the mutational process in the colon tumors we analyzed (51, 52). RT-PCR analysis was performed using primers flanking the highly conserved regions I-III, IV-VI, and IX and X of the MTase gene (Fig. 5), which are involved in AdoMet binding and catalysis. Portions of the MTase gene were screened for mutations by SSCP. Analysis of 10 tumor samples showed no mutations by SSCP. To confirm these results, PCR prod

TUMOR TISSUE ucts amplified from these conserved regions using cDNA derived NORMAL MUCOSA from seven colon carcinoma samples as a template were directly ABCDEFG sequenced. No mutations or polymorphisms were detected (data not FACTOR shown). Although the number of samples analyzed was small, the data T/N 1.9 0.7 showed no evidence for a mutated MTase, which might confer a Fig. 3. Expression of methyltransferase mRNA in human colon tissue. Total RNA was isolated from human colon tissue (N. normal: T, tumor) and HeLa cells. RT-PCR was mutator phenotype in human colon cancer. performed with primers specific for methyltransferase (MTase) and GAPDH. respectively, U:G Mismatch Repair. MTase-induced deamination of cytosine and products were separated by agarose gel electrophoresis (A). Results were summarized leads to a premutagenic U:G mismatch. Repair of this mismatch is as the ratio of MTase:GAPDH (ÃŸ). initiated by UDG, which excises uracil, leaving an a basic site. Base as an internal control. The concentrations of both substances were higher in the tumors than in normal tissue, and the concentrations of AdoMet were 8.8 Â±4.4 nmol/g wet tissue in samples from normal mucosa and 30.6 Â±12.3 nmol/g wet tissue in tumor tissue (Fig. 4). LDVESGCG ENVRNFVS QVGNAVPPPL Average AdoHcy concentrations were 4.2 Â±2.0 nmol/g wet tissue in \ EMWD LCGGPPCQGFS RWSVRECARSQGFPDTY normal mucosa and 10.5 Â±7.3 nmol/g wet tissue in tumor tissue. The ratio of AdoMet:AdoHcy was not significantly different comparing Fig. 5. Conserved regions in the catalytic domain of the human MTase cDNA. The catalytic domain starting at an amino acid position of approximately 900 contains 10 normal (2.1 Â± 2.2) and tumor (2.9 Â± 1.7) tissue. Extracts from conserved regions (rectangles). Amino acids thai are involved in AdoMet binding in the mucosal tissue from two patients without cancer showed AdoMet and bacterial MTase M.Hhal (26) by forming hydrogen bonds to this compound are under lined. The catalytic cysteine, which forms a covalent bond to the 5-position of the target AdoHcy concentrations in the same range compared with values in C, is bold. Primers used for RT-PCR were rl and r2; primers for mutational analysis of normal tissue from cancer patients (Fig. 4, / and 2). We conclude the MTase cDNA were ml-m6.

Fig. 4. Concentration of AdoMet and AdoHcy in human colon tissue samples. Extracts from human colon carcinomas and sur rounding mucosa were analyzed for AdoMet and AdoHcy using HPLC. (A-G. tissue from cancer patients: / and 2, tissue from patients without cancer).

TUMOR TISSUE NORMAL MUCOSA

2378

In this study, we focused on mechanisms for the generation of C â€”¿Â» 100 - T mutations and measured parameters that might favor the induction of these mutations during the DNA methylation process by human

80 - MTase (Fig. 1). This pathway, which has been validated for several bacterial MTases (16, 27-29), is particularly attractive, because it is specific for methylation sites and may be triggered by a diet low in

Â£ 60 - methyl group-providing substances. The mechanism is also consistent with the observation that inactivation of MTase and reduction of methylation reduces the incidence of polyp formation in a mouse 40 -J model system (38), and that levels of MTase expression and MTase activity have been reported to be elevated in colorectal tumors (35, 36). Analysis of normal and transformed colonie tissues confirmed 20 - that sites that are hot spots for C â€”¿Â»Tmutations in the p53 tumor suppressor gene are methylated and were, therefore, targeted by MTase. RT-PCR analysis revealed that the average increase of MTase expression was 3.7-fold in colon carcinomas compared with normal 0.0001 0.001 0.01 adjacent mucosa (Fig. 3). These findings are much lower than what has been reported earlier by El-Diery et al. (35). This group found that HgProtein values for MTase mRNA expression ranged up to several hundred Fig. 6. G:U mismatch repair activity in human colon tissue. pSV2Â«ecÃ'plasmiliwith fold higher in human colon cancer tissue than in tissue samples from one U:G mismatch in a kanamycin resistance gene was incubated with human colon tissue patients with benign disorders. Our data are similar to the 5.4-fold extract (A-C, tissue extract from three different patients). Unrepaired, the plasmid is able higher MTase enzyme activity detected by Issa el al. (36) in colon to confer kanamycin resistance to E. coli deficient in UDG. Repair activity was scored by comparing the reversion frequency of bacteria transformed with the plasmid. which was cancers compared with normal mucosa. In hepatomas of LEG rats, incubated with the colon extract, to bacteria transformed with the untreated plasmid (see MTase mRNA expression was also only 2-fold higher compared with "Materials and Methods"). normal liver, and MTase activity was increased approximately 3-fold (64). excision is the rate-limiting step in this repair pathway. Subsequently, The decisive parameter that determines whether the MTase methy- several enzymes, including DNA polymerase ÃŸandDNA ligases, are lates its target cytosine or induces deamination (Fig. 1, upper and involved in restoring the C:G bp (53). We used a genetic assay based lower pathwa\s) is the concentration of AdoMet available to the on the reversion to neomycin resistance to determine the activity of enzyme during the methylation process. In our analysis, AdoMet UDG in colon tissue samples. Site-directed mutagenesis was used to concentrations were increased in some tumors or not significantly create a pSV2Â«Â«/plasmid, in which the sequence GTGC was con changed in carcinoma tissue compared with normal mucosa from the verted to GCGC, inactivating the neomycin resistance conferred by same patient or from patients without cancer. Assuming that the this plasmid and creating a target for M.Hhal. A U:G mismatch was intracellular water content is in the range of 70-85% (65, 66), the formed at this site by incubation of the plasmid with Hluil MTase in average intracellular concentration of AdoMet in normal mucosa can the absence of AdoMet (16, 27). This plasmid was then incubated be calculated to be approximately 11 ^.M, which is similar to that with different concentrations of extracts from normal and cancerous which has been found in rat colon (17 nmol/mg protein; Ref. 67), and tissue and subsequently transfected into bacteria deficient in UDG to average concentrations in the carcinoma tissue were approximately 38 measure U:G mismatch repair efficiencies in human colon tissues. /J.M(Fig. 4). The Km of human MTase from HeLa cells for AdoMet The results for three different tumor samples and adjacent normal has been determined to be 3.25 /UM,and Km values of other eukaryotic mucosa from the same patient showed that repair of U:G mismatches MTases are in the same range (rat, 0.5-6.6 /J.M;Refs. 68 and 69; rice, were very efficient, and no significant difference between normal and 2.6-6 /UM;Ref. 70; and wheat, 5-6 /XM;Ref. 71). Although this is cancerous tissue was found (Fig. 6). Interestingly, we found large approximately 10-100-fold higher than the Km of bacterial MTases interindividual variations in the efficiency of uracil excision. This for AdoMet (24, 72), it is unlikely that extensive induction of cytosine observation has been described previously for human colon and small deamination by MTase is possible under these high concentrations of intestine tissue samples (54). These data, together with our recent AdoMet. In addition, we found that the concentration of AdoHcy that results using an alternative assay (23), indicate that UDG activity is has been shown to inhibit the deamination reaction (27) was higher in not decreased in colon tumors. the tumor tissue as well. It should be noted that a direct comparison of concentration data obtained from tumor and mucosal tissue is DISCUSSION difficult, because mucosal tissue is heterogeneous, and rapidly prolif erating cells are located only in the crypt base (73). Therefore, caution The mechanism by which DNA methylation contributes to the should be used in using MTase expression levels measured for the development of human tumors is currently a subject of intense interest entire mucosa as indicators for MTase levels in colon cells at the bases (55). In particular, the recent observations that widespread methyla of the crypts. Thus, we cannot exclude that cytosine deamination tion changes occur during tumor development (30-32, 34, 56).4 and occurs as a side reaction at very low efficiency and might contribute that mice deficient in MTase activity have a decreased rate of polyp to the burden of DNA repair. Moreover, it has been shown that a diet formation (38), have stimulated interest in this field. Hypomethylation low in methionine, which is a direct precursor in the biosynthesis of of proto-oncogenes (57-59) and hypermethylation of tumor suppres AdoMet, and low in other methyl group-providing factors can reduce sor genes (47, 60, 61) can alter their expression levels, providing an intracellular AdoMet levels and also reduce DNA methylation (40- epigenetic model for tumor development (8, 62). On the other hand, 42). Therefore, it is possible that special conditions in human tissue methylation of cytosine to 5mC leads to a higher rate of C â€”¿Â»T might result in intracellular concentrations of AdoMet below the Km mutations compared with that in unmethylated sites (9, 63), thus of MTase, creating conditions favorable for the induction of cytosine contributing to a genetic pathway of carcinogenesis. deamination (Fig. 1, upper pathway). Because it is not possible to 2379

follow fluctuations of AdoMet or AdoHcy concentrations over time in Rideout. W. M. I.. Coetzee. G. A.. Olumi. A. F.. and Jones. P. A. 5-Methylcytosine tissue during the initial or later stages of tumor development, a as an endogenous mutagen in the human LDL receptor and p53 genes. Science (Washington DC). 249: 1288-1290. 1990. transient shortage of AdoMet might enable the MTase to induce Greenblatt. M. S.. Bennett, W. P.. Hollstein. M., and Harris. C. C. Mutations in the deaminations at its target cytosine in colon tissue. However, the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res.. 54: 4855-4878. 1994. concentrations of AdoMet we measured in the normal colon or tumor Magewu. A. N.. and Jones. P. A. Ubiquitous and tenacious methylation of the CpG tissue exclude the possibility that a continuous shortage of AdoMet site in codon-248 of the pSJ gene may explain its frequent appearance as a mutational caused by a defect in AdoMet synthesis exists in these cells. In hot-spot in human cancer. Mol. Cell. Biol.. 14: 4225-4232. 1994. Tornaletti. S.. and Pfeifer. G. P. Complete and tissue-independent methylation of CpG addition, a drop of AdoMet concentration by a factor of approximately sites in the p53 gene: implications for mutations in human cancers. Oncogene. IO: 4 to concentrations below the Km for MTase would be expected to 1493-1499. 1995. affect all enzymes using this cofactor for transmethylation reactions Bottema. C. D.. Ketterling. R. P.. Vielhaber. E.. Yoon. H. S.. Gostout. B., Jacobson. D. P.. Shapiro. A., and Sommer. S.S. The pattern of spontaneous germ-line mutation: and, thus, would be more cytotoxic than mutagenic for a cell. relative rales of mutation at or near CpG dinucleotides in the factor IX gene. Hum. Recently, we have shown (50) that bacterial MTases carrying Genet.. 91: 496-503, 1993. mutations in the AdoMet-binding pocket, which presumably prevents Schorderet. D. F.. and Gartier. S. M. Analysis of CpG suppression in methylated and nonmethylated species. Proc. Nati. Acad. Sci. USA. 89: 957-961, 1992. efficient binding of the cofactor, can induce C â€”¿>Udeaminations in Sved, J.. and Bird, A. The expected equilibrium of the CpG dinucleotide in vertebrate vitro and in vivo (Fig. 1, upper pathway) at normal concentrations of genome under a mutation model. Proc. Nati. Acad. Sci. USA. 87: 4692-4696. 1990. Yang. A. S., Shen, J. C.. Zingg. J. M., Mi. S.. and Jones. P. A. Hhal and Hpall DNA AdoMet, even in the presence of a functional UDG repair system. methyltransferases bind DNA mismatches, methylate uracil and block DNA repair. Therefore, we screened tumors for the presence of similar mutations Nucleic Acids Res.. 23: 1380-1387. 1995. in the AdoMet-binding pocket of the human MTase gene. The fact that Spruck. C. H.. Rideout. W. M.. and Jones. P. A. DNA methylation and cancer. In: 1. P. Jost and H. P. Saluz (eds.), DNA Methylation: Molecular Biology and Biological such mutations were not observed in the colon tumors argues against Significance, pp. 487-509. Basel: BirkhÃ¤userVerlag. 1993. this mechanism being of importance in colon carcinogenesis. 18. Coulondre. C., Miller, J. H., Farabaugh. P. J., and Gilbert. W. Molecular basis of base An additional factor determining whether a MTase-induced deami- substitution hotspots in Escherichia coli. Nature (Lond.). 274: 775-780, 1978. 19. Wang, R. Y.. Kuo. K. C.. Gehrke. C. W.. Huang. L. H.. and Ehrlich. M. Heat- and nation reaction contributes to the generation of C â€”¿Â»Tmutationsis the alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA. Bio- ability of cells to repair U:G mismatches generated by this process. chim. Biophys. Acta. 697: 371-377. 1982. 20. Shen. J. C.. Rideout. W. M., and Jones. P. A. The rate of hydrolytic deamination of Therefore, we measured the ability of colon extracts to repair these 5-methylcytosine in double-stranded DNA. Nucleic Acids Res.. 22: 972-976. 1994. mismatches. Excision of uracil was efficient in all samples tested (Fig. 21. Lindahl. T. Instability and decay of the primary structure of DNA. Nature (Lond.). 6; Ref. 23). Moreover, UDG expression has been shown to be max 362: 709-715. 1993. 22. Shenoy, S.. Ehrlich. K. C.. and Ehrlich. M. Repair of thymine.guanine and uracil.- imal in S-phase (74, 75), when MTase activity peaks. Although it is guanine mismatched base-pairs in bacteriophage M13mpl8 DNA heteroduplexes, J. possible that MTase forms a tight complex with the U:G mismatch Mol. Biol.. Â¡97:617-626, 1987. formed by the deamination reaction, or that a small fraction of uracil 23. Schmutte. C.. Yang. A. S.. Bean. R. W.. and Jones. P. A. Base excision repair of U:G mismatches at a mutational hotspot in the Â¡i53gene is more efficient than base gets methylated by MTase, thus escaping repair by UDG (16, 76), the excision repair of T:G mismatches in extracts of human colon tumors. Cancer Res.. high efficiency of UDG repair found in the human colon extracts 55: 3742-3746, 1995. 24. Wu, J. C., and Santi, D. V. Kinetic and catalytic mechanism of Hhal methyltrans- makes it unlikely that a U:G mismatch persists in DNA until replica ferase. J. Biol. Chem.. 262: 4778-4786. 1987. tion completes the C â€”¿Â»Tmutation. 25. Chen. L.. MacMillan. A. M.. Chang. W.. Ezaz, N. K.. Lane, W. S.. and Verdine, G. L. None of the parameters tested suggest that biochemical conditions Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyl- favor a MTase-induced pathway for the formation of C â€”¿Â»Uâ€”¿>T transferase. Biochemistry, 30: 11018-11025, 1991. 26. Klimasauskas. S.. Kumar. S., Roberts, R. J.. and Cheng. X. Hhal methyltransferase transitions in human tissue (Fig. 1, upper pathway). Spontaneous flips its target base out of the DNA helix. Cell. 76: 357-369, 1994. deamination of 5mC, therefore, is the most likely mechanism by 27. Shen. J. C.. Rideout. W. M.. and Jones. P. A. High-frequency mutagenesis by a DNA methyltransferase. Cell. 71: 1073-1080. 1992. which the majority of these mutations are formed (Fig. 1, lower Wyszynski. M.. Gabbara, S., and Bhagwat, A. S. Cytosine deaminations catalyzed by pathway). Nevertheless, it is difficult to exclude the possibility that DNA cytosine methyltransferases are unlikely to be the major cause of mulalional hot low-efficiency side reactions of enzymatic methylation do not con spots at sites of cytosine methylation in Escherichia cali. Proc. Nati. Acad. Sci. USA, 91: 1574-1578. 1994. tribute to the generation of these mutations. The mechanisms by 29. Bandaru. B.. Wyszynski. M.. and Bhagwat. A. S. Hpall methyltransferase is muta which methyl-deficient diets contribute to carcinogenesis and the genic in Escherichia coli. J. Bacteriol.. 177: 2950-2952. 1995. 30. Goelz. S. E.. Vogelstein, B.. Hamilton. S. R., and Feinberg, A. P. Hypomethylation reason for the decreased number of polyps formed in transgenic mice of DNA from benign and malignant human colon neoplasms. Science (Washington with lower MTase levels (38) may, therefore, be best explained by an DC), 22Â«:187-190, 1985. epigenetic pathway. 31. Feinberg. A. P.. Gehrke, C. W.. Kuo. K. C.. and Ehrlich. M. Reduced genomic 5-methylcytosine content in human colonie neoplasia. Cancer Res.. 48: 1159-1161. 1988. 32. Diala. E. S.. Cheah. M. S., Rowith, D., and Hoffman. R. M. Extent of DNA REFERENCES methylation in human tumor cells. J. Nati. Cancer Inst., 71: 755-764, 1983. 33. de Bustros. A.. Nelkin, B. D.. Silverman. A.. Ehrlich. G.. Poiesz. B.. and Baylin. S. B. 1. Bird. A. P. CpG-rich islands and the function of DNA methylation. Nature (Lond.), The short arm of chromosome 11 is a "hot spot" for hypermethylation in human 321: 209-213, 1986. neoplasia. Proc. Nati. Acad. Sci. USA, Â«5:5693-5697. 1988. 2. Gardiner-Garden. M.. and Frommer. M. CpG islands in vertebrate genomes. J. Mol. 34 Makos. M.. Nelkin, B. D.. Lerman. M. I.. Latif, F., Zbar. B., and Baylin, S. B. Distinct Biol.. 196: 261-282. 1987. hypermethylation patterns occur at altered chromosome loci in human lung and colon 3. Riggs. A. D.. and Pfeifer. G. P. X-chromosome inactivation and cell memory. Trends cancer. Proc. Nati. Acad. Sci. USA, 89: 1929-1933. 1992. Genet.. 8: 169-174, 1992. 35. El-Deiry, W. S., Nelkin. B. D.. Celano. P.. Yen, R. W.. Falco. J. P.. Hamilton, S. R., 4. Bartolomei. M. S.. Zenel. S., and Tilghman, S. M. Parental imprinting of the mouse and Baylin, S. B. High expression of the DNA melhyltransferase gene characterizes H19 gene. Nature (Lond.), 365: 764-767, 1991. human neoplastic cells and progression stages of colon cancer. Proc. Nati. Acad. Sci. 5. Gama-Sosa, M. A., Slagel, V. A., Trewyn, R. W., Oxenhandler, R., Kuo, K. C. USA. 88: 3470-3474. 1991. Gehrke. C. W., and Ehrlich, M. The 5-methylcytosine content of DNA from human 36. Issa, J. P., Venino. P. M.. Wu, J.. Sazawal, S., Celano, P.. Nelkin. B. D.. Hamilton. tumors. Nucleic Acids Res.. //: 6883-6894. 1983. S. R., and Baylin, S. B. Increased cytosine DNA-methyltransferase activity during 6. Baylin. S. B.. Hoppener, J. W., de Bustros, A., Steenbergh. P. H.. Lips, C. J.. and colon cancer progression. J. Nat!. Cancer Inst., 85: 1235-1240, 1993. Nelkin. B. D. DNA methylation patterns of the calcitonin gene in human lung cancers 37. Kautiainen. T. L., and Jones. P. A. DNA methyltransferase levels in tumorigenic and and lymphomas. Cancer Res.. 46: 2917-2922. 1986. nontumorigenic cells in culture. J. Biol. Chem.. 26/: 1594-1598. 1986. 7. Baylin, S. B., Makos, M., Wu. J. J., Yen. R. W., de Bustros, A., Venino, P.. and 38. Laird. P. W., Jacksongrusby. L., Fazeli, A.. Dickinson. S. L.. Jung, W. E.. Li, E.. Nclkin. B. D. Abnormal patterns of DNA melhylation in human neoplasia: potential Weinberg. R. A., and Jaenisch. R. Suppression of intestinal neoplasia by DNA consequences for tumor progression. Cancer Cells (Cold Spring Harbor), 3: 383-390, hypomethylation. Cell, 81: 197-205. 1995. 1991. 39. Wu. J.. Issa. J. P.. Herman, J.. Bassett. D. J.. Nelkin. B. D.. and Baylin, S. B. 8. Jones. P. A., and Buckley. J. D. The role of DNA methylation in cancer. Adv. Cancer Expression of an exogenous eukaryotic DNA methyltransferase gene induces trans Res., 54: 1-23, 1990. formation of NIH 3T3 cells. Proc. Nati. Acad. Sci. USA. 90: 8891-8895, 1993. 2380

40. Wainfan, E., and Poirier. L. A. Methyl groups in carcinogenesis: effects on DNA 667-672,lation of the 1992. c-mvc ' gene in human colorectal cancer progression. Br. J. Cancer., 65: methylation and gene expression. Cancer Res., 52 (Suppl.).- 2071s-2077s. 1992. 41. Christman. J. K., Sheikhnejad. G.. Dizik, M.. Abileah, S.. and Wainfan. E. Revers 59. Bhave, M. R., Wilson, M. J.. and Poirier, L. A. c-H-ras and c-K-ra.v gene hypo ibility of changes in nucleic acid methylation and gene expression induced in rat liver methylation in the livers and hepatomas of rats fed methyl-deficient, amino acid- by severe dietary methyl deficiency. Carcinogenesis (Lond.), 14: 551-557, 1993. defined diets. Carcinogenesis (Lond.), 9: 343-348. 1988. 42. Pogribny. I. P., Basnakian, A. G., Miller, B. J.. Lopalina. N. G., Poirier. L. A., and 60. Merlo. A.. Herman, J. G., Mao, L.. Lee, D. J., Gabrielson, E.. Burger. P., Baylin. S. B., and Sidransky, D. 5' CpG island methylation is associated with transcriptional James, S. J. Breaks in genomic DNA and within the p53 gene are associated with hypomethylation in livers of folate/methyl-deficient rats. Cancer Res.. 55: 1894- silencing of the tumour suppressor pl6/CDKN2/MTSl in human cancers. Nat. Med., 1901. 1995. /: 686-692. 1995. 43. Smith, M. L.. Yeleswarapu, L.. Scalamogna, P.. Locker, J., and Lombardi, B. p53 61. Herman, J. G., Merlo. A.. Lapidus. R. G.. Issa, J-P. J.. Davidson. N. E.. Sidransky, D., mutations in hepatocellular carcinomas induced by a choline-devoid diet in male and Baylin, S. B. Inactivation of the CDKN2/pl6/MTSI gene is frequently associated Fischer 344 rats. Carcinogenesis (Lond.), 14: 503-510, 1993. with aberrant DNA methylation in all common human cancers. Cancer Res., 55: 44. Giovannucci, E., Rimm, E. B., Ascherio, A., Stampfer. M. J., Colditz, G. A., and 4525-4530, 1995. Willett, W. C. Alcohol, low-methionine-low-folate diets, and risk of colon cancer in 62. Greger, V.. Passarge, E.. Hopping, W., Messmer. E., and Horsthemke. B. Epigenetic men. J. Nati. Cancer Inst.. 87: 265-273, 1995. changes may contribute to the formation and spontaneous regression of retinoblas- 45. Coalson. D. W.. Mechara. J. O., Stern, P. H.. and Hoffman. R. M. Reduced avail toma. Hum. Genet., 83: 155-158, 1989. ability of endogenously synthesized methionine for S-adenosylmethionine formation 63. Duncan, B. K.. and Miller, J. H. Mutagenic deamination of cytosine residues in DNA. in melhionine-dependent cancer cells. Proc. Nati. Acad. Sci. USA, 79: 4248-4251, Nature (Lond.). 287: 560-561. 1980. 1982. 64. Miyoshi. E., Jain, S. K., Sugiyama, T., Fujii, J.. Hayashi, N., Fusamoto. H.. Kamada, 46. Feinberg. A. P.. and Vogelstein. B. A technique for radiolabelling DNA restriction T., and Tanaguchi, N. Expression of DNA methyltransferase in LEC rat during endonuclease fragments to high specific activity. Anal. Biochem., 132: 6-13, 1983. hepatocarcinogenesis. Carcinogenesis (Lond.), 14: 603-605, 1993. 47. Gonzalez-Zulueta, M., Bender. C. M.. Yang, A. S., Nguyen, T. T.. Bear!, R. W., Van 65. Wheatley, D. N., Inglis, M. S., Foster, M. A., and Rimington, J. E. Hydration, volume Tornout. J. M.. and Jones, P. A. Methylation of the 5' CpG island of the pl6/CDKN2 changes and nuclear magnetic resonance proton relaxation times of HeLa S-3 cells in tumor suppressor gene in normal and transformed human tissues correlates with gene M-phase and the subsequent cell cycle. J. Cell Sci., 88: 13-23, 1987. silencing. Cancer Res.. 55.- 4531-4535, 1995. 66. Hoffman, D., Kumar, A. M.. Spitzer. A., and Gupta. R. K. NMR measurement of 48. Wagner, J., Claverie, N., and Danzin, C. A rapid high-performance liquid Chromato intracellular water volume in rat kidney proximal tubules. Biochim. Biophys. Acta, graphie procedure for the simultaneous determination of methionine, ethionine, 889: 355-360, 1986. S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues. 67. Halline, A. G., Dudeja, P. K.. and Brasitus, T. A. 1,2-Dimethylhydrazine-induced Anal. Biochem.. 140: 108-116, 1984. premalignant alterations in the S-adenosylmethionine/S-adenosylhomocysteine ratio 49. Harris, C. C., and Hollstein, M. Clinical implications of the p53 tumor-suppressor and membrane lipid lateral diffusion of the rat distal colon. Biochim. Biophys. Acta. gene. N. Engl. J. Med., 329: 1318-1327, 1993. 944: 101-107, 1988. 50. Shen. J. C., Zingg, J. M., Yang, A. S., Schmutte. C.. and Jones. P. A. A mutant DNA 68. Arnaud, M., Dante, R., and Niveleau, A. DNA methyltransferases in normal and avian (cytosine-5)-methyltransferase functions as a mutator enzyme. Nucleic Acids Res., sarcoma virus-transformed rat cells. Quantitation of 5-methyldeoxycytidine in DNA 32: 4275-4282, 1995. and enzyme kinetics study. Biochim. Biophys. Acta, 826: 108-112, 1985. 51. Kumar, S., Cheng, X., Klimasauskas, S., Mi, S., Posfai, J., Roberts, R. J., and Wilson, 69. Ruchirawat, M., Becker, F. F., and Lapeyre, J. N. Indistinguishable physical and G. G. The DNA (cytosine-5) methyltransferases. Nucleic Acids Res., 22: 1-10, 1994. catalytic properties of DNA methyltransferase from normal rat liver and a transplant- 52. Yen, R. W., Venino, P. M., Nelkin, B. D., Yu, J. J., El-Deiry. W., Cumaraswamy, A., able rat hepatocellular carcinoma. Carcinogenesis (Lond.), 6: 877-882, 1985. Lennon. G. G.. Trask, B. J., Celano, P., and Baylin. S. B. Isolation and characteriza 70. Giordano. M.. Mattachini. M. E.. Cella. R.. and Pedrali, N. G. Purification and tion of the cDNA encoding human DNA methyltransferase. Nucleic Acids Res., 20: properties of a novel DNA methyltransferase from cultured rice cells. Biochem. 2287-2291, 1992. Biophys. Res. Commun., 777: 711-719, 1991. 53. Singhal, R. K., Prasad, R., and Wilson, S. H. DNA polymerase ÃŸconducts the 71. Theiss, G., Schleicher, R., Schimpff, W. G.. and Follmann. H. DNA methylation in gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear wheat. Purification and properties of DNA methyltransferase. Eur. J. Biochem.. /67: extract. J. Biol. Chem., 270: 949-957, 1995. 89-96. 1987. 54. Myrnes, B., Giercksky, K. E., and Krokan, H. Intel-individual variation in the activity 72. Rubin. R. A., and Modrich, P. EcoRI methylase. J. Biol. Chem.. 252: 7265-7272, of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase. Carcino 1977. genesis (Lond.), 4: 1565-1568, 1983. 73. O'Brien. M. J., O'Keane. J. C., Zauber. A., Gottlieb. L. S.. and Winawer. S. J. 55. Balmain, A. Exploring the bowels of DNA methylation. Curr. Biol.. 5: 1013-1016. Precursors of colorectal carcinoma. Biopsy and biologic markers. Cancer (Phila.). 70: 1995. 1317-1327. 1992. 56. Feinberg. A. P.. and Vogelstein, B. Hypomethylation distinguishes genes of some 74. Slupphaug, G., Olsen, L. C., Heiland. D.. Aasland. R., and Krokan, H. E. Cell cycle human cancers from their normal counterparts. Nature (Lond.), 301: 89-92, 1983. regulation and in vitro hybrid arrest analysis of the major human uracil-DNA 57. Munzel, P. A., Pfohl, L. A., Rohrdanz, E., Keith, G., Dirheimer, G., and Bock, K. W. glycosylase. Nucleic Acids. Res.. 19: 5131-5137, 1991. Site-specific hypomethylation of c-mvc protooncogene in liver nodules and inhibition 75. Weng, Y., and Sirover. M. A. Developmental regulation of the base excision repair of DNA methylation by Â¿V-nitrosomorpholine. Biochem. Pharmacol., 42: 365-371, enzyme uracil DNA glycosylase in the rat. MutÃ¢t.Res., 293: 133-141, 1993. 1991. 76. Klimasauskas, S., and Roberts, R. J. M. Hhal binds tightly to substrates containing 58. Sharrard, R. M., Royds. J. A., Rogers, S., and Shorthouse, A. J. Patterns of methy mismatches at the target base. Nucleic Acids Res.. 23: 1388-1395. 1995.

2381

Christoph Schmutte, Allen S. Yang, TuDung T. Nguyen, et al.

Cancer Res 1996;56:2375-2381.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/10/2375

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/56/10/2375. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.