O6-Methylguanine DNA methyltransferase(MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-Methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at rasgenes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35%(55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation(P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.
Although ras mutation is the most common oncogenic alteration in human cancer (1, 2), the incidence of K-ras activation varies widely among carcinomas. K-ras mutation is rare in human primary breast carcinomas but occurs in approximately one-half of colorectal carcinomas. ras oncogenes are transforming after single-point mutations within their coding sequences. Mutations in naturally occurring ras oncogenes have been localized in codons 12, 13, 59, and 61. Alteration of codon 12, GGT, is the most common change. Substitution of the wild-type glycine 12 by any other amino acid results in oncogenic activation of this molecule (1, 2).
The mutational spectrum of critical genes in cancer is often not completely random. The tumor suppressor p53, the most commonly mutated gene in human cancer, commonly undergoes C to T changes from deamination of methylated cytosines (3). However, in the case of K-ras, the absence of a CpG dinucleotide at codon 12 prevents this alteration on either strand. In contrast, K-ras mutations often result from G to A transitions. In animal models, chemical carcinogens can target ras oncogene sequences. Some modified bases are highly mutagenic because of their miscoding properties, as is the case for O6-methylguanine, an adduct produced by several carcinogens, including N-MNU.3O6-methylguanine is read as adenine by DNA polymerases, thus leading to the frequent generation of G to A transitions in K-ras (4). Avoidance of this mutagenic effect is directly related to the presence of a functional DNA repair protein, MGMT (5). MGMT removes alkyl groups, as well as larger adducts involving chloroethylations, at the O6 position of guanine in a reaction that inactivates one MGMT molecule for each lesion repaired. In vitro assays also show that endogenous MGMTexpression protects mammalian cell lines from spontaneous G:C to A:T transitions in the aprt gene (6). Further insight into the role of MGMT as a keeper of genome integrity comes from animal models. Transgenic mice overexpressing MGMT are protected against O6-methylguanine-DNA adducts caused by MNU (7) and against G to A mutations in K-rasin aberrant colorectal crypt foci (8) and lung tumors(9) in mice. No studies connecting these events in human cancer have been reported.
Genetic defects in MGMT have not been found in cancer, but hypermethylation of the MGMT CpG island as the cause of MGMT transcriptional silencing in cell lines defective in O6-methylguanine repair has been recognized (10, 11, 12, 13). Recently, we have reported that the MGMT gene is epigenetically inactivated by promoter hypermethylation in many primary tumor types with specific patterns(12). Although this change is not present in breast carcinomas, in which K-ras mutations are extremely rare, it occurs in approximately 40% of cases of colorectal carcinomas associated with the loss of MGMT expression (12, 14) and is also frequent in non-small cell lung carcinoma (12). In the present study, we examined whether the epigenetic silencing of MGMT might be linked to the presence of K-rasmutations in human colorectal tumorigenesis. We determined the MGMT methylation status and the presence and type of K-ras mutations in codons 12 and 13 in a large collection of primary colorectal carcinomas and adenomas. Our data show that MGMT promoter hypermethylation is an early event in human colorectal tumorigenesis, independent of the aberrant methylation of other genes, and linked to the appearance of G to A mutations in the K-ras oncogene.
Materials and Methods
Initially, 79 colorectal carcinomas and 65 colorectal adenomas were obtained from surgical resection specimens of surgical patients at the Johns Hopkins Hospital. Specimen collection procedures were approved by the Joint Committee on Clinical Investigation (Institutional Review Board) of the Johns Hopkins University School of Medicine. Subsequently, 120 additional colorectal carcinoma samples were obtained from surgical patients at the Hospital de Sant Pau in Barcelona between July 1991 and July 1993 under the supervision of Dr. Gabriel Capella. The study protocol was approved by the Ethics Committee. This last set of samples has been previously characterized for K-ras and p53 mutations (15) and p14ARFpromoter hypermethylation (16). All of the samples were frozen in liquid nitrogen immediately after resection and stored at−70°C until processing. DNA was extracted by standard methods.
DNA methylation patterns in the CpG island of the MGMT gene were determined by chemical modification of the unmethylated, but not the methylated, cytosines to uracil, and subsequent PCR using primers specific for either the methylated or the modified unmethylated DNA(17). Primer sequences of MGMT were for the unmethylated reaction 5′-TTT GTG TTT TGA TGT TTG TAG GTT TTT GT-3′(upper primer) and 5′-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3′ (lower primer) and for the methylated reaction 5′-TTT CGA CGT TCG TAG GTT TTC GC-3′ (upper primer) and 5′-GCA CTC TTC CGA AAA CGA AAC G-3′ (lower primer). The annealing temperature was 59°C. Placental DNA treated in vitro with Sss I methyltransferase (New England Biolabs)was used as positive control for methylated alleles of MGMT,and DNA from normal lymphocytes was used as negative control for methylated alleles of MGMT.
Briefly, 1 μg of DNA was denatured by NaOH and modified by sodium bisulfite. DNA samples were then purified using Wizard DNA purification resin (Promega), again treated with NaOH, precipitated with ethanol,and resuspended in water. Controls without DNA were performed for each set of PCR. Ten μl of each PCR reaction was directly loaded onto nondenaturing 6% polyacrylamide gels, stained with ethidium bromide,and visualized under UV illumination.
RT-PCR was performed as described previously (16), using 3μg of total cellular RNA to generate cDNA. One hundred ng of this cDNA were amplified by PCR with primers for exon 5′-GCC GGC TCT TCA CCA TCC CG-3′ and exon 5′-GCT GCA GAC CAC TCT GTG GCA CG- 3′ of MGMT, which amplify a 211-bp product spanning sequence 339–527 from GenBank accession number M29971. RT-PCR for GAPDH served as a positive control (16). Ten μl of each PCR reaction was directly loaded onto nondenaturing 6% polyacrylamide gels, stained with ethidium bromide, and visualized under UV illumination. The colorectal cancer cell lines HT-29 and RKO, unmethylated at the MGMT promoter (12, 13), and MGMTproficient were used. The colorectal cell line SW48, hypermethylated at the MGMT promoter that lacks MGMT expression,(12) was used as negative control.
Detection of K-ras Mutations.
Mutations at codons 12 and 13 of the K-ras gene were detected and characterized by an artificial RFLP/PCR approach(18) and mutant allele-specific amplification(19). Mutations were confirmed by direct cycle sequencing of the PCR products using the AmpliCycle Sequencing kit (Perkin-Elmer,Branchburg, NJ).
MGMT Promoter Hypermethylation and the Occurrence and Type of K-ras Mutations.
Among 244 colorectal lesions, 179 carcinomas, and 65 adenomas, 103(42%) demonstrated hypermethylation of MGMT. Representative examples of the methylation analysis in colorectal carcinomas and adenomas are shown in Fig. 1 A. Of these 244 samples, 88 (36%) had mutant K-ras. These two events were related because 48 (55%) of the 88 tumors with mutant K-ras had methylated MGMT, whereas only 55 (35%) of the 156 tumors without K-ras mutations had methylated MGMT (Fischer’s exact test, two-tailed, P = 0.003).
One explanation for this association might be that K-rasalterations predispose to hypermethylation of genes including MGMT because ras activity may lead to increases in DNA methyltransferase (20). To address this issue, we have also examined the methylation of other genes hypermethylated in colorectal cancer. K-ras mutations were independent of the promoter methylation status of the tumor suppressor genes p16INK4a or p14ARF, previously analyzed in a subset of these tumors (16). MGMT promoter hypermethylation was also not associated with aberrant methylation of either p16INK4a or p14ARF (16), showing the independence of these events.
Alternatively, an association between MGMT promoter hypermethylation and K-ras mutations could result from epigenetic inactivation of MGMT leading to K-rasmutation through the lack of O6-methylguanine repair. This relationship would be predicted to result in predominantly G to A transitions. Our data clearly support this latter mechanism: 71% (36 of 51) of tumors with G to A mutation in K-ras showed MGMT epigenetic inactivation, whereas only 32% (12 of 37)of the tumors with other K-ras mutations and 35% (55 of 156) of those without K-ras mutations exhibited aberrant methylation of the MGMT promoter. K-ras G to A transitions in codon 12 versus codon 13 had a similar distribution of MGMT aberrant methylation, 69% (29 of 42) versus 78% (7 of 9), respectively. Fig. 1 B displays in a graphic way the distribution of MGMT promoter hypermethylation according to the K-ras status. The frequency of MGMT methylation in the G to A mutation group was statistically different from tumors with non-G to A K-ras mutations (Fischer’s exact test two-tailed, P < 0.0005) or tumors without K-ras mutations (Fischer’s exact test two-tailed, P < 0.0001). Thus, MGMTmethylation is tightly linked only to the presence of G to A mutations in K-ras and not to other K-ras mutations.
Timing of MGMT Hypermethylation-associated Inactivation.
To further explore the role for MGMT deficiency in the genesis of G to A K-ras mutations, we examined the timing of both events. If the inactivation of MGMT leads to G to A mutations in K-ras, it would be expected that the loss of MGMT expression would precede K-ras alterations in colorectal tumorigenesis. Although MGMT promoter hypermethylation has been associated with a loss of MGMTfunction in primary colorectal carcinomas (12, 14) and cancer cell lines (10, 11, 12, 13), we confirmed this tight correlation examining the expression of MGMT using RT-PCR in colorectal adenomas. Ten colorectal adenomas without MGMTmethylation expressed high levels of MGMT mRNA, whereas 19 polyps with MGMT methylation expressed very little(n = 5) or no detectable MGMT mRNA(n = 14; Fig. 1,C). Thus, just as in the invasive tumors, transcriptional loss was associated with MGMT hypermethylation (12, 14). To further demonstrate that the promoter methylation leads to loss of MGMT expression, the treatment of the colorectal cell line SW48—methylated at MGMT and without MGMTexpression—with the demethylating agent 5-aza-2′-deoxycytidine restored MGMT expression (Fig. 1 C).
Within the colorectal lesions described above, we then investigated these changes in relation to stage of the disease. MGMTmethylation was present in equal frequency in small adenomas <1 cm (9 of 21, 43%), large adenomas ≥1 cm (23 of 44, 52%), and carcinomas(71 of 179, 40%), which suggests that this change occurs early in neoplastic progression. However, in large adenomas and carcinomas,hypermethylation of MGMT was associated with G to A mutations in K-ras (9 of 12 large adenomas and 27 of 39 carcinomas with G to A mutations had MGMT methylation),whereas in small adenomas, no G to A mutations were observed, even in the 9 adenomas with MGMT methylation. Thus, MGMTpromoter hypermethylation (and its consequence, MGMTtranscriptional inactivation) is an early event in colorectal tumorigenesis, often appearing before the development of large adenomas, and this event precedes the appearance of K-rasmutations in colorectal tumors.
A large amount of information concerning the genetic alterations found in human cancer has been compiled in recent years. However, we still know very little about the causes behind the DNA instability and enhanced mutational rates of a cancer cell. For the tumor suppressor p53, the deamination of CpG dinucleotides has been established as one of its important mutagenic events (8). Other genes with potential relevant roles in cancer, such as BAX or TGFBRII, suffer very specific mutations in sporadic tumors with compromised mismatch repair (21, 22). Silencing of the DNA repair gene hMLH1 by promoter hypermethylation is the probable event behind these defects in colorectal and endometrial cancer (23, 24, 25). In this report, we provide another example of the interaction between epigenetic and genetic events in human cancer. Our data suggest that epigenetic silencing of the DNA repair gene MGMT by hypermethylation is strongly associated with, and precedes, G to A mutations in K-ras in colorectal tumorigenesis. This change would result from persistence of the promutagenic adducts in the O6 position of guanine that are not repaired by MGMT. Alkylating agents causing the promutagenic lesion may be provided from dietary nitrates reduced in the proximal colon by bacteria, by nitrosation of amines and amides derived of protein catabolism (26, 27, 28). A previous study had suggested that loss of enzyme activity in the normal mucosa was found in patients with G to A mutations in K-ras but could not make this association in tumors (29). However, in our study, the temporal relationship between inactivation of MGMT and the appearance of K-ras mutation in tumors with MGMT inactivation suggests a strong link between these two events.
It is of interest to take note of the tumor types for which MGMT promoter hypermethylation is a frequent event. It is known that in mouse models, the mammary tumors induced by MNU harbor characteristically G to A mutations in H-ras(30). Strikingly, the absence of K-rasmutations is a common feature of sporadic human breast carcinomas. Concordant with these phenomena, MGMT is not inactivated by promoter hypermethylation in breast tumors (12). On the other hand, for example, colorectal and non-small cell lung carcinomas have both changes: K-ras mutations (1, 2) and MGMT promoter hypermethylation (12). An interesting case arises from the brain tumors. Among the glial tumors, MGMT epigenetic inactivation (12) and MGMT loss or reduced activity (31, 32) are common features, but ras mutations are infrequent. These data may reflect a different spectrum of carcinogen exposure, i.e., O6-ethylguanine is removed faster than the O6-methylguanine that prevents ras mutations (33), or a different target gene more important for the biology of brain tumors than K-rasmutations. Thus, whereas the present study comments solely on G to A transitions in the K-ras gene, one important aspect of our data concerns the fact that each guanine in the human genome may generate a promutagenic lesion in the absence of correct repair. Other genetic alterations and mutations in target genes in human cancer that might be induced by MGMT epigenetic inactivation should receive close attention, and a search for these changes should be the focus of future investigations.
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.
M. E. is a recipient of a Spanish Ministerio de Educacion y Cultura Award.
The abbreviations used are: MNU,methylnitrosourea; MGMT, O6-methylguanine DNA methyltransferase; RT-PCR, reverse transcription-PCR; GAPDH,glyceraldehyde-3-phosphate dehydrogenase.
We thank Bert Vogelstein for the review of the manuscript.