Smoking-related increase of O6-methylguanine-DNA methyltransferase expression in human lung carcinomas

Eighty-three non-small cell lung carcinomas (NSCLC) of previously untreated patients were analysed for expression of O6-methylguanine-DNA methyltransferase (MGMT) by means of immunohistochemistry. Expression of MGMT was detected in 62 of 83 tumours (75%). There was a significant difference in MGMT staining between smokers and non-smokers (P = 0.001). Tumours of smokers expressed more frequently MGMT than tumours of non-smokers. There was a trend of MGMT expression to be higher in tumours of patients smoking >20 cigarettes/day than patients smoking <20 cigarettes/day. Abstinence from smoking resulted in a significant decrease in MGMT expression (lin reg r = -0.59, P < 0.05). These results demonstrate that MGMT expression in human lung carcinomas is influenced by smoking habits of the patients.      

O6-methylguanine-DNA methyltransferase gene: epigenetic silencing and prognostic value in head and neck squamous cell carcinoma.

BACKGROUND: Alkylating N-nitroso compounds can interact directly with DNA, forming O(6)-alkylguanine, a DNA adduct proved to be mutagenic and carcinogenic if not sufficiently repaired. A specific DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT), can remove the alkyl group from the O(6)-position of the guanine, thereby preventing its mutagenic and carcinogenic effects. Inactivation of the MGMT gene in association with promoter hypermethylation results in persistence of O(6)-alkylguanine in DNA, leading to G:C to A:T transition mutation and these G:C to A:T transition mutations can inactivate p53 tumor suppressor gene or activate ras proto-oncogene. METHODS: We analyzed MGMT promoter hypermethylation and protein expression patterns in 94 cases of primary head and neck squamous cell carcinoma (HNSCC) by methylation-specific PCR (MSP) and immunohistochemical staining. The results were then correlated with clinical follow-up data. RESULTS: MGMT promoter hypermethylation was present in 17 of 94 patients (18.1%) and apparent loss of protein expression was seen in 19 of 93 HNSCC patients (20.4%). The presence of MGMT promoter hypermethylation was significantly correlated with loss of MGMT protein expression in HNSCC. Both MGMT promoter hypermethylation and loss of protein expression were significantly correlated to increased tumor recurrences and decreased patient survival, independent of other risk factors, such as tumor site, tumor size, nodal status, age, and chemoradiation therapy. CONCLUSIONS: MGMT promoter hypermethylation and apparent loss of protein expression are reliable and independent prognostic factors in HNSCC. The above study may also provide guideline or basis for applying alkylating antitumor agents to patients with HNSCC that display MGMT promoter hypermethylation and/or loss of MGMT protein expression.     

Promoter hypermethylation and inactivation of O(6)-methylguanine-DNA methyltransferase in esophageal squamous cell carcinomas and its reactivation in cell lines

The purpose of this study was to characterize the role of DNA hypermethylation in the loss of expression of O(6)-methylguanine-DNA methyltransferase (MGMT) during the development of esophageal squamous cell carcinoma (ESCC) and to investigate its reactivation in cell lines. Samples were collected from Linzhou City of the Henan province in northern China, a high-risk area of this disease. The hypermethylation of promoter CpG islands of the MGMT gene was examined by methylation-specific PCR in ESCC and neighboring non-tumorous tissues, as well as in laser capture microdissected samples with normal epithelium, basal cell hyperplasia (BCH), and dysplasia (DYS). The MGMT mRNA and protein expression were determined with RT-PCR and immunohistochemistry, respectively. Five of 17 (29%) normal, 10 of 20 (50%) BCH, 8 of 12 (67%) DYS, and 13 of 18 (72%) ESCC samples had DNA hypermethylation in the MGMT promoter region, showing a gradual increase with the progression of carcinogenesis. The frequency of the loss of MGMT mRNA and protein expression progressively decreased from normal to BCH, DYS, and ESCC, and it was highly correlated with MGMT promoter hypermethylation according to Fisher's exact tests. When each individual sample was considered, good concordance between DNA hypermethylation and the loss of expression of MGMT was also observed. In samples from the same patient, if hypermethylation was detected in earlier lesions, it was usually observed in more severe lesions. In the ESCC cell line KYSE 510, treatment with a DNA methyltransferase inhibitor, 2'-deoxy-5-azacytidine partially reversed the CpG hypermethylation status and restored mRNA expression of the MGMT gene. Similar reactivation of MGMT gene by dietary polyphenols, (-)-epigallocatechin-3-gallate and genistein, has also been observed. The results suggest that the DNA hypermethylation of MGMT is an important mechanism for MGMT gene silencing in the development of ESCC, and this epigenetic event may be prevented or reversed by these polyphenols for the prevention of carcinogenesis.     

Inactivation of O(6)-methylguanine-DNA methyltransferase by glucose-conjugated inhibitors.

The DNA-repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a decisive determinant of resistance of tumor cells to methylating and chloroethylating anti-cancer drugs. Therefore, selective inhibition of MGMT in tumors is expected to cause tumor sensitization. Several inhibitors of MGMT have been developed which function in both tumors and normal tissue. To deplete MGMT preferentially in tumors, strategies to target the inhibitor to the tumor tissue need to be developed. Here, we report on the properties of glucose-conjugated MGMT inhibitors that might be useful for tumor targeting since tumor cells frequently over-express glucose transporter. O(6)-Benzylguanine (O6BG), 8-aza-O(6)-benzylguanine, O(6)-(4-bromothenyl)-guanine (O6BTG) and the corresponding spacer-linked beta-D-glucose conjugates were analyzed comparatively for MGMT-inhibitory activity. Substitution at the N9 position of the purine moiety resulted generally in a reduction in the efficiency with which the inhibitors blocked MGMT. However, the inhibitory activity of the O6BTG conjugates increased with increasing spacer length, and O6BTG conjugated with a C8 spacer with beta-D-glucose was nearly as effective as O6BTG on its own. MGMT was inhibited by the conjugates both in crude cell extracts and upon treatment of intact HeLa cells, indicating efficient uptake of the glucose conjugates into cells. Since the O6BTG-C8-D-glucose conjugate 8-[O(6)-(4-bromothenyl)-guan-9-yl]-octyl-beta-D-glucoside was highly efficient at MGMT inhibition in a non-toxic concentration range, the drug might be a useful tool for specific tumor sensitization.     

O(6)-methylguanine-DNA methyl transferase gene expression and prognosis in breast carcinoma

O(6)-methylguanine-DNA methyl transferase (MGMT) in human carcinomas has been associated with tumor resistance to alkylating agents. The aims of this study were: i) to correlate tumor MGMT expression and patient and tumor characteristics in malignant breast carcinomas treated with induction chemotherapy including cyclophosphamide (CPM) and ii) to study the predictive and prognostic values of tumor MGMT gene expression. We used RT-PCR to measure the levels of tumor MGMT expression in 107 patients with breast carcinomas prior to neoadjuvant chemotherapy. Sixty patients (56%) received anthracyclines and CPM and 47 (44%) received only anthracyclines. Low levels of MGMT expression correlated with Scarff-Bloom-Richardson grade III (p<0.005), elevated S-phase (p<0.05), negative estrogen receptors (p<0.05), metastatic status (p<0.05) and occurrence of death (p=0.01). MGMT expression was not predictive of treatment response. Unexpectedly, survival was longer when tumor MGMT expression was high (p<0.005). The 4-year survival rate was 76% for high level MGMT patients and only 55% for others. This difference is also significant using the COX model (p<0.05). In breast cancer, tumor MGMT expression was not predictive of response to CPM. A low MGMT expression was significantly related to poor survival.     

Changes in O6-methylguanine-DNA methyltransferase expression during immortalization of cloned human fibroblasts

Suppressed expression of the DNA repair enzyme O⁶-methylguanine-DNAmethyltransferase (MGMT), characterized as the Mer^– phenotype,occurs only in malignant or transformed cell lines. To investigatethe relationship between the transformation process and lossof MGMT expression, we derived 20 cloned lines of IMR90 normalfibroblasts transfected with the plasmid pSV3neo expressingthe SV40 large-T antigen. Of the five lines that were grownuntil crisis phase, four emerged as continuously proliferatingimmortal lines. Of these, only one retained MGMT, the otherthree having become Mer^–. In every case the loss of MGMTcoincided with the final phase of immortalization followingcrisis. Because these were cloned cell lines it is clear thatthe phenotypic change to Mer^– is not merely due to selectionof a Mer^– cell from the initial population, but must involvea cellular change in MGMT regulation, but must involve a cellularchange in MGMT regulation. It is not clear if increased mutationrate associated with loss of MGMT results in increased frequencyof an immortalization event or if an immortalization event,such as telomere disruption, results in MGMT suppression. Inaddition, we have shown that, consistent with previous observations,both hypermethylation in promoter sequences and hypomethylationof downstream sequences in the body of the gene were closelyassociated with loss of MGMT expression. These studies alsoillustrate the utility of these new cloned cell lines for characterizingmolecular events associated with transformation and immortalization.     

Expression of O6-methylguanine-DNA methyltransferase in childhood medulloblastoma

Medulloblastomas (MB) are the most common malignant brain tumors in childhood. Alkylator-based drugs are effective agents in the treatment of patients with MB. In several tumors, including malignant glioma, elevated O⁶-methylguanine-DNA methyltransferase (MGMT) expression levels or lack of MGMT promoter methylation have been found to be associated with resistance to alkylating chemotherapeutic agents such as temozolomide (TMZ). In this study, we examined the MGMT status of MB and central nervous system primitive neuroectodermal tumor (PNET) cells and two large sets of primary MB. In seven MB/PNET cell lines investigated, MGMT promoter methylation was detected only in D425 human MB cells as assayed by the qualitative methylation-specific PCR and the more quantitative pyrosequencing assay. In D425 human MB cells, MGMT mRNA and protein expression was clearly lower when compared with the MGMT expression in the other MB/PNET cell lines. In MB/PNET cells, sensitivity towards TMZ and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) correlated with MGMT methylation and MGMT mRNA expression. Pyrosequencing in 67 primary MB samples revealed a mean percentage of MGMT methylation of 3.7?C92% (mean: 13.25%, median: 10.67%). Percentage of MGMT methylation and MGMT mRNA expression as determined by quantitative RT-PCR correlated inversely (n = 46; Pearson correlation r ² = 0.14, P = 0.01). We then analyzed MGMT mRNA expression in a second set of 47 formalin-fixed paraffin-embedded primary MB samples from clinically well-documented patients treated within the prospective randomized multicenter trial HIT??91. No association was found between MGMT mRNA expression and progression-free or overall survival. Therefore, it is not currently recommended to use MGMT mRNA expression analysis to determine who should receive alkylating agents and who should not.     

Inhibition of O(6)-methylguanine-DNA methyltransferase increases azoxymethane-induced colonic tumors in rats

Azoxymethane (AOM) causes O(6)-methylguanine adduct formation which leads to G-->A transitions. Their repair is carried out by O(6)-methylguanine-DNA methyltransferase (MGMT). To evaluate the importance of this repair event in AOM-induced carcinogenesis, we examined the effect of O(6)-benzylguanine (BG), a potent inhibitor of MGMT, on colonic tumor development. Rats were treated weekly for 2 weeks at 0 and 24 h with BG (60 mg/kg body wt i.p.) or vehicle (40% polyethylene glycol, PEG-400), followed 2 h after the first dose of BG with AOM (15 mg/kg body wt) or vehicle (saline) i.p. Rats were killed 35 weeks later and tumors harvested and DNA extracted. In the AOM-treated groups, BG caused a significant increase in tumor incidence with tumors in 65.9%, versus 30.8% in the AOM/PEG-treated group (P < 0.05). In the BG/AOM group there was also a significant increase in tumor multiplicity, with 2.3 tumors/tumor-bearing rat, versus 1.6 tumors/tumor- bearing rat in the AOM/PEG group (P < 0.05). Since O(6)-methylguanine adducts can cause activating mutations in the K-ras and beta-catenin genes, we examined the effects of BG on these mutations. In the BG group there were seven mutations in codon 12 or 13 of exon 1 of the K-ras gene in 51 tumors examined, compared with no K-ras mutations in 17 tumors analyzed in the AOM/PEG group (P = 0.12). In the BG/AOM group there were 10 mutations in exon 3 of the beta-catenin gene among 48 tumors evaluated, compared with six mutations in 16 tumors analyzed in the PEG/AOM group (P = 0.16). In summary, MGMT inhibition increases AOM-induced colonic tumor incidence and multiplicity in rats.     

Combined loss of expression of O6-methylguanine-DNA methyltransferase and hMLH1 accelerates progression of hepatocellular carcinoma

BACKGROUND AND OBJECTIVES: O(6)-methylguanine-DNA methyltransferase (MGMT) and human Mut L homologue 1 (hMLH1) are proteins that play an important role in DNA repair. No reports have yet described whether deficient MGMT and hMLH1 expression correlates with tumor progression and the prognosis of patients with hepatocellular carcinoma (HCC). METHODS: Using immunohistochemical analysis, we evaluated the expression status of MGMT and hMLH1 protein in 60 paraffin-embedded samples from consecutive patients with curatively resected HCC. RESULTS: The lack of expression of both MGMT and hMLH1 in HCCs (n = 7) correlated with advanced pTNM stage (P = 0.039), as compared with HCCs expressing both proteins (n = 25). The absence of both MGMT and hMLH1 was a significant indicator of malignant potential. The expression status of both MGMT and hMLH1 was a predictive factor for overall survival in patients with HCC (P < 0.001). CONCLUSIONS: HCC lacking both MGMT and hMLH1 is correlated with an advanced stage and a poor prognosis. The expression status of both repair proteins is a predictive prognostic marker in patients with HCC after surgical resection.     

O6-methylguanine-DNA methyltransferase activity in human liver tumors

Previous studies have shown that in about one-fifth of human tumor cell strains, the activity of O6-methylguanine-DNA methyltransferase (MGMT), which can repair O6-alkylguanine in DNA produced by alkylating agents, is deficient. These strains are termed Mer- cells. To see if there is any human tumor lacking MGMT activity, we measured the MGMT activity in extracts from liver tumors of 21 patients, and compared it to the activity in normal peritumoral tissues derived from the same patients. The MGMT activity was assayed by measuring the 3H radioactivity transferred from the substrate DNA containing [methyl-3H]-labeled O6-methylguanine to an acid-insoluble protein fraction. There was considerable variation in MGMT activity among individual extracts; the interindividual variation was approximately 6-fold in normal liver tissue and much larger in liver tumors. Although in many cases similar high levels of MGMT activity were found both in liver tumors and in the normal counterpart, six tumors had greater than 3-fold less activity compared with the normal liver tissue from the same patient. Liver tumors from two patients did not have any detectable level of MGMT activity by the present method used, in spite of the fact that the corresponding normal liver samples demonstrated significant activities. We also measured in the same tissue extracts the activities of two common enzymes, glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH). The activities of GPT and LDH in the liver tumor samples that showed undetectable levels of MGMT activity were similar to those in the surrounding normal liver tissues. These results may suggest the existence of human Mer- tumors, deficient or very low MGMT activity.     

O6-methylguanine-DNA methyltransferase activity in human tumors.

The distribution of O6-methylguanine-DNA methyltransferase (MGMT) activity in extracts of tumors from 74 patients was measured. The results demonstrated that there was considerable variation of MGMT activity in different human tumor tissues as well as in different individuals. The mean values (X +/- SD, pmol/mg of protein) in breast cancer, stomach cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, esophageal carcinoma, brain tumors, colon carcinoma and malignant melanoma were 1.071 +/- 0.374 (9), 0.515 +/- 0.107 (5), 0.509 +/- 0.251 (5), 0.461 +/- 0.227 (24), 0.329 +/- 0.246 (5), 0.273 +/- 0.376 (5), 0.244 +/- 0.175 (14), 0.242 +/- 0.308 (5) and 0.201 +/- 0.161 (2) respectively. It was notable that six samples (1/24 non-small cell lung cancer, 3/5 esophageal carcinoma, 1/14 brain tumors and 1/5 colon carcinoma) did not have any detectable level of MGMT activity. Activity of glutamine pyruvic transaminase (GPT) was also measured in the same extracts used for the assay of MGMT activity. The activity of GPT in these samples with undetectable level of MGMT activity was similar to those with significant MGMT activity. These results further strengthen the assumption that a certain fraction of human tumors are Mer-.     

O6-methylguanine-DNA methyltransferase promoter hypermethylation in colorectal carcinogenesis

Epigenetic alterations have been reported in colorectal neoplasia which can either complement or in some cases be predisposed to genetic alterations such as K-ras mutations. We examined the promoter methylation status of the CDKN2A and O6-methylguanine-DNA methyltransferase (MGMT) genes, after sodium bisulfite conversion and DNA amplification with methylation specific PCR. Moreover, we searched for G to A transitions in codons 12 and 13 of the K-ras oncogene in normal colorectal mucosae, aberrant crypt foci (ACF, early premalignant lesions) and carcinomas. CDKN2A hypermethylation was an infrequent event in ACF (2 of 26, 7.7%). On the contrary, MGMT hypermethylation was found in the normal mucosae (3 of the 12 samples, 25%), in 14 of the 26 ACF (53.8%) and in 7 of the 9 (77.8%) carcinomas examined. K-ras mutations were evident in 6 ACF (23%) and in 3 carcinomas (33.3%), mostly associated with MGMT promoter hypermethylation. These findings strongly support the hypothesis that epigenetic mechanisms play an important role in the early steps of colorectal carcinogenesis.     

Aberrant MGMT (O6-methylguanine-DNA methyltransferase) promoter methylation in choroid plexus tumors

Aberrant methylation of the MGMT (O6-methylguanine-DNA methyltransferase) DNA-repair gene is a predictive marker for the response to chemotherapy with alkylating agents (e.g., temozolomide) in malignant gliomas. Since temozolomide is considered for the treatment of choroid plexus tumors, MGMT promoter methylation status was retrospectively assessed in 36 choroid plexus tumors using methylation specific PCR, combined bisulfite restriction analysis (COBRA), and clone sequencing. By methylation specific PCR, all samples demonstrated a signal for MGMT methylation. COBRA confirmed >10% methylation of CpGs 17 and 31 in 58% of tumors. Clone sequencing of six cases methylated by COBRA confirmed aberrant methylation including a previously recognized enhancer element. In conclusion, MGMT promoter methylation is frequent in choroid plexus tumors and can be quantified using COBRA. Determination of MGMT promoter methylation status might be useful for the stratification of patients for alkylator-based treatments in future clinical trials. The authors M. Hasselblatt and J. Mühlisch have contributed equally.     

Inactivation of DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relation to p53 mutations in esophageal squamous cell carcinoma

The development of esophageal squamous cell carcinoma (ESCC) has been linked to exposure to carcinogens such as nitrosamines that cause various alkyl DNA damages and O6-methylguanine-DNA methyltransferase (MGMT) is a primary defence against alkylation-induced mutagenesis and carcinogenesis. This study was to investigate the role of inactivation of MGMT by promoter hypermethylation and its relation to p53 mutations in ESCC. Methylation of MGMT promoter was determined by methylation-specific polymerase chain reaction in 119 ESCC specimens, 22 corresponding tissue samples adjacent to the tumors, and 21 normal epithelial specimens of the esophagus. The levels of MGMT protein in ESCC with methylated or unmethylated MGMT were analyzed by quantitative immunohistochemistry. Mutations of p53 in 119 ESCC were detected by denaturing high-performance liquid chromatography and sequencing. We found that all 21 normal esophageal tissues had unmethylated MGMT; however, among 119 ESCC, 46 (38.7%) had hypermethylated MGMT. This epigenetic change also occurred in some normal tissues adjacent to the tumors. The level of MGMT protein in MGMT-methylated ESCC was significantly lower than that in MGMT-unmethylated ESCC, whereas great inter-individual variation and poor expression was also observed among MGMT-unmethylated ESCC. Fifty-one percent (61/119) ESCC showed p53 mutations but the distribution of the mutations did not differ significantly between MGMT-methylated ESCC (44%) and MGMT-unmethylated ESCC (56.2%; P = 0.18). MGMT promoter hypermethylation was neither associated with overall G:C to A:T mutations nor associated with this type of mutations in non-CpG dinucleotides in p53. Our results demonstrate that inactivation of MGMT by aberrant promoter methylation is a frequent molecular event in ESCC. This epigenetic alteration is an important, but may not be the sole, mechanism leading to the impaired expression of MGMT. Aberrant MGMT methylation seemed not to be associated with overall frequency and spectrum of p53 mutations in ESCC.