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 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.     

Influence of O6-methylguanine-DNA methyltransferase activity on chloroethylnitrosourea chemotherapy in brain tumors

Chloroethylnitrosoureas (CENUs) alkylate DNA at specific sites and inhibit DNA replication in tumor cells. O⁶-Alkylguanine moieties resulting from alkylation of guanine bases are thought to be one of most lethal adducts in living cells. Effectiveness of CENUs is known to relate well with an enzymic activity of the DNA repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT), which recognizes and removes O⁶-alkylguanine. To improve therapeutic results of CENUs, we have measured MGMT activity of human brain tumors and studied the relationship between MGMT activity and clinical responsiveness to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). Thirty-seven patients with brain tumors were entered into the study. The neoplasms included gliomas, non-glial tumors, and brain metastases. The MGMT activity of gliomas was significantly lower than that of non-glial tumors and brain metastases. No significant difference in the enzyme activity was noted between low- and high-grade gliomas. Out of the 22 gliomas 5 tumors indicated a value below 60 fmol/mg, suggestive of a methyl excision repair minus (Mer^?) tumor. Two out of 3 evaluable patients with a Mer^? tumor responded well to post-operative ACNU adjuvant chemotherapy. Our results suggest that brain tumors include a certain percentage of Mer^? phenotype tumors, and that CENUs such as ACNU should be applied selectively on tumors with a low MGMT activity in order to increase the therapeutic effectiveness. © 1993 Wiley-Liss, Inc.     

Expression of O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea resistance of human brain tumors.

Northern blot analysis with O6-methylguanine-DNA methyltransferase (MGMT) cDNA as a probe was used to analyze the MGMT activity regulating drug resistance of human cells to chloroethylnitrosoureas (CENUs). By this method, the expression levels of MGMT mRNA in six human glioma cell lines and 12 human brain tumor tissues from surgical specimens were determined. These MGMT mRNA levels were compared with the SD10 values of the tumor cells, estimated by cell survival assay, which indicated their resistance to the anticancer drug, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Human brain tumors that were highly resistant to ACNU, such as glioblastoma Gbl1 and metastatic brain tumor Col1 with SD10 values (microM) of above 100, expressed markedly increased amounts of 0.95 kb MGMT mRNA. In contrast, tumor cells such as U-87MG, U-251MG, U-343MG, U-373MG and SF-126 with SD10 values of under 14 indicating low resistance to ACNU scarcely synthesized any MGMT mRNA. These results indicated that the level of expression of MGMT mRNA in human brain tumors determined by Northern blot analysis truly reflects their cellular resistance to ACNU. Thus the Northern method with MGMT cDNA probe reported here is a practical and reliable method for estimation of cellular resistance to CENUs such as ACNU and for screening the chemotherapeutic response to CENUs of human brain tumors.     

Epigenetic silencing of the DNA repair enzyme O6-methylguanine-DNA methyltransferase in Mex- human cells.

Regulation of the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) has been investigated in a number of human lymphoblastoid cell lines. In a number of Mex- cell lines that do not express methyltransferase activity, CpG sequences in the mgmt gene were hypomethylated with respect to methyltransferase-expressing Mex+ lines. In the cell line GM1953(S), in which the mgmt gene is coregulated with the thymidine kinase and galactokinase genes, reexpression of all three activities was experimentally induced. In this case, the mgmt gene in the nonexpressing cells was found to be hypermethylated and underwent a demethylation at CpG sequences that was coincident with the reappearance of the mgmt mRNA and the three enzyme activities. The simultaneous silencing of three activities in these cells was correlated with an increase in DNA 5-methylcytosine that was widespread throughout the genome. The data indicate that MGMT expression can be controlled epigenetically in human lymphoid cell lines, although the relationship between cytosine methylation and MGMT expression is complex. Furthermore, the rapid alterations in methylation in GM1953(S) cells indicate the existence of signals that can induce widespread and abrupt alterations in cytosine methylation in human cells in culture.     

O6-methylguanine-DNA methyltransferase activity and sensitivity to cyclophosphamide and cisplatin in human lung tumor xenografts

The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) is a main determinant of resistance of cells to the cytostatic effects of O⁶-alkylguanine-generating alkylating agents. The purpose of our study was to assay MGMT activity in cells of lung cancers and to correlate MGMT levels with chemotherapy response to cyclophosphamide (CTX) and cisplatin (DDP). MGMT levels were determined in 14 human lung tumor xenografts. There was a wide variation of MGMT expression in these tumors, ranging from 10 to 984 fmol/mg protein. There was also a wide range in the sensitivity of the xenografts to CTX and DDP, as measured by specific growth delay. When the MGMT levels of the different xenograft lines were compared with the corresponding responses to CTX and DDP, a close correlation was found between MGMT activity and CTX (lin reg., r = –0.83, p < 0.05). The higher the MGMT activity, the less pronounced was the growth-inhibiting effect of CTX. With DDP, no such correlation was found. Our results indicate that the in vivo response of tumors to CTX is related to the level of MGMT expression. Int. J. Cancer 77:919–922, 1998. © 1998 Wiley-Liss, Inc.     

Effect of 5-azacytidine on expression of the human DNA repair enzyme O6-methylguanine-DNA methyltransferase

The role of methylatlon of CpG dinucleotides in the regulationof O⁶-methylguanine-DNA methyltransferase (MGMT) gene expressionhas been investigated. A previous observation, that cell linesdeficient in MGMT (Men^– are methylated in a SmaI sitein the MGMT gene promoter whereas MGMT expressing cells (Mer⁺)are unmethylated in the same site, has been extended to a totalof 30 cell lines, tumors and normal tissues. To examine furtherthe association between methylation in the MGMT promoter andthe Mer^– phenotype we have treated Mer⁺ and Mer^–cell lines with 5-azacytidine to inhibit DNA methylation. Reducedmethylation in the SmaI site coincided with induction of MGMTexpression for one of three Mer^– cell lines. MGMT increasedseveral-fold further upon continued culture of the induced cellsin the absence of 5-azacytidlne, coincident with an abrupt increasein methylation in the body of the MGMT gene even though theSmaI site remained demethylated. These results, and those ofother previous studies, suggest that methylation of sequenceswithin the MGMT gene promoter and methylation within the bodyof the gene have opposite effects.     

O6-methylguanine-DNA methyltransferase in human normal and tumor tissue from brain, lung, and ovary

The resistance of human tumor strains in culture to cell killing by alkylating nitrosoureas is correlated with their levels of the DNA repair activity O6-methylguanine-DNA methyltransferase. Strains with the Mer- phenotype have no activity and are extremely sensitive. However, the relationship between the sensitivity of human tumors in vivo and transferase levels is not known, and even the existence of Mer- human tumors in vivo has been questioned. In this study 73 human tumor and normal tissue samples from brain, lung, and ovary were assayed for transferase levels and methylpurine glycosylase activity. For each organ, transferase levels varied over 100-fold, and Mer- tumors were detected in each group. There was no correlation between transferase and glycosylase levels, indicating that the absence of transferase in some tumor samples was not an artifact due to necrosis or inactivation of enzymes in the extract.     

Combined effect of temozolomide and hyperthermia on human melanoma cell growth and O6-methylguanine-DNA methyltransferase activity

Hyperthermic isolated limb perfusion (HILP) with L-phenylalanine mustard (L-PAM) represents an effective treatment for locally advanced melanoma of the limbs. However, regional chemotherapy of melanoma still needs to be improved. Temozolomide (TMZ) is a methylating agent that spontaneously decomposes into the active metabolite of dacarbazine, the most effective agent for the systemic treatment of melanoma. Tumor cells with high levels of O6-methylguanine-DNA methyltransferase (MGMT) and/or with a defective DNA mismatch repair (MMR) are resistant to TMZ. Inhibition of MGMT activity increases TMZ sensitivity of MMR-proficient, but not of MMR-deficient cells, while inhibition of base excision repair (BER) potentiates TMZ cytotoxicity in both cell types. Recent studies, performed in an animal model, have shown that TMZ is more effective than L-PAM when applied regionally and that hyperthermia can increase the antitumor activity of TMZ. In this study, three thermoresistant human melanoma cell lines, endowed with different MGMT activity and functional status of the MMR system, were treated with TMZ at 37 degrees C or 41.5 degrees C for 90 min, and then analyzed for cell growth and MGMT activity. Hyperthermia significantly enhanced TMZ cytotoxicity in MMR-proficient cells, either endowed or not with MGMT activity, and in MMR-deficient cells. Endogenous MGMT activity was not affected by hyperthermia that, however, enhanced the enzyme depletion induced by TMZ treatment. Moreover, MGMT recovery after drug removal was delayed in cells that had been treated at 41.5 degrees C. Taken together, these findings confirm the therapeutic potential of a combined treatment of hyperthermia and TMZ. They also suggest that inhibition of BER and/or increased DNA methylation may be involved in the thermal enhancement of TMZ cytotoxicity. Additional studies are necessary to better clarify the mechanisms underlying hyperthermia-induced potentiation of TMZ activity. However, the present investigation provides further support to the development of clinical trials of HILP with TMZ.     

O6-methylguanine-DNA methyltransferase activities in biopsies of human melanoma tumours.

Tumour samples obtained from one primary melanoma and several lymph node and skin metastases were analysed for O6-methylguanine-DNA methyltransferase (MGMT) activity. While lymph node and skin metastases had similar average MGMT activity, the variance was significantly higher in lymph node metastases. Variability in MGMT activity was frequently observed in different metastases in the same individual and to a lesser extent within metastases.     

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.      

Production and characterization of antipeptide antibodies against human O6-methylguanine-DNA methyltransferase

Four synthetic peptides from the sequence of human O6-methylguanine-DNA methyltransferase (MGMT), three corresponding to different hydrophilic regions and one corresponding to the sequence containing the alkyl acceptor residue cysteine 145, were used to immunize rabbits. The antibody against Peptide III (residues 171-184) was highly specific, and MGMT protein could be detected on Western blots of soluble protein extracts containing as little as 1 fmol of active MGMT. Antibodies against all of the peptides were able to immunoprecipitate denatured MGMT, while only the antibody against Peptide III was able to react with active enzyme. The antibody against Peptide III did not cross-react with methyltransferase from mice. The use of synthetic peptides has led to the production of a highly sensitive, specific antibody that recognizes native and denatured human MGMT. This antibody should prove useful in studies involving the detection, purification, and characterization of this enzyme.     

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.     

O6-methylguanine-DNA methyltransferase in human fetal tissues: fetal and maternal factors

O6-Methylguanine methyltransferase (O6-MT) was measured and compared in extracts of 7 human fetal tissues obtained from 21 different fetal specimens as a function of fetal age and race and of maternal smoking and drug usage. Liver exhibited the highest activity followed by kidney, lung, small intestine, large intestine, skin, and brain. Each fetal organ homogenate exhibited a 3- to 5-fold level of interindividual variation of O6-MT. There did not appear to be any significant differences of O6-MT as a function of fetal race and age and in the tissues obtained from mothers who smoked cigarettes during pregnancy. The fetal tissues obtained from an individual using phenobarbital exhibited 4-fold increases in O6-MT activity. The tissues obtained from another individual on kidney dialysis were 2- to 3-fold higher than the normal population. These data suggest a possible enhancement of human fetal O6-MT by certain xenobiotics, with little if any modulation by racial factors and maternal smoking habits.     

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.     

O6-Alkylguanine-DNA alkyltransferase activity in human brain and brain tumors

It was found that extracts from human brain catalyzed the transferof methyl groups from O⁶-methylguanine in methylated double-strandedDNA to a cysteine residue in a protein of mol. wt. 22 000. ThisO⁶-alkylguanine-DNA alkyltransferase had properties similarto those previously characterized from rodent Liver, human liver,cultured human cells and E. coli. The alkyltransferase activityof human brain was considerably greater than that reported forrat brain, but was significantly less than the activities foundin human Liver and other tissues. The activity was found inboth normal brain samples (peritumoral material which containedno tumor infiltration) and in a variety of brain tumors. Thehighest activity was found in meningeomas and neurinomas, butmost tumors with the exception of some gliomas had higher activitiesthan the normal brain. All 23 tumor samples examined in thisstudy had alkyllramferase activity in contrast to publishedreports showing that :35% of human brain-tumor-derived linesgrown in culture lacked this activity. This discrepancy maybe due to the celluar polymorphism of the tumors, but also suggeststhat complete lack of the alkyltransferase is not a common occurrencein human brain tumors.     

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.     

Regulation of O6-methylguanine-DNA methyltransferase by methionine in human tumour cells.

Methionine (MET)-dependent cell lines require MET to proliferate, and homocysteine (HCY) does not act as a substitute for this requirement. From six O6-methylguanine-DNA methyltransferase (MGMT)-efficient (mer+) cell lines tested, two medulloblastomas (Daoy and D-341) and a lung non-small-cell adenocarcinoma with metastatic potential (H-1623) were most sensitive to MET deprivation, while two glioblastomas (U-138, D-263) and a small-cell lung carcinoma H-1944 were moderately to weakly dependent. Regardless of the degree of MET dependence, all of these lines down-regulated their MGMT activity within 48-72 h of transfer from MET+HCY- to MET-HCY+ media, long before the eradication of the culture. Reduction of MGMT activity was due to a decline of both MGMT mRNA and protein levels. However, the reduction was not related to the methylation status of the MGMT promoter at the SmaI site or the HpaII sites in the body of the gene; such sites have been shown to be associated in MGMT regulation and in defining the mer phenotype. MET-dependent, mer+ tumour cells cultured in MET-HCY+ were more sensitive to BCNU (IC50 = 5-10 microM) than those cultured in MET+HCY-(IC50 = 45-90 microM), while MET-independent or mer- cell lines were unaffected. This indicates that reduction of MGMT, imposed by the absence of MET, renders mer+ tumour cells more susceptible to alkylating agents. The relatively selective suppression of MGMT activity in mer+ MET-dependent tumour cells, in combination with the inability of such cells to proliferate in the absence of MET, may lead to the development of more effective treatment strategies for mer+ MET-dependent tumours.