O6-methylguanine-DNA-methyltransferase expression and gene polymorphisms in relation to chemotherapeutic response in metastatic melanoma.

In a retrospective study, O(6)-methylguanine-DNA-methyltransferase (MGMT) expression was analysed by immunohistochemistry using monoclonal human anti-MGMT antibody in melanoma metastases in patients receiving dacarbazine (DTIC) as single-drug therapy or as part of combination chemotherapy with DTIC-vindesine or DTIC-vindesine-cisplatin. The correlation of MGMT expression levels with clinical response to chemotherapy was investigated in 79 patients with metastatic melanoma. There was an inverse relationship between MGMT expression and clinical response to DTIC-based chemotherapy (P=0.05). Polymorphisms in the coding region of the MGMT gene were also investigated in tumours from 52 melanoma patients by PCR/SSCP and nucleotide sequence analyses. Single-nucleotide polymorphisms (SNPs) in exon 3 (L53L and L84F) and in exon 5 (I143V/K178R) were identified. There were no differences in the frequencies of these polymorphisms between these melanoma patients and patients with familial melanoma or healthy Swedish individuals. Functional analysis of variants MGMT-I143V and -I143V/K178R was performed by in vitro mutagenesis in Escherichia coli. There was no evidence that these variants decreased the MGMT DNA repair activity compared to the wild-type protein. All melanoma patients with the MGMT 53/84 polymorphism except one had tumours with high MGMT expression. There was no significant correlation between any of the MGMT polymorphisms and clinical response to chemotherapy, although an indication of a lower response rate in patients with SNPs in exon 5 was obtained. Thus, MGMT expression appears to be more related to response to chemotherapy than MGMT polymorphisms in patients with metastatic melanoma.

Novel O6-methylguanine-DNA methyltransferase SNPs: a frequency comparison of patients with familial melanoma and healthy individuals in Sweden.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is involved in the cellular defense against alkylating agents. Genetic alterations in the MGMT gene may impair the protein's ability to remove alkyl groups from the O6-position of guanine, thereby raising the mutation rate and increasing the risk of cancer. We assessed polymorphisms in the promoter region and the 5 exons of the MGMT gene by PCR/SSCP and nucleotide sequence analysis of DNA extracted from blood samples. The population studied consisted of 89 melanoma patients, each belonging to a different family with a hereditary predisposition for melanoma, and 76 healthy individuals (blood donors). A total of 11 single nucleotide polymorphisms (SNPs) were detected, five in the promoter region, one in exon 1, two in exon 3 and three in exon 5. Six of the alterations were novel polymorphisms, of which five were located in the promoter region and one in exon 5. When the distribution of specific SNPs in cases and controls with only one variant was calculated; C575A was present only in melanoma patients (p=0.072). Moreover, while 20% of the healthy individuals had no SNPs this was the case in only 12.4% of the melanoma patients. However, no statistically significant differences were seen between cases and controls for any of the 11 SNPs.

Analysis of O(6)-methylguanine-DNA methyltransferase in melanoma tumours in patients treated with dacarbazine-based chemotherapy.

In a retrospective study we analysed the levels of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) in melanoma metastases in patients receiving dacarbazine (DTIC) either as a single drug or as part of combination chemotherapy regimens, and related the expression levels to the clinical response to treatment. Biopsies of subcutaneous and lymph node metastases obtained before chemotherapy in 65 patients with disseminated malignant melanoma were examined for MGMT protein levels by immunohistochemistry using a monoclonal anti-human MGMT antibody. All patients received chemotherapy with DTIC, given either as a single drug or in combination with vindesine and in some cases cisplatin. DTIC as single agent was given to 44 patients, while 21 received combination chemotherapy. Objective responses to chemotherapy were seen in 12 patients, while 53 patients failed to respond to treatment. The expression of MGMT was determined according to the proportion of antibody-stained tumour cells, using a cut-off level of 50%. In 12 of the patients more than one metastasis was analysed, and in seven of these cases the MGMT expression differed between tumours in the same individual. Among the responders a larger proportion (six out of 12, 50%) had tumours containing less than 50% MGMT-positive tumour cells than among the non-responders (12 out of 53, 23%). These data are consistent with the hypothesis that MGMT contributes to resistance to DTIC-based treatment, although the difference between responders and non-responders with respect to the proportion of MGMT-positive tumour cells was not statistically significant (P = 0.077).

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

Role of O6-methylguanine-DNA methyltransferase, glutathione transferase M3-3 and glutathione in resistance to carmustine in a human non-small cell lung cancer cell line.

O6-methylguanine-DNA methyltransferase (MGMT), glutathione transferase (GST) M3-3 and glutathione (GSH) have all been implicated in the resistance of cells to the cytostatic drug carmustine. U1810, a human non-small cell lung cancer cell line, expresses all of these putative resistance factors. The U1810 cells show a 4.4-fold lower sensitivity to carmustine compared with the U1690 cell line, a human small cell lung cancer cell line lacking detectable levels of both MGMT and GST M3-3. We investigated the effect of the MGMT inhibitor O6-benzylguanine, the GST inhibitor ethacrynic acid and the GSH synthesis inhibitor D,L-buthionine-S,R-sulfoximine (BSO) on the cytotoxicity of carmustine to U1810 cells. No potentiation to carmustine was observed after treatment with ethacrynic acid, while a 2-fold potentiation was found after exposure to O6-benzylguanine. Depletion of GSH with BSO showed a similar sensitising effect as that obtained with O6-benzylguanine. Thus, MGMT and GSH are the predominant resistance factors to carmustine in the U1810 cell line, whereas it is unclear whether GST M3-3 plays any role.

Immunohistochemical examination of the expression of O6-methylguanine-DNA methyltransferase in human melanoma metastases.

In tumour cell lines, an inverse relationship has been shown between susceptibility to the cytotoxic effects of the O6-alkylating agents and the expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). One of the most effective single agents in chemotherapy of metastatic melanoma is the O6-alkylating drug, dacarbazine. We therefore examined the distribution of MGMT in 37 skin and lymph node melanoma metastases using rabbit antihuman MGMT antiserum. MGMT expression was undetectable in tumours from 2 out of 34 patients and low in 4 further patients. When present, staining was mainly nuclear and showed a marked variation both among tumour cells within the same metastases, between separate metastases in the same patient and between tumours in different patients. MGMT expression determined by immunohistochemistry showed a relation to MGMT activity measurements, but was not related to the number of proliferating cells, as identified by staining with MIB-1 antibody. Tumour cells with moderate to strong immunostaining with MGMT antiserum were significantly more abundant in metastases excised after dacarbazine-based chemotherapy (n = 8) than in those excised before treatment (n = 29).

Analysis of O6-methylguanine-DNA methyltransferase mRNA in fine needle biopsies from human melanoma metastases by reverse transcription and polymerase chain reaction.

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein, which removes alkyl groups from the O6 atom of guanine residues. Tumour cells which lack MGMT are sensitive to cytostatic drugs such as dacarbazine (DTIC), whose active species bind to this site. To explore whether analyses of MGMT expression can be used as a predictive test for clinical sensitivity to DTIC in melanomas, we developed a method to assay MGMT mRNA levels in cells obtained by fine needle aspiration biopsies of metastases. cDNA was synthesised from mRNA prepared from biopsy material. Polymerase chain reaction was performed using primers complementary to MGMT cDNA and to beta-actin, which served as an internal control. Analyses of 44 biopsies from 35 patients showed a considerable variation in MGMT mRNA, with 15 samples (34%) lacking detectable mRNA. In 6 out of 8 patients in whom more than one tumour was analysed, separate metastases had different levels of MGMT mRNA. There was no correlation between MGTM activity studied by a biochemical assay and MGMT mRNA levels when these were compared in 10 surgical biopsies.

Increased cisplatin sensitivity of human fibroblasts from a subject with inherent glutathione deficiency.

Fibroblasts from an individual with 5-oxoprolinuria are deficient in glutathione (GSH) as compared to cells from a healthy sibling. Glutathione transferase (GST) levels also differ for some isoenzymes: GSH deficient cells have more GST A1-1, but lack GST M1-1. However, the contents of the quantitatively dominating isoenzyme GST P1-1 show no significant difference. While there is equal sensitivity to ultaviolet irradiation (UV), the GSH deficient cells are more sensitive to cisplatin, carboplatin and melphalan. Depletion of GSH with buthionine sulfoximine sensitizes the normal fibroblasts to these drugs. Only slightly more cisplatin-induced DNA cross-links are detected in the GSH deficient cells, and there is no difference in the removal of DNA cross-links. Similar amounts of DNA strand breaks are seen in normal and GSH deficient cells after UV exposure, but there is a reduced rates of resealing of strand breaks in GSH deficient cells. The main conclusion from the studies of these fibroblasts is that GSH plays a significant role in the cellular resistance to cytostatic drugs such as platinum compounds and melphalan.

Apoptosis and c-jun induction by cisplatin in a human melanoma cell line and a drug-resistant daughter cell line.

Cisplatin resistance was developed in the human melanoma cell line RPMI8322 by repeated short-term exposures to cisplatin. The most resistant daughter cell line, RPMI8322/CDDP-300, was 4-fold resistant to cisplatin, and partially cross-resistant to carboplatin, melphalan and UV, but not to BCNU. RPMI8322/CDDP-300 cells showed less apoptosis after cisplatin than the parental cells. The cisplatin resistance was not paralleled by a similar reduction in cellular cisplatin accumulation or DNA cross-links in RPMI8322/CDDP-300 cells, and these cells exhibited no increase in cellular glutathione or in mRNA encoding the DNA excision repair protein ERCC1 and XPB. Induction of c-jun mRNA by cisplatin was considerably lower in RPMI8322/CDDP-300 cells than in RPMI8322 cells, consistent with the possibility that c-jun induction may be involved in a pathway that triggers apoptosis after exposure to DNA damaging agents. However, c-jun induction is not necessary for apoptosis, since cisplatin also induced apoptosis in A14 rat embryo fibroblasts, cells in which the c-jun gene is deleted.

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.

Contribution of glutathione transferase M3-3 to 1,3-bis(2-chloroethyl)-1-nitrosourea resistance in a human non-small cell lung cancer cell line.

The glutathione transferase (GST) isoenzyme profile was determined in two human tumor cell lines, U1690 derived from a small cell lung cancer and U1810 derived from a non-small cell lung cancer. U1810 cells are 3.2-fold more resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) than are U1690 cells, a finding ascribable in part to the expression of O6-alkylguanine-DNA alkyltransferase activity in the U1810 cells. GST P1-1 and GST A1-1 were determined quantitatively by enzyme-linked immunoassay and were found to be 1.3- and 15-fold higher in the cytosol fraction of U1690 cells as compared to U1810 cells, respectively. The higher BCNU resistance in U1810 cells can, therefore, not be correlated with the expression of these isoenzymes. However, sodium dodecyl sulfate/polyacrylamide gel electrophoresis in combination with immunoblot analysis demonstrated a class Mu GST, which was identified as GST M3-3 on the basis of electrophoretic mobility and cross-reaction with anti-rat GST 3-3 antibodies. This isoenzyme was detectable in U1810 cells but not in U1690 cells. Studies with purified human GST A1-1, GST M1-1, GST M3-3, and GST P1-1 demonstrated that GST M3-3, but not the other isoenzymes, catalyzed the denitrosation of BCNU. Such inactivation of BCNU has previously been demonstrated with rat class Mu GSTs (M. T. Smith et al., Cancer Res., 49: 2621-2625, 1989) but not with any human GST. These findings suggest that GST M3-3 contributes to BCNU resistance in the U1810 cells.

Carmustine-induced toxicity, DNA crosslinking and O6-methylguanine-DNA methyltransferase activity in two human lung cancer cell lines.

O6-methylguanine-DNA methyltransferase (O6-MT) probably plays an important role in the repair of chloroethylnitrosourea-induced DNA damage. O6-MT was studied as a possible drug resistance factor in two human lung cancer cell lines, one small cell lung cancer (U1690) and one non-small cell lung cancer (U1810), with different sensitivities to carmustine. The U1810 cell line was 3.4-fold more resistant to carmustine than U1690 cells, although the two cell lines were equally sensitive to mustine, melphalan and cisplatin. A 23-fold higher level of DNA interstrand crosslinks was observed following exposure of U1690 cells to carmustine compared with U1810 cells. The O6-MT activity of U1810 cells was 11 times higher than that of U1690 cells. The O6-MT activity in the U1810 cells showed a dose-dependent decrease after exposure to carmustine. These results show a correlation between increased O6-MT activity, decreased drug induced DNA interstrand crosslinking and cellular resistance to carmustine.