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Purpose. VNP40101M (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine) is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy in murine models. VNP40101M activation generates chloroethylating species that alkylate DNA at the O6-position of guanine, and a carbamoylating agent, methyl isocyanate, which inhibits O6-alkylguanine-DNA alkyltransferase (AGT) in model systems. We determined whether expression of AGT in Chinese hamster ovary (CHO) cells decreased sensitivity to VNP40101M and explored the mechanism of VNP40101M cytotoxicity by employing analogs of VNP40101M that generate reactive intermediates with either carbamoylating or chloroethylating activity. Methods. AGT was overexpressed in CHO cells by transfection with an expression vector containing the human AGT gene. Cell lines expressing AGT were employed in clonogenic assays to determine the cytotoxicity of VNP40101M and its analogs. Results. VNP40101M was more active against AGT-expressing CHO cells than 90CE (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine), a chloroethylating generator devoid of carbamoylating activity. Furthermore, the greater the degree of AGT expression the more resistance to VNP40101M cytotoxicity. Combination chemotherapy experiments support the conclusions that methyl isocyanate and the chloroethylating species generated from the activation of VNP40101M function synergistically to kill cells. Conclusions. The findings support the concept that alkylation of the O6-position of guanine residues in DNA is the predominant lesion created by VNP40101M, and that methyl isocyanate resulting from the base-catalyzed activation of VNP40101M inhibits AGT and presumably other enzymes involved in DNA repair, thereby enhancing the yield of the DNA G-C interstrand crosslinks responsible for the antitumor activity of this agent. [ABSTRACT FROM AUTHOR]

Purpose. To investigate the interaction of the electrophilic species generated by the decomposition of the antineoplastic prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M) on the ability of O6-alkylguanine-DNA alkyltransferase (AGT) to repair alkylated O6-chloroethylguanine and/or N1,O6-ethanoguanine DNA lesions. Materials and methods. The contributions of inhibitory electrophilic species generated from VNP40101M towards AGT was assessed using analogues that selectively generated either the chloroethylating or the carbamoylating components of VNP40101M. The activity of AGT was determined from the inhibition of crosslink formation from O6-chloroethylguanine and/or N1,O6-ethanoguanine lesions. The half-lives of sulfonylhydrazine derivatives and isocyanates were measured using an acidification assay which gives a change in absorbance proportional to the release or consumption of small quantities of protons. Results. Both of the reactive components produced by VNP40101M directly inactivated cloned human AGT; the carbamoylating moiety (IC50 about 13 μM) was approximately seven- to eight-fold more potent than the alkylating component(s) (IC50 about 100 μM). These inhibitory actions were moderated by the addition of naked T5 bacteriophage DNA. Thus, AGT bound to DNA was markedly more resistant than free AGT to these electrophilic species. DNA also blocked the spontaneous loss of AGT activity which occurred upon incubation of this protein under mild conditions. Conclusions. The reaction of AGT with the methyl isocyanate generated from the decomposition of VNP40101M increased the net number of crosslinks generated by VNP40101M compared to a sulfonylhydrazine prodrug that formed the equivalent alkylating species in the absence of the cogeneration of methyl isocyanate. These actions may be of significance to the antineoplastic activity of VNP40101M. [ABSTRACT FROM AUTHOR]

Transcriptional silencing of the DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), occurs only in malignant or transformed cell lines, and such MGMT-deficient cells are hypersensitive to chemotherapeutic alkylating agents such as 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Previously we demonstrated in a panel of established cell lines that the lack of gene expression correlated with methylation within the CpG island in the MGMT 5' gene flank. Now, we investigated the relationship between CpG methylation, MGMT suppression and drug-sensitivity in normal, diploid MGMT-expressing IMR90 cells and five immortalized sublines (AA, EE, J, KK and Pool), four of which have silenced MGMT. As expected, the MGMT-expressing parental cells were most drug-resistant and free of promoter methylation, whereas the MGMT-silenced immortal sublines were more drug-sensitive and promoter-methylated. Surprisingly, the sole MGMT-positive immortal subline, (AA) showed some promoter methylation although it was relatively drug-resistant; and an apparently MGMT-negative subline, (EE) showed unexpectedly low levels of methylation. We determined if these discrepancies were due to heterogeneity (cellular or allelic) and if this reflected transitional states between expressing and silenced phenotypes. Analysis of the methylation status of CpGs by genomic sequencing of cloned single copy DNA confirmed heterogeneity in both these sublines. With increasing cell culture passage, CpG methylation progressively increased with a concomitant trend to a completely MGMT-silenced phenotype in these sublines.

We previously found that 2-chloroethyl-3-sarcosin-amide-1-nitrosourea (SarCNU), a new chloroethylnitrosourea analogue presently in phase I clinical trials, is a selective cytotoxin that enters cells via the extraneuronal transporter for monoamine transmitters (EMT). In this study, we assessed whether EMT expression correlates with SarCNU cytotoxicity by determining EMT expression in 23 human tumor cell lines with reverse-transcription PCR. Western blot analysis was used to measure protein levels of the DNA repair genes, O6-methylguanine-DNA methyltransferase (MGMT), and excision repair cross-complementing rodent repair deficiency gene 2 (ERCC2). SarCNU cytotoxicity was determined by the sulforhodamine B colorimetric anti-cancer-drug screening assay and correlated with gene expression. Almost all of the cell lines screened were positive for EMT expression. However, seven cell lines (MGR-1, MGR-2, T98-G, SKI-1, SKNSH, 297, and GBM) expressed low levels of EMT. Although there was no linear correlation between SarCNU cytotoxicity and EMT expression, SarCNU cytotoxicity significantly correlated with ERCC2 protein levels, and MGMT-rich (Mer+) cell lines (MGMT protein level >0.1) were more resistant to SarCNU than MGMT-poor (Mer-) cell lines (MGMT protein level <0.1). Moreover, multiple regression analysis indicated that the best correlation with SarCNU cytotoxicity was attainable with EMT plus MGMT and ERCC2 expression. This study suggests that in human tumor cell lines both EMT and DNA repair factors, specifically, MGMT and ERCC2, are important determinants of SarCNU activity. Because EMT is expressed in a wide variety of human tumors, SarCNU should be a more widely effective alternative chemotherapeutic agent.

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation-sensitive restriction enzymes (BssHII, SacII, Eagl, Nael, and Smal) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of Eagl and Nael sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P<0.01). Methylation-sensitive single-nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by Eagl and Nael digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the Eagl and Nael sites and nt -128 than in the 13 tumors lacking the methylation pattern (P<0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression.

We examined the O6-methylguanine-DNA methyltransferase (MGMT) protein as well as MGMT activity levels and the excision repair cross-complementing rodent repair deficiency gene, ERCC2 (XPD), protein levels in 14 human tumor cell lines not selected for chloroethylnitrosourea (CENU) resistance. These results were compared with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity and UV light sensitivity. MGMT protein correlated significantly with MGMT activity (r = 0.9497, p = 0.0001). There was no significant linear correlation between BCNU cytotoxicity and MGMT content as determined by both Western analysis (r = 0.139, p = 0. 6348) and activity assay (r = 0.131, p = 0.6515). However, MGMT-rich cell lines were found to be more resistant than MGMT-poor cell lines to BCNU (t = 2.2375, p = 0.0225) but not to UV (t = 1.1734, p = 0.1317). Furthermore, the most BCNU-sensitive cell lines were all MGMT-poor. UV sensitivity was significantly correlated to BCNU cytotoxicity (r = 0.858, p = 0.0001). Significant correlations were found between ERCC2 protein levels and BCNU cytotoxicity (r = 0.786, p = 0.0009) or UV sensitivity (r = 0.874, p = 0.0001). Our results confirm that MGMT plays an important role in CENU resistance, but not in UV resistance. The correlation of UV sensitivity with BCNU cytotoxicity suggests that nucleotide excision repair is an important modifying factor of MGMT-mediated innate CENU resistance in human tumor cell lines, especially in highly resistant cell lines. ERCC2 may be implicated in this process.

O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer+) and -deficient (Mer-) cells, correlated with the endogenous MGMT activity in Mer+ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5'-CTGGGTCGC-3') for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer+ cells tested but was apparently restricted to the cytoplasm of Mer- cells. We conclude that the MEBP-enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer+ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer- cells.

High-level expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) correlates with cellular resistance to the chloroethylnitrosourea (CENU) class of alkylating agents. Consequently, tumors expressing low levels of MGMT are sensitive to CENU chemotherapy, and any mechanism that can be used to reduce MGMT levels could sensitize resistant tumors. We have demonstrated that transient transfection of wild-type, but not mutant, p53 protein into a p53-null cell line, Saos-2, suppresses MGMT promoter activity in a reporter gene system. In addition, following a 24-h transduction of IMR90 fibroblasts with a wild-type p53-adenoviral vector, endogenous MGMT protein is down-regulated and is no longer detectable 5 days following infection. Because p53 is inducible by ionizing radiation, we propose that existing cancer therapy regimens that combine radiotherapy with CENU chemotherapy may be improved by altering scheduling and allowing enough time between the two therapies for the relatively stable MGMT protein to degrade.

The DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT) is responsible for repair of damage induced by alkylating agents that produce adducts at O6-guanine in DNA. Although the MGMT gene promoter has housekeeping gene promoter characteristics, unlike these genes which are expressed at a constant level, MGMT transcriptional activity varies between cell types. During an attempt to identify regions of the MGMT regulatory sequence sensitive to variations in transcription factors between cell types, we have identified a 59 bp enhancer which is required for efficient MGMT promoter function. This fragment produced increased transcriptional activity in reporter gene constructs containing either the MGMT or UMP-synthase promoter when transfected into either of two cell lines; it seems therefore that this enhancer may interact with relatively common trans-acting factors. Functional activity is only detected when the enhancer is in 'cis' with respect to the promoter, suggesting that complexes are formed between proteins bound to the enhancer and promoter sequences. We propose that the enhancer-binding protein may be a novel transcription factor since there are no obvious consensus sequences within the 59 bp sequence for known DNA-binding proteins.

Approx. 20% of human tumor cell lines (termed Mer-) are deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT; E.C.2.1.1.63). Such cells possess the MGMT gene and promoter sequences but have virtually no mRNA or protein. Cytosine methylation of gene sequences has been proposed as a mechanism by which MGMT could be suppressed in Mer- cells; however, the experimental evidence does not uniformly support this idea. We therefore investigated the effect of in vitro methylation of the MGMT promoter in a reporter gene construct transfected into cultured human cells. DNA methylation by HpaII or HhaI methylases suppressed the activity of the promoter, although the effect was not absolute. The occurrence of partial intracellular demethylation of promoter sequences may account for the incomplete inhibition of transcription. A model that attempts to reconcile the opposing views on the role of cytosine methylation in MGMT gene expression is presented.

A synthetic oligonucleotide containing ribozyme sequences targeted to the 5' region of the human O6-methylguanine-DNA methyltransferase (MGMT) mRNA has been constructed. This ribozyme demonstrates cleavage activity in vitro in the presence of Mg2+. To determine whether this ribozyme can function in vivo, HeLa CCL2 cells were transfected with a mammalian expression vector containing the ribozyme sequence. Following selection and expansion of individual transfectants, a stable clone was isolated that lacks both MGMT mRNA and protein. Molecular analysis of this cell line indicates that in vivo cleavage of MGMT mRNA is responsible for the lack of MGMT activity.

The activity of the human O6-methylguanine-DNA methyltransferase (MGMT) gene promoter was determined in eight human cell lines by measuring chloramphenicol acetyltransferase activity in a reporter gene system. MGMT promoter activities in cells that do not express MGMT (Mer-) fell within the range of activities seen in cells that do express MGMT (Mer+). The promoter region contains 11 potential binding sites for the transcription factor Sp1, but no correlation was seen between cellular Sp1 protein and MGMT promoter chloramphenicol acetyltransferase activity. Because Mer- cells are not deficient in the factors needed for transcription of MGMT, we suggest that at least two mechanisms regulate MGMT expression. One suppresses MGMT mRNA and protein in Mer- cells, and another regulates the levels of constitutive expression in Mer+ cells. Sp1 is not a limiting factor in MGMT expression.

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.

Repair of chloroethylnitrosourea (CENU)-induced precursors of DNA interstrand cross-links by O6-alkylguanine-DNA alkyltransferase (GAT or GATase) appears to be a factor in tumor resistance to therapy with this class of antineoplastic drugs. Since human GAT is highly specific for O6-guanine, yet the probable cross-link structure is N'-Guanine N3-cytosine ethane, rearrangement of the initial O6-guanine adduct via O6,N1ethanoguanine has been proposed. We suggested that GAT reaction with this intermediate would produce DNA covalently linked to protein through an ethane link from N1-guanine to the alkylacceptor site on GAT. In preliminary studies we demonstrated a covalent complex between GAT and carmustine (BCNU)-treated DNA by a precipitation assay method. We have now developed a method for isolating the reaction product of BCNU-treated synthetic 14-mer [32P]-labeled oligodeoxynucleotide and GAT using polyacrylamide gel electrophoresis. This approach can be used to characterize the adducts induced by CENUs that lead to complex formation with GAT. Results obtained to date are consistent with these adducts being precursors of DNA interstrand cross-links.

The reaction of partially purified human O6-alkylguanine-DNA alkyltransferase with 1,3-bis(2-chloroethyl)-1-nitrosourea-treated DNA produces a covalent DNA-protein complex. Evidence that the complex-forming and the alkyltransferase activities are one and the same includes their copurification, identical heat inactivation kinetics, and similar responses to inhibitors. This novel reaction thus provides a sensitive alternative assay for detecting the alkyltransferase. It also provides support for a model in which N1,O6-ethanoguanine is an intermediate in formation of chloroethylnitrosourea-induced DNA interstrand cross-links.

The formation of DNA interstrand cross-links was compared in DNA treated with either 1,3-bis(2-chloroethyl)-1-nitrosourea or 2-chloroethyl(methylsulfonyl)methanesulfonate. DNA that was pulse treated briefly with either of these drugs continued to form cross-links at 37 degrees C for over 8 h after drug removal, indicating that such DNA contained stable precursors of cross-links. When human O6-alkylguanine-DNA alkyltransferase was added to the drug-treated DNA further cross-link formation was prevented at all points during this protracted time course, indicating that these stable cross-link precursors also remained substrates for this repair enzyme. Although the pattern of 2-chloroethyl(methylsulfonyl)methanesulfonate-induced cross-link formation and susceptibility to suppression by O6-alkylguanine-DNA alkyltransferase resembled that for 1,3-bis(2-chloroethyl)-1-nitrosourea, quantitative differences in the rates of cross-link formation and in the amounts of O6-alkylguanine-DNA alkyltransferase required to suppress cross-link formation suggest that critical differences exist between these agents.

The reaction of partially purified human O6-alkylguanine-DNA alkyltransferase with 1,3-bis(2-chloroethyl)-1-nitrosourea-treated DNA resulted in formation of a DNA-protein covalent complex. Complex formation required active alkyltransferase and brief treatment of DNA with the drug. DNA lost its capacity to form the complex once drug-induced DNA interstrand cross-links were completely formed. These results are consistent with a model in which the transferase catalyzes cleavage at O6-guanine and transfer of the alkyl moiety in a putative O6, N1-ethanoguanine intermediate of cross-link formation. DNA-protein complex formation presumably results when the transferase accepts the N1-ethanoguanine-DNA structure, analogous to its acceptance of simple alkyl groups.