The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) is inducible by genotoxic stress. MGMT induction results from transcriptional activation of the MGMT gene which is a speci®c response to DNA damage. A possible factor involved in triggering MGMT induction might be p53, because both p53 and MGMT are activated by DNA breaks. To study the e ect of p53 on induction of the MGMT gene, we compared the presence of functional wild-type (wt) and mutant p53 with MGMT expression level in various mouse ®broblasts and rat hepatoma cell lines upon genotoxic treatment. Cells which responded to ionizing radiation (IR) by MGMT induction displayed functional p53, whereas in cells not expressing wt p53, MGMT induction was not observed. Also, the cloned MGMT promoter was inducible by IR upon transfection into p53 wt cells, but not in cells de®cient for p53. Thus, expression of wt p53 appears to be required for induction of MGMT mRNA and protein by IR. On the other hand, transfection of a MGMT-promoter-CAT construct together with p53 (either wt or mutant) in cells expressing wt p53 markedly reduced the basal activity of the MGMT promoter whereas cotransfection with a p53 antisense construct slightly increased MGMT promoter activity. Furthermore, cotransfection of MGMT promoter with wt or mutant p53 in p53 wt cells reduced radiation evoked MGMT promoter induction. Thus, transfection mediated high level expression of p53 has inhibitory e ect both on basal MGMT promoter activity and its activation by IR. The results give evidence for involvement of p53 in DNA damage-induced MGMT promoter activation.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.

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.

Methylating and chloroethylating agents have been used in the therapy of various kinds of cancer for a decade. 1 Methylating drugs (e.g., procarbazine, dacarbazine, streptozotocin and temozolomide) and chloroethylating agents (e.g., carmustine, lomustine, semustine and fotemustine) alkylate DNA at various sites, among them the O 6 position of guanine. The resulting DNA lesions, i.e., O 6 -methylguanine and O 6 -chloroethylguanine, are highly pro-mutagenic 2 and pro-carcinogenic 3,4 and act as a trigger of cytotoxicity 5,6 and apoptosis. 7,8 Cytotoxicity and apoptosis caused by O 6 -methylguanine are mediated by mismatch repair, 9,10 whereas when caused by O 6 -chloroethylguanine the result is chemical re-arrangement of the chloroethyl group forming cross-links between N1 of guanine and N3 of cytosine. 11 These cross-links are supposed to block DNA replication and trigger the cytotoxic response.The primary cytotoxic lesions O 6 -methylguanine and O 6 -chloroethylguanine are subject to repair by the DNA-repair protein O 6 -methylguanine-DNA methyltransferase (MGMT; also designated alkyltransferase), which transfers the alkyl group from the O 6 position of guanine onto an internal cysteine residue in its active site, leading to an alkylthioether that cannot be re-activated. Thus, guanine in the DNA is restored and the MGMT molecule inactivated. Due to the suicide mechanism of action, the repair reaction is stoichiometric, depending on the initial number of MGMT molecules per cell and the rate of resynthesis upon alkylation. 12,13 The MGMT-repair protein plays a key role in the resistance of tumor cells to methylating and chloroethylating drugs. This has been demonstrated by comparison of naturally occurring tumor cell variants, 14 by transfection experiments in which MGMT was over-expressed, 6 by MGMT knockout strategies 15 and by MGMTdepletion experiments with anti-sense oligonucleotides 16 or specific inhibitors. 17,18 In isogenic cell lines transfected with MGMT cDNA, the level of resistance was a linear function of cellular MGMT activity up to a maximal saturation level, where nonspecific effects of the drugs come into play. A significant protective effect of MGMT can be achieved with quite low expression levels. 6

To study molecular aspects of cytotoxicity of the anticancer drug b-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since b-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D 10 values) to the cytotoxic effects of b-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by b-D-glucoseifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.