Human MGMT Research Articles

Abstract 5505: Potent redox-based inhibition of human MGMT for improving the efficacy of anticancer alkylating agents

Abstract There is an urgent need for new strategies for inhibiting the DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase) and improving the efficacy of alkylating agents. MGMT is highly expressed in human cancers and prevents the formation of cytotoxic lesions in alkylated DNA. Current clinical trials involving MGMT depletion by O6-benzylguanine (BG), although promising, are beset with severe toxicity to the bone marrow, which has necessitated the transduction of BG-resistant MGMT gene into hematopoietic stem cells. Here, we exploited the highly reactive nature of Cysteine 145 of MGMT which accepts the alkyl groups for pharmacological intervention. Cys145 has a pKa of 4.8 due to its microenvironment and is susceptible for glutathionylation (Niture & Srivenugopal, Proc of AACR 47, abst. 789, 2006) and nitrosylation (Liu et al. Cancer Res. 62, 3037, 2002). S-Thiolation and S-nitrosylation are reversible posttranslational mechanisms that gauge the intracellular redox and transduce them into functional responses. This study investigated the effect of two nontoxic small molecules which readily react with reactive cysteines, namely, the NCX-4016 (nitro-aspirin capable of S-nitrosylation) and disulfiram (capable of thiol-conjugation). NCX-4016, also called a fatty aspirin, is degraded by plasma and tissue esterases to release NO in a sustained manner. In three MGMT-proficient human cancer cell lines (HT29, T47D, and HCT116), nitro-aspirin at very low concentrations (5-10 µM) caused a 90% inhibition of MGMT activity within 1 h of exposure. Interestingly, the MGMT protein also disappeared with similar kinetics after NCX-4016 treatment; approx. 80-90% of MGMT was degraded after 5 µM NO-aspirin treatment for 2 h. These data are highly comparable or better than those reported for BG. Further, purified MGMT or tumor cell extracts exposed to NCX-4016 failed to bind the biotin-labeled BG, indicating Cys145 to be the site of nitrosylation. >60% of MGMT protein was regenerated at 24 h when NO-aspirin treated HT29 cells were post-incubated in drug-free medium, indicating a transient inhibition and rapid repletion. Disulfiram (DS), the alcohol deterrent drug, also curtailed MGMT activity in HT29 and HCT116 cells with a 20 h 400 µM treatment causing a 95% inhibition. DS at 200 µM induced about 70% degradation of MGMT at 12 h. Other redox-sensitive proteins such as the wild-type and mutant p53, NF-κB, and ubiquitin E1 were all degraded by DS in a dose- dependent manner. Studies to evaluate MGMT inhibition by NO-aspirin and DS in animal tissues are in progress. Because NO-aspirin, is non-toxic (IC50>500 µM for cell lines), yields a chemopreventive by-product, unlikely to elicit tumor resistance, and is lipophilic enough to cross the BBB, we believe it can be exploited for glioma therapy. Our studies show that the redox-regulated proteins are ‘druggable’ and highlight options for redox- driven therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2011-5505

Comparison of MGMT and ERCC₂ expression in temozolomide for the treatment of malignant glioma drug resistance and their genetic relationship

To study the impact of two human glioma tissue resistance genes MGMT and ERCC(2) on the temozolomide-based treatment of malignant gliomas and detect the relationship of their expressions. A total of 58 malignant glioma patients aged 19 - 68 years old receiving a chemotherapy of temozolomide were followed up and classified as non-sensitive group (n = 30) and sensitive group (n = 28). Immunohistochemistry was employed to detect the expression rates of MGMT and ERCC(2). And the correlation between the expressions of two genes was analyzed by immunohistochemistry and RT-PCR (reverse transcription-polymerase chain reaction). The expression rates of MGMT and ERCC(2) were 10.71% and 3.57% in the sensitive group and 63.33% and 56.67% in the non-sensitive group. It had an obvious correlation with the expressions of MEGT and ERCC(2) through an analysis of immunohistochemistry and RT-PCR (both P < 0.01). The expressions of MGMT and ERCC(2) in the sensitive group are markedly lower than those in the non-sensitive group. The expression of two genes may be related to tumor prognosis. Maybe these two genes have an intrinsic link between their expressions. Both participate in the repair of cellular DNA damage and the formation of tumor drug resistance. And the prognosis has obvious relevance.

342 POSTER 2-[18F] fluoro-2-deoxy-d-glucose positron emission tomography is an early biomarker for tumor growth inhibition of human Colo205 xenografts by the novel and selective CENP-E inhibitor, GSK923295A

Recent evidence suggests that human O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT–Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase δ, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21waf1/cip1), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1α), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90α and β, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

A phase I trial of lomeguatrib and irinotecan in metastatic colorectal cancer

2535 Background: The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) contributes to cellular resistance to irinotecan (IR) and its metabolite SN-38. In human cancer cell lines MGMT expression correlates with SN-38 sensitivity, inactivation of MGMT enhances cell killing and increasing MGMT expression by transfection increases resistance to IR. This study evaluated the safety, tolerability and pharmacokinetics (PK) and pharmacodynamic (PD) effects of lomeguatrib (LM), an MGMT inactivator, in combination with IR. Methods: Patients with stage 4 colorectal cancer, performance status 0–2 and adequate bone marrow and biochemical function took part in the trial, which followed a standard 3 patient cohort dose escalation design. LM was given daily PO for 5 days, with IR by IVI on day 4 of each 21 day cycle. Starting doses were 10 mg/day LM and 350 mg/m2 IR. Tumor response to treatment was measured by RECIST criteria. Blood was taken for PK &amp; PD measurements in cycle 1. Results: 24 patients (14M/10F; median age 62) were enrolled. The first dose level was not tolerated, with 2 of 3 patients experiencing dose limiting haematological toxicity. The IR dose was decreased to 250 mg/m2 for subsequent cohorts, and the LM dose escalated from 10 to 80 mg/day. With LM 80 mg/day a final 6 patient cohort received 300 mg/m2 IR, establishing this as the maximum tolerated dose. Non-hematological toxicity matched that of single agent IR. No partial responses have been seen amongst 22 evaluable patients. 10 patients had prolonged disease stabilisation, completing all 6 treatment cycles, with another 3 patients still on treatment. There was no PK interaction between the drugs. Conclusion: LM and IR is well tolerated, requiring a modest reduction in the single agent dose of IR. Clinical activity, in this pre-treated population, is modest. PD data on MGMT levels and DNA-topoisomerase 1 complex formation in peripheral blood mononuclear cells, measured by bioassay and RIA respectively, will be presented. No significant financial relationships to disclose.

MGMT: Key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents

O 6-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against alkylating agents that generate, among other lesions, O 6-alkylguanine in DNA (collectively termed O 6-alkylating agents [O 6AA]). The defense is highly important, since O 6AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O 6AA induced DNA damage is subject to repair, which is executed by MGMT, AlkB homologous proteins (ABH) and base excision repair (BER). Although this review focuses on MGMT, the mechanism of repair by ABH and BER will also be discussed. Experimental systems, in which MGMT has been modulated, revealed that O 6-methylguanine (O 6MeG) and O 6-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O 6MeG-induced clastogenicity and cell death require MutSα-dependent mismatch repair (MMR), whereas O 6-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O 6MeG to provoke cytotoxicity. In MGMT depleted cells, O 6MeG induces apoptosis almost exclusively, barely any necrosis, which is presumably due to the remarkable ability of secondarily formed DNA double-strand breaks (DSBs) to trigger apoptosis via ATM/ATR, Chk1, Chk2, p53 and p73. Depending on the cellular background, O 6MeG activates both the death receptor and the mitochondrial apoptotic pathway. The inter-individual expression of MGMT in human lymphocytes is highly variable. Given the key role of MGMT in cellular defense, determination of MGMT activity could be useful for assessing a patient's drug sensitivity. MGMT is expressed at highly variable amounts in human tumors. In gliomas, a correlation was found between MGMT activity, MGMT promoter methylation and response to O 6AA. Although the human MGMT gene is inducible by glucocorticoids and genotoxins such as radiation and alkylating agents, the role of this induction in the protection against carcinogens and the development of chemotherapeutic alkylating drug resistance are still unclear. Modulation of MGMT expression in tumors and normal tissue is currently being investigated as a possible strategy for improving cancer therapy.

MGMT inhibitors—The Trinity College–Paterson Institute experience, a chemist's perception

The DNA repair protein, O 6-alkylguanine-DNA alkyltransferase (MGMT) can confer resistance to the cancer chemotherapeutic effects of the class of DNA damaging drugs generally referred to as the O 6-alkylating agents. Inactivation of MGMT is thus a practical approach to improving the efficacy of such agents. An account is given of the collaboration between groups at Trinity College, Dublin and the Paterson Institute, Manchester which led to the development of the MGMT inactivating drug, Patrin™ (PaTrin-2, Lomeguatrib). The development of a simpler method of synthesis of O 6-arylmethylguanines opened up the way to make a series of O 6-heteroalkylmethyl analogues of the archetypal MGMT pseudosubstrate, O 6-methylguanine. Of these, the furfuryl and thenyl compounds were the most active against recombinant Human MGMT in an in vitro assay. The 4-bromothenyl derivative was chosen for clinical trial as the most active compound. The MGMT active site tolerates O 6-substituted guanines where the side chain can be quite large, but does not tolerate those with an aromatic or heteroaromatic ring with an ‘ ortho’ substituent.

Hypermethylation of RASSF1A in Human and Rhesus Placentas

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

Chemopreventative strategies targeting the MGMT repair protein: augmented expression in human lymphocytes and tumor cells by ethanolic and aqueous extracts of several Indian medicinal plants

O6-alkylguanines are potent mutagenic, pro-carcinogenic and cytotoxic lesions induced by exogenous and endogenous alkylating agents. A facilitated elimination of these lesions by increasing the activity of O6-methylguanine-DNA methyltransferase (MGMT) is likely to be a beneficial chemoprevention strategy, which, however, has not been examined. Because, a marginal enhancement of this protein may be adequate for genomic protection, we studied alterations in MGMT activity and expression in human peripheral blood lymphocytes and cancer cell lines induced by water-soluble and alcohol-soluble constituents of several plants with established antioxidant and medicinal properties. Both the ethanolic and aqueous extracts from neem (Azadirachta indica), holy basil (Ocimum sanctum), winter cherry (Withania somnifera), and oregano (Origanum majorana) increased the levels of MGMT protein and its demethylation activity in a time-dependent manner with a maximum of 3-fold increase after 72-h treatment. The extracts from gooseberry (Emblica officinalis), common basil (Ocimum basilicum), and spearmint (Mentha viridis) were relatively less efficient in raising MGMT levels. Increased levels of MGMT mRNA accounted at least, in part, for the increased activity of the DNA repair protein. The herbal treatments also increased glutathione S-transferase-pi (GSTP1) expression, albeit to a lesser extent than MGMT. These data provide the first evidence for the upregulation of human MGMT by plant constituents and raise the possibility of rational dietary approaches for attenuating alkylation-induced carcinogenesis. Further, they reveal the putative antioxidant responsiveness of the MGMT gene in human cells.

Genetic Variation at the Human MGMT Locus and its Biological Consequences

O6-alkylguanine-DNA alkyltransferase (MGMT) repairs DNA adducts that result from alkylation at the O6 position of guanine. These lesions are mutagenic and toxic and can be produced by a variety of agents ranging from carcinogens present in cigarette smoke to drugs used in cancer chemotherapy. There is a considerable amount of interindividual variation of MGMT activity and recent work has uncovered a series of polymorphisms that affect protein activity or are associated with differences in expression levels. On the other hand, current evidence for an association with cancer risk is tenuous: while some studies find such associations, others fail to support them. However, all published studies are based on small sample numbers. Not yet addressed are issues such as the relationship between sequence variation and the extent of the side effects of chemotherapy with O6-alkylating agents. This review summarises the current state of knowledge in these areas.

A novel DNA damage recognition protein in Schizosaccharomyces pombe

Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.

459. Murine Study To Evaluate Long-Term Transgene Expression of the MSCV-MGMTP140K wc Retroviral Vector in Support of a Phase I Gene Transfer Trial-Limitations of the Murine Model as a Pre-Clinical Tool

Retroviral gene therapy vectors expressing the MGMTP140K transgene have been shown to protect hematopoietic cells from toxicity associated with a combined cancer treatment using 6-benzylguanine (6-BG) and an alkylating agent such as Temozolomide (TEM). In a Phase I gene transfer trial, high grade astrocytoma patients having poor prognoses using standard therapies, will undergo escalating dose treatments with 6-BG and TEM following MGMTP140K gene transfer into autologous hematopoietic stem cells. Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted preclinical model defined to assess their safety, and, in particular their risk related to insertional mutagenesis. This study was designed as a murine pre-clinical study to assess the long term effects of the transduction of hematopoietic cells with the retroviral vector to be used in the clinical trial, MSCV-MGMTP140Kwc.

Proteomic analysis of human O6-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

Recent evidence suggests that human O 6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT–Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase δ, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21 waf1/cip1), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1α), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90α and β, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

Inactivated MGMT by O6-Benzylguanine is associated with prolonged G2/M arrest in cancer cells treated with BCNU

HCT116 and HCT15 cells that highly express O(6)-methylguanine-DNA-methyltransferase (MGMT) displayed a transient cell cycle G2/M arrest in response to exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone; however, 70-80% of cells were arrested in G2/M after treatment with O(6)-benzylguanine (BG) and BCNU. Cells accumulated in G2/M showed elevated levels of an inactive form of cyclin B1/p-Cdc2 (Tyr15) complex that was not associated with activation of Chk1/p-Cdc25C and was independent of p53/p21 status. The most prominent feature of cell death was the appearance of enlarged and multinucleated cells that was related to the inhibition of mitotic entry. In contrast, BG-resistant cell lines, HCT116 BBR and HCT15 BBR cells that contain mutations K165E and K165N of MGMT, respectively, displayed a normal cell cycle progression with a slight and transient increase in G2/M arrest at 24 h after treatments with either BCNU alone or BG combined with BCNU. The differences in the ability to progress toward G2/M after treatment with BG and BCNU between cells expressing wild-type MGMT and mutated MGMT were confirmed in CHO cells transfected with human wild type and K165E mutant MGMT cDNA, respectively. Thus, our findings suggest that BG-inactivated MGMT may be linked to cell signaling events, forcing cells into a permanent G2/M arrest in response to the DNA damages induced by BCNU.

404. Cloning and Expression of Canine O6-Methylguanine–DNA Methyltransferase Gene in Ex Vivo Cultured Cells

The human O6-methylguanine-DNA-methyltransferase (MGMT) gene and its mutants have been used previously for in vivo selection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination with O6-benzylguanine (BG). To allow similar in vivo selection in dogs, without the risk of inducing of an immune response, we have cloned the canine MGMT drug resistance gene (GenBank Accession No. AY423553). Comparison of canine and human MGMT coding regions indicated that there was about 62% amino acid identity and 78% similarity between the two MGMTs. The canine MGMT was also longer by 9 amino acids. Proline at position 140 and the surrounding amino acids of the human MGMT were highly conserved in the canine sequence. To determine if mutation of the proline residue at position 144 to lysine (P144K) in the canine MGMT would provide a similar advantage for selection with BG and BCNU of transduced cells as the human P140K mutant, we created the corresponding P144K mutant of canine MGMT. Bicistronic Moloney murine leukemia virus and human immunodeficiency type 1 vectors were used to express separately canine and human MGMTs in cultured canine CTAC cells. Drug-resistance assays demonstrated that the wild-type and the P144K mutant canine MGMTs provided resistance to the BCNU or BG together with BCNU selection, respectively that was comparable to the human MGMT counterparts.

Lack of association between Caucasian lung cancer risk and O6-methylguanine-DNA methyltransferase-codon 178 genetic polymorphism

The formation of DNA adducts is thought to be a critical step for the induction of chemically induced cancer. O 6-Methylguanine-DNA methyltransferase (MGMT) is a ubiquitously expressed enzyme that repairs DNA adducts formed by alkylating carcinogens. Thus, genetic polymorphisms of the MGMT that could result in differences in MGMT activity are potential risk factors for cancer. In the present study, we established a convenient and reliable genotyping method for the MGMT codon 178 polymorphism, a Lys (A AG) to Arg (A GG) substitution, using restriction fragment length polymorphism (RFLP), and studied differences in the distribution of this polymorphism in 92 Caucasian lung cancer patients and 85 controls. Frequencies of the “A” and “G” alleles ( MGMT codon 178, A AG and A GG, respectively) were 0.91 and 0.09, respectively. The genetic polymorphism of the MGMT codon 178 was linked with that of the MGMT codon 143 ( P<0.05). The distribution of the MGMT codon 178 genetic polymorphism was not significantly different between lung cancer patients and controls. Thus, our study suggests that the MGMT codon 178 (and possibly 143) polymorphisms do not appear to markedly affect lung cancer risk for this population. In addition, we found an apparent 10 bp-deletion in the intron before exon 5 by DNA sequencing. Because this “deletion” was observed in all sequenced samples ( N=20), the previously reported human (Caucasian) MGMT gene sequence should be revised to exclude this 10 bp segment.

DNA repair and cancer: lessons from mutant mouse models.

DNA damage, if the repair process, especially nucleotide excision repair (NER), is compromised or the lesion is repaired by some other error-prone mechanism, causes mutation and ultimately contributes to neoplastic transformation. Impairment of components of the DNA damage response pathway (e.g., p53) is also implicated in carcinogenesis. We currently have considerable knowledge of the role of DNA repair genes as tumor suppressors, both clinically and experimentally. The deleterious clinical consequences of inherited defects in DNA repair system are apparent from several human cancer predisposition syndromes (e.g., NER-compromised xeroderma pigmentosum [XP] and p53-deficient Li-Fraumeni syndrome). However, experimental studies to support the clinical evidence are hampered by the lack of powerful animal models. Here, we review in vivo experimental data suggesting the protective function of DNA repair machinery in chemical carcinogenesis. We specifically focus on the three DNA repair genes, O(6)-methylguanine-DNA methyltransferase gene (MGMT ), XP group A gene (XPA) and p53. First, mice overexpressing MGMT display substantial resistance to nitrosamine-induced hepatocarcinogenesis. In addition, a reduction of spontaneous liver tumors and longer survival times were evident. However, there are no known mutations in the human MGMT and therefore no associated cancer syndrome. Secondly, XPA mutant mice are indeed prone to spontaneous and carcinogen-induced tumorigenesis in internal organs (which are not exposed to sunlight). The concomitant loss of p53 resulted in accelerated onset of carcinogenesis. Finally, p53 null mice are predisposed to brain tumors upon transplacental exposure to a carcinogen. Accumulated evidence in these three mutant mouse models firmly supports the notion that the DNA repair system is vital for protection against cancer.

834 Second-line treatment of disseminated malignant melanoma with Fotemustine

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O6-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer− (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC50 values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N7 guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis

To examine involvement of mismatch repair system in alkylation-induced apoptosis and mutagenesis, cell lines defective in the Mgmt gene encoding a DNA repair enzyme, O 6-methylguanine-DNA methyltransferase, and/or the Mlh1 gene encoding a protein involved in mismatch repair were established from gene-targeted mice. Mgmt −/− cells are hypersensitive to the killing effect of N-methyl- N-nitrosourea (MNU) and this effect of MNU was overcome by introducing an additional mutation in the Mlh1 gene. Mgmt −/− Mlh1 −/− cells are more resistant to MNU than are wild-type cells. When the human Mgmt cDNA sequence with a strong promoter was introduced, the wild-type cells acquired the same high level of resistance to MNU as that of Mgmt −/− Mlh1 −/− cells. Although no apparent increase in MNU-induced mutant frequency was observed in such methyltransferase-overproducing wild-type cells, mutant frequency of Mgmt −/− Mlh1 −/− cells became 10-fold higher after being treated with MNU. Mgmt −/− Mlh1 +/− cells carrying approximately half the normal level of MLH1 protein showed a normal level of spontaneous mutant frequency, yet were still highly responsive to the mutagenic effect of the alkylating carcinogen. This haploinsufficient character of Mlh1 mutation was also observed in cell survival assays; Mgmt −/− Mlh1 +/− cells were as resistant to MNU as were Mgmt −/− Mlh1 −/− cells. While caspase-3 was induced in Mgmt −/− Mlh1 +/+ cells after treatment with MNU, no induction occurred in Mgmt −/− Mlh1 +/− cells or in Mgmt −/− Mlh1 −/− cells. The cellular content of MLH1 protein seems to be critical for determining if damaged cells enter into either a death or mutation-inducing pathway. The haploinsufficient phenotype of Mlh1-heterozygous cells may be explained by competition in heterodimer formation between MLH1 homologues.

DNA Alkylation and Repair in the Large Bowel: Animal and Human Studies

O6-methylguanine (O6-MeG), a procarcinogenic DNA adduct that arises from exposure to methylating agents, has been detected in human colorectal DNA at levels comparable to those that cause adverse effects in model systems. O6-MeG levels vary within the colon, being higher in the cancer-prone regions of the large bowel. In rats and mice, O6-MeG persistence in colon DNA is associated with the induction of colon tumors after treatment with methylating agents. These tumors frequently contain K-ras GC-->AT transition mutations, which is consistent with the mutagenic properties of O6-MeG: such mutations are also commonly found in human colorectal cancers. O6-Alkylguanine adducts are removed by the DNA repair protein, O6-alkylguanine DNA-alkyltransferase (MGMT). MGMT overexpression in transgenic mice reduces the formation of K-ras GC-->AT mutations and tumors induced by methylating agents. Interindividual variations in human colon MGMT activity are large and large bowel tumors can occur in regions of low activity. Low MGMT activity in normal mucosa has been associated with the occurrence of K-ras GC-->AT mutations, whereas reduced MGMT expression and an increased frequency of K-ras GC-->AT mutations in colorectal cancers have been linked to MGMT promoter methylation. MGMT activity is also lower in adenomas than in adjacent normal tissue but only in those adenomas with this specific mutation. These results are entirely consistent with the hypothesis that GC-->AT mutations in the K-ras oncogene result from the formation and persistence of O6-alkylguanine lesions in colorectal DNA. Human exposure to endogenous or exogenous alkylating agents may thus be an environmental determinant of colorectal cancer risk.

Improvement of combination chemotherapy tolerance by introduction of polycistronic retroviral vector drug resistance genes MGMT and MDR1 into human umbilical cord blood CD34 + cells

We obtained a full-length cDNA fragment encoding human O 6-methylguanine-DNA-methyltransferase (MGMT) from the liver tissue of a patient with cholelithiasis by RT-PCR and confirmed by DNA sequencing. The polycistronic retrovirus vector G1Na-MGMT-Neo r-IRES-MDR1 was constructed and verified by restriction endonuclease analysis and DNA sequencing. The vector was transfected into packaging cells GP+E86 and PA317 by the LipofectAMINE method. Cord blood CD34 + cells were transfected with the supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hematopoietic growth factors. PCR, RT-PCR, Southern Blot, Western Blot, FACS and MTT analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34 + cells and expressed efficiently. The transgene cord blood CD34 + cells conferred 5.8–6.3-fold stronger resistance to P-glycoprotein effluxed drugs and 5-fold to BCNU than untransduced cells. The polycistronic retrovirus vector mediated transfer of two different types of drug resistance genes into human cord blood CD34 + cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.