A novel fluorometric oligonucleotide assay to measure O 6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase

DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O⁶-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.     

Repair of O(6)-methylguanine is not affected by thymine base pairing and the presence of MMR proteins

Methylation at the O(6)-position of guanine (O(6)-MeG) by alkylating agents is efficiently removed by O(6)-methylguanine-DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O(6)-MeG-inducing agents. If O(6)-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O(6)-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O(6)-MeG mispaired with thymine (O(6)-MeG/T)? (2) Do MMR proteins interfere with the repair of O(6)-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O(6)-MeG? Using an in vitro assay we show that oligonucleotides containing O(6)-MeG/T mismatches are as efficient as oligonucleotides containing O(6)-MeG/C in competing for MGMT repair activity, indicating that O(6)-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSalpha, to the in vitro repair assay did not affect the repair of O(6)-MeG/T lesions by MGMT. This indicates that the presence of MutSalpha still allows access of MGMT to O(6)-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O(6)-benzylguanine (O(6)-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O(6)-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.     

DNA repair protein O6-methylguanine-DNA methyltransferase in testis and testicular tumors as determined by a novel nonradioactive assay

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT, alkyltransferase) is an important suicide enzyme involved in defense against O6-alkylating endogenous metabolites and environmental carcinogens. It also plays a pivotal role in primary and acquired resistance of tumors to alkylating anticancer drugs targeting the O6-position of guanine (i.e., methylating and chloroethylating agents). MGMT can thus be considered a crucial biomarker for individual susceptibility to alkylating carcinogens and tumor drug resistance. This implies a need for a fast and convenient method for determination of MGMT. Routinely, MGMT is being quantified by radioactive assays which are relatively laborious. Here we report a nonradioactive MGMT enzyme-linked immunosorbent assay (ELISA) for quantification of MGMT in cell and tissue homogenates. We compared the MGMT-ELISA with the standard radioactive assay and found it to be as sensitive but less time consuming. Therefore, it represents an alternative for the quantification of MGMT in cell and tissue homogenates. We applied the assay for determining MGMT in normal and tumor tissue of testes. In both normal and tumor tissue MGMT was quite variable, ranging from zero to 1300 fmol/mg protein. In various tumor samples MGMT was lower than MGMT in the normal tissue from the same patient or was even not detectable. The MGMT-ELISA might become a useful tool for MGMT determination in clinical routine and health control.     

Role of MGMT in protecting against cyclophosphamide-induced toxicity in cells and animals

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells from the biological consequences of alkylating agents by removing alkyl groups from the O(6)-position of guanine. Cyclophosphamide and ifosfamide are oxazaphosphorines used clinically to treat a wide variety of cancers; however, the role of MGMT in recognizing DNA damage induced by these agents is unclear. In vitro evidence suggests that MGMT may protect against the urotoxic oxazaphosphorine metabolite, acrolein. Here, we demonstrate that Chinese hamster ovary cells transfected with MGMT are protected against cytotoxicity following treatment with chloroacetaldehyde (CAA), a neuro- and nephrotoxic metabolite of cyclophosphamide and ifosfamide. The mechanism by which MGMT recognizes damage induced by acrolein and CAA is unknown. CHO cells expressing a mutant form of MGMT (MGMT(R128A)), known to have >1000-fold less repair activity towards alkylated DNA while maintaining full active site transferase activity towards low molecular weight substrates, exhibited equivalent CAA- and acrolein-induced cytotoxicity to that of CHO cells transfected with plasmid control. These results imply that direct reaction of acrolein or CAA with the active site cysteine residue of MGMT, i.e. scavenging, is unlikely a mechanism to explain MGMT protection from CAA and acrolein-induced toxicity. In vivo, no difference was detected between Mgmt-/- and Mgmt+/+ mice in the lethal effects of cyclophosphamide. While MGMT may be important at the cellular level, mice deficient in MGMT are not significantly more susceptible to cyclophosphamide, acrolein or CAA. Thus, our data does not support targeting MGMT to improve oxazaphosphorine therapy.     

Development of a new application of the comet assay to assess levels of O6-methylguanine in genomic DNA (CoMeth)

O⁶-methylguanine (O⁶meG) is one of the most premutagenic, precarcinogenic, and precytotoxic DNA lesions formed by alkylating agents. Repair of this DNA damage is achieved by the protein MGMT, which transfers the alkyl groups from the O⁶ position of guanine to a cysteine residue in its active center. Because O⁶meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O⁶meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O⁶meG is not repaired, and during replication, O⁶meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O⁶meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O⁶meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O⁶meG toxicity by removing the methyl residue from the guanine. Although removal of O⁶meG makes MGMT an important anticarcinogenic mechanism of DNA repair, its activity significantly decreases the efficacy of cancer chemotherapeutic drugs that aim at achieving cell death through the action of the MMR system on unrepaired O⁶meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O⁶meG lesions after their conversion to strand breaks in proliferating MMR-proficient cells after MGMT inhibition. This functional assay allows the testing of compounds with effects on O⁶meG levels, as well as on MGMT or MMR activity, in a proliferating cell system. The expression of MGMT and MMR genes is often altered by promoter methylation, and new epigenetically active compounds are being designed to increase chemotherapeutic efficacy. The CoMeth assay allows the testing of compounds with effects on O⁶meG, MGMT, or MMR activity. This proliferating cell system complements other methodologies that look at effects on these parameters individually through analytical chemistry or in vitro assays with recombinant proteins.     

Induction of repair enzyme O6-methylguanine-DNA methyltransferase gene expression under the influence of cytokine EMAP II in human cells in vitro

The aim of our study was to investigate the effect of recombinant human cytokine EMAP II (endothelial monocyte-activating polypeptide II) on the expression of MGMT gene, encoding repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) in human cell cultures. The influence of EMAP II on cell proliferation was performed using routine MTT assay. Identification of MGMT in cell extracts was performed using Western blot analysis. We used cell lines: A102 (fibroblasts), CB-1 (umbilical cord blood stromal cells), 4BL6 (cells derived from peripheral blood). It was shown that cytokine EMAP II caused induction of MGMT expression in studied human cell lines. There was a decrease in cell number at high concentrations of this cytokine. It was found that the presence of cytokine EMAP II in serum-free growth medium leads to increasing of repair enzyme MGMT expression level in human cells in vitro.     

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(6)AA]). The defense is highly important, since O(6)AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O(6)AA 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(6)MeG) and O(6)-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O(6)MeG-induced clastogenicity and cell death require MutS alpha-dependent mismatch repair (MMR), whereas O(6)-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O(6)MeG to provoke cytotoxicity. In MGMT depleted cells, O(6)MeG 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(6)MeG 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(6)AA. 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.     

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.     

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.     

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.     

An O6-methylguanine-DNA methyltransferase-like protein from Thermus thermophilus interacts with a nucleotide excision repair protein

The major damage to DNA caused by alkylating agents involves the formation of O(6)-methylguanine (O(6)-meG). Almost all species possess O(6)-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O(6)-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O(6)-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O(6)-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O(6)-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O(6)-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways.     

Differential role of base excision repair proteins in mediating cisplatin cytotoxicity

Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions.     

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 delta, 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-1alpha), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90alpha and beta, 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.     

Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1

APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC¹²⁸⁰), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.