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

Abstract

O⁶-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against alkylating agents that generate, among other lesions, O⁶-alkylguanine in DNA (collectively termed O⁶-alkylating agents [O⁶AA]). The defense is highly important, since O⁶AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O⁶AA 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 O⁶-methylguanine (O⁶MeG) and O⁶-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O⁶MeG-induced clastogenicity and cell death require MutSα-dependent mismatch repair (MMR), whereas O⁶-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O⁶MeG to provoke cytotoxicity. In MGMT depleted cells, O⁶MeG 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, O⁶MeG 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 O⁶AA. 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.

Introduction

Mutagens in the environment [1], in tobacco smoke [2] and food [3], as well as endogenous metabolic products [4] generate reactive electrophilic species that alkylate DNA. Despite their carcinogenic potential, monofunctional alkylating agents are used, due to their cytotoxic properties, in chemotherapy of various cancers such as Hodgkin's disease, non-Hodgkin's lymphoma, malignant melanoma, neuroblastoma, soft tissue sarcomas, pancreatic (islet cell) cancer, carcinoid tumors, astrocytoma, glioblastoma and brain metastasis from solid tumors. Examples of methylating anticancer drugs are procarbazine (PCB, PCZ, N-methyl-hydrazine, Natulan^®, Matulane^®), dacarbazine (DIC, imidazole carboxamide, dimethyl-triazeno-imidazole-carboxamide, DTIC^®-Dome), streptozotocin (STZ, NSC 85998, Zanosar^®) and temozolomide (TMZ, SCHS2.365, NSC 362856, Temodal^®, Temodar^®). PCB and DTIC require metabolic activation to form their DNA-reactive metabolite [5], [6] while STZ alkylates DNA without metabolic activation [7]. TMZ undergoes spontaneous hydrolysis at physiological pH to generate the active metabolite, 5-(3-methyl triazen-1-yl)imidazole-4-carboxamide (MTIC) [8] (Fig. 1). The O⁶-chloroethylating agents, which include carmustine (BCNU, BiCNU^®), lomustine (CCNU, CeeNU^®), nimustine (ACNU) and fotemustine (Muphoran^®) are used in the treatment of glioblastoma and to a lesser extent in treating malignant melanoma, gastrointestinal and pancreatic cancer, Hodgkin's and non-Hodgkin's lymphoma. Various combinations of methylating and chloroethylating agents together with other anticancer drugs have been used [9], [10], [11] and trials are still ongoing, e.g. in glioma therapy [12].

All of the above-mentioned mutagens and chemotherapeutics react with DNA via an S_N1 mechanism to form 12 base adducts and phosphotriester (Fig. 2). The S_N1 reaction follows a first-order kinetics that is dependent on the formation of an electrophilic carbonium ion, which covalently binds to nucleophilic sites on DNA. Alkyl DNA base adducts have different stabilities. Thus, N3-methyladenine (N3MeA) and N3-methylguanine (N3MeG) are readily hydrolyzed, while other adducts, e.g. N7-methylguanine (N7MeG) are stable for longer times (T_1/2 in vitro: 40–80 h) [13]. O⁶MeG is more stable and persists in DNA in the absence of O⁶-methylguanine-DNA methyltransferase (MGMT) [14], [15], [16]. N7MeG and N3MeA are the most frequent methyl adducts comprising 80–85% and 8–18% of total alkyl adducts, respectively. However, O⁶MeG accounting for O.3 (for methyl methanesulfonate) up to 8% (for methylnitrosourea) of the total DNA methyl adducts is the most critical lesion since it is pre-mutagenic and pre-toxic. Another pre-mutagenic methylation lesion is O⁴-methylthymine (O⁴MeT), which is induced at a much lower level (<0.4%) [13]. In this review, agents inducing O⁶-alkylguanine in DNA are collectively termed O⁶-alkylating agents (O⁶AA).

O⁶MeG and O⁴MeT are repaired by MGMT (for extensive reviews on MGMT see [17], [18], [19], [20], [21]). If not repaired, O⁶MeG can give rise to cell death, chromosomal aberrations, mutations and cancer. How much O⁴MeT contributes to these end points is not precisely known. This review will focus on the important role this DNA repair protein plays in preventing these detrimental endpoints. We will also briefly discuss other alkylation damage defense and processing functions (ABH, BER, MMR) and, finally, the mechanisms behind these endpoints triggered by the MGMT repaired lesions O⁶MeG and O⁶-chloroethylguanine.