Genome stability is affected by environmental, endogenous, and inherited factors. DNA alkylating agents are genotoxic chemicals that cause DNA damage, playing a crucial role in mutagenesis and carcinogenesis [1]. Depending on their cytotoxicity, DNA guanine O6-alkylating agents such as carmustine (BCNU), nimustine (ACNU), lomustine (CCNU), and temozolomide (TMZ) are used in chemotherapy of particular types of cancer, such as glioblastoma, Hodgkin’s disease, and malignant melanoma (Table 1) [2].

Genetic alterations through DNA damage in proto-oncogenes and tumor suppressor genes are considered initiating events in chemical carcinogenesis [3]. O6-alkylguanine (O6-AlkylG), such as O6-methylguanine (O6-MG), causes highly toxic and mutagenic lesions with impactful biological effects, including DNA point mutations, chromosomal aberrations, sister chromatid exchange (SCE), tumorigenesis, and cell death [4]. The O6-MG adduct mispairs thymine instead of the correct cytosine, inducing a GC→AT transition mutation [5]. In fact, mutations in tumor suppressor genes such as p53, can trigger malignant cell transformations [6], [7], [8]. Therefore, the continuous accumulation of O6-MG during cell replication is the rate-limiting step in tumor initiation. Another unique DNA damage is DNA cross-linking generated by chloroethylating agents, resulting in the chloroethylation of guanine at the O6 position, followed by intramolecular cyclization of guanine at the N1 position, as shown in Fig. 1 [2]. This unstable intermediate reacts with cytosine at the N3 position of the opposite DNA strand, forming DNA interstrand cross-links (ICLs) [9]. If not repaired, these DNA adducts inhibit strand separation during DNA replication and transcription, eventually leading to cell death [6].

Mammalian cells exposed to alkylating agents can initiate defensive pathways by activating diverse proteins related to cell cycle checkpoint control, apoptosis, and DNA repair [3]. These key cellular processes are crucial for recognizing and repairing DNA damage, to maintain genomic stability and avoid mutations and/or cell death [10].

O6-methylguanine-DNA methyltransferase (MGMT/AGT), an important DNA repair protein, mainly restores DNA alkylation damage. MGMT repairs O6-MG or other O6-AlkylG adducts via a ‘Glu172-His146-water-Cys145’ hydrogen bond network (Fig. 2), the adducted groups are transferred to the Cys145 residue in a stoichiometric manner [11]. This irreversible reaction results in an MGMT conformational change, triggering its degradation through the ubiquitin–proteasome system [12], [13], and conveying its denomination as a “suicide enzyme” [14]. MGMT rarely repairs O4-methylthymine (O4-MT) in mammalian cells because of the unmatched nature of their conformational structures [15]. Nonetheless, MGMT repairs larger DNA adducts. MGMT does not only transfer the chloroethyl group from O6-chloroethylguanine (O6-ClEtG) to the Cys145 residue, but also reacts with the N1,O6-ethanoguanine (N1,O6-EtG) intermediate prior to ICL formation, thereby preventing detrimental DNA cross-linking (Fig. 1) [2], [16], [17]. Additionally, the Fanconi anemia (FA) pathway also plays an important role in replication-dependent ICL repair [18]. MGMT repair capacity is directly proportional to the number of active molecules per cell and the resynthesis rate. Therefore, owing to MGMT exhaustion, the repair rate may present as a rapid initial phase followed by a slower stage [6].

The ability of MGMT to repair O6-MG-induced lesions in normal cells is important for the prevention of malignant transformation, the activation of proto-oncogenes, and apoptosis caused by methylating agents [19]. Transgenic and knockout mouse models highlight the protective role of MGMT against O6-MG-induced cancer development [20], [21], [22], [23].

The most used guanine O6-alkylating agents used in cancer treatment are listed in Table 1. The efficacy of tumor treatment can be impaired by the activity of MGMT [24]. When O6-alkylating agents are used to treat tumor cells with relatively high MGMT expression, MGMT can enable tumor cells to escape apoptosis, eventually leading to drug resistance [6]. Currently, the combination of MGMT inhibitors, such as O6-benzylguanine (O6-BG), and chemotherapeutics is used to increase the cytotoxicity of alkylating agents against tumor cells [25]. However, MGMT inhibitors also sensitize normal tissues to chemotherapy, giving rise to harmful effects, such as myelosuppression, leukemia, and myelodysplastic syndrome [26]. MGMT also plays a crucial cellular defense role in proliferating tissues, notably in stem cells. Therefore, transferring the MGMT gene into blood stem cells before chemotherapy could confer protection against chemotherapy-induced side effects [25], [27]. Strategies aimed at enhancing MGMT expression in non-target tissues show great prospects. Local drug delivery and prodrug approaches are emerging methods being explored [25]. MGMT is important for protection of normal cells from the adverse effects of chemotherapeutic alkylating agents, however, the MGMT status becomes a major factor for tumor resistance when using guanine O6-alkylating drugs.

The assessment of MGMT level or activity is helpful for epidemiological diagnosis and cancer treatment. Currently, methods for detecting MGMT expression or activity include immunoblotting, methylation-specific polymerase chain reaction assay (MSP), HPLC-MS, enzyme-linked immunosorbent assay (ELISA), radioactive labeling assay, and fluorescein-labeling assay [28], [29], [30], [31], [32].

This review explores the dual role of MGMT in tumor defense and treatment in the past decades. Tissues with low MGMT activity tend to be more susceptible to guanine O6-alkylating agents, which may lead to enhanced chemotherapeutic efficacy; however, these agents also induce stronger side effects, such as bone marrow suppression [33]. Higher MGMT activity blocks tumor initiation induced by alkylating agents but also promotes drug resistance in tumor treatment, as shown in Fig. 3. The dual property of MGMT is not only a critical factor in the defense against tumorigenesis, but also the key to relieving pain (and side effects) in patients undergoing chemotherapy. Therefore, studying the role of MGMT has determinant potential for new strategies for cancer prevention and treatment.