Repair of O6-carboxymethylguanine adducts by O6-methylguanine-DNA methyltransferase in human colon epithelial cells

Abstract

The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine adduct back to guanine. In this context, it may protect against colorectal cancer (CRC) formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-methylguanine (O6-MeG) and O6-carboxymethylguanine (O6-CMG) adducts. It is well-established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCEC). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated CRC.

Discrimination between Different DNA Lesions by Monitoring Single-Molecule Polymerase Stalling Kinetics during Nanopore Sequencing

“…O 6 -alkG results from the reaction of electrophilic alkylating agents with the O 6 -position of guanine. O 6 -Carboxymethylguanosine (O 6 -CMG) and O 6 -methylguanosine (O 6 -MeG) ( Figure A), which are mutagenic and carcinogenic, are two intensively studied O 6 -alkG lesions. , Their occurrence in humans is found to be positively associated with the consumption of red or processed meat and may lead to colorectal cancer. − An AP site (Figure A), another common DNA lesion, is formed by alkylating agents via the initiation of the base excision repair (BER) pathway. , In this pathway, the AP site acts as an important intermediate generated via excision of the damaged nucleobases of DNA . In addition, the AP site can result from other endogenous and exogenous sources including spontaneous hydrolysis of nucleobases in DNA .…”

“…O 6 -Carboxymethylguanosine (O 6 -CMG) and O 6 -methylguanosine (O 6 -MeG) (Figure 1A), which are mutagenic and carcinogenic, are two intensively studied O 6 -alkG lesions. 12,13 Their occurrence in humans is found to be positively associated with the consumption of red or processed meat and may lead to colorectal cancer. 13−15 An AP site (Figure 1A), another common DNA lesion, is formed by alkylating agents via the initiation of the base excision repair (BER) pathway.…”