CONTRIBUTION OF O-6-ALKYLGUANINE AND N-ALKYLPURINES TO THE FORMATION OF SISTER-CHROMATID EXCHANGES, CHROMOSOMAL-ABERRATIONS, AND GENE-MUTATIONS - NEW INSIGHTS GAINED FROM STUDIES OF GENETICALLY-ENGINEERED MAMMALIAN-CELL LINES

O-6-methyl- and O-6-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkylating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O-6-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O-6-methylguanine-DNA methyltransferase (MGMT). Cells that overexpress MGMT (or the bacterial Ada protein) gained resistance to the formation of alkylation-induced SCEs and aberrations, as compared to MGMT deficient cells. A correlation was apparent between the level of protection for SCEs and cell killing, indicating that both phenomena are interrelated. The protective effects were dependent on the level of MGMT expression, the agent used for alkylation, and cell cycle progression. Our data suggest that at least 2 kinds of lesions are responsible for SCE and aberration formation, namely, O-6-alkylguanine and one or various N-alkylation products. The probability that O-6-methylguanine is converted into cytogenetic effects has been estimated to be about 1:30 for SCEs, and 1:147,000 and 1:22,000 for chromosomal aberrations in the first and second post-treatment mitosis, respectively. The induction of SCEs and likely also of aberrations by O-6-methylguanine requires two replication cycles and is supposed to involve the formation of secondary DNA lesions. Increased repair of 3-methyladenine and 7-methylguanine in CHO cells that overexpress the N-methylpurine-DNA glycosylase (MPG) after transfection with the human MPG-cDNA did not give rise to protection against methylation-induced SCEs and aberrations, probably because of incomplete excision repair. MPG overexpressing cells reacted even more sensitively to methylating agents, suggesting apurinic sites formed as a result of MPG action to be SCE and aberration-inducing lesions. (C) 1993 Wiley-Liss, Inc.