Exogenous O6-methylguanine inhibits adduct removal and sensitizes human cells to killing by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine

Abstract

We partially depleted the O⁶-methylguanine-DNA methyltransferase activity in four O⁶-methylguanine (O⁶-mGua) repair-proficient (Mer +) human cell strains with exogenous O⁶-mGua (2 mM for 3 h, a non-toxic regimen) and then challenged them with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT-partially depleted HT29 cells removed O⁶-mGua from DNA at about half the rate of control cells, while removal of 3-methyladeninwas unaffected. In spite of partial depletion, of MT, however, cell killing by MNNG in a colony-forming assay with HT29, A549, A498 or KD cells was not greatly affected. (This is in contrast to the dramatic potentiation of CNU cytotoxicity observed previously.) In an attempt to sensitize Mer + strains to killing by MNNG, we treated cells with O⁶-mGua following MNNG exposure (0.4 mM for 4 days), in addition to the pre-MNNG treatment of 2 mM O⁶-mGua for 3 h. This sensitized KD and HT29 cells 2-fold to killing by MNNG, based on the dose at 10% survival, but did not sensitive Mer - A1336. However, post-treatment alone was as effective as combined pre- and post-treatment in sensitizing KD cells to killing. Thus, when the O⁶-mGua post-tretment was begun, > 50% of O⁶-mGua was already removed from cell DNA. Our finding may be accounted for by at least two schemes, one in which nonlethal O⁶-mGua are removed from DNA rapidly, while potentially lethal O⁶-mGua are repaired later. The other scheme proposes that exogenous O⁶-mGua increases the lethality of a non-O⁶-mGua lesion by reducing its repair both in Mer + and Mer − cells. Both schemes are consistent with the hypothesis that O⁶-mGua may be a lethal DNA lesion in human cells.