In previous studies, we have observed distinct cycle phase susceptibilities for toxicity and neoplastic transformation in 10T1/2 cells treated with methylating chemicals. Although G1 and S phase cells are susceptible to cytotoxicity (and, apparently, mutation), only S phase cells are markedly susceptible to neoplastic transformation. Although the period of maximal susceptibility for all of these cellular responses is S phase, our observations suggest that different types of DNA damage and/or different rates of DNA repair, and different DNA targets are responsible for different pathological responses. In previous studies we have that total alkylations produced in DNA by MNNG do not vary with the cell cycle, whereas, repair of selected specific adducts varies widely both in rate and with cycle phase. We hypothesize that cytotoxicity and mutation are produced by a relatively common adduct(s) that is (are) repaired slowly enough so that many lesions introduced during G1 persist into S phase. We posit that transformation is caused either by a lesion(s) that is (are) repaired rapidly so that lesions produced in G1 are repaired virtually completely before S phase, or by a unique lesion introduced into DNA only during S phase. Furthermore, we also hypothesize that both neoplastic transformation and mutation are related to damage incurred in specific DNA sites (targets), whereas cell death results from more random damage to DNA.
The aim of this proposal is to test these hypotheses in synchronized C3h 10T1/2 cells exposed in vitro to chemicals that introduce methyl or ethyl groups (MMS, MNNG, EMS, ENNG) into DNA by Sn1 of Sn2 reations, producing different spectra of adducts. Quantitation of DNA damage and repair and of cytotocxicity, mutation, and neoplastic transformation when populations of 10T1/2 cells are exposed to chemicals at specific points in the G1 and S phases of the cycle should allow us to corroborate or refute our hypotheses.
