Repair of DNA alkylation products formed in 9L cell lines treated with 1-(2-chloroethyl)-1-nitrosourea

Abstract

The purpose of this study has been to measure the formation and repair of individual DNA alkylation products in 9L, 9L-2 and BTRC-19 cell lines after treatment with 1-(2-chloroethyl)-1-nitrosourea (CNU). The levels of seven DNA adducts N7-(2-hydroxyethyl)-guanine, N7-(2-chloroethyl)-guanine; 1,2-(diguan-7-yl)-ethane, N1-(2-hydroxyethyl)-2-deoxyguanosine, 1-( N1-2-deoxyguanosinyl), 2-( N3-2-deoxycytidyl)-ethane, O 6-(2-hydroxyethyl)-2-deoxyguanosine and phosphotriesters were separated by HPLC and quantified by liquid scintillation counting. The levels of N7-(2-hydroxyethyl)-guanine, N7-(2-chloroethyl)-guanine; O 6-(2-hydroxyethyl)-2-deoxyguanosine and phosphotriesters were not significantly different in the three glioma lines. Furthermore, comparison of the levels of these products in treated cells with the levels formed in purified DNA suggest that they were not actively repaired over the 6 h interval. The levels of 1,2-(diguan-7-yl)-ethane and N1-(2-hydroxyethyl)-2-deoxyguanosine were reduced in 9L-2 and significantly reduced in BTRC-19 ( P=0.003) compared to 9L. Analysis of the data suggests that the reduction in the level of N1-(2-hydroxyethyl)-2-deoxyguanosine was due to repair of its precursor O 6-ClEtdG by O 6-alkylguanine-DNA-alkyltransferase (AGT). The level of the crosslinked product 1-( N1-2-deoxyguanosinyl), 2-( N3-2-deoxycytidyl)-ethane was significantly reduced ( P<0.001) in both 9L-2 and BTRC-19 as compared to 9L. Reduction in the level of 1-( N1-2-deoxyguanosinyl), 2-( N3-2-deoxycytidyl)-ethane in 9L-2 and BTRC-19 are consistent with repair of the precursor alkylation product O 6-ClEtdG by AGT. This study demonstrates that there are very significant differences in the rates of removal of individual DNA adducts formed by CNU treatment of the glioma cell lines.