Effect of alkyl-N-purine DNA glycosylase overexpression on cellular resistance to bifunctional alkylating agents

Increased activity of alkyl-N-purine DNA glycosylase (ANPG; a.k.a. N3-methyladenine DNA glycosylase) has been correlated with resistance to both chloroethylnitrosoureas and nitrogen mustards. Also, overexpression of the human glycosylase in Escherichia coli results in resistance to alkylating agents. To determine how overexpression of the protein affects resistance to these bifunctional alkylating agents in mammalian cells, wild-type CHO-AA8 cells were transfected with an expression construct containing the human ANPG cDNA. Several clonally isolated lines that expressed increasing levels of glycosylase activity were selected. None of these lines displayed increased resistance to either bis-chloroethylnitrosourea or melphalan. To determine how overexpression of this protein affects cells in the absence of nucleotide excision repair, the mutant CHO-UV20 cell line was transfected with the same expression construct. This cell line lacks functional ERCC-1 protein and displays extreme hypersensitivity to bifunctional alkylating agents. Again, none of the UV20 transfectants displayed increased resistance. The results of these experiments indicate that unlike E. coli, overexpression of the glycosylase alone is not sufficient to confer resistance to bifunctional alkylating agents in this system. Structural differences between mammalian cells and E. coli may explain the interesting result that a mammalian gene can confer drug resistance in E. coli but not in mammalian cells.