Modulation of cyclophosphamide activity by O-6-alkylguanine-DNA alkyltransferase

Purpose: The human medulloblastoma cell line D283 Med (4-HCR), a line resis tant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of D NA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR ) cells are cross-resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, bur pa rtial sensitivity is restored after elevated levels of O-6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O-6-benzylguanine (O-6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxyidechlorocy clophosphamide against D283 Med (4-HCR) after AGT is depleted by O-6-BG, Me thods: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxyidechlorocyclophosphamide with or withou t O-6-BG. Results: The activity of 4-HC and 4-hydroperoxydidechlorocyclopho sphamide against D283 Med (4-HCR) was increased after AGT depletion by O-6- BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protei n, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrole in caused a decrease in AGT levels, Conclusions: We propose that a small bu t potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.