Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

Abstract

DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.