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. 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.Key terms: Monoclonal antibodies, flow cytometry, human ovarian carcinoma cell line A2780 Emergence of tumor cell populations which have resistance to anticancer drugs is a major problem in the successful application of cancer chemotherapy. Detection of resistant cells in heterogeneous populations may, therefore, have a critical role for selection and monitoring of tumor treatment.DNA damage is considered to be a target for antitumor and cytotoxic actions of alkylating agents. Biochemical methods were applied extensively for the analysis of DNA damage induced by these drugs (2,5,11,12,15,19). However, no methods for the measurement of DNA damage a t a cellular level applicable to heterogeneous cell populations were developed.In previous reports, the detection of DNA damage in individual cells using anti-DNA monoclonal antibody (MOAB) was described (7,s). The binding of MOAB F7-26 to DNA, in fixed EDTA-treated cells, correlated well with the decrease in cell viability induced by alkylating agents. However, the fluorescence intensity was not sufficient for complete separation between populations of control and drug-treated cells in fluorescence histograms or for the detection of cells with clinically relevant levels of drug resistance.In the current work, a modified highly sensitive procedure for FCM analysis of DNA damage was applied. The new procedure is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mgzi. The effects of drug treatment on the binding of MOAB F7-26 to DNA in the cells sensitive and resistant to alkylating agents were compared in order to evaluate the ability of staining procedure to predict drug resistance. Since fluorescence difference in sensitive and resistant cells was sufficient to distinguish between two cell types, the method was then applied to heterogeneous populations of tumor cells. Mixed populations of sensitive and resistant cells were used as a model of tumors containing minor subsets of resistant cells.

Effects of 1-(Halogenalkoxy)Alkyl-5-Fluorouracil Derivatives on Cell Growth, Cell Volume and Nucleus Size of Mouse Lymphoma Cells

“…tophotom eter [22], DNA damage can be dem onstrated by flow cytometry, too [23]. An increase in nucleus size can be explained by scries of DNA single strand breaks.…”

Application of anti‐ssDNA monoclonal antibody to study exogenous and apoptosis‐associated DNA damage

“…Validity of the assay was demonstrated by a linear relationship between fluorescence intensity and cell killing measured by the colony forming assay (8,10), by detection of drug resistant cells (10,11), by evaluation of drug interaction and measurement of DNA repair (11,12), and by analysis of the intercellular transfer of drug resistance (13).…”

Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry

“…Cytometric techniques based on nucleoid preparation (13,23), or on the alkaline unwinding of DNA within cells (241, are destructive, and thus do not allow the correlation of the damage with additional cellular parameters. Non-destructive techniques making use of monoclonal antibodies have been recently proposed for detecting UV-induced pyrimidine dimers (15), alkylated DNA (5,6), or strand breaks (30). However, the usefulness of this approach may be limited by: lack of specificity (9); modification of the antigenic characteristics of the target molecule (17); unavailability of antibodies directed against other types of damage.…”