Flow cytometric analysis of DNA damage and repair in the cells resistant to alkylating agents.
DNA damage in the cells sensitive and resistant to alkylating agents was determined by flow cytometry analysis of cells stained with anti-DNA monoclonal antibody (MOAB) F7-26. MOAB F7-26 interacted with single-stranded regions in alkylated DNA, and the binding of antibody to the cells increased in proportion to the decrease in cell viability. Development of resistance to L-phenylalanine mustard (L-PAM) in A2780 cells was associated with decreased immunoreactivity of DNA with MOAB F7-26. Fluorescence was significantly lower in resistant cells than in sensitive cells, and the difference in the binding of MOAB between two cell types increased with the dose of L-PAM. The enhancement of L-PAM cytotoxicity to resistant cells by buthionine sulfoximine and hyperthermia was accompanied by a proportional increase of MOAB F7-26 binding to DNA. The same relative potential of sensitization regimens was established by cell survival and MOAB staining. The time course of DNA repair established by decrease of MOAB binding after L-PAM removal was similar in sensitive and resistant cells. Resistance of A2780 cells to L-PAM was associated with low initial level of DNA damage and with decreased cytotoxicity per unit of damage. We conclude that resistant cells could be distinguished from sensitive cells by staining with MOAB F7-26 and that the sensitization of resistant cells could be quantitatively predicted by flow cytometry analysis of MOAB binding.
Frankfurt
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Oncology Laboratory, Cedars Medical Center, Miami, Florida 33136.
Seckinger
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Sugarbaker
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EV
D000477:Alkylating Agents / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D019328:Buthionine Sulfoximine; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007700:Kinetics; D008558:Melphalan / Q000494:pharmacology*; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010051:Ovarian Neoplasms; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects*
D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D008558:Melphalan