title: Flow cytometric enumeration of drug-resistant tumor cells.
abstract: In an attempt to elucidate the role of somatic mutation in the development of resistance to cancer chemotherapy, an assay was sought to measure the frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants in human tumors. Based on the same principle as [3H]thymidine/autoradiography, a method was developed to identify cell proliferation using the thymidine analog 5-bromo-2'-deoxyuridine (BrdUrd). BrdUrd incorporation into DNA was measured following the immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analog. The human leukemic cell line, CCRF-CEM, was used to investigate the conditions necessary for the stringent selection of HPRT- mutants using 6-thioguanine (6TG). The appropriate 6TG exposure necessary to inhibit BrdUrd incorporation in wild-type cells, while allowing proliferation of spontaneous HPRT- mutants, was greater than or equal to 30 microM 6TG for 72 h (10 microM BrdUrd added 24 h prior to harvest). BrdUrd did not affect the growth of HPRT- mutants in the presence of 6TG. BrdUrd-labeled 6TG-resistant cells were enumerated flow cytometrically using fluorescent microspheres as an internal standard and the nonparametric, Kolmogorov-Smirnov test was used for independent statistical analysis of the subpopulations of fluorescent, 6TG-resistant cells. Evidence that CCRF-CEM cells which incorporated BrdUrd in the presence of 6TG were, in fact, HPRT- mutants was sought. It was demonstrated that spontaneous 6TG-resistant cells from the CCRF-CEM population were reduced by growth in medium containing aminopterin. The mutant frequency in the CCRF-CEM cell line was found to be 4.28 x 10(-5) +/- 2.04 x 10(-5) using the BrdUrd/flow cytometric technique.
authors.affiliation: Ludwig Institute for Cancer Research, University of Sydney, Australia.
authors.forename: A
authors.initials: A
authors.lastname: deFazio
authors.forename: E A
authors.initials: EA
authors.lastname: Musgrove
authors.forename: M H
authors.initials: MH
authors.lastname: Tattersall
mesh_terms: D001973:Bromodeoxyuridine / Q000378:metabolism; D003470:Culture Media; D004351:Drug Resistance; D005434:Flow Cytometry; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009154:Mutation; D013866:Thioguanine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D003470:Culture Media; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine; D001973:Bromodeoxyuridine