Enumeration of 6-thioguanine-resistant tumour cells using flow cytometry and comparison with a microtitration cloning assay

• Published in: Mutation Research/Environmental Mutagenesis and Related Subjects Vol. 216; no. 1; pp. 57 - 64
• Main Authors: deFazio, A., Heneine, N., Musgrove, E.A., Tattersall, M.H.N.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.1989
• Subjects: 6-Thioguanine resistance; Animals; Anti-bromodeoxyuridine antibody; Bromodeoxyuridine; Drug Resistance; Flow Cytometry; Fluorescent Antibody Technique; Hypoxanthine Phosphoribosyltransferase - genetics; In Vitro Techniques; Leukemia L1210 - genetics; Mutagenicity Tests - methods; Mutation; Mutation frequency; Thioguanine - toxicity; Mutation frequency; Anti-bromodeoxyuridine antibody; 6-Thioguanine resistance
• ISSN: 0165-1161; 0027-5107
• DOI: 10.1016/0165-1161(89)90023-X

Measurement of mutant frequency in tumour specimens has been hampered by low cloning efficiency in soft agar. A method was developed to detect cell proliferation using the thymidine analogue 5-bromo-2′-deoxyuridine (BrUdR). BrUdR incorporation was monitored by immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analogue. The 6-thioguanine (6TG) exposure conditions which inhibited DNA synthesis, as measured by BrUdR incorporation, in wild-type cells while allowing proliferation of spontaneous hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants were investigated using tumour cell lines. It was shown that exposure to 10 −5 M BrUdR for the equivalent of 1 cell cycle time did not affect growth of wild-type cells, nor did it affect the growth of HPRT − mutants in the presence of 6TG. Methods for rapid flow cytometric enumeration of BrUdR-labelled 6TG-resistant cells were developed using fluorescent microspheres as an internal standard. To validate the BrUdR mutation assay, the 6TG mutant frequency (M F) was measured in L1210 R/S, a mouse leukaemic cell line (BrUdR 6TG M F = 7.0 × 10 −5) and the results directly compared with those from a microtitration cloning assay (M F = 4.6 × 10 −5). The results were similar and within the range reported for HPRT M F in mammalian cells.