Enumeration of 6-thioguanine-resistant tumour cells using flow cytometry and comparison with a microtitration cloning assay.
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 (MF) was measured in L1210 R/S, a mouse leukaemic cell line (BrUdR 6TG MF = 7.0 X 10(-5] and the results directly compared with those from a microtitration cloning assay (MF = 4.6 X 10(-5]. The results were similar and within the range reported for HPRT MF in mammalian cells.
deFazio
A
A
Ludwig Institute for Cancer Research (Sydney Branch), University of Sydney, N.S.W., Australia.
Heneine
N
N
Musgrove
E A
EA
Tattersall
M H
MH
D000818:Animals; D001973:Bromodeoxyuridine; D004351:Drug Resistance; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D066298:In Vitro Techniques; D007939:Leukemia L1210 / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods; D009154:Mutation; D013866:Thioguanine / Q000633:toxicity*
D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine; D001973:Bromodeoxyuridine