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Flow Cytometric Enumeration of Drug-resistant Tumor Cells

Ludwig Institute for Cancer Research (Sydney Branch), Blackburn Building, University of Sydney, NSW 2006, Australia

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 30 µM 6TG for 72 h (10 µM 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.

¹ To whom requests for reprints should be addressed, at the Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, NSW, 2010, Australia.

² Present address: Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, NSW, 2010, Australia.

Received 8/ 3/87. Revised 11/30/87. Accepted 7/19/88.

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