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Flow cytometric enumeration of drug-resistant tumor cells
A deFazio1, E A Musgrove, M H Tattersall
1Ludwig Institute for Cancer Research, University of Sydney, Australia.
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.
Insights
This study developed a new assay to measure hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants in cancer cells. The method uses 5-bromo-2'-deoxyuridine (BrdUrd) and flow cytometry to detect drug-resistant cells, aiding cancer chemotherapy research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Somatic mutations play a role in cancer chemotherapy resistance.
- Accurate measurement of mutation frequency is crucial for understanding resistance mechanisms.
Purpose of the Study:
- To develop and validate a novel assay for quantifying hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants in human tumors.
- To investigate the conditions for stringent selection of HPRT-deficient (HPRT-) mutants using 6-thioguanine (6TG).
Main Methods:
- Developed a method using 5-bromo-2 eal-deoxyuridine (BrdUrd) incorporation to identify proliferating cells.
- Utilized immunofluorescent staining and flow cytometry for enumerating BrdUrd-labeled, 6TG-resistant cells.
- Employed the Kolmogorov-Smirnov test for statistical analysis of mutant subpopulations.
Main Results:
- Established optimal conditions for selecting HPRT- mutants: ≥30 μM 6TG for 72 hours with BrdUrd.
- Demonstrated that BrdUrd incorporation in the presence of 6TG specifically identifies HPRT- mutants.
- Quantified the spontaneous mutant frequency in CCRF-CEM cells as 4.28 x 10⁻⁵ ± 2.04 x 10⁻⁵.
Conclusions:
- The BrdUrd/flow cytometric assay is effective for measuring HPRT mutant frequency.
- This assay provides a valuable tool for studying somatic mutations in cancer chemotherapy resistance.