Flow cytometric screening for selective toxicity to multidrug-resistant cells

Abstract

Mixed cultures originally containing doxorubicin [(DOX) NCS-123127]-sensitive leukemia P388 (P388/S) cells and DOX-resistant leukemia P388 (P388/R) cells were treated with drugs for 1 hour or with radiation, grown until the cell numbers had increased 250 times (8 cell doublings), and incubated with 2 micrograms daunorubicin (NCS-82151)/ml for 1 hour. The fluorescence of intracellular anthracycline measured on a flow cytometer was used as a marker to distinguish P388/S and P388/R cells. The proportions of low-fluorescent P388/R cells and high-fluorescent P388/S cells were determined from fluorescence histograms. Selective toxicity for multidrug-resistant P388/R cells was indicated by the decreased proportion of these cells in the mixed cultures. In untreated cultures grown from suspensions containing equal proportions of P388/R and P388/S cells, the mean proportion of P388/R cells after 4 days' growth was 34.4%. DOX eliminated P388/S cells from mixed cultures. Nitrogen mustard [(HN2) NCS-762] and x-rays were selectively toxic to P388/R cells. The selectivity of HN2 and x-rays was observed in a narrow dose range by the absence of selective inhibition at low doses, the decreased proportion of P388/R cells at moderate doses, and the killing of all cells in mixed cultures at high doses. Combination treatment of mixed cultures with DOX plus x-rays, or HN2 plus x-rays, produced complete inhibition of growth. Mixed cultures recovered after treatment with DOX plus HN2 contained only P388/R cells. Results obtained by colony-formation assay showed the same patterns of relative sensitivity for P388/R and P388/S cells as the data obtained by flow cytometry (FCM) selectivity assay. FCM analysis of mixed cultures detected selective toxicity for P388/R cells after treatments, characterized by approximately 1 log higher cell killing of P388/R cells than of P388/S cells. It was concluded that FCM analysis of mixed cultures provided a sensitive and reliable assay for fast screening of drugs for selective toxicity to multidrug-resistant cells.