Optimization of PKH67 labeling conditions for proliferation monitoring in daunorubicin-treated leukemic cells.

Daunorubicin (DNR) is commonly used to treat acute myeloid leukemia (AML). The aim of this study was to determine whether PKH67 dye dilution could be used for differential proliferation monitoring in chemosensitive (K562S) and chemoresistant (K562R) leukemic sublines after drug treatment. Cells were labeled with PKH67 and treated for 2 h with a sublethal dose of DNR or vincristine either immediately or after 3 h in fresh medium. Viability (TOTO-3 exclusion) and DNR uptake (total cellular DNR fluorescence) were assessed by flow cytometry, and nuclear DNR accumulation was determined by confocal laser microspectrofluorimetry. Immediate DNR treatment led to enhanced DNR uptake and decreased viability in PKH67-labeled K562S, whereas no excess toxicity was seen if DNR treatment was delayed for 3 h. Treatment with vehicle control (Diluent C) gave similar results. In contrast, PKH67 labeling had no effect on K562S viability after vincristine treatment. For K562R, DNR uptake measured at 120 min was unaltered by prior exposure to PKH67 or vehicle, but viability was again significantly reduced after immediate DNR treatment. As with K562S, delaying DNR treatment for 3 h normalized viability in K562R. The excess DNR toxicity seen for PKH67-labeled K562S appears to be drug related, since it is not seen with vincristine, and may be due to the daunosamine sugar moiety present in DNR. However, with the addition of a 3-h incubation in fresh medium prior to drug exposure, PKH67 dye dilution can be used for proliferation monitoring of both K562S and K562R cells after treatment with DNR.