PKH26 probe in the study of the proliferation of chemoresistant leukemic sublines

Proliferative status and multidiug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia. Although classical methods f or proliferative assessment such as tritiated thymidine or BrdUrd incorpora tion, are correlated with treatment outcome, they are time consuming and di fficult to standardize. As an alternative, we have evaluated the use of a d ye dilution method using PKH26 to determine rate and extent proliferation i n drug sensitive and resistant cell lines. When cells labelled with this fl uorescent membrane intercalating dye divide, each resulting daughter cell r eceives half of the dye. Using flow cytometric analysis, it is possible to estimate the number of cells having undergone different numbers of cell div isions. Four different questions were addressed in these studies: a) does P KH26 give stable and reproducible labelling? b) does labelling with PKH26 a lter cellular proliferation characteristics ? c) is PKH26 a substrate for P GP and MRP ? d) does PKH26 labelling alter PGP expression and/or PGP activi ty ? We found that PKH26 labelling is stable, reproducible and has no effec t on cell proliferation. It does not modify PGP activity or expression, nor does it appear to be a substrate for PGP or MRP, since the rate of decreas e in fluorescence intensity is similar for sensitive and resistant cells wh ich are proliferating at the same rate. Using the dye dilution method, it i s possible to simultaneously assess PGP, proliferative status, and level of PGP expression. We conclude that the methods developed here provide a simp ler, more complete means for assessment of the effects of the drug therapy on sensitive and resistant cell populations in patients with hematologic ma lignancies.