Use of liquid culture and cell cycle analysis to compare drug damage following in vitro treatment of normal human bone marrow cells with adriamycin, arabinosyl-cytosine, and etoposide

The effects of adriamycin (ADM), arabinosyl-cytosine (ARA-C) and etoposide (VP16) were studied on human bone marrow mononucleated cells using colony formation in agar, a modified liquid culture system, and flow cytometry analysis of the cell cycle. Drug concentrations tested during a l-h incubation ranged from 0.1 to 4 μg/ml for ADM, from 0.3 to 30 μg/ml for VP16, and from 10-7 to 10-3 M for ARA-C. Regression analysis of the dose-response curves was used to to assess the drug concentration that inhibited 90% ± 5% (LD90) of colony growth. LD90s were 0.4 μg/ml for ADM, 20 μg/ml for VP16, and 10-4 M for ARA-C. LD90-surviving cells were cultured in liquid medium for 3 weeks. Surviving cells over this time were 13% of the control for ADM, 22% for VP16, and 95.7% for ARA-C. Although cells decreased drastically in ADM- and VP16-treated samples, granulocyte-macrophage colony-forming units (CFU-GM) per 10*5 surviving cells rose to twice the control fro ADM, to 60% for VP16, and to 150% for ARA-C. Flow cytometry analysis of the cell cycle was performed at day 0 and at day 4 after treatment with the LD90 dose. It showed a rapid and reversible effect of ARA-C on cells in the S-phase, whereas the action of VP16 concerned all cells, regardless of their cycle phase. We conclude that the direct effects of the three drugs on CFU-GM in agr are poorly predictive of hematopoietic reconstitution capacity, except for VP16. Liquid culture gives a much more accurate appraisal of the long-term damage and recovery due to anticancer drugs.