Purging in autologous hematopoietic stem cell transplantation using adenosine triphosphate (ATP) and 4-hydroperoxycyclophosphamide (4-HC)

Abstract In this study, we show potent in vitro purging induced by adenosine triphosphate (ATP) for leukemic cells. The treatment of murine L1210 leukemic cells with 2 mM of ATP in vitro for 3 h was able to reduce the number of leukemic clonogenic cells by about one order of magnitude presumably by changing the permeability of the leukemic cell membrane. Furthermore, the incubation of L1210 cells with ATP (2 mM) and low dose 4-hydroperoxycyclophosphamide (4-HC, 2 μg/ml) for 3 h resulted in at least a four-log reduction of clonogenic L1210 cells. Only a slight degree of toxicity to pluripotent hematopoietic stem cells (CFU-S) was observed in both treatment protocols. To determine the efficacy of pharmacological purging by ATP, we designed a murine system to mimic the conditions expected in the clinical setting of autologous transplantation using simulated partial remission marrow (SPRM) which was prepared by mixing normal marrow cells and L1210 cells at a ratio of 9:1. After the SPRM cells were incubated in vitro at a concentration of 1×10 6 /ml with both ATP (2 mM) and low dose 4-HC (2 μg/ml) for 3 h, 5×10 4 of the cells were then injected into lethally irradiated 9 weeks male BDF1 mice. All the mice given untreated-SPRM died of leukemia by day 27, whereas none of the recipients transplanted treated-grafts had died by day 70, thus suggesting that the combination use of ATP and 4-HC may be a potentially effective way to purge leukemic cells in autologous stem cell transplantation. The mechanism of the selective killing of leukemic cells is assumed that 4-HC is effectively incorporated into leukemic cells by increasing the permeability of the cell membrane by ATP. Taken together, this simple and rapid procedure is able to purge leukemic cells from autologous bone marrow grafts.