19. Difference in Calcium Mobilization between Human Leukemia Cell Line, HL60, and Its Hydrogen Peroxide Resistant Mutant Cell Line, HP100

Kazunori Anzai and Guo-Jiang Zhang

Keywords: HL60, HP100, fura-2, ATP, hydrogen per oxide, intracellular calcium

HP 100 is a hydrogen peroxide (H₂O₂) -resistant cell line, which was constructed from HL60, a human leukemia cell line. In the present study, we have measured the change in the intracellular catcium concentration ([Ca²⁺]_in) responding to exogenous ATP or H₂O₂ stimulation with fura-2 fluorescence technique.

HL60 and HP100 cell culture were maintained in (I -MEM supplemented with ION FBS. 10 M H₂O₂ was added to the medium for HP100. Fura-2 was loaded inside the cells by mixing fura-2 AM (1 -2 /2M) and the cell suspension (1-2 x 10⁶ cells/ml) following incubation at 37C for 1 h. The fura-2 loaded cells were washed with a buffer containing 141 mM Nail, 5 mM KCI, 1 mM Na₂ HPO₄, 1 mM CaCL₂, 0.5 mM MgSO₄, 5 mV glucose, and 10 mM Hepes-Na (pH 7.4) and resuspended in the same buffer at 1-1.5 x TO⁶ cells/ml. The sample was kept at OC under dark until use within 4 h. The change in [Ca²⁺]_in was measured at 37C with an intracellular ion concentration monitor (JASCO CAFIIO)

ATP caused transient increase in [Ca²⁺]_in both for Hl,60 and HP100 cells. The sensitivity to ATP in HL60 cells (EC₅₀0.5M) was about five times higher than that in HP100 cells (EC₅₀2.5M) ADP also caused similar transient increase in [Ca²⁺]_in, but the sensitivity of these cells to ADP (ECHO : 16 p M for Hl,60) was lower than that to ATP. On the other hand, H₂O₂ caused smaller increase in [Ca²⁺]_in, and the recovery of [Ca²⁺]_in to the basal level was slower than that observed by the stimulation with ATP. The maximum change in [Ca²⁺]_in observed in HP100 cells was about twice larger than that observed in H1.60 cells. Based on the results of time-dependent change in [Ca²⁺]_in after extracellular calcium chelation with EGTA, existence of at least three compartments for calcium ion storage was suggested. The consecutive stimula tion of ATP and H₂O₂ or vice versa suggested that intracellular calcium pools responding to ATP and H₂O₂ are at least partially different.