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.