Effects of arotinoid acid on induction of apoptosis and differentiation and telomerase activity and cell cycle in the HL-60 cell line

Objective To investigate the effects of arotinoid acid (Ro13-7410) on the morphological and functional alterations of leukemia HL-60 cell line and compared with those of RA.
Methods Differentiation of HL-60 cells was assessed by morphology and by NBT reduction.Trypan blue exclusion was used to determine viability.Apoptosis was assessed by changes in cell morphology and by measurement of fragmented DNA using the PCD-assay-kit.Telomerase PCR ELISA-kit tested telomerase activity.The cell cycle was analyzed by flow cytometry.
Results Incubation of the HL-60 cells with 10^-6-10^-8 mol/L Ro13-7410 resulted in suppression of cell growth.Apoptotic cells were detected following exposure to 10^-6 mol/LRo13-7410 for 3 hours by measurement of the "in situ" enzymatic labeling of DNA breaks with biotinylated dUTP.Ultrastructural examination of Ro13-7410-treated samples showed cells with chromatin compaction and cytoplasm condensation and the presence of "apoptotic bodies".Cells induced into apoptosis were accompanied by increase of intracellular free Ca2+].Percentage of HL-60 cells reduced NBT following incubation with Ro13-7410 was lower than with all-trans retinoic acid (RA) (27% vas 85%).Telomerase PCR-ELISA assay showed that HL-60 cells cultured in the absence of inducing agents had significant telomerase activity.Telomerase activity declined gradually after 10^-6 mol/L Ro13-7410 treatment, and changes becoming evident at 1 day.The inhibition of telomerase activity at day 5 of treatment with Ro13-7410 was less effective than with RA.DNA flow cytofluorimetric analysis revealed that Ro13-7410 caused partial cell arrest in the G₂/M phase after a 2-day treatment and the percentage of cells arrested in the G₂/M phase increased after 4-days treatment.With RA-treated cells, a reduction in the percentage of cells in the G₂/M phase was observed after 2-day of treatment.
Conclusion Our study shows that Ro13-7410 suppresses HL-60 cells growth mainly via the induction of apoptosis and is less effective than RA in induction differentiation.Ro13-7410 dramatically inhibits telomerase activity during the course of induction and results in G₂/M arrest within 2 days.These findings suggest that Ro13-7410 is worthy of further study for its effects on leukemic cells.