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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 Department of Hematology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China (Liu XS, Lou LS, Zeng SR and Tang ZH) Department of Clinical Biochemistry, Chongqing University of Medical Sciences, Chongqing 400046, China (Jiang JK, Zhang Y, Xu XG, Liu BZ, He YJ and Kang GF) increase of intracellular free Ca 2+ 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 G 2/M phase after a 2 day treatment and the percentage of cells arrested in the G 2/M phase increased after 4 days treatment With RA treated cells, a reduction in the percentage of cells in the G 2/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 G 2/M arrest within 2 days These findings suggest that Ro13 7410 is worthy of further study for its effects on leukemic cells
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 determined viability of 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 in 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 prese nce of “apoptotic bodies” Cells induced into apoptosis were accompanied by Department of Hematology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China (Liu XS, Lou LS, Zeng SR and Tang ZH) Department of Clinical Biochemistry, Chongqing University of Medical Sciences, Chongqing 400046, China (Jiang JK, Zhang Y, Xu XG, Liu BZ, He YJ and Kang GF) increase of intracellular free Ca 2+ Percentage of HL 60 cells reduced NBT following incubation with Ro 13 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 decreased 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 G 2 / M phase after a 2 day treatment and the percentage of cells arrested in the G 2 / M phase increased after 4 days treatment with RA treated cells, a reduction in the percentage of cells in the G 2 / M phase was observed after 2 days of treatment Conclusion Our study shows that Ro13 7410 suppresses HL 60 cells growth primarily via the induction of apoptosis and is less effective than RA in induction differentiation Ro13 7410 dramatic inhibits telomerase activity during the course of induction and results in G 2 / M arrest within 2 days These findings suggest that Ro13 7410 is worthy of further study for its effects on leukemic cells