Effect of a novel vitamin D-3 analog, EB1089, on G(1) cell cycle regulatory proteins in HL-60 cells

Progression of cell cycle in eukaryotes is regulated by a series of the cyc lin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs) . It has been shown that 1,25(OH)(2)D-3 is able to arrest cell cycle at G(1 ) phase in malignant cells including HL-60 cells. EB1089 is a novel 1,25(OH )(2)D-3 analog that has more potent antileukemic properties with reduced hy percalcemic effect in vine and in vivo than 1,25(OH)(2)D-3. In the present study, we examined the effect of EB1089 on HL-60 cells at the protein level s of several G(1) regulatory proteins. Exposure of HL-60 cells to EB1089 (1 x10(-8) M) for 3 days showed the G(1) block: by FAGS analysis. The level of p21 was markedly induced in HL-60 cells treated with EB1089 at 24 h, and p 27 were progressively increased in a time-dependent manner. The expressions of CDK2 and CDK6 were down-regulated during G(1) block of HL-60 cells, and CDK4 is progressively elevated. In addition, level of cyclin D1 was increa sed in a time-dependent manner, however, no change of cyclin E was noted th rough the G(1) to S traverse. Immunoprecipitation study demonstrated that p 27 did not bind to CDK2, CDK4 and CDKG in EB1089-treated HL-60 cell extract s. In contrast, complexes immunoprecipitated from EB1089-treated HL-60 cell s with antibodies CDK2 and CDKG contained higher amounts of immunodetectabl e p21 protein compared to untreated HL-60 cells, whereas no detectable chan ge was noted with anti-CDK4 antibody. Furthermore, the kinase activities of CDK2 and CDK6 were decreased while little change was observed in CDK4 acti vity. These data indicated that p21 protein is a strong candidate for the c ontrol of G(1) progression in EB1089-treated HL-60 cells, and its major tar get molecules are CDK2 and CDK6.