Progression through eukaryotic cell division cycle is regulated by synergistic activities of both positive and negative regulatory factors. The active form of
vitamin D(3) (1alpha,25(
OH)(2)D(3), 1,25D) and a number of its synthetic analogs have been shown to arrest cells in the G(1) phase of the cell cycle. In the present study, 1alpha,25(
OH)(2)D(3) and the analogs
KH1060,
EB1089, and CB1093 were used to study the mechanism of the cell cycle arrest and to compare the effectiveness of these compounds in human MG-63
osteosarcoma cells. The 20-epi analogs
KH1060 and CB1093, as well as the 20-normal analog
EB1089, were found to be more potent than 1alpha,25(
OH)(2)D(3) in inhibiting cell proliferation and arresting the MG-63 cells in the G(1) phase. These analogs were more active than 1alpha,25(
OH)(2)D(3) in increasing the
cyclin dependent kinase inhibitor p27 protein levels (approximately 2.3-2.5-fold compared to 1alpha,25(
OH)(2)D(3)) by both increasing its formation and decreasing its degradation rate. The increased p27 formation was accompanied by stabilization of binding of
nuclear proteins to the Sp1+NF-Y responsive promoter region of the p27 gene. The increase in p27
protein levels and the simultaneous decrease in
cyclin E protein levels was accompanied by decreased Cdk2
kinase activity,
retinoblastoma (
Rb) protein hypophosphorylation and, finally, cell cycle arrest in the G(1) phase. In summary, the analogs
KH1060,
EB1089, and CB1093 keep
Rb protein in its growth-suppressing, hypophosphorylated form and prevent cell cycle progression through the restriction point. Therefore, these synthetic
vitamin D(3) analogs may be potential candidates for treating diseases, where cell cycle regulation is needed.