Lowering of p27^Kip1 levels by its antisense or by development of resistance to 1,25-dihydroxyvitamin D₃ reverses the G1 block but not differentiation of HL60 cells

Abstract

Cyclin-dependent kinase inhibitors are proteins with functions which appear to involve regulation of cell cycle traverse, and have been suggested to have a role in cell differentiation. However, there is as yet no rigorous proof that this is the case. We have addressed the participation of one of these inhibitors, p27^Kip1, in the induction of differentiation and the subsequent G1 block induced in HL60 cells by 1,25-dihydroxyvitamin D₃ (1,25D₃). First, it was noted that sublines of HL60 cells able to grow rapidly in the presence of 1,25D₃ have protein levels of p27^Kip1 lower than the levels in cells subjected to 1,25D₃-induced growth inhibition, but higher than in untreated parental cells. In contrast, there was no discernible relationship between the levels of p27^Kip1 and the expression of differentiation markers. Further, HL60 cells treated with 1,25D₃ and an oligonucleotide antisense, but not mismatched, to p27^Kip1 showed an almost complete elimination of the 1,25D₃-induced G1 block, but no decrease in the expression of differentiation markers. Similar results were obtained following transient transfection with an expression vector bearing the entire p27^Kip1 coding sequence in the anti-sense orientation. This is the first direct demonstration that p27^Kip1 plays a role in the 1,25D₃-induced G1 arrest, and that partial reduction in its levels has no effect on the induction of differentiation in HL60 cells.