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

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.