Relationship between cyclin D1 and p21 Waf1/Cip1 during differentiation of human myeloid leukemia cell lines

Abstract

Expression of cell cycle-regulating genes was studied in human myeloid leukemia cell lines ML-1, ML-2 and ML-3 during induction of differentiation in vitro. Myelomonocytic differentiation was induced by phorbol ester (12- o-Tetradecanoyl-phorbol-13-acetate, TPA), tumor necrosis factor α (TNFα) or interferon γ (INFγ), or their combination. Differentiation (with the exception of TNFα alone) was accompanied by inhibition of DNA synthesis and cell cycle arrest. Inhibition of proliferation was associated with a decrease in the expression of cdc25A and cdc25B, cdk6 and Ki-67 genes, and with increased p21 Waf1/Cip1 gene expression, as measured by comparative RT-PCR. Expression of the following genes was not changed after induction of differentiation: cyclin A1, cyclin D3, cyclin E1 and p27 Kip1. Surprisingly, cyclin D1 expression was upregulated after induction by TPA, TNFα with IFNγ or BA. Cyclin D2 was upregulated only after induction by BA. The results of the expression of the tested genes obtained by comparative RT-PCR were confirmed by quantitative real-time (RQ) RT-PCR and Western blotting. Quantitative RT-PCR showed as much as a 288-fold increase of cyclin D1 specific mRNA after a 24 h induction by TPA. The upregulation of cyclin D1 in differentiating cells seems to be compensated by the upregulation of p21 Waf1/Cip1. These results, besides others, point to a strong correlation between the expression of cyclin D1 and p21 Waf1/Cip1 on the one hand and differentiation on the other hand in human myeloid leukemic cells and reflect a rather complicated network regulating proliferation and differentiation of leukemic cells.