CHANGES OF G1 CYCLINS, CDK2, AND CYCLIN-A DURING THE DIFFERENTIATION OF HL-60 CELLS INDUCED BY TPA

Differentiation induction by 12-o-tetradecanoyl 13-acetate (TPA) resul ts in the growth arrest of HL60 cells in the G1 phase. However, little is known about the changes of cell cycle-regulating genes during this differentiation process. We investigated the changes of mRNA for vari ous cyclins (A, C, D1, D2, D3 and E) and cdk2. Synchronized HL60 cells began to proliferate immediately after release from cell cycle block and cell cycle synchrony was obvious until the second S phase. TPA-tre ated cells accumulated in G1 phase within 24 h and most of the cells w ere arrested in this phase at 36 h. The expression of cyclins and cdk2 was studied by Northern blot hybridization or the reverse-transcripti on polymerase chain reaction (RT-PCR). TPA treatment altered the expre ssion of all genes studied. The expression of cdk2 and cyclin A mRNA w as markedly down-regulated. Cyclin E mRNA expression was also prominen tly downregulated from 12 h to 36 h, at which time a second increase o f its expression was observed in control cells. In contrast, the expre ssion of cyclin D1 mRNA was induced by TPA, while its expression in co ntrol cells was undetectable by Northern blot hybridization throughout the cell cycle. Cyclin C expression was faint and fluctuated irreleva nt of cell cycle, but its expression in both control and TPA-treated c ells was higher than at baseline. Cyclin D2 expression remained stable in control cells and TPA treatment resulted in slight down-regulation at 12 h, but no difference was observed after 24 h. Cyclin D3 mRNA ex pression was slightly induced at 6 h, a time when its expression was d ownregulated in control cells. At 48 h, these cyclins (C, D2, and D3) showed almost same level of expression as the control. These findings suggest that the down-regulation of cyclin A and cdk2 expression contr ibutes to the G1 arrest of HL60 cells during monocytic differentiation induced by TPA and that cyclin D1 plays an additional role other than the regulation of cell cycle progression.