Cyclin-dependent Kinase Inhibitor p27 as a Mediator of the G1-S Phase Block Induced by 1,25-Dihydroxyvitamin D3 in HL6O Cells1

Progressionof mammalian cells through G1 is controlled by the con certed action of protein kinases,the activities of which are modulated in both positive (cyclins) and negative [cydlin-dependent kinase inhibitors (CDIs)] manners by families of regulatory proteins. In differentiation of leukemiacells,a G1arrest is a common,if not invariable, occurrenceand takes placeafter the appearanceof markers of monocytic differentiation in human leukemia HL6O cells treated with 1,25 dihydroxyvitamin D3 (1,25D3)at low to moderately high concentrations(F. Zhang et aL, Cell Proliferation 27: 643—654, 1994).In the presentstudy,we investigatedthe proteinlevelsof severalG1regulatoryproteinsthat are potentialmedia tors of the 1,251)3-inducedG1block. During the first 24 h of exposureto a highconcentration(4 x iO@ M)of l,25D3,noincreasewasnotedin the immunodetectablelevelsof cydlinsDl or E, or CDISp16@@@k4, p2lCIPlJW@fl, or p27@', eventhoughmonocyticdifferentiationmarkerswereevident, and a prolongation of G, wasnoted.After 48 h of exposure4 x iO@ Mto 1,25D3, a G1 to S-phase block progressively increased in parallel with the abundanceof the p27'°― CDI. A transient increasein p2l@―@' was noted only at 48 hr. The increasein p27―@' protein level wasdependent on the concentrationof 1,25D3and wasaccompaniedby an increasein cydlin D and E proteins, which normally peak in mid-G1 and at the G1 to S-phasetransition, respectively.Theseresultsindicatethat p27@@' protein is a strong candidatefor the cell cycleregulator that blocks the entry into the S-phasein 1,25D3-treatedHL6Ocells.