INCREASED STRINGENCY OF THE 1,25-DIHYDROXYVITAMIN-D-3-INDUCED G1-PHASE TO S-PHASE BLOCK IN POLYPLOID HL-60 CELLS

Treatment of mammalian cells with 1,25-dihydroxyvitamin D-3 (1,25D(3)) produces a G1 to S (G1/S) phase cell cycle block. In addition, it has been noted that a smaller proportion of cells accumulates in the G2/M compartment in 1,25D(3)-treated cultures. Since cyclins have a major influence on the regulation of cell cycle progression, we determined t he expression of cyclins A and B as markers of the G2 phase and of cyc lin E as the marker of G1/S transition. No increase in the steady-stat e levels of cyclin A or cyclin B mRNA was detected in the total cell p opulation or in the cyclin B1 protein in the G2/M cell cycle compartme nt. In contrast, immunodetectable cyclin E protein was increased in ce ll cultures as a whole and specifically in the G2/M compartment cells. Determination of BrdU incorporation into DNA by flow cytometry showed marked inhibition of DNA replication in cells with DNA content higher than 4C, and autoradiography of H-3-TdR-pulsed cells showed that poly nucleated cells did not replicate DNA after 96 h of treatment with 1,2 5D(3) or analogs. Taken together, these experiments show that at least a portion of the G2/M compartment in 1,25D(3)-arrested cultures of HL 60 cells represents G1 cells at a higher ploidy level, which are block ed from entering the high ploidy S phase. (C) 1996 Wiley-Liss, Inc.