Phosphorylation of Retinoblastoma Protein Assayed in Individual HL-60 Cells during Their Proliferation and Differentiation

Expression of pRb and its state of phosphorylation were immunocytochemically assayed in individual HL-60 cells during their proliferation and after induction of differentiation, using mAb which detects hypophosphorylated pRb (pRbP-) combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT). Correlated measurements of pRbP-, pRbT, a ratio of pRbP-/pRbT, and cellular DNA content by flow cytometry revealed expression of total pRb and its phosphorylation state vis-à-vis the cell cycle position. Following mitosis (during the exponential phase of cell growth) a mixture of hypo- and hyperphosphorylated pRb was present within the cell for less than 2 h, i.e., early in G1; no hypophosphorylated pRb was detected throughout remainder of the cycle. Cellular pRb content was increasing primarily during G1and the cell entrance to S was correlated with attainment of a distinct threshold level of pRb. No correlation was seen between the content of pRb per cell and its state of phosphorylation during G1. Cell differentiation whether induced by 1,25-dihydroxyvitamin D3, retinoic acid, or phorbol myristate acetate led to cell arrest primarily in G0/1. The G0/1cells in these cultures, compared to G1cells from the untreated cultures, had increased level of both pRbTand pRbP-. However, because the relative increase of pRbP-was disproportionally greater than of pRbT, the pRbP-/pRbTratio of the differentiating cells was markedly elevated. The cells that still were in S and G2/M in the differentiating cultures also showed the presence of hypophosphorylated pRb. Our data suggest that the mechanism of irreversible cell cycle arrest during terminal differentiation involves both the increase in content of pRb and dephosphorylation of pRb already present within the cell. This provides a large pool of hypophosphorylated pRb that can effectively remove all free E2F, thereby precluding activation of the genes whose transcription is needed to pass the G1restriction point. In contrast to terminal differentiation the transient quiescence (G0state) manifests only by dephosphorylation of pRb, without a change in its cellular level.