CONCENTRATION OF RB PROTEIN IN NUCLEUS VS CYTOPLASM IS STABLE AS PHOSPHORYLATION OF RB CHANGES DURING THE CELL-CYCLE AND DIFFERENTIATION

Unphosphorylated RE (retinoblastoma tumor suppressor) protein is known to bind isolated nuclear matrix in vitro, whereas phosphorylated RE h as a lower affinity, suggesting a mechanism which might contribute to differential nuclear/cytoplasmic localization as part of its regulator y activity, This motivates interest in the in vivo localization of the endogenous RE protein as its phosphorylation state changes during the cell cycle and cell differentiation. It is known that in proliferatin g HL-60 cells all the RE protein is phosphorylated, but the extent of phosphorylation increases with progression from G1 to S to G2+M. It ha s also been previously shown that retinoic acid and 1,25-dihydroxy vit amin D3 shift the RE protein to the unphosphorylated state with cell d ifferentiation (Yen, A., S. Varvayanis, Exp, Cell Res. 214, 250-257 (1 994)). The dependence of cell cycle progression and differentiation on RE nuclear versus cytoplasmic localization, as well as the dependence of RE localization on phosphorylation state can thus be tested, Confo cal image analysis of the RE protein in vivo shows that the ratio of t he concentration of the RE tumor suppressor gene protein in the nucleu s versus the cytoplasm remains stable as the RE protein undergoes eith er phosphorylation during cell cycle progression or dephosphorylation during cell differentiation induced by retinoic acid or 1,25-dihydroxy vitamin D3, For the cell cycle analysis, HL-60 human promyelocytic le ukemia cells were fluorescently stained for DNA and for the RE protein , G1, S, and G2+M subpopulations were isolated by fluorescence-activat ed cell sorting. For each subpopulation, the relative concentration of RE protein in the nucleus and the cytoplasm was measured by laser con focal image analysis, To determine the effect of retinoic acid-induced myeloid differentiation or 1,25-dihydroxy vitamin D3-induced monocyti c differentiation, the same cell sorting and image analysis was perfor med on cells treated with these inducers, In all cases the concentrati on of the RE protein in the nucleus was approximately 2 times that in the cytoplasm, Thus, the ratio of nuclear versus cytoplasmic RE protei n concentration is stable and independent of phosphorylation or dephos phorylation of RE during both the cell cycle and cell differentiation.