12-O-TETRADECANOYLPHORBOL-13-ACETATE AND STAUROSPORINE INDUCE INCREASED RETINOBLASTOMA TUMOR-SUPPRESSOR GENE-EXPRESSION WITH MEGAKARYOCYTICDIFFERENTIATION OF LEUKEMIC-CELLS

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced increased expression of the retinoblastoma (RB) tumor suppressor gene product in the course of megakaryocytic differentiation of the K562 h uman leukemia cell line, a differentiatively multipotent hematopoietic precursor cell. The induced increase in RB protein per cell occurred early, by 8 h of treatment, preceding any significant phenotypic diffe rentiation evidenced by cellular expression of the CD41 differentiatio n-specific megakaryocytic cell surface marker, but not inhibition of c ell cycle transit, leading to a cell population arrested with 2 n, 4 n , and 8 n DNA content. The increase in RB protein per cell occurred fo r cells in all cell cycle phases. Staurosporine (STSP) was found to in duce a similar course of cell cycle arrest and differentiation. Furthe rmore, STSP caused an up-regulation of RB expression similar to that c aused by TPA. Almost all of the RB protein is phosphorylated in untrea ted cells, but TPA and STSP both caused the late appearance of hypopho sphorylated RB protein following cell cycle arrest. The STSP-caused hy pophosphorylation was much later than the TPA effect. Hypophosphorylat ion of RB is, thus, not necessarily a prerequisite for cell cycle arre st but may be a consequence of G0. Given that TPA can be an activator and STSP an inhibitor of protein kinase C, it appears that the induced processes of tumor suppressor gene regulation and growth and differen tiation control are not necessarily protein kinase C dependent in K562 cells. Furthermore, the findings that these two presumably divergent inducing agents caused a similar increase in RB gene expression sugges ts that the up-regulation of RB associated with differentiation is not a coincidence of just one specific inducer but may be a common essent ial feature of the induced differentiation. The amount of RB protein p er cell increased within hours of exposure to TPA or STSP and may have a role in the induced metabolic cascade producing the new phenotype.