Involvement of PKC in TPA-potentiated apoptosis induction during hemin-mediated erythroid differentiation in K562 cells.

Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied the effects of PKC pathway with an activator of the protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), during hemin-induced erythroid differentiation of K562 erythroleukemia cells. K562 cell line has been used as a model of common progenitor of erythroblasts and magakaryocytes and can be differentiated into erythroid and megakaryocytic lineages by hemin and TPA, respectively. TPA induced almost complete loss of proliferation during megakaryocytic differentiation in K562 cells. However, upon hemin-mediated erythroid differentiation, the growth rate was slightly decreased at the subtoxic concentrations. Cotreatment with TPA at the hemin-treated K562 cells produced a concentration-dependent increase in cell injuries with apoptotic changes and significantly diminished the erythroid phenotype. To better understand the events implicated, we have used the PKC inhibitors such as bisindolylmaleimide II, RO318220, and the PKCbeta inhibitor. Our data showed that TPA-potentiated apoptosis in hemin-treated K562 cells was rescued by the application of the PKC inhibitors. Taken together, our results suggested the involvement of PKC in TPA-potentiated apoptosis induction during hemin-mediated erythroid differentiation in K562 cells.

Involvement of nPKC-MAPK pathway in the decrease of nucleophosmin/B23 during megakaryocytic differentiation of human myelogenous leukemia K562 cells.

Human myelogenous leukemia K562 cells were induced to undergo megakaryocytic differentiation by long-term treatment with phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The protein level of nucleophosmin/B23 (NPM/B23), a nucleolar protein, was substantially decreased upon TPA treatment. In this study, we found that the proteasome inhibitors blocked the decrease of NPM/B23 protein in response to TPA, suggesting the proteasomes were involved in the downregulation of NPM/B23 upon megakaryocytic differentiation. To investigate the signaling pathway in the downregulation of NPM/B23 during early TPA-induced megakaryocytic differentiation of K562 cells, K562 cells were treated with TPA in the presence of the PKC isozyme-selective inhibitors, GF109203X and Gö 6976, or MEK1 inhibitor, PD98059. The decrease of NPM/B23 protein in the TPA-treated K562 cells was blocked by GF109203X but not by Gö 6976, suggesting the involvement of novel PKCs in the downregulation of NPM/B23 during TPA-induced megakaryocytic differentiation of K562 cells. The application of MEK1 inhibitor PD98059 upon TPA treatment blocked the TPA-induced decrease of NPM/B23 protein and aborted the megakaryocytic differentiation but not to break through the cell growth arrest. Unlike NPM/B23, the degradation of nucleolin in the TPA-treated K562 cells could not be blocked by PD98059 while the TPA-induced megakaryocytic differentiation was abrogated. The decrease of NPM/B23 protein seems to be more correlated with the novel PKC-MAPK-induced megakaryocytic differentiation than another nucleolar protein, nucleolin. Taken together, our results indicated that novel PKC-MAPK pathway was required for the decrease of NPM/B23 during TPA-induced megakaryocytic differentiation.