Effect of novel modulators of protein kinase C activity upon chemotherapy-induced differentiation and apoptosis in myeloid leukemic cells

Abstract

Modulation of protein kinase C (PKC) activity has been demonstrated to either prevent or enhance drug-induced apoptosis in various tissue types. We tested four novel modulators of PKC activity in comparison to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for the capability to affect differentiation, cell cycle progression and apoptosis in the human myeloid leukemia cell lines U937 and HL-60. Farnesyl thiotriazole (FTT) and N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) are both direct activators of PKC, whereas 6-(2-(4-[(4-fluorophe-nyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quin-azolinone (R59949) are diacyl glycerol kinase inhibitors that activate PKC by enhancing the levels of the endogenous ligand diacyl glycerol. U937 cells displayed a slight reduction in the number of cells in G(2)/M cell cycle phase after exposure to FTT, SC-10, R59022 and R59949, respectively. In contrast, HL-60 cells demonstrated a largely unaltered cell cycle distribution. Whereas TPA treatment resulted in a strong induction of p21(WAF/CIP1), c-Fos and c-Jun levels, neither one of the novel PKC activators altered expression of these proteins. Consequently, we tested the ability of the activators to cause membrane translocation of PKC. While TPA treatment resulted in translocation of the PKC isoforms alpha, delta and epsilon, SC-10 and FTT failed to induce alterations in the PKC content of the membrane and cytosolic fractions, respectively. Expression of the beta(2)-integrin CD11c that is induced during TPA-mediated differentiation remained unaltered after exposure to SC-10 and was partly reduced after treatment with FTT. To further investigate the effect of these activators upon apoptosis in leukemic cells, HL-60 and U937 cells were treated with 1-beta-D-arabinofuranosylcytosine (Ara-C) or etoposide (VP-16). Whereas TPA strongly reduced apoptosis in Ara-C- or VP-16-treated U937 cells, little if any reduction was observed after pretreatment with either FTT, SC-10, R59022 or R59949, respectively, in these cells. In contrast, TPA enhanced apoptosis in Ara-C- or VP-16-treated HL-60 cells. Interestingly, FTT and SC-10 demonstrated a protective effect in Ara-C-treated HL-60 cells. Taken together, these data suggest that the novel PKC activators FTT, SC-10, R59022 and R59949 exhibit modest biological effects upon leukemic blast cells, and are not capable of enhancing the apoptotic response of these cells to cytotoxic drugs.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alters Mitochondrial Membrane Lipids

“…We additionally used R59949, a specific inhibitor of DAG kinase, which blocks metabolic conversion of DAG. R59949 produces an increase in the cellular level of DAG, which is analogous to that derived from increased phospholipase activity (85). At the standard concentration of 50 Amol/L, D609 and R59949 comparably inhibited TRAIL-induced translocation of PKCy to mitochondria (Fig.…”

“…PKCy is a member of a novel PKC subfamily that is activated by DAG and relocates to the mitochondria in response to apoptosis stimuli (46,47,80,82,(84)(85)(86)(87). Although phosphorylation of PKCy on distinct tyrosine residues is pivotal to its function during apoptosis (83), it is not known whether PKCy is activated in response to TRAIL.…”

Fos modulates myeloid cell survival and differentiation and partially abrogates the c-Myc block in terminal myeloid differentiation

“…Consistent with this notion are data showing that expression of c-Fos correlates with leukemogenicity and cytokine responsiveness in myeloid and lymphoid leukemias in humans. [25][26][27] Thus, better understanding of the molecular nature of the interaction between c-Fos and c-Myc may have prognostic and predictive values for treatment of certain subtypes of myeloid leukemias involving alterations in the expression and/or function of these genes.…”

The HMG-CoA reductase inhibitor simvastatin overcomes cell adhesion–mediated drug resistance in multiple myeloma by geranylgeranylation of Rho protein and activation of Rho kinase

“…Cells were stained by the manufacturer's recommendations as described 7 and expression was determined by flow cytometry.…”