Activation of Signaling Pathways in HL60 Cells and Human Neutrophils by Farnesylthiosalicylate

Abstract

Effects of the farnesylcysteine mimetic, farnesylthiosalicylate on the activation of myeloid cells were studied. In dimethyl-sulfoxide-differentiated HL60 cells and in human neutrophils farnesylthiosalicylate (G20 pM) dose-dependently elevated cytosolic Ca" concentrations, suggesting phospholipase-c-mediated release of the ion from intracellular stores. In human neutrophils, in addition to the production of inositol trisphosphate, farnesylthiosalicylate induced activation of the NADPH oxidase and translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane. The calcium signal, inositol-trisphosphate production and superoxide generation elicited by farnesylthiosalicylate were partially blocked by treatment of the cells with pertussis toxin, consistent with participation of pertussistoxin-sensitive and pertussis-toxin-resistant elements. In HL60 cells, farnesylthiosalicylate ( c 2 0 pM) did not activate NADPH oxidase but dose-dependently augmented PMA-elicited activity of the enzyme. This effect was resistant to pertussis-toxin treatment. In vitro augmentation of PKC-mediated phosphorylation of histone and cytosolic p47-phox by farnesylthiosalicylate and the finding that downregulation of PKC abrogated potentiation of NADPH oxidase activity by farnesylthiosalicylate were compatible with the involvement of PKC in the response of HL60 cells to farnesylthiosalicylate. It is suggested that the effects of farnesylthiosalicylate on myeloid cells reflect interaction of the analog with prenylcysteine-docking sites on cellular signaling elements.Keywords: neutrophil ; HL60 cells ; NADPH oxidase ; farnesylcysteine; farnesylthiosalicylate.Neutrophils constitute the first line of defense against invading microorganisms. They respond to exogenous signals by chemotaxis, activation of the respiratory-burst oxidase, exocytosis of their cytoplasmic granules, and phagocytosis of foreign particles. Studies of responses of neutrophils to extracellular stimuli contributed to a better understanding of diverse signal-transduction pathways and their elements. In many studies, the promyelocytic leukemia cell line HL60 (Collins, 1987) was employed as a model of neutrophils. HL60 cells induced to myelocytic differentiation by dimethyl sulfoxide (Me,SO) or retinoic acid acquire most of the features of circulating blood neutrophils.Activation of the neutrophil NADPH oxidase (Morel et al., 1991 ;Chanock et al., 1994) in response to exogenous stimuli is characterized by a rapid onset and is amenable to easy experimental analysis. The release of superoxide radicals reflects assembly at the cell membrane of the multicomponent enzymatic

Characterization of the Regulation and Functional Consequences of p21ras Activation in Neutrophils by Antineutrophil Cytoplasm Antibodies

“…In nontransformed cells, concentrations of 25 to 50 M are required for inhibition (12). These concentrations have no effect on either G␤␥ subunits (12) or NADPH oxidase (14) directly.…”

Selective Role for β-Protein Kinase C in Signaling for O⨪2 Generation but Not Degranulation or Adherence in Differentiated HL60 Cells

“…The suspension was then centrifuged for 10 min at 300 ϫ g, and the cell pellet was resuspended in buffer containing 131 mM NaCl, 1 mM EGTA, 100 mM potassium phosphate buffer, pH 7.0, 2 mM PMSF, 20 g/ml leupeptin, 20 g/ml aprotinin, and 20 g/ml pepstatin (buffer A). (24). The cells were disrupted using three 5-s bursts of a microprobe sonicator at low power at 4°C and centrifuged at low speed (500 ϫ g, 5 min) to remove unbroken cells and nuclei.…”

Prevention of farnesylation of c‐Ha‐Ras protein enhances synergistically the cytotoxic action of doxorubicin in cycling but not in quiescent cells

“…Rat cells expressing a temperature‐sensitive p53 mutant [Eliyahu et al, 1985] provided the experimental system to test under the same genetic background the susceptibility of exponentially growing transformed cells versus quiescent cells to doxorubicin (DOX), a frequently used anti‐cancer drug, alone or in combination with the inhibitor of FPTase. To ensure that the observed effects are attributable to inhibition of farnesylation, we additionally used inhibitors of carboxy‐methylation of Ras such as AFC [Tisch et al, 1996] and FTA [Marom et al, 1995]. Inhibition of protein farnesylation resulted in the loss of the membrane anchoring of the de novo synthesized c‐Ha‐Ras protein in both proliferating and quiescent cells.…”