P53-Independent induction of Fas and apoptosis in leukemic cells by an adenosine derivative, Cl-IB-MECA

Abstract

A 3 adenosine receptor (A 3AR) agonists have been reported to influence cell death and survival. The effects of an A 3AR agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9 H-purin-9-yl]-1-deoxy- N-methyl-β- d-ribofuranonamide (Cl-IB-MECA), on apoptosis in two human leukemia cell lines, HL-60 and MOLT-4, were investigated. Cl-IB-MECA (≥30 μM) increased the apoptotic fractions, as determined using fluorescence-activated cell sorting (FACS) analysis, and activated caspase 3 and poly-ADP-ribose-polymerase. Known messengers coupled to A 3AR (phospholipase C and intracellular calcium) did not seem to play a role in the induction of apoptosis. Neither dantrolene nor BAPTA-AM affected the Cl-IB-MECA-induced apoptosis. Cl-IB-MECA failed to activate phospholipase C in HL-60 cells, while UTP activated it through endogenous P2Y 2 receptors. Induction of apoptosis during a 48 hr exposure to Cl-IB-MECA was not prevented by the A 3AR antagonists [5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate] (MRS 1220) or N-[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5- c]quinazolin-5-yl]benzeneacetamide (MRS 1523). Furthermore, higher concentrations of MRS 1220, which would also antagonize A 1 and A 2A receptors, were ineffective in preventing the apoptosis. Although Cl-IB-MECA has been shown in other systems to cause apoptosis through an A 3AR-mediated mechanism, in these cells it appeared to be an adenosine receptor-independent effect, which required prolonged incubation. In both HL-60 and MOLT-4 cells, Cl-IB-MECA induced the expression of Fas, a death receptor. This induction of Fas was not dependent upon p53, because p53 is not expressed in an active form in either HL-60 or MOLT-4 cells. Cl-IB-MECA-induced apoptosis in HL-60 cells was augmented by an agonistic Fas antibody, CH-11, and this increase was suppressed by the antagonistic anti-Fas antibody ZB-4. Therefore, Cl-IB-MECA induced apoptosis via a novel, p53-independent up-regulation of Fas.