p53-Independent induction of Fas and apoptosis in leukemic cells by an adenosine derivative, Cl-IB-MECA

doi:10.1016/S0006-2952(02)00839-0

Biochemical Pharmacology

Volume 63, Issue 5, 1 March 2002, Pages 871-880

https://doi.org/10.1016/S0006-2952(02)00839-0 Get rights and content

Abstract

A₃ adenosine receptor (A₃AR) agonists have been reported to influence cell death and survival. The effects of an A₃AR agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-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 A₃AR (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 P2Y₂ receptors. Induction of apoptosis during a 48 hr exposure to Cl-IB-MECA was not prevented by the A₃AR 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 A₁ 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 A₃AR-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.

Introduction

Following the introduction of A₃AR-selective ligands [1], [2], [3], the A₃AR has been demonstrated to have diverse physiological functions, including its effects on inflammation [4], hypotension [5], mast cell degradation [6], protection of brain and heart [7], [8], [9], and apoptosis [10], [11], [12], [13], [14], [15]. Specifically, potent A₃AR agonists showed dual effects leading to either cellular protection or death. In rat astroglial and human astrocytoma cells, micromolar concentrations of A₃AR agonists reduced the cell number, while nanomolar concentrations promoted cytoskeletal changes that were associated with cytoprotection [13], [16]. In chick ventricular myocyte culture [14] and in the isolated rabbit heart [17], the activation of the A₃AR showed a preconditioning-like effect that improved the outcome following an ischemic injury. Similar effects were observed in vivo in a gerbil model of global ischemia [8]. In promyelocytic human leukemia HL-60 cells [11], the A₃AR agonists IB-MECA (1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-β-d-ribofuranuronamide) and Cl-IB-MECA induced apoptosis at high concentrations. At lower concentrations, they protected the cells from apoptosis induced by A₃AR antagonists. In CHO cells transfected with the human A₃AR, effects on the cell cycle were induced with high concentrations of Cl-IB-MECA in those cells expressing the A₃AR, but not in control cells [18].

This study sought to probe the mechanism of the apoptotic cell death induced by Cl-IB-MECA in the HL-60 and MOLT-4 leukemic cell lines.

Section snippets

Materials

HL-60 and MOLT-4 cells were obtained from the ATCC. RPMI 1640 medium, fetal bovine serum, penicillin, streptomycin, and glutamine were purchased from Gibco BRL. Cl-IB-MECA and MRS 1220 (N-[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-yl]benzeneacetamide) were obtained from RBI-Sigma. MRS 1523 [5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate] was synthesized as described previously [19]. A TACS™ 2 TdT-DAB in situ apoptosis detection kit was obtained

Induction of apoptosis by Cl-IB-MECA

HL-60 or MOLT-4 cells were treated with 0–30 μM Cl-IB-MECA for 24 or 48 hr, and the fraction of cells undergoing apoptosis was determined using FACS. Significant apoptosis was induced by 30 μM Cl-IB-MECA in both cell lines (36 and 58% in HL-60 cells, and 29 and 48% in MOLT-4 cells, at 24 and 48 hr, respectively), as indicated in Fig. 1. This apoptosis rate was confirmed using the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method (TACS™ 2 TdT-DAB in situ

Discussion

It has been reported that A₃AR agonists induce cell death in leukemic and other cells [10], [11], [12], [13], [14], [15], while the mechanisms remain unknown. The present study was undertaken to determine how Cl-IB-MECA, an A₃AR agonist, induced apoptosis in leukemic cells.

Intracellular calcium has been known to modulate or transduce many intracellular signals including those of programmed cell death or apoptosis. The increase in intracellular free calcium following the activation of the A₃AR

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p53-Independent induction of Fas and apoptosis in leukemic cells by an adenosine derivative, Cl-IB-MECA

Abstract

Introduction

Section snippets

Materials

Induction of apoptosis by Cl-IB-MECA

Discussion

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Neurochem. Int.

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Biochem. Biophys. Res. Commun.

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J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Exp. Cell. Res.

Biorg. Med. Chem. Lett.

J. Biol. Chem.

Cell

Biochem. Biophys. Res. Commun.

2-Substitution of N6-benzyladenosine-5′-uronamides enhances selectivity for A3 adenosine receptors

J. Med. Chem.

Adenosine A1- and A3-selective N-alkoxypurines as novel cytokine modulators and neuroprotectants

Drug. Dev. Res.

2-Substitution of N⁶-benzyladenosine-5′-uronamides enhances selectivity for A₃ adenosine receptors

Adenosine A₁- and A₃-selective N-alkoxypurines as novel cytokine modulators and neuroprotectants