A PURINE NUCLEOSIDE PHOSPHORYLASE (PNP) INHIBITOR INDUCES APOPTOSIS VIA CASPASE-3-LIKE PROTEASE ACTIVITY IN MOLT-4 T-CELLS

Children with congenital homozygous deficiency of purine nucleoside ph osphorylase (PNP) have abnormalities in purine metabolism that result in T-cell selective immune deficiency. The mechanism of action for cel l death has been attributed to intracellular accumulation of dGTP, a p otent inhibitor of ribonucleotide reductase and subsequently DNA synth esis, in thymocytes and T-cells but not B-cells, However, the mode of cell death has not been determined to be either necrosis or apoptosis. To examine the involvement of apoptosis in T-cells following PNP inhi bition, MOLT-4 cells, a human T cell leukemia cell line, were co-treat ed with the PNP inhibitor, CI-1000 (2-amino 3-thienylmethyl)-4H-pyrrol o[3,2-d]-pyrimidin-4-one HCl), and 2'-deoxyguanosine (dGuo) which resu lted in a concentration-dependent loss of cell viability (trypan blue) and inhibition of tritiated thymidine ([H-3]-TdR) uptake. Staining of cells with the DNA dye Hoechst 33258 showed nuclear morphology charac teristic of apoptosis. Western blots (24 h lysates) were probed with a ntibodies against several proteins implicated in apoptosis. Anti-PARP revealed the presence of an 85 kD PARP breakdown product while, anti-a lpha-spectrin revealed the accumulation a 120 kD breakdown product, bo th suggestive of CPP32 cleavage (caspase-3; an ICE-like cysteine prote ase). Western blots also detected the loss of the intact 32 kD caspase -3 isoform, a biochemical event associated with caspase-3 activation. Corresponding fluorometric activity assays detected a marked increase in caspase-3-like activity using the substrate Ac-DEVD-MCA. Lastly, a pan caspase inhibitor (Z-D-DCB) and 2'-deoxycytidine (dCyd), which is known to prevent dGTP accumulation following PNP inhibition, were able to prevent cell death and all indicators of caspase-3-like activity i n MOLT-4 cells co-treated with dGuo and CI-1000. In summary, we provid ed several lines of evidence for the role of apoptosis and the contrib ution of caspase-3-like proteases in T-cell death following PNP inhibi tion. (C) 1997 Elsevier Science B.V.