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 phosphorylase (PNP) have abnormalities in purine metabolism that result in T-cell selective immune deficiency. The mechanism of action for cell death has been attributed to intracellular accumulation of dGTP, a potent inhibitor of ribonucleotide reductase and subsequently DNA synthesis, 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 inhibition, MOLT-4 cells, a human T cell leukemia cell line, were co-treated with the PNP inhibitor, CI-1000 (2-amino 3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]-pyrimidin-4-one HCl), and 2'-deoxyguanosine (dGuo) which resulted in a concentration-dependent loss of cell viability (trypan blue) and inhibition of tritiated thymidine ([3H]-TdR) uptake. Staining of cells with the DNA dye Hoechst 33,258 showed nuclear morphology characteristic of apoptosis. Western blots (24 h lysates) were probed with antibodies against several proteins implicated in apoptosis. Anti-PARP revealed the presence of an 85 kD PARP breakdown product while, anti-alpha-spectrin revealed the accumulation a 120 kD breakdown product, both suggestive of CPP32 cleavage (caspase-3; an ICE-like cysteine protease). 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 in MOLT-4 cells co-treated with dGuo and CI-1000. In summary, we provided several lines of evidence for the role of apoptosis and the contribution of caspase-3-like proteases in T-cell death following PNP inhibition.
Posmantur
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Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Department of Immunopathology, Ann Arbor, MI 48105, USA. Posmanr@aa.wl.com
Wang
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KK
Nath
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Gilbertsen
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RB
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D003849:Deoxyguanosine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors*; D013049:Spectrin / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D013936:Thymidine / Q000493:pharmacokinetics
D002843:Chromatin; D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; D011506:Proteins; D013049:Spectrin; C076778:9-deaza-9-(3-thienylmethyl)guanine; D006147:Guanine; D004247:DNA; D007770:L-Lactate Dehydrogenase; D011683:Purine-Nucleoside Phosphorylase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D003849:Deoxyguanosine; D013936:Thymidine