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A purine nucleoside phosphorylase (PNP) inhibitor induces apoptosis via caspase-3-like protease activity in MOLT-4 T cells.

Abstract
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.
AuthorsR Posmantur, K K Wang, R Nath, R B Gilbertsen
JournalImmunopharmacology (Immunopharmacology) Vol. 37 Issue 2-3 Pg. 231-44 (Oct 1997) ISSN: 0162-3109 [Print] NETHERLANDS
PMID9403342 (Publication Type: Journal Article)
Chemical References
  • Chromatin
  • Enzyme Inhibitors
  • Protease Inhibitors
  • Proteins
  • Spectrin
  • 9-deaza-9-(3-thienylmethyl)guanine
  • Guanine
  • DNA
  • L-Lactate Dehydrogenase
  • Purine-Nucleoside Phosphorylase
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Cysteine Endopeptidases
  • Deoxyguanosine
  • Thymidine
Topics
  • Apoptosis (drug effects, physiology)
  • Caspase 3
  • Caspases
  • Cell Line
  • Cell Survival (drug effects)
  • Chromatin (drug effects, metabolism)
  • Cysteine Endopeptidases (drug effects, metabolism)
  • DNA (drug effects, metabolism)
  • Deoxyguanosine (pharmacology)
  • Enzyme Inhibitors (pharmacology)
  • Guanine (analogs & derivatives, pharmacology)
  • Humans
  • L-Lactate Dehydrogenase (secretion)
  • Protease Inhibitors (pharmacology)
  • Proteins (metabolism)
  • Purine-Nucleoside Phosphorylase (antagonists & inhibitors)
  • Spectrin (metabolism)
  • T-Lymphocytes (cytology, drug effects, enzymology)
  • Thymidine (pharmacokinetics)

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