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.