Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.

Hereditary tyrosinemia type I is the most severe metabolic disease of the tyrosine catabolic pathway mainly affecting the liver. It is caused by deficiency of fumarylacetoacetate hydrolase, which prevents degradation of the toxic metabolite fumarylacetoacetate (FAA). We report here that FAA induces common effects (i.e., cell cycle arrest and apoptosis) in both human (HepG2) and rodent (Chinese hamster V79) cells, effects that seem to be temporally related. Both the antiproliferative and apoptosis-inducing activities of FAA are dose dependent and enhanced by glutathione (GSH) depletion with L-buthionine-(S,R)-sulfoximine (BSO). Short treatment (2 h) with 35 microM FAA/+BSO or 100 microM FAA/-BSO induced a transient cell cycle arrest at the G2/M transition (20% and 37%, respectively) 24 h post-treatment. In cells treated with 100 microM FAA/-BSO, an inactivation, followed by a rapid over-induction of cyclin B-dependent kinase occurred, which peaked 24 h post-treatment. Maximum levels of caspase-1 and caspase-3 activation were detected at 3 h and 32 h, respectively, whereas release of mitochondrial cytochrome c was maximal at 24-32 h post-treatment. The G2/M peak declined 24 h later, concomitantly with the appearance of a sub-G1, apoptotic population showing typical nucleosomal-sized DNA fragmentation and reduced mitochondrial transmembrane potential (Deltapsi(m)). These events were prevented by the general caspase inhibitor z-VAD-fmk, whereas G2/M arrest and subsequent apoptosis were abolished by GSH-monoethylester or N-acetylcysteine. Other tyrosine metabolites, maleylacetoacetate and succinylacetone, had no antiproliferative effects and induced only very low levels of apoptosis. These results suggest a modulator role of GSH in FAA-induced cell cycle disturbance and apoptosis where activation of cyclin B-dependent kinase and caspase-1 are early events preceding mitochondrial cytochrome c release, caspase-3 activation, and Deltapsi(m) loss. -Jorquera, R., Tanguay, R. M. Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.