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 t yrosine catabolic pathway mainly affecting the liver. It is caused by defic iency of fumarylacetoacetate hydrolase, which prevents degradation of the t oxic 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 temporall y related. Both the antiproliferative and apoptosis-inducing activities of FAA are dose dependent and enhanced by glutathione (GSH) depletion with L-b uthionine-(S,R)-sulfoximine (BSO). Short treatment (2 h) with 35 mu M FAA/BSO or 100 mu M FAA/-BSO induced a transient cell cycle arrest at the G2/M transition (20% and 37%, respectively) 24 h post-treatment. In cells treate d with 100 mu M FAA/-BSO, an inactivation, followed by a rapid over-inducti on 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 m aximal at 24-32 h post-treatment. The G2/M peak declined 24 h later, concom itantly with the appearance of a sub-G I, apoptotic population showing typi cal nucleosomal-sized DNA fragmentation and reduced mitochondrial transmemb rane potential (Delta psi(m)). These events were prevented by the general c aspase inhibitor z-VAD-fmk, whereas G2/M arrest and subsequent apoptosis we re abolished by GSH-monoethylester or N-acetylcysteine. Other tyrosine meta bolites, maleylacetoacetate and succinylacetone, had no antiproliferative e ffects and induced only very low levels of apoptosis. These results suggest a modulator role of GSH in FAA-induced cell cycle disturbance and apoptosi s Ft here activation of cyclin B-dependent kinase and caspase-1 are early e vents preceding mitochondrial cytochrome c release, caspase-3 activation, a nd Delta psi(m) loss.