Abstract
Caspase-9 is a component of the apoptosome that mediates cell death following release of cytochrome c from mitochondria. Inhibition of Caspase-9 with a dominant negative construct (Casp9DN) blocks apoptosome function, promotes viability and has been implicated in carcinogenesis. Inhibition of the apoptosome in vitro impairs mitochondrial function and promotes mitophagy. To examine whether inhibition of the apoptosome would enhance mitophagy and promote oncogenesis in vivo, transgenic mice were generated that express Casp9DN in the T cell lineage. The effects of Casp9DN on thymocyte viability, mitophagy and thymic tumor formation were examined. In primary thymocytes, Casp9DN delayed dexamethasone (Dex)-induced cell death, altered mitochondrial structure, and decreased oxidant production. Transmission electron microscopy (TEM) revealed that inhibition of the apoptosome resulted in structurally abnormal mitochondria that in some cases were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitochondria being engulfed by autophagosomes (mitophagy), confocal microscopy showed colocalization of LC3-GFP and mitochondria. However, Casp9DN did not significantly accelerate T-cell lymphoma alone, or in combination with Lck-Bax38/1, or with Beclin 1+/− mice, two tumor-prone strains in which altered mitochondrial function has been implicated in promoting tumor development. In addition, heterozygous disruption of Beclin 1 had no effect on T-cell lymphoma formation in Lck-Bax38/1 mice. Further studies showed that Beclin 1 levels had no effect on Casp9DN-induced loss of mitochondrial function. These results demonstrate that neither inhibition of apoptosome function nor Beclin 1 haploinsufficiency accelerate T-cell lymphoma development in mice.
Highlights
Apoptosis is a highly conserved cell death pathway that regulates tissue homeostasis and normal development
Expression of Casp9DN markedly reduced cell death following Dex treatment almost as effectively as Q-VD-OPh. These results demonstrate that Casp9DN expression significantly delays in vitro apoptotic cell death in thymocytes
Recent studies suggest that rare cells that have undergone mitochondrial outer membrane permeabilization (MOMP) may retain proliferative capability
Summary
Apoptosis is a highly conserved cell death pathway that regulates tissue homeostasis and normal development. Bcl-2 family members are critical regulators of the apoptosis pathway. The Bcl-2 family primarily regulates apoptosis through their effects on the mitochondrial outer membrane permeabilization (MOMP). Bax and Bak form a multimeric complex that mediates the release of cytochrome c, Smac/Diablo, and other factors from the intermembrane space of mitochondria. Cytochrome c binds to Apaf-1 and proCaspase-9 forming the apoptosome and activating Caspase-9. This initiator Caspase activates downstream effector Caspases such as Caspase 3/7 that lead to apoptotic cell death. In contrast to Bcl-2, IAP family proteins act downstream of mitochondrial permeabilization as direct inhibitors of Caspase function
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