Regulation of Cytochrome C Release in UV-Induced Apoptosis
Apoptosis, or programmed cell death, is vitally important for maintaining cellular homeostasis. When damaged cells do not appropriately enact the cell death program they have the potential to accumulate genetic mutations and become cancerous. Therefore, a mechanistic understanding of how cells decide to die would provide the foundation for drug discovery to specifically target cancer cells. Current genotoxic chemotherapeutics, and other sources such as ultraviolet (UV) radiation, damage DNA, leading to induction of apoptosis through the mitochondrial pathway. Cytochrome c is released from the mitochondria, binds dATP and Apaf-1, and together they form a structure called the apoptosome. The apoptosome then binds and activates caspase-9, which cleaves caspase-3 and other caspases resulting the morphological changes characteristic to apoptosis. To understand how UV causes apoptosis, an assay was developed to reproduce cytochrome c release in vitro. This assay revealed that UV radiation causes a rapid decrease of the anti-apoptotic protein Mcl-1 in HeLa cells. Reduction of Mcl-1 on the mitochondria causes in vitro release of cytochrome c from mitochondria three hours before in vivo release of cytochrome c is observed, i.e. the mitochondria are primed to release cytochrome c. Removal of Mcl-1 is required for UV-induced apoptosis, but it is not sufficient to induce apoptosis. This means that, in addition to mitochondrial priming, there is another required event, a second hit. It was reasonable to think that this required second hit might be bound specifically by Mcl-1. To that end, Mcl-1 was found to bind BimEL with far greater affinity than any other pro-apoptotic Bcl-2 family member. In addition, BimEL is dephosphorylated after UV in an ERK1/2-dependent manner. Despite perfectly fitting the profile of the second hit, BimEL and ERK1/2 phosphorylation do not have any affect on induction of caspases after UV. These results have helped gain insight into the regulation of cytochrome c release, which remains the most perplexing and important question in apoptosis.