Abstract
The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-xAK, a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-xAK may trigger apoptosis.For efficient overexpression, Bcl-xAK was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-xAK appeared as an essential and initial step. Bcl-xAK was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-xL. A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.
Highlights
Apoptosis is a defined genetic death program that leads to ordered destruction of cellular components while membrane integrity is preserved [1]
We show that Bcl-xAK clearly activated the mitochondrial pathway, and its activity was critically controlled by both proand anti-apoptotic Bcl-2 proteins, despite the lack of BH3
Other proapoptotic Bcl-2 proteins induced apoptosis already at 24 h, as shown here for the BH3-only protein Bik/Nbk subcloned in the same adenoviral background (Fig. 1G)
Summary
Apoptosis is a defined genetic death program that leads to ordered destruction of cellular components while membrane integrity is preserved [1]. It represents a safeguard mechanism against tumor formation, due to the elimination of altered and mutated cells. Two major apoptosis pathways (extrinsic and intrinsic) have been described in detail. Extrinsic pathways are initiated by binding of death ligands (TNF-a, CD95L and TRAIL) to cell surface receptors, leading to the formation of death-inducing signaling complexes, where initiator caspases 8 and 10 are activated [3,4]. Intrinsic/mitochondrial apoptosis pathways are triggered by intracellular signals such as by cellular or DNA damage. Initiator caspases cleave and activate downstream effector caspases, which target a large number of death substrates to set apoptosis into work [6,7]
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