Molecular dissection of the role of cell survival and apoptotic signaling in normal and cancer cells.

摘要

Apoptosis, triggered by various cytotoxic cancer therapies, is a physiological form of cell death and is tightly controlled via complex interactions between cell survival and cell death regulatory molecules. Using Apoptin as a model molecule, we demonstrate the role of different cell survival and cell death pathways during apoptosis in normal and cancer cells. Apoptin, a chicken anemia virus derived protein, induces cell death selectively in cancer cells. The signalling pathways of Apoptin induced cancer cell-selective apoptosis are not well understood. Apoptin triggers caspase dependent apoptosis by activating the mitochondrial/intrinsic pathway, which is regulated by Bcl-2 family members. In addition, Apoptin, via its proline rich motif, interacts with the SH3 domain of p85 regulatory subunit of PI3-kinase leading to the constitutive activation of PI3-kinase and Akt. Downstream of PI3-kinase, Apoptin also interacts with Akt and mediates the nuclear translocation of active Akt. In the nucleus, Akt activates CDK2 and mediates its cytoplasmic translocation, where it phosphorylates Bcl-2 specifically at Thr-56 residue, thus mediating its degradation, which contributes to the activation of the mitochondrial death pathway. In addition, CDK2 phosphorylates Apoptin and regulates its tumor specific nuclear retention. Among additional targets of Apoptin's signaling events in the nucleus, Akt directly phosphorylates p27kip1 and enhances its proteosome-dependent degradation. Apoptin also indirectly mediates the cytoplasmic translocation of Nur77. These events are crucial for the activation of mitochondrial apoptotic pathway.;A direct link between Akt and CDK2 in the nucleus has also been demonstrated, which is important both for apoptosis and cell cycle progression. Akt phosphorylates CDK2 at threonine 39 residue, both in vitro and in vivo. The Akt mediated CDK2 phosphorylation occurs during the specific S/G2 boundary of the cell cycle via a transient nucleo-cytoplasmic shuttling of Akt during S and G2 phases. The phosphorylated CDK2 translocates to the cytoplasm temporarily, which is required for cell cycle progression from S to G2/M phase as the CDK2 T39A mutant lacking the phosphorylation site and defective in cytoplasmic localization severely affects the cell cycle progression at S-G2/M transition. Interestingly, we have also shown that the Akt/CDK2 pathway is constitutively activated by some anticancer drugs, like methotrexate and docetaxel, and under these conditions it promotes, rather than suppress cell death. Thus, the constitutive activation of the Akt/CDK2 pathway and altered sub cellular localization, promotes apoptosis. In contrast, transient physiologic Akt/CDK2 activation is necessary for cell cycle progression. These results reveal a novel function for the PI3-kinase/Akt pathway during an apoptotic process that could serve as a target for novel strategies for modulating apoptosis in cancer therapies.