Regulation Of Cell Survival By Host And Viral Anti Apoptotic Bcl 2 Proteins PDF Download

Apoptosis is very critical for the maintenance of homeostasis, tissue differentiation, and removal of damaged/infected cells, which serves as a defense strategy against emergence of diseases such as cancer. Dysregulation of apoptotic pathways have been implicated in many diseases such as cancer and neurodegeneration. Therefore regulation of apoptosis to maintain a balance between apoptosis and cell proliferation is critical for the well being of all living organisms. The Bcl-2 family of proteins is one of the major regulators of apoptotic pathways. They consist of the anti-apoptotic subgroup, including Bcl-XL, and proapoptotic subgroup, including Bcl-XS. Previous studies have shown that mouse embryos that are deficient in the Bcl-X gene, a gene producing both Bcl-XL and Bcl-XS proteins, die before birth. Both Bcl-XL and Bcl-XS have been found to regulate cellular responses to apoptotic stimuli. The exact molecular and biochemical mechanism still remains unclear and controversial. We therefore hypothesize that Bcl-XL and Bcl-XS regulate apoptosis by differentially interacting with other members of the Bcl-2 protein family . Specific aims to test this hypothesis include (1) Investigate how endogenous Bcl-XL regulates apoptosis, and (2) Determine the molecular mechanisms by which Bcl-XS regulates apoptosis. To investigate how endogenous Bcl-XL regulates apoptosis, we developed mouse embryonic fibroblasts (MEFs) with (wild-type, WT) or without Bcl-X gene expression (Bd-X-KO). Both WT and Bcl-X-KO MEFs were treated with various apoptotic stimuli, including chemotherapeutic drugs and a panel of BH3-only Bcl-2 proteins, and cell viability was measured over time. Preliminary results showed that Bcl-X-KO MEFs were more sensitive to chemotherapeutic drugs than WT MEFs indicating that endogenous Bcl-XL plays a more prominent role in modulating cellular responses to these chemotherapeutic drugs. Furthermore, based on their effects on the viability of Bcl-X-KO and WT MEFs, the BH3-only Bcl-2 proteins were categorized into three groups: (a) neither Bcl-XKO nor WT MEFs underwent apoptosis upon Bad, Bnip3, Nix, Bim, or Bik treatment, implicating that these proteins do not directly interact with Bcl-XL, (b) both Bcl-X-KO and WT MEFs died at the same rate upon Tbid, Bmf, Hrk, or Puma treatment, showing that Bcl-XL was not able to prevent cell death initiated by these proteins, and (c) Noxa treatment induced significant cell death in Bcl-XKO but not in WT MEFs at all, indicating that endogenous Bcl-XL is able to completely inhibit the pro-apoptotic activities of Noxa. Therefore, endogenous Bcl-XL mediates cellular responses to BH3-only proteins expression by differentially antagonizing different BH3-only Bcl-2 proteins. Future study will involve identifying BH3-only pro-apoptotic Bcl-2 proteins that interact with Bcl-XL and characterizing the interaction between endogenous Bcl-XL and the BH3-only Bcl-2 protein(s). To determine the molecular mechanism by which Bcl-XS regulates apoptosis, Bcl-XS was re-expressed in both WT and Bcl-X-KO MEFs at a level similar to endogenous Bcl-XL level. All MEFs were treated with different apoptotic stimuli and cell viability was measured. Preliminary results showed that Bcl-XS expression had no effect on cell viability upon vincristine, etoposide, or doxorubicin treatment, but upon actinomycin D treatment, Bcl-XS protected cells from apoptosis. Future study involves examining the effects of Bcl-XS on apoptosis initiated by the different BH3-only Bcl-2 proteins, the activities of Bcl- XS in vitro, and the role of the intracellular localization of Bcl-XS during apoptosis.