Expression of Evil (Starvin), a Drosophila melanogaster homologue of human BAG-family proteins, in mammalian carcinoma cells suppresses human Hsp70 expression.

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Genes that regulate programmed cell death are conserved widely across the evolutionary spectrum. A family of proteins, the Bcl-2 associated athanogene (BAG) proteins, in mammals is anti-apoptotic, enhances cell proliferation, and is over-expressed in certain human cancers. BAG family proteins have been demonstrated to protect cells against cell injury, such as heat shock, and cell death. High levels of BAG protein expression have been demonstrated to associate with cell survival and cell proliferation in several cancer cell lines in vitro. Sequence elucidation has identified BAG domain containing proteins in humans (hBAGs 1-6), mouse, X. laevis, C. elegans, S. cerevisiae, Arabidopsis thaliana, and Drosophila melanogaster. While plant BAG family-proteins functions as regulators of apoptosis-like processes, similar to the mammalian counter parts, Drosophila BAG family-proteins, Starvin/Evil, plays an essential role in the morphological development of muscles and tendons in fly embryos and larvae with lethality in embryos. While the developmental function of Evil has been studied, its functional role in adult flies has not been described. The Drosophila homologue Starvin/Evil shares 57% and 62% protein sequence similarity to the BAG domains of BAG-3 and 4 respectively. We have previously demonstrated BAG domain dependent protection from heat shock of MDA435 human breast cancer cells overexpressing BAG-3. In vitro analysis demonstrated that the BAG-domain of Evil binds to both human and Drosophila chaperone proteins, Hsp70 and Hsc70, indicating that Evil shares a conserved function with the human BAG family of proteins. We expressed both V5 tagged isoforms of Evil/Starvin-1 and 2 with and without the 26 amino acid N-terminal inset sequence in MDA435 cells. Observation using immunohistochemistry shows that Evil-1 has a diffused cytosolic expression pattern while Evil-2 has a focal perinuclear pattern. Preliminary data shows a differential influence on Hsp70 induction after a 44^oC heat shock for 40 minutes, with more Hsp70 in Evil-2 expressing cells and less Hsp70 in Evil-1 expressing cells compared to control cells. We have observed that Evil-1 grows more than Evil-2 in culturing conditions. More analyses are under way looking at various forms of cell death such as using cleaved-caspase 3 and PARP detection.