Regulation of cell fate by caspase-3 and PKCδ and Hsp 27

Biological Sciences: 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)Regulation of cell fate by caspase-3 and Protein Kinase Cδ and heat shock protein 27 Objectives of the study. Apoptosis is an evolutionarily conserved mechanism essential for normal development and defense against viral infections and cancer. Cancer initiation and progression has been associated with a dysregulation of the homeostatic balance between cell proliferation and apoptosis. A key regulator of apoptosis is the cysteine protease, caspase-3. The inability to activate caspase-3 has been associated with resistance to chemotherapy and the progression of leukemia. However, the mechanisms of caspase-3 activation are still largely unknown. Thus, the purpose of this study was to determine how the direct regulators of caspase-3, Protein Kinase Cδ (PKCδ) and heat shock protein 27 (hsp27), mediate apoptosis. Methodology. Primary human monocytes, macrophages and transiently transfected monocytic-like-leukemia cells (THP-1) were used in immunoprecipitations (IP), immunofluorescence (IF), and short inferences RNA to determine the mechanisms involved in apoptosis. Apoptosis and differentiation was determined using DEVD-AFC caspase-3 activity assays and cell flow cytometry analysis respectively. Human purified recombinant proteins were used in caspase-activity assays and in vitro kinase assays to identify the molecular mechanism that regulate caspase-3. Results & Conclusions. We previously found that caspase-3 associates with PKCδ and hsp27. We showed that PKCδ phosphorylates caspase-3 and hsp27 and that PKCδ-dependent phosphorylation of caspase-3 promotes caspase-3 activity. We investigated the role of hsp27 and PKCδ on caspase-3 activation. We found using IF and subcellular fractionation that caspase-3, PKCδ and hsp27 are differentially localized during monocyte apoptosis and monocytes/macrophage differentiation. . We showed in monocytes that, unlike other classical heat shock proteins, the expression of hsp27 was constitutive and unaltered and increased dramatically during monocyte/macrophage differentiation. To determine the role of hsp27 in apoptosis, monocytes overexpressing hsp27 were induced to undergo cell death. We found a significant decrease in apoptosis in cells overexpressing hsp27. Consistently, silencing of hsp27 showed an increase in apoptotic cells upon treatment with chemotherapeutic drugs. We next investigated the mechanisms by which PKCδ and hsp27 regulate caspase-3. We mapped the domains of the association of caspase-3 with its two direct regulators hsp27 and PKCδ. Using IP, we showed that the carboxy-terminal domain of hsp27 associates with the amino-terminal domain of caspase-3 and found that binding of hsp27 inhibited the autocatalytic cleavage required for caspase-3 activation. Together our findings support that caspase-3 activation is regulated by anti- and pro-apoptotic factors. Our results suggest that the caspase-3:hsp27:PKCδ complex localizes in the cytoplasm. Upon activation of cell death, hsp27 and PKCδ relocalized to the nucleus freeing caspase-3 allowing its full activation. High levels of hsp27 inhibit apoptosis and may play a role in monocyte/macrophage differentiation. Significance. In an attempt to induce cell death in cancer cells, we identified a mechanism responsible for the regulation of caspase-3. Our studies demonstrated that hsp27 is a negative regulator of apoptosis and may contribute to the resistant of cancer cells to chemotherapeutic drugs. These findings contributed to the basic understanding of apoptosis and identified potential therapeutic targets for cancer treatment.No embarg