This is a revised application focused on evaluating the role of calmodulin (CaM) kinase III as a possible key intermediate in the mitogenic response of brain tumors. Calmodulin, a major calcium binding protein in lung muscle tissues, is known to be involved in numerous aspects of cellular physiology that are critical for the control of cell replication and viability. Disrupting calmodulin signaling pathways could lead to inhibition of cell growth and enhanced cell death (apoptosis). The hypothesis to be tested is that mitogenic stimulus leads to the proliferation of glioma cells through the activation of CaM kinase III and that interference with this pathway could result in inhibition of cell growth and loss of cell viability. The proposed function of CaM kinase III is that this enzyme phosphorylates a unique substrate elongation factor-2 (eEF-2) which is required for protein synthesis. Inhibition of phosphorylation of eEF-2 kinase (also known as CaM kinase III) reduces its binding to ribosomes, hence resulting in inhibition of protein synthesis. The decreased protein synthesis, perhaps through degradation of short-lived repressor proteins, allows cell division to proceed in a variety of experimental systems. The applicant provided evidence to suggest that CaM kinase III is activated in glioma cells in response to mitogens and that this enzyme serves as a target for inhibition. Blocking CaM kinase III could result in inhibition of glioma cell growth in vitro.
Two specific aims are proposed for this project. First is to elucidate the factors that control actively how CaM kinase III in glioma cells is responsive to growth stimuli. The basic design of this aim is to purify the CaM kinase III from malignant glioma and characterize its activity in rat and human glioma cell lines by immunochemical and cell sorting techniques. The kinetics, pH optima, affinity for ATP, Ca2+ / CaM, and eEF-2 will be determined in CaM kinase III isolated from rat and human glioblastoma. Both the activity and the subcellular distribution of CaM kinase III in each phase of the cell cycle will be measured with a newly developed antibody against CaM kinase III. To elucidate mechanisms responsible for activation of CaM kinase III, the applicant proposes to determine if protein and/or RNA synthesis may be required for the increase of CaM kinase III activity following exposure to mitogens. If new protein synthesis is not required for CaM kinase III activation, the applicant will explore the cytoplasmic calcium mobilization hypothesis to identify whether increased cytosolic calcium by mobilization of calcium stores in the endoplasmic reticulum may attribute to CaM kinase III reactivation. In addition, the applicant proposes to determine whether CaM kinase III may be activated through a post-translational modification. The second specific aim is to determine the inhibitors of CaM-dependent signaling on the growth of glioma cells. The rationale of this approach is that if the proliferative response of glioma cells is mediated through Ca2+ / CaM-dependent activation of CaM kinase III, then inhibition of this pathway should block cell replication. A number of approaches were outlined in this aim. First, CaM inhibitors and antisense oligonucleotides will be used to inhibit CaM-mediated signaling of the growth of glioma cells. Next the applicant will test a newly described inhibitor of CaM kinase III, rottlerin, and its anti-tumor activity in vitro. It is hypothesized that CaM kinase III is an attractive therapeutic target that could lead to new drug development for anticancer therapies.
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