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Defining the Role of a Novel CDK-Related Kinase in Cell Cycle Progression
Cyclin-dependent kinases (CDKs) are key regulators of the eukaryotic cell cycle whose activities are frequently aberrant in breast cancer cells. While the role of the prototypic CDK, CDK1/cdc2, in driving mitosis is well established, as is the role of CDK2 in S-phase regulation, there exist a number of CDK-related genes whose functions are entirely unknown. Among these is a gene loosely termed KKIALRE that has two other very close human homologs and is conserved in metazoans. Drosophila has just one highly homologous gene, RKIALRE, that we recovered in a screen for genes whose depletion leads to an increased incidence of bi-nucleate cells. More specifically, depletion of RKIALRE function by RNA interference (RNAi) in Drosophila S2 cells promoted aberrant telophase figures in which the two DNA masses had failed to separate properly resulting in bi-lobed nuclei and/or failures in cytokinesis. These cell cycle defects represent the first functional data for any of the KKIALRE family of kinases in any system. The proposed work aims to build on these findings and elucidate the role of this CDK-related gene in Drosophila. Initial emphasis will be placed on defining the precise nature of the RNAi phenotype in S2 cells, using a combination of flow cytometry and both live and fixed microscopy. In addition, I will test the hypothesis that the kinase is regulated in a manner akin to the well-characterized CDKs. I will test for physical interactions with known or putative CDK regulators and I will test the abilities of various (RNAi-insensitive) RKIALRE mutant constructs to rescue the RNAi phenotype. Among them will be putative phosphorylation site mutants and an ATP analog-sensitive version of the kinase, whose activity can be specifically blocked pharmacologically. In parallel with these cell culture studies, I will recapitulate the mutant phenotype in the Drosophila embryo. This is a very powerful experimental setting since the cell-cycles are extremely well characterized and they are amenable to powerful genetic and biochemical manipulations. I will examine RKIALRE mutant phenotypes induced by hairpin RNAi transgene expression and antibody injections. Having tested its efficacy in cell culture, I will also express the ATP analog-sensitive RKIALRE kinase in the RNAi background in vivo and monitor the effects of its chemical inhibition. Collectively, these studies are likely to uncover previously unknown regulatory features of the metazoan cell cycle. Since such mechanisms are usually remarkably well conserved, the findings should be directly relevant to human cell physiology and pathophysiology, and could lead to new avenues for therapeutic intervention in the treatment of breast cancer.
All cancers, including breast cancers, display uncontrolled proliferation arising from errors in cell division. A full molecular understanding of the normal control of cell division and its subsequent misregulation will facilitate the development of the best strategies for combating breast cancers. The cell division cycle is so well conserved throughout evolution that much of our current understanding of its workings in humans has resulted from the study of model organisms such as yeast and flies, where powerful genetic approaches can be used. Enzymes called cyclin-dependent kinases (CDKs) were thus identified and are universally accepted as master regulators of the cell division cycle, making them attractive drug targets for cancer therapy. However, despite their central importance, only a subset of the genes in the CDK family have been extensively characterized. Among those for which no functional information is available is a sub-group of closely related genes including one family called KKIALRE kinases. The fruitfly Drosophila melanogaster has one equivalent gene and our preliminary experiments have strongly implicated it in cell cycle regulation. Specifically, cells depleted of the gene’s function showed striking defects in separating their chromosomes and in completing cell division: anomalies that can lead to the genomic instability frequently exhibited by breast cancer cells. The proposed work therefore aims to define the function of this gene using both cultured cells and Drosophila embryos. Drosophila embryos are an excellent system for cell cycle research since the cells within them divide in a well-defined and stereotypic manner (meaning that irregularities are easy to detect), and they are amenable to very powerful genetic and biochemical manipulations. Probing the function of this highly conserved, yet previously unexamined, CDK-related gene is likely to uncover new aspects of cell cycle regulation, thus allowing for a better understanding of the etiology of breast cancer and thus facilitating a directed approach to developing novel therapies for its treatment.