Balance of human choline kinase isoforms is critical for cell cycle regulation Implications for the development of choline kinase-targeted cancer therapy

The enzyme choline kinase (CK), which catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP, has an essential role in the biosynthesis of phosphatidylcholine, the major constituent of all mammalian cell membranes. CK is encoded by two separate genes expressing the three isoforms CKa1, CKa2 and CK beta that are active as homodimeric or heterodimeric species. Metabolic changes observed in various cancer cell lines and tumors have been associated with differential and marked up-regulation of the CKa genes, and specific inhibition of CKa activity has been proposed as a potential anti-cancer strategy. As a result, less attention has been given to CK beta and its interaction with CKa. With the aim of profiling the intracellular roles of CKa and CK beta, we used RNA interference (RNAi) as a molecular approach to down-regulate the expression of CK in HeLa cells. Individual and simultaneous RNAi-based silencing of the CK a and beta isoforms was achieved using different combinations of knockdown strategies. Efficient knockdown was confirmed by immunodetection using our isoform-specific antibodies and by quantitative real-time PCR. Our analyses of the phenotypic consequences of CK depletion showed the expected lethal effect of CKa knockdown. However, CK beta- and CKa + CK beta-silenced cells had no aberrant phenotype. Therefore, our results support the hypothesis that the balance of the a and beta isoforms is critical for cancer cell survival. The suppression of the cancer cell killing effect of CKa silencing by simultaneous knockdown of both isoforms implies that a more effective CK-based anti-cancer strategy can be achieved by reducing cross-reactivity with CK beta.