Abstract 1664: Disrupting Calmodulin-Cyclin E Interactions with a Novel Peptide Inhibits Cell Cycle Progression and Proliferation of Vascular Smooth Muscle Cells
Background: Molecular mechanisms underlying cell cycle regulation of vascular smooth muscle cells (VSMC) may inform the design of novel therapies aimed at vaso-occlusive diseases. We previously showed that a coordingaed elevation in [Ca2+]i is required for G1-to-S phase cell cycle transitions in VSMC. Recently, we showed that Calmodulin (CaM)-dependent cyclin E/CDK2 activity mediates Ca2+-sensitivity of this transition, and requires direct binding of CaM to cyclin E via a CaM-binding motif in human cyclin E1. A peptide composed of this CaM-Binding Sequence (CBS) inhibited in vitro cyclin E/CDK2 activity by ~40% and abrogated Ca2+-sensitive cyclin E/CDK2 activity. We now test whether treatment with this peptide can inhibit proliferation of VSMC, and cancer cell lines with dysregulated cell cycle regulation.
Methods & Results: The delivery of a fluorescent-tagged CBS-peptide into the nucleus of cells by nucleofection (nucleus-targeting electroporation, Amaxa) was confirmed by confocal microscopy. Thiazolyl blue tetrazolium bromide (MTT) assay showed that peptide delivery inhibited proliferation of primary aortic SMC in a dose-dependent manner (IC50: 187 μM). A similar level of inhibition was observed in cancer cell lines HeLa and Saos-2. FACS analysis revealed that the CBS-peptide caused a G1-arrest greater than that observed in non-specific peptide-treated controls (70% vs. 53%). Western blot and co-immunoprecipitation analysis revealed that the CBS-peptide inhibited phosphorylation of CDK2 on Thr160 (65% inhibition), which is required for activation of CDK2, by selectively inhibiting CaM-cyclin E interactions. Importantly, binding of CaM to another target protein, calcineurin, was not altered by the CBS-peptide.
Implications: These data suggest that interactions between CaM and cyclin E may represent a novel therapeutic target for proliferative vascular diseases, and possibly also for certain cancers. CBS-peptide appears selectively to inhibit the binding of CaM to cyclin E, and thus may not interfere with other calcium/CaM-dependent pathways.