The Role of a Cyclin D2 Splice Variant in the Regulation of the Cell Cycle: Connecting Cell Cycle to Cancer Biology
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CycD2SV is a splice variant of cyclin D2 (cycD2) which has been implicated in cell cycle exit in embryonic cardiomyocytes. To further characterize the role of this splice variant, we overexpressed cycD2SV in a multitude of immortalized and transformed cell lines. We also monitored the expression profile of cycD2SV during cell confluence and serum deprivation, two states of growth arrest. Overexpression of cycD2SV resulted in a marked decrease in cell proliferation as determined by [3H]-thymidine S-phase labeling. TUNEL staining analysis indicated that cycD2SV overexpression does not induce apoptotic cell death. However, compared to control cells, cycD2SV expressing cells were more sensitive to cell death induced by external stressors, such as trypsinization. Deletion experiments identified the 54-136 amino acid sequence of cycD2SV as the cell cycle inhibitory domain while immunoprecipitation and immunofluorescence co-localization experiments elucidate a mechanism for cycD2SV mediated cell cycle exit where by the splice variant sequesters endogenous cell cycle proteins, such as CDK4 and cycD2, into aggresomal structures and targets them to autophagy mediated degradation. Cell cycle arrays completed for cycD2SV transfected cells associated the splice variant with changes in transcriptional profile of G2/M regulatory genes. During cell confluence and serum deprivation, we observed a significant increase in cycD2SV protein levels (2.5 fold) accompanied by a decrease in cycD1, D2 and D3 protein levels. Interestingly, while D-type cyclin mRNA levels decreased, cycD2SV mRNA levels did not change. Knock down of cycD2SV during confluence rescued cycD2 levels but not cycD1 or cycD3 levels suggesting that cycD2SV upregulation is responsible for cycD2 protein downregulation. Furthermore, knockdown of cycD2SV increased the percentage of cells entering S-phase suggesting that endogenous cycD2SV might be responsible for arresting cells at the G1 phase of the cell cycle. Collectively, our results are consistent with a universal negative cell cycle regulatory role for cycD2SV in both non-cardiac and cardiac cell types. Additionally, for the first time we provide evidence that cycD2SV is expressed endogenously and plays a role in cell cycle exit during states of growth arrest. Further studies on cycD2SV structure, function and regulation may facilitate development of novel anticancer strategies.