ROSCOVITINE, A SMALL PURINE-LIKE CDK INHIBITOR, TRIGGERS APOPTOSIS IN HUMAN EMBRYONIC STEM CELLS

VERÓNICA FURMENTO; CAROLINA GARCIA; GUILLERMO VIDELA RICHARDSON; LEONARDO ROMORINI; SANTIAGO MIRIUKA; GUSTAVO SEVLEVER; MARÍA ELIDA SCASSA

San Francisco

Congreso; ISSCR Annual Meeting; 2016

ISSCR

Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst, during a stage of development defined by rapid cell division rates. These cells possess the unique characteristic of indefinite self-renewal while retaining an undifferentiated state. Protein kinase complexes formed by the association of cyclins and their catalytic subunits called cyclin dependent kinases (CDKs) represent the key molecules that orderly regulate progression through the cell cycle. In hESCs, elevated cyclin activity combined with lack of endogenous CDK inhibitors results in increased activity of CDK1 and CDK2 and consequently in diminished G1 and G2 cell cycle phases. Acute inhibition of CDK1 or CDK2 in proliferating somatic cells generally results in reversible arrest of the cell cycle without significant cell death. However, in other cellular contexts, CDK inhibitors could induce cell differentiation or apoptosis. Therefore, CDKs have cell-type specific functions, and compensatory roles exist among different CDK isoforms, which in turn could play pivotal roles in the regulation of proliferation and apoptosis. Herein, we used Roscovitine (ROSC), a small purine-like CDK inhibitor, to examine the role of CDK1 and CDK2 inhibition in WA09 hESC line. Initially, by XTT/PMS vital dye assays we found that ROSC exposure reduces WA09 cells viability in a dose-dependent manner.Interestingly, by western blot analysis we determined that the loss in cell viability was accompanied by apoptotic features such as caspase-9 and caspase-3 activation and PARP-1 cleavage. Flow cytometric analysis of DNA content revealed that ROSC treatment led to an increase in the percentage of cells in G2/M phase, which coincided with nuclear accumulation and site-specific phosphorylation of p53 at Serine 46. Frequently, molecular inhibition of CDKs protects normal noncycling cells from programmed cell death, however in the present study we found that, similar to what occurs in many types of cancer cells, in rapidly proliferating hESCs ROSC triggers apoptosis. Our findings further support the concept that CDKs activities might not be universally required for apoptosis in the same way that CDKs are universally required for cell cycle progression in all eukaryotic cells studied.