PNUTS knockdown potentiates the apoptotic effect of Roscovitine in breast and colon cancer cells

The phosphorylation suite of Retinoblastoma protein (Rb) plays a role in cell proliferation and apoptosis. Within cells. cyclin dependent kinases (cdks) phosphorylate Rh in response to growth stimulatory signals. whereas protein phosphatase 1 (PP1) dephosphorylates Rb when cells stop pro! iterating or undergo apoptosis in response to anti-proliferative or stress signals Stimulation of PP1 activity via siRNA mediated knockdown of its interacting protein PNUTS (Phosphatase Nuclear Targeting Subunit) leads to Rb dephosphorylation and apoptosis in cancer cells We utilized two separate methods to modulate the phosphorylation state of Rh in cancer cells. Kinase activity toward Rb is inhibited by the clinically relevant cdk inhibitor, Roscovitine In addition. siRNA mediated PNUTS knockdown stimulates phosphatase activity toward Rb. Either of these treatments in cancer cells causes a 2-fold stimulation of apoptosis When activation of phosphatase activity is combined with inhibition of cdk activity toward Rb. however, cells exhibit a 4-fold increase in apoptosis. The mechanism by which PNUTS knockdown mediated PP1 activation leads to apoptosis was determined to be dependent on the activity of the transcription factor E2F1 The Rb phosphorylation profiles resulting from each treatment were analyzed and found to be similar but not identical. In addition. the two treatments differentially effect the expression of bcl-2 family proteins. Thus inhibition of cdk activity and activation of PP I activity toward pRb are functionally distinct processes that together increase the apoptotic effect in cells.