(A) Small intestinal epithelial cells responded to irinotecan by activating a p53-dependent response pathway leading to the transcriptional activation of genes encoding cell cycle arrest (p21 and 14-3-3σ) and apoptotic (PUMA) proteins. Activation of cyclin-dependent protein kinases (Cdks) was prevented by at least two mechanisms including p21 binding and tyrosine phosphorylation (pY). Two pools of p21 were present in these cells (those that existed prior to irinotecan-treatment (basal or p53-independent, teal colored) and those that accumulated after irinotecan-treatment (p53-induced, salmon colored). Although equivalent levels of DNA damage were observed in p53 proficient and null cells, only p53 proficient cells underwent apoptosis, due to the accumulation of cell death proteins such as PUMA. In p21 null cells, DNA damage failed to efficiently inactivate Cdks due to loss of p21 (both p53-dependent and –independent pools) and cells did not efficiently arrest their cell division cycles under these conditions. Higher levels of DNA damage and therefore apoptosis (p53-dependent) were observed under these conditions. Cells lacking both p21 and p53 sustain high levels of DNA damage (due to loss of basal and induced pools of p21) but underwent less apoptosis (due to loss of p53-dependent apoptosis).
(B) Treating small intestinal epithelial cells with irinotecan followed by a Chk1 inhibitor, resulted in the tyrosine dephosphorylation of Cdks due to the accumulation of the Cdc25A phosphatase (). However, both basal and induced p21 pools were able to maintain low Cdk activity under these conditions. Thus, Chk1 inhibition did not synergize with irinotecan to induce more DNA damage in normal intestinal epithelial cells. However, in p53 null cells treated with the combination therapy, loss of tyrosine phosphorylation coupled with loss of p21 synergized to induce apoptosis. Finally, levels of apoptosis were even higher in cells lacking p21 because these cells lack Cdk tyrosine phosphorylation as well as basal and induced pools of p21.