(A) Small intestinal crypts isolated from wild-type (WT), p53 null and p21 null mice that had been subjected to the indicated treatments were examined by IHC for γH2AX (brown) and nuclei were counterstained with hematoxylin (blue). Scale bar =100 μM. Arrows indicate γH2AX positive cells that migrated up into the villi. (B) γH2AX positive nuclei were counted in images similar to those shown in panel A. Three mice were evaluated for each treatment group and a total of 250–400 crypt nuclei were scored per treatment group. Data are presented as mean +/− SEM. Asterisks indicate significantly different using one-way ANOVA. *p between 0.01 and 0.05; **p between 0.01 and 0.001; ***p< 0.001. Statistical significance for comparisons between irinotecan and the combination treatment across genotypes is indicated in .

(A) Small intestinal crypts isolated from treated wild-type (WT) mice or mice null for either p53 or p21 were examined by IHC for cleaved caspase-3 (CC3) (brown) and nuclei were counterstained with hematoxylin (blue). Scale bar =100 μM. (B) The number of cells staining positive for CC3 was determined in a total of 50 crypts per treatment group (n = 3 to 5 mice per treatment group). Data are presented as mean +/− SEM. Asterisks indicate significantly different using a one way ANOVA. **p between 0.01 and 0.001, ***p < 0.001. Statistical significance for comparisons between irinotecan and the combination treatment across genotypes is indicated in .

(A) Small intestinal crypts isolated from treated wild-type (WT) mice or mice null for either p53 or p21 were examined for Geminin by immunofluorescent staining. Right panel illustrates Geminin positive cells in small intestinal crypts of irinotecan treated WT mice. Geminin (green); nuclei (blue, DAPI). Scale bar = 50 μM. Percentage of crypt epithelial cells staining positive for Geminin was determined by scoring 200 to 300 nuclei per mouse (n = 3 to 5 mice per treatment group). Data are presented as mean +/− SEM. Asterisks indicate significantly different as determined by a one-way ANOVA. *p between 0.01 and 0.05; **p between 0.01 and 0.001; ***p< 0.001. Significant difference was also observed between the combination treatments for WT compared to p53 null with p< 0.001and WT compared to p21 null with p< 0.001 (B) Small intestinal crypts isolated from treated mice were stained for phosphohistone H3 (pHH3) and the number of pHH3 positive cells per 75 crypts per mouse (n = 3 to 4 mice per treatment group) was determined (panel C). pHH3 (red); nuclei (blue, DAPI). Scale bar = 200 μM. Data are presented as mean +/− SEM. Asterisks indicate significantly different by a one-way ANOVA. *p between 0.01 and 0.05; **p between 0.01 and 0.001; ***p< 0.001. Statistical significance for pHH3 comparisons between irinotecan and the combination treatment across genotypes is indicated in .

(A) Small intestines were isolated from treated WT mice (lanes 1–4), p53 null (lanes 5–8) and p21 null mice (lanes 9–12), and analyzed by Western blotting for Cdk1 (total levels), Cdk1 phosphorylated on tyrosine 15 (pY15) and tubulin. (B) Small intestinal crypts isolated from treated mice were examined for cleaved caspase-3 (CC3) (brown) and nuclei were counterstained with hematoxylin (blue) by IHC. Scale bar = 50 μM. (C) Cells staining positive for CC3 were counted in 50 crypts per mouse per treatment group (3 to 4 mice per treatment group). Data are presented as mean +/− SEM. Asterisks indicate significantly different using a two-tailed T-test. *p between 0.01 and 0.05, ***p < 0.001.

(A) Small intestinal crypts isolated from treated mice null for both p53 and p21 were examined by IHC for γH2AX (brown) and nuclei (hematoxylin, blue). Scale bar = 50 μM. (B) Three mice were evaluated for each treatment group and a total of 150–200 crypt nuclei were scored for γH2AX positive nuclei in each treatment group. (C) Small intestines were isolated from treated mice and analyzed by Western blotting for Cdk1 (total levels), Cdk1 phosphorylated on tyrosine 15 (pY15) and tubulin. (D) Small intestinal crypts isolated from treated mice were stained for phistone H3 (pHH3, red) and nuclei (blue, DAP I) and (E) the number of pHH3 positive cells per 75 crypts was determined. Scale bar = 100 μM. Three mice were analyzed per treatment group. (F) Small intestinal crypts isolated from treated mice were examined for cleaved caspase-3 (CC3) (brown) and nuclei (hematoxylin, blue) by IHC. Scale bar = 100 μM. (G) The number of cells staining positive for CC3 was determined by counting CC3 positive cells in a total of 50 crypts per treatment group (n = 3 mice per treatment group). Data in panels B, E and G is presented as mean +/− SEM. Asterisks indicate significantly different by a one-way ANOVA.*p between 0.01 and 0.05; **p between 0.01 and 0.001; ***p< 0.001. Statistical significance for comparisons between irinotecan and the combination treatment across genotypes is indicated in .

(A) Parental HCT116 cells stably expressing click beetle red luciferase (CBRLuc) and control (Ctrl) shRNAs were either mock irradiated or exposed to 20 J/m² UV. Lysates were prepared and analyzed by Western blotting 24 h later. (B) Lysates prepared from p53 null HCT116 cells stably expressing CBRLuc and either Ctrl or p21 specific shRNAs were analyzed by Western blotting. (C) p53 null HCT116 cells stably expressing CBRLuc and Ctrl shRNAs were implanted into the cecum of immunocompromised mice. One week later, mice were injected with D-luciferin and subjected to bioluminescence imaging using the IVIS Lumina system (Caliper). Representative images of mice are shown. Photon flux is indicated by pseudocolored heatmap and tumor specific luciferase activity was measured by drawing a rectangular region of interest (ROI) as indicated (units: photons/sec × 10⁶). (D) Treated mice engrafted with the indicated HCT116 cell lines were sacrificed and tumors were isolated and analyzed for pHH3 by immunofluorescence. Total and pHH3 positive cells were determined in three separate fields per tumor section per treatment group (n = 3 mice per treatment group). (E) Treated mice engrafted with the indicated HCT116 cell lines were sacrificed and tumors were isolated and analyzed by IHC for CC3 (brown) and nuclei were counterstained with hematoxylin (blue). (F) Total and CC3 positive cells were determined in four to five separate fields per tumor section per treatment group (n = 3 to 4 mice per treatment group). Scale bar =100 μM. Data in panels D and E is presented as mean +/− SEM. Asterisks indicate significantly different by a one-way ANOVA. *p = 0.01 to 0.05, **p= 0.01 to 0.001.

(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.