DNA damage response to the Mdm2 inhibitor Nutlin-3

Abstract

Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethaneinduced mouse AJ02-NM 0 colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin). Quantification of γH2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/ M were mounting a DNA damage response (compared to a ~5 0% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events.

Revisiting DNA damage repair, p53-mediated apoptosis and cisplatin sensitivity in germ cell tumors

“…It should also be noted that in some cellular contexts nutlin-3 treatment of somatic tumor cell lines might have genotoxic effects, as it induces the activation of the DDR and formation of gH2AX foci (Valentine et al, Verma et al, 2010). Although still controversial, such ability seems to be independent of p53 status, and unrelated to the role of nutlin-3 as MDM2 antagonist (Valentine et al, 2011, Verma et al, 2010. Using Flow cytometry, we extended these observations to TGCTs and found that, in the EC cell lines 2102Ep, Tera-1 and NT2D1, though nutlin-3 treatment induced a comparable stabilization of p53 protein (Fig 2A), the magnitude of their apoptotic response was not uniform (Fig.…”

Novel Piperazine-based Compounds Inhibit Microtubule Dynamics and Sensitize Colon Cancer Cells to Tumor Necrosis Factor-induced Apoptosis

“…Flow Cytometry-HT29 and WI38 were analyzed for DNA content by ethanol fixation and staining with propidium iodide as described previously (19). Floating and adherent cells were combined and analyzed by flow cytometry.…”

Identification of novel compounds that enhance colon cancer cell sensitivity to inflammatory apoptotic ligands

“…HT29 and HCT116 cells were analyzed for DNA content by ethanol fixation and staining with propidium iodide as previously described. 72 Floating and adherent cells were combined and analyzed by flow cytometry. Adherent cells were harvested using a trypsin-EDTA solution, centrifuged together with the floating cells at 100 × g for 5 min and resuspended in 1 ml of cold saline GM.…”