Circumvention and reactivation of the p53 oncogene checkpoint in mouse colon tumors

Aizu, Wataru; Belinsky, Glenn S.; Flynn, Christopher; Noonan, Emily J.; Boes, Colleen C.; Godman, Cassandra A.; Doshi, Bindi M.; Nambiar, Prashant R.; Rosenberg, Daniel W.; Giardina, Charles

doi:10.1016/j.bcp.2006.07.009

Cited by 10 publications

(12 citation statements)

References 39 publications

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“…This finding suggests an intimate cross-talk between various domains of p53 that acts to precisely coordinate its biological activity. Recent report showed that a Mdm2 inhibitor, nutlin-3, can activate p53 binding to a level comparable with doxorubicin and 5-FU and trigger G 2 -M arrest compared with G 1 arrest triggered by doxorubicin and 5-FU (36). This report provides further evidence that different genotoxic stress on p53 interacting protein Mdm2 can also cause unique differences in p53 activation kinetics and/or its posttranslational modification status.…”

Section: Discussionsupporting

confidence: 53%

Regulation of p53 Expression in Response to 5-Fluorouracil in Human Cancer RKO Cells

Schmitz

Song

et al. 2007

Clinical Cancer Research

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Purpose:The purpose of the study is to investigate the regulation of p53 expression in response to 5-fluorouracil (5-FU) in human colon cancer cells. Experimental Design: Human colon cancer RKO cells were used as our model system. The levels of p53 expression and p53 protein stability in response to 5-FU and doxorubicin were investigated. In addition, the acetylation and phosphorylation status of p53 after 5-FU and doxorubicin treatment was analyzed by Western immunoblot analysis. Results:Treatment of human colon cancer RKO cells with10 Amol/L 5-FU resulted in significantly increased levels of p53 protein with maximal induction observed at 24 h. The level of acetylated p53 after 5-FU exposure remained unchanged, whereas the phosphorylated form of p53 was expressed only after 24 h drug treatment. Northern blot analysis revealed no change in p53 mRNA levels after 5-FU treatment. No differences were observed in the half-life of p53 protein in control and 5-FU^treated cells, suggesting that the increase in p53 was the direct result of newly synthesized protein. In contrast, the maximal induction of p53, in response to doxorubicin, occurred at an earlier time point (4 h) when compared with cells treated with 5-FU (24 h). No corresponding change in p53 mRNA was observed. Levels of both the acetylated and phosphorylated forms of p53 were markedly increased upon doxorubicin exposure when compared with treatment with 5-FU, resulting in a significantly prolonged half-life of p53 (120 versus 20 min). Conclusion: These results, taken together, suggest that the regulatory mechanisms controlling p53 expression, in response to a cellular stress, are complex and are dependent upon the specific genotoxic agent. With regard to 5-FU, we show that translational regulation is an important process for controlling p53 expression. Studies are under way to define the specific mechanism(s) that control 5-FU^mediated translational regulation of p53.

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Section: Discussionsupporting

confidence: 53%

Regulation of p53 Expression in Response to 5-Fluorouracil in Human Cancer RKO Cells

Schmitz

Song

et al. 2007

Clinical Cancer Research

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“…For instance, p53 activated in this manner lacks many p53 modifications resulting from a DNA damage response [26], and these post-translational modifications may be important for regulating some aspects of p53’s functions. We have been employing the mouse AOM colon cancer model as a pre-clinical model of Nutlin-3 efficacy [25]. This model is particularly well-suited for studying Nutlin-3 since colon tumors in this model are typically p53-normal.…”

Section: Discussionmentioning

confidence: 99%

“…Since a DNA damage response induced by Nutlin-3 occurs in late S and G2/M, proliferating cells may be more sensitive to this activity. We previously reported that the ex vivo treatment of mouse colonic tissues with Nutlin-3 generated a significantly more robust p53 response in AOM-induced tumors than in normal adjacent mucosa [25]. In these experiments, tumors and normal tissue were removed from AOM-treated mice, bisected, and cultured in control or Nutlin-3 containing medium.…”

Section: Discussionmentioning

confidence: 99%

“…The elevated expression of the Mdm2 in AOM-induced tumors appears to be partly responsible for suppressing p53 activity [20]. Treatment of these lesions ex vivo with the Mdm2 inhibitor Nutlin-3 generates a robust and selective activation of p53 target genes, relative to normal adjacent tissue [25]. These findings suggest that Mdm2 inhibitors may provide an effective method for cancer prevention or treatment in this model, and potentially in early human colonic lesions that possess an intact p53 gene.…”

Section: Introductionmentioning

confidence: 99%

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DNA damage response to the Mdm2 inhibitor Nutlin-3

Verma

Rigatti

Belinsky

et al. 2010

Biochemical Pharmacology

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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.

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“…In this regard, additional infliction of damage to tumor cells by standard genotoxic chemotherapy is known to enhance the effects of nutlin (13)(14)(15)(16)(17). Second, normal cells, by having intact checkpoints and low stress signaling, are thought to undergo a mild and transitory p53-dependent cell cycle arrest after treatment with nutlin, and this transient arrest, in fact, may protect normal cells from the toxicity of standard chemotherapy (18,19). The latter approach implies that nutlin, by protecting normal tissues, could increase the therapeutic window to standard chemotherapy even in the treatment of p53-deficient tumors.…”

Section: Introductionmentioning

confidence: 99%

Induction of p53-Dependent Senescence by the MDM2 Antagonist Nutlin-3a in Mouse Cells of Fibroblast Origin

et al. 2007

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Cellular senescence is emerging as an important in vivo anticancer response elicited by multiple stresses, including currently used chemotherapeutic drugs. Nutlin-3a is a recently discovered small-molecule antagonist of the p53-destabilizing protein murine double minute-2 (MDM2) that induces cell cycle arrest and apoptosis in cancer cells with functional p53. Here, we report that nutlin-3a induces cellular senescence in murine primary fibroblasts, oncogenically transformed fibroblasts, and fibrosarcoma cell lines. No evidence of druginduced apoptosis was observed in any case. Nutlin-induced senescence was strictly dependent on the presence of functional p53 as revealed by the fact that cells lacking p53 were completely insensitive to the drug, whereas cells lacking the tumor suppressor alternative reading frame product of the CDKN2A locus underwent irreversible cell cycle arrest. Interestingly, irreversibility was achieved in neoplastic cells faster than in their corresponding parental primary cells, suggesting that nutlin-3a and oncogenic signaling cooperate in activating p53. Our current results suggest that senescence could be a major cellular outcome of cancer therapy by antagonists of the p53-MDM2 interaction, such as nutlin-3a. [Cancer Res 2007;67(15):7350-7]

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Circumvention and reactivation of the p53 oncogene checkpoint in mouse colon tumors

Cited by 10 publications

References 39 publications

Regulation of p53 Expression in Response to 5-Fluorouracil in Human Cancer RKO Cells

Regulation of p53 Expression in Response to 5-Fluorouracil in Human Cancer RKO Cells

DNA damage response to the Mdm2 inhibitor Nutlin-3

Induction of p53-Dependent Senescence by the MDM2 Antagonist Nutlin-3a in Mouse Cells of Fibroblast Origin

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