Phosphorylation of p53 serine 18 upregulates apoptosis to suppress Myc-induced tumorigenesis

Abstract

ATM and p53 are critical regulators of the cellular DNA damage response and function as potent tumor suppressors. In cells undergoing ionizing radiation, ATM is activated by double-strand DNA breaks and phosphorylates the NH(2) terminus of p53 at serine residue 18. We have previously generated mice bearing an amino acid substitution at this position (p53S18A) and documented a role for p53 phosphorylation in DNA damage-induced apoptosis. In this present study, we have crossed E mu myc transgenic mice with our p53S18A mice to explore a role for ATM-p53 signaling in response to oncogene-induced tumorigenesis. Similar to DNA damage induced by ionizing radiation, expression of c-Myc in pre-B cells induces p53 serine 18 phosphorylation and Puma expression to promote apoptosis. E mu myc transgenic mice develop B-cell lymphoma more rapidly when heterozygous or homozygous for p53S18A alleles. However, E mu myc-induced tumorigenesis in p53S18A mice is slower than that observed in E mu myc mice deficient for either p53 or ATM, indicating that both p53-induced apoptosis and p53-induced growth arrest contribute to the suppression of B-cell lymphoma formation in E mu myc mice. These findings further reveal that oncogene expression and DNA damage activate the same ATM-p53 signaling cascade in vivo to regulate apoptosis and tumorigenesis.

Figure 1

Myc overexpression induces p53 serine 18 phosphorylation and upregulates Puma expression and p53-dependent apoptosis in B cells. (A) Western analysis of spleen samples from wildtype (WT) or p53S18A mice either untreated (Un) or 4 hours after whole body irradiation with 4Gy (IR). Phosphorylation of p53 at serine 18 occurs in response to DNA damage in spleen. (B) Western analysis of untreated spleens reveals upregulation of Puma expression in Eμmyc transgenic mice. (C) Histochemical staining of representative sections of pre-malignant spleens using a p53-serine18 phospho-specific antibody. Samples were isolated from untreated wildtype mice and Eμmyc transgenic mice, and from IR-treated wildtype mice or p53S18A homozygous mice. Sporadic p53 phosphorylation is detected in Eμmyc transgenic mouse spleen.

Figure 2

Myc expression induces B cell proliferation as well as B cell apoptosis that is regulated by p53 serine 18 phosphorylation. (A) Histochemical staining of representative sections of pre-malignant spleens isolated from WT mice, from Eμmyc transgenic mice, and from Eμmyc transgenic, p53S18A mice. Ki-67 staining indicates that Myc expression upregulates cell proliferation in vivo regardless of p53 status, whereas TUNEL staining reveals that Myc expression upregulates splenic apoptosis in a p53 serine 18 phosphorylation-dependent manner. (B) B cell growth in Eμmyc transgenic mice. Left panel: Myc-induced proliferation is not regulated by p53 serine18 phosphorylation in B cells. Average values for proliferation are derived from four mice per genotype, with bars representing SD. P values are given above brackets. Right panel: Myc-induced apoptosis is regulated by p53 Serine 18 phosphorylation. Average values for apoptosis are derived from four mice per genotype, with bars representing SD. P values are given above brackets. (C) Western analysis of p53 and Puma levels in premalignant spleens harvested from Eμmyc transgenic mice, or Eμmyc transgenic, p53S18A mice. Tubulin was used as a loading control. Values for protein amounts in Eμmyc transgenic, p53S18A mice are given in parentheses, as determined by densitometry, with p53 or Puma levels in Eμmyc transgenic spleens adjusted for Tubulin value and set at 1.

Figure 3

Myc-induced, B cell tumorigenesis is suppressed by p53 serine18 phosphorylation in mice. (A) Kaplan-Meier survival curve of Eμmyc transgenic mice that are either wildtype for p53 (S18/S18), heterozygous for the p53S18A allele (S18/A18), or homozygous for the p53S18A allele (A18/A18). (B) Representative tumor sections from Eμmyc transgenic mice heterozygous for the p53S18A allele (S18/A18) or homozygous for the p53S18A allele (A18/A18) stained with hematoxylin and eosin, or with B220 antibody. (C) Model for Myc activation of p53 tumor suppression. Inappropriate levels of Myc may activate ATM, p53 Serine18 phosphorylation, and p53-mediated apoptosis either by inducing DNA damage within the cell or by directly activating ATM-p53 signaling.