Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase

Abstract

The ataxia telangiectasia mutated (ATM) kinase is a key tumor suppressor that regulates numerous cell cycle checkpoints as well as apoptosis. Here, we report that ATM is a critical player in the regulation of apoptosis and lymphomagenesis in the presence of c-myc. In turn, deletion of the inhibitory ATM phosphatase, Wip1, results in ATM up-regulation and suppression of Emicro-myc-induced B cell lymphomas. Using mouse genetic crosses, we show that the onset of myc-induced lymphomas is dramatically delayed in Wip1-null mice in an ATM- and p53-, but not p38 MAPK- or Arf-, dependent manner. We propose that Wip1 phosphatase is critical for regulating the ATM-mediated tumor surveillance network.

Figure 1.

Wip1 deficiency suppressed myc-induced lymphomagenesis. (A) In vitro association of ATM fragments with Wip1. Bacteria- expressed purified His-Wip1 was incubated with purified glutathione S-transferase (GST) or different GST-ATM fragments. Wip1 bound to ATM was detected after pull-down with glutathione-sepharose. The appropriate GST fragments are marked with asterisks. (B) Levels of phosphorylation and the expression of different proteins were analyzed in splenocytes from two separate Wip1^+/+ Eμ-myc and Wip1^−/− Eμ-myc mice. (C) The occurrence of lymphomas in Wip1^+/+, Wip1^+/−, and Wip1^−/− mice bearing the Eμ-myc transgene was analyzed over 400 d. The MLS for different genotypes is shown at the top. (D) The levels of expression of different proteins were analyzed in tumor samples obtained from Wip1^+/+ Eμ-myc (–6) or Wip1^−/− Eμ-myc (–12) mice. (E) The level of ATM was analyzed in cultured lymphoma cells with or without caspase inhibitor zVAD-fmk.

Figure 2.

p38 MAPK is not required to render Wip1-deficient mice resistant to Myc-induced lymphomas. (A) The levels of phosphorylation of p38 and its target HSP27 were analyzed in dermal fibroblasts purified from wild-type, Wip1^−/−, or Wip1^−/− p38α^Ki/+ mice. Cells were treated with different doses of ultraviolet C and harvested 1 h later. (B) The occurrence of lymphomas in Wip1^+/+ p38α^Ki/+ or Wip1^−/− p38α^Ki/+ mice bearing the Eμ-myc transgene was analyzed, and the MLS for the different genotypes is shown at the top.

Figure 3.

Wip1 deficiency suppressed myc-induced lymphomagenesis in an ATM/p53-dependent, Arf-independent manner. (A) The occurrence of lymphomas in Wip1^+/+ p53^+/− or Wip1^−/− p53^+/− mice bearing the Eμ-myc transgene was analyzed, and the MLS for different genotypes is shown at the top. (B) The occurrence of lymphomas in Wip1^+/+ Arf^+/− and Wip1^−/− Arf^+/− mice bearing the Eμ-myc transgene was analyzed, and the MLS for different genotypes is shown at the top. (C) The occurrence of lymphomas in Wip1^+/+ ATM^−/− and Wip1^−/− ATM^−/− mice bearing the Eμ-myc transgene was analyzed, and the MLS for different genotypes is shown at the top.

Figure 4.

ATM is critical in the activation of myc-induced apoptosis. (A) The mitotic index based on analysis of phospho-histone H3⁺ cells was determined in tumors that arose in different backgrounds. (B) Apoptosis based on analysis of TUNEL⁺ cells was measured in tumors that arose in different backgrounds. (C) The levels of p53, p19^Arf, and p38 (loading control) were analyzed in ATM^−/− Eμ-myc tumors with wild-type (–4) or Wip1^−/− (–8) backgrounds. (D) The levels of p53 and p53 phosphorylation at Ser18 were analyzed in wild-type, Wip1, and Wip1/ATM double-deficient splenocytes expressing Eμ-myc. (E) A diagram illustrating the role of the Wip1 phosphatase in suppressing lymphomagenesis in the presence of myc.