P1+/+ mice have been crossed to Atm+/+Wip1+/- mice, and double heterozygous F1 progeny were re-crossed to receive F2 Atm+/+Wip1+/+, Atm-/-Wip1+/+, Atm-/-Wip1+/-, and Atm-/-Wip1-/- mice. A minimum of 43 mice for every single genotype were monitored more than their entire lifespan. As anticipated, Atm+/+Wip1+/+ mice live fairly typical lifespans of over two years (Fig. 1A). Constant with prior reports, 95 of Atm-/-Wip1+/+ mice developed thymic lymphomas by 150 days of age, and all are dead by 300 days of age (Fig. 1A). Conversely, only 11 of Atm-/-Wip1-/- mice create thymic lymphomas by 150 days of age, and hardly ever developed tumors soon after 180 days (6 months). The majority on the double knockout mice exhibited considerably Fluoroglycofen Autophagy enhanced longevities when compared with Atm null mice, with median lifespans of 620 and 110 days, respectively (Fig. 1A). No Wip1 dosage effect was observed, as Atm-/-Wip1+/- mice developed tumors at the identical price as Atm-/-Wip1+/+ mice. As a result, the absence of Wip1 largely rescues tumor susceptibility phenotypes observed in Atm null mice. To establish if there were any differences among the tumors that developed within the Atm-/-Wip1+/+, Atm-/-Wip1+/-, and Atm-/-Wip1-/- mice, tumors have been collected in the mice. Gross necropsies revealed only thymic tumors in Atm-/-Wip1+/+, Atm-/-Wip1+/-, and Atm-/-Wip1-/- mice. Analysis of hematoxylin and eosin (H E) stained tumor sections confirmed that all tumors have been thymic lymphomas of probably T-cell origin, and no histopathological Cibacron Blue 3G-A Formula variations were observed among the Atm-/-Wip1+/-, Atm-/-Wip1-/- and Atm-/-Wip1+/+ lymphomas (Fig. 1B-D). Atm-/-Wip1-/- mice exhibit enhanced p53 and DNA damage responses The reduced tumor incidence inside the Atm-/-Wip1-/- mice when compared with Atm null mice is constant with enhanced DNA harm and p53 responses. To examine this further, Atm+/+Wip1+/+, Atm+/+Wip1-/-, Atm-/-Wip1+/+, and Atm-/-Wip1-/- eight week old mice were irradiated with five Gy of ionizing radiation (IR). Thymi were harvested six hours following IR and analyzed for phosphorylation status of recognized Wip1 dephosphorylation targets. Lysates from regular thymi and spleens were assessed by Western blot analysis with antibodies to p53 and H2AX at the same time as phospho-specific antibodies for p53 (pS18) and H2AX (pS140). Each of these phosphorylation events are markers for an activated DNA harm response. Basal levels of -H2AX and phospho-p53 have been low in unirradiated Atm+/+Wip1+/+ lymphoid tissues but had been induced to moderate levels six hours immediately after IR therapy (Fig. 2A; Fig. S1). Irradiated Atm+/+Wip1-/- thymi and spleens exhibited enhanced phosphorylation of H2AX and p53 compared to irradiated Atm+/+Wip1+/+ thymi and spleens. Surprisingly, deletion of Atm didn’t impair IR-induced phosphorylation of H2AX and p53 and was comparable to Atm+/+Wip1+/+ levels (Fig. 2A-C). This is most likely a result of compensatory phosphorylation by other PIKKs. Within the presence of IR damage, the Atm-/-Wip1-/- thymi exhibited higher phosphorylation levels of H2AX and p53 comparable to Atm+/+Wip1-/- thymi (Fig. 2A-C). Furthermore, IR therapy resulted in improved p53 protein levels across all 4 genotypes, as anticipated. Absence of Wip1 in Atm+/+ and Atm-/- mice conferred modestly increased p53 protein stability right after IR in comparison with wildtype and Atm null mice (Fig. 2A). Lastly, irradiation of your various Atm/Wip1 genotype mice resulted in similar patterns of enhanced phosphorylation of Brca1 Ser1423 inside the absence ofAuthor Manuscript Author Manuscript Author.