Deletion of Puma and p21Waf1 In Mice Deactivates p53-Induced Cell Death and Cell Cycle Arrest, but Protects Mice From Irradiation-Induced Lymphomagenesis by a Mechanism Involving Hemopoietic Stem Cell Quiescence

Abstract 90 Introduction: The p53 gene is non-functional in >50% of human tumors. In mice deletion of p53 leads to a high incidence of tumors and to a significant acceleration of tumorigenesis induced by repeated gamma-irradiation. While a large number of effects have been described for p53, current concepts of p53-mediated tumor suppression discuss the roles of p53 in regulation of cell cycle and apoptosis as being essential. Two main targets have been identified in this respect: p21Waf1 as an essential regulator of cell cycle arrest downstream of p53 and Puma as the largest single contribution towards p53 induced cell death. Methods: We have generated p21Waf1/Puma doubly deficient (i.e. double-knockout – DKO) mice on a pure C57BL/6 background to investigate the effects on tumorigenesis. Results: In ex vivo irradiation studies DKO thymocytes expectedly showed reduced cell death and loss of a G1/S arrest upon irradiation. When following a cohort of mice for spontaneous tumor development, the DKO mice did not differ from wild-type (WT) controls. Since this may be explained by additional p53 down-stream effectors essential for tumor suppression, we set out to challenge the mice with an established repeated irradiation protocol (4 × 1.75 Gy over 4 weeks) in order to increase the likelihood of uncovering a defect in tumor suppression not apparent in unchallenged mice. While irradiated WT mice developed thymic lymphomas at an expected rate and p53 deficiency accelerated the lymphoma formation as published, irradiated DKO mice did not develop any thymic lymphoma at all. During the irradiation protocol WT mice followed a series of depletion and regrowth cycles in thymic cellularity with a high rate of cell death early post irradiations in TUNEL assays and a surge of proliferation on day 5 after irradiations detected by in vivo BrdU labeling. By contrast in DKO mice thymic cellularity dropped only slightly during the first irradiation cycle. This was followed by a slow and steady decline in cellularity over the following 3 cycles of irradiation. No late apoptotic wave or loss of proliferative capacity of remaining thymocytes could explain the loss of cellularity, nor could senescence of thymocytes be detected by SA-β-Gal staining in situ, suggesting that thymic influx was defective. It had previously been reported for the repeat-irradiation lymphomagenesis model, that the irradiation of hemopoietic precursor cells was essential for tumorigenesis. In contrast to thymic cellularity, DKO LSK numbers stayed relatively stable over the course of the 4 irradiations. By comparison WT LSK numbers dropped to about 50% by the time 4 irradiations were completed. Indeed, short-term repopulating (ST) cells dropped significantly, while long-term repopulating (LT) and multipotent progenitor (MPP) cell populations stayed more stable. In DKO marrows the relative content of LT, ST and MPP cells proved very stable across the irradiation schedule. In vivo BrdU labelling showed that WT LSK had a higher fraction of labelled cells at baseline and a >100% increase in the proliferative fraction during irradiation, while in DKO LSK the proliferation index was lower and stayed stably low over time, compatible with the replenishment defect observed in the thymus. DKO stem cells were only slightly more efficient (1.6-fold) than WT in bone marrow reconstitution experiments without challenge. However, when mixed chimeras were then subjected to the irradiation protocol with 4 × 1.75 Gy a clear advantage of the DKO cells became apparent (28-fold). Moreover, when reconstituting lethally irradiated mice with a mixture of WT and DKO marrow taken from repeatedly irradiated donors the efficacy ratio was 1:152. Conclusion: Our data contrast observations made in cell lines, where loss of Puma and p21Waf1 led to a p53-resistant outgrowth of cells. We present in an animal model that loss of Puma and p21Waf1 is not tumorigenic and in fact protects mice from irradiation carcinogenesis. Together with our recently published findings in irradiated Puma singly-deficient mice (Labi G&D 2010), our data suggest that tumorigenesis in irradiated DKO mice is inhibited by effects on hemopoietic stem cell reactivity to DNA damage. A combination of lack of generation of free niche space through protection of hemopoietic stem cells from cell death and a stem cell quiescence state retained in DKO stem cells after irradiation seems responsible for the phenotype. Disclosures: No relevant conflicts of interest to declare.