Reoxygenation Induces Premature Senescence of FA Hematopoietic Progenitor/Stem Cells through a Pathway Involving ATM, P53, and P21^CIP1/WAF1.

Hematopoietic cells are often exposed to transient hypoxia as they develop and migrate between blood and tissues. We sought to test the role of continuous cycles of hypoxia and reoxygenation in the pathogenesis of bone marrow failure in Fanconi anemia (FA). We isolated Lin⁻ BM cells from wild-type (WT) and Fancc−/− mice, and subjected them to two cycles of hypoxic (1% O₂) then reoxygenation (20% O₂). Expansion of Fancc−/− Lin⁻ BM cells was significantly decreased in response to the oxidative stress of reoxygenation, as compared with WT Lin⁻ BM cells (1.3 vs. 4.9-fold by day 6, respectively). This inhibition was attributable to a marked decrease of Fancc−/− progenitor (Lin⁻ ScaI⁻ c-kit⁺) cells, as well as a slower expansion of Fancc−/− stem (Lin⁻ ScaI⁺ c-kit⁺) cells than the WT stem cells following reoxygenation. Fancc−/− Lin⁻ BM cells transduced with FANCC retroviruses exhibited a significant growth advantage to untransduced cells, particularly in hypoxia and reoxygenation conditions where the predominance of FANCC/GFP⁺ progenitors was observed in both the short-term liquid culture and clonogenic assays. There was no evidence of increased apoptotic death in these reoxygenated Fancc−/− BM progenitor and stem cells compared to their WT counterparts, as assessed by a flow cytometric method for caspase 3 activation (early apoptosis). Interestingly, evaluation of the apoptotic profile and cell cycle of stroma-supported long-term BM culture (LTBMC; two weeks after second reoxygenation) revealed that reoxygenated Fancc−/− LTBMC cells had reduced apoptosis compared to reoxygenated short-term culture. However, a vast majority (70.6%) of reoxygenated Fancc−/− LTBMC cells was residing in the G₀ + G₁ phases compared with 55.8% in WT LTBMC cells. Fancc−/− LTBMC cells stained intensely for SA-b-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21^CIP1/WAF1 and to a lesser extent, p16^INK4A. Moreover, reoxygenation-induced phosphorylation of p53^ser15 was dependent on the ATM kinase but not ATR, as inhibition of ATM signaling by the kinase inhibitor 2-aminopurine or siRNA knockdown of ATM decreased p53^ser15 phosphorylation and reduced cell senescence. Taken together, these results suggest that reoxygenation induces premature senescence in Fancc−/− BM hematopoietic progenitor and stem cells by signaling through the ATM to p53, upregulating p21 and causing senescent cell cycle arrest. In addition, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic stem cell depletion and BM failure in FA. <B style=%