Regulation of Oxidative Stress by ATM Is Required for the Self-Renewal of Haematopoietic Stem Cells.

It has been hypothesized that a signaling pathway for regulating aging and longevity may be involved in stem cell functions. To address the hypothesis we investigate roles of molecules that regulate aging on self-renewal capacity of HSC.

Ataxia telangiectasia (A-T) is an autosomal recessive disorder caused by mutational inactivation of the ATM. ATM has a central role in maintenance of genomic stability via regulating cell cycle checkpoint in response to DNA damage, stability of telomere and oxidative stress. A-T patients display variety of sympotoms including high incidence of lymphoma and premature aging. In this study we investigated the effects of ATM deficiency on the hematopoietic system.

ATM^−/− mice have normal numbers of peripheral blood cells and colony-forming cells in the bone marrow (BM) at the age of 8 weeks. A frequency of KSL in the ATM^−/− BM was similar to that of wild-type mice. However, the number of colony-forming cells derived from ATM^−/− KSL cells after 6 weeks co-culture of stromal cells was significantly decreased. To directly assess a repopulating ability of the HSC in vivo, we performed a competitive reconstitution assay with congenic mice. Short-term (4-weeks) repopulation was not affected. However, there were dramatically fewer hematopoietic cells derived from ATM^−/− BM at 16 weeks post-transplant, indicating that ATM has an essential role in the self-renewal of adult HSCs, but is not required for the differentiation or proliferation of hematopoietic progenitor cells.

We next evaluated the effects of ATM deficiency on hematopoiesis in older mice. All ATM^−/− mice older than 20 weeks exhibited a progressive pancytopenia with hypocellularity in bone marrow. The numbers of myeloid and erythroid precursors among ATM^−/− BM MNCs were markedly decreased, KSL cells had disappeared, and co-culture on stromal cells showed that ATM^−/− cells were no longer able to form any colonies after 2 weeks of culture. Taken together, our data indicate that chronic ATM deficiency in vivo results in progressive multi-lineage BM failure due to defective maintenance of the adult HSC pool.

ATM is involved in oxidative defense, and the loss of ATM results in oxidative damage in several tissues. To elucidate the mechanism underlying the regulation of the HSC pool by ATM, we next evaluated ROS generation in HSC, and were able to demonstrate that the intracellular concentration of radical oxygen was higher in KSL cells from ATM^−/− mice than from WT animals. Two members of CDK inhibitors, p16 and p19, were highly elevated in ATM^−/− KSL cells after 2 days in vitro incubation with cytokines. Treatment with the permeant thiol N-acetylcysteine (NAC) abrogated the upregulation of p16 and p19 expression and BM failure corresponding to decrease in the level of intracellular ROS.

The numbers of colonies formed from ATM^−/− HSCs were restored to near-WT levels by treatment with either NAC or catalase in long term culture. Furthermore we found that NAC treatment of ATM^−/− mice dramatically restored the repopulation capacity comparable to that of the WT. We conclude that self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress. Our data support a model in which master regulator molecules govern the disparate processes of stem cell self-renewal, normal aging and tumor development.