The Role of Apoptosis and DNA Repair in Normal and Abnormal Hematopoiesis.

Deciphering the networks controlling apoptosis and DNA repair in the hematopoietic system is essential for understanding normal homeostasis and identifying dysregulation during leukemogenesis. To gain a molecular understanding of the apoptotic machinery during steady-state hematopoiesis, we performed a quantitative RT-PCR (qRT-PCR) survey of the expression levels of regulators and components of apoptotic pathways at distinct stages of hematopoietic differentiation. We found that hematopoietic stem cells (HSC: Lin-/c-Kit+/Sca-1+/Thy1.1+/Flk2-) had high expression levels of both anti-apoptotic genes (Bcl2, Bcl-xl, Mcl1, A1) and BH3 only pro-apoptotic genes (Puma, Bad, Noxa, Bim), low levels of BH3 multidomain genes (Bax and Bak) and specific expression of various components of the apoptosis machinery. In contrast, granulocyte/macrophage progenitors (GMP: Lin-/c-Kit+/Sca-1-/CD34+/FcγR+) had much lower levels of all anti-apoptotic genes, similar levels of BH3 only genes, and higher levels of Bax, Bak and Apaf1. These molecular signatures suggest HSC may be more resistant to death than myeloid progenitors. In fact, using AnnexinV and PI staining in combination with cell surface markers, we found that HSC had undetectable levels of apoptosis while ∼ 20% of GMP were apoptotic. These results suggest that while the overall levels of apoptosis in the bone marrow is low (≤10%), each subpopulation has distinct levels that are likely to be important for their specific turnover rates. We also found that HSC were more capable than GMP of withstanding higher doses of DNA damaging ionizing radiation (IR) and of resuming proliferation and differentiation. Taken together, these data suggest that HSC may be more resistant to apoptosis and capable of repairing DNA damage than committed myeloid progenitors. Dysregulation of HSC and GMP homeostasis is often observed in myeloid leukemia. We used the junB-deficient model of myeloproliferative disorder (MPD) to assess the effects of leukemic transformations on apoptosis and DNA repair machineries in the HSC and GMP compartments. Investigation of leukemic HSC by microarray analysis did not reveal major changes in the expression levels of apoptosis-related genes. In contrast, qRT-PCR analysis of leukemic GMP indicated a significant down-regulation in the expression levels of several apoptosis-related genes (Bcl2, Mcl1, Bax, Puma) and components of the DNA repair pathway (Atm, P53, Brca1, Pten). To determine the functional consequences of these molecular changes, we evaluated the apoptosis levels in junB-deficient mice using AnnexinV and PI staining in combination with cell surface markers. We found a significant decrease in the apoptosis levels of all junB-deficient stem and progenitor cells compared to control cells (2 to 7 fold), except for the megakaryocyte/erythrocyte progenitors (MEP). JunB-deficient granulocytes also displayed a 2-fold decreased in their apoptosis levels following 4 Gy of DNA damaging IR. These results indicate that junB-deficient cells are more resistant to apoptosis than control cells, and suggest that defects in apoptosis could be central to the MPD development in junB-deficient mice. Taken together, these data provide a link between the molecular networks controlling apoptosis and genomic stability and the cellular response of hematopoietic populations that will be applicable to understand tissue homeostasis and to develop targeted anti-leukemia therapies.