Abstract
DNA damage can be caused by intrinsic and extrinsic sources, and unrepaired or imprecisely repaired DNA can lead to mutagenesis, cell death, or malignant transformation. Here, we have used highly purified hematopoietic stem cells (HSC) and myeloid progenitor populations (MP: CMP & GMP) to understand how they respond to ionizing radiation (IR) and to establish how they recognize and repair damaged DNA. Our overall goal is to determine whether HSC and MP differ in their ability to deal with genotoxic stress and to identify the type of deregulations that each population requires for accumulating mutations and becoming transformed. By following their ability to form colonies in methylcellulose and to grow in liquid culture, we found that HSC are more capable of withstanding increasing doses of IR than MP. BrdU incorporation pulses revealed that irradiated HSC have an initial and transient pause in proliferation, while irradiated MP cycled faster than untreated MP. By monitoring apoptosis with AnnexinV/7AAD and cleaved caspase 3 staining, we showed that both irradiated HSC and MP have an immediate apoptotic response but that HSC quickly recover and restore low baseline levels of apoptosis while MP maintain high levels of apoptosis. These results suggest that HSC may pause cycling to repair damaged DNA while MP increase cycling to replenish cells cleared by apoptosis. At the molecular level, irradiated HSC displayed a strong and transient induction of several pro-apoptotic genes (bax, puma, noxa) but minimal change in expression of pro-survival genes, which are already high in those cells. In contrast, irradiated MP displayed minimal induction of pro-apoptotic genes (except for puma) but decreased expression of pro-survival genes, which are already low in those cells. Taken together, these results suggest that HSC can withstand genotoxic stress better than MP due to differences in the regulation of their apoptotic machinery leading to protection of HSC and elimination of MP. To monitor the ability of HSC and MP to recognize and repair damaged DNA, we used immunofluorescence techniques to study IR-induced DNA damage foci. Irradiated HSC and MP displayed similar kinetics of DNA damage recognition as monitored by the formation and resolution of gamma-H2AX and 53BP1 foci. In contrast, the kinetics of Rad51 foci – which signal the initiation of homologous recombination (HR) DNA repair – significantly differed between these populations. While Rad51 foci were immediately induced and quickly resolved in MP, very few Rad51 foci were formed in HSC up to 12 hours post-IR. Since HR only occurs during S/G2/M phases of the cell cycle, it is possible that the largely quiescent HSC can not utilize HR and instead use the more error prone DNA repair mechanism non-homologous end joining (NHEJ). At the molecular level, both HSC and MP displayed similar levels of genes associated with DNA damage recognition and repair (atm, rad50) and specific to HR (brca1), while MP display significantly lower levels of genes specifically involved in NHEJ (ku80) compared to HSC. These results indicate that both HSC and MP can recognize damaged DNA but might preferentially use different repair mechanisms. They also suggest that to become transformed and drive leukemia development, HSC might only require DNA damage, while MP might also need deregulation affecting both DNA repair mechanisms and apoptosis machinery. These findings provide insights into the mechanisms that maintain homeostatic function of hematopoietic stem and progenitor cells in normal tissues, and the deregulations that can occur during aging and cancer development. Ultimately, they could identify molecular targets to prevent therapy-related organ damage or secondary leukemia.
Turning the Mre11/Rad50 DNA repair complex on its head: lessons from SMC protein hinges, dynamic coiled-coil movements and DNA loop-extrusion?
