Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells.

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis.

Most adult stem cells, including hematopoietic stem cells (HSCs), are maintained in a quiescent or resting state in vivo. Quiescence is widely considered to be an essential protective mechanism for stem cells that minimizes endogenous stress caused by cellular respiration and DNA replication. We demonstrate that HSC quiescence can also have detrimental effects. We found that HSCs have unique cell-intrinsic mechanisms ensuring their survival in response to ionizing irradiation (IR), which include enhanced prosurvival gene expression and strong activation of p53-mediated DNA damage response. We show that quiescent and proliferating HSCs are equally radioprotected but use different types of DNA repair mechanisms. We describe how nonhomologous end joining (NHEJ)-mediated DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. Our results demonstrate that quiescence is a double-edged sword that renders HSCs intrinsically vulnerable to mutagenesis following DNA damage.

Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma.

In response to DNA damage, the p53 tumor suppressor can elicit either apoptosis or cell-cycle arrest and repair, but how this critical decision is made in specific cell types remains largely undefined. We investigated the mechanism by which the transcriptional repressor Slug specifically rescues hematopoietic progenitor cells from lethal doses of gamma radiation. We show that Slug is transcriptionally induced by p53 upon irradiation and then protects the damaged cell from apoptosis by directly repressing p53-mediated transcription of puma, a key BH3-only antagonist of the antiapoptotic Bcl-2 proteins. We established the physiologic significance of Slug-mediated repression of puma by demonstrating that mice deficient in both genes survive doses of total-body irradiation that lethally deplete hematopoietic progenitor populations in mice lacking only slug. Thus, Slug functions downstream of p53 in developing blood cells as a critical switch that prevents their apoptosis by antagonizing the trans-activation of puma by p53.

p53 loss of function enhances genomic instability and accelerates clonal evolution of murine myeloid progenitors expressing the p(210)BCR-ABL tyrosine kinase.

BACKGROUND AND OBJECTIVES: The p210 bcr-abl fusion protein has a key role in the pathogenesis of chronic myeloid leukemia (CML). However, its influence on disease progression to blast crisis is marginal and mostly due to its effect of impairing the genomic stability of clonal myeloid progenitors through pathways still largely unknown. DESIGN AND METHODS: To elucidate the role of p53 in CML progression we generated, from the 32D murine myeloid cell line, several clones co-expressing the E6 product gene of human papilloma virus (HPV) 16, which abrogates p53 function, and a temperature-sensitive bcr-abl construct encoding a fully active p210 protein only at the permissive temperature of 33 degrees C. RESULTS: Co-expression of the two proteins resulted in a significant enlargement of the G(2)/M phase of cell cycle and in the appearance of a poly-aneuploid cell population. Furthermore, with continuous in vitro passages the p210 tyrosine kinase became dispensable for growth. Increased levels of cyclin B(1) and enhanced activity of its associated cyclin-dependent kinase (cdc2) became apparent during the clonal evolution of p210 bcr-abl-transduced 32D cell clones lacking p53. INTERPRETATION AND CONCLUSIONS: The acceleration of clonal evolution of p210 bcr-abl-transduced 32D myeloid progenitors associated with p53 functional abrogation is consistent with oncosuppressor loss having a key role in CML progression. This would allow emergence of additional genomic aberrations which would lead to the fully transformed phenotype of blast crisis. Deregulated activity of the cyclin B1-cdc2 complex may be involved in the loss of temporal co-ordination of mitotic events and further free the barrier to genomic instability of CML clonal myeloid progenitors lacking p53.

Futile caspase-8 activation during the apoptotic cell death induced by DNA damaging agents in human B-lymphoblasts.

Caspase-8 plays an essential role in apoptosis induced by Fas activation. Moreover, caspase-8 can be processed also in response to exposure to genotoxic agents. To decipher the role of caspase-8 in DNA damaging agent (DDA)-induced apoptosis as well as the pathway(s) leading to its activation in response to genotoxic stress, we investigated caspase-8 processing induced by ionizing radiation (IR) or mitomycin C (MMC) treatment in human B-lymphoblasts. Altogether, our observations establish that caspase-8 is actively processed in both receptor-mediated and DDA-induced cell death. However, while Fas-dependent apoptosis absolutely required caspase-8 activity, it is not necessary for completion of the apoptotic program induced by IR and MMC. Experiments performed to understand the molecular pathway(s) of the caspase-8 activation after DDA demonstrated that for both IR and MMC, the Fas/Fas-L interaction is dispensable. Data obtained from caspase inhibitors and from lymphoblasts carrying mutations in ATM and FANCC proteins, involved in DDA response, clearly showed that distinct mechanisms are responsible for caspase-8 activation by IR and MMC in B-lymphoblasts. IR-dependent processing of caspase-8 involves ATM, mitochondrial collapse, FANCC, and caspase-3 activation. Caspase-8 activation by MMC evokes the mitochondrial pathways involving FANCC but not ATM. Collectively, our data indicate that caspase-8 activation is essentially a bystander effect and not a major determinant of the behavior of DDA-exposed cells.