Radioprotection of hematopoietic progenitors by low dose amifostine prophylaxis.

PURPOSE: Amifostine is a highly efficacious cytoprotectant when administered in vivo at high doses. However, at elevated doses, drug toxicity manifests for general, non-clinical radioprotective purposes. Various strategies have been developed to avoid toxic side-effects: The simplest is reducing the dose. In terms of protecting hematopoietic tissues, where does this effective, non-toxic minimum dose lie? MATERIAL AND METHODS: C3H/HEN mice were administered varying doses of amifostine (25-100 mg/kg) 30 min prior to cobalt-60 irradiation and euthanized between 4-14 days for blood and bone marrow collection and analyses. RESULTS: Under steady-state, amifostine had little effect on bipotential and multi-potential marrow progenitors but marginally suppressed a more primitive, lineage negative progenitor subpopulation. In irradiated animals, prophylactic drug doses greater than 50 mg/kg resulted in significant regeneration of bipotential progenitors, moderate regeneration of multipotential progenitors, but no significant and consistent regeneration of more primitive progenitors. The low amifostine dose (25 mg/kg) failed to elicit consistent and positive, radioprotective actions on any of the progenitor subtypes. CONCLUSIONS: Radioprotective doses for amifostine appear to lie between 25 and 50 mg/kg. Mature, lineage-restricted progenitors appear to be more responsive to the protective effects of low doses of amifostine than the more primitive, multipotential progenitors.

Role of NF-kappaB in hematopoietic niche function of osteoblasts after radiation injury.

OBJECTIVE: Hematopoietic tissue is very sensitive to ionizing radiation (IR). In adult mammalian bone marrow, hematopoietic stem and progenitor cells (HSPC) reside next to the endosteal bone surface, which is lined primarily by osteoblastic cells. In the present study, we proposed to investigate the mechanisms by which osteoblasts in the hematopoietic niche regulate survival, proliferation, and differentiation of HSPC after radiation injury. MATERIALS AND METHODS: Human primary CD34+ HSPC were cultured with human fetal osteoblast (hFOB) cell line cells or conditioned medium (CM) from hFOB cells with or without irradiation. Survival, apoptosis, and cell cycle were analyzed using clonogenic and flow cytometric assays. Cytokine and chemokine expression were measured by cytokine array and enzyme-linked immunosorbent assay. Their regulatory activities were assessed by quantitative real-time polymerase chain reaction, small interfering (si)RNA transfection, immunoblotting, and transbinding assays. RESULTS: Survival of gamma-irradiated CD34+ HSPC was significantly enhanced by coculture with hFOB cells or by CM from hFOB cells. There were six factors in hFOB cell lysates and five factors released into hFOB CM enhanced by IR. IR induced phosphorylation of p53, c-Jun, and p38 and downstream p21 expression, as well as cell cycle arrest and apoptosis in hFOB cells. However, IR also induced phosphorylation of nuclear factor (NF)-kappaBp65 (ser536) and NF-kappaB activation in hFOB cells. Inhibition of NF-kappaB expression with siRNA upregulated p21, inhibited release of cytokines and chemokines, and induced hFOB and CD34+ cell apoptosis. CONCLUSIONS: NF-kappaB is a radiation-induced prosurvival factor in human osteoblastic cells. NF-kappaB gene knockdown abrogated the hematopoietic niche function of hFOB cells in supporting survival of CD34+ cells after IR.

5-Androstenediol promotes survival of gamma-irradiated human hematopoietic progenitors through induction of nuclear factor-kappaB activation and granulocyte colony-stimulating factor expression.

5-Androstenediol (5-AED) stimulates hematopoiesis and enhances survival in animals exposed to ionizing radiation (IR), suggesting that this steroid may act on hematopoietic progenitor cells. We used gamma-irradiated primary human CD34(+) hematopoietic progenitor cells to show that 5-AED protects hematopoietic cells from IR damage, as shown by enhanced cell survival, clonogenicity, proliferation, and differentiation. Unlike in tumor cells, IR did not induce nuclear factor-kappaB (NFkappaB) activation in primary progenitors. However, IR stimulated IkappaB(beta) release from NFkappaB/IkappaB complexes and caused NFkappaB1 (p50) degradation. 5-AED stabilized NFkappaB1 in irradiated cells and induced NFkappaB gene expression and NFkappaB activation (DNA binding). 5-AED stimulated interleukin-6 and granulocyte colony-stimulating factor (G-CSF) secretion. The survival-enhancing effects of 5-AED on clonogenic cells were abrogated by small interfering RNA inhibition of NFkappaB gene expression and by neutralization of G-CSF with antibody. The effects of 5-AED on survival and G-CSF secretion were blocked by the NFkappaB inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). 5-AED had no effect on accumulation of the proapoptotic factor p53 after IR, as determined by Western blot. The results indicate that NFkappaB1 degradation after IR may be responsible for the radiation sensitivity of CD34+ cells compared with tumor cells. 5-AED exerts survival-enhancing effects on irradiated human hematopoietic progenitor cells via induction, stabilization, and activation of NFkappaB, which results in increased secretion of hematopoietic growth factor G-CSF.

Effects of whole-body gamma irradiation and 5-androstenediol administration on serum G-CSF.

5-Androstenediol (5-AED) is a natural circulating adrenocortical steroid hormone that interconverts in vivo with other members of the 5-androstene family of steroids: dehydroepiandrosterone and 5-androstenetriol. These steroids stimulate immune responses and resistance to infection. 5-AED has been identified as a systemic radiation countermeasure that enhances survival in mice exposed to gamma irradiation and ameliorates radiation-induced neutropenia in mice and nonhuman primates. 5-AED mitigates radiation-induced decreases in platelets, natural killer (NK) cells, red blood cells, and monocytes. Administration of 5-AED causes functional activation of circulating granulocytes (phagocytic ability), monocytes (oxidative burst), and NK cells (surface CD11b expression). The effects of 5-AED on survival and hematological parameters are consistent with induction of hematopoietic cytokines. To test this hypothesis, we measured serum cytokines by ELISA, Luminex, and a cytokine array. A cytokine array was used for 62 different cytokines, chemokines, growth factors, and soluble receptors. 5-AED caused significant increases in circulating granulocyte colony-stimulating factor (G-CSF) in irradiated and unirradiated animals as observed with ELISA and Luminex. The cytokine array results suggest induction of G-CSF and additional cytokines, and related molecules. Since G-CSF is an important hematopoietic cytokine, the results support our hypothesis that the previously observed increases in numbers of hematopoietic progenitors, circulating innate immune cells and platelets, and functional activation of granulocytes, monocytes, and NK cells result from a cytokine cascade induced by 5-AED.