Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog.

The function of Hedgehog signaling in hematopoiesis is controversial, with different experimental systems giving opposing results. Here we examined the role of Desert Hedgehog (Dhh) in the regulation of murine erythropoiesis. Dhh is one of 3 mammalian Hedgehog family proteins. Dhh is essential for testis development and Schwann cell function. We show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In addition, we found that erythroblast populations were Dhh-responsive in vitro and ex vivo and that Dhh negatively regulated erythroblast differentiation. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT.

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents.

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.

Sublethal radiation injury uncovers a functional transition during erythroid maturation.

OBJECTIVE: Clastogenic injury of the erythroid lineage results in anemia, reticulocytopenia, and transient appearance of micronucleated reticulocytes. However, the micronucleated reticulocyte dose-response in murine models is only linear to 2 Gy total body irradiation and paradoxically decreases at higher exposures, suggesting complex radiation effects on erythroid intermediates. To better understand this phenomenon, we investigated the kinetics and apoptotic response of the erythron to sublethal radiation injury. MATERIALS AND METHODS: We analyzed the response to 1 and 4 Gy total body irradiation of erythroid progenitors and precursors using colony assays and imaging flow cytometry, respectively. We also investigated cell cycling and apoptotic gene expression of the steady-state erythron. RESULTS: After 1 Gy total body irradiation, erythroid progenitors and precursors were partially depleted. In contrast, essentially all bone marrow erythroid progenitors and precursors were lost within 2 days after 4 Gy irradiation. Imaging flow cytometry analysis revealed preferential loss of phenotypic erythroid colony-forming units and proerythroblasts immediately after sublethal irradiation. Furthermore, these populations underwent radiation-induced apoptosis, without changes in steady-state cellular proliferation, at much higher frequencies than later-stage erythroid precursors. Primary erythroid precursor maturation is associated with marked Bcl-xL upregulation and Bax and Bid downregulation. CONCLUSIONS: Micronucleated reticulocyte loss after higher sublethal radiation exposures results from rapid depletion of erythroid progenitors and precursors. This injury reveals that erythroid colony-forming units and proerythroblasts constitute a particularly proapoptotic compartment within the erythron. We conclude that the functional transition of primary proerythroblasts to later-stage erythroid precursors is characterized by a shift from a proapoptotic to an antiapoptotic phenotype.

Radiosensitization of murine normoblasts in vivo by bromodeoxyuridine to the genotoxicity and cytotoxicity of the bone-seeking radiopharmaceutical 153Sm-EDTMP.

Abstract To establish a basis for a possible strategy for bone marrow ablation or therapy, we examined the effect of bromodeoxyuridine (BrdU) incorporation into DNA on the genotoxic and cytotoxic effects of samarium-153 ethylenediaminetetramethylene phosphonate ((153)Sm-EDTMP) in normoblasts in vivo. Cytotoxicity and genotoxicity were established by time-response curves of polychromatic erythrocyte (PCE) and micronucleated polychromatic erythrocyte (MN-PCE) frequencies, respectively, in mouse peripheral blood samples. The group treated with (153)Sm-EDTMP showed a clear induction of MN-PCEs; however, the group treated with BrdU plus (153)Sm-EDTMP paradoxically showed only a slight increase with respect to untreated controls. Treatment with (53)Sm-EDTMP caused a small reduction in PCE frequency, but exposure to BrdU or to BrdU plus (53)Sm-EDTMP reduced the PCE frequency significantly from 32 h to the end of the experiment. The PCE frequencies in the BrdU plus (53)Sm-EDTMP group were significantly lower than in the BrdU control group at the final time and were much lower than the group treated with only (53)Sm-EDTMP, which returned to basal values. The results suggest the radioinduction of a lethal lesion in BrdU-substituted DNA that cannot be repaired easily and does not permit cell division and micronucleus formation.

Effect of gamma-linolenic acid and prostaglandins E1 on gamma-radiation and chemical-induced genetic damage to the bone marrow cells of mice.

The effect of gamma-linolenic acid (GLA) and prostaglandin E1 (PGE1) on gamma-radiation, diphenylhydantoin (DPH), benzo(a)pyrene (BP), and 4-alpha-phorbol-induced genetic damage to the bone marrow cells of mice, using the sensitive micronucleus (MN) test was investigated. PGE1 and its precursor GLA prevented gamma-radiation, DPH, BP, and 4-alpha-phorbol-induced genetic damage.

Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)--mediated cytotoxicity by selective up-regulation of TRAIL-R1.

Cytotoxic activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand), used alone or in different combinations with either a low (1.5 Gy) or a high (15 Gy) single dose of ionizing radiation (IR), was investigated on erythroleukemic cells (K562, HEL, Friend, primary leukemic erythroblasts) and on primary CD34(+)-derived normal erythroblasts. Human recombinant TRAIL alone variably affected the survival/growth of erythroleukemic cells; K562 cells were the most sensitive. Moreover, all erythroleukemic cells were radio-resistant, as demonstrated by the fact that cytotoxicity was evident only after treatment with high-dose (15 Gy) IR. Remarkably, when IR and TRAIL were used in combination, an additive effect was noticed in all erythroleukemic cells. Augmentation of TRAIL-induced cell death by IR was observed with both low and high IR doses and required the sequential treatment of IR 3 to 6 hours before the addition of TRAIL. Conversely, both TRAIL and IR showed a moderate cytotoxicity on primary CD34(+)-derived normal erythroblasts when used alone, but their combination did not show any additive effect. Moreover, the cytotoxicity of IR plus TRAIL observed in erythroleukemic cells was accompanied by the selective up-regulation of the surface expression of TRAIL-R1 (DR4), and it was completely blocked by the z-Val-Ala-Asp (OMe)-CH(2) (z-VAD-fmk) caspase inhibitor. On the other hand, the surface expression of TRAIL-R1 in CD34(+)-derived normal erythroblasts was unaffected by IR, which induced the up-regulation of the decoy TRAIL-R3. These data demonstrate that treatment with IR provides an approach to selectively sensitize erythroleukemic cells, but not normal erythroblasts, to TRAIL-induced apoptosis through the functional up-regulation of TRAIL-R1.

Radioprotective effects of the thiols GSH and WR-2721 against X-ray-induction of micronuclei in erythroblasts.

The frequency of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow and peripheral blood of adult male Swiss mice treated with reduced glutathione (GSH) and S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721), at a dose of 400 mg/kg body weight, and exposed to 6 Gy X-rays. GSH or WR-2721 was applied alone, or 60 and 30 min, respectively, prior to X-ray-exposure. The number of MNPCEs was determined at 24 h after the thiol treatment and X-irradiation. The radioprotection and toxicity caused in the mouse erythroblasts by GSH and WR-2721, as indicated by the number of MNPCEs were dependent on the thiol applied. The stronger radioprotective effect is obtained following WR-2721 administration than after GSH application. WR-2721 showed greater toxicity than GSH. The combination of GSH and WR-2721 given before X-ray-exposure resulted in the most radioprotective effect as compared to the respective single-drug treatment of mice. Application of the both thiols, without subsequent X-irradiation appeared to be the most toxic, compared with administration of WR-2721 or GSH alone. The effective radioprotection by the combined action of GSH and WR-2721 against genomic instability induced in the mouse erythroblasts by X-rays was shown.

Endogenous p53 regulation and function in early stage Friend virus-induced tumor progression differs from that following DNA damage.

Erythroleukemia induced by the anemia strain of Friend virus occurs in two stages. The first stage results in rapid expansion of pre-leukemic proerythroblasts (FVA cells) dependent on erythropoietin (Epo) for differentiation and survival in vitro. The second stage is characterized by emergence of erythroleukemic clones (MEL cells) which typically bear activation of the ets-oncogene, PU.1/spi.1, and loss of functional p53. We developed a Friend virus-sensitive, p53-deficient mouse model to investigate the biological advantage conferred by p53-loss during tumor progression. Here we report p53 was not required for cell survival or growth arrest during differentiation of FVA cells, nor was p53 required for induction of apoptosis upon Epo withdrawal. However, we detected induction of the p21Cip1 cyclin-dependent kinase inhibitor gene during differentiation, which was markedly enhanced in the presence of p53. p53-dependent expression of p21Cip1 occurred in the absence of an increase in p53 mRNA and protein levels and was specific for p21Cip1, since expression of gadd45, mdm-2, cyclin G and bax were unaffected by p53. In contrast, treatment of FVA cells with DNA damaging agents led to rapid accumulation of p53 protein resulting in transcription of multiple p53-regulated genes, leading to either apoptosis or growth arrest, depending on the agent used. These data demonstrate that p53-dependent activities during differentiation of preleukemic erythroblasts are distinct from those observed in response to genotoxic agents. We propose that enhancement of p53-dependent gene expression during differentiation may represent a tumor suppressor function which is necessary to monitor differentiation of preleukemic cells and which is selected against during tumor progression.

Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.

Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock.

Radiation-induced micronucleus formation in mouse bone marrow after low dose exposures.

The incidence of micronucleus formation was studied at 12, 24 and 36 h post-irradiation in the polychromatic (PCE) and normochromatic (NCE) erythrocytes of the bone marrow of mice whole-body exposed to 0, 3, 9, 18, 36, 54 and 72 cGy of 60Co gamma-radiation. It was observed that the frequency of micronucleated polychromatic erythrocytes (MPCE) increased with the increase in exposure dose at all the post-irradiation time periods studied. Similarly, the frequency of micronucleated normochromatic erythrocytes (MNCE) also increased with the increase in exposure dose and the increase for both MPCE and MNCE was dose related. The dose-response relationship was linear-quadratic for both MPCE and MNCE. The study of mitotic index revealed that a dose as low as 9 cGy is capable of reducing the mitotic index significantly at 24 h post-irradiation and the dose response was linear-quadratic. However, no significant decline in the mitotic index was observed at 12 and 36 h post-irradiation.

Residual adriamycin (AdR)-induced hematopoietic damage: a consideration for subsequent radiotherapy.

The long-term effect of adriamycin (AdR) on the radiation response of hematopoietic marrow was studied at 16 weeks after treatment with a MTD (10 mg/kg) for the BDF1 mouse. The radiation response was monitored in both the "stem cell" (CFUs-8) and myeloid (CFU-gm, granulocyte, WBC) compartments, as well as the erythroid (BFUe, CFUe, HcT) compartments of the marrow for 14 days following a whole body dose (TBI) of 4.5 Gy X ray. At the time of irradiation, animal and spleen weight of AdR treated animals were reduced while HcT and WBC remained at control levels. At the same time the granulocyte and CFUs-8d compartments were depressed, while the BFUe compartment was expanded. The CFUe and CFU-gm compartments remained at control levels. For all marrow compartments, treatment with AdR 16 weeks prior to 4.5 Gy resulted in a radiation response deficit determined from the temporal recovery curves. The data suggest that manifestation of long-term AdR injury, at least through 16 weeks following treatment, is dependent on a subsequent stress of sufficient magnitude to enhance the proliferative activity associated with hematopoietic cell production and differentiation. A comparison is made between these observations and previously reported long-term drug-induced hematopoietic injury.

Self-renewal capacity of murine hemopoietic stem cells under internal contamination with 239Pu and 241Am.

The self-renewal capacity of murine pluripotent hemopoietic stem cells (CFU-S) of vertebral bone marrow was studied under conditions of short-term and long-term internal contamination with 239Pu or 241Am in female mice. Measurement of the CFU-S self-renewal capacity was carried out using double transplantation assay. To evaluate the production of differentiated progeny of stem cells average erythroblast numbers/visible spleen colony and 59Fe-uptake/colony were computed. The marrow cellularity/vertebra and the number of CFU-S/vertebra were decreased and affected more by 239Pu than by 241Am. The production of erythroblasts per a single CFU-S and the 59Fe-uptake/colony were reduced, similarly the numbers of secondary spleen colonies and of secondary CFU-S in primary colonies. The above changes resulting from impaired functions of surviving CFU-S were more serious with 241Am than with 239Pu. The biological effects of plutonium and americium appeared independent of the phase of contamination.

The effects of low dose (less than 1 rad) X-rays on the erythropoietic marrow.

Adult female Sprague-Dawley rats were exposed (200 kvp X-rays) to whole body doses of 22-1320 mrad and examined for changes in the level of red blood cell precursors (RBCp) in the marrow at 5-30 weeks post-irradiation, under nonbled and phlebotomy-induced anemic stress conditions. Increases in the RBCp %, RBCp/mg marrow, and RBCp/skeleton under nonbled conditions, and a suppressed erythroid response to an induced anemia, were found after acute doses in the range of at least 70 mrad. Dosages of 22 or 44 mrad that induced no measurable changes when applied only once were found to be effective when they were employed 4 or 2 times/week, respectively. The results suggested the presence of a linear-quadratic dose-response relationship in which the quadratic function exists between 88 and 981 mrad, and the linear dependency, below 88 mrad.

Radiation protection of pronormoblasts and normoblasts by 2-mercaptopropionylglycine (MPG).

The pronormoblasts and normoblasts in Swiss albino mice were found to be very sensitive to radiation and their percentage was reduced drastically after exposure to gamma-rays. The degree of damage increased with increase in radiation dose. MPG reduced the initial damage and brought about an early and fast recovery. It is concluded that the drug protects the stem cells and thereby reduces the depletion of the regenerating pool which causes a more efficient and accelerated recovery.

[Ineffective erythropoiesis in myeloid splenomegaly: the 59Fe test, bone marrow histologic and cytologic data]. / L'érythropoïèse inefficace au cours des splénomégalies myéloïdes: test au 59Fe, données histologiques et cytologiques médullaires.

Eight patients presenting primary myelofibrosis or spent polycythemia were submitted to a ferrokinetic study, a triphine bone marrow biopsy and a bone marrow puncture. In all cases before treatment (splenic radiotherapy) ferrokinetic measurements demonstrated an ineffective erythropoiesis, the intensity of which was precised on bone marrow biopsies, in numbering the erythroblasts per surface unit. Bone marrow biopsies permitted also, through a semiquantitative estimate of the bone marrow cellularity, to evaluate an aplasia component not always expected with ferrokinetic study. Study of bone marrow allowed to precise the site of the erythroblastic abortion; cellular death might occur during the last nitosis of the erythroblastic series (transition from the polychromatophil erythroblasts to the acidophil erythroblasts). In order to delineate the general pattern of erythropoiesis before treatment and when assessing the results of a treatment, besides ferrokinetic measurements, the study of bone marrow biopsy and smears appears relevant.

The effects of continous irradiation on histological picture and deoxyribonucleoprotein content in spleen of rats.

The histological picture and deoxyribonucleoprotein (DNP) content in the spleen of the white male Wistar rats continuously irradiated with the daily dose rates 9.57 mGy (1 R), 95.7 mGy (10 R), 478.5 mGy (50 R) and 657.0 mGy (100 R) were studied. The animals were examined within the day until 60 of irradiation. The number of lymphocytes permanently decreased at the daily dose rates 95.7 mGy and higher ones. The increased activity of reticulum with the simultaneous multiplication of the erythroid cells in the red pulp of spleen was observed at day 10 and 60 of irradiation with 9.57 mGy and 95,7 mGy. Simultaneously DNP content in spleen increased in accordance with the increased activity of reticulum and extramedullar erythropoiesis. The significant decrease in DNP content alter the onset of irradiation with the daily dose rates 478.5 mGy and 957.0 mGy was caused mainly by the fall of lymphocytes. The incidence of eosinophilic leukocytes increased from day 40 irradiation with the daily dose rates 9.57 mGy, 95.7 mGy and 478.5 mGy and decreased with the daily dose rate 957.0 mGy. The plasma cells tended to increase in accordance with the increasing accumulated dose of irradiation with all daily dose rates.

Dose response at low doses of X-irradiation and MMS on the induction of micronuclei in mouse erythroblasts.

The test of induced micronuclei in erythrocytes of mammalian bone marrow constitutes, because of its high experimental resolution power, a suitable method for the screening of induced chromosomal lesions at very low dosages of chemicals or irradiations. This test was used for a comparative investigation of the effect of low dose levels of X-irradiation and of the alkylating agent methyl methanesulphonate (MMS). The dose-effect curve of X-irradiation indicated a deviation from linearity at 10 rad, showing a significantly stronger effect than expected on extrapolation from the control to 100 rad. This deviation from linarity, however, only appeared at a low dose rate (18 R/min), whereas a linear dose-effect relation was indicated with a high dose rate (95 R/min). Experiments at 10 rad with different dose rates at two different current potentials suggested that this effect of the dose rate is more pronounced with soft than with hard X-irradiation. The induction of micronuclei with MMS follows a drastically different dose-effect curve as compared with X-irradiation. The relative efficiency of the treatment is lowest at low concentrations, presumably as a result of the efficient repair process at such dose levels. Simultaneous treatment with X-rays and MMS at low dose levels only resulted in an additive effect. This suggests that X-irradiation does not interfere with the repair process operating with MMS. The difference in the dose-effect relations of X-irradiation as compared with MMS may be brought back to the fact that X-rays, in contrast with MMS, produce double-strand breaks.

Effect of irradiation in the space environment on the blood-forming system in rats.

A special experiment was carried out on board Cosmos 690 [correction of 605] in order to study the modifying influence of weightlessness on the radiobiological effect. On the tenth flight day 35 rats were exposed to irradiation using an on-board gamma-ray source. They were irradiated for 24 hours at a dose rate of 32 rads/hr with a total dose of 220 +/- 25 rads and 800 +/- 53 rads. On the 1st and 26th post-flight days haemopoiesis in bone marrow (determined from myelograms and the total count of myelo-karyocytes) was examined and the blood composition was analysed. The total count of thymocytes and spleenocytes was measured in the thymus and spleen. Identical measurements were made in the control animals exposed to irradiation on the earth. The haematological findings indicate an enhancement of the radiobiological effect in the rats irradiated in space flight.