Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity.@*METHODS@#Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively.@*RESULTS@#Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001).@*CONCLUSION@#DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

Protective effects of deferasirox and N-acetyl-L-cysteine on iron overload-injured bone marrow

Using an iron overload mouse model, we explored the protective effect of deferasirox (DFX) and N-acetyl-L-cysteine (NAC) on injured bone marrow hematopoietic stem/progenitor cells (HSPC) induced by iron overload. Mice were intraperitoneally injected with 25 mg iron dextran every 3 days for 4 weeks to establish an iron overload (Fe) model. DFX or NAC were co-administered with iron dextran in two groups of mice (Fe+DFX and Fe+NAC), and the function of HSPCs was then examined. Iron overload markedly decreased the number of murine HSPCs in bone marrow. Subsequent colony-forming cell assays showed that iron overload also decreased the colony forming capacity of HSPCs, the effect of which could be reversed by DFX and NAC. The bone marrow hematopoiesis damage caused by iron overload could be alleviated by DFX and NAC.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Counteracting the enzymatic activity of dipeptidylpeptidase 4 for potential therapeutic advantage, with an emphasis on cord blood transplantation

Dipeptidylpeptidase (DPP) 4, also known as CD26, is an enzyme present on the surface of a number of different cell types. It is also found within cells and as a soluble protein in body fluids. It can specifically truncate proteins at the penultimate N-terminus residue for some amino acids, such as alanine, proline, serine, and perhaps others. DPP4 has been implicated in regulating the in vitro and in vivo functional activities of a number of hematopoietically active molecules, and this information, along with that on inhibition of DPP4, has been studied in efforts to enhance hematopoietic cell transplantation (HCT), hematopoiesis after stress in mouse models, and in the clinical setting of single-unit cord blood (CB) HCT. This article reviews the current status of this compound's effects on regulatory proteins, the field of CB HCT, a potential role for modulating DPP4 activity in enhancing single-unit CB HCT in adults, and future aspects in context of other cellular therapies and the area of regenerative medicine.

Effect of Human Parathyroid Hormone on Hematopoietic Progenitor Cells in NOD/SCID Mice Co-Transplanted with Human Cord Blood Mononuclear Cells and Mesenchymal Stem Cells

PURPOSE: We evaluated the effect of human parathyroid hormone (hPTH) on the engraftment and/or in vivo expansion of hematopoietic stem cells in an umbilical cord blood (UCB)-xenotransplantation model. In addition, we assessed its effect on the expression of cell adhesion molecules. MATERIALS AND METHODS: Female NOD/SCID mice received sublethal total body irradiation with a single dose of 250 cGy. Eighteen to 24 hours after irradiation, 1x107 human UCB-derived mononuclear cells (MNCs) and 5x106 human UCB-derived mesenchymal stem cells (MSCs) were infused via the tail vein. Mice were randomly divided into three groups: Group 1 mice received MNCs only, Group 2 received MNCs only and were then treated with hPTH, Group 3 mice received MNCs and MSCs, and were treated with hPTH. RESULTS: Engraftment was achieved in all the mice. Bone marrow cellularity was approximately 20% in Group 1, but 70-80% in the hPTH treated groups. Transplantation of MNCs together with MSCs had no additional effect on bone marrow cellularity. However, the proportion of human CD13 and CD33 myeloid progenitor cells was higher in Group 3, while the proportion of human CD34 did not differ significantly between the three groups. The proportion of CXCR4 cells in Group 3 was larger than in Groups 1 and 2 but without statistical significance. CONCLUSION: We have demonstrated a positive effect of hPTH on stem cell proliferation and a possible synergistic effect of MSCs and hPTH on the proportion of human hematopoietic progenitor cells, in a xenotransplantation model. Clinical trials of the use of hPTH after stem cell transplantation should be considered.

Amifostine exerts anti-angiogenic activity and suppresses vascular endothelial growth factor secreted by hemopoietic stem/progenitor cells

To assess, in vitro, the effect of Amifostine [AMF, WR-2721] on angiogenesis and levels of vascular endothelial growth factor [VEGF] secreted from hemopoietic stem/progenitor cell populations. We conducted the study in the research laboratories of the Hashemite University, Jordan between September 2003 and January 2005 where we took samples were from Myelodysplastic syndrome [MDS] patients and healthy donors attending Al-Hussein Cancer Center and We determined the proliferation of human umbilical vein endothelial cells [HUVECs] in cultures supplemented with media conditioned with AMF-treated and AMF-untreated pure hemopoietic cells [CD34+ cells, and erythroid, myeloid and megakaryocytic progenitors]. Furthermore, in the same conditioned media, we evaluated levels of elaborated VEGF by a sensitive enzyme linked immunosorbent assay. Biologically, media conditioned with AMF-treated cells reduced proliferation of HUVECs compared to media conditioned with untreated control cells [p<0.05]. In cultures of AMF-untreated cells, elaboration of VEGF was higher [p<0.05] in media conditioned with cells from MDS patients compared to healthy donors. A 30 minutes pre-exposure of cells to AMF [500 mM] suppressed levels of VEGF secreted within 24 hours in 63 of 89 evaluated cultures. The percentage of reduction of VEGF in AMF-sensitive cultures was comparable in cultures of MDS cells [18%, 2-37%; median, range] and normal cells [12%, 2-45%]. The results showed that AMF exerts an anti-angiogenic activity and suppresses the secretion of VEGF in hemopoietic stem/progenitor cells obtained from both healthy individuals and patients with MDS

Leukotriene B4 pathway regulates the fate of the hematopoietic stem cells

Leukotriene B4(LTB4), derived from arachidonic acid, is a potent chemotactic agent and activating factor for hematopoietic cells. In addition to host defense in vivo, several eicosanoids have been reported to be involved in stem cell differentiation or proliferation. In this study, we investigated the effect of LTB4 on human cord blood CD34+ hematopoietic stem cells (HSCs). LTB4 was shown to induce proliferation of HSC and exert anti-apoptotic effect on the stem cells. Blockade of interaction between LTB4 and its receptor enhanced self-renewal of the stem cells. Effect of LTB4 on differentiation of CD34+ HSCs were confirmed by clonogenic assays, and induction of the expression of BLT2 (the low- affinity LTB4 receptor), during the ex vivo expansion was confirmed by reverse transcription-PCR. Our results suggest that LTB4-BLT2 interaction is involved in the cytokine-induced differentiation and ex vivo expansion of hematopoietic stem cells.

Allogeneic Peripheral Blood Stem Cell Rescue of Late Graft Failure after Bone Marrow Transplantation in Patients with Aplastic Anemia

We investigated the effect and outcome of allogeneic peripheral blood stem cell (PBSC) rescue for aplastic anemia (AA) patients with graft failure after allogeneic bone marrow transplantation (BMT). Seven (28%) of 25 AA patients who received BMT from HLA-identical sibling donors developed late graft failure at a median of 7 months (range, 2.0-9.3 months) after transplantation. The patients with graft failure were treated with PBSC collected from the original donor after mobilization with granulocyte-colony stimulating factor (G-CSF). The median boost dose of peripheral blood mononuclear cells was 3.1 x 10(8)/kg (range, 1.4-11.9 x 10(8)/kg). Median times to reach an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet count greater than 50 x 10(9)/L were 7 days (range, 4-14 days) and 9 days (range, 3-41 days), respectively. There was sustained graft function in 6 of 7 patients, with a median follow-up duration of 3.3 yr (range, 1.0-6.2 yr). Grade-I acute graft-versus-host disease (GVHD) occurred in 2 patients, while extensive chronic GVHD developed in 3 patients. This report shows that G-CSF-mobilized allogeneic PBSC rescue is very effective in achieving complete and sustained engraftment in patients with AA after graft failure. However, more efficacious measures to prevent extensive chronic GVHD remain to be developed.

Effects of ligustrazine on hematopoiesis in the early phase of bone marrow transplantation mice / 华中科技大学学报（医学）（英德文版）

To investigate the effects of Ligustrazine on histogenesis of bone marrow in the early phase of hematopoietic reconstruction in bone marrow transplantation (BMT) mice. The syngeneic BMT mice model was established. The syngeneic BMT mice were orally given 2 mg Ligustrazine twice a day. 1, 3, 5, 7, 10, 15 and 21 day(s) after BMT, peripheral blood granulocytes and bone marrow nucleated cells (BMNC) were counted and the diameter of central vein and the area of micro-vessel in femur were measured. The effect of Ligustrazine on hematopoietic stem cells was observed by colony forming unit of spleen (CFU-S). The effect of Ligustrazine on hemopoietic progenitors was studied by observing the number of progenitors of Granulocytes/Macrophage on day 10 and day 20 after BMT. In Ligustrazine-treated group, the diameter of center veins and the area of micro-vessel of femur were all significantly less than the control group 7, 10, 15, 21 days after BMT (P < 0.01). In addition, Ligustrazine significantly increased the number of CFU-S on day 10 and the number of CFU-GM on day 10, 20 after BMT. These results indicate that Ligustrazine can accelerate the histogenesis of hemopoietic bone marrow, which may be one mechanism by which Ligustrazine promotes hematopoietic reconstitution after BMT.

Expressão de receptor para o fator de crescimento insulina-símile tipo I (IGF-IR) na fase crônica da leucemia mielóide crônica (LMC) / Expression of the receptor to the insulin-like growth factor type I (IGF-IR) in chronic phase of the chronic myelogenous leukemia (CML)

Com o objetivo de verificar se o fator de crescimento insulina símile tipo I (IGF-I) e seu receptor (IGF-IR) estão implicados na instalação da leucemia mielóide crônica (LMC) foram estudados 35 pacientes portadores de LMC na fase crônica antes ou durante o tratamento com interferon `ALFA' ou hidroxiurea e 16 indivíduos sadios como grupo controle. A análise do IGF-IR realizada através da citometria de fluxo e expressão de seu RNAm pelo ensaio molecular de RT-PCR nas células sanguíneas dos pacientes com LMC não tratada não mostrou diferenças estatísticas em relação ao grupo controle. Pacientes tratados com hidroxiurea apresentaram expressão diminuída do receptor em granulócitos, monócitos e linfócitos (P`MENOR'0,01) quando comparados aos demais grupos analisados...

Role of endogenous polyamines in the proliferation of normal hematopoietic progenitor cells: high-proliferative potential colony-forming cells (HPP-CFC), and low-proliferative potential colony-forming cells (LPP-CFC). Studies "in vitro"

This study concerns the role of endogenous polyamines in the proliferation of normal hematopoietic progenitor cells: high-proliferative potential colony-forming cells (HPP-CFC), and low-proliferative potential colony-forming cells (LPP-CFC) in CFW/ep mouse bone marrow cells in the agar culture system. DL-a-difluoromethylornithine (DFMO) was used as a selective and irreversible inhibitor of ornithine decarboxylase which is the first limiting step in polyamine biosynthesis. The polyamines have been implicated in cell proliferation and differentiation. The results showed that DFMO significantly inhibited the formation of LPP-CFC colonies and completely inhibited the growth of the HPP-CFC colonies. Addition of exogenous putrescine at the concentration of 10(-7) M reverses the suppressive action of DFMO in both progenitor cells. At this concentration putrescine alone had no affect on the number or the size of the colonies. It was then concluded that endogenous polyamines appear to be essential for HPP-CFC proliferation and an important requirement for LPP-CFC proliferation.

Effects of deltamethrin on the growth and differentiation of bone marrow hematopoietic stem cells

1. The effects of deltamethrin on mouse bone marrow and spleen progenitor cell responsiveness to granulocyte and macrophage colony-stimulating factors (CSFs) were evaluated. 2. Deltamethrin (1-5 mg/kg) was administered four times subcutaneously on alternate days for one week to male BALB/c mice, 5-8 weeks old (N = 6 mice/group), raised under pathogen-free conditions and maintained in conventional animal rooms for four weeks before use. Soft agar colony formation (CFU-C), marrow and spleen cell counts as well as body, spleen and thymus weights were determined. 3. Although treatment with the lowest dose (1 mg kg-1 48 h-1) produced no significant effect on CFU-C, the administration of 3 and 5 mg kg-1 48 h-1 caused a more than two-fold increase in the formation of granulocyte and macrophage colonies in the marrow, but not in the spleen (control value = 100.5 +/- 12 for N = 6). Colony numbers returned to normal values within five days after the end of deltamethrin administration. 4. No changes were observed in the total (range: 1-3 x 10(8) per spleen) and differential marrow and spleen cell counts, nor was there any alteration in spleen weight. However, treatment with the three doses resulted in a dramatic reduction in thymus weight. 5. These effects were not due to the liberation of endotoxin, because if endotoxin had been present it would have been < 0.060 ng/ml, a concentration that would not have a biological effect. 6. In vitro addition of 0.10 to 10 microM deltamethrin to marrow cell cultures obtained from untreated mice did not induce any response. 7. These data indicate that the CSF-driven granulocyte and macrophage development provides a useful model for the study of the effects of toxicants on myelopoiesis

Comparative studies of erythroid growth factors

Se evaluó la aparición de factores séricos capaces de estimular la proliferación de progenitores eritroides mamíferos. Sueron anémicos y normales de ambos e jemplares, fraccionados por tratamiento alcohólico, se ensayaron por el método del ratón post-hipóxico, no detectándose incorporación de 59Fe. Al ser ensayados en cultivos semisólidos de médula ósea murina a diferentes tiempos de incubación (colonias CFU-E y BFU-E), el suero anémico aviario mostró alta actividad estimulatória, mientras que el suero anémico anfibio resultó incapaz de incrementar la proliferación eritroide. La muestra aviaria parcialmente purificada por tratamiento alcohólico fue cromatografiada en Sephadex G-150 revelando tres entidades moleculares respondables de la actividad biológica in vitro, con pesos moleculares aparentes de 29, 14 y 10 KD respectivamente. Los factores séricos aviarios estimulantes de la línea eritroide fueron sometidos a diversos tratamientos físico-químicos y luego se evaluó la conservación de su actividad biológica. Todos ellos resultaron termoestables, sensibles al tratamiento con neuraminideasa, mientras que el ditiotreitol provocó la pérdida de la actividad biológica de las proteínas de bajo peso molecular. Estos resultados sugieren, al menos bajo estas condiciones experimentales, la presencia de factores análogos estimulantes del crecimiento eritroideo entre amniotas homeotermos

A quantitative method for evaluating the effects of drugs on bone marrow cellularity using the same experimental animal: quantification of the effects of a single dose busulphan

Se describe un método que permite cuantificar la celuradidad de la médula ósea de la rata por unidad de peso. A tal efecto se determinaron en diferentes tiempos los números absolutos de cada tipo celular presentes en el fémur derecho e isquierdo del mismo animal de experimentación y los resultados se expresan en número de células por mg de médula ósea. En la rata normal se demostró que el número absoluto de cada tipo celular por mg de médula ósea es muy similar en el fémur derecho e izquierdo. Esto ocurre cuando el estudio de la celularidad medular se realiza simultáneamente en ambos huesos, y también cuando se comparan los resultados del estudio cuantitativo del fémur izquierdo con los del fémur derecho del mismo animal con 10 y 20 días de intervalo. Con el objeto de ratificar la validez del método para el estudio de la acción de drogas sobre la hematopoyesis medular, se observaron, además, los efectos de una dosis de busulfán (20 mg/Kg, vía oral), administrada inmediatamente después de que se realizó el estudio cuantitativo de la celularidad de la médula ósea del fémur izquierdo de una rata normal. Los resultados se compararon con los obtenidos de la médula ósea del fémur derecho de los mismos animales, 10 días después. Se demostró una marcada y significativa disminución del total de células nucleadas por mg de médula ósea, luego de ese período de observación. Los efectos celulares del busulfán fueron particularmente intensos sobre la progenie mieloide de médula ósea, con una reducción del