Stathmin 1 Is Involved In The Highly Proliferative Phenotype Of High-Risk Myelodysplastic Syndromes and Acute Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 861-861
Author(s):  
João Agostinho Machado-Neto ◽  
Paula de Melo Campos ◽  
Patricia Favaro ◽  
Mariana Lazarini ◽  
Irene Lorand-Metze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Low Risk ◽  
Leukemia Cells ◽  
Healthy Donors ◽  
Stathmin 1 ◽  
Marrow Cells

Abstract Introduction : Stathmin 1, also known as Oncoprotein 18 (OP18) or Leukemia-associated phosphoprotein p18 (LAP18), is an important cytoplasmic microtubule-destabilizing protein that plays a critical role in the process of mitosis, proliferation and accurate chromosome segregation through regulation of microtubule dynamics. High levels of Stathmin 1 have been reported in solid tumors and have been associated with poor prognosis in various types of cancers. The identification of overactive proteins in leukemia cells, compared to normal hematopoietic cells, as well as understanding the molecular and cellular basis of the disease may provide new therapeutic opportunities. Aims: To evaluate Stathmin 1 expression in proliferating and non-proliferating hematopoietic cells, in bone marrow cells from healthy donors and from patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). In addition, we evaluated the effect of Stathmin 1 silencing on proliferation and apoptosis in the U937 acute myeloid leukemia cell line. Materials and Methods: A panel of human leukemia cell lines that included myeloid (K562, KU812, NB4, HL60, P39, HEL, U937, KG1 and THP1) and lymphoid cells (Jurkat, MOLT4, Daudi, Raji, Namalwa and Karpas 422) in exponential growth was used. Peripheral blood lymphocytes (PBL) were induced, or not, to proliferate upon PHA stimulation for 72 hours. A total of 30 healthy donors and 117 patients at diagnosis (MDS=52 [low-risk=36, high-risk=16], AML=49, and ALL=16) were included in the study. Stathmin 1 gene and protein expression was evaluated by qPCR and Western blot. Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector and cell growth was examined by MTT assay, clonogenicity by colony formation and apoptosis by AnnexinV/PI. Appropriate statistical analyses were performed; results are expressed as median (minimum- maximum). Results: A higher expression of Stathmin 1 was observed in all leukemia cell lines, when compared with normal non-proliferating hematopoietic cells. We also observed a marked increase in Stathmin 1 expression in PBL induced to proliferate with PHA after 72 hours. Stathmin 1 transcripts were significantly increased in total bone marrow cells from patients with AML (2.01 [0.35-8.88]; p=.0009) and ALL (2.94 [1.16-10.82]; p=.0004), compared with healthy donors (1.01 [0.38-4.08]). No difference in Stathmin 1 expression was observed between healthy donors and MDS patients. When the MDS group was stratified by the WHO classification into low and high-risk MDS, Stathmin 1 expression was significantly higher in the high-risk, when compared with low-risk MDS (1.62 [0.42–3.28] vs. 1.13 [0.36–2.61], p=.03). Similar results were found in isolated CD34+ bone marrow cells, Stathmin 1 transcripts were significantly increased in CD34+ AML cells compared with CD34+ normal cells, and in high-risk compared with low-risk MDS (all p≤.02). Interestingly, 3 out of 5 MDS patients showed a significant increase in Stathmin 1 transcripts after disease progression. Also, a significant positive correlation was observed between percentage of bone marrow blasts and Stathmin 1 expression in MDS patients (p=.03; r=.31). In U937 leukemia cells, Stathmin 1 silencing significantly reduced cell proliferation (p=.02) and clonal growth (p<.0001), but did not modulate apoptosis. Conclusions: Stathmin 1 is overexpressed in high-risk MDS and acute leukemia cells, and is upregulated during MDS progression, suggesting that Stathmin 1 plays a role in the highly proliferative phenotype. Our study adds new insights to the role of Stathmin 1 in leukemogenesis. Future studies are necessary to validate whether Stathmin 1 is a predictive marker for MDS progression, and to determinate whether Stathmin 1 is a “driver” or a “passenger” during malignant transformation. Disclosures: No relevant conflicts of interest to declare.

BNIP3 Is Downregulated in Myelodysplastic Syndromes and Acute Myeloid Leukemia and Is a Potential Molecular Target for Decitabine

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1708-1708
Author(s):  
Mariana Lazarini ◽  
Joao Machado-Neto ◽  
Adriana S. S. Duarte ◽  
Fernando V Pericole ◽  
Patricia Favaro ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
High Risk ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Low Risk ◽  
U937 Cells ◽  
Healthy Donors ◽  
Marrow Cells

Abstract Abstract 1708 In myelodysplastic syndromes (MDS), changes in the balance between pro- and anti-apoptotic proteins are associated with disease progression and evolution towards acute myeloid leukemia (AML). BNIP3 is a pro-apoptotic protein, member of the Bcl-2 superfamily, associated with the pathogenesis of many diseases, including cancer. More recently, BNIP3 was identified as a potent inducer of autophagy. In primary leukemia cells, BNIP3 expression has been shown to be reduced due to epigenetic modifications; however, there is a lack of studies of BNIP3 in MDS. The aim of this study was to characterize BNIP3 mRNA expression levels and DNA methylation status of its 5' CpG island in bone marrow cells from MDS and AML patients, healthy donors and in leukemia hematopoietic cell lines. We also investigated the effects of BNIP3 silencing upon decitabine (DAC) treatment in U937 cells regarding colony formation, apoptosis and autophagy. Bone marrow aspirates were obtained from 21 healthy donors, 55 patients with MDS and 44 patients with AML at diagnosis. MDS patients were grouped into low-risk and high-risk groups according to FAB (RA/RARS=30; RAEB/RAEBt=25), WHO (RCUD/RCMD/RARS/del5/=29; RAEB1/RAEB2=17) and IPSS (low/INT-1=38; INT2/high=16). The study was approved by the ethics committee and informed written consent was provided. Gene expression was evaluated by qPCR and DNA methylation was performed by Combined Bisulfite Restriction Analysis. BNIP3 knockdown was performed in U937 cells with specific shRNA-expressing lentiviral vector. ShRNA encoding no specific sequence was used as control. Colony formation was carried out in semisolid methylcellulose medium. Apoptosis and autophagy were accessed by flow cytometry. All assays were performed in lentiviral transduced cells treated or not with 5μM DAC for 72 hours. Mann-Whitney and Student's t-test were used for statistical analyses. We observed a significant decrease in BNIP3 mRNA expression in total bone marrow cells from AML and MDS compared to healthy donors (AML= 0.52 [5.27-0.00]; MDS=0.52 [5.25-0.02] versus healthy donors=1.09 [6.04-0.18]; P<0.05). BNIP3 expression did not differ between low-risk and high-risk MDS patients according to FAB and WHO classifications, and IPSS. Methylation of BNIP3 promoter was detected in 19% (4/21; 2/14 low-risk and 2/7 high-risk) MDS and 26% (4/15) AML patients, but not in any of the 6 healthy donors evaluated. BNIP3 expression was detected in all myeloid cell lines studied (K562, HL60, U937 and P39 cells). The lowest expression was found in U937, which presented methylation of BNIP3 promoter. Interestingly, in U937 cells, DAC treatment resulted in upregulation of BNIP3 expression (3.7-fold), total inhibition of colony formation, increased apoptosis and autophagy (50% for both). The effect of DAC on cell apoptosis was reduced by BNIP3 silencing; apoptosis was reduced by 48%±4.3 in shBNIP3 cells compared to 59%±5.7 in shControl cells (p<0.05). Autophagy was not modulated by DAC treatment in BNIP3 silenced cells. In bone marrow cells from MDS and AML patients, the low frequency of methylated BNIP3 promoter suggests that DNA methylation is not the single reason for the decreased BNIP3 expression in these patients. Other mechanisms, such as histone deacetylation, may be involved in aberrant BNIP3 expression. DAC treatment in BNIP3 silenced cells had no effect on autophagy, suggesting that other mechanisms besides increasing BNIP3 expression is involved in this cell function. On the other hand, DAC treatment in U937 cells was able to induce apoptosis, especially in cells expressing BNIP3, supporting the hypothesis that BNIP3 methylation is a molecular target for DAC. Disclosures: No relevant conflicts of interest to declare.

Hedgehog Pathway Is Deregulated In Myelodysplastic Syndrome Progenitor Bone Marrow Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1533-1533 ◽  
Author(s):  
Juliana M. Xavier ◽  
Adriana Silva Santos Duarte ◽  
Fernando V Pericole ◽  
Paulo Latuf-Filho ◽  
Fabiola Traina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Hedgehog Signaling ◽  
Bone Marrow Cells ◽  
Low Risk ◽  
Hedgehog Pathway ◽  
Shh Pathway ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Total Bone Marrow

Abstract The sonic hedgehog (Shh) pathway is a regulatory network involved in the development of cancer. The Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) bind to the receptors Patched1 and Patched2, resulting in the inhibition of their repression on Smoothened (Smo). Smo is a seven-transmembrane protein, which inhibits Suppressor of Fused (SUFU), resulting in the stabilization and activation of the Gli family of transcription factors, followed by a transcriptional response to the “canonical Hh signaling”. SUFU is a negative regulator of the Hedgehog signaling pathway, through the restriction of Gli activity through cytoplasmic sequestration. Germline mutations in SUFU are involved in medulloblastoma. Dysregulation of Shh signaling has been reported to be involved in several malignancies, including chronic myeloid leukemia. To our knowledge, however, only one study has been carried out in myelodysplastic syndrome (MDS) and acute leukemia so far, and the role of this pathway in leukemogenesis is still unknown. Recently we showed an increased protein expression of Sonic and Dessert Hedgehog in addition to increased mRNA levels of PTCH and SMO in MDS bone marrow. Here, we investigated whether this increase was capable of activating Hedgehog pathway in total and in progenitor bone marrow cells. Bone marrow (BM) samples were collected from 39 MDS (25 low-risk, 14 high-risk, according WHO 2008) patients and 26 healthy donors. CD34+ cells were isolated from peripheral blood of 9 healthy donors and 20 (12 low-risk and 8 high risk) MDS patients. Relative expressions of PTCH, SMO, SUFU and GLI1 were obtained by Real time-PCR. Increased expression observed in ligands and receptors were not sufficient to upregulate the activity of Hedgehog pathway in MDS total bone marrow. The downstream effector GLI1 was under expressed in the disease as median [min-max] as follows: healthy donors 1.04[0.02-4.01]; low risk MDS 0.21[0.03-2.26]; high risk MDS 0.07[0.03-1.48] healthy donors vs low risk p=0.007; healthy donors vs high risk p=0.006. Furthermore, SUFU expression was similar in healthy donors 3.3[0.35-7.84] and in myelodysplastic (low risk 3.76[0.75-13, 2] and high risk 1.8[0.58-16.4]) bone marrows. Different from total bone marrow, the receptors Patched and SMO showed decreased expression in MDS CD34+ cells: Patched healthy donors 2.24[0.74-4.36]; low risk 0.6[0.1-3.34]; high risk 0.98[0.29-1.22] and SMO healthy donors 1.18[0.62-2.16]; low risk 0.79[0,11-1.87]; high risk 0.86[0.37-1.02]; healthy donors vs high risk MDS p=0.049. SUFU was overexpressed (healthy donors 1.44[0.75-2.39]; low risk 2.77[0.93-4.11]; high risk 1.68[1.07-3.43]; healthy donors vs low risk MDS p=.005). However, myelodysplastic progenitors had GLI1 overexpression (healthy donors 1.09[0.6-2.91]; low risk 2.93[0.75-14.52]; high risk 1.32[0.71-2.58]; healthy donors vs low risk p=0.03), indicating an activation of the Shh pathway in MDS progenitors. Recently, Kobune et al (Blood Cancer J. 2012 Sep 7) showed activation of Hedgehog signaling in primary CD34+ blasts from acute myeloid leukemia and MDS, as those cells expressed IHh, PTCH, SMO, GLI1 and GLI. Taken together, these results and ours led us to conclude that the Hedgehog pathway is activated in MDS. Since Shh pathway is being explored as a new opportunity for targeted therapies against tumors, our study suggests that the inhibition of this pathway could be strategic in the control of leukemic stem cell and MDS treatment. Disclosures: No relevant conflicts of interest to declare.

Study on the Membrane Hemopoietic Cytokine Receptor Expression on CD34+ Bone Marrow Cells of the Patients with Myelodysplastic Syndromes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2908-2908
Author(s):  
Zonghong Shao ◽  
Lijuan Li ◽  
Rong Fu ◽  
Huaquan Wang ◽  
Lanzhu Yue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Bone Marrow Cells ◽  
Low Risk ◽  
Receptor Expression ◽  
Cytokine Receptor ◽  
Cytokine Receptors ◽  
Control Group ◽  
The Mean ◽  
Marrow Cells

Abstract Abstract 2908 Objectives This study was to detect if there were abnormalities of membrane hemopoietic cytokine receptor expression on CD34+ bone marrow cells in MDS. Methods 34 newly diagnosed MDS(12 in low-risk and 22 in high-risk) cases and 32 normal controls were enrolled in this study. Their CD34+CD38+ and CD34+CD38- bone marrow cells and the expressions of stem cell factor receptor(SCF-R),erythropoietin receptor (EpoR), granulocyte colony-stimulating factor receptor (G-CSFR) and thrombopoietin receptor (TpoR) on those cells were measured by flow cytometry. Results The mean percentage of CD34+ BMMNCs of MDS cases in high risk[(2.94±4.79)%)] was significantly higher than that of control group[(0.95±1.06)%](P<0.05). The mean percentages of CD34+CD38+ cells were significantly lower in low risk and high risk groups[(86.98±6.83)% and (83.57±9.86)% respectively] than that in control group [(92.41±3.43)%], thus the percentage of CD34+CD38- cells was significantly higher in either low-risk or high-risk group[(13.03±6.84)% and (16.42±9.85)% respectively]than that in control group[(7.59±3.43)%](P<0.05). In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+ cells [(17.72±20.24) %] than that in CD34+CD38- cells [(64.65±21.02)%](P<0.01), The expressions of SCF-R,G-CSFR and TpoR on CD34+CD38- cells were not significantly different from these on CD34+CD38+ cells. The expression of EpoR on CD34+CD38+ cells of low-risk and high-risk MDS groups[(7.01±6.82)% and (7.16±9.45)% respectively] were significantly lower than that of control group[(17.72±20. 24) %] (P<0.05), The expression of G-CSFR on CD34+CD38+ cells of low-risk and high-risk MDS groups[(22.65±12.14)% and (26.50±19.65)% respectively] were significantly lower than that of control group[(45.13±23.41)%](P<0.01). The amount of EpoR on CD34+CD38-cells of low-risk and high-risk MDS groups[(40.18±20.38)% and (28.58±17.00)% respectively] were significantly lower than that of control group[(64.65±21.02)%](P<0.01), The expression of TpoR on CD34+CD38- cells of low-risk and high-risk MDS groups[(4.46±7.45)% and (3.23±4.55)% respectively] were significantly lower than that of control group[(15.33±14.95)%](P<0.01). The incidence of cytopenia of MDS cases with low expression rates of hemopoietic cytokine receptors on CD34+cells were higher than that of MDS with high expression rates of hemopoietic cytokine receptors. Conclusions There were abnormalities of differentiation and membrane hemopoietic cytokine receptors expression of CD34+ bone marrow cells in MDS, which were associated with MDS cytopenia and might be useful for MDS diagnosis. Disclosures: No relevant conflicts of interest to declare.

Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3553-3560 ◽  
Author(s):  
Wolf-K. Hofmann ◽  
Sven de Vos ◽  
Martina Komor ◽  
Dieter Hoelzer ◽  
William Wachsman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
High Risk ◽  
Bone Marrow Cells ◽  
Low Risk ◽  
Control Group ◽  
Expression Data ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Marrow Cells

Gene patterns of expression in purified CD34+ bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34+ bone marrow cells from 4 healthy control subjects. CD34+ cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid–induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34+cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.

scholarly journals Friend Leukemia Virus Infection Enhances DNA Damage-Induced Apoptosis of Hematopoietic Cells, Causing Lethal Anemia in C3H Hosts

2002 ◽  
Vol 76 (15) ◽  
pp. 7790-7798 ◽  
Author(s):  
Masanobu Kitagawa ◽  
Shuichi Yamaguchi ◽  
Maki Hasegawa ◽  
Kaoru Tanaka ◽  
Toshihiko Sado ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Knockout Mice ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Hematopoietic Cells ◽  
Friend Leukemia ◽  
C3h Mice ◽  
Friend Leukemia Virus ◽  
Marrow Cells

ABSTRACT Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53−/− ) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53−/− → C3H p53+/+ bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.

scholarly journals Augmented Production of Interleukin-6 by Normal Human Osteoblasts in Response to CD34+ Hematopoietic Bone Marrow Cells In Vitro

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1165-1172 ◽  
Author(s):  
Russell S. Taichman ◽  
Marcelle J. Reilly ◽  
Rama S. Verma ◽  
Stephen G. Emerson
Keyword(s):  
Bone Marrow ◽  
Interleukin 6 ◽  
Transforming Growth Factor ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Colony Stimulating Factor ◽  
Cd34 Cells ◽  
Stimulating Factor ◽  
Marrow Cells

Abstract Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-β1 , but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% ± 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1β or tumor necrosis factor-α, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.

Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 488-499 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard A. Ashmun ◽  
Donald Orlic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Blood Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Immunoblot Analysis ◽  
Marrow Cells

Abstract The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

Over-Expression of Cancerous Inhibitor of PP2A (CIP2A) in Bone Marrow Cells from Patients with a Group of High-Risk Myelodysplastic Syndromes

2013 ◽  
Vol 20 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Na Li ◽  
Shinya Abe ◽  
Morito Kurata ◽  
Shiho Abe-Suzuki ◽  
Iichiroh Onishi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndromes ◽  
Bone Marrow Cells ◽  
Over Expression ◽  
Marrow Cells

Combined Use of Dendritic Cells Enhances Specific Anti-Leukemia Effect of Leukemia Cell-Derived-HSP70 in a Mouse Model with Minimal Residual Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3065-3065
Author(s):  
Kazuya Sato ◽  
Yoshihiro Torimoto ◽  
Yasuyuki Iuchi ◽  
Yasuaki Tamura ◽  
Junko Jimbo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Bone Marrow Cells ◽  
Leukemia Cell ◽  
Class I ◽  
Biochemical Data ◽  
Leukemia Cells ◽  
Combined Use ◽  
Minimal Residual

Abstract Background: Heat shock proteins (HSPs) are molecular chaperones binding a broad repertorie of endogenous antigenic peptides and carrying them to the MHC. Because the identification of each tumor specific antigen is not necessary, the immunotherapy using HSPs is more practical than other immunological procedures. Meanwhile, relapse due to minimal residual disease (MRD) is a big problem of autologous stem cell transplantation (SCT) against leukemia. We previously reported that immunotherapy using leukemia cell-derived HSPs is effective against MRD after syngeneic bone marrow transplantation (BMT) in mice (Sato et al. Blood, 2001). However, patients receiving SCT are usually immunocompromised due to repeated anti-cancer therapies. Accordingly, it is important to enhance the cytotoxicities (CTXs) against leukemia. Dendritic cells (DCs) are known as professional antigen-presenting cells with a specific receptor for HSPs and are expected to play a major role in immunotherapy. In this study, we evaluated whether the vaccination of DCs pulsed with HSP70 enhances the anti-leukemia effect induced by leukemia cell-derived HSP70 after syngeneic BMT and evaluated the safety of this immunotherapy. Methods: Three class-I-identical mouse tumor cell lines (A20: B-cell leukemia; T27A: myeloid leukemia; colo26: colonic carcinoma) and syngeneic balb/c mice were used in this study. HSP70 was purified from tumor cells. DCs were generated from bone marrow cells cultured with GM-CSF. DCs were pulsed with HSP70 (HSP70-pulsed-DCs) in vitro. Mice were received total body irradiation (TBI) and transplanted bone marrow cells after TBI, then inoculated 2.5 x 104 A20 cells intravenously. HSP70 or HSP70-pulsed-DCs was subcutaneously administrated. Survival days of immunized mice were compared using Kaplan and Meier methods. CTXs of splenocytes against A20 cells were determined by 51Cr release assay. Histological findings of liver and knee joint and biochemical data of serum of immunized mice were investigated. Results: All mice without immunization or immunized with DCs alone died from leukemic dissemination within 90 days after A20 inoculation, whereas mice immunized with A20-derived HSP70 (A20-HSP70) or A20-HSP70-pulsed-DCs survived long significantly. Additionally, although only 60% of the mice immunized with A20-HSP70 survived on day 120, all the mice immunized with A20-HSP70-pulsed-DCs survived with no residual leukemia cells over 120 days. Moreover, splenocytes of mice immunized with A20-HSP70-pulsed-DCs showed significantly higher CTXs against A20 cells in vitro compare to those with A20-HSP70 alone. However, no CTXs against A20 cells were induced by immunization with colo26-or T27A-HSP70-pulsed-DCs. These CTXs against A20 cells were significantly blocked by anti-CD8 and anti-MHC class-I, but not by anti-CD4. Additionally, no abnormal findings were detected either in biochemical data of serum or in histopathology of liver and joint tissue in long term immunized mice. Conclusions: Combined use of dendritic cells with leukemia cell-derived HSP70 enhances anti-leukemia effect by inducing specific cytotoxic activities against leukemia cells, and eradicates MRD effectively and safely even for immunocompromised status after syngeneic BMT. This approach would be useful for a further application of HSP in leukemia-patients after autologous SCT.

FLIP (Long) and FLIP (Short) Expression in Bone Marrow Cells in Patients with Myelodysplastic Syndrome: Correlation with FAB and WHO Classification.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4926-4926
Author(s):  
Paula Campos ◽  
Fabiola Traina ◽  
Adriana Duarte ◽  
Bruno Benites ◽  
Marcelo Brandao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Mrna Expression ◽  
Who Classification ◽  
Fas Ligand ◽  
Low Risk ◽  
World Health ◽  
Caspase 8 ◽  
Marrow Cells

Abstract The paradox of peripheral cytopenias despite of normo/hypercellular marrow in myelodysplastic syndrome (MDS) has been ascribed to excessive intramedullary hematopoietic cell apoptosis. Several apoptosis-inducing systems, including Fas/Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) and its receptors, are upregulated in MDS. FLIP (FLICE (FAS-associated death-domain-like IL-1β-converting enzyme)-inhibitory protein) was identified as a FAS and TRAIL signal inhibitor. The largest variant FLIPLong (FLIPL) was originally characterized as a molecule with inhibitory activity on caspase-8. The short splice form termed FLIPShort (FLIPS) has also been characterized as a potent (TRAIL-induced) apoptosis inhibitor. However, whereas FLIPL and FLIPS have been described as death receptor pathway inhibitors, recent data suggest that physiologically, FLIPL may have caspase-8-activating properties. This study aims to characterize the expression of FLIPL and FLIPS based on mRNA, by Real-time quantitative PCR, in marrow cells from MDS patients and to correlate the expression with French-American-British (FAB) and World Health Organization (WHO) classification. For each sample, results were first calculated as a ratio of the total transcript number of FLIPL or FLIPS and the total transcript number of the endogenous reference gene (β-actin) to obtain a normalized target value. Transcript ratios of each sample were normalized against the respective ratio of a pool of 6 normal bone marrow donors (NBM), and the ratio between the two was used as measure for the relative FLIPL or FLIPS level. We hypothesized that FLIPL and FLIPS expression differed between low and high risk of MDS. Marrow aspirates were obtained from 6 NBM and 16 patients with MDS out of treatment (7 males, 9 females; 23–78 (median 64) yo). The National Ethical Committee Board approved this study, informed-written consent was obtained from all patients and donors. According to FAB classification, patients were distributed as: 10 RA, 2 RARS and 4 RAEB. According to WHO classification: 10 RCMD, 2 RCMD-RS, 3 RAEB-1 and 1 RAEB-2. FLIPS mRNA expression were significantly higher in high risk DS according to FAB and WHO classification; RA/RARS compared with AREB (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03). However, FLIPL mRNA expression also tended to be higher in high risk MDS according to FAB and WHO classification, though not significantly different: RA/RARS compared with AREB (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46). Lower FLIPS level in low risk MDS marrows, in addition to the well described upregulation of extracellular proapoptotic signals, would explain the increased susceptibility of hematopoietic cells in low risk MDS marrow to death-inducing stimuli. The fact that FLIPL expression did not differ according to FAB and WHO classification could be related to the hypothesis that FLIPL may have caspase-8-activating properties rather than anti-apoptotic activity. Differential regulation of FLIPL and FLIPS according to risk groups in MDS patients might result in different rates of apoptosis. Further studies are needed to elucidate the mechanisms controlling and regulating FLIP expression in normal and malignant hemopoietic cells.

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