The Potent Deacetylase Inhibitor Trichostatin a (TSA) Increases CXCR4 Expression in Hematopoietic Stem/Progenitor Cells by Chromatin Remodelling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3487-3487 ◽  
Author(s):  
Hilal Gul ◽  
Leah A. Marquez-Curtis ◽  
Jennifer Lo ◽  
Nadia Jahroudi ◽  
A. Robert Turner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Trichostatin A ◽  
Mrna Level ◽  
Hdac Inhibitors ◽  
Cxcr4 Expression ◽  
Chromatin Remodelling ◽  
Hematopoietic Stem ◽  
Chip Analysis

Abstract Stromal-cell derived factor (SDF)-1α/CXCL12 and its cognate receptor, CXCR4, play a crucial role in the trafficking of normal hematopoietic stem/progenitor cells (HSPC) and their homing/retention in bone marrow. Consequently, modulation of CXCR4 expression in HSPC could be applied therapeutically to improve the efficiency of HSPC transplantation. It is known that gene expression can be regulated by chromatin remodelling. Two groups of histone modifying enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) participate in the regulation of chromatin structure, and hence gene expression. Disruption of normal HAT or HDAC activities has been found in many human cancers. Recently, several structurally diverse and highly specific HDAC inhibitors (HDI) have been reported. They act as strong modulators of growth, differentiation and apoptosis in several types of cancer, particularly acute myeloid leukemia (AML). However, very little is known regarding the effects of HDI on HSPC. We have previously shown that a specific short-chain fatty acid HDI, valproic acid (VPA), enhances CXCR4 expression and function in normal HSPC (Blood2007: 110; 425a). In order to determine whether other structurally diverse classes of HDI are able to influence CXCR4 expression in HSPC through chromatin remodelling, we investigated the effect of potent hydroxamic acid HDI Trichostatin A (TSA) on CXCR4 in normal HSPC. We examined the effect of TSA on CXCR4 expression (by FACS and real-time RT-PCR), modulation of CXCR4 transcription (chromatin immunoprecipitation (X-ChIP) analysis) and on functional response towards an SDF-1α gradient (by chemotaxis assay) of HSPC (CD34+ cells from cord blood (CB) and the models of immature hematopoietic cells expressing CD34 antigen, namely AML cell lines KG-1a and KG-1). Cells were incubated for 24 h in IMDM supplemented with 20% FCS in the presence of TSA (0.1 μM). We found that TSA increases the percentage of CXCR4-expressing CB CD34+, KG-1a, KG-1 cells (2.5-, 8- and 3-fold, respectively). This effect was also confirmed at the mRNA level in CB CD34+, KG-1a and KG-1 cells (by about 2.5-, 5- and 2.5-fold up-regulation, respectively). Moreover, X-ChIP analysis showed a significant increase in association of acetyl-histone H4 binding to the CXCR4 promoter in CB CD34+ and KG-1 cells (2- and 1.7-fold, respectively). TSA was also shown to significantly increase the chemotaxis of KG-1a cells towards SDF-1α (20 ng/mL), which was inhibited by AMD3100, a potent antagonist of CXCR4. We conclude that other HDI such as TSA regulate CXCR4 expression in HSPC by chromatin remodelling and we suggest that priming of HSPC with HDI may improve their homing and engraftment into bone marrow, especially in CB transplantation.

Lenalidomide and CC-4047 Inhibit the Proliferation of Namalwa Cancer Cells While Expanding CD34+ Progenitor Cells. New Insights on the Combination Therapy with Certain HDAC Inhibitors for Hematological Cancers.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1120-1120
Author(s):  
Dominique Verhelle ◽  
Laura G. Corral ◽  
Kevin Wong ◽  
Jessica H. Mueller ◽  
Laure Moutouh de Parseval ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Valproic Acid ◽  
Combination Therapy ◽  
Progenitor Cells ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
Malignant Cells ◽  
Cell Models ◽  
Proliferative Effect ◽  
Hematological Cancers

Abstract Clinical studies involving patients with Myelodysplastic Syndrome and Multiple Myeloma have demonstrated the efficacy of lenalidomide (CC-5013), a drug recently approved by the FDA under the commercial name of Revlimid®, by reducing and often eliminating malignant cells while restoring bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide, and its analog CC-4047, on the proliferation of two different hematopoietic cell models: the Namalwa cancer cell line and CD34+ progenitor cells. We found that both compounds have anti-proliferative effect on Namalwa cells and pro-proliferative effect on CD34+ cells while p21WAF−1 expression was upregulated in both cell models. In Namalwa cells, we determined that p21WAF−1 is responsible for the inhibition of CDK2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest. In contrast, in CD34+ progenitor cells, despite upregulated p21WAF−1 expression, we observed an increase of the cell division rate, leading to the enhancement of CD34+ expansion. Finally, we found that CC-4047 and lenalidomide have synergistic effects with two different HDAC inhibitors (Valproic acid and Trichostatin A) in both increasing the apoptosis of Namalwa cells and enhancing CD34+ cell expansion. Taken together, our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus progenitor cells and could explain, at least in part, the reduction of malignant cells and the restoration of the bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, and proposes new potential clinical uses such as bone marrow regeneration. Finally, our vitro experiments showing the efficacy of the combination of CC-4047 and lenalidomide with Valproic acid and Trichostatin A suggest that HDAC inhibitors might be ideal candidates for combination therapy by elevating the therapeutic index, versus monotherapy, to treat hematological malignancies.

scholarly journals Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

2019 ◽  
Author(s):  
Paulina Gil-Kulik ◽  
Ewa Dudzińska ◽  
Elżbieta Radzikowska-Büchner ◽  
Joanna Wawer ◽  
Mariusz Jojczuk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Mrna Level ◽  
Hematopoietic Stem ◽  
Genes Expression ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow, which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at the mRNA level in the cells of the hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization.Results: The results found that the bone marrow cells of patients with acute myeloid leukemia (AML) show over expression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed the positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

scholarly journals Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

2020 ◽  
Author(s):  
Paulina Gil-Kulik ◽  
Ewa Dudzińska ◽  
Elżbieta Radzikowska-Büchner ◽  
Joanna Wawer ◽  
Mariusz Jojczuk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Mrna Level ◽  
Hematopoietic Stem ◽  
Genes Expression ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression.Methods: The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. Results: The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

scholarly journals Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

2020 ◽  
Author(s):  
Paulina Gil-Kulik ◽  
Ewa Dudzińska ◽  
Elżbieta Radzikowska-Büchner ◽  
Joanna Wawer ◽  
Mariusz Jojczuk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Mrna Level ◽  
Hematopoietic Stem ◽  
Genes Expression ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. Methods The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. Results The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML. Conclusions Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

Differential hMLH1 Gene Expression after Temozolomide Selection Linked to Microsatellite Instability in a Subset of Hematopoietic Stem/Progenitor Cells in Old Versus Young and Cancer Versus Normal Patient Samples.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 509-509
Author(s):  
Jonathan Kenyon ◽  
Emily Thomas ◽  
Karen Lingas ◽  
Stanton L. Gerson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Microsatellite Instability ◽  
Progenitor Cells ◽  
Mismatch Repair ◽  
Dna Methyltransferase ◽  
Apoptotic Cell ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract The etiology of hematologic pathologies such as leukemia, myelodysplasia, anemia, bone marrow failure, altered immune function, and how they are associated with aging, remains unclear. Our hypothesis is that these diseases are caused or aggravated by a subset of hematopoietic stem/progenitor cells (HSC) lacking effective mismatch repair (MMR) and therefore exhibiting a hypermutator phenotype. Microsatellite instability (MSI) is a marker of MMR deficiency. We used cord blood, bone marrow, and bone core samples to isolate and then clonally expand HSC for MSI analysis. Five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250) were analyzed for insertions and deletions. We have analyzed 38 patient samples between the ages of 0 and 86 years, including 8 cancer patients. These data show an age-dependent increase in the frequency of high grade microsatellite instability (MSI-H), i.e. those CFU with microsatellite instability at >20% of loci tested. Additionally, samples obtained from individuals older than 50 years were 6 times more likely to have a > 10% frequency of MSI-H CFU than samples obtained from younger individuals, suggesting an inflection point for the onset of hematopoietic diseases. In all instances this instability is seen only within a subset of human HSC clones. To further characterize the origin of this deficiency, a method to select for MMR deficient hematopoietic cells was developed that first selected for survival of MMR deficient HSC, and then allowed for the examination of expression status of key MMR pathway genes hMLH1 and hMSH2 and their protein products. First, CD34+ HSC were isolated from various aged patient samples. To avoid possible effects of other repair pathways, the cells were treated with O6-Benzylguanine (BG) to remove O6-methylguanine DNA-methyltransferase (MGMT) activity and prevent removal of O6-methylguanine lesions. Next, temozolomide (TMZ) at concentrations of 50–125 μM was used to induce O6-methylguanine (O6-mG) lesions that persist in the presence of BG. These O6-mG lesions mispair with cytosine and are recognized as DNA mismatches by the mismatch repair (MMR) pathway inducing apoptotic cell death. TMZ selected cells that fail to recognize the mispair due to a lack of MMR survive this selection. In these TMZ resistant clones, RT-PCR amplification of hMLH1 transcripts from total RNA isolated reveal a defect in hMLH1 but not hMSH2 expression. In the one AML sample obtained thus far HSC treated with 200 uM TMZ we have observed 0 to 30% of hMLH1 expression within TMZ resistant CFU was observed when compared to untreated controls. Together this data links MSI to MMR defects of a subpopulation of hematopoietic precursors in older individuals. This is the first examination of MMR gene expression in clones of HSC that has shown specific MMR functional deficiencies. Our study suggests that a MMR pathway deficiency in a subset of stem cells could contribute to age related hematopoietic disease processes including stem cell failure and malignant transformation.

A Novel Observation That Heme Oxygenase-1 (HO-1) Deficient Mice Are Easy Mobilizers and That HO-1 Plays An Important Role in Maintaining Expression of SDF-1 in Bone Marrow (BM) Stroma and Promotes Retention of Hematopoietic Stem/Progenitor Cells (HSPCs) in the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 315-315
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Heme Oxygenase ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Heme Oxygenase 1 ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Cell Counts

Abstract Abstract 315 Background. Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. Two distinct HO isoforms have been identified: HO-2, which is constitutively expressed, and HO-1, which is stress-responsive and plays an important function in various physiological and pathophysiological states associated with cellular stress. HO-1 plays a role in ischemic/reperfusion injury, atherosclerosis, and cancer. It has also been reported that HO-1 regulates expression of a-chemokine stromal derived factor-1 (SDF-1) in myocardium (J Mol Cell Cardiol.2008;45:44–55). Aim of study. Since SDF-1 plays a crucial role in retention and survival of hematopoietic stem cell/progenitor cells (HSPCs) in BM, we become interested in whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach. To address this issue, we employed several complementary strategies to investigate HO-1−/−, HO+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) chemotactic responsiveness of HSPCs to an SDF-1 gradient, iv) adhesiveness of clonogenic progenitors, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100 assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. Results: Our data indicate that under normal, steady-state conditions, HO-1−/− and HO+/– mice have normal peripheral blood cell counts and numbers of circulating CFU-GM. Interestingly, lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. Next, BMMNCs from HO-1−/−have normal expression of the SDF-1-binding receptor, CXCR4, but a 5-times lower level of CXCR7, which is another SDF-1-binding receptor. Of note, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1−/−animals responded more robustly to an SDF-1 gradient, and HO-1−/−animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into peripheral blood in response to G-CSF and AMD3100. Conclusions: Our data demonstrate for the first time that heme oxygenase plays an important and underappreciated role in BM retention of HSPCs and may affect their trafficking. Since small non-toxic molecular inhibitors of HO-1 have been developed for clinical use (e.g., metaloporhirins), blockage of HO-1 could be a novel strategy for mobilizing HSPCs. Our recent in vivo mobilization studies lend support to this hypothesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Autism-Associated Chromatin Remodeler CHD8 Governs the Survival and Differentiation of Hematopoietic Stem/Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1191-1191
Author(s):  
Zhaowei Tu ◽  
Chen Wang ◽  
Chuntao Zhao ◽  
Lei Huang ◽  
Nathan Salomonis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
P53 Gene ◽  
Autism Spectrum ◽  
Cycle Phase ◽  
Chromatin Remodeler ◽  
Hematopoietic Stem ◽  
Direct Binding

Hematopoiesis is a strictly regulated process which depends on regulated proliferation, differentiation, and survival of hematopoietic stem/progenitor cells (HSPCs). Multiple signaling pathways, transcription factors and epigenetic machineries are involved in this important process. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in stem/progenitor cell regulation. Among them, the CHD8 gene encodes a member of chromodomain helicase DNA-binding (CHD) family of SNF2H-like ATP-dependent chromatin remodeler, mutations of which define a subtype of autism spectrum disorders. Whether CHD8 plays a role in hematopoietic cells remains unknown. In this study, to define the role of CHD8 in hematopoiesis and HSPC regulation, we carried out conditional deletion of CHD8 in the hematopoietic lineages using an interferon inducible Mx1-Cre; Chd8F/Fmouse model. As early as one week after polyI:C injection, the CHD8 knockout mice experienced drastic pancytopenia and bone marrow failure with a reduction of bone marrow (BM) cellularity to ~1/3 of that of WT controls. In the HSPC compartment, loss of CHD8 resulted in a depletion of Lin-c-Kit+ (LK) and Lin-Sca1+c-Kit+ (LSK) progenitors and a severe reduction of the GMP, MEP, MPP and CD150+CD48-LSK HSC populations. Accompanying the loss of HSPCs were dramatically increased apoptosis rate and cell cycle arrest in Chd8-/- cells. An examination of HSPC function by transplantation found that the BM cells from Chd8-/- mice could not engraft in the recipient mice that died ~20 days post-transplantation. The chimerism of Chd8-/- BM cells also reduced drastically in a competitive BM transplantation assay when induction of Chd8 deletion occurred after Mx1-Cre; Chd8F/Fcells were pre-engrafted at 1:1 ratio with WT BM cells. These results indicate that CHD8 is essential for hematopoiesis and HSPC survival and proliferation. To elucidate the underlying mechanism of CHD8 function in HSPCs, we performed complementary biochemical, genomic and genetic analyses of the WT (Chd8F/F) and Chd8-/-HSPC cells. Firstly, we found blood progenitor cells dominantly express the short form of CHD8 containing residues 1 to 751 which retains the N-terminal P53 binding domain, rather than the long form of the CHD8 protein. Secondly, we revealed by RNA-seq and subsequent RT-PCR analyses that the P53 signaling pathway signatures including P53, P21, Bax and Noxa are aberrantly activated in Chd8-/-HSPCs. At the protein level, P53 and P21 are significantly elevated in Chd8-/- LK cells. Thirdly, ATAC-seq analysis of the LSK cells revealed increased global chromatin accessibilities after CHD8 deletion, including the promoter regions of P21 and Noxa genes. By a "Cut & Run" assay using H3K4me3 antibody in Chd8F/Fand Chd8-/- LSK cells we have also seen significantly enhanced epigenetic H3K4me3 mark on the promoter region of P21 and Noxa. An anti-CHD8 "Cut & Run" further detected direct binding sites of CHD8 on P53 and P21, but not Noxa or Bax gene. Fourthly, we observed a direct binding interaction by co-immunoprecipitation between P53 and CHD8 proteins. Finally, genetic deletion of one allele of P53 gene in Chd8-/- HSPCs (P53+/-;Chd8-/-) was able to completely suppress the Bax/Noxa expression and apoptosis phenotype and rescue most of the HSC and early progenitor defects but not the later stage of differentiation defects and BM cellularity loss. Interestingly, deletion of both alleles of P53 gene (P53-/-;Chd8-/-) was able to further remove the differentiation block between LSK and LK cells observed in the P53+/-;Chd8-/-background which is associated with a P21 mediated G0 cell cycle phase arrest. These combined results suggest a mechanism of action that CHD8 works through a direct complex with P53 to regulate P21 gene expression which mediates cell cycle control of HSPC differentiation and an indirect CHD8 mediated P53 expression and subsequent P53 regulated Noxa/Bax expression which regulates HSPC survival. Thus, we identify the autism-associated CHD8 as an essential chromatin suppressor in regulating hematopoiesis through restricting the expression of P53 and chromatin accessibility of P53 downstream targets, Bax/Noxa and P21, to enable selective control of HSPC survival and differentiation, respectively. Disclosures No relevant conflicts of interest to declare.

scholarly journals Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

2020 ◽  
Author(s):  
Paulina Gil-Kulik ◽  
Ewa Dudzińska ◽  
Elżbieta Radzikowska-Büchner ◽  
Joanna Wawer ◽  
Mariusz Jojczuk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Mrna Level ◽  
Hematopoietic Stem ◽  
Genes Expression ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression.Methods: The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. Results: The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

Leukemogenic Fusion Gene (p190 BCR-ABL) Transduction Into Hematopoietic Stem/Progenitor Cells In the Common Marmoset

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4323-4323
Author(s):  
Yamin Tian ◽  
Yan Dong ◽  
Seiichiro Kobayashi ◽  
Manabu Ozawa ◽  
Kiyoko Izawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Fusion Gene ◽  
Lentiviral Vector ◽  
Lymphoblastic Leukemia ◽  
Common Marmoset ◽  
Hematopoietic Stem ◽  
The Common

Abstract Abstract 4323 [Introduction] Patients with Philadelphia chromosome (p190 BCR-ABL fusion gene)-positive acute lymphoblastic leukemia have a poor prognosis despite intensive therapeutic intervention. Although a rodent model of this leukemia was previously established, the genetic and physiological differences between humans and rodents make it difficult to extrapolate the results from these models and apply these findings to human cases. Primates are more genetically related to humans than rodents. In this study, we attempted to develop a leukemia non-human primate model that mimics various human systems. [Methods and results] (1) A third-generation VSV.G pseudotyped lentiviral vector expressing the p190 BCR-ABL fusion gene driven by CMV or PGK promoter was produced (HIV-CMV/PGK-BCR-ABL). Ba/F3 cells, a mIL-3-dependent murine hematopoietic cell line, were transfected with this vector and cultured without mIL-3. These cells rapidly expanded after 12 days, indicating that p190 BCR-ABL gene expression allowed the Ba/F3 cells to grow autonomously. Next, using a biotin-labeled anti-marmoset CD34 monoclonal antibody (clone MA24) which was produced in our laboratory, MACS-sorted bone marrow CD34+ cells were transduced with the lentiviral vector (HIV-CMV/PGK-BCR-ABL) and subjected to the colony formation assay. In the majority of examined colonies, p190 BCR-ABL gene was detected regardless of the promoter. Taken together, the above findings indicate that p190 BCR-ABL gene was efficiently transduced into marmoset hematopoietic stem/progenitor cells. (2) Peripheral blood mononuclear cells (PBMNCs) were collected from individual marmosets after mobilizing the hematopoietic stem/progenitor cells with G-CSF. These cells were stimulated with cytokines (hIL3, hSCF and hTPO), followed by the transduction with the lentiviral vector. These cells were transplanted into marmosets preconditioned with busulfan. In this ex vivo transduction method, p190 BCR-ABL gene expression which was detected in PBMNCs by nested RT-PCR disappeared after day 56 and 100 in two marmosets. (3) Concentrated lentiviral vector was directly injected into the bone marrow cavity of individual marmosets pretreated with 5-fluorouracil and prednisolone. In this in vivo direct injection method, p190 BCR-ABL gene expression was maintained for more than one year and a half. Transduction of p190 BCR-ABL gene into hematopoietic stem/progenitor cells was confirmed by colony forming assay. In this model, one marmoset unexpectedly developed myelofibrosis-like disease. However, none of the marmosets have developed leukemia to date. [Conclusion] We successfully achieved sustained p190 BCR-ABL gene expression in vivo. This novel in vivo approach will help to develop a marmoset leukemia model in the future. Because a multiple-hit model of oncogenesis has been proposed for various human cancers, a genetic mutation in addition to p190 BCR-ABL may be required for the malignant transformation of hematopoietic stem/progenitor cells in the common marmoset. Disclosures: No relevant conflicts of interest to declare.

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