scholarly journals Delineation of target expression profiles in CD34+/CD38− and CD34+/CD38+ stem and progenitor cells in AML and CML

2020 ◽  
Vol 4 (20) ◽  
pp. 5118-5132 ◽  
Author(s):  
Harald Herrmann ◽  
Irina Sadovnik ◽  
Gregor Eisenwort ◽  
Thomas Rülicke ◽  
Katharina Blatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Array ◽  
Nsg Mice ◽  
Stem And Progenitor Cells ◽  
Membrane Antigens

Abstract In an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38− and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38− and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38− LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably expressed “aberrant” membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication–mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38− LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.

Persisting Chronic Myeloid Leukemia Stem and Progenitor Cells From Patients in Major Molecular Remission Under Imatinib Are Characterized by Low BCR/ABL Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2207-2207
Author(s):  
Ashu Kumari ◽  
Cornelia Brendel ◽  
Thorsten Volkmann ◽  
Sonja Tajstra ◽  
Andreas Neubauer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Soft Agar ◽  
Molecular Remission ◽  
Stem And Progenitor Cells ◽  
First Diagnosis

Abstract Abstract 2207 Poster Board II-184 Introduction: Treatment with the Abl-kinase specific inhibitor imatinib (IM) is very effective in chronic myeloid leukemia (CML). However, IM presumably fails to eradicate CML stem cells (HSC) leading to disease persistence and relapse after IM-discontinuation. Although causes of CML persistence under imatinib remain ill defined, quiescence and BCR/ABL-overexpression of CML stem and progenitor cells have been suggested as underlying mechanisms. We here set out to identify means to directly study persistence mechanisms in residual BCR/ABL-positive progenitor and stem cell clones from chronic phase CML patients in major molecular remission (mmR) under imatinib. Methods: Bone marrow specimens of twenty-one CML patients in at least major molecular remission (mmR) according to the international scale, first diagnosis (FD) patients (n=5) and healthy donors (n=4) were sorted into HSC, common myeloid progenitors (CMP), granulocyte/macrophage progenitors (GMP) and megakaryocate-erythrocyte progenitors (MEP) and BCR-ABL mRNA expression was directly assessed by quantitative real time (qPCR) and/or nested PCR (mRNA of 4.000 sorted cells). Alternatively, HSC, CMP, GMP and MEP were seeded into soft agar and mRNA was extracted from individual colony forming units (CFU) to assess BCR/ABL-mRNA expression by qPCR. Moreover, CFU of sub-fractions of first diagnosis CML patients were treated in vitro with IM at 3mM and BCR/ABL-expression of surviving CFU was compared with the BCR/ABL expression levels of mock-treated CML-CFU. In total, 595 soft agar colonies were analyzed. Results: By nested PCR, BCR/ABL-mRNA was readily detectable in the HSC compartments of 7 of 10 (7/10) CML patients in mmR. BCR/ABL was also detected in the CMP-, GMP-, and MEP-compartments in 6, 10 and 8 of the 10 patients, respectively. Real time qRT-PCR suggested only a trend toward stronger BCR/ABL positivity of the HSC compartment when compared to the other progenitor compartments (table 1). A detailed analysis of the BCR/ABL-expression of individual CFU from HSC-, CMP-, GMP-, and MEP-compartments of mmR patients revealed that persisting CML-CFU expressed significantly less BCR/ABL than first diagnosis CML-CFU obtained before imatinib therapy (table 1). This finding could be recapitulated in vitro: primary CML-CD34+ cells of first diagnosis CML patients (n=4) were seeded into soft agar in the presence or absence of 3 uM imatinib. After 14 days BCR/ABL expression only of BCR/ABL-positive CFU was compared. BCR/ABL-positive CML-CFU (n=30) that had survived imatinib exposure expressed significantly less BCR/ABL than mock-treated CML-CFU (n=175) (p<0.001). Work is in progress providing in vitro evidence that selection/induction of low BCR/ABL expression in immature progenitor and stem cells is a new mechanism of imatinib persistence in mmR patients via reducing oncogenic addiction from BCR/ABL. Conclusions: We showed that BCR/ABL-persistence is not confined to the quiescent CML-stem cell compartment, but seems to affect also the highly proliferative progenitor compartments. More intriguingly, persisting CML-HSC and -precursor cells express remarkably low levels of BCR/ABL when compared to first diagnosis HSC and progenitors, implying that low BCR/ABL expression reduces imatinib sensitivity in vivo. The simple model of selection / induction of low BCR/ABL expression as mechanism of imatinib persistence in CML would explain the low propensity of disease progression after achieving mmR, and the low genetic instability of CML clones from mmR patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Global gene expression profiles of hematopoietic stem and progenitor cells from patients with chronic myeloid leukemia: the effect of in vitro culture with or without imatinib

2017 ◽  
Vol 6 (12) ◽  
pp. 2942-2956 ◽  
Author(s):  
Sócrates Avilés-Vázquez ◽  
Antonieta Chávez-González ◽  
Alfredo Hidalgo-Miranda ◽  
Dafne Moreno-Lorenzana ◽  
Lourdes Arriaga-Pizano ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Global Gene Expression ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

scholarly journals Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e66-e73 ◽  
Author(s):  
Chih-Wen Ni ◽  
Haiwei Qiu ◽  
Amir Rezvan ◽  
Kihwan Kwon ◽  
Douglas Nam ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Expression Profiles ◽  
Disturbed Flow ◽  
A Genome ◽  
Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

scholarly journals Mechanism of Action of Diallyl Sulphide in Ameliorating the Hematopoietic Radiation Injury

2021 ◽  
Author(s):  
Omika Katoch ◽  
Mrinalini Tiwari ◽  
Namita Kalra ◽  
Paban K. Agrawala
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
Radiation Induced ◽  
Medicinal Properties ◽  
Marrow Cellularity

AbstractDiallyl sulphide (DAS), the pungent component of garlic, is known to have several medicinal properties and has recently been shown to have radiomitigative properties. The present study was performed to better understand its mode of action in rendering radiomitigation. Evaluation of the colonogenic ability of hematopoietic progenitor cells (HPCs) on methocult media, proliferation and differentiation of hematopoietic stem cells (HSCs), and transplantation of stem cells were performed. The supporting tissue of HSCs was also evaluated by examining the histology of bone marrow and in vitro colony-forming unit–fibroblast (CFU-F) count. Alterations in the levels of IL-5, IL-6 and COX-2 were studied as a function of radiation or DAS treatment. It was observed that an increase in proliferation and differentiation of hematopoietic stem and progenitor cells occurred by postirradiation DAS administration. It also resulted in increased circulating and bone marrow homing of transplanted stem cells. Enhancement in bone marrow cellularity, CFU-F count, and cytokine IL-5 level were also evident. All those actions of DAS that could possibly add to its radiomitigative potential and can be attributed to its HDAC inhibitory properties, as was observed by the reversal radiation induced increase in histone acetylation.

scholarly journals High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1517-1524 ◽  
Author(s):  
Marjan J. T. Veuger ◽  
M. Willy Honders ◽  
Jim E. Landegent ◽  
Roel Willemze ◽  
Renée M. Y. Barge
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Deoxycytidine Kinase ◽  
Wild Type ◽  
Healthy Donors ◽  
Alternatively Spliced ◽  
Acute Myeloid

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

Expression of ERG11 and efflux pump genes CDR1, CDR2 and SNQ2 in voriconazole susceptible and resistant Candida glabrata strains

2019 ◽  
Vol 58 (1) ◽  
pp. 30-38
Author(s):  
Patricia Navarro-Rodríguez ◽  
Adela Martin-Vicente ◽  
Loida López-Fernández ◽  
Josep Guarro ◽  
Javier Capilla
Keyword(s):  
Gene Expression ◽  
Candida Glabrata ◽  
Efflux Pump ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Clear Correlation ◽  
Synonymous Mutations ◽  
First Time

AbstractCandida glabrata causes difficult to treat invasive candidiasis due to its antifungal resistance, mainly to azoles. The aim of the present work was to study the role of the genes ERG11, CDR1, CDR2, and SNQ2 on the resistance to voriconazole (VRC) in a set of C. glabrata strains with known in vitro and in vivo susceptibility to this drug. Eighteen clinical isolates of C. glabrata were exposed in vitro to VRC, and the expression of the cited genes was quantified by real time quantitative polymerase chain reaction (q-PCR). In addition, the ERG11 gene was amplified and sequenced to detect possible mutations. Ten synonymous mutations were found in 15 strains, two of them being reported for the first time; however, no amino acid changes were detected. ERG11 and CDR1 were the most expressed genes in all the strains tested, while the expression of CDR2 and SNQ2 was modest. Our results show that gene expression does not directly correlate with the VRC MIC. In addition, the expression profiles of ERG11 and efflux pump genes did not change consistently after exposure to VRC. Although individual analysis did not result in a clear correlation between MIC and gene expression, we did observe an increase in ERG11 and CDR1 expression in resistant strains. It is of interest that considering both in vitro and in vivo results, the slight increase in such gene expression correlates with the observed resistance to VRC.

scholarly journals High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1517-1524 ◽  
Author(s):  
Marjan J. T. Veuger ◽  
M. Willy Honders ◽  
Jim E. Landegent ◽  
Roel Willemze ◽  
Renée M. Y. Barge
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Deoxycytidine Kinase ◽  
Wild Type ◽  
Healthy Donors ◽  
Alternatively Spliced ◽  
Acute Myeloid

Abstract Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

scholarly journals Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells

Blood ◽  
2020 ◽  
Vol 136 (13) ◽  
pp. 1507-1519 ◽  
Author(s):  
Simon Raffel ◽  
Daniel Klimmeck ◽  
Mattia Falcone ◽  
Aykut Demir ◽  
Alireza Pouya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Transcript Level ◽  
Characteristic Energy ◽  
Hematopoietic Stem ◽  
Improve Patient Survival ◽  
Stem And Progenitor Cells ◽  
Blast Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.

Differentiation-independent retinoid induction of folate receptor type β, a potential tumor target in myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3529-3536 ◽  
Author(s):  
Hui Wang ◽  
Xuan Zheng ◽  
Frederick G. Behm ◽  
Manohar Ratnam
Keyword(s):  
Retinoic Acid ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Transforming Growth Factor ◽  
Retinoic Acid Receptor ◽  
Folate Receptor ◽  
Leukemia Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Construct

Abstract Folate receptor (FR) type β is expressed in the myelomonocytic lineage, predominantly during neutrophil maturation and in myeloid leukemias. FR-β expression was elevated up to 20-fold by all-trans retinoic acid (ATRA) in KG-1 myeloid leukemia cells in a dose-dependent and reversible manner in the absence of terminal differentiation or cell growth inhibition. ATRA also increased FR-β expression in vitro in myeloid leukemia cells from patient marrow. FR-β was not up-regulated in KG-1 cells treated with phorbol ester, dexamethasone, 1,25-dihydroxy vitamin D3, or transforming growth factor β. ATRA did not induce FR-β expression in receptor negative cells of diverse origin. The ATRA-induced increase in FR-β expression in KG-1 cells occurred at the level of messenger RNA synthesis, and in 293 cells containing a stably integrated FR-β promoter–luciferase reporter construct, ATRA induced expression of the reporter. From experiments using retinoid agonists and antagonists and from cotransfection studies using the FR-β promoter and expression plasmids for the nuclear receptors retinoic acid receptor (RAR)α, RARβ, or RARγ, it appears that the retinoid effect on FR-β expression could be mediated by ligand binding to RARs α, β, or γ, but not to retinoid X receptors. Furthermore, there was apparent cross-talk between RARα and RARγ selective agonists or antagonists, suggesting a common downstream target for RAR isoforms in inducing FR-β expression. Thus, blocks in the RARα-specific pathway of retinoid-induced differentiation may be bypassed during retinoid induction of FR-β expression. The results suggest that to facilitate FR-targeted therapies, retinoids may be used to modulate FR-β expression in myeloid leukemia cells refractory to retinoid differentiation therapy.

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