The Role of Cbx Proteins in Human Benign and Malignant Hematopoiesis

Abstract Introduction: Hematopoietic stem cells (HSCs) need to properly balance self-renewal and differentiation to prevent malignant transformation or HSC exhaustion. We recently showed that the presence of specific Cbx proteins in the Polycomb Repressive Complex 1 (PRC1) plays a crucial role in balancing self-renewal and differentiation of murine HSCs. Whereas Cbx7 induces self-renewal, Cbx4 and Cbx8 instead induce differentiation. In this project, we investigate the role of various CBX Polycomb proteins in human normal and malignant hematopoiesis. We aim to identify genes which are controlled by CBX7 in normal and malignant human hematopoiesis. Methods: We overexpressed CBX2, -4, -6, -7 and -8 in human CD34+HSPCs. We assessed functional consequences by measuring cobblestone area-forming cell (CAFC), colony-forming unit (CFU) frequencies, and performed stem cell xenotransplantation studies in NSG mice. To identify genes which are controlled by CBX7 or CBX8, we performed RNA- and Chip-Seq in CD34+ HSPCs. To explore the role of CBX7 in leukemic cells, we performed short hairpin RNA-mediated knockdown of protein expression in leukemic cell lines. Results: Overexpression of CBX7 and CBX8 in human CD34+ HSPCs led to a significant 5-10-fold increase of week 5 CAFC (fold increase: 9,62 for CBX7; 4,92 for CBX8) and 1,5-fold increase CFU-frequencies (fold increase: 1,32 for CBX7; 1,41 for CBX8) after 14 days. In contrast, overexpression of CBX4 and CBX2 led to equal or decreased CAFC- and CFU-frequencies. Transplantation of CBX7 overexpressing human CD34+ HSPCs in NSG mice led to higher long-term engraftment in comparison to the empty vector control (p<0,05 after 18 weeks). Even after one week of in vitro culture of CBX7-overexpressing CD34+cells significant engraftment levels were obtained. Conversely, downregulation of CBX7 in various leukemic cell lines resulted in markedly decreased proliferation and strongly induced differentiation. Expression analysis showed that genes that are upregulated upon CBX7 overexpression are preferentially expressed in primitive stem cells, and are repressed in more differentiated cell types. Conclusions: Our study indicates that CBX7 and CBX8 regulate self-renewal of human HSPCs. Furthermore, our data show that repression of CBX7 markedly inhibits proliferation of leukemic cells and can release the differentiation block of leukemic cells in vitro. Collectively our results indicate that targeting CBX7 may be a viable strategy to induce terminal differentiation of leukemic cells. Disclosures No relevant conflicts of interest to declare.

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Targeting Aberrant Self-Renewal of Leukemic Cells with a Novel CBP/p300 Bromodomain Inhibitor

Blood ◽

10.1182/blood.v124.21.3750.3750 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3750-3750

Author(s):

Angeliki Thanasopoulou ◽

Katharina Dumrese ◽

Sarah Picaud ◽

Oleg Fedorov ◽

Stefan Knapp ◽

...

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Colony Formation ◽

Leukemia Cell ◽

Leukemic Cell ◽

Leukemic Cells ◽

Human Leukemia ◽

Self Renewal ◽

Hematopoietic Stem ◽

Leukemic Cell Lines

Abstract The CBP/p300 histone acetyltransferases are key transcriptional regulators of hematopoiesis that have been found to be involved in AML-associated recurrent chromosomal translocations and shown to function as co-activators of leukemogenic fusion oncogenes, suggesting that specific targeting of CBP/p300 may be beneficial for therapy. We characterized the anti-leukemic potential of I-CBP112, a novel chemical inhibitory probe targeting the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. I-CBP112 represents a new, potent and selective class of BRD inhibitors (oxazepines) binding to recombinant CBP/p300 BRDs with a KD of 151nM and 157nM respectively. Initial characterization by FRAP and BRET assays revealed that I-CBP112 displaced the isolated BRD construct from chromatin but not the full length CBP. I-CBP112 also impaired the interaction of CBP/p300 with p53, resulting in reduced p53-K382 acetylation, reduced p21 expression, and high sensitivity to Doxorubicin-induced DNA damage. We started to explore the effects of the compound on leukemic cells by exposing a series of murine cell lines immortalized by the MLL-CBP fusion and other potent leukemia-associated oncogenes including the MLL-AF9, MLL-ENL, or the NUP98-HOXA9 fusion to increasing doses of I-CBP112. Interestingly, no significant cytotoxicity was observed up to concentrations of 5μM. However, in all cell lines we observed a significant reduced number of colonies formed in methylcellulose, associated with morphological differentiation as observed in Giemsa stained cytospots. Similar to the murine leukemic cell lines we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Reduced colony formation upon I-CBP112 treatment was also observed of human primary AML blasts but not of CD34+ hematopoietic stem cells from two healthy donors. I-CBP112 effects were studied in more detail in three human leukemia cell lines: SEM (MLL-AF4+), MOLM13 (MLL-AF9+) and Kasumi-1 (AML1-ETO+). Long-term exposure of these cells to I-CBP112 in liquid medium, resulted in a dose-dependent G1 cell cycle arrest, with Kasumi-1 being the most sensitive to the inhibitor, demonstrating further morphological signs of differentiation and apoptotic cell death. Importantly, combination of I-CBP112 with the BET-BRD inhibitor JQ1 or Doxorubicin revealed a clear synergistic effect on cell survival of the AML cell lines except for the combination of I-CBP112 with Doxorubicin on MOLM13. Surprisingly only modest effects of I-CBP112 exposure on the transcriptional programs of SEM, MOLM13 and Kasumi-1 cells were found by microarray expression profiling. Genes found affected were mainly immune response regulators or NFkappaB targets suggesting that attenuation of NFkappaB downstream signals might impair the leukemia initiation capacity reflected by reduced colony formation. Extreme limited dilution assays (ELDA) in methylcellulose, as well as bone marrow transplantations in limiting dilutions using MLL-AF9-transformed murine leukemic blasts revealed that I-CBP112 significantly impaired self-renewal of the leukemic stem cell compartment in vitro and reduced the leukemia-initiating potential in vivo. Taken together, these data demonstrate that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML and other human cancers. Disclosures No relevant conflicts of interest to declare.

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Reactive Oxygen Species (ROS) Levels Define Functional Heterogeneity in Human Leukemia Stem Cells and Represent a Critical Parameter for Therapeutic Targeting

Blood ◽

10.1182/blood.v118.21.639.639 ◽

2011 ◽

Vol 118 (21) ◽

pp. 639-639

Author(s):

Eleni Lagadinou ◽

Kevin Callahan ◽

Sarah J Neering ◽

ShanShan Pei ◽

Mohammad Minhajuddin ◽

...

Keyword(s):

Stem Cells ◽

Redox Regulation ◽

Leukemic Cells ◽

Oxygen Species ◽

Self Renewal ◽

Nsg Mice ◽

Intracellular Ros ◽

Oxidative State

Abstract Abstract 639 Failure to eliminate leukemia stem cells (LSC) appears to be a major limitation of standard regimens and a significant challenge in the development of improved therapies. To effectively target LSC, it is essential to unravel the unique functional properties of these cells with respect to bulk leukemic and normal cells. Reactive oxygen species (ROS) are oxygen-containing chemicals continuously generated in cells through various biochemical pathways. Normal stem cells appear to tightly control intracellular ROS at low levels so as to maintain long-term self-renewal and survival. Although cancer cells are generally considered to have increased ROS levels due to oncogene activation and acquisition of a tumor-specific metabolic profile, the redox regulation in cancer stem cells remains largely unknown. We hypothesized that, similar to normal stem cells, that redox state is important for LSC survival. By employing primary human acute myeloid leukemia (AML) samples and several redox-sensitive dyes that provide information on different aspects of intracellular oxidative state, we show that leukemic cells display a range of intracellular ROS, with functionally defined LSC being distributed across the ROS gradient. However, leukemic cells prospectively isolated by flow cytometry from the lowest end of the ROS gradient contain the highest concentration of leukemic stem cell activity, as defined by their preferentially quiescent cell cycle profile, enhanced potential in colony forming assays, increased engraftment in immune deficient NSG mice, and capacity to re-establish in the xenograft model an oxidative state heterogeneity similar to that observed in the primary tumor. Further, in serial transplantation assays, which evaluate the long-term maintenance of self-renewal capacity, the ROS-low leukemic population is preferentially able to engraft NSG mice in most cases. In contrast, leukemic cells residing in higher oxidative levels (ROS-high) are more actively cycling; show reduced in vivo self-renewal potential in NSG mice, and evidence of increased DNA damage. Upon therapeutic challenge, prospectively isolated ROS-low primary leukemic cells are less responsive to in vitro drug treatment with both conventional (daunorubicin) and experimental (parthenolide, temsirolimus) anti-leukemic agents, and are better able to resist induction towards an increased oxidative state after pro-oxidant treatment (hydrogen peroxide, BSO). To further investigate the relative sensitivity of ROS-low vs high primitive populations to anti-leukemic therapies, we treated sorted primary AML subsets with anti-leukemic drugs for 18hr and then transplanted into NSG mice. In this functional assay, only the ROS-low LSC fraction retains its self-renewal capacity after therapy, thereby indicating that ROS-low cells may be a critical target for therapy. To better understand mechanisms controlling redox heterogeneity in leukemia, we performed biochemical analyses on ROS-low vs high subsets. Our studies indicate that ROS-low cells adopt unique metabolic and redox regulation properties. Whereas ROS-high cells exhibit robust activation of pathways implicated in cell cycle promotion, metabolism, inflammation and senescence, the respective gene and protein expression in ROS-low cells is either markedly decreased or diminished, and ROS-low cells also show a dramatically reduced dependence on mitochondrial oxidative phosphorylation, as determined by bio-energetic analyses. Further, ROS-low cells seem to contain increased levels of glutathione, a critical cellular antioxidant. Taken together, these findings suggest that ROS-low LSC represent an important target for therapy, but as a consequence of their unique biological properties may be very difficult to eradicate. To this end, we have begun to characterize agents with the ability to enhance leukemia selective targeting of the ROS-low population. Intriguingly, our initial studies show that the naturally occurring compound rocaglamide is a potent inducer of cell death in the ROS-low compartment, as determined by both in vitro culture and xenograft analyses. Importantly, rocaglamide is much less toxic to normal stem and progenitor, indicating that this approach may have a significant therapeutic index. Thus, we propose that regimens designed to include compounds of this class may result in more effective elimination of human LSCs. Disclosures: No relevant conflicts of interest to declare.

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CDX2 regulates Human Leukemic Cell Clonogenicity and in Vivo Repopulation Capacity

Blood ◽

10.1182/blood.v124.21.1005.1005 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1005-1005

Author(s):

Anna Paczulla ◽

Sarah Grzywna ◽

Matthias Grauer ◽

Ursula Kohlhofer ◽

Leticia Quintanilla-Martinez ◽

...

Keyword(s):

Cell Cycle ◽

Leukemic Cell ◽

Leukemic Cells ◽

Qrt Pcr ◽

Nsg Mice ◽

Leukemic Cell Lines ◽

Cdx2 Expression ◽

Human Leukemic Cells

Abstract Objectives: The caudal-type homeobox (CDX) gene family has been mainly studied during early development for its role in axial elongation and antero-posterior patterning. More recently, CDX genes were shown to regulate embryonic hematopoiesis via downstream HOX genes and interactions with the WNT signaling pathway. The role of CDX genes in adult hematopoiesis is poorly understood and almost no data exists on human cells. Healthy bone marrow (BM) derived hematopoietic cells express low levels of CDX1 and CDX4 but lack CDX2 expression. However, CDX2 expression is found in >80% of human acute myeloid (AML) and lymphoid leukemia (ALL) and its induction in murine BM cells results in myeloid leukemia. Here, we explore the role of CDX2 in human healthy hematopoietic and leukemic cells. Methods: CDX2 expression was modulated via lentiviral treatment in human BM CD34+ and SKM-1, EOL-1 and NALM16 human leukemic cell lines. CDX2 knockdown experiments were performed using two different shRNAs against CDX2 to control for potential off-target effects. To generate cultures displaying a very strong knockdown, individual clones were generated from single transduced cells and analyzed also separately. Efficient modulation of gene and protein CDX2 expression was analyzed by qRT-PCR and respectively immunoblot analyses. CDX2-modified (overexpressing or knockdown) or control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry and qRT-PCR assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rγnull (NSG) mice. Human recombinant DKK-1 protein was supplemented in CFU assays to CDX2 knockdown and respectively control cells. Results: shRNA-mediated CDX2 knockdown performed on SKM-1 as well as EOL-1 cells strongly reduced clonogenic capacity in CFU assays while only slightly reducing growth. Consistently, proliferation, apoptosis sensitivity and cell cycle were not influenced by CDX2 downregulation in any of the three analyzed leukemic cell lines. Importantly, CDX2 knockdown SKM-1 as well as NALM16 cells transplanted into immunopermissive NSG mice showed profoundly suppressed in vivo leukemogenic properties compared to control cells. However, overexpression of the human CDX2 gene using an SFFV-promotor driven lentiviral vector in human healthy CD34+ bone marrow-derived and also in leukemic cells resulted in a G0/G1 cell cycle arrest, reducing in vitro growth and CFU formation. Consistent with these results and in contrast to previous results reported in mice, CDX2 overexpression did not confer in vitro serial replating capacity or in vivo leukemogenic properties to healthy human CD34+ cells. To further investigate the molecular mechanisms underlying these CDX2-mediated effects in human leukemia, we analyzed the expression of HOX and Wnt-pathway associated genes via qRT-PCR. Modulation of HOX gene expression was indeed observed upon CDX2 knockdown or overexpression. Previously reported repressive effects of CDX2 on Klf4 gene expression were confirmed. Surprisingly however, we found that CDX2 expression positively regulates the expression of the WNT-inhibitory molecule DKK-1 in both leukemic and healthy CD34+ stem/progenitor cells. Supplementation of DKK-1 was able to rescue the clonogenic capacity of CDX2 knockdown leukemic cells in CFU-assays while opposite effects were noted in control cells. Conclusion: Our data suggest that CDX2 regulates in vivo leukemogenesis by inducing clonogenic properties in human leukemic cells. Its downstream molecular pathways in human leukemic cells may include, next to Klf4, HOX gene family members and the Wnt-inhibitor DKK-1. Leukemic cells might use DKK-1 expression to fine-tune their Wnt-signaling activity to an optimal dosage required for leukemia initiation and growth. Work underway in our laboratory is further investigating this hypothesis. Disclosures No relevant conflicts of interest to declare.

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Crispr Engineering in CD34+ Progenitors Reveals Cis-Acting Regulatory Regions Mediating 3D Interactions and Stem Cell Fate Decisions

Blood ◽

10.1182/blood.v128.22.1466.1466 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1466-1466

Author(s):

Mira Jeong ◽

Yong Lei ◽

Ivan Bochkov ◽

Muhammad S Shamim ◽

Anna G Guzman ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

T Cells ◽

Hoxa Cluster ◽

Self Renewal ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract DNA methylation is a critical regulator of cis-regulatory elements that can impact the distribution of epigenetic regulators and transcription factors. We have mapped DNA methylation changes in human CD34+ cells and downstream progeny to detect changes in DNA methylation with differentiation. Some large regions of low DNA methylation emerge with differentiation, which we call dynamic (d-) canyons. In order to determine whether these correlate with changes in the 3D genome, we generated 6 high-resolution Hi-C contact maps from CD34+CD38- cells, Adult bone marrow CD34+ cells, 7 days cultured CD34+ cells, CD71+CD36+CD235+ Erythroid cells, CD4+ T cells and AML patient blast cells. We generated ~ 1 billion mapped reads in order to create each 3D map of the genome at kilobase resolution. We identified several sites of d-canyons which correlate with changes in Hi-C loops. To study the functional role of d-canyons, we performed CRISPR/CAS9 mediated genome engineering targeting three sites of interest in CD34+ progenitors. We designed sgRNAs to delete several d-canyon regions that exhibited cell-type specific methylation and dynamic H3K27ac marking. First, we targeted a novel putative regulatory region of the RUNX1 gene, a critical master regulator of hematopoiesis that is frequently mutated in human leukemia. From genome-wide DNA methylation profiling, we identified a d-canyon located in the first intron of RUNX1, which overlaped with a H3K27ac peak in human CD34+CD38- hematopoietic progenitor cells. DNA methylation in this region is further depleted in T cells and increased in AML cells, suggesting a role for regulating RUNX1 in specific cell types. To study its function, we designed 2 sgRNAs to delete a 1.8 kb d-canyon. CRISPR/Cas9-mediated deletion of this region resulted in ablation of RUNX1 expression in cord blood hematopoietic stem cells and a significant decrease of engraftment activity in NSG mice along with an increase of erythroid colony forming ability in in vitro assays. In addition, we identified d-canyons upstream of the master regulators GATA2 and in the HOXA cluster. We deleted 1.7kb d-canyon upstream of GATA2 gene, finding that it resulted in increased self-renewal of CD34+CD38- cells in NSG xenografts. In contrast, when we deleted one a d-canyon located ~2Mb upstream of the HOXA cluster, within a HiC loop signal present only in CD34+CD38- cells, we observed in decreased CD34+CD38- cell self-renewal but a significant increase of CD34+CD38+ differentiated progenitors in an in vitro culture system, as well as in NSG mice. Colony forming assay showed decreased colony size and numbers. Finally, we identified a d-canyon associated with TCF3, also known as E2A, a transcription factor involved in B and T cell lineage differentiation. We designed 4 sgRNAs to delete 1.5kb d-canyon edge regions within the second intron of TCF3. Removal of this region resulted in a significant decrease of CD19+ B cells, but an increase of CD3+ T cells in NSG mice. Taken together, these results suggest the functional importance of d-canyons for orchestrating genome architecture and fate decisions of hematopoietic stem cells. These findings advance our understanding of the relationship between DNA methylation changes and loop interactions, providing new insights into the potential impact of potential aberrant DNA methylation and chromatin structure. Disclosures No relevant conflicts of interest to declare.

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YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway

10.21203/rs.3.rs-414853/v1 ◽

2021 ◽

Author(s):

Hong-Chen Yan ◽

Yu Sun ◽

Ming-Yu Zhang ◽

Shu-Er Zhang ◽

Jia-Dong Sun ◽

...

Keyword(s):

Stem Cells ◽

Signaling Pathway ◽

Adult Stem Cells ◽

Self Renewal ◽

Human Ascs ◽

Regulation Mechanisms ◽

Interfering Rna ◽

Pluripotency Genes

Abstract Background Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of ASCs is a hot topic. Porcine models have close similarities to humans and porcine SDSCs (pSDSCs) offer an ideal in vitro model to investigate human ASCs. To date, studies concerning the role of yes-associated protein (YAP) in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this paper, we explore the link between the transcriptional regulator YAP and the fate of pSDSCs. Results We found that YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Sox2, Oct4. The overexpression of YAP prevented the differentiation of pSDSCs and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/β-catenin signaling pathway. When an activator of the Wnt/β-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939 an inhibitor of Wnt/β-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Conclusions our results suggested that, YAP and the Wnt/β-catenin signaling pathway interact to regulate the fate of pSDSCs.

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MicroRNA-28-5p Regulates Liver Cancer Stem Cell Expansion via IGF-1 Pathway

Stem Cells International ◽

10.1155/2019/8734362 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 2

Author(s):

Qing Xia ◽

Tao Han ◽

Pinghua Yang ◽

Ruoyu Wang ◽

Hengyu Li ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Liver Cancer ◽

Critical Role ◽

Self Renewal ◽

Sorafenib Treatment ◽

The Impact

Background. MicroRNAs (miRNAs) play a critical role in the regulation of cancer stem cells (CSCs). However, the role of miRNAs in liver CSCs has not been fully elucidated. Methods. Real-time PCR was used to detect the expression of miR-miR-28-5p in liver cancer stem cells (CSCs). The impact of miR-28-5p on liver CSC expansion was investigated both in vivo and in vitro. The correlation between miR-28-5p expression and sorafenib benefits in HCC was further evaluated in patient-derived xenografts (PDXs). Results. Our data showed that miR-28-5p was downregulated in sorted EpCAM- and CD24-positive liver CSCs. Biofunctional investigations revealed that knockdown miR-28-5p promoted liver CSC self-renewal and tumorigenesis. Consistently, miR-28-5p overexpression inhibited liver CSC’s self-renewal and tumorigenesis. Mechanistically, we found that insulin-like growth factor-1 (IGF-1) was a direct target of miR-28-5p in liver CSCs, and the effects of miR-28-5p on liver CSC’s self-renewal and tumorigenesis were dependent on IGF-1. The correlation between miR-28-5p and IGF-1 was confirmed in human HCC tissues. Furthermore, the miR-28-5p knockdown HCC cells were more sensitive to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrated that the miR-28-5p may predict sorafenib benefits in HCC patients. Conclusion. Our findings revealed the crucial role of the miR-28-5p in liver CSC expansion and sorafenib response, rendering miR-28-5p an optimal therapeutic target for HCC.

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Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger

Blood ◽

10.1182/blood.v95.4.1427.004k48_1427_1434 ◽

2000 ◽

Vol 95 (4) ◽

pp. 1427-1434 ◽

Cited By ~ 119

Author(s):

Ivan N. Rich ◽

Diana Worthington-White ◽

Oliver A. Garden ◽

Philip Musk

Keyword(s):

Cell Cycle ◽

Intracellular Ph ◽

Annexin V ◽

Leukemic Cell ◽

Differential Sensitivity ◽

Leukemic Cells ◽

Hematopoietic Tissue ◽

Leukemic Cell Lines ◽

Targeted Inhibition

The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.

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Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽

10.1182/blood.v85.5.1237.bloodjournal8551237 ◽

1995 ◽

Vol 85 (5) ◽

pp. 1237-1245 ◽

Cited By ~ 51

Author(s):

G Manfioletti ◽

V Gattei ◽

E Buratti ◽

A Rustighi ◽

A De Iuliis ◽

...

Keyword(s):

Cell Lines ◽

Constitutive Expression ◽

Leukemic Cell ◽

Leukemic Cells ◽

Maturation Stage ◽

Lymphoid Tissues ◽

Specific Cell ◽

B Cell Differentiation ◽

Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

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Pioglitazone Inhibits the Growth of Human Leukemic Cell Lines and Primary Leukemic Cells in Vitro.

Blood ◽

10.1182/blood.v104.11.4493.4493 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4493-4493 ◽

Cited By ~ 1

Author(s):

Yoshihiro Hatta ◽

Minoru Saiki ◽

Yuko Enomoto ◽

Shin Aizawa ◽

Umihiko Sawada ◽

...

Keyword(s):

Cell Lines ◽

Colony Formation ◽

Mononuclear Cells ◽

Hematopoietic Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Peroxisome Proliferator ◽

Leukemic Cell Lines ◽

Ppar Γ

Abstract Troglitazone and pioglitazone are one of thiazolidinediones that are high affinity ligand for the nuclear receptor called peroxisome proliferator-activated receptor gamma (PPAR-γ). Troglitazone is a potent inhibitor of clonogenic growth of acute myeloid leukemia cells when combined with a retinoid. However, the effect of pioglitazone to neoplastic cells and normal hematopoietic cells has not been studied yet. Adult T-cell leukemia (ATL), prevalent in western Japan, is a highly aggressive malignancy of mature T lymphocyte. Therefore, we studied antitumor effect of pioglitazone against leukemic cells including ATL as well as normal hematopoietic cells. With 300 μM of pioglitazone, colony formation of ATL cell lines (MT1, MT2, F6T, OKM3T, and Su9T01) was completely inhibited. Colony formation of HUT102, another ATL cell line, was 12 % compared to untreated control. Clonogenic cells of other leukemic cell lines (K562, HL60, U937, HEL, CEM, and NALM1) was also inhibited to 0–30% of control. Colony formation of primary leukemic cells from 5 AML patients was decreased to 15 %. However, normal hematopoietic cells were weakly inhibited with 300 μM pioglitazone; 77 % of CFU-GM, 70 % of CFU-E, and 33 % of BFU-E survived. Cell cycle analysis showed that pioglitazone decreased the ratio of G2/M phase in HL60 cells, suggesting the inhibition of cell division. By Western blotting, PPAR-γ protein level was similar in all leukemic cells and normal bone marrow mononuclear cells. Taken together, pioglitazone effectively eliminate leukemic cells and could be used as an antitumor agent in vivo.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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