BMI1 collaborates with BCR-ABL in leukemic transformation of human CD34+ cells

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. 4621-4630 ◽  
Author(s):  
Aleksandra Rizo ◽  
Sarah J. Horton ◽  
Sandra Olthof ◽  
Bert Dontje ◽  
Albertina Ausema ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Blast Crisis ◽  
Severe Combined Immunodeficiency ◽  
Chronic Phase ◽  
Cancer Therapies ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract The major limitation for the development of curative cancer therapies has been an incomplete understanding of the molecular mechanisms driving cancer progression. Human models to study the development and progression of chronic myeloid leukemia (CML) have not been established. Here, we show that BMI1 collaborates with BCR-ABL in inducing a fatal leukemia in nonobese diabetic/severe combined immunodeficiency mice transplanted with transduced human CD34+ cells within 4-5 months. The leukemias were transplantable into secondary recipients with a shortened latency of 8-12 weeks. Clonal analysis revealed that similar clones initiated leukemia in primary and secondary mice. In vivo, transformation was biased toward a lymphoid blast crisis, and in vitro, myeloid as well as lymphoid long-term, self-renewing cultures could be established. Retroviral introduction of BMI1 in primary chronic-phase CD34+ cells from CML patients elevated their proliferative capacity and self-renewal properties. Thus, our data identify BMI1 as a potential therapeutic target in CML.

The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 516-516 ◽  
Author(s):  
Daniel Goff ◽  
Alice Shih ◽  
Angela Court Recart ◽  
Larisa Balaian ◽  
Ryan Chuang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chemotherapy Resistance ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4081-4091 ◽  
Author(s):  
Laure Gilles ◽  
Romain Guièze ◽  
Dominique Bluteau ◽  
Véronique Cordette-Lagarde ◽  
Catherine Lacout ◽  
...  
Keyword(s):  
Ploidy Level ◽  
Molecular Mechanisms ◽  
Transcriptional Level ◽  
Moderate Increase ◽  
Protein Levels ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Ploidy Class

Abstract The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34+ cells, we observed that p19INK4D expression was increased both at the mRNA and protein levels during ploidization. p19INK4D knockdown led to a moderate increase (31.7% ± 5%) in the mean ploidy of MKs suggesting a role of p19INK4D in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41highCD42high) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19INK4D overexpression in CD34+ cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19INK4D KO mice exhibited an increase in mean ploidy level from 18.7N (± 0.58N) to 52.7N (± 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19INK4D promoter. Moreover, AML-1 inhibition led to the p19INK4D down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.

Novel Findings of Anti-cancer Property of Propofol

2018 ◽  
Vol 18 (2) ◽  
pp. 156-165 ◽  
Author(s):  
Jiaqiang Wang ◽  
Chien-shan Cheng ◽  
Yan Lu ◽  
Xiaowei Ding ◽  
Minmin Zhu ◽  
...  
Keyword(s):  
General Anesthesia ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Intravenous Anesthetic ◽  
The Past ◽  
Anti Cancer ◽  
Underlying Mechanisms ◽  
Recent Attention

Background: Propofol, a widely used intravenous anesthetic agent, is traditionally applied for sedation and general anesthesia. Explanation: Recent attention has been drawn to explore the effect and mechanisms of propofol against cancer progression in vitro and in vivo. Specifically, the proliferation-inhibiting and apoptosis-inducing properties of propofol in cancer have been studied. However, the underlying mechanisms remain unclear. Conclusion: This review focused on the findings within the past ten years and aimed to provide a general overview of propofol's malignance-modulating properties and the potential molecular mechanisms.

scholarly journals Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2063-2063
Author(s):  
Naoya Uchida ◽  
Claire Drysdale ◽  
Morgan Yapundich ◽  
Jackson Gamer ◽  
Tina Nassehi ◽  
...  
Keyword(s):  
Rhesus Macaques ◽  
Fetal Hemoglobin ◽  
Selective Advantage ◽  
Erythroid Cells ◽  
Post Transplant ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Positive feedback between lncRNA FLVCR1-AS1 and KLF10 may inhibit pancreatic cancer progression via the PTEN/AKT pathway

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jiewei Lin ◽  
Shuyu Zhai ◽  
Siyi Zou ◽  
Zhiwei Xu ◽  
Jun Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Positive Feedback ◽  
Molecular Mechanisms ◽  
Tumor Tissues ◽  
And Migration

Abstract Background FLVCR1-AS1 is a key regulator of cancer progression. However, the biological functions and underlying molecular mechanisms of pancreatic cancer (PC) remain unknown. Methods FLVCR1-AS1 expression levels in 77 PC tissues and matched non-tumor tissues were analyzed by qRT-PCR. Moreover, the role of FLVCR1-AS1 in PC cell proliferation, cell cycle, and migration was verified via functional in vitro and in vivo experiments. Further, the potential competitive endogenous RNA (ceRNA) network between FLVCR1-AS1 and KLF10, as well as FLVCR1-AS1 transcription levels, were investigated. Results FLVCR1-AS1 expression was low in both PC tissues and PC cell lines, and FLVCR1-AS1 downregulation was associated with a worse prognosis in patients with PC. Functional experiments demonstrated that FLVCR1-AS1 overexpression significantly suppressed PC cell proliferation, cell cycle, and migration both in vitro and in vivo. Mechanistic investigations revealed that FLVCR1-AS1 acts as a ceRNA to sequester miR-513c-5p or miR-514b-5p from the sponging KLF10 mRNA, thereby relieving their suppressive effects on KLF10 expression. Additionally, FLVCR1-AS1 was shown to be a direct transcriptional target of KLF10. Conclusions Our research suggests that FLVCR1-AS1 plays a tumor-suppressive role in PC by inhibiting proliferation, cell cycle, and migration through a positive feedback loop with KLF10, thereby providing a novel therapeutic strategy for PC treatment.

Fas-Mediated Modulation of Bcr/Abl in Chronic Myelogenous Leukemia Results in Differential Effects on Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 981-989 ◽  
Author(s):  
Carmine Selleri ◽  
Jaroslaw P. Maciejewski ◽  
Fabrizio Pane ◽  
Luigia Luciano ◽  
Anna Maria Raiola ◽  
...  
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Poor Response ◽  
Inhibitory Response ◽  
Myelogenous Leukemia ◽  
Poor Responders ◽  
Cd34 Cells ◽  
In Cells

Abstract Fas-R is expressed constitutively in CD34+ cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that α-interferon (IFN-α) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-α can prime CML cells for the effects of Fas, the response to IFN-α in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34+ CML cells after Fas-R triggering and the clinical response to IFN-α. After priming with IFN-α, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-α; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-α. In responders to IFN-α, Fas-R agonist induced dose-dependent apoptosis of CD34+ cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-α, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and nobcr/abl downmodulation was observed. Finally, we measuredbcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/ablprotein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-α treatment. © 1998 by The American Society of Hematology.

scholarly journals An Update on the Role of Dietary Phytochemicals in Human Skin Cancer: New Insights into Molecular Mechanisms

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 916 ◽  
Author(s):  
Salman Ul Islam ◽  
Muhammad Bilal Ahmed ◽  
Haseeb Ahsan ◽  
Mazharul Islam ◽  
Adeeb Shehzad ◽  
...  
Keyword(s):  
Skin Cancer ◽  
Human Skin ◽  
Cancer Progression ◽  
Fruits And Vegetables ◽  
Molecular Mechanisms ◽  
In Vivo Studies ◽  
Skin Malignancy ◽  
Dietary Phytochemicals

Human skin is continuously subjected to environmental stresses, as well as extrinsic and intrinsic noxious agents. Although skin adopts various molecular mechanisms to maintain homeostasis, excessive and repeated stresses can overwhelm these systems, leading to serious cutaneous damage, including both melanoma and non-melanoma skin cancers. Phytochemicals present in the diet possess the desirable effects of protecting the skin from damaging free radicals as well as other benefits. Dietary phytochemicals appear to be effective in preventing skin cancer and are inexpensive, widely available, and well tolerated. Multiple in vitro and in vivo studies have demonstrated the significant anti-inflammatory, antioxidant, and anti-angiogenic characteristics of dietary phytochemicals against skin malignancy. Moreover, dietary phytochemicals affect multiple important cellular processes including cell cycle, angiogenesis, and metastasis to control skin cancer progression. Herein, we discuss the advantages of key dietary phytochemicals in whole fruits and vegetables, their bioavailability, and underlying molecular mechanisms for preventing skin cancer. Current challenges and future prospects for research are also reviewed. To date, most of the chemoprevention investigations have been conducted preclinically, and additional clinical trials are required to conform and validate the preclinical results in humans.

scholarly journals The polypeptide GALNT6 Displays Redundant Functions upon Suppression of its Closest Homolog GALNT3 in Mediating Aberrant O-Glycosylation, Associated with Ovarian Cancer Progression

2019 ◽  
Vol 20 (9) ◽  
pp. 2264 ◽  
Author(s):  
Razan Sheta ◽  
Magdalena Bachvarova ◽  
Elizabeth Macdonald ◽  
Stephane Gobeil ◽  
Barbara Vanderhyden ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Survival Rates ◽  
Functional Redundancy ◽  
Tumor Formation ◽  
Migration And Invasion ◽  
Gene Ablation

Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy; a better understanding of the molecular mechanisms associated with EOC etiology could substantially improve EOC management. Aberrant O-glycosylation in cancer is attributed to alteration of N-acetylgalactosaminyltransferases (GalNAc-Ts). Reports suggest a genetic and functional redundancy between GalNAc-Ts, and our previous data are indicative of an induction of GALNT6 expression upon GALNT3 suppression in EOC cells. We performed single GALNT3 and double GALNT3/T6 suppression in EOC cells, using a combination of the CRISPR-Cas9 system and shRNA-mediated gene silencing. The effect of single GALNT3 and double GALNT3/T6 inhibition was monitored both in vitro (on EOC cells roliferation, migration, and invasion) and in vivo (on tumor formation and survival of experimental animals). We confirmed that GALNT3 gene ablation leads to strong and rather compensatory GALNT6 upregulation in EOC cells. Moreover, double GALNT3/T6 suppression was significantly associated with stronger inhibitory effects on EOC cell proliferation, migration, and invasion, and accordingly displayed a significant increase in animal survival rates compared with GALNT3-ablated and control (Ctrl) EOC cells. Our data suggest a possible functional redundancy of GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective of using both these enzymes as novel EOC biomarkers and/or therapeutic targets.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

Soluble Interleukin-6 (IL-6) Receptor With IL-6 Stimulates Megakaryopoiesis From Human CD34+ Cells Through Glycoprotein (gp)130 Signaling

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
Xingwei Sui ◽  
Kohichiro Tsuji ◽  
Yasuhiro Ebihara ◽  
Ryuhei Tanaka ◽  
Kenji Muraoka ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Ex Vivo ◽  
Serum Free ◽  
Human Cd34 ◽  
Receptor Component ◽  
Cd34 Cells ◽  
Interleukin 6 Receptor ◽  
Clonal Cultures

Abstract We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34+ cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34+ cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34+ cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34+ cells were subfractionated into two populations of IL-6R–negative (CD34+ IL-6R−) and IL-6R–positive (CD34+ IL-6R+) cells by fluorescence-activated cell sorting. The CD34+IL-6R− cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34+ IL-6R+cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

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