bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Role of Activated Ras in Leukaemogenesis: Induction of Angiogenesis.
Authors: Knight Robert J. , O’Brien Tracey A. , Lindeman Robert , Dolnikov Alla 
Published: April 2020

Abstract
Mutations in Ras genes are among the most frequent genetic alterations found in leukaemia. Our previous results have indicated that mutant N-ras (N-rasm) expression in the murine haematopoietic progenitor cells results in the development of myeloproliferative disorders that resemble human leukaemia. Our group has recently established a ‘humanised’ NOD/SCID mouse model of Ras-induced leukaemogenesis. N-rasm was transduced into normal human primitive haematopoietic progenitor cells (HPC) using recombinant GFP-expressing retrovirus to initiate the leukaemogenic transformation. When injected in NOD/SCID mice, N-rasm/GFP-expressing HPCs induce a pre-leukaemic condition characterised by the increased expansion of human N-rasm/GFP-positive cells with concomitant myeloid lineage expansion in the bone marrow of the recipient mice. In long-term culture N-rasm induces expansion of primitive CD34+/CD117+ and CD34+CXCR4+ progenitor cells enriched with NOD/SCID repopulating cells as well as myelomonocytic CD34−/CD14+ cells within the expanse of erythroid and lymphoid lineages. Microarray expression analysis revealed the induction of a number of the angiogenic factors and extracellular matrix proteins including VEGF, SDF-1, IL3, PDGF 1 and 2, metalloproteinases 9, 11, 12 and 14 that promote angiogenesis. The expansion of the primitive endothelial (CD34+VEGFR2+) and lymphatic (CD34+VEGFR3+) progenitor cells was consistently observed in N-rasm-transduced cultures. In addition, myelomonocytic CD34−CD14+ cells expanded by N-rasm also appear to contribute to neo-angiogenesis. N-rasm-induced pro-angiogenic factors also act as mitogenic and survival factors for pre-leukaemia HPCs by inhibiting apoptosis and promoting leukaemia-inititating-cell engraftment into the bone marrow of the recipient mice. N-rasm acts to modulate the interaction of leukaemia-initiating-cells with the bone marrow stroma by the induction of extracellular matrix degradation. We propose that the induction of pro-angiogenic factors by N-rasm acts to promote leukaemogenic transformation of leukaermia-inititating stem cells and needs to be investigated as a new candidate therapeutic target for leukaemia.
Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein.
Authors: Suzuki Takahiro , Yokoyama Yasuhisa , Kumano Keiki , Takanashi Minoko , Kozuma Shiro , Takato Tsuyoshi , Nakahata Tatsutoshi , Nishikawa Mitsuo , Sakano Seiji , Kurokawa Mineo , Ogawa Seishi , Chiba Shigeru 
Published: April 2020

Abstract
[Background and purposes] Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. The use of Notch ligands or soluble IL-6 receptor combined with IL-6 has been a major technique that revealed several fold expansion of human cord blood SCID repopulating cells (SRCs). These studies, however, have been conducted in an independent manner, which hampered direct comparison and evaluation of the effect of combination of these methods. Our purpose of this study is to clarify these issues in a chemically defined serum-free medium that allows us to develop clinical usage of the culture condition. We also compared the efficiencies of SRC isolation with magnetic beads targeting CD34 and CD133.
[Methods] Human cord blood CD133-sorted cells were cultured on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, flt-3 ligand, IL-3 and IL-6/soluble IL-6 receptor chimeric protein (FP6) for three weeks, and cultured cells were transplanted into NOD/SCID mice after limiting dilution to calculate the number of SRCs. To confirm whether full multipotency and self-renewal capacity of SRCs were maintained during the culture, cells were transplanted serially into NOD/SCID/γcnull (NOG) mice, and hematopoietic reconstitution was examined. To compare the efficiencies of CD34- and CD133-sorting, we divided each sample into two aliquots and separated CD34+ and CD133+ cells, and calculated the SRC numbers recovered by both separation methods.
[Results and discussion] The frequencies of SRCs in the culture-initiating CD133-sorted cells and cultured progeny were calculated as one out of 1,020 and one out of 175 (adjusted to culture-initiating cells), respectively, indicating 6-fold expansion of SRCs that was statistically significant. Delta1 significantly enhanced the expansion rate of SRCs, and addition of IL-3 to this condition further promoted the expansion. In the serial transplantation assays, we found human myeloid and lymphoid reconstitution both in the primary and secondary NOG recipients, verifying the SRC capacity in the cultured cells. Notably, the CD133-sorting was approximately 4.5 times more efficient in collecting SRCs than the CD34-sorting from the same number of mononuclear cells (MNCs) (308 and 254 SRCs by CD133-sorting vs. 67 and 59 SRCs by CD34-sorting from 108 MNCs). Our study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.
Bmi-1-Green Fluorescent Protein (GFP)-Knock-In Mice Reveal the Dynamic Regulation of Bmi-1 Expression in Normal and Leukemic Hematopoietic Cells.
Authors: Hosen Naoki , Clarke Michael F. , Weissman Irving L. 
Published: April 2020

Abstract
The ability to self-renew is essential for all kinds of stem cells regardless of tissue types. One of the best candidate genes involved in conferring self-renewal capacity is Bmi-1, which has been proven to be essential for the maintenance of both normal adult hematopoietic and leukemia stem cells as well as adult neural stem cells. Furthermore, it has been reported that Bmi-1 is expressed at high level in immature hematopoietic cells and its expression is down-regulated along with cell differentiation. In order to investigate the possible role of Bmi-1 expression as a marker of stem or progenitor cells in several types of normal or malignant tissues, we generated two types of Bmi-1-green fluorescent protein (GFP)-knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of the Bmi-1 gene. In one strain, GFP was inserted into the transcription start site, resulting in the heterozygous allele for Bmi-1. In another strain, GFP was expressed as a fusion protein with Bmi-1 from the targeted allele. Using these two types of targeted reporter mice, we demonstrate that Bmi-1 expression levels are well-correlated with cell differentiation in normal hematopoietic cells. Bmi-1 is expressed in CD150+(or Flk2/Flt3−) CD34− c-kit+ lin- Sca-1+ (KLS) cells, which is highly enriched with long-term hematopoietic stem cells (LT-HSCs), at its highest levels and down-regulated upon commitment to differentiation. The Bmi-1high/lineage-marker- cells contained 33.8% of KLS cells, indicating that HSCs can be highly enriched according to the Bmi-1 expression levels combined with lineage-markers. Then, we analyzed the Bmi-1 expression levels in acute myeloiud leukemia (AML) models induced by the combination of TEL/PDGFβR and AML1/ETO, or chronic myelogenous leukemia (CML) models induced by P210 BCR/ABL. Bmi-1 is also expressed at its highest levels in undifferentiated KLS leukemia cells. In addition, the Bmi-1 expression levels in leukemic KLS cells are similar to normal KLS cells, suggesting that the Bmi-1 over-expression in leukemia samples might simply reflect the accumulation of immature cells. Furthermore, in several other non-hematopoietic tissues, cells could be separated into distinct subpopulations with differential Bmi-1 expression. Thus, these mice allow for the isolation of viable Bmi-1-expressing cells and have the potential to become a useful tool for understanding the role of Bmi-1 in normal and cancer stem cells in multiple tissue types.
Enforced RUNX1 Expression Increased the Numbers of CD34+ and CD45+ Cells from Human Embryonic Stem Cell-Derived Embryoid Bodies.
Authors: D’Costa Jenice , Friedman Alan D. , Civin Curt I. 
Published: April 2020

Abstract
RUNX1 is an essential gene for mouse hematopoietic development. Mouse knockout models for either RUNX1 or its binding partner core binding factor(CBFβ) are embryonic lethal and lack definitive hematopoiesis. Dominant negative inhibitors of RUNX1 protein function CBFβ-SMMHC (INV), encoded by t(16;16) and AML1-ETO, encoded by t(8;21) are seen in approximately 8% and 15% of acute myeloid leukemia cases, respectively. Recently we reported that dominant inhibition of endogenous RUNX1 function by dual promoter lentivector-expressed INV inhibited proliferation and caused G1 arrest in both human and mouse myeloid progenitors. Conversely, exogenous expression of RUNX1 in mouse primary hematopoietic stem-progenitor cells resulted in increased proliferation [DCosta 2005]. In order to determine if exogenous expression of RUNX1 would enhance HESC-derived hematopoiesis, we used conditionally-regulated RUNX1 in human embryonic stem cells (HESC) as a model system. We transduced H1 (WA01) HESC with dual promoter lentivectors expressing modified estrogen receptor (ER) fusions: RUNX1-ER (EF-RUNX1-ER/PGK-GFP) or control GFP (Ub-GFP). Microscopically GFP+ HESC colonies were plucked and expanded on primary mouse embryonic fibroblasts to enrich for transduced cells. During growth under “pluripotent conditions,” no changes in HESC proliferation or differentiation (as assessed by staining for pluripotent surface markers SSEA-4, Tra-160 and CD9) were observed in cells induced to express RUNX1. Transduced HESC were allowed to undergo embryoid body (EB) formation. EB, cultured with or without 4-hydroxy-tamoxifen (4HT), were harvested at selected time points, dissociated with trypsin/EDTA, counted, and immunostained for CD34 and CD45 [Zambidis 2005; DCosta 2005]. Upon EB formation, there were 2–3-fold higher percentages and numbers of transduced (GFP+) cells in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture induced with 4HT than in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture without 4HT. In contrast, no 4HT-dependent difference was seen in the Ub.GFP control lentivector-transduced cell cultures. At these time points, 2–4% of the GFP+ cells in the EF.RUNX1-ER/PGK.GFP lentivector-transduced cell culture containing 4HT expressed CD34 and there were similar percentages of CD45+ cells, whereas there were lower numbers of cells expressing these markers in the control cultures. Thus, enforced expression of RUNX1 boosted the numbers of CD45+ and CD34+ cells in EB derived from HESC.
Genetic Uncoupling of the Regulation of Hematopoietic Stem Cell Quiescence and Self-Renewal.
Authors: Liu Yan , Miyata Yasuhiko , Sashida Goro , Debalsio Anthony , Liu Yuhui , Koff Andrew , Nimer Stephen D. 
Published: April 2020

Abstract
It is usually stated that HSCs must choose to either self-renew or to differentiate and lose some of their multi potentiality. Recently, we demonstrated that MEF, an ETS family of transcription factor, played an important role in regulating HSC quiescence, illustrating a third choice for the HSC, namely to make an “active” choice and remain quiescent, without undergoing either self-renewal, or differentiation. MEF null HSCs are more quiescent than normal HSCs. In addition, MEF null mice exhibit greater numbers of hematopoietic stem cells and show resistance to chemotherapy and radiation. Little is known about the regulation of self-renewal vs. quiescence of HSCs, however the cdk inhibitor p21 has been implicated in regulating both HSC quiescence and proliferation. In the absence of p21, hematopoietic stem cell numbers are reported to be increased, but so is proliferation, leading to stem cell exhaustion. This implies that while p21 may maintain HSCs in their quiescent state, MEF functions to facilitate the entry of quiescent HSCs into the cycle, To investigate the potential opposing roles of MEF and p21 in HSC quiescence and self-renewal and to test whether the quiescent state of MEF null HSCs is dependent on the presence of p21, we have generated MEF / p21 double-knockout (DKO) mice. These mice are viable and born at normal mendelian frequency. MEF / p21 DKO mice have a higher than normal proportion of HSCs in the G0 phase, based on Pyronin Y/Hoechst staining and staining for the proliferation antigen Ki-67. Thus, the increased quiescence is not dependent on the presence of p21. However, by measuring LSK cells, we have observed a normal number of HSCs in the bone marrow of MEF / p21 DKO mice, in contrast to the increased number of HSCs in the bone marrow of MEF null mice. This suggests that the increased number of hematopoietic stem cells in MEF null mice is dependent on p21. Ongoing studies will further address the unique mechanisms that control HSC vs. stem cell expansion.
Genetic Deletion of Bcl-x Reveals a Potential Role in Governing Stem Cell Homeostasis.
Authors: Chyla Brenda J. , Harb Jason G. , Huettner Claudia S. 
Published: April 2020

Abstract
Germline deletion of bcl-x demonstrated the importance of this gene for hematopoiesis. Bcl-x deficient embryos failed to thrive due to massive apoptosis of immature hematopoietic and neural cells. Deletion of this gene in the adult has uncovered its requirement for the final maturation of erythroid cells, the transition of pre-B cells to the pro-B cell stage and recently, we showed an important role in the development of natural killer cells. While the role of bcl-x in hematopoietic stem cells (HSC) has yet to be addressed, a role for other members of anti-apoptotic bcl-2 family of proteins has been established. Overexpression of bcl-2 led to accumulation of HSC, whereas genetic deletion of mcl-1 resulted in ablation of HSC. In the present study, we determined by quantitative Real-Time PCR, that in addition to mcl-1, bcl-x is also highly expressed in hematopoietic stem cells, whereas the level of bcl-2 is threefold lower. Moreover, we found the expression of bcl-x to be down regulated with differentiation of HSC into progenitor populations (i.e. GMP, CMP and MEP). Therefore, we established a model to delete bcl-x in the stem cells of adult mice. We bred the Mx1-cre transgenic line with mice that carry the bcl-x gene flanked by loxP sites. Deletion of bcl-x occurred following administration of pIpC. A total of 10 bcl-x deficient mice and 7 age-matched control animals (Mxi-cre/wildtype bcl-x) were examined. Quantitative real-time PCR revealed a 55 – 95% reduction of bcl-x mRNA in Lin−, Sca-1+, c-kit+ cells 8 days post the first administration of pIpC. This population consists of phenotypically and functionally defined long-term HSC (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitors. We used FACS analysis to identify LT-HSC versus ST-HSC populations and to further assess the effects of bcl-x deletion. LT-HSC were defined by staining, for the following cell surface markers as Lin−, Sca-1+, c-kit+, Flt3−, CD34−; ST-HSC were characterized as Lin−, Sca-1+, c-kit+, Flt3−, CD34+. We found a 2 fold shift in the ratio of LT-HSC versus ST-HSC in bcl-x deficient mice compared to the ratio in control animals. This result raises the hypothesis that bcl-x regulates entry of HSC into the cell cycle. A notion that is supported by the observation that overexpression of bcl-x in fibroblasts delays transition from the Go/G1 to the S phase of the cell cycle. Future experiments will determine if the observed phenomenon is mediated through the anti-apoptotic function or cell cycle activity of the bcl-x protein.
NK Cells Contribute Significantly to the Innate Immune Effector Role of CD37-Specific SMIP in CLL and NHL.
Authors: Zhao Xiaobin B. , Trupti Joshi , Lapalombella Rosa , Cheney Carolyn , Gowda Aruna , Hayden-Ledbetter Martha S. , Baum Peter R. , Lin Thomas S. , Jarjoura David , Lehman Amy , Lee Robert J. , Caligiuri Michael A. , Tridandapani Susheela , Muthusamy Natarajan , Byrd John C. 
Published: April 2020

Abstract
CD37 is a lineage-specific B-cell antigen that represents an attractive target for immunotherapy in B-cell malignancies, especially in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL). CD37-specific small modular immuno pharmaceutical (CD37-SMIP™) drug is an engineered protein therapeutic directed to the CD37 antigen using a single chain variable region (scFv) linked to a modified human IgG1 hinge, CH2, and CH3 domains. We have previously presented that CD37-SMIP™ drug induces both ADCC and apoptosis against primary CLL cells and B-cell lymphoma cells and therapeutic efficacy was observed in a Raji cell disseminated leukemia xenograft model. Herein, we sought to determine the effector cell type(s) mediating ADCC and explore if agents that deplete NK cell inhibitory T-regulatory cells influence CD37-SMIP™ efficacy. Immunostaining of human peripheral blood mononuclear cells (PBMCs) from CLL patients demonstrated no expression of CD37 on CD3+ T cells, CD16+ or CD56+ NK cells, or CD64+ monocytes whereas CD37 was highly expressed on CD19+ B cells. Using purified human PBMCs as effector cells and Cr-51 labelled CLL B cells as targets, we found the CD37-SMIP™ dependent ADCC was predominantly mediated by NK cells, but not naïve or activated monocytes. Consistent with these in-vitro results, the in-vivo therapeutic efficacy of CD37-SMIP™ drug was significantly compromised by depletion of NK cells in the Raji cell disseminated leukemia xenograft SCID mouse model. The median survival time of CD37-SMIP™ treated mice decreased from 51 days (95% CI: 38, 78) to 27 days (95% CI: 25, 37) (p=0.017) with the depletion of NK cells. Consistent with previous studies, ADCC is not diminished by fludarabine, an agent that may deplete T-regulatory cells, suggesting that the anti-CD37 protein and fludarabine might combine for increased efficacy in-vivo. We therefore examined the effect of fludarabine on CD37-SMIP™ in-vitro apoptotic activity. These data demonstrate that direct cell death mediated by CD37-SMIP™ drug (5 ug/mL) synergizes with fludarabine-induced caspase-dependent apoptosis, as measured by both the MTT assay and annexin V/PI staining of CLL cells (combination index, CI=0.44). Overall, these data suggest that the CD37-SMIP™ is a promising therapeutic agent against CD37+ B-cell malignancies as either monotherapy or in combination with fludarabine. Further clinical development is warranted to investigate the effect of CD37-SMIP™ drug in CLL and NHL.
(SMIP trademark is owned by Trubion Pharmaceuticals).
Heme Oxygenase 1 Deficiency Compromises Stress Responses of Hematopoietic Stem Cells.
Authors: Cao Yu-An , Wagers Amy J. , Karsunky Holger , Zhao Hui , Reeves Robert , Wang Ronald J. , Stevenson David K. , Weissman Irving L. , Contag Christopher H. 
Published: April 2020

Abstract
Hematopoietic stem cells (HSCs) must be able to balance their self-renewal and differentiation activities in order to preserve their compartment in response to hematopoietic insults for efficient and life-long hematopoiesis while ensuring sufficient blood production to meet the increased hematopoietic demand. Mechanism(s) that regulate this balance during stress hematopoiesis remain to be fully understood. Heme oxygenase 1 (HO-1) is an important stress-inducible protein and a key enzyme of heme degradation that produces iron, bilirubin, and carbon monoxide (CO). CO is a gaseous regulator of cellular responses to a variety of insults. HO-1 deficiency results in reduced stress defense due to inadequate levels of its products. We report here that mice lacking one allele of HO-1 (HO-1−/+ mice) recovered more rapidly from myelotoxic injury and that sufficient HO-1−/+ bone marrow (BM) transplants engrafted lethally irradiated hosts with accelerated kinetics. These effects of HO-1 deficiency on regenerative hematopoiesis were associated with accelerated cell division of lineage-depleted (lin-) Sca-1+ cells. However, in other experimental settings, the converse can be seen in HO-1 deficiency. Repeated treatment of HO-1−/+ mice with 5-FU significantly reduced the number of HSCs in the marrow, relative to wild-type animals, and following adoptive transfer, the HO-1−/+ HSC compartment was smaller compared to that of HO-1+/+ in recipient animals. As a consequence, limited numbers of HO-1−/+ BM cells failed to provide adequate radio-protection of lethally irradiated recipients, and HO-1−/+ HSCs showed significantly compromised capacity to serially repopulate myeloablated hosts. We also noted that expression levels of the cyclin-dependent kinase inhibitor p21Cip/WAF1 were lower in lin- HO-1−/+ BM cells in contrast to that in HO-1+/+ cells under steady-state conditions, and that p38 mitogen-activated protein kinase (p38MAPK) was insufficiently activated in HO-1−/+ multipotent progenitors (MPPs) after heme challenge, compared to that in wild-type MPPs. We propose a model wherein HO-1 plays an intracellular regulatory and cyto-protective role in maintaining HSC compartment, especially during stress hematopoiesis. It follows that reduced levels of p38MAPK activation, presumably due to insufficient production of CO when HO-1 is deficient, result in accelerated proliferation of MPPs. This would potentially skew the balance between self-renewal and differentiation of HSC and deplete the primitive cell compartment, leading to ultimate hematopoietic exhaustion.
The Oral PKC-β Inhibitor Enzastaurin (LY317615) Suppress Phosphorylation and Induces Apoptosis in Multiple Myeloma Cell Lines by Inhibition of AKT Pathway.
Authors: Neri Antonino , Marmiroli Sandra , Tassone Pierfrancesco , Lombardi Luigia , Nobili Lucia , Verdelli Donata , Civallero Monica , Cosenza Maria , Bertacchini Jessika , Federico Massimo , De Pol Anto , Deliliers Giorgio Lambertenghi , Sacchi Stefano 
Published: April 2020

Abstract
The PKC pathway has been shown to play a role in the regulation of cell proliferation in several hematologic malignancies. In this study we tested the oral PKC-β inhibitor, Enzastaurin (LY317615 - Eli Lilly) for its therapeutic efficacy in Multiple Myeloma (MM). We first analyzed PKC-β I and II expression by Western blot in a panel of 19 human MM cell lines, showing that 9 cell lines express either 1 or both isoforms. We next examined the growth inhibition effect of Enzastaurin in the same panel of MM cell lines using either WST-1 or MTT assay and cell viability assessment by Tripan Blue exclusion. Eighteen cell lines have IC50 value ranging from 1,2 μM to 12,5 μM. To examine molecular mechanisms whereby Enzastaurin induces cytotoxicity, we performed cell cycle profiling using PI and observed a significant increase of the percentage of cells in the sub G0–G1 fraction. To determine whether Enzastaurin-induced cell death is mediated by apoptosis, we studied by ELISA and Western blot caspase 3 and PARP cleavage. We observed induction of caspase 3 and PARP cleavage in a dose and time dependent fashion. Notably, the broad caspase (Z-VAD-FMK) inhibitor reduced Enzastaurin-induced cytotoxicity. We next determined whether Enzastaurin could inhibit AKT phosphorylation in MM cell lines with constitutive phosphorylation of AKT.
Enzastaurin decreased AKT phosphorylation in a dose and time dependent fashion. Phosphorylation of GSK3β, a downstream target protein of AKT, was also markedly inhibited. Phosphorylation of PDK-1, a known upstream activator of AKT, was not affected by Enzastaurin.
In conclusion, our results indicate that Enzastaurin-induced cytotoxicity is mediated via activation of caspase. This effect is associated with significant inhibition of AKT activity and its downstream target GSK3 β. Enzastaurin does not alter the phosphorylation of the upstream AKT activator PDK-1. These data suggest that Enzastaurin inhibit AKT signalling pathway and support its evaluation in a murine model of human MM.