bluuebunny/crossref_metadata_2025 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

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

[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

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

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

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

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

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

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

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.
p38-MAP Kinase Activation Followed by BIM Induction Is Critical for Glucocorticoid-Induced Apoptosis in Lymphoblastic Leukemia Cells.
Authors: Harada Hisashi , Lu Jianghua , Quearry Bonnie
Published: April 2020

Glucocorticoids (GC) are common components in chemotherapeutic protocols for lymphoid malignancies. GC-induced apoptosis requires the BCL-2 family-regulated pathway, but it remains largely undefined how the signaling pathways and their downstream target molecules, including the BCL-2 family members, participate in GC-induced cell death. BIM (BCL-2-interacting mediator of cell death) identified as a BH3-only protein induces apoptosis and its activity can be modulated at the transcriptional and post-translational levels. BIM is required for apoptosis induced by Taxol, dexamethasone, Gleevec, and HDAC inhibitors, indicating that this protein has a role in anticancer therapy against human malignancies. However, the molecular mechanisms how BIM is regulated and how BIM regulates downstream cell death machinery are not entirely clear.

In this study, we used a T cell acute lymphoblastic leukemia (T-ALL) cell line, CCRF-CEM to examine the molecular mechanisms of Dexamethasone (Dex)-induced cell death. Dex treatment induces the mRNA and protein of BIM in CCRF-CEM cells before the onset of apoptosis. Bim mRNA induction is completely inhibited by a transcription inhibitor, actinomycin D. However, a protein synthesis inhibitor, cycloheximide treatment augments Dex-induced Bim mRNA accumulation (super-induction), suggesting that de novo protein synthesis is not required for the induction of Bim mRNA. BIM protein induction is inhibited by actinomycin D or cycloheximide, thus new BIM protein synthesis is required for its accumulation. These data indicate that Dex treatment increases BIM levels mainly by new mRNA synthesis (transcription). In contrast, the overall expression of the multi-domain pro-apoptotic effectors, BAX and BAK, are constant. The anti-apoptotic members, BCL-2, MCL-1 and BCL-XL, also show constant expression. Down-regulation of BIM by shRNA strongly inhibits Dex-induced apoptosis. Thus, BIM is an essential molecule in Dex-induced apoptosis in CCRF-CEM cells. We then investigated the upstream signaling pathways to induce BIM. p38-mitogen activated protein kinase (p38-MAPK) is activated by Dex treatment prior to BIM induction. Treatment with a p38-MAPK inhibitor, SB203580, dramatically reduces Dex-induced apoptosis as well as BIM induction.

These findings indicate that BIM induction through p38-MAPK activation is a critical pathway in GC-induced cell death.
Novel Functions for a Fibrinolytic Pathway in Controlling the Stem Cell Niche.
Authors: Akiyama Haruyo , Lund Leif R. , Morita Yohei , Nakauchi Hiromitsu , Ogawa Hideoki , Okumura Ko , Dano Keld , Werb Zena , Heissig Beate , Hattori Koichi
Published: April 2020

Hematopoietic stem cells (HSCs) reside within specific niches, where they are maintained by self-renewal and can be mobilized into circulation, but the underlying mechanisms are still unknown. The hematopoietic niche comprises of a local network of stromal cells (like fibroblasts, endothelial cells), accessory cells (T lymphocytes, monocytes), their products (extracellular matrix (ECM) and cytokines, capable of influencing self-renewal, proliferation and differentiation of HSCs. One such ECM molecule is fibrin, which can be found along surfaces in the bone marrow. Its precursor fibrinogen can maintain hematopoiesis in long-term bone marrow liquid cultures and increase the colony size of bone marrow cells cultured in semisolid medium containing serum. Plasmin, a serine protease, proteolytically degrades fibrin and fibrinogen. Plasminogen (Plg), its inactive proenzyme is present in the plasma, interstitial fluid or adherent to ECM proteins and can be activated by tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and other serine proteasesWe demonstrated previously that matrix metalloproteinase-9 (MMP-9) controls stem cell recruitment from the bone marrow niche. During fibrinolysis plasmin participates in the consecutive activation of MMPs. We initially wished to determine if this fibrinolytic cascade might be involved in regulating the stem cell niche. In this study we provide novel mechanistic data demonstrating that classical fibrinolytic factors-Plg/Plasmin are involved in recruitment of HSCs into the cell cycle following myelosuppression and their mobilization from the BM niche influencing cell differentiation, proliferation and migration by activating niche cells to secrete typical niche factors, including Kit-ligand and stromal cell-derived factor-1 (SDF-1, CXCL12). Deletion of Plg in mice prevented hematopoietic stem cells from entering the cell cycle and undergoing differentiation after myelosuppression, leading to the death of the mice. By Plg-dependent and Plg-independent pathways, tissue-type plasminogen activator activated matrix metalloproteinases and released Kit ligand and stromal cell-derived factor-1 from niche cells, thereby promoting stem cell recruitment and proliferation. We conclude that the fibrinolytic cascade plays a critical role in controlling the exit from the stem cell niche and that this represents a general stem cell regulatory axis.
The Hematopoietic Supportive Potential of Human Mesenchymal Stromal Cells Is Associated with Expression of Cadherins.
Authors: Roderburg Christoph , Diehlmann Anke , Wein Frederik , Faber Anne , Krause Ulf , Frankhauser Maria , Seckinger Anja , Ho Anthony D. , Wagner Wolfgang
Published: April 2020

Self renewal and differentiation of hematopoietic stem cells (HSC) are governed by interaction with the supportive microenvironment of the bone marrow. Secreted factors as well as specific cell adhesion proteins are involved in this interaction. As an in vitro model system, the hematopoietic microenvironment can be mimicked by supportive mesenchymal stromal cells (MSC).

We have compared the supportive potential of human MSC from bone marrow (BM) isolated under two different culture conditions (BM-MSC-M1 and BM-MSC-M2), from adipose tissue (AT) and umbilical cord blood (CB) that were all cultivated as described before (Wagner et al. Exp Hematol.2005;11:1402–1416.). As controls we have used the human fibroblast cell line HS68 and the murine fetal liver cell line AFT024. CD34+ cells were isolated from human cord blood and cultured in direct contact with irradiated stromal cells. After four, seven and twelve days the immunophenotype of the hematopoietic cells was analyzed by flow cytometry. Many progenitor cells cultured on BM-MSC or AFT024 maintained a primitive phenotype of CD34+/CD38- cells whereas the proportion of these cells was reduced upon cultivation with CB-MSC and cells cultured on AT-MSC and HS68 displayed a significantly higher expression of CD38 and lower expression of CD34. Furthermore, long term culture initiating-cell (LTC-IC) assays were performed on the different feeder layer. LTC-IC frequency was significantly higher on BM-MSC that were isolated under the two different culture conditions (BM-MSC-M1 1,15 ±0.11%; BM-MSC-M2 1.14±0.08%) and on CB-MSC (1.10±0.13%) compared to AT-MSC (0.32±0.09%) and HS68 (0.67±0.12%). We have compared gene expression profiles of BM-MSC-M1, BM-MSC-M2, CB-MSC, AT-MSC and HS68 by cDNA microarray analysis (51,144 different cDNA clones of the RZPD3 Unigene Set). Differential expression of various genes correlated with the observed differences in supportive potential. Among these were adhesion proteins like N-cadherin, cadherin11, fibronectin1, various integrins (ITGA1, ITGA5 and ITGB1) and VCAM1 as well as secreted proteins including osteonectin, CTGF and SDF1. Westerblot analysis verified on protein level that cadherin11, N-cadherin, and ITGB1 were highly expressed on BM-MSC as compared to AT-MSC and HS68 fibroblasts.

In conclusion MSC from human bone marrow or from umbilical cord blood support to a significantly higher degree the maintenance and proliferation of primitive hematopoietic progenitors than MSC derived from adipose tissue. This affinity correlated with up-regulation of cadherin11, N-cadherin and intergrin-beta1 on BM-MSC and CB-MSC.
Identification of Meis1 Target Genes Involved in the Induction of AML in Collaboration with NUP98-HOXD13.
Authors: Palmqvist Lars , Argiropoulos Bob , Humphries R. Keith
Published: April 2020

Meis1, a homeodomain-containing HOX cofactor, cooperates with multiple native and NUP98-HOX fusion proteins to accelerate the onset of AML. We have recently shown that enforced overexpression of Meis1 in NUP98-HOX transduced bone marrow (bm) cells leads to a marked elevation in the tyrosine receptor molecule, Flt3 transcription and subsequent activation of Flt3 signaling pathways (Blood. Aug 1, 2006). Furthermore, overexpression of wildtype Flt3 is sufficient to collaborate with NUP98-HOX fusions to induce AML in mice, albeit with a longer disease latency than NUP98-HOX+Meis1-mediated AML. To further study the molecular mechanisms underlying Meis1’s strong pro-leukemic effect together with HOX proteins, we conducted a structure-function analysis of Meis1 in the context of NUP98-HOXD13 (ND13) leukemogenesis. We show that the homeodomain (ΔHD, amino acids 272–335) is required for Meis1 collaboration with ND13 while the N-terminal domain (ΔN, amino acids 1–67) is dispensable for leukemogenesis but decelerates the disease onset. Interestingly, primary bm cells transduced with ND13-Meis1ΔHD or ND13-Meis1ΔN revealed no significant upregulation of Flt3 mRNA levels in either case. These results suggest that Meis1 triggers additional Flt3-independent pathways to accelerate leukemia development. Thus, the retained leukemogenic properties of ND13-Meis1ΔN and the longer latency observed for NUP98-HOX fusions and Flt3 overexpression to induce leukemia point to additional roles for Meis1 in induction of leukemia independent of its ability to upregulate Flt3. To search for these additional Meis1-induced leukemic pathway(s), we used the Affymetrix GeneChip MOE430 to compare the gene expression profiles of bm cells transduced with ND13, ND13+Meis1, ND13+Meis1ΔHD and Meis1ΔN. Interestingly, the gene array results indicate that only a relatively small number of genes (72 increased and 64 decreased) differed significantly between the non-leukemic Meis1ΔHD and the leukemic Meis1 bm cells and these genes are all strong candidates as to be direct Meis1 target genes. A significant number of these genes are involved in cytokine receptor pathways as revealed by gene ontology analysis and, furthermore, promoter analysis revealed the presence of putative Hox, Meis1 and Pbx binding sites in several of these. The gene array results also indicate that only a small fraction of genes differed significantly between the non-leukemic Meis1ΔHD and the leukemic Meis1ΔN bm cells suggesting that these genes could be involved in Flt3-independent Meis1 leukemogenic activity.
The Groucho Corepressors, TLE1 and TLE4, Are Candidate Del(9q) AML Tumor Suppressor Genes That Can Complement AML1-ETO.
Authors: Sweetser David A. , Dayyani Farshid , Wang Jianfeng , Zhang Yuntian , Zaidi Tanweer
Published: April 2020

Deletion of the long arm of chromosome 9, del(9q), is one of the most common cytogenetic abnormalities seen with t(8;21) AML. This close association suggests that that the loss of a critical gene(s) on chr9q cooperates with the AML1-ETO (RUNX1-MTG8) fusion gene produced by t(8;21) in leukemogenesis. We recently mapped the commonly deleted region of del(9q) AML to less than 2.4 Mb and identified all the genes mapping to this region. Extensive sequence analysis of these genes failed to reveal obvious mutations in del(9q) AML samples although the expression of a number of these genes appeared low specifically in del(9q) samples (Genes Chr and Ca 44, 279–291). To help determine the critical affected genes in the del(9q) CDR we used an siRNA expression library directed against all del(9q) CDR gene transcripts in an in vitro complementation assay with AML1-ETO. Inducible expression of AML1-ETO in U937 cells (U937T-A/E, Mol Cell Biol 21, 5577–5590) leads to cell cycle arrest and apoptosis. We introduced the del(9q) CDR siRNA library into U937T-A/E cells with the goal of finding siRNAs that rescued these cells after induction of AML1-ETO expression. The most commonly recovered siRNA using flanking PCR primers was directed against TLE1. Transducin-like enhancer of split (TLE) 1 and TLE4, are two members of the Groucho family of co-repressors that map adjacent to the del(9q) CDR. We subsequently demonstrated that knockdown of either TLE1 or TLE4 with specific siRNAs were able to overcome the induced cell cycle arrest in U937T-A/E cells. We further demonstrate that knockdown of either TLE in the AML1-ETO+ Kasumi-1 cell line increased cell division, increased expression of cyclin D1 and Ki67 and reduced the G0/G1 fraction, while expression of TLE1 or TLE4 in Kasumi-1 cells induced cell cycle arrest and apoptosis. Understanding how various mutations work cooperatively to produce a malignant phenotype is one of the great challenges in oncology. The TLEs are known to inhibit NFKB as well as Wnt signaling, two pathways implicated leukemic stem cell expansion. TLE inhibition may represent an important cooperating mutation with AML11-ETO in AML that links Wnt signaling and core binding protein transcription factors. Studies are currently underway to demonstrate this cooperativity and more directly evaluate the role of TLE inhibition in leukemogenesis.
Ceramide Kinase Promotes Store Dependent Ca2+ Signals and Enhances Phagolysosomal Fusion in COS-1 Cells.
Authors: Hinkovska-Galcheva Vania T. , Clark Andrea J. , Kindzelskii Andrei L. , VanWay Susan M. , Huang JiBiao , Shanley Thomas P. , Shayman James A. , Petty Howard R. , Boxer Laurence A.
Published: April 2020

We showed that ceramide-1-phosphate (C1P) is formed by ceramide kinase (CERK). In this study the mechanism by which C1P enhanced phagocytosis and phagolysosomal formation in COS-1 cells expressing hCERK was evaluated. We hypothesized that CERK has a unique role in inducing Ca2+ signaling during Fc-mediated phagocytosis via its generation of C1P. Furthermore, we also hypothesize that CERK mediated changes in Ca2+ levels were not direct but required the participation of a store operated channels (SOC). In some systems, evidence suggests that SOC might be related to transient receptor potential channel (TRP) homologues. This hypothesis was tested using cell lines with different levels of expression of CERK and by using series of pharmacological inhibitors. To monitor subcellular localization of hCERK, red fluorescent protein (RFP)-tagged hCERK was created and confocal microscopy was performed. To study dynamic changes in Ca2+ during phagocytosis, imaging microscopy was used. Phagolysosome formation was evaluated by labeling the cells with lysotracker red DND-99 at 37oC and followed by imaging. When challenged with EIgG, hCERK transfected cells increased phagocytosis and simultaneously increased C1P. During subcellular fractionation of hCERK transfected cells, CERK translocates during activation from the cytosol to a lipid raft fraction. We also observed that TRP-1 accumulated at the site of CERK translocation in lipid rafts. Microfluorimetry of indo-1 revealed that Ca2+ oscillations in COS-1 cells, stably transfected with Fcγ RIIA/hCERK, were much higher compared to Fcγ RIIA transfected cells. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. hCERK transfected cells had a mean rate of fusion of 90%, compare to 76% ( catalytic inactive mutant of CERK-G198DhCERK), 65% (Fcγ RIIA), and 70% (Vector) transfected cells. The indo-1 intensity spikes demonstrated significantly higher Ca2+ intensities for phagosomes associated with the hCERK transfectants. The SOC inhibitor SKF96365 blocked enhanced Ca2+ signaling in the hCERK transfected cells. Similarly SOC inhibitors reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. Our data showed that there is a co-localization of EIgG and CERK as well as caveolin-1 and CERK on the surface of the phagocytic cells. Site directed mutagenesis demonstrated that the kinase activity of hCERK was required for higher Ca2+ signals. Pharmacological inhibitors revealed that this Ca2+ signal operates through SOC. Our results show that introduction of the hCERK gene in cells alters the functional behavior of intracellular signals and supports the role of C1P in promoting membrane fusion by modulating Ca2+ signaling through activation of SOC
The Gene Locus Distribution of HLA in the Chinese Population by High Resolution Typing of HLA-A,B,C,DR,DQ and Its Effect on Stem Cell Transplantation.
Authors: Hou Jian Q. , He Jun , Chen Zi X. , Bao Xiao J. , Yao Li , Di Wen Y. , Zhang Hui , Zhang Jian
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (AHSCT)can potentially cure a variety of hematopoietic and congenital metabolic disorders. Ideally, one would hope to find identical unrelated donors who are genotypically identical to the patients at all HLA loci. To explore distribution of HLA gene subsites in the Chinese population and its effect on AHSCT. High resolution typing of HLA-A,B,Cw,DR,DQ were performed on 101 cases from the data base of China Marrow Donor Program (CMDP) donors and 76 patients (ALL 23cases, CML 32cases, AML 23cases)by using PCR-SSP and PCR-SSO. The HLA subsites with highest gene frequency were A*0201, A*0207, A*1101, A*2402, B*4001,B*4601, C*0102, C*0702, DR*0901, DR*1202,DQ*0301and DQ*0303(PF>0.2). The subsites with secondary high frequency were A*3101,A*3303,B*1301,B*1302,B*1501, B*5101,B*5801,C*0303,C*0304,C*0602,C*0801, DR*0405, DR*0406,DR*0701,DR*0803, DR*1501,DQ*0202,DQ*0302,DQ*0401, DQ*0502, DQ*0601(PF>0.1). The phenotypic frequency of A*1101,B*4601,C*0102,DR*0901, DQ*0303 in the Chinese population was as high as 0.38, Among which B*4601and DR*0901 were the single allele with highest frequency. The gene locus in HLA with linkage disequilibrium were A*0207-B*4601- DR*0901-C*0102-DQ*0301or0303;A*0201-B*4601-DR*0405 or DR*0803- C*0102-DQ*0302orDQ*0502;A*3001-B*1302-DR*0701-C*0602-DQ*0202 and A*3303- B*5801-DR*0901-C*0302-DQ*0303 or DQ*0302, respectively. CMDP demonstrated the effect of HLA-A and B allele matching on the development of severe acute GVHD and on the overall survival rate. HLA-DR matching are less effective than classIfor clinical outcomes. Additionally, the HLA-C mismatch was found to have a synergistic effect on the acute GVHD and survival rate when another HLA locus mismatch coexisted. Our results demonstrated that the frequency distribution of HLA gene subsites is important for selecting more suitably matched donor for HSCT and is helpful for making donor recruitment strategy. As the HLA-Cw played important role on aGVHD occurrence and the failure of BMT, more attention should be paied on HLA-Cw allele in clinical HSCT.
The Role of Rab38 in Rat and Human Platelet Function Defects.
Authors: Datta Yvonne H. , Rangel-Filho Artur , Wu Francisca C. , Stephens Kenyatta W.
Published: April 2020

The fawn-hooded hypertensive (FHH) rat is a model of a moderate platelet function defect manifest by severely prolonged tail bleeding times, decreased platelet aggregation to ADP and collagen, and decreased ADP release from dense granules. The FHH rat has a mutation at the ATG start site of the gene encoding the small GTP-binding protein Rab38, which we have identified as being strongly associated with the bleeding phenotype. We have further characterized expression of Rab38 in the FHH rat and in humans. As assessed by reverse transcriptase PCR, we found that Rab38 is expressed in a wide variety of tissues from FHH and control Brown-Norway (BN) rats. However, by western blotting there was no protein expression of Rab38 in the FHH tissues, confirming that the mutation results in a severe defect at the protein level. Confocal immunofluorescence microscopy demonstrated Rab38 localized in punctuate cytoplasmic granules in human megakaryocytic cell lines. The Rab38 staining colocalized with von Willebrand factor (VWF) found in alpha-granules. However, we did not demonstrate any significant colocalization of Rab38 with serotonin, a marker of dense granules or Lamp-1, a marker of lysosomes, in the megakaryocytic cell lines. We also demonstrated colocalization of Rab38 with VWF in normal human platelets. We hypothesized that some humans with mild to moderate platelet dysfunction may have abnormalities of Rab38. We sequenced the entire coding region and intron-exon boundaries of the Rab38 gene in six patient samples collected at Emory University for the CDC Women with Bleeding Disorders and Menorrhagia Study. Four of these patients had detectable platelet aggregation abnormalities and two did not. In this limited initial study, no obvious mutations or new polymorphisms were detected in the Rab38 gene. Numerous known polymorphisms were detected, but no clear pattern was associated with the platelet aggregation abnormalities as of yet. These results along with our previous findings in the FHH rat suggest that Rab38 may be involved in a general secretion mechanism in platelets. It remains to be seen whether mutations in the Rab38 gene account for some of the platelet function defects seen in humans, but we are actively pursuing this hypothesis.
Blood Platelets Organize Pro- and Anti-Angiogenic Factors into Separate, Distinct Alpha Granules: Implications for the Regulation of Angiogenesis.
Authors: Italiano Joseph , Richardson Jennifer L. , Folkman Judah , Klement Giannoula
Published: April 2020

In addition to contributing to hemostasis, platelets are increasingly being viewed as major contributors to wound healing and tumor growth. This can be attributed to the many angiogenesis regulatory proteins selectively taken up and stored within the alpha granules of platelets and deposited in the local environment of a tumor or wound upon platelet aggregation and adhesion. Platelets contain both pro and anti-angiogenesis regulators, and the mechanisms by which they modulate angiogenesis are unclear. To address this question, we examined the localization of both pro-and anti-angiogenic factors in platelets and megakaryocytes using immunostaining and confocal microscopy. Double immunofluorescence microscopy revealed that VEGF (an angiogenesis stimulator) and endostatin (an inhibitor), are localized in separate and distinct alpha granules. Immunofluorescence for thrombospondin 1, basic FGF, platelet factor 4, and placental growth factor subsequently confirmed the separate and distinct alpha granules. These observations, in combination with a recent report that proteinase-activated receptors (PARs) selectively modulate the release of angiogenesis regulators from platelets (Ma et al., PNAS 102:216), motivated the hypothesis that these distinct populations of alpha granules could undergo selective release as well. To investigate whether PARs facilitate differential release of alpha granules, we treated human platelets with PAR agonists or antagonists and assayed for differential alpha granule release. The selective PAR1 agonist stimulated the release of endostatin-containing alpha granules and suppressed the release of VEGF-containing alpha granules. Furthermore, confocal microscopy studies revealed that in the presence of the PAR1 antagonist (SCH79797), thrombin stimulated the release of endostatin-containing alpha granules, but not VEGF-containing granules. These observations suggest that platelets may be organizing angiogenesis regulatory proteins into pharmacologically and morphologically distinct populations of alpha granules, which are then susceptible to differential regulation upon platelet activation.