bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.
Authors: Tipping Alex J. , Pina Cristina , Castor Anders , Atzberger Ann , Hong Dengli , Lazzari Lorenza , May Gillian , Jacobsen Sten Eirik , Enver Tariq 
Published: April 2020

Abstract
Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.
Competing Influences of Genetic and Leukaemia-Specific Factors on P-Glycoprotein Expression in Blasts from 817 Patients Entered into the NCRN AML14 and 15 Trials.
Authors: Seedhouse Claire H. , Grundy Martin , White Paul , Li Yun , Fisher Janet , Yakunina Darya , Moorman Anthony , Russell Nigel , Burnett Alan , Pallis Monica 
Published: April 2020

Abstract
Background. P-glycoprotein (pgp), expressed on acute myeloid leukaemia (AML) blasts, is associated with failure to respond to chemotherapy in AML. This study aimed to determine whether expression and function of pgp may be linked to polymorphisms of the encoding gene (MDR1, also known as ABCB1) and whether leukaemia-specific changes in cell biology may override genetic factors in predicting pgp expression. G1199A, G2677T and C3435T polymorphisms in MDR1 (RFLP analysis) as well as pgp protein expression (using MRK-16) and function (modulation of R123 accumulation by PSC 833) were studied in leukaemic blast samples from 817 patients with AML entered into the NCRN AML14 and AML15 clinical trials.
Age, low white blood cell count (WBC), high bcl2, secondary AML and MDS and adverse cytogenetics all correlated with high pgp expression. However, ABCB1 3435TT homozygosity had a negative impact on pgp. Overall, pgp expression was high in 41% of the cohort, but in elderly patients high pgp expression was found in 54% with the 3435C allele versus 32% in the 3435TT group (P=0.009). This is similar to the frequency of pgp in younger patients (34%) and indicates that the 3435TT polymorphism affects pgp expression in elderly patients by preventing the upregulation of pgp protein normally seen in this group. No association was found between genetic factors and pgp function. In a subset of 341 patients, pgp protein levels were analysed on a quantitative scale in relation to C3435T and G2677T gene polymorphisms. The lowest protein expression occurred in the variant TT group for both polymorphisms. Differences were even more marked in the elderly subset (using Mann-Whitney test, P = 0.003 and 0.002 respectively). Linkage disequilibrium occurs between positions 2677 and 3435, and there was a highly significant association between the haplotype and pgp protein expression (P=0.001). ABCB1 mRNA was also measured in 81 patients and was found to be high in patients with the G1199A allele (median expression 0.36 ABCB1/b2M ratio v 0.06 in the GG group, P=0.04), suggesting that the 1199A polymorphism affects ABCB1. However no further correlations were found between the 1199A polymorphism and pgp protein or function. The association between pgp and a low white WBC, (P&lt;0.001) was further examined by cell cycle analysis in 40 patients confirming that pgp is associated with a low proportion of cycling cells - median % cycling cells: pgp negative/low 4.0%, pgp intermediate/high 1.0% (P=0.02).
Conclusions. We conclude that there is an extended MDR phenotype of indolent, pgp positive, bcl2-high cells in AML, particularly in the elderly, and that pgp is affected by an interaction between genetic factors and acquired, leukaemia-specific factors.
Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function.
Authors: Yarde Danielle N. , Hazlehurst Lori A. , Oliveira Vasco A. , Chen Qing , Dalton William S. 
Published: April 2020

Abstract
The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.
Anti-VEGF Antibody Treatment Markedly Inhibits Tumor Growth in SCID-hu Models of Human Multiple Myeloma.
Authors: Campbell Richard A. , Sanchez Eric , Chen Haiming , Turker Lauren , Trac Olivia , Li Mingjie , Shalitin Dror , Gordon Melinda S. , Pang Shen , Bonavida Benjamin , Said Jonathan , Plowman Gregory D. , Fuh Germaine , Berenson James R. 
Published: April 2020

Abstract
Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. Based on these findings, we evaluated a new mouse/human anti-VEGF antibody using our SCID-hu mouse models of human MM. Each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGκ-1A tumor fragment into the left superficial gluteal muscle. The tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into one of two treatment groups. In one group, anti-VEGF antibody was administered via intraperitoneal injection twice per week at a dose of 5 mg/kg. In the other cohort, control mice were given a control IgG antibody (5 mg/kg) on the same schedule. Mice receiving the anti-VEGF antibody showed marked inhibition of tumor growth (P = 0.0005) and reduction of paraprotein levels (P = 0.0002) compared to mice receiving control antibody. On day 42, LAGκ-1A-bearing mice receiving the anti-VEGF antibody showed a 70% reduction in human paraprotein levels and an 80% decrease in tumor volume compared the control antibody treated animals. Treatment with the anti-VEGF antibody was not associated with any observed toxicity. We are currently evaluating this anti-VEGF antibody in several of our mouse models of human MM and plasma cell leukemia. Based on these data with anti-VEGF monotherapy, we are currently investigating the anti-tumor activity of anti-VEGF antibody plus bortezomib as well as other available anti-MM agents using our in vivo SCID-hu myeloma murine models. Preliminary results are encouraging with single agent anti-VEGF antibody and additional studies may be used to direct the clinical development of anti-VEGF antibody treatment alone and in combination regimens for patients with relapsing or refractory MM.
Cytotoxic CD8 T Cell Immune Responses to Wilms Tumor Protein (WT-1) Characterizes Immunosuppression-Responsive Myelodysplasia (MDS).
Authors: Sloand Elaine M. , Rezvani Katayoun , Yong Agnes , Douek Daniel , Kurlander Roger , Price David , Barrett John , Young Neal S. 
Published: April 2020

Abstract
Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated immunologically-mediated pathophysiology in some patients. Indeed, among 129 patients treated with immunosuppression at NIH, trisomy 8 was associated with hematologic response to immunosuppression. We have demonstrated that trisomy 8 patients have oligoclonal CD8 T cell expansion, as determined by increased proportions of one or more T cell receptor (TCR) V beta subfamilies (Sloand et al. Blood106:841; 2005) which had cytotoxic activity toward trisomy 8 cell progenitors. Here we examine the response of cytotoxic T cells to cyclin D1 and Wilms tumor protein (WT-1); these candidate autoantigens had earlier been observed to be over-expressed in trisomy 8 CD34 cell in our microarray analysis (Chen et al. Blood 104:4210, 2004 ). Bone marrow mononuclear cells from 19 trisomy 8 patients (defined by cytogenetics and FISH) had significantly increased levels of WT-1 mRNA and protein expression compared to normal CD34 cells by real time PCR and immunoblot, respectively (p&lt;0.001). When patient and control CD8 cells were cultured in the presence of WT-1- and cyclin D1-loaded HLA-A2-restricted antigen-presenting cells (using three different HLA-A0201- restricted epitopes of WT-1 and four different epitopes for CD1), eight patients’ CD8 cell tested positive for interferon gamma, indicating a cytotoxic response. Staining with labeled MHC class I A2-restricted tetramers used to measure WT-1-specific cells showed concordant results in all eight patients. Tetramer staining was increased in 12 of 14 trisomy 8 patients examined. Cytotoxic T cells specific for WT-1 were present within the expanded V β subfamilies previously found to suppress trisomy 8 cell proliferation in vitro. CD8 cells from another eight MDS patients without trisomy 8 but who had responded to immunosuppression, sampled prior to treatment, also demonstrated WT1-specific cytotoxic activity, but only two of 18 non-responders showed measurable levels of WT-1-specific cytotoxic T cells (entire treated cohort seen in Fig 1). We infer from these data an autoimmune pathophysiology for trisomy 8 MDS with: 1) over-expression of WT-1 by the aneuploid clone; 2) a specific cytotoxic CD8 T cell immune response to WT-1; 3) subsequent cytotoxic T cell marrow suppression by apoptosis, either by cross reactivity or a bystander effect; and 4) improvement in hematopoiesis after immunosuppression. Other MDS which is responsive to immunosuppression may also be mediated by WT1.
Figure Figure
Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.
Authors: Janzen Viktor , Fleming Heather E. , Waring Michael T. , Milne Craig D. , Scadden David T. 
Published: April 2020

Abstract
The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.
Zfx Is an Essential and Specific Regulator of Hematopoietic Stem Cell Self-Renewal.
Authors: Reizis Boris , Harel Sivan , Galan Jose M. , Arenzana Teresita L. 
Published: April 2020

Abstract
Mammalian hematopoiesis is based on the activity of multipotent hematopoietic stem cells (HSC) that undergo continuous self-renewal throughout the adult life. The self-renewal represents a unique property of adult HSC, and appears distinct from the proliferation of hematopoietic progenitors or from the initial specification and expansion of embryonic HSC. Despite recent progress, little is known about the molecular mechanisms governing HSC self-renewal. Zfx is a broadly expressed zinc finger-containing transcriptional activator that is highly conserved in vertebrates. Using conditional gene targeting in mice, we demonstrate that Zfx is an essential and specific regulator of HSC self-renewal. The expression of Zfx was elevated in HSC compared to progenitors and differentiated cells. An inducible deletion of Zfx from the adult bone marrow (BM) resulted in the specific loss of the HSC population. The short-term proliferation and lodging of Zfx-deficient HSC in the BM were normal, suggesting a specific defect of long-term self-renewal. The deletion of Zfx in pre-established BM chimeras completely abrogated HSC maintenance as reflected by the rapid loss of HSC contribution to hematopoiesis. Furthermore, a constitutive pan-hematopoietic deletion of Zfx spared embryonic HSC in the fetal liver, yet resulted in the loss of adult BM HSC. In contrast, adult erythromyeloid progenitors or differentiated cells were not affected by the absence of Zfx. Genome-wide expression analysis identified candidate target genes of Zfx, some of which were controlled by Zfx in HSC but not in their differentiated progeny. Finally, several immediate-early and/or stress-inducible genes were upregulated specifically in Zfx-deficient HSC, suggesting that the latter undergo increased stress-related signaling. Thus, functional and gene expression analysis establishes Zfx as a novel specific regulator of adult HSC self-renewal. Further studies of the target genes and pathways controlled by Zfx should provide novel insights into the molecular basis of HSC function.
Acquisition of FOXP3 Expression by Human Effector CD4+ T Cells Is a Natural Consequence of Antigen Recognition.
Authors: Melenhorst J. Joseph , Scheinberg Phillip , Ambrozak David R. , Hensel Nancy F. , Douek Daniel C. , Price David A. , Barrett A. John 
Published: April 2020

Abstract
Recent data suggest that CD4+CD25+FOXP3+ regulatory T cells (TR) can be generated from CD4+CD25−FOXP3− T cells in the periphery. We studied the induction of CD4+CD25+FOXP3+ T cells during the course of immune responses to cytomegalovirus, tetanus toxoid, purified protein derivative and streptokinase. Peripheral blood mononuclear cells (PBMC) from healthy donors were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA) and stimulated with antigen for 6–7 days. Cells were stained for CD3, CD4, CD25, HLA-DR, CD38, and intracellular FOXP3. Responders to antigens had proliferating (CFDA[dim]) CD4+ T cells expressing CD25 and the activation markers CD38 and HLA-DR. In PBMC from five donors, a median of 37% (range 9–57%) of the proliferating fraction expressed FOXP3 (figure) which mostly co-expressed CD25, HLA-DR and CD38, suggesting that FOXP3 expression is the consequence of cellular activation triggered through the T cell receptor. Since all of these healthy subjects had circulating, pre-existing CD4+CD25+FOXP3+ T cells in the absence of ex vivo antigen stimulation, however, it was possible that these gave rise to the FOXP3+ responder cells. Antigen stimulation of PBMC from three donors aged 69–70 years with no detectable CD4+CD25+FOXP3+ T cells also induced proliferating activated CD4+CD25+FOXP3+ T cells, excluding the possibility that a pre-existing pool of FOXP3+ T cells gave rise to this population. Subsequently, we sorted CD4+ T cells that proliferated in response to antigen by flow cytometry on the basis of FOXP3 expression to sequence the T cell receptor-β CDR3 regions and to establish the T cell clonotype structure of proliferating FOXP3-positive and -negative T cells. These data also indicate that effector CD4+ T cells acquire FOXP3 expression during the course of an immune response. Based on these and previously reported findings, we propose that the acquisition of FOXP3 expression by effector CD4+ T cells is a natural consequence of antigen recognition that serves as a specific regulatory feedback system.
Induction of FOXP3 expression in proliferating CD4+ T cells in response to T cell receptor triggering Induction of FOXP3 expression in proliferating CD4+ T cells in response to T cell receptor triggering