bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heightened Activation of the p53-ATM Axis in Fanconi Anemia Mutant-Derived Primary Cells: Implications for Carcinogenesis.
Authors: Yamamoto Kazuhiko , Ouchi Toru , Liu Johnson M. 
Published: April 2020

Abstract
Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects and cancer, particularly leukemia and squamous cell carcinomas involving the cervix or head and neck. Cancer predisposition suggests that at least one function of the FA gene products is tumor suppression, however, the molecular basis for this is unclear. FA gene products promote chromosome integrity, and p53-mediated apoptosis is known to provide a crucial tumor suppressive mechanism to eliminate cells susceptible to genomic instability. Consequently, we have sought to analyze the integrity of the p53-ATM axis in primary (untransformed) fibroblasts derived from FA-A and FA-C patients or Fanca-null mouse embryonic fibroblasts (MEFs). We found that in FANCA (two different) or FANCC-mutant primary fibroblasts, p53 expression, Ser15 phosphorylation of p53, ATM expression and phosphorylation were all upregulated following irradiation or mitomycin C treatment, when compared to identically treated isogenic mutant cells transduced with wild-type FANCA or FANCC. ATM is activated in response to DNA double-strand breaks, but the sequence and mechanism of its activation are only partially understood. Checkpoint kinase-2 (CHK2) expression and phosphorylation were not significantly different, nor were there consistent differences in putative upstream regulators of ATM activation such as MRE11/RAD50/NBS1, 53BP1, MDC1, or TRF2. To extend our analysis of the p53 axis to primary murine cells, we first transduced human p53 into Fanca-null MEFs, with the intent of creating cells with defined basal levels of p53 (since endogenous murine p53 was nearly absent in the MEFs). As in the human patient fibroblasts, p53 expression and Ser15 phosphorylation of p53 were markedly upregulated in Fanca −/− MEFs transduced with human p53 when compared to Fanca +/+ MEFs transduced with human p53. These latter experiments also imply that upregulation of p53 levels may be due to enhanced protein stability rather than transcription. In previous work, we had reported that FANCC-mutant primary fibroblasts transduced with wild-type FANCC were at least 10-fold less prone (than parental cells) to form SV40 virus-induced transformation foci. By contrast, our present data, which indicate p53-ATM upregulation in mutant cells, suggest intact tumor suppressor proapoptotic pathways in mutant cells responding to DNA damage. Acquired functional (qualitative) p53 defects or defects in p53-independent mechanisms of tumor suppression may therefore underlie carcinogenesis in FA.
Clinical Scale Expansion of Human Pluripotent Stem Cells.
Authors: Perry Robert , Miller David , Deans Robert 
Published: April 2020

Abstract
Research studies of adherent stem cell populations, primarily isolated from bone marrow but also cord blood, adipose and other tissues, have shown cultures capable of regenerating more than one primitive germ layer in vitro or in vivo. This raises exciting possibilities for tissue repair in acute injury or degenerative disease, provided scalable processes for cell expansion can be developed. We have optimized an expansion process based on the Multi-potent Adult Progenitor Cell (MAPC) platform first described by Dr. C. Verfaillie. Termed Multistem, these adherent stem cell cultures have the capacity to differentiate into many different primary tissue types including but not limited to endothelial, hepatic, neuronal, adipogenic, and osteogenic lineages cells. Three fresh marrow aspirates were obtained from healthy donors and the mononuclear cells isolated using the COBE 2991 Cell Processor. Once the mononuclear cells were isolated, they were subsequently placed into Nunc cell factories that had been coated with 10 ng/ml Fibronectin A (Day 0). Serum containing media supplemented with PDGF and EGF was used, with cells incubated under low oxygen tension (5%). Cells were expanded to Day six, and then every two to three days until greater than 100 million cells were obtained (routinely Day 15–17). The growth rate was linear through approximately 24 population doublings, (yielding a theoretical 1011 cells) with a doubling rate of 24–30 hours. Release criteria qualifying successfully expansion to clinical scale include sterility, identity/purity, and tissue lineage potency. In addition, the cells were analyzed at approximately population doubling 20 for cytogenetic abnormalities and out of 20 spreads, 0 abnormal cells were detected for each of the three samples. All cultures tested positive by qPCR for expression of Oct4, a marker of pluripotency in ES cell systems. Telomerase levels were analyzed using a TRAP QPCR assay and the levels ranged from between 15 and 22 times higher than that measured in commercially purchased mesenchymal stem cells (MSC) at about population doubling 20 FACS analysis was performed on cells expanded using these conditions, with all cultures being positive for CD49c, CD90 and negative for CD45 and HLA class II surface markers. Current process improvements have allowed for the growth of human cells out to greater than 60 population doublings. These results demonstrate that large numbers of human stem and progenitor cells can be obtained using these culturing techniques, and that the cell products are consistent between donors. Donor ages ranged between 19 and 57. This is the first report of a scalable and consistent process for expansion of human stem cell cultures with pluripotent lineage capacity.
Isolation and Characterization of Umbilical Cord Blood-Derived Multipotent Stem Cells Arising from Adherent CD45+/CD34+ Cell Subset.
Authors: McKenna David H. , Adams Sheryl , Emrick Eileen , Sprague Stacey , Tigges Barbara M. , Collins Daniel P. 
Published: April 2020

Abstract
Introduction: Using a cell separation medium (PrepaCyte-MLPC, BioE, Inc.) and plastic adherence, umbilical cord blood (UCB)-derived multipotent stem cells were isolated, expanded, and characterized. These stem cells, termed multi-lineage progenitor cells (MLPCs), are the only such cell type from any source demonstrated to be CD45+/CD34+ upon initial isolation.
Methods: PrepaCyte-MLPC, an antibody-based cell separation medium, is added to an UCB unit (American Red Cross Cord Blood Program). Following homo- and heterophilic aggregation of undesired cell populations and subsequent sedimentation to gravity, the supernatant containing stem cells is expressed. After overnight incubation in a T-flask in MSCGM (Cambrex, Inc.), non-adherent cells are washed, leaving adherent cells to expand in culture. As MLPC colonies are observed, cells are further enriched by detachment (PBS/0.1% EGTA) and transfer to a new T-flask.
Results: MLPCs were isolated and expanded from 3 of 10 UCB units. Cells were initially positive for CD45, CD34, CD133, SSEA-3, SSEA-4, and several MSC markers. Several days into culture, expression of HSC/ESC markers was lost. MSC marker expression was maintained; cells remain positive for CD9, CD13, CD29, CD44, CD73, CD90, and CD105. These MLPC lines have been cultured through at least 16 passages with no apparent impact on expansion or differentiation potential. Cell lines have been successfully differentiated into cell types representative of each embryonic layer (i.e., adipocytes, osteocytes, myocytes, hepatocytes, and endothelial and neural cells). Additional experiments and limited-dilution cloning are underway, and further studies (cytogentic/molecular and animal teratoma studies) are planned.
Conclusions: UCB-derived MLPCs were isolated as a rare population of adherent, initially CD45+/CD34+ cells with lymphoid morphology. Following successful expansion in culture, multi-potency was demonstrated. MLPCs may serve a role in bone marrow transplantation (for malignant and non-malignant disease) and regenerative medicine; clinical studies are in an early phase of development.
Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.
Authors: Takebe Naoko , MacVittie Thomas , Cheng Xiangfei , Farese Ann M. , Welty Emily , Meisenberg Barry 
Published: April 2020

Abstract
Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.
Percoll Gradient Separation of Cord Blood Mononuclear Cells Reveals the Presence of a Novel Population of CXCR4+ Oct-4+ Small Embryonic-Like Stem Cells.
Authors: Kucia Magda , Oldak Krzysztof , Ratajczak Mariusz Z. , Ratajczak Janina , Pojda Zygmunt 
Published: April 2020

Abstract
Cord blood (CB) mononuclear cells (MNC) were demonstrated to contribute to organ/tissue regeneration, however, the identity of the specific cell type(s) involved remains unknown. Recently, it had been identified in adult bone marrow (BM) a mobile, SDF-1-, HGF/SF- and LIF-responsive population of CXCR4+ non-hematopoietic MNC that expresses markers (RQ-PCR, immunhistochemistry) of early pluripotent/tissue committed stem cells (TCSC) for skeletal muscles, heart, neural tissue, liver, pancreas, epidermis and intestinal epithelium (Leukemia2004:18;29–40). We hypothesized that a similar population of these rare cells may also be present in CB, however, their final yield may depend on the method of MNC preparation. We hypothesized that since these cells are very small (~3–5 μm), they may cross a Ficoll-Paque gradient or sedimentate more rapidly than other CB MNC and as result of this are lost during routine CB preparations. Thus, taking in consideration their small size, in order to evaluate if these cells are present in CB, we isolated small CB MNC by employing Percoll gradient (1.078–1.095). We found that this allows us to isolate from CB a population of small cells (2.5% of the total number of MNC) that is enriched in a population of TCSC/PSC(~0.002% of MNC) that we have originally identified in BM. Accordingly, these CB-derived TCSC/PSC CXCR4+ cells are very small (~3μm), posses large nuclei that contain embryonic stem cell type open chromatin (euchromatin), and express several markers for skeletal muscles, heart, neural tissue, liver, pancreas, epidermis and intestinal epithelium as well as pluripotent stem cell (PSC) transcription factors such as Oct-4, Nanog and Rex-1. In vitro cultures of CB-derived small TCSC/PSC are able to grow neurospheres that gave rise to neuronal (β-III tubulin+, nestin+) and macroglia (O4+, MBP+, GFAP+) lineages and cardiomyocytes (β-myosin heavy chain+, α-sarcomeric actin+). Based on this we conclude that CB contains embryonic-like stem cells which may be lost during routine procedures to isolate MNC. Thus, Percoll gradient centrifugation allows for optimal isolation of these small CXCR4+ PSC/TCSC and we postulate that the CB tissue/organ regenerating potential may be much higher than initially postulated and we are currently testing this hypothesis in vivo in animal models.
Low Baseline Intrinsic Sensitivity to Imatinib (High IC50) in CML Patients Is Due to Reduced Oct-1 Mediated Influx. Intrinsic Sensitivity to AMN107 Does Not Correlate with That of Imatinib and Uptake of AMN107 Is Not Oct-1 Mediated.
Authors: White Deborah L. , Saunders Verity A. , Engler Jane , Dang Phuong , Zannettino Andrew C.W. , Cambareri Antony , Quinn Steven , Manley Paul , Hughes Timothy P. 
Published: April 2020

Abstract
We have recently demonstrated that there is variation in the intrinsic sensitivity of de novo CML patients to imatinib. Patients with low intrinsic sensitivity to imatinib, have a lower probability of achieving major molecular response by 12 months. Intrinsic sensitivity was determined pre-therapy using western blot, with the IC50imatinib defined as the dose of drug required in vitro to reduce phosphorylation of the adaptor protein Crkl (p-Crkl) by 50%. In the current study we show that the observed variation in IC50imatinib between patients can be explained by the intracellular concentration of imatinib. In 16 de novo CML patients the IC50imatinib strongly correlated (R2 = −0.6, p = 0.002) with the intracellular concentration of 14C-labelled imatinib after 2 hours exposure at 37°C. In keeping with the notion that the organic cation transporter Oct-1 is responsible for imatinib influx, the addition of Prazosin, an inhibitor of Oct-1, to 14C imatinib experiments greatly reduced the inter-patient variation in intracellular concentration.
Intracellular drug concentration. Median IC50imatinib uM [imatinib] in-vitro * [imatinib] in-vitro* + Prazosin Median IC50AMN107 uM [AMN107] in-vitro * [AMN107] in-vitro* + Prazosin * ng of intracellular imatinib or AMN107 per 200000 cells when 2uM drug added in vitro. low IC50 imatinib (n=8) 0.5 28.4 6.9 0.035 28.6 27.4 p&lt;0.001 p&gt;0.05 high IC50 imatinib (n=8) 1.375 14.8 8.4 0.04 28.5 27.6 p value &lt;0.001 0.04 &gt;0.05 &gt;0.05 &gt;0.05 &gt;0.05 p=0.03 p&gt;0.05
We have now demonstrated that IC50 values for the novel, more selective Bcr-Abl inhibitor AMN107 are on average 22-fold lower than those observed for imatinib and there is no correlation between the IC50 values for imatinib and AMN107 (R2 = 0.375, p&gt;0.05). There was also less inter-patient variation in AMN107 IC50 (range 0.07uM) when compared to imatinib (range 3.1uM). In contrast to imatinib, there is no correlation between IC50AMN107 and the intracellular concentration of drug achieved in 14C-uptake studies (R2 = 0.337, p&gt;0.05). Further to this the addition of prazosin had no effect on influx of AMN107 suggesting that Oct-1 may not be a rate limiting determinant of intracellular drug concentration. In conclusion, variable Oct-1 mediated influx of imatinib may explain the heterogeneous IC50imatinib values we have observed in de novo CML patients. Oct-1 mediated influx may therefore be a key determinant of molecular response to imatinib. The causes of inter-patient variability in Oct-1 mediated influx requires further investigation. By contrast, poor Oct-1 mediated uptake is unlikely to have any impact on response to AMN107. This may be of relevance to patients with low intrinsic sensitivity to imatinib.
Constitutive Activation of SRC-Family Kinases in Chronic Myelogenous Leukemia Patients Resistant to Imatinib Mesylate in the Absence of BCR-ABL Mutations: A Rationale for Use of SRC/ABL Dual Kinase Inhibitor-Based Therapy.
Authors: Donato Nicholas J. , Wu Ji , Kong Ling-Yuan , Meng Feng , Lee Francis , Talpaz Moshe 
Published: April 2020

Abstract
BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of a reciprocal chromosomal translocation that is common in chronic myelogenous and acute lymphocytic leukemia. BCR-ABL induces transformation of hematopoetic stem cells through tyrosine phosphorylation of multiple substrates. The src-family kinases (SFKs), Lyn and Hck, are highly activated by BCR-ABL in leukemic cells and recent studies suggest that they are substrates and essential mediators of BCR-ABL signal transduction and transformation. In cells selected for resistance to the BCR-ABL inhibitor, imatinib mesylate, Lyn kinase is overexpressed and its activation is not dependent on or regulated by BCR-ABL, suggesting that autonomous regulation of SFKs may play a role in imatinib resistant. In this report, activation of Lyn and Hck was compared in CML specimens derived from imatinib responsive and resistant patients that did not express a mutant BCR-ABL protein as their primary mediator of resistance. In imatinib sensitive cell lines and specimens derived from imatinib responsive CML patients imatinib effectively reduced activation of both BCR-ABL and SFKs. However, in multiple specimens from resistant patients, imatinib reduced BCR-ABL kinase activation but failed to reduce SFK activation. The dual ABL/SRC inhibitor, BMS-354825, blocked activation of both BCR-ABL and SFKs expressed in leukemic cells and correlated with clinical responsiveness to this agent. Animal models demonstrated that loss of imatinib-mediated inhibition of Lyn kinase activation significantly impaired its anti-tumor activity which was recovered by treatment with BMS-354825. Direct silencing of Lyn or Hck reduced CML cell survival in imatinib resistant patient specimens and cell models, suggesting a direct role for these kinases in cell survival. Our results show that SFK activation is mediated by BCR-ABL in imatinib responsive cells but these kinases escape control by BCR-ABL in CML patients that develop imatinib resistance in the absence of BCR-ABL point mutations. This form of resistance can effectively be overcome by BMS-354825 through its dual SRC and ABL kinase inhibitory activities. Dual specificity kinase inhibitors may be indicated for the treatment and prevention of imatinib resistance in CML when it is associated with constitutively activated src-family kinases.
Targeting Bcl-2 as Therapy for Multiple Myeloma.
Authors: Chauhan Dharminder , Velankar Mugdha , Hideshima Teru , Mitsiades Constantine , Catley Laurence , Raje Noopur , Schlossman Robert , Richardson Paul , Anderson Kenneth C. 
Published: April 2020

Abstract
Mitochondrial signaling is an important component of chemotherapy-induced apoptosis in cancer cells (Korsmeyer, SJ, 2004Cell, 111:205–219). Members of the Bcl-2 family of proteins regulate, in part, the release of apoptogenic factors, such as cytochrome-c from the intermembrane space into the cytosol, thereby initiating caspase-9-mediated cell-death signaling cascade (Reed, SJ 1997, 34: 9–19; Kirkin et al, 2004, Biochim Biophys Acta. 2204, 1644:229–249). Overexpression of Bcl-2 or Bcl-x(L) confers resistance to chemotherapy in cancer cells, including MM (Letai, A 2004Cancer cell, 6:241–249; Chauhan et al, 2005, Annu Rev Pharmocol Toxicol, 45:465–476). Here we utilized ABT-737 (Abbott Laboratories), a potent small-molecule inhibitor of anti-apoptotic proteins Bcl-2, Bcl-x(L) and Bcl-w with markedly higher affinity than previously reported compounds (Oltersdorf, Elmore, &Shoemaker et al, Nature2005, 435; 677–81). We show that ABT-737 inhibits the growth of MM cells, including those resistant to conventional agents dexamethasone, melphalan or doxorubicin. Examination of purified patient MM cells demonstrated similar results. Importantly, ABT-737 also decreases the viability of CD138+ tumor cells obtained from Bortezomib-refractory MM patient. In contrast, no significant toxicity of ABT-737 was observed against peripheral blood mononuclear cells from normal healthy donors or CD138− MM patient cells. Moreover, ABT-737 does not affect the viability of MM patient-derived bone marrow stromal cells (BMSCs). Mechanistic studies show that ABT-737-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, and caspase-3, followed by proteolytic cleavage of poly (ADP) ribose (PARP) enzyme. Finally, ABT-737 enhances the anti-MM activity of Bortezomib or Melphalan, without significant toxicity in normal cells. Collectively, these findings provide the rationale for clinical evaluation of ABT-737 alone, or in combination with Bortezomib or Melphalan, to enhance MM cell killing, overcome drug-resistance, and improve patient outcome in MM.