bluuebunny/crossref_metadata_2025 huggingface dataset:
B Domain of Coagulation Factor VIII Regulates Exposure of Its Heparin-Binding Site.
Authors: Makogonenko Evgeny M. , Sarafanov Andrey G. , Ananyeva Natalya M. , Radtke Klaus-Peter , Strickland Dudley K. , Saenko Evgueni L.
Published: April 2020

B domain of coagulation factor VIII (fVIII) was previously considered to be dispensable for fVIII function. Recently, it was found that the B domain is important for fVIII intracellular interaction with its chaperon and likely involved in fVIII clearance via asialoglycoprotein receptor. At the same time, the major clearance mechanism of fVIII involves initial interaction with heparan sulfate proteoglycans (HSPGs) followed by internalization via low-density lipoprotein receptor-related protein (LRP), member of low-density lipoprotein receptor (LDLR) family (Saenko et al, 1999; Sarafanov et al, 2001). It is possible that recently discovered clearance of fVIII via LDLR (Bovenschen et al, 2005) occurs in the same way. Since it was previously shown that fVIII binding sites for LRP are not located within B domain, we investigated if the latter regulates fVIII interaction with HSPGs. To explore this role of B domain, we compared the binding of plasma-derived fVIII (pd-fVIII) and recombinant B domain-deleted fVIII (BDD-fVIII) to immobilized LRP and heparin (a model of HSPGs) in surface plasmon resonance-based assay. The corresponding affinities were assessed by processing the binding signals obtained for five different concentrations of each analyte. Both pd-fVIII and BDD-fVIII showed similar affinities for LRP (KD 42–60 nM). The LRP-binding site of BDD-fVIII was partially blocked by pre-incubation with its carrier protein von Willebrand factor (vWf) indicating that it is only partially accessible within fVIII/vWf complex. This was further confirmed by the finding that monoclonal antibody 413, which recognizes a high-affinity LRP-binding site within the fVIII A2 domain, interacted with ~25% of BDD-fVIII molecules bound to immobilized vWf. The affinities of pd-fVIII and recombinant BDD-fVIII for immobilized heparin were similar (KD ~20 nM) and 2-fold higher than that for purified A2 domain (KD ~46 nM). Noteworthy, the maximal binding level (Rmax) proved to be 10-fold lower for pd-fVIII in comparison with BDD-fVIII indicating that in the circulation intact fVIII may have limited interaction with HSPGs. Importantly, pre-incubation with vWf did not interfere with the interaction between BDD-fVIII and heparin (KD ~ 19.5 and 21.8 nM, Rmax ~ 194 and 354 RU for BDD-fVIII and BDD-fVIII/vWf, respectively) thus revealing that heparin-binding site of fVIII is completely exposed in BDD-fVIII/vWf complex. These findings suggest that the presence of B domain in circulating fVIII/vWf complex may regulate fVIII clearance by preventing its interaction with HSPGs. The absence of B domain leads to exposure of heparin-binding site within fVIII and binding of fVIII/vWf complex to HSPGs. This binding may be a driving force in fVIII clearance which involves subsequent exposure of LRP (LDLR)-binding site(s) and internalization of fVIII from its complex with vWf via these receptors.
pH-Dependent Association of Factor VIII Chains: Enhancement of Affinity at Physiological pH by Cu2+.
Authors: Wakabayashi Hironao , Zhou Qian , Nogami Keiji , Ansong Charles , Varfaj Fatbardha , Miles Stephen , Fay Philip J.
Published: April 2020

Factor VIII circulates as a heterodimer composed of a heavy chain (HC) and light chain (LC). The structural integrity of the heterodimer is crucial for factor VIII function. In this study we found that the reconstitution of factor VIII from isolated HC and LC was pH-dependent. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH values. The affinity of HC and LC was maximal at pH 6.5-6.75 (Kd ~ 4 nM), whereas a Kd of ~20 nM was observed at physiological pH (7.25) (Figure). The effect of Cu2+ (0.5 μM total Cu2+) on maximal activity regenerated was negligible at pH 6.25–8.0. However, this level of Cu2+ increased the inter-chain affinity by ~5 fold at pH 7.25 (Figure). This effect resulted from an ~1.5-fold increased association rate constant (kon) and an ~3-fold reduced dissociation rate constant (koff). Addition of Cu2+ to a mixture of HC and LC at equilibrium resulted in an increase in cofactor activity that followed a single exponential, suggesting that association was defined by a simple reaction model. High affinity (Kd = 5.3 fM) of the factor VIII chains for Cu2+, as estimated by increases in cofactor activity, suggested a rapid association rate for the metal ion. Together, these results suggest that the protonation state of specific residues, His in particular, modulate inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.

Figure Figure
Mapping Factor VIIIa Inter-Subunit Interactions Using Chemical Modification and Zero-Length Cross-Linking.
Authors: Miles Stephen M. , Freas Jan , Fay Philip J.
Published: April 2020

Activated factor VIII (FVIIIa) is a complex of three subunits, designated A1, A2, and A3C1C2, that serves as a cofactor for factor IXa in the proteolytic activation of factor X. The structure and surface interactions between factor VIIIa subunits have yet to be fully defined, but a homology-based model is available as a predictive tool (Stoylova-McPhie et al, Blood, 2002). To investigate FVIIIa inter-subunit interactions we used a chemical modification approach to determine surface-exposed and potentially interactive residues. The protein modification reagents acetic anhydride and diethylpyrocarbonate (DEPC), that modify lysine and histidine residues, respectively, were used to identify residues that are modified in the free subunits but protected in the bound complex. The modified samples were digested with various proteases and the resulting digests were analyzed by MALDI-TOF mass spectrometry to determine sites of peptide modification (Figure 1). The majority of data observed was in agreement with the A1/A3C1C2 interface in the predicted model. Protection was observed at Lys-89, Lys-142, and Lys-230 in the A1 subunit, all of which are near the A3C1C2 interface. While most His residues were completely modified by DEPC, partial protection was seen at His-1954, His-1957, and His-1961 in A3C1C2, which we have previously shown to be an A1-interactive region (Ansong and Fay, Biochemistry, 2005). In addition to this data, we attempted to define inter-subunit contacts by covalently cross-linking the FVIIIa complex. Using the zero-length cross-linker 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), which cross-links Glu/Asp residues to Lys, four distinct cross-linked bands were identified by SDS-PAGE (Figure 2). The composition of the cross-linked products was determined using proteolytic digestion and MALDI analysis. The four products correspond to each possible combination of the three subunits. Interestingly, there are no candidate cross-link sites between A1 and A2 in the model, yet this cross-link is the most predominant of the four. One alternative is that the A1–A2 cross-link may involve the A1 337–372 region that is not represented in the model. Taken together, the data are directed toward physically defining interactive regions between FVIIIa subunits and serve to test and supplement current structure models.

FIGURE 1, FIGURE 2 FIGURE 1, FIGURE 2.
Identification of a Plasmin-Interactive Site within the A2 Domain of the Factor VIII Heavy Chain.
Authors: Nogami Keiji , Shima Midori , Nishiya Katsumi , Saenko Evgueni L. , Takeyama Masahiro , Tatsumi Kohei , Tanaka Ichiro , Yoshioka Akira
Published: April 2020

Factor VIII (FVIII) is inactivated by limited proteolytic cleavage by plasmin immediately after the activation. However, the plasmin-interactive region(s) in FVIII remain to be determined. Recently, we reported that the A2 domain may interact with plasmin during FVIII inactivation by this protease (Abst #1991, BLOOD102, 2002). In the current study, several approaches were employed to examine the localization and role of plasmin-interactive region(s). Activation and inactivation rate constants of plasmin-catalyzed FVIII and FVIIIa by the addition of isolated A2 subunit were reduced by ~4 and ~13-folds, respectively, in dose-dependent manners using one-stage clotting assay. The addition of Glu-Gly-Arg active-site modified factor IXa, interacts with the A2 domain, also reduced the rate constant of FVIIIa inactivation by ~4-fold. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody 413, recognizing residues 484–509 in factor IXa-interactive site, blocked the plasmin-catalyzed cleavages at Arg336, Arg372, and Arg740 in the heavy chain. Surface plasmon resonance-based assay using anhydro-plasmin, catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that FVIII and isolated A2 subunit bound to anhydro-plasmin with Kd values of 4 and 21 nM, respectively. The binding assay using ELISA with immobilized anhydro-plasmin also showed the similar binding affinities. Monoclonal antibody 413 blocked the A2 subunit binding to anhydro-plasmin by ~80% (IC50: 151 nM). Furthermore, synthetic peptide with sequences 479–504 inhibited this binding by ~55% (Ki: 3 microM), however, peptide with sequences 489–514 had a very weak inhibition (by <20%). To investigate the responsible residues in A2 domain for plasmin binding, the mutant forms of the A2 domain were expressed in baculovirus system and purified. Compared with wild type (23 nM), the affinity of R484A mutant was dramatically decreased by ~250-fold, and the affinities of K377A, K466A, R471A, and K523A mutants also were decreased by 10~40-folds, respectively. Especially, the addition of R484A mutant was reduced inactivation rate constant of plasmin-catalyzed FVIIIa by only ~40% of that of wild type. These findings demonstrate that Arg484 in the A2 domain contains plasmin-binding site responsible for plasmin-catalyzed FVIII(a) inactivation.
Fragments of Activated Coagulation Factor VIII Bind to Multiple Sites within Low-Density Lipoprotein Receptor-Related Protein.
Authors: Sarafanov Andrey G. , Makogonenko Evgeny M. , Andersen Olav M. , Khrenov Alexey V. , Mikhailenko Irina A. , Ananyeva Natalya M. , Strickland Dudley K. , Saenko Evgueni L.
Published: April 2020

Catabolism of coagulation factor VIII (fVIII) is mediated by the hepatic multiligand receptor low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CRs) organized in four clusters, among which clusters II and IV bind most of LRP ligands. In turn, fVIII contains two major LRP-binding sites, located in A2 and A3 domains (Saenko et al, JBC 1999; Bovenschen et al, JBC 2003). In present work, we characterized binding sites in LRP for A2 domain (A2) and heterodimer A1/A3-C1-C2 (HD), the products of dissociation of activated fVIII. Using a baculovirus expression system, we generated CR clusters II, III and IV, along with eight overlapping CR triplets encompassing clusters II and IV. Surface plasmon resonance-based assays demonstrated that both A2 and HD bind to clusters II and IV, and to the same sets of their CR triplets with similar affinities (KDs 25–50 nM). The same kinetic parameters of interaction of both A2 and HD were observed for several CR doublets from cluster II, shown previously to be minimal binding sites for a classical ligand of LRP, receptor associated protein (RAP) (Andersen et al, JBC 2000). The specificity of A2 and HD interactions with all tested fragments of LRP was confirmed by the ability of RAP to inhibit these interactions, and by the ability of these fragments to inhibit binding of 125I-A2 and 125I-HD to immobilized LRP in a solid-phase assay, and LRP-mediated catabolism of 125I-A2 and 125I-HD in cell culture. Notably, some mutations of the LRP-binding site in A2 resulted in significant reduction or abolishment of its binding to certain fragments of LRP, while the binding to other LRP fragments was less affected. In summary, we demonstrated that i) A2 and HD interact with LRP via its multiple binding sites spanning CRs 3–8 in cluster II and CRs 24–29 in cluster IV, and ii) the elementary binding unit of LRP is formed by at least two adjacent CRs, similar to that shown for RAP. The above data also suggest that besides regulating fVIII levels, LRP also plays a role in clearance of the products of dissociation of activated fVIII.
Loss of TM-Dependent PC-Activation Predisposes to Diabetic Nephropathy: Potential Role of Endothelial Apoptosis.
Authors: Isermann Berend , Thati Madhusudhan , Vinnikov Ilya , Herzog Stefanie , Huntscha Sina , Bünder Robert , Weiler Hartmut , Bierhaus Angelika , Nawroth Peter P.
Published: April 2020

Increased levels of soluble thrombomodulin (TM) in patients with diabetes mellitus are considered a marker of endothelial damage. It is unknown whether the loss of endothelial TM-function contributes to the progression of vascular complications in diabetes mellitus. To address whether the loss of TM-dependent protein C (PC) activation contributes to diabetic complications such as diabetic nephropathy two animal models were employed: (1) TMPro mice, which have been previously described and carry a point mutation in the TM-gene (E404P), resulting in a loss of TM-dependent PC-activation, and (2) hPC mice, which carry a transgene resulting in the expression of a mutant “hyperactivatable” PC, which can be activated by thrombin in the absence of TM. The mutant PC could be captured from plasma samples of hPC mice and activated ex vivo by thrombin in the absence of TM. hPC mice had a prolonged bleeding time. Following induction of diabetes by streptozotocin TAT levels were increased in diabetic control (wild type) mice and to a larger extent in TMPro mice, but not in diabetic hPC mice. In comparison to diabetic control mice the kidney weight was increased in diabetic TMPro mice, but not in diabetic hPC mice. Albuminuria was increased in diabetic TMPro mice and reduced in diabetic hPC mice in comparison to diabetic control mice, indicating increased glomerular damage in TMPro mice and partial protection from glomerular damage in hPC mice. Consistently, using a histological score glomerular damage was more severe in diabetic TMPro mice in comparison to diabetic control mice, while diabetic hPC mice were protected. Preliminary data suggest that the observed changes are associated with increased apoptosis in glomeruli of diabetic TMPro mice. Using HUVECs we were able to establish that high glucose concentrations (30 mM) reduce TM-dependent PC activation. The reduced TM-dependent PC activation is associated with increased apoptosis. Glucose induced apoptosis in HUVECs is associated with an increased Bax/Bcl-2 ratio, increased translocation of Bax into mitochondria, and increased caspase-3 activation. Activated PC normalizes the Bax/Bcl-2 ratio, prevents translocation of Bax, and reduces caspase-3 activity. Further studies using TRAPs and inhibitory antibodies established that the antiapoptotic effect of aPC in glucose stressed endothelial cells is mediated through a Par-1 and EPCR-dependent mechanism. The current data strongly suggest that the loss of the endothelial TM-PC system is not just a marker of endothelial damage in diabetic patients, but rather contributes to the progression of diabetic vascular complications.
Genome-Wide Identification of Prednisolone-Responsive Genes in Primary Acute Lymphoblastic Leukemia Cells.
Authors: Tissing Wim J.E. , den Boer Monique L. , Meijerink Jules P.P. , Menezes Renee X. , Swagemakers Sigrid , van der Spek Peter J. , Sallan Stephen E. , Armstrong Scott , Pieters Robrecht
Published: April 2020

Glucocorticoids (GC) are keystone drugs in the treatment of childhood ALL and therefore it is important to get more insight in signal transduction pathways involved in GC induced apoptosis. Affymetrix U133A GeneChips were used to identify genes that are transcriptionally regulated upon 3 and 8 hours of prednisolone exposure in leukemic cells of 13 children newly diagnosed with ALL. After 3 hours of exposure no changes in gene expression could yet be found. After 8 hours exposure, 57 probesets (51 unique genes) were differentially expressed (p<0.0005 and false discovery rate <0.1) of which 44 probesets (39 genes) were upregulated (median 2.4-fold), whereas 13 probesets (12 genes) were downregulated (median 1.7-fold). Twenty-one genes were not previously found to be transcriptionally regulated by GC. Two tumor suppressor genes, Thioredoxin interacting protein (TXNIP) and Zinc finger and BTB domain containing 16 (ZBTB16), were 3.7-fold and 8.8-fold upregulated. Genes were functionally categorized in three major routes: i.e. MAPK pathways (9 genes), NF-k B signaling (11 genes) and carbohydrate metabolism (6 genes).

Biological characterization of these genes and pathways might elucidate the action of GC in leukemic cells. This knowledge may point to causes of GC resistance, ways to circumvent GC resistance and new potential targets for therapy.
DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.
Authors: Ikeda Kazuhiko , Shichishima Tsutomu , Yamashita Yoshihiro , Maruyama Yukio , Mano Hiroyuki
Published: April 2020

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of >22,000 probe sets (corresponding to >14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P <0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.
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

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

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

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

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

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

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<0.001 p>0.05 high IC50 imatinib (n=8) 1.375 14.8 8.4 0.04 28.5 27.6 p value <0.001 0.04 >0.05 >0.05 >0.05 >0.05 p=0.03 p>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>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>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

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

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.
Chromosomal Abnormalities in Philadelphia Chromosome (Ph)-Negative Metaphases Appearing during Imatinib Mesylate (IM) Therapy in Patients (pts) with Newly Diagnosed Chronic Myeloid Leukemia (CML) in Chronic Phase.
Authors: Jabbour Elias , Kantarjian Hagop , O’Brien Susan , Verstovsek Srdan , Garcia-Manero Guillermo , Rios Mary Beth , Cortes Jorge
Published: April 2020

The development of chromosomal abnormalities in the Ph-negative metaphases during IM therapy of CML has been recognized mostly in pts who failed prior therapy. Prior exposure to cytarabine has been suggested to be a predisposing factor. This phenomenon has not been yet assessed to date in patients with newly diagnosed CML and treated with IM. This is different from clonal evolution where the abnormalities are observed in the Ph-positive metaphases. We assessed the frequency and the significance of this event among 258 newly diagnosed pts with CML receiving IM (800 mg/d n=207, 400 mg/d n=51) as first line of therapy between March 2001 and April 2005. After a median follow-up of 30 months (range, 6–48 months), 19 pts (7%) developed 21 chromosomal abnormalities in Ph-negative metaphases. Thirteen (62%) of these abnormalities have been seen in 2 or more metaphases. The median time from the start of IM to appearance of abnormalities was 18 months (range, 3–36 months). The most common cytogenetic abnormalities were: loss of chromosome Y (n=7, 33%), trisomy 8 (n=3, 14%), and deletion of chromosome 7 (n=2, 10%). Excluding loss of chromosome Y abnormalities, the incidence was 5%. All pts achieved a major (Ph < 35%) cytogenetic (CG) response (complete cytogenetic response [CCGR] in 17 [89%] pts). Major molecular response (BCR-ABL/ABL ratio <0.05) was observed in 13 (68%) pts (including 2 with complete molecular response). In all but 4 pts these events have been transient and disappeared after a median of 4 months (range, 3–9 months). In 4 pts (loss of chromosome Y n=3, trisomy 8 n=1), they persisted for a median of 13+ months (range, 6+–24+ months). One pt developed acute myeloid leukemia (associated with -7); none of the other pts has any feature of myelodysplasia. After a median follow-up of 13 months (range, 1–42 months), 17 of the 19 pts are alive. One pt died after allogeneic stem cell transplantation, and one died after 6 months of CCGR from myocardial infarction. One pt lost response to IM. The remaining 16 pts are in major CG response at the last follow-up. We conclude that: 1) cytogenetic abnormalities occur in Ph-negative cells in a small fraction of patients (7%; 5% if loss of Y excluded) in newly diagnosed CML on IM; 2) in the majority of cases, they are transient with no clear clinical consequences; 3) in rare instances (loss of chromosome 7 only in our study) they could reflect the emergence of a new malignant clone necessitating and a close follow-up.
Feasibility of Administering Oblimersen (G3139; Genasense) with Imatinib Mesylate in Patients with Imatinib Resistant Chronic Myeloid Leukemia (CML) - Cancer and Leukemia Group B Study 10107.
Authors: Wetzler Meir , Yu Daohai , Odenike Olatoyosi , Feldman Eric J. , Hurd David D. , Hoke Eva , Hars Vera , Larson Richard A.
Published: April 2020

The CALGB studied the feasibility and effect of oblimersen (G3139; Genasense) with imatinib mesylate in patients with primary imatinib resistant CML. We hypothesized that CML cells that are resistant to imatinib mesylate (i.e., less than a complete hematologic remission after 8 weeks of treatment or less than a major cytogenetic remission after 6 months of treatment) are no longer being driven to proliferate by Bcr/Abl tyrosine kinase activity alone. Instead, the anti-apoptotic protein Bcl-2 would regulate one of the pathways controlling growth and/or viability. Thus, blocking both Bcr/Abl and Bcl-2 simultaneously with imatinib mesylate and G3139, respectively, would result in hematologic and cytogenetic improvement. Preclinical data suggested synergy. G3139 was administered via continuous intravenous infusion with an ambulatory pump over 10 days every 21 days, along with daily oral imatinib. Doses of both drugs were escalated in 3 cohorts; the initial dose of imatinib was 600 mg/day. The median age of the 21 enrolled patients (13 females and 8 males) who received any therapy was 54 years (range, 25–74), with 13 Caucasians and 8 African Americans. Twelve patients had primary imatinib resistance and 8 had secondary imatinib resistance (data are not yet available on 1 patient). Five patients had never achieved complete hematologic remission while only two patients had achieved major cytogenetic remission while on a prior imatinib therapy. Nine patients received 4 mg/kg/day G3139 and 600 mg of imatinib, 7 patients received 7 mg/kg/day G3139 and 600 mg of imatinib, and 5 patients received 7 mg/kg/day G3139 and 800 mg of imatinib. Progressive CML after the first (4 patients), second (1 patient), third (2 patients) or fourth course (1 patient) was noted in 8 patients whose imatinib dose had been reduced from 800 mg to 600 mg/day to comply with study entry requirements. Nine patients completed all 4 planned courses. Grade IV thrombocytopenia and fatigue (both at 7 mg/kg G3139 and 600 mg of imatinib) and deep vein thrombosis (at 7 mg/kg G3139 and 800 mg of imatinib) were noted in one patient each. One patient with primary imatinib resistance achieved complete hematologic remission following 2 courses of therapy at 4 mg/kg G3139 and 600 mg of imatinib. No patients had cytogenetic responses. Although the combination of G3139 and imatinib is safe and feasible, we did not detect clinical benefit in these patients with imatinib-resistant CML using these doses and schedule.