bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pro-Apoptotic Synergistic Interactions between ERK1/2 and Bcl-2 Inhibitors in Acute Myeloid Leukemia Cells.
Authors: Ricciardi Maria Rosaria , Konopleva Marina , Ruvolo Peter P. , McQueen Teresa , Milella Michele , Andreeff Michael 
Published: October 2019

Abstract
We previously reported that activation of the Ras/MEK/ERK signaling pathway alone or in combination with high levels of Bcl-2 confers poor prognosis in patients with acute myeloid leukemias (AML). In this study, we investigated whether the simultaneous disruption of these two pathways by pharmacological inhibition of ERK1/2 and Bcl-2 utilizing novel small molecule inhibitors would induce pro-apoptotic activity in myeloid leukemic cells. For inhibition of ERK1/2 signaling, we utilized the MEK inhibitor CI-1040 (Pfizer Global Research and Development). Functional Bcl-2 inhibition was achieved by the novel BH3 peptide binding domain inhibitor A438744.7 (Abbott Laboratories). A dose-dependent increase in apoptosis as assessed by an increase in phosphatidylserine externalization was observed in OCI-AML3 cells after 24h incubation with CI-1040 or A438744.7 (both in a range between 0.25 and 2 μM). When these agents were used in combination at a fixed 1:1 ratio, a dramatic enhancement of cell killing was observed especially at lower concentrations (70.63%±9.95 Annexin V+ cells at 0.25 μM of each compound). Isobologram analysis (Chou and Talalay method) revealed a Combination Index (CI) < 1 (CI=0.114 and 0.436 at 0.25 μM and 2 μM, respectively) suggesting the strongly synergistic nature of these interactions. In addition, combined treatment with CI-1040 and A438744.7 resulted in a substantial increase of mitochondrial damage and caspase cleavage. Pre-incubation (1-hour) with a pan-caspase inhibitor (IDN-1965) was able to completely abrogate sensitivity to the inhibitors. We further demonstrated that bcl-2 overexpression prevented induction of apoptosis by low doses of both, MEK and BH3 inhibitors, whereas enforced Bcl-XL expression essentially abrogated the lethal effects of A438744.7, but not of CI-1040. Finally, to evaluate whether the combined strategy of ERK1/2 and Bcl-2 inhibition would be potentially applicable to leukemia patients, primary cells were isolated from AML samples and exposed to CI-1040 and A438744.7, as single agents or in combination at a 1:1 ratio for up to 96 hours. In each sample, drugs administrated individually were minimally toxic. Co-administration of CI-1040 and BH3 inhibitors displayed strong synergistic lethal effects toward primary cells from AML patients (CI=0.45±0.42 at 0.25 μM and CI=0.20±0.22 at 0.5 μM after 48h incubation). In conclusion, simultaneous exposure to nanomolar concentrations of MEK and bcl-2 inhibitors induced mitochondrial dysfunction, caspases activation and striking synergistic pro-apoptotic activity in myeloid leukemic cells. Similar synergistic interactions occured in primary AML samples. Together, these data strongly suggest that therapeutic strategies combining MEK and Bcl-2 inhibitors warrant further examination in AML.
Highly Synergistic Interaction between Farnesyltransferase Inhibitors and the Chk1 Inhibitor UCN-01 to Induce Apoptosis in Human Leukemia Cells through Interruption of Both Akt and MEK/ERK Pathways and Activation of SEK1/JNK.
Authors: Dai Yun , Rahmani Mohamed , Pei Xin-Yan , Khanna Payal , Dent Paul , Grant Steven 
Published: October 2019

Abstract
Ras mutations, which result in constitutive Ras activation, occur frequently in human malignancies, including leukemia. This finding has prompted the development of farnesyltransferase inhibitors (FTIs), which interfere with Ras farnesylation and membrane translocation necessary for Ras function. However, FTIs alone have not yet fulfilled their clinical potential, raising the possibility that their role may lie in combination with other agents. The present studies examined interactions between the farnesyltransferase inhibitor L744832 and the Chk1 inhibitor UCN-01 in human leukemia cells. Combined exposure of U937 cells to sub-toxic concentrations of UCN-01 (100nM) and the FTI L744352 (10μM) resulted in a dramatic and highly synergistic increase in mitochondrial dysfunction, apoptosis, and loss of clonogenicity. Similar interactions were noted in other leukemia cells (HL-60, Raji, Jurkat) and primary AML blasts, and with other farnesyltransferase inhibitors (e.g., FTI-277). These events were accompanied by cleavage of the anti-apoptotic proteins Bcl-2, XIAP, and Mcl-1. Co-administration of L744832 blocked UCN-01-mediated phosphorylation of MEK/ERK, leading to down-regulation of phospho-CREB and -p90RSK, and activation of p34cdc2 and SEK/JNK. Combined treatment also resulted in pronounced reductions in levels of phospho-Akt, -GSK, -p70S6K, -mTOR, -FKHR, -caspase-9, and -Bad. Ectopic expression of Bcl-2 or Bcl-xL but not dominant-negative caspase-8 blocked UCN-01/L744832-mediated mitochondrial dysfunction and apoptosis, but did not prevent activation of p34cdc2 and JNK, or inactivation of MEK/ERK and Akt. Enforced expression of myristolated Akt but not constitutively-active MEK significantly attenuated UCN-01/L744832-induced apoptosis. However, dual transfection with constitutively active Akt and MEK resulted in further protection from UCN-01/L744832-mediated lethality. Finally, down-regulation of JNK1 by siRNA significantly reduced the lethality of the UCN-01/L744832 regimen. Together, these findings suggest that farnesyltransferase inhibitors interrupt the cytoprotective Akt and MAPK pathways while reciprocally activating SAPK/JNK in leukemia cells exposed to UCN-01, and in so doing, dramatically increase mitochondria-dependent apoptosis. They also raise the possibility that combined treatment with FTIs and UCN-01 may represent a novel therapeutic strategy in leukemia.
The Role of the Cytoplasmic Domain of the FcγRI α Chain in FcγRI Signaling.
Authors: Hunter Sharon , Huang Zhen-Yu , Chien Paul , Kim Moo-Kyung , Worth Randall G. , Daniels Anthony B. , Kim-Han Tai-Hee , Indik Zena K. , Schreiber Alan D. 
Published: October 2019

Abstract
The high affinity Fcγreceptor FcγRI, consists of a ligand binding α chain and an associated γ chain subunit. Signaling by FcγRI is thought to be mediated through the cytoplasmic domain (CYT) of the γ chain which contains the ITAM sequence essential for many Ig receptor signaling events. For example, we have demonstrated that the γ chain is required for FcγRI mediated phagocytosis. The FcγRI α chain CYT lacks known signaling motifs and consists of 64 amino acids whose function has not yet been defined. Evidence is beginning to accumulate, however, for the participation of the FcγRIα CYT in FcγRI mediated signaling events. For example, we previously demonstrated that the FcγRIα CYT contributes to modulation of FcγRI mediated cytokine release (eg IL-6) and calcium flux. We have now observed that in the absence of γchain expression, FcγRIα efficiently induces the internalization of aggregated IgG complexes in transfected COS-1 cells. Taken together, the data suggest that the FcγRIα CYT contributes to signaling by FcγRI. We, therefore, utilized yeast two-hybrid screening to identify effector molecules that bind to the FcγRIα CYT. Of especial interest among the identified binding molecules is β arrestin-2, recently defined as an adaptor molecule linking receptors such as the LDL and type III TGF-β receptors to the endocytic pathway. Also of interest is Snapin, an adaptor molecule involved in membrane trafficking. These studies should help to define sequences in the cytoplasmic domain of FcγRIα that mediate and/or modulate important FcγR functions.
Proviral Tagging Combined with Human Microarray Data Reveals Genes and Pathways That Cooperate in Disease Induction.
Authors: Dave Utpal P. , Akagi Keiko , Dupuy Adam , Suzuki Takeshi , Jenkins Nancy A. , Copeland Neal G. 
Published: October 2019

Abstract
Retroviruses induce hematopoietic cancer by integrating into the genome and insertionally mutating cancer causing genes and provide powerful molecular tags for cancer gene identification. Retroviruses induce cancer following several rounds of insertional mutagenesis and clonal selection; as a result, each tumor contains multiple retroviral integrations that mark the location of cooperating cancer genes. Some retroviral integrations occur early in tumor formation and initiate tumorigenesis while others occur late and promote tumor progression. Using ligation-mediated PCR (LM-PCR), a new technique that permits the automated cloning and sequencing of thousands of retroviral integration sites per day, we are now able to rapidly identify the many initiation and progression events that cooperate to produce a mouse tumor. We have used LM-PCR to identify the myriad of genes that cooperate with activating mutations at [itallic]Lmo2 in leukemia induction. We compare our mouse results with the published microarray data for 19 human tumors that overexpress LMO2 and demonstrate a remarkable correlation between the genes causally associated with murine Lmo2 leukemias and the genes overexpressed in human LMO2 leukemias. Our studies highlight the power of LM-PCR for sampling tumor microheterogeneity and provide a powerful new means for interpreting and annotating human microarray data.
Effect of Factor XI or Factor IX Deficiency on an Arterial Occlusion Model.
Authors: Gailani David , Cheng Qiufang , Xu Lin , Ogletree Martin , Wang Xinkang 
Published: October 2019

Abstract
Factor XI (fXI) and factor IX (fIX) are zymogens of plasma proteases that are required for normal formation and maintenance of a blood clot. Recent work has implicated these proteins in the pathogenesis of vascular thrombosis. Epidemiologic studies indicate that high levels (top 10% of normal distribution) of fXI or fIX are independent risk factors for venous thromboembolism, increasing risk ~2-fold. Recently, it was shown that fXI deficiency protects mice from carotid artery occlusion in a ferric chloride (FeCl3 ) injury model. FeCl3-induced thrombus formation involves thrombin generation, in addition to platelet activation and von Willebrand factor. We used a modified version of the FeCl3 model to study the antithrombotic effects of complete fXI or fIX deficiency. In wild type C57Bl/6 mice, carotid artery flow measured by Doppler flow probe is completely blocked within 10 minutes of applying 3.5% FeCl3 to the vessel. 3.0% FeCl3 induced occlusion in some (5 of 8) mice by 30 minutes, while no animal treated with 2.5% FeCl3 experienced occlusion. FXI and fIX deficient mice were fully protected from occlusion induced by 3.5% or 5% FeCl3. Some fXI (4 of 8) and fIX (4 of 6) deficient animals developed occlusion with 7.5% FeCl3, while occlusion occurred in all mice at 10% FeCl3. To put the effect of fXI or fIX deficiency on this model into perspective, it requires a very high dose of heparin (1000 U/kg) to produce similar protection. With 5% FeCl3, heparin at 200 U/kg only protects 50% of wild type mice from occlusion, despite prolonging the activated partial thromboplastin time beyond the upper limit of the assay (> 500 secs). High dose aspirin (100 mg/kg) did not prevent occlusion induced by 5% FeCl3, despite producing a nearly complete block of arachidonic acid-induced platelet aggregation in vitro. While fXI and fIX deficiency affect the FeCl3 model similarly, they have significantly different impacts on a tail bleeding time (TBT) assay. FXI deficient and wild type mice have similar mean TBTs (265 ± 68 and 287 ± 92 secs, respectively), while fIX deficiency causes prolonged bleeding (1561 ± 125 secs, p < 0.01). In comparison, heparin (200 units/kg) causes the TBT to exceed the upper limit of the assay (1800 seconds), while aspirin (30 mg/kg) modestly increases the TBT (~2.2-fold). The data indicate that fXI and fIX are involved in thrombus formation in the FeCl3 model, and support a growing body of evidence that thrombin formation through the fIX/fXI axis contributes to thrombotic disease. Given the mild bleeding diathesis associated with fXI deficiency, inhibition of fXI may be a useful component of therapy for treating or preventing thrombus formation, and would be associated with a relatively low risk of bleeding.
The Three-Dimensional Structure of the cGMP-Dependent Protein Kinase I - α Leucine Zipper Domain and Its Interaction with the Myosin Binding Subunit.
Authors: Zhou Guo-Ping , Surks Howard K. , Schnell Jason R. , Chou James J. , Mendelsohn Michael E. , Rigby Alan C. 
Published: October 2019

Abstract
Vascular smooth muscle contractile state is tightly coupled to myosin light chain phosphorylation. Myosin light chain phosphorylation is regulated by the counter regulatory activities of myosin light chain kinase and myosin phosphatase. Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase. This in turn functions to dephosphorylate myosin light chains. Mechanistically this involves a protein-protein interaction mediated by a leucine zipper (LZ) domain that is localized within the N-terminus of cGK1α and the C-terminus of myosin-binding subunit (MBS). To better understand the importance of this interaction in regulating smooth muscle vascular tone we have determined the three-dimensional structure of the LZ domain of cGK1α, (cGKIα1–59) using triple resonance, multidimensional nuclear magnetic resonance (NMR) spectroscopy. We have expressed the LZ domain (cGKIα1–59) in E. coli under conditions facilitating the uniform incorporation of 13C/15N and 2H isotopic labels and purified this expressed protein. Circular dichroism data supported that cGK1α is comprised of an α-helix, while our NMR data identified that this helix commences at residue Ala9 and extends through to residue Leu47 of the LZ domain. 15N backbone longitudinal and transverse relaxation rates are best fit to a rotational diffusion anisotropy model that is consistent with a coiled-coil dimmer structure, not a monomeric helix. This suggests that cGKIα1–59 is comprised of a coiled-coil conformation over this same region. A time course investigation carried out with the LZ domain and a homobifunctional amine reactive crosslinker [dithio-bis(succinimidyl propionate)] (DSP) supported the presence of a homodimer. Furthermore, 15N heteronuclear single quantum correlation (HSQC) chemical shift mapping data has been used to identify residues that are critical for interacting with the C-terminal region of the myosin binding subunit (MBS). These data have allowed us to identify those residues necessary for cGK1α dimerization, while simultaneously permitting a better understanding of the critical determinants of cGK1α and MBS that are involved in regulating the contractile state of smooth muscles within blood vessels. The spatial and temporal contractile state of these blood vessels is important in the physiologic control of blood vessel tone and plays a role in the pathogenesis of hypertension and atherosclerosis a leading cause of death in the US and worldwide.
Proteomics of Membrane from Human Sickle and Normal Fractioned Red Cells Identifies Different Expression of Stress-Response Proteins.
Authors: Turrini Franco , Carta Franco , Biondani Andrea , Roncada Paola , Cappellini Maria D. , Corrocher Roberto , De Franceschi Lucia 
Published: October 2019

Abstract
Sickle cell disease (SCD) is characterized by the presence of sickle hemoglobin (HbS), which has the unique property of polymerizing when deoxygenated and thereby triggering erythrocyte sickling and dehydration. Repeated HbS polymerization is associated with generation of dense, distorted red cells characterized by an extensive membrane oxidative damage and abnormal activation of membrane cation transport pathways. The recently developed proteomic technologies represent a useful tool for identification of new functional pathways in sickle red cells. We studied the membrane proteoma of sickle red cells from homozygous patients (SS), not carrying alpha-thalassemia, not undergoing hydroxyurea treatment and not transfused at least three months before the study. The buffy-coat was removed and the red cells were separated into two fractions corresponding to the reticulocytes enriched fraction and to the dense red cell fraction (n= 8). Following osmotic lysis, the red cell ghost proteins were separated by one- dimensional or by two-dimensional gel electrophoresis (1-DE, 2-DE). Using the ImageMaster 2D Platinum (5.0 Amersham-Biosciences) software the 2-DE gels from control and sickle cells were compared, allowing us to identify spots differently expressed in sickle cells compared to normal cells. The selected spots were excised and trypsinized before mass spectrometric analysis and compared with the SWISS-PROT and NCBI non-redundant databases. In sickle reticulocytes enriched fraction compared to the control one, we selectively identified the following cystokeleton proteins: four spots for beta actin, two spots for myosin light chain and a train of four proteins identified as protein 4.1, one of which was phosphorylated on Tyr13, a protein similar to flotillin 2, carbonic anhydrase I and the following stress-response proteins: heat shock protein 70 (HSP70) protein 9B, HSPC108 and peroxiredoxin II (PrxII), which was phosphorylated in Thr142. In dense sickle red cell fraction compared to normal one we selectively identified a train of two spots for 4.1R, band 3 and aldolase and two spots identified as HSP70 protein 5.
The identified proteins were then confirmed by western-blotting analysis.
These data suggest that in sickle red cells HSP70, HSP108 function may be required by the extensive assistance in the refolding of misfolded proteins most likely related to abnormal sickle red cell oxidative environment as also supported by the presence of phosphorylated peroxiredoxin II, which has been recently proposed as regulator of redox-sensitive signaling in other cell types.
PDE-4 Inhibitor Rolipram Prevents Hypoxia Induced Pulmonary Hypertension in Transgenic Sickle Cell Sad Mice.
Authors: De Franceschi Lucia , Malpeli Giorgio , Janin Anne , Scarpa Aldo , Leboeuf Christhophe , Platt Orah , Leboulch Philippe , Payen Emmanuel , Beuzard Yves , Brugnara Carlo 
Published: October 2019

Abstract
Pulmonary hypertension (PH) is one of the major causes of morbidity and mortality for young-adult patients with sickle cell disease (SCD). Little is known about the role of vaso-active agents, pro-inflammatory and pro-fibrotic mediators in the genesis of PH in SCD.
We developed a model for studying PH in transgenic sickle cell SAD mice. Wild-type (WT) and SAD mice aged between 4 and 6 months were divided in groups of 7 animals each. One group from each strain served as control under normoxia, while a second group from each strain was exposed to 7 days hypoxia (8% oxygen). The following parameters were evaluated in normoxic and hypoxic groups: lung histopathology, complete blood count, reticulocytes, bronchoalveolar lavage (BAL) total leokocyte and neutrophil count, BAL levels of IL-6, IL-10, IL-1β and TNF-α. The expression of nine genes was examined by quantitative RT-PCR. These included endothelin-1 (ET-1), endothelin-1 receptor (ET-1R), cycloxygenase-2 (COX2), NF-kBp65, transforming-growth-factor-1β (TGF-1β), metalloproteinase-2 and 9 (MMP-2, MMP-9), IL-10 and IL-1β.
Two of the seven SAD mice exposed to hypoxia died at day fourth. The remaining five mice were sacrified at day seven and showed: a) increased pulmonary arterial wall thickness and α-actin staining, suggesting a vascular remodeling; b) lung injury compatible with PH in SCD; c) increased BAL total leukocyte and neutrophil count; d) increased BAL IL1β, IL10, IL6 and TNF-α; e) increased peripheral neutrophil and reticulocyte counts, hematocrit and hemoglobin levels. In lungs of hypoxic SAD mice, ET-1, ET-1R, TGF-1β, MMP-2, MMP-9, IL-1β and IL-10 gene expression were induced, with no significant changes in NF-Kbp65 gene expression. These data were consistent with a model of PH in SCD.
We then assessed the effect of the PDE-4 inhibitor ROLIPRAM on the PH in seven hypoxic SAD mice. ROLIPRAM was administrated at the dosage of 30 mg/Kg/day by gavage. No deaths were observed in this group. ROLIPRAM a) protected SAD mice from hypoxia induced lung injury; b) decreased BAL total leukocyte and neutrophil count; c) decreased BAL IL-1β, IL-6 and IL-10 levels; d) reduced peripheral neutrophil count; d) normalized and/or modulated the expression of the hypoxia-induced genes. These data suggest that the PDE-4 inhibitor ROLIPRAM has anti-inflammatory properties and could have beneficial effects on the hypoxia-induced airway-remodeling and possibly PH in sickle cell disease.
Arginine Supplementation of Sickle Transgenic Mice Leads to a Rapid Decrease in MCHC and Percent Dense Cells and a Slow Increase When Supplementation Is Discontinued.
Authors: Fabry Mary E. , Etzion Zipora , Bookchin Robert M. , Suzuka Sandra M. , Nagel Ronald L. 
Published: October 2019

Abstract
Nitric oxide (NO) is produced from arginine by nitric oxide synthase (NOS) and is essential to the maintenance of vascular tone. Plasma arginine levels are reduced in sickle cell anemia patients, and we have previously reported that S+S− Antilles mice have lower plasma arginine than control mice (C57BL). Long term arginine supplementation (5% arginine in chow) restored plasma arginine levels in S+S− Antilles mice to that found in C57BL mice, and, in S+S− Antilles mice, MCHC and the percent high density red cells were reduced. Our observation that Ca++-activated K+ channel [K(Ca) channel or Gardos channel] activity is reduced in supplemented vs non-supplemented S+S− Antilles mice can account for reduced red cell density and dense cell formation (Blood99: 1103, 2002). The time course of arginine induced changes in red cell indices in sickle transgenic mice has not yet been studied. Using the Advia 120 Hematology System, we found that, within 7 days after the onset of arginine supplementation, MCHC* (CHCM in the Advia system) fell from 33.4±0.5 g/dL to 31.0±0.2 (mean ±SD, P<0.000002, N=6) and the percent high density red cells (MCHC>37 g/dL) decreased from 10.5±4.3 % to 4.5±1.6 (P<0.01, N=6). MCV rose from 43.6±0.8 fL to 46.4±0.5 (P<0.00003, N=6). Reticulocyte counts and mean corpuscular hemoglobin did not vary significantly. Although plasma arginine levels increase within a few hours of oral administration and persist for only a few hours, no change in red cell indices was observed 7 days after discontinuing arginine supplementation, and the indices slowly returned to baseline levels after 40 days. In summary, MCHC* and the percent high density RBCs decrease significantly within 7 days of the onset of arginine supplementation. When arginine supplementation is withdrawn, the return to baseline values is much slower and required more than 40 days to complete. These observations have important implications for arginine supplementation in sickle cell anemia patients. In particular, the persistence of decreased MCHC* and reduced percent high density RBCs for an extended period of time after supplementation was withdrawn parallels our previously reported observation of persistent improvement of perfusion in sickle transgenic mice after supplementation was withdrawn and suggests that daily administration of arginine may not be necessary once an induction period has passed.