bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Cell Cycle Arrest and Apoptosis by Dephosphorylation of ERK, Akt, and Bcl-2 and Phosphorylation of JNK by LY293111 in Human Anaplastic Large Cell Lymphoma (ALCL).
Authors: Zhang Weiguo , Konopleva Marina , McQueen Teresa , Schober Wendy , Andreeff Michael
Published: October 2019

The prognosis of patients with anaplastic large cell lymphoma (ALCL) treated with standard chemotherapy remains poor. Leukotriene B4 (LTB4) receptor has been reported to exhibit a growth-promoting activity in lymphoma, since it augments DNA synthesis and increases cell proliferation of lymphocytes. LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a Leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. LY293111 inhibits pancreatic cancer cell growth, induces apoptosis, and reduces growth of colon cancer in vivo. In the present study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in an ALCL cell line. We utilized the Sup/M2 cell line derived from human anaplastic large cell lymphoma as a model. Dose-response studies demonstrated that the IC50 of LY293111 was 4μM after 5 days of treatment. LY293111 caused retardation of Sup/M2 cell entry into S phase in a dose-dependent fashion as measured by BrdU/propidium iodide flow cytometry. LY293111 at 2.5μM induced complete G1-S cell cycle arrest in cells synchronized by serum starvation. LY293111 upregulated p21 and p27 protein expression and reduced cyclin E mRNA and protein. Pre-treatment with LY293111 for 4 hours resulted in profound inhibition of serum-induced phosphorylation of ERK1/2, Akt, and Bcl-2. Concomitantly, phosphorylation of stress-activated kinase JNK dramatically increased following LY293111 treatment. LY293111 induced pronounced apoptosis of Sup/M2 cells and caspase inhibition by Benzyloxycarbonyl -Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) abrogated this effect. LY293111 induced cleavage of the caspase-9, -3, PARP and XIAP, but had no effect on caspase-8, suggesting activation of the intrinsic apoptotic pathway. Accordingly, early loss of mitochondrial membrane potential was observed in a dose-dependent fashion. At 72 hrs, LY293111 decreased Bcl-2 expression levels. These findings provide the first evidence that LY293111 inhibits ALCL proliferation by arresting cells in G1 phase of the cell cycle, as a result of cyclin E downregulation and induction of cell-cycle inhibitory proteins p21 and p27. LY293111 induces apoptosis in Sup/M2 lymphoma cells, with activation of the mitochondrial apoptosis pathway, including cleavage of caspase 9, caspase 3, PARP, XIAP, and Bcl-2. Furthermore, LY293111 treatment markedly shifts signaling toward the JNK stress-related pathway and away from cytoprotective MEK/ERK and AKT signaling. These results suggest that LY293111 may have a utility in the management of aggressive non-Hodgkin’s lymphomas.
Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.
Authors: Naumovski Louie , Ramos Jason , Chen Jun , Sirisawad Mint , Lucas David , Byrd John C. , Miller Richard A.
Published: October 2019

Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.
Differential Targeting of Pro-Survival Bcl-2 Proteins by Their Pro-Apoptotic BH3-Only Ligands: Implications for Targeted Therapies.
Authors: Wei Andrew , Chen Lin , Roberts Andrew , Huang David
Published: October 2019

Chemoresistance is a significant barrier in the management of hematological malignancies. Despite some striking successes, many malignancies remain incurable. The efficacy of cytotoxic drugs is often blunted by loss of p53 or overexpression of pro-survival proteins like Bcl-2 or its functional homologs Bcl-xL and Mcl-1. Bcl-2-like proteins promote cell survival until antagonized by BH3-only proteins such as Bim. A potential way of overcoming tumor resistance is to engineer BH3-only mimetics to trigger cell death. Although such drugs may have a useful role in overcoming chemoresistance, a serious concern is the potential deleterious effects on normal cells. To assess the feasibility of such an approach, we studied the binding of mammalian pro-apoptotic BH3-only proteins to their pro-survival Bcl-2-like targets. The targeting of pro-survival Bcl-2-like proteins has been considered to be promiscuous, but unexpected selectivity emerged from our analysis. The interactions varied over 10,000-fold in affinity and, accordingly, certain protein pairs associated selectively inside cells. Some death ligands that bound promiscuously were potent killers when overexpressed, whereas Noxa which only targets some pro-survival proteins, but not Bcl-2, had weak activity. Importantly, although expression of Noxa by itself caused limited cytotoxicity, it markedly enhanced sensitivity to cytotoxic drugs such as etoposide, even in those cells normally highly chemoresistant because of Bcl-2 overexpression. Thus, selective targeting of key pro-survival proteins in a malignancy may improve the efficacy of currently used cytotoxic agents with reduced undesirable side effects. In particular, our results suggest that it should be feasible to discover BH3 mimetic drugs with better therapeutic profiles and with the potential to overcome chemoresistant cancers overexpressing pro-survival proteins.
Characterization of the Resistance Mechanisms of HL60 Leukemia Cell Derivatives to Apo2L/TRAIL.
Authors: Cheng Jinrong , Hylander Bonnie L. , Baer Maria R. , Repasky Elizabeth A.
Published: October 2019

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a recently identified member of the TNF superfamily. Recombinant Apo2L/TRAIL is a promising immunotherapeutic agent for treating malignant diseases since this molecule preferentially induces apoptosis in a variety of tumor cells with apparently little toxicity to normal cells. However, it has also been shown that some tumor cells are resistant to this molecule. We hypothesized that resistance to Apo2L/TRAIL occurs through defects in the Apo2L/TRAIL-mediated apoptotic signaling pathway. To address this hypothesis, we developed several Apo2L/TRAIL-resistant HL60 derivatives (HL60/TR) by exposure of Apo2L/TRAIL-sensitive HL60 human leukemia cells to escalating levels of Apo2L/TRAIL, followed by subcloning. Two of these resistant clones (a moderately resistant clone-R1 and a highly resistant clone-R3) were selected for further study. Molecules in the Apo2L/TRAIL-mediated apoptotic pathway of R1 and R3 cells were analyzed by Western blot analysis, flow cytometry and gene sequencing and compared to those in parental HL60 cells. In the R1 cells, the activation of caspase-8 and -10 by Apo2L/TRAIL was significantly inhibited. However, R1 cells were still sensitive to Fas agonistic monoclonal antibody treatment, indicating that the FAS-mediated apoptosis-inducing pathway was intact. In the R3 cells, caspase-8 expression was completely lost and activation of caspase-10 in response to Apo2L/TRAIL was totally inhibited; R3 cells were therefore also resistant to FAS antibody treatment. Although the total protein level of death receptors DR4 and DR5 was equal in HL60 cells and in the Apo2L/TRAIL-resistant derivatives, the cell surface levels of DR4 were significantly decreased in both R1 and R3 cells, while the surface expression level of DR5 in these two clones was comparable to that on HL60 cells. No mutation in either the DR4 or DR5 genes was found in these cells. These results suggest that defective targeting of DR4 molecules to the cell surface occurs in these Apo2L/TRAIL resistant cells. Blocking cell surface DR4 significantly attenuated the sensitivity of parental HL60 cells to Apo2L/TRAIL, indicating that cell surface expression of DR4 plays a crucial role in regulating susceptibility of tumor cells to Apo2L/TRAIL. Taken together, our results demonstrate that malignant cells can develop resistance to Apo2L/TRAIL by several different mechanisms and multiple resistance mechanisms may develop in a single tumor cell (such as R3 cells). Understanding the basis of Apo2L/TRAIL resistance will help to predict sensitivity and to develop strategies to circumvent resistance.
JS-K, a Novel Nitric Oxide (NO) Generator, Shows Potent Anti-Angiogenic Activity.
Authors: Shami Paul J. , Kaur Gurmeet , Thillainathan Jagadambal , Jia Lee , Saavedra Joseph E. , Keefer Larry K.
Published: October 2019

NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity in vitro and in vivo (Molecular Cancer Therapeutics 2:409-417,2003). The purpose of this study was to determine the effect of JS-K on angiogenesis. The anti-angiogenic properties of JS-K were tested in 3 different in vitro assays: proliferation, cord formation (reflecting new vessel formation) and migration using Human Umbilical Vein Endothelial Cells (HUVEC). JS-K inhibited the proliferation of HUVEC’s with a 50% inhibitory concentration (IC50) of 0.432, 0.466, and 0.505 μM at 24, 48, and 72 hours, respectively. At concentrations of 1 μM or above, HUVEC proliferation was totally inhibited. In the cord formation assay, treatment with JS-K lad to a decrease in both the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 μM, respectively. At a concentration of 1 μM, JS-K inhibited cord formation completely. JS-K inhibited cell migration at 5 hours using 10 ng/mL VEGF as a chemoattractant. At that time point, migration inhibition occurred at JS-K concentrations that did not affect cell growth with an IC50 of 0.493 μM. We conclude that JS-K is a potent inhibitor of 3 important elements of angiogenesis, namely endothelial cell proliferation, cord formation, and endothelial cell migration. These experiments identify a new mechanism by which JS-K and similar compounds may inhibit leukemia and solid tumor cell growth in vivo. Determining whether the anti-angiogenic effects of JS-K are NO-dependent will require further studies. (NO1-CO-12400).
The ERG Gene in Normal and Malignant Megakaryopoiesis.
Authors: Rainis Liat , Rosenthal Esther , Strehl Sabine , Haas Oskar A. , Izraeli Shai
Published: October 2019

About 10% of patients with Down syndrome (DS) are born with a transient megakaryoblastic leukemia. We (Rainis et al. Blood2003; 102:981) and others have demonstrated acquired intrauterine inactivating mutations in the X-linked gene GATA1 in these leukemias. The gene(s) on chromosome 21 that promote the proliferation of these abnormal megakaryoblasts are presently unknown. We hypothesize such a role to the ets transcription factor ERG that is located at the critical DS region on chromosome 21. This hypothesis is based on the close homology between ERG and FLI-1, a transcription factor that induces megakaryopoiesis through cooperation with GATA1. ERG occasionally replaces FLI-1 in the translocation with EWS in Ewing sarcoma and is also involved in the leukemogenic translocation FUS-ERG. However no role for ERG in hematopoiesis has been demonstrated so far. Here we show that (a) ERG is expressed in platelets and in megakaryoblastic cell lines, including those derived from Down Syndrome. (b) ERG is expressed in primary leukemic cells from DS patients. (c) Its expression is induced upon megakaryocytic differentiation (d) ERG collaborates with GATA1 in activation of megakaryocytic promoters in reporter assays; and (e) Forced ectopic expression of ERG in K562 cells induces megakaryocytic differentiation. Thus we provide the first evidence that the ERG gene participate in megakaryopoiesis. Promotion of megakaryopoiesis caused by its overexpression in DS coupled with the differentiation arrest induced by the acquired mutation in GATA1 may explain the high incidence of a congenital megakaryoblastic proliferation disorder in Down Syndrome.
The Kinase Inhibitor Imatinib Mesylate Potently Inhibits TNF-α Production by Myeloid Cells and Prevents T-Cell-Mediated Hepatic Injury in Mice.
Authors: Wolf Anna M. , Wolf Dominik G.F. , Rumpold Holger F. , Enrich Barabara , Gastl Guenther A. , Eberhard Gunsilius , Tilg Herbert
Published: October 2019

Imanitib mesylate (Gleevec®) exhibits potent anti-leukemic effects in vitro and in vivo. Despite of it`s well known anti-leukemic effects, the potential of Imatinib in the treatment of inflammation remains elusive so far. Our current report provides strong evidence that Imatinib indeed exerts anti-inflammatory effects. It potently inhibits LPS- and ConA-induced TNF-α and IFN-γ production of human myeloid cells in vitro (PBMNC, CD14-selected monocytes and monocyte-derived macrophages). Of note, the production of the anti-inflammatory cytokine IL-10 was only slightly affected by Imatinib. In line with this observation, the activation of NF-κB, which has recently been shown to be critically involved in TNF-α but not IL-10 expression, was significantly impaired by Imatinib. In addition, Imatinib reduced either LPS or ConA-induced intracellular phophotyrosine content. For in vivo testing of the anti-inflammatory role of Imatinib in a murine model of inflammation, we injected BALB/c mice with either 75 mg/kg body weight Imatinib or solvent before administration of ConA. The latter has recently been shown to induce T-cell, macrophage and TNF-α-dependent inflammatory damage of the liver. Imatinib pre-treatment prevented the development of acute hepatic injury, which was paralleled by reduced intrahepatic TNF-α mRNA and circulating TNF-α protein levels. Improvement of liver pathology by Imatinib was further assessed by light microscopy and TUNEL staining. Of note, Imatinib protected mice also from GalN/LPS- but not from GalN/TNF-induced liver pathology, which corroborates our in vitro findings that Imatinib potently inhibits TNF-α production of myeloid cells. These findings point out to a potent anti-inflammatory role of the tyrosine kinase inhibitor Imatinib, which might be of therapeutic benefit for the treatment of TNF-α-mediated immune-pathologies, such as inflammatory bowel disease (IBD), aGvHD or immune-mediated liver injury.
The Effects of Imatinib on the Function of Monocyte Derived Dendritic Cells Are Mediated Via the Inhibition of Akt and NF-kappaB Signal Transduction Pathways.
Authors: Rupf Anette , Appel Silke , Weck Markus M. , Grünebach Frank , Kanz Lothar , Schoor Oliver , Brümmendorf Tim H. , Weinschenk Toni , Brossart Peter
Published: October 2019

Imatinib is a novel tyrosine kinase inhibitor effective against Abl kinases, c-Kit and platelet-derived growth-factor receptor (PDGF-R) and is currently used for the treatment of patients with CML and GIST. However, little is known about the effects of imatinib on function and differentiation of non-transformed normal cells. Using this compound, we show that human monocyte derived DC generated in the presence of therapeutic concentrations of imatinib show a concentration dependent reduced expression of CD1a, HLA and co-stimulatory molecules as well as decreased activation-induced secretion of chemokines and cytokines involved in T cell activation. Moreover, exposure to imatinib reduces the capacity of DC to prime T cell responses that cannot be restored by the addition of IL-12 and which is not due to induction of apoptosis or IL-10 secretion. Using Western blot analyses we found that these effects are mediated by a pronounced downregulation of nuclear localized protein levels of NF-kB family members RelB, RelA and NF-kB p50. Furthermore, imatinib treatment inhibited the phosphorylation of Akt, indicating the involvement of the PI3 kinase pathways while not affecting the phosphorylation state of p38 and ERK1 MAP kinase. In line with these results, incubation of monocytes with PI3 kinase inhibitors resulted in a similar phenotype of DC as described above. Gene expression profiling utilizing DNA microarrays revealed upregulation of lysosomal genes and molecules preferentially expressed in monocytes/macrophages. However, in contrast to these observations, imatinib treatment had no effect on the incorporation of latex beads by DC and resulted in a reduced FITC-labeled dextran uptake. Importantly, utilizing blocking antibodies and tyrosine kinase inhibitors we demonstrate that the inhibitory effects of imatinib on DC differentiation are not mediated by PDGF-R and c-Kit but most likely via c-Abl tyrosine kinase. These results demonstrate that imatinib affects the antigen presenting function of DC on several levels: their phenotype, antigen uptake and processing as well as production of cytokines and chemokines.
Long Term Follow-Up of Patients with Hairy Cell Leukemia (HCL).
Authors: Ravandi-Kashani Farhad , Kantarjian Hagop , Faderl Stefan , O’Brien Susan , Thomas Deborah , Koller Charles , Cortes Jorge , Pierce Sherry , Keating Michael , Estey Elihu
Published: October 2019

Interferon-a (IFN-a) and, more recently, nucleoside analogs (NA) such as 2-CDA and pentostatin have been used with significant success to treat HCL. A single course of 2-CDA produces response rates over 75%, with responses maintained for 4 years in 75% of pts. Similar results have been observed with pentostatin. (Dearden et al, Br J Haematol1999;106:515). To learn more about long-term outcome, we reviewed 234 pts with untreated HCL seen at the MDACC over a 30-year period; their median follow-up was 122 months from diagnosis (up to 387 months). Their median age at diagnosis was 50 years (range 23–83 years). Survival was superior among the 173 pts given either 2-CDA (n = 161) or pentostatin (n = 12) compared to those never given either NA (p = 0.042).

Figure Figure

Relapse occurred in 32 of 176 pts who received 2-CDA as their first chemotherapy either at MDACC or before presenting to MDACC. Therapy for relapse after 2-CDA included rituximab (N=9), a second course of 2-CDA (n=9), a second course of 2-CDA followed by rituximab (n=8), and other (n=6). No statistically significant differences in survival were seen possibly due to small pt numbers. The estimated 10-year and 20-year survival (from diagnosis) for the 176 pts who received 2-CDA as first chemotherapy was 84%, and 65%, respectively.

Figure Figure

The majority of pts who died were not in remission at time of death and thus presumably died of HCL. Hence alternatives to NA are needed in at least some pts, and we are evaluating whether covariates exist that might distinguish pts who do and do not relapse and, among the former, pts whose remissions are short. Pending identification of the latter and keeping in mind the length of the average remission, alternatives to NA should have very little toxicity. We have begun to add rituximab to 2-CDA to lengthen remission duration and thus extend survival in pts with untreated HCL.
A New Method of Retroviral Lineage Specific Expression Utilizing the Cre/lox System Induces a T-Lymphoid Malignancy in a Mouse Model of ALCL.
Authors: Miething Cornelius , Grundler Rebekka , Mugler Claudia , Peschel Christian , Duyster Justus
Published: October 2019

A t(2;5) (p23;q35) chromosomal translocation can be found in more than 50% of all cases of anaplastic large cell lymphoma (ALCL), which belong to the group of aggressive Non-Hodgkin-Lymphomas (NHL). The fusion of the nucleophosmin gene (NPM) on chromosome 5 to the anaplastic lymphoma kinase (ALK) on chromosome 2 leads to a deregulated, constitutively active kinase which has been shown to play an important role in lymphomagenesis of ALCL. Mouse models, where the NPM-ALK fusion oncogene is retrovirally expressed in whole bone marrow (BM) cells which are subsequently transplanted into lethally irrradiated recipients have shown that NPM-ALK is able to induce histiocytic and plasmacytoid malignancies. Since ALCLs are in the vast majority either of T- or ‘Null’-cell phenotype, we have now developed a murine model of T-lymphoid ALCL. By employing a retroviral vector incorporating a translational stop-cassette flanked by loxP recombination sites recognized by the Cre recombinase, we established a new method of lineage specific gene expression in-vivo. We tested the vector by infecting BM cells harvested from mice expressing Cre from different lineage specific promotors. We have infected BM cells from mice expressing Cre under the control of the lysozyme M-promotor (lysM-mice), which is active in the myeloid compartment, from mice expressing Cre from the granzyme B-promotor (GrzmB-mice), which is mainly active in T-cells, and finally we also used BM cells from C57B6 wildtype mice as a control. Mice transplanted with BM from lysM-mice succumbed to a histiocytic malignancy after 4–6 weeks, whereas mice transplanted with BM from GrzmB-mice developed a mixed T-NHL/histiocytic phenotype with a similar latency. These mice displayed infiltration of malignant, mostly CD4/CD8 DP T-cells in the thymus, lymphnodes and spleen, as well as a histiocytic infiltration in the spleen and BM. Mice transplanted with control BM from wt mice did not show signs of disease after up to 6 months. The results indicate that the hematopoetic lineage where an oncogene is expressed is paramount to the malignant phenotype

The described model represents a versatile tool for the analysis of NPM-ALK signaling pathways in-vivo. With the help of this method, any gene can be rapidly expressed in a defined lineage or in a developmental stage. Thus, also more subtle phenotypes may become amenable to investigation in-vivo.
Survival Factor Stimulates ERK-Dependent Phosphorylation of BIM Inhibiting Its Association with BAX and Pro-Apoptotic Activity.
Authors: Harada Hisashi , Quearry Bonnie , Korsmeyer Stanley J.
Published: October 2019

Growth factors and cytokines signal survival through their cognate receptors, activating signaling pathways often composed of protein kinase cascades. On the other hand, inadequate growth factor initiates a signaling cascade that leads to cell death. Both pro-and anti-apoptotic BCL-2 family members lie downstream of these proximal signals. The “multi-domain” BCL-2 members either suppress (e.g., BCL-2, BCL-XL) or promote apoptosis (e.g., BAX, BAK), whereas the “BH3-only” subfamily members identified to date (e.g., BAD, BID, BIM) function to promote cell death. It has become clear that BH3-only proteins are upstream initiators of apoptosis. BIM was identified as a BH3-only protein that induces apoptosis and can be antagonized by anti-apoptotic BCL-2 family members. Studies in BIM-deficient mice/cells indicate important roles for BIM in hematopoietic cell homeostasis. In the present study, an siRNA knockdown of BIM confirms this BH3-only member is important for the cytokine-mediated homeostasis of hematopoietic cells. We show that the phosphorylation status of BIM controls its pro-apoptotic activity. Interleukin-3 (IL-3), a hematopoietic survival factor, induces ERK/MAPK-mediated phosphorylation of BIM on three serine sites (Ser55, Ser65, Ser100). IL-3 withdrawal results in the dephosphorylation of BIM and only non-phosphorylated BIM interacts with the multi-domain pro-apoptotic effector BAX. Phosphorylation of BIM upon exposure of cells to IL-3 dramatically reduces the BIM/BAX interaction. A non-phosphorylatable BIM molecule (S55A, S65A, S100A) demonstrates enhanced interaction with BAX and enhanced pro-apoptotic activity. Thus, ERK/MAPK-dependent phosphorylation of BIM in response to survival factor regulates BIM/BAX interaction and the pro-death activity of BIM.
Post-Thrombotic Syndrome, Functional Disability and Quality of Life after Upper Extremity Deep Venous Thrombosis in Adults.
Authors: Kahn Susan R. , Elman Elyssa , Bornais Chantal , Blostein Mark , Wells Philip S.
Published: October 2019

Background: The post-thrombotic syndrome (PTS) after upper extremity deep venous thrombosis (UEDVT) has not been well characterized.

Objectives: To describe and quantify residual symptoms, functional disability and quality of life associated with post-thrombotic syndrome after UEDVT in adults.

Methods: Twenty-four patients with objectively diagnosed UEDVT (bilateral in 1 patient) at least 6 months previously were recruited from two thrombosis clinics in Canada (Montreal and Ottawa). Data were collected by interview and chart review on demographic characteristics, DVT risk factors and affected venous segments. The Villalta PTS scale, modified for the upper extremity, was used to diagnose PTS and to quantify its severity (score >4=PTS; score >14=severe PTS). Patients completed questionnaires on type and severity of symptoms, degree of functional disability (DASH questionnaire) (higher scores=greater disability), and generic (SF-36) and disease-specific (VEINES-QOL) quality of life (lower scores=worse QOL). Results of these measures were compared in patients with and without PTS.

Results: The mean age of study patients was 51 years and 54% were female. The average time since diagnosis of UEDVT was 13 months and median duration of warfarin treatment was 5 months. At the time of assessment, daily ipsilateral arm or hand swelling was reported by 52% of patients and daily ipsilateral arm pain by 20% of study patients, compared with 0% and 0%, respectively, in the contralateral arm. PTS was present in 11/25 (44%) limbs (11/24 patients). One patient had severe PTS. Patients with PTS had significantly higher mean DASH scores than those without PTS (23.3 vs. 3.9, p= 0.009) and poorer quality of life than those without PTS (mean VEINES-QOL score 45.6 vs. 53.6; p=0.001; mean SF-36 Physical Component Score (PCS) 40.8 vs. 50.2; p= 0.12). Although the overall frequency of PTS did not differ according to whether the initial UEDVT was in the dominant arm, PTS scores were higher and quality of life was poorer when PTS involved the dominant arm.

Conclusion: PTS occurs frequently after UEDVT at a rate that is similar to that of lower limb DVT. PTS of the upper extremity is associated with significant functional disability and has a measurable, adverse impact on quality of life. Patients with dominant arm PTS appear to fare worse than those with non-dominant arm PTS. Larger, prospective studies to identify prognostic factors that lead to PTS after UEDVT are warranted.
Megakaryocyte Specific Cre Transgenic Mouse.
Authors: Gushiken Francisca C. , Adachi Roberto , Lopez Jose A.
Published: October 2019

Here we report the first animal model with specific expression of Cre recombinase in megakaryocytes. Cre is an enzyme from bacteriophage-P1 that catalyzes reciprocal DNA recombination between pairs of loxP sites that results in excision of the DNA fragment located between them. The expression of Cre can be genetically manipulated by tissue specific promoters. When a tissue specific Cre mouse is crossed with a mouse line who has a gene flanked by loxP sites, the flanked gene can be deleted in a tissue specific manner. To target selective expression in megakaryocytes we used the promoter region of platelet factor-4 (PF4). The DNA transgenic construct consisted of the 387-bp of the promoter region of human PF4 (nucleotides −312 to +75) followed by the cDNA of Cre cloned at the start codon of the native gene, and the polyA sequence of the human β-globin gene. Transgenic animals were generated using routine pronuclear injection of fertilized oocytes. Transgenic mice were genotyped by a combination of Southern blot analysis and PCR. To evaluate functionality and tissue specificity of Cre expression, hPF4-Cre transgenic mice were crossed with ROSA26-lacZ reporter mice (B6.129S4-Gt(ROSA)26Sortm1Sor). In this reporter the cDNA for lacZ has been introduced into the Rosa26 locus preceded by an intervening sequence flanked by two loxP sites. In cells expressing Cre, recombination at the two loxP sites produces excision of the intervening sequence allowing the expression lacZ, thus β-galactosidase activity is a surrogate for Cre activity. β-galactosidase activity was analyzed by X-gal assay in megakaryocytes of bone marrow harvested from animals harboring the Cre transgene and reporter gene. We used the littermates that were either ROSA26-lacZ reporter/WT or WT/hPF4- Cre as negative controls, and Tg-ROSA26 (B6;129S-Gt(ROSA)26Sor), a strain that expresses lacZ in all tissues, as positive control. We have documented β-galactosidase activity that is present only in megakaryocytes and platelets in 6 established transgenic lines. In conclusion, we have generated megakaryocyte specific Cre mice using the promoter region of human platelet factor 4 (hPF4). Crossing this mouse with a loxP mouse will result in deletion of the loxP-flanked gene. This deletion will happen only in megakaryocytes. This tissue specific transgenic line is a powerful research tool for gene targeting experiments in platelets.

Figure Figure
Thrombin Regulates the Camp Level in Human Platelets through Phosphorylation /Activation of Akt.
Authors: Zhang Wei , Nale Gary , Colman Robert W.
Published: October 2019

Phosphorylation /activation of Akt is a critical event in platelet activation stimulated by thrombin. We previous demonstrated that the activation of PDE3A was increased concomitantly with the phosphorylation /activation of Akt. In order to demonstrate a link between these two events, in this study, we monitored thrombin-induced cAMP changes in washed platelets. We confirmed that the platelet cAMP level decreased following thrombin stimulation. The thrombin-induced decrease of cAMP was inhibited by an Akt specific inhibitor (1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate). This Akt inhibitor exhibited a concentration-dependent inhibition of thrombin-induced decrease of cAMP. The Akt inhibitor blocked 70 % of thrombin-induced decrease of cAMP. Moreover, a phosphoinositide 3-kinase inhibitor wortmannin also reduced the thrombin effect, consistent with its upstream position relative to Akt. These results suggested that phosphorylation /activation of Akt is required for decrease of cAMP in platelets. In addition, we found that thrombin-induced cAMP level changes were markedly reduced following preincubation of platelets with milrinone, a selective PDE3A inhibitor. We therefore examined PDE3A activity after stimulation of platelets with thrombin with or without the Akt inhibitor or wortmannin. Either the Akt inhibitor or wortmannin inhibited the thrombin-induced activation of PDE3A. Our data indicated that phosphorylation/activation of Akt plays a major role in regulation of cAMP by thrombin. The activation of Akt by thrombin results in activation of PDE3A and a consequent decrease of the intracellular cAMP level, which serves as a positive feedback enhancing the thrombin activation of platelet function. Understanding the mechanisms that are involved in regulation of PDE3A in platelets could provide new targets for therapeutic advances in the treatment of thrombotic disorders.
Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.
Authors: Murugappan Swaminathan , Shankar Haripriya , Kunapuli Satya
Published: October 2019

Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.
FOG-1 Directly Binds to a Chromatin Remodeling and Deacetylase-Containing Complex To Mediate Transcriptional Repression by GATA-1.
Authors: Hong Wei , Nakazawa Minako , Chen Ying-Yu , Kori Rajashree , Rakowski Carrie , Blobel Md Gerd A.
Published: October 2019

Terminal erythroid maturation requires coordinated activation of erythroid marker genes and repression of genes associated with the undifferentiated state. These gene expression patterns are mediated by the concerted action of the erythroid transcription factor GATA-1 and its cofactor FOG-1 that can activate or repress transcription depending on promoter context. We and others showed previously that one mechanism by which FOG-1 functions is to facilitate GATA-1 association with certain DNA target sites in vivo. Using gene complementation studies of GATA-1-ablated erythroid cells, we show that at several GATA-1-repressed target genes (c-kit, c-myc and GATA-2) FOG-1 is dispensable for GATA-1 occupancy in vivo but essential for gene repression and histone deacetylation. To examine how FOG-1 functions as co-repressor we performed affinity chromatography, conventional protein purification and in vitro binding studies to identify proteins that bind FOG-1. We discovered that FOG-1 directly associates with the nucleosome remodeling and histone deacetylase complex NURD. This interaction is mediated by a small conserved domain at the N-terminus of FOG-1 and the MTA-1 subunit of NURD. Association of FOG-1 with NURD occurs in vivo and depends on an intact N-terminus of FOG-1. A series of point mutations across the N-terminus of FOG-1 revealed a tight correlation between NURD binding and transcriptional repression. In particular, a single point mutation at the N-terminus of FOG-1 that abrogated NURD binding also blocked gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing a mutant form of FOG-1 that is defective for NURD binding. Together, these studies show that FOG-1 and very likely other FOG proteins are bona fide co-repressors that link GATA proteins to histone deacetylation and nucleosome remodeling via a novel protein interaction module.
Protective Effects of No-Albumin and Albumin on Lung Injury Induced by Hypoxia/Reoxygenation in a Mouse Model of Sickle Cell Disease.
Authors: De Franceschi Lucia , Malpeli Giorgio , Janin Anne , Muchitsch Eva , Scarpa Aldo , Leboulch Philippe , Leboeuf Christhope , Beuzard Yves , Brugnara Carlo
Published: October 2019

Clinical manifestations of sickle cell disease are related to vaso-occlusive (VOC) events, which are responsible for acute and chronic organ damages. We have recently shown the beneficial effects of inhaled nitric oxide (NO) in both transgenic sickle cell (SAD) mice exposed to hypoxia/reoxygenation (H/R) and in sickle cell children with acute painful crisis (Blood102: 1097, 2003; JAMA289: 1136,2003). Polynitroxyl-albumine (PNA), nitrosylated at position 34, may represent a new therapeutic tool in acute sickle cell VOCs. In SAD mice, PNA was intraperitoneally (ip) administrated and a dose-response curve was determined. Methemoglobin (MetHb) levels and blood pressure were measured under normoxia. PNA, 300 mg/Kg twice a day (equal to 4.5 mM/Kg twice a d.), induced a significant but transitory decrease in blood pressure 60 min after ip injection, while MetHb levels did not change. The effects of PNA and human serum albumine (hsAlb) on ischemia/reperfusion (H/R) lung injury related to sickle cell disease were then studied. SAD mice were divided into 4 groups of 6 animals each: one served as normoxia untreated control. The other groups were exposed to 46 hrs hypoxia (8% O2) followed by 2 hrs normoxia: one served as untreated hypoxic group, one was treated with hsAlb (300 mg/Kg twice a day) and one with PNA (300 mg/Kg twice a day). We evaluated lung histopathology, complete blood counts, bronchoalveolar (BAL) fluid neutrophil counts, cytokine levels, which are activated by H/R stimulus and are modulated by inhaled NO (Blood102:1087, 2003).

In addition to histological lung damage, H/R induced an increase in blood neutrophils, a market increase in BAL total leukocyte and neutrophil counts, an increase in BAL IL-6 and IL-1β, with no significant changes in BAL IL-10 and TNF-α levels. PNA prevented the H/R lung injury, determined a reduction in H/R mediated increase in blood neutrophils, prevented the increase in BAL total leukocyte and neutrophil counts and in BAL IL-6 and IL-1β levels, while no change was observed in BAL IL-10, TNF-α and MetHb levels. hsAlb ameliorates the H/R lung injury but it did not affect either the H/R induced blood neutrophils, and BAL cells and cytokines. These results show that the beneficial effects of PNA are similar to those observed with inhaled NO and that PNA could be therapeutically efficacious in sickle cell disease.
An Animal Model Suitable for Predicting In Vivo Behavior of Platelets in Humans: A Tool for New Product Development.
Authors: Blajchman Morris A. , Li Junzhi , Kulczycky Myron , Woskowska Zofia , Pieters Henry , Roodt Jan , Badenhorst Philip , Crookes Robert , Heyns Anthon , Goodrich Raymond
Published: October 2019

Testing platelet in vivo activity in an animal model remains an attractive approach for the pre-clinical evaluation of platelets that have been treated or manipulated by a new technology or product. Mirasol PRT™ is a novel pathogen reduction technology based on riboflavin photochemistry for the safety of blood component transfusion. In this study, rabbit platelets were tested for viability after exposure to 2, 3 and 5 J/cm2 of UV light in the presence of 50 μM riboflavin in a polypropylene bag (Sangewald). In order to minimize the potential variations caused by different isotope labeling and by individual rabbit differences, a pool of platelets was split into two for the treatment and control groups. Eight to ten rabbits were used in each group as recipients for each dose of light. Half of the rabbits in the group received concurrent injections of 111In-labeled PRT treated platelets and 51Cr-labeled control platelets. The other half received 51Cr-labeled PRT-platelets and 111In-labeled control platelets. The recovery and survival times were determined post-infusion for both treated and control platelets using the multiple hit model (γ-function) and data from the various groups were pooled and analyzed. The platelets treated with 2, 3 and 5 J/cm2 retained recoveries of 63.2% (SD=17, n=9), 55.3% (SD=10.8, n=10) and 44.0% (SD=12.8, n=8) of control platelets, respectively. The survival times of the platelets treated with 2, 3 and 5 J/cm2 were 59.5% (SD=15.7, n=9), 60.1% (SD=19.5, n=10) and 55.1% (SD=18.1, n=8) of control, respectively. Both values of recovery and survival times were correlated with UV light dose with correlation coefficients of 0.90 (r2) and 0.73 (r2) respectively. In a subsequent single site human clinical study, human platelets were treated with UV light at a dose of 3 J/cm2 in a Sangewald bag, radiolabelled with Indium at day 5 of storage and infused into autologous subjects. Recovery and survival values for these subjects were obtained. At a dose of 3 J/cm2, recovery and survival activities retained 54.9% (SD=3.1, n=4) and 71.7% (SD=21.5) of control platelets, respectively. Good agreement with observations made in the rabbit model was thus obtained. Based on this work, a light dose was selected for standard treatment of human platelets using the Mirasol PRT system. In conclusion, this kinetic study on UV light dose in an animal model was predictive of human platelet in vivo activity, making it a potentially valuable model for estimating human clinical trial outcomes for new platelet storage and treatment methodologies.
An Increase in the Immature Platelet Fraction Predicts Reconstitution of the Platelet Count and May Reduce Prophylactic Platelet Transfusions after Stem Cell Transplantation.
Authors: Machin Samuel J. , Hart Dan , Kunka Stefan , Briggs Carol , Corash Laurence
Published: October 2019

A new automated method to reliably quantitate reticulated platelets, expressed as the immature platelet fraction (IPF), has been developed on an automated cell counter (XE-2100, Sysmex). The IPF is identified by flow cytometery using a polymethine dye, staining platelet RNA, in the reticulocyte channel; the results are available at the same time as the CBC. The IPF normal range is 1.1–6.1%, mean 3.4%, 2 SD 2.3%. Reproducibility and stability results over 48 hours were acceptable. The IPF is raised when there is increased peripheral consumption/destruction. In untreated idopathic thrombocytopenic purpura, n = 12, mean 22.3%, range 9.2–33.1% and active thrombotic thrombocytopenic purpura, n = 5, mean 17.2%, range 11.2–30.9%. Patients who may require prophylactic platelet transfusion, usually at threshold counts less than 10 x 109/L, to support periods of marrow aplasia were monitored daily for platelet count and IPF%. The recovery phase of thrombocytopenia in most chemotherapy (n=13) and stem cell/bone marrow transplant patients (n=15) was preceded by a rise in IPF% several days prior to platelet recovery, mean IPF 13.7%, range 7–27.3%. In particular, patients undergoing autologous transplantation (n=8) using peripherally collected stem cells have a very characteristic IPF% motif, with a rise 1 day prior to engraftment for all patients except one where it was 2 days prior. For bone marrow derived transplant patients the increase in IPF was more variable, the rise preceded the rise in platelet count by 2–7days. These patients suffer more septic episodes where there is a rise in the IPF with no immediate increase in the platelet count, and require more regular platelet transfusions. Following a platelet transfusion there is a 24-hour transitory fall in the IPF response, which may impede platelet recovery. A parameter that could predict the timing of platelet recovery could be used clinically to reduce the use of prophylactic platelet transfusion in these patients, thus minimising donor exposure, infection risk and allowing substantial financial savings. The IPF is a useful parameter in the evaluation of the thrombocytopenic patient and has the potential to allow more optimal transfusion of platelet concentrates.