bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting Pim Kinases in Hematological Malignancies.
Authors: Gourley Eric S. , Liu Xiao-Hui , Lamb Jeremy D. , Vankayalapati Hariprasad , Grand Cory L. , Bearss David J.
Published: October 2019

The three Pim kinases represent a small subfamily of serine/threonine kinases known to be involved in a number of signaling pathways as downstream effectors and potent inhibitors of apoptosis. Unlike most other kinases, Pim kinases lack a regulatory domain which means they are controlled largely at the transcriptional level and that the mRNA expression levels of Pim kinases in cells correlate with their activity. While normal expression of Pim-1 kinase is seen in cells of hematopoietic origin, examination of gene expression of Pim-1 in different malignancies using cDNA microarray analysis suggests that Pim-1 is overexpressed in a large percentage of ALL, AML, CML, DLBCL, Prostatic Adenocarcinoma, Bladder, and Oral cancers. Pim-2 is also largely expressed in ALL, AML, Squamous Cell Lung and Adenocarcinoma of the Lung while Pim-3 is restricted to Melanoma, Pancreatic Adenocarcinoma, Gastric cancers. This implicates Pim-1 and Pim-2 in the onset and progression in several of hematological malignancies and therefore they represent interesting potential targets for drug development. To evaluate the potential of Pim-1 and -2 as a drug targets we have identified and synthesized a series of Pim kinase inhibitors using our proprietary CLIMB™ drug discovery process. Through the use of CLIMB™, the published Pim-1 kinase crystal structure was used to build several models that were then used to predict potential small molecule inhibitors of Pim-1 and Pim-2 from a large virtual library. A subset of leads, based on calculated binding energies as well as additional physical chemical properties, were screened using a number of in silico physicochemical and ADMET prediction algorithms to determine which compounds were most likely to be successful in a biological context. Lead candidates were initially screened using biochemical enzyme-based and cell-based assays. Cell-based activity was determined in HEL (acute megakaryocytic leukemia), K562 (chronic myeloid leukemia), MO7e (myeloid leukemia), and other human leukemia and lymphoma cell lines. From several lead candidates a series of analogs were produced with improved inhibitory activity and pharmacokinetic characteristics. In the Pim-1 and Pim-2 in vitro kinase assay and in the cell-based assay a number of leads exhibited inhibitory activity with IC50 concentrations in the low nanomolar range. Here we present the details of the biochemical and cell-based assay results as well as the activity in tumor xenograft models of our Pim kinase inhibitors.
Targeting the JAK2 Kinase in Hematological Malignancies.
Authors: Olsen Colin E. , Gourley Eric S. , Lui Xiao-Hui , Walker Jeff , Grand Cory L. , Warner Steven L. , Vankayalapati Hariprasad , Bearss David J.
Published: October 2019

JAK2 is an intracellular protein tyrosine kinase whose dysregulation has been implicated in leukemia, lymphoma, and myeloproliferative disorders (MPD). Increased kinase activity of JAK2, caused by point mutation of the JH2 autoinhibitory region or formation of JAK2 fusion proteins, causes increased activation of downstream signaling pathways affecting cell differentiation, proliferation, migration, and apoptosis. Through the use of CLIMB™, our proprietary drug discovery process, the published JAK2 crystal structure was used to build several models that were then used as a substrate for in silico docking of 2.3 million virtual small molecule compounds to generate a subset of leads based on calculated binding energies. These leads were further screened using a number of in silico physicochemical and ADMET prediction algorithms to determine “druggable” leads which were most likely to be successful in a biological context. Lead JAK2 inhibitor candidates exhibit low nanomolar IC50 activity against the JAK2 and JAK2 V617F mutant enzymes. Cancer cell lines expressing either the wild-type or mutant JAK2 enzyme demonstrate sensitivity to these inhibitors resulting in IC50 values in low micromolar to nanomolar range. Consistent with the inhibition of the JAK2 enzyme, activity of downstream signaling partners are severely decreased. The phosphorylation level of STAT5, a downstream modulator of JAK2 signaling, in treated HEL cell lysates was analyzed by western blot analysis. These results showed that lead JAK2 candidates caused an inhibition of STAT5 phosphorylation at a low nanomolar EC50. This series of compounds are currently being tested in in vivo xenograft models. Evaluation of lead candidates in biochemical assays against the hERG and CYP450 enzymes showed that these compounds have little inhibitory activity against these enzymes. SuperGen’s lead selective JAK2 inhibitors exhibit potent inhibition of wild-type and mutant JAK2 kinase activity translating into potent inhibition of cellular signaling pathways and cancer cell proliferation.
Rapid and Sensitive Single Tube Screen for Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Aplastic Anemia, Myelodysplastic Syndrome and Bone Marrow Failure Syndromes by Multiparameter Flow Cytometry.
Authors: Movalia Mayur K. , Illingworth Andrea
Published: October 2019

Several studies have shown that the presence of glycosylphosphatidylinositol (GPI) anchored protein-deficient PNH-type cells in patients with aplastic anemia and myelodysplastic syndrome may indicate favorable response to therapy and an overall favorable prognosis. Multiparameter flow cytometry is a powerful tool in the detection of even minimal populations of these cells. Evaluation for PNH-type clones has typically required in-depth analysis of red blood cells, granulocytes and monocytes using three tubes looking for the loss of multiple GPI-anchored proteins. Our laboratory has used this method in over 2,100 patients with known aplastic anemia and myelodysplastic syndrome. Accurate PNH-type clone evaluation requires careful backgating, strict gating strategies and a certain level of experience as immature cells, including blasts, may mimic PNH-type clones. The aim of this study was to develop a single tube five-color assay that could be used as a rapid and sensitive screen for the presence of PNH-type cells. We prospectively analyzed the granulocytes in the peripheral blood of 27 patients with known aplastic anemia and myelodysplastic syndrome using a single tube 5-color assay with FLAER-Alexa488 / CD24-PE / CD14-ECD / CD15-PC5 / CD45-PC7. This one-tube approach allows lineage specific gating on granulocytes looking for CD24 and FLAER deficiency. Also, to a more limited extent, the monocytes can be evaluated for CD14 and FLAER deficiency. The results were compared to a more in-depth three tube analysis for PNH-type cells analyzing the red blood cells (GPA-FITC/CD59-PE), granulocytes (CD66b-FITC/ CD24-PE/ CD45-ECD/ CD15-PC5/ CD16) and monocytes (CD64-FITC/ CD55-PE/ CD45-ECD/ CD33-PC5/ CD14). The results are summarized in Table 1. Using a detection sensitivity of 0.05% among the granulocytes showing loss of CD24 and FLAER, the screening assay was able to detect all six patients with PNH-type clones (range 0.4% – 79.1%), which was confirmed by the multiple tube panel. Of the 21 negative samples, all were below the 0.05% limit of detection. Four samples did show a population of CD24-negative, FLAER-negative granulocytes ranging from 0.01%–0.02%. The overall sensitivity and specificity of the single tube screen was 100%. This single tube five-color approach allows for a rapid and simple approach to screening patients for PNH-type clones with a detection sensitivity of 0.05%. Although an extended multiple tube analysis may still be required on positive screening samples to evaluate for the type of red blood cell PNH clone, the single tube screen optimizes the testing approach for PNH-type cells.

Single tube screen versus multiple tube analysis for PNH-type Clones Multiple tube Multiple tube Positive Negative Total Positive = greater than or equal to 0.05% PNH-type cells detected Single tube Positive 6 (22%) 0 (0%) 6 Single tube Negative 0 (0%) 21 (78%) 21 Total 6 21
Evidence for HLA Class II Susceptible and Protective Haplotypes for Osteomyelitis in Pediatric Patients with Sickle Cell Anemia.
Authors: Almawi Wassim Y. , Mahdi Najat , Al-Ola Khadija , Ali Muhallab E. , Al-Subaie Abeer M.
Published: October 2019

Background and Objective: Osteomyelitis (OM) is a common complication and significant cause of morbidity in patients with sickle cell anemia (SCA); however, its mechanisms are poorly understood. In view of their link to inflammatory diseases and autoimmune disorders, we investigated the association of HLA class II alleles and haplotypes with the pathogenesis of OM in Bahraini SCA patients.

Patients and Methods. SCA patients comprised 42 with and 150 patients without OM; HLA-DRB1* and -DQB1* genotyping was done by PCR-SSP. HLA allele frequency was determined by the gene counting method, and haplotype frequency was determined by the maximum-likelihood method.

Results. At the allele level, only DRB1*100101 (0.229 vs. 0.082, Pc = 0.003) was positively associated with OM after applying the Bonferrni correction factor. At the haplotype level, DRB1*070101-DQB1*0201 (Pc = 0.001), and DRB1*100101-DQB1*050101 (Pc = 0.001) were more prevalent among patients, while DRB1*030101-DQB1*0201 (Pc = 0.020) and DRB1*040101-DQB1*0302 (Pc = 0.039) was more prevalent among controls, thereby conferring disease susceptibility or protection to these DRB1*-DQB1* haplotypes, respectively.

Conclusion. These results demonstrate that specific HLA halpotypes influence osteomyelitis risk in SCA, thereby suggesting that specific HLA types may serve as a marker for identifying SCA patients at high risk for osteomyelitis.

HLA-DRB1* and DQB1* Haplotype Frequencies Haplotype OM Patients Controls P Pc OR DRB1*030101-DQB1*0201 0.015 0.141 0.002 0.020 0.07 DRB1*030201-DQB1*0401 0.042 0.016 0.212 0.908 2.95 DRB1*040101-DQB1*0302 0.016 0.126 0.004 0.039 0.08 DRB1*070101-DQB1*0201 0.219 0.060 <0.001 0.001 4.58 DRB1*110101-DQB1*030101 0.056 0.108 0.278 0.962 0.53 DRB1*100101-DQB1*050101 0.208 0.063 <0.001 0.001 4.03 DRB1*130101-DQB1*060101 0.042 0.034 0.773 1.000 1.45 DRB1*150101-DQB1*060101 0.085 0.068 0.774 1.000 1.27 DRB1*160101-DQB1*0201 0.064 0.020 0.121 0.725 3.10 DRB1*160101-DQB1*050101 0.011 0.099 0.016 0.149 0.11
Evaluation of a Dye Binding Method for Determining the Solution Concentration of Low Molecular Weight Heparins.
Authors: Kim Yumi , John Boggs , Megan Hunt , Seneviratne Janaki , Yamamoto Ralph
Published: October 2019

Low Molecular Weight Heparins are carbohydrate-based therapeutics with anticoagulant activities. Common brands include: Lovenox, Fragmin and Arixtra, which are currently used to treat Deep Vein Thromobosis. Unlike protein or peptide-based drugs, easy test methods for measuring the concentration of carbohydrates in solution are not available. In this report, we extend the use of commercially available dye-binding reagents and protocol (Biocolor Ltd, Ireland) for rapidly measuring the solution concentration of LMWHs. The assay was evaluated according to typical validation parameters: Linearity, Accuracy, Precision (Reproducibility and Intermediate), Solution Stability and Specificity. The assay is also capable of monitoring LMWH “stability” as changes in the dye binding response appears associated with changes in Anti-fXa and anti-fIIa activity, Molecular Weight and Free Sulfate levels. The assay was also used to measure the concentration of two Products, Lovenox (99.6% of claim) and Fragmin (94% of claim), from pre-filled syringes.
A 21-Year-Old Woman with Thrombotic Thrombocytopenic Purpura as Initial Symptom of Systemic Lupus Erythematosus and a Literature Review.
Authors: Zhou Rongfu , OuYang Jian , Zhou Ming , Zhang Qiguo , Xu Yong
Published: October 2019

We describe a 21-year-old woman with systemic lupus erythematosus (SLE) who first presented with an thrombotic thrombocytopenic purpura (TTP), and compare the clinical manifestations and prognosis between SLE patients with thrombotic thrombocytopenic purpura in the reported literature. At the time of admission, she was suffering from petechia, purpura and had neurological symptoms. Laboratory findings demonstrated haemolytic anaemia, thrombocytopaenia and high levels of fragmentocytes. Serological test results were highly positive for antinuclear antibodies (ANA) with a particle fluorescent pattern, and also detected antibodies against Smith antibody, RNP antibody and SSA antibody but was negative for dsDNA. Direct Coombs-test was positive and D-dimer was negtive. The activity of ADAMTS13 was significantly reduced even after 4 times plasmapheresis. TTP as presenting sign in the patient with SLE was diagnosed. After 5 times plasmapheresis treatments and immunosuppressive therapy she recovered and abnormal laboratory tests were gradually returned to normal. With a follow-up of 20 months, she had a normal life. Compared with other reports, TTP can be differentiated from other thrombotic microangiopathic syndromes by its normal levels of prothrombin time, partially activated thromboplastin time (APTT), fibrinogen and direct Coombs-test. But TTP might also be a complication of SLE and the manifestations of TTP are similar to those in SLE. The detection of the fragmentation of peripheral red blood cells helped the early diagnosis of TTP. Coomb’s test might be positive when patient with TTP had SLE. ADAMTS13 and its inhibitory antibody had an important role in the pathogenesis of TTP. Plasma exchange combined with corticosteroid and cyclophsphamide should be used as early as possible in TTP patient with SLE. The prognosis was related to the treatment time and methods including plasmapheresis.
Determination of ADAMTS13 Antigen and Activity Levels in Patients with Acute Myocardial Infarction and Acute Ischemic Stroke.
Authors: Dong Ningzheng , Liu Fang , Ji Shundong , Ruan Changgeng
Published: October 2019

von Willebrand factor-cleaving protease (ADMTS13) is a metalloproteases regulating von Willebrand factor multimers size in plasma, its activity decrease has been reported in many physiological and pathological conditions. But information on ADAMTS13 in arterial thrombosis disease and in the general population is limited. In this study, we investigated the ADAMTS13 antigen and activity levels in 87 healthy control subjects of different ages, in 93 patients with acute myocardial infarction (AMI) and in 30 patients with acute ischemic stroke (AIS). ADAMTS13 Antigen levels were measured using a sandwich ELISA, and ADAMTS13 activity levels were analyzed by the FRETS-VWF73 method. A pool of plasma obtained from 40 blood donors was used as reference and was defined to contain 100% of the protease. The individual subject of the pool was not included in the study. Compared with 50 sex- and age-matched control subjects, ADAMTS13 antigen and activity levels were significantly lower in patients with arterial thrombosis diseases. Antigen levels were 87.8±19.8%, 61.8±18.8%, and 70.2±15.5% respectively in control, AMI and ASI patients. ADAMTS13 activity were 81.7±13.9%, 59.2±22.1% and 65.4±15.8% respectively. In the 87 healthy subjects, the range of ADAMTS13 activity is from 46.3% to 119.0%, and antigen is from 60.8% to 141.9%. In different age groups (17 to 20 years old, 21 to 40 years old, 41 to 60 years old, and older than 60 years old), ADAMTS13 antigen levels were 76.0%, 109.7%, 99.3%, and 74.5% respectively, and activity levels were 80.8%, 81.1%, 77.9%, and 87.3% respectively. It seems the antigen reached the highest level in people from 21 to 40 years old, then gradually decreased with age. The antigen and activity mean in males (87.1% and 80.8%) was lower than that in females (94.7% and 86.1%), but the difference is not significant (p=0.145 and p=0.124). In conclusion, the decrease of ADAMTS13 antigen and activity in AMI and AIS patients indicated ADMATS13 might contribute to arterial thrombosis, and the plasma ADAMTS13 antigen level in the general population was significantly affected by age.
Rituximab in Warfarin Resistance Treatment in Patients with Thrombophilia Due to Primary Antiphospholipid Syndrome: A Pilot Study.
Authors: Chavez Jaime Garcia , Mendoza-Torres Moises L. , Vela-Ojeda Jorge , Reynoso Eduardo E.
Published: October 2019

Introduction: The inability to obtain a therapeutic value of the INR regardless of the warfarin high doses administration represents a significant problem to resolve in the oral anticoagulation therapy, this phenomena is called “Warfarin Resistance”. Based on the experience and success of Rituximab in the treatment of autoimmune diseases like Immune Thrombocytopenic Purpura, we treated 7 patients with Primary Antiphospholipid Syndrome (PAS) and Warfarin Resistance.

Objective: To assess if Rituximab can revert the warfarin resistance in patients with thrombophilia associated to PAS

Material and Methods: We evaluated 7 patients with PAS who were receiving 15 mg of warfarin and had persistent low INR values. Rituximab was given weekly at dose of 375 mg/m2 for 4 doses. Effectiveness and safety were registered weekly during 2 months since the start of treatment with clinical evaluation and INR, aPTT, warfarin serum levels and CD20 blood lymphocytes count determinations.

Results: All patients were women with a mean of 35 years old (37– 48) and INR of 1.11 (0.79–1.50). All the patients (100%) reached the therapeutic level of INR between days 8 to 15, but the response lasted only a mean of 24 days (8 – 40). The maximum INR values were observed through the 2nd to the 3rd doses of Rituximab. The warfarin serum levels did not change during the assessment period.

Conclusion: Rituximab was effective in reverting resistance to Warfarin therapy in patients with thrombosis for primary Antiphospholipid Syndrome, however it lasted a very short time and eventually all patients relapsed.
Hemoglobin Levels Prior to Blood Transfusions in Oncology Patients Receiving Chemotherapy and Erythropoiesis-Stimulating Agents (ESAs): Observational Data from the DOSE Registry.
Authors: Larholt Kay M. , Burton Tanya , Pashos Chris L. , Bookhart Brahim K. , Piech Catherine Tak , McKenzie Scott R.
Published: October 2019

Objective: Blood transfusions in oncology patients (pts) occur at various hemoglobin (Hb) levels depending on each pt’s characteristics and situation, including underlying malignancy, treatment and comorbidities. Minimal data on the Hb levels at which transfusions are given in these pts are available. To characterize those Hb levels, or transfusion triggers, in oncology pts receiving ESAs, observational data from an ongoing registry were analyzed.

Methods: Real-world data on ESA-treated oncology pts in United States oncology clinics were analyzed from an ongoing prospective, observational registry (Dosing and Outcomes Study of Erythropoiesis-Stimulating Therapies [DOSE}). Data were collected from participating hospital- and community-based outpatient practices between December 2003 and July 2007. Hb values within 3 days prior to transfusion were assessed and summarized. If multiple Hb values were recorded, the Hb value closest to the transfusion was reported. Similarly, if two Hb values occurred on the same day, the lower of the values was reported.

Results: Of 334 transfusions, 314 had a Hb reported within 3 days. While the mean transfusion trigger was 8.55g/dL (SD 1.1), the trigger ranged from <7 to >10g/dL. Sixty-nine percent of transfusions occurred with a Hb trigger of ≥8g/dL, including 9% with a transfusion trigger >10g/dL, as described in the table below. Subgroup analyses based on age (< 65 v ≥65) and payer type (Medicare v other) reported similar findings.

Conclusion: Data from this ongoing observational registry found that the Hb transfusion trigger in oncology pts treated with ESAs varies considerably, with the majority of transfusion events occurring at Hb ≥8.0g/dL and approximately 9% occurring at Hb>10g/dL. Further analyses should explore pt characteristics as well as clinic-specific or other factors that may help to explain this variation.

Transfusion trigger <7g/dL 7–7.9g/dL 8–8.9g/dL 9–9.9g/dL >10g/dL All patients Number of transfusions (%) (N=314) 12 (4%) 89 (28%) 140 (45%) 46 (15%) 27 (9%) Subset by age Age≥65 (n=155) 7(5%) 45 (29%) 70 (45%) 19 (12%) 14 (9%) Age <65 (n=159) 5 (3%) 44 (28%) 70 (44%) 27 (17%) 13 (8%) Subset by payer type Medicare (n=150) 7 (5%) 45 (30%) 62 (41%) 19 (13%) 17 (11%) Other (n=164) 5 (3%) 44 (27%) 78 (48%) 27 (16%) 10 (6%)
Augmentation of Vasculogenesis by Injected Human Hematopoietic Stem Cells in Response to Ischemia Follows Early Activation of Innate Immune Response.
Authors: Finney Marcie R. , Fanning Laura R. , Kozik Margaret , Matsuyama Shigemi , Joseph Matthew E. , Goldberg Johnathan L. , Greco Nicholas J. , Haviernik Peter , Fu Pingfu , Pompili Vincent J. , Laughlin Mary J.
Published: October 2019

The immunocompromised NOD.SCID murine model of hindlimb ischemia has been used extensively to test in vivo the impact of human hematopoietic stem cell (HSC) therapy on neovascularization. Although NOD.SCID lack T and B cell adaptive immune responses, innate responses are intact. We utilized this model to test the hypothesis whether injection of human HSC following femoral ligation impacts neovascularization within the ischemic bed and whether early murine innate cellular responses differ in animals treated with HSC. NOD.SCID underwent femoral ligation and were divided into 3 study groups: controls (media alone), human HSC (CD133+ HSC selected from umbilical cord blood-UCB), or human peripheral blood mononuclear cells. Laser Doppler studies day +28 after femoral ligation showed significantly improved blood flow ratio in animals treated with human HSC vs. controls, 0.55 ± 0.06 (n=9) and 0.37 ± 0.03 (n=12, p<0.05) respectfully. Histology day +42 confirmed increased capillary density in animals treated with human HSC (320±18 cells/mm2) when compared to control animals (131±.6.9 cells/mm2; p<0.0001). Gene expression microarrays (Affymetrix Murine GeneChip 430 2.0) of RNA extracted from tissues harvested from the ischemic limbs 3 days following femoral ligation and injection of cells (n=3 each experimental group) comparing control mice to those receiving human HSC vs. controls revealed 414 genes with 2-fold or greater change. Included in these were genes regulating innate immunity: monocyte chemoattractant protein-5 precursor (MCP-5), CCL11, CXCL5, CXCL9, IL4 receptor, CXCL10, CCL5 and toll-like receptor 3 (TLR3). Of note large expression differences were noted comparing animals treated with human HSC including kruppel-like factor 15 (4 fold increase), IL6 receptor (3.25 fold increase), cyclin A2 (4.25 fold decrease). No significant increases in genes regulating angiogenesis were noted comparing animals treated with human HSC vs controls, including VEGF, angiopoietin, or bFGF. In summary, NOD.SCID treated with human HSC after femoral ligation demonstate significantly improved capillary density and blood flow recovery at later time points (day 28–42). Proteins regulating innate immunity are significantly higher at early time points in animals treated with human HSC suggesting an important role for inflammatory responses leading to augmentation of vasculogenesis after acute vascular injury.
Peripheral Localization and Regulation of the Murine β-Globin Locus.
Authors: Ragoczy Tobias , Telling Agnes , Byron Rachel , Bender M.A. , Groudine Mark
Published: October 2019

The interphase cell nucleus is structurally and functionally compartmentalized, making the subnuclear position of genes an important determinant of their activity. During cellular differentiation, as tissue-specific expression programs unfold, dynamic reorganization of the nucleus positions sets of genes in active or repressive compartments. The nuclear periphery has emerged as an unusually complex compartment in this process. While it is marked by facultative heterochromatin and has been considered primarily as a repressive compartment, recent work suggests that active genes may also associate with the periphery. Moreover, the nuclear envelope is riddled with nuclear pore complexes, the gateways for molecular exchange between the cytoplasm and the nucleus, resulting in substantial traffic through this compartment. Using murine erythropoiesis as a model system, our recent three dimensional analysis of the positioning of the β-globin locus revealed that, when inactive in undifferentiated embryonic stem cells and erythroid progenitor cells, the locus is positioned predominantly at the nuclear periphery and appears to contact the lamina. This association is lost with progressing erythroid maturation, and the locus is repositioned towards the nuclear interior concomitant with increasing β-globin gene expression. Importantly, however, β-major globin expression begins at the nuclear periphery prior to relocalization, suggesting that associations of the locus at the periphery may shift from repressive to activating complexes. We are investigating the interactions of the β-globin locus with the nuclear periphery by two approaches:

Using enhanced imaging permitted by Cryo-ImmunoFISH, we are analyzing the position of the locus relative to specific components of the nuclear lamina and nuclear pore complexes at distinct differentiation stages. In addition, we are probing the physical interactions of the locus with the periphery by biochemical means.

To this end we are using ChIP-chip to identify lamina associated proteins binding the β-globin locus and to determine what sequence elements within the locus mediate these interactions. Ultimately these experiments will shed further light on mechanisms regulating β-globin gene expression during erythropoiesis and how stage-specific nuclear localization contributes to this process.
IL-12 Producing Capacity of Cultivated Dendritic Cells of Aplastic Anemia Patients during Disease Manifestation and Immunosuppressive Treatment.
Authors: Archuadze Shorena , Mikhailova Elena , Parovichnikova Elena , Kulikov Sergei , Savchenko Valeri
Published: October 2019

Dendritic cells (DCs) play principal role in the induction of antigen-specific T-cell immune response and in the development of antigen-specific self-tolerance. Moreover, they promote either Th1 or Th2 polarization of naive lymphocytes by production of immunoregulatory cytokines (IL-12 and IL-10). AA is characterized by increased Th1/Th2 ratio and by Th1 mediated antigen-specific suppression of hematopoiesis. DCs might be responsible for the activation of T-clone in AA patients. However, functional characteristics such as IL-12 production by DC in AA patients have not been studies yet. Therefore, IL-12 secretion capacity of DCs generated from peripheral mononuclear cells (PMNC) of 30 AA patients before and during the immunosuppressive therapy (IST) was compared to that of DCs of 7 donors. DC were derived from PMNC in the presence of GM-CSF and IL-4 for 5–7 days and further stimulated by exposition to 3T3-CD40L fibroblasts for 48 hours. Supernatants of DC cultures were subjected to ELISA for evaluation of IL-12 production. 70,8% of AA patients exhibited significantly (p=0,02) increased baseline level of IL-12 production by DCs compared to that of donors. Programmed IST resulted in the diminution of IL-12 production level in 67% of AA patients. Moreover, significant decrease in IL-12 production level correlated (Rs=0,79) with the achievement of independence from transfusions. Patients with high baseline level of IL-12 production by DCs required continuous (median - 24 months, p<0,05, see pict. 1) IST including repeated courses of antithymocyte globulin and cyclosporin-A. These data suggest that increase in IL-12 production by DCs of AA patients might contribute to cytotoxic T-clone expansion and consequently, to AA development. Patients that showed high initial levels of IL-12 secretion by DCs comprised the worst prognostic group.

Figure Figure
Effects of Sodium Valproate on Proliferation and Apoptosis of Human Myelodysplastic Syndromes Cell Line MUTZ-1.
Authors: Chen Bao-An , Zhao Hui-Hui , Xia Guo-Hua , Shao Ze-Ye , Gao Feng , Chen Jian , Sun Yun-Yu , Wang Jun , Zhao Gang , Ding Jia-Hua , Chen Ning-Na , Pei Xiao-Ping
Published: October 2019

OBJECTIVE: Myelodysplastic syndromes (MDS) are a heterogeneous group with the expansion of a malignant clone. No satisfactory treatment for MDS is available. Sodium valproate (VPA), can induce G0–G1 phase arrest and cell apoptosis, inhibit proliferation of tumor cells in vitro significantly. The effects and possible mechanism of VPA on MUTZ-1 cell line of MDS were studied in this experiment.

METHODS: Cell proliferation was determined by MTT assay. Apoptotic morphological features were observed under microscope and transmission electronmicroscope. Cell apoptosis and cell cycle were analyzed by flow cytometry (FCM). The expression of p21WAF1(cyclin-dependent kinase inbihitor) was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis.

RESULTS: VPA could inhibit the proliferation of MUTZ-1 cells in dose-and time-dependent manners. The typical apoptotic morphological features appeared in cells treated with 4 mmol/L VPA for 72 hours. Condensation of cells and nuclear chromatin, disintegration of nuclear chromatin, and apoptotic body could be observed under microscope. Aggregation and margination of apoptotic nuclear chromatin, cytoplasm condensation, and irregular chromatin masses could be observed under transmission electronmicroscope. The percentage of apoptotic cells which were treated with 1,2 and 4 mmol/L VPA for 72 hours increased from 1.39% to 2.18%, 16.03%, 22.02%, and cell cycle arrest at G0–G1 phase could be caused by VPA as shown by FCM. The expression level of p21WAF1 mRNA and p21WAF1 protein were up-regulated in a dose dependent manner in MUTZ-1 treated with VPA for 72 hours (P<0.05).

CONCLUSION: VPA induced a G0–G1 cell cycle arrest and have an effect on proliferation and apoptosis of MUTZ-1 cells in vitro. The mechanism may be associated with the up-regulation of p21WAF1.
Ectopic Expression of Mer on Jurkat Cells and Its Anti-Apoptosis Effect.
Authors: Fan Lei , Xie Liqian , Shen Fei , Dai Lan , Li Jianyong , Ruan Changgeng
Published: October 2019

BACKGROUND & OBJECTIVE: Mer is one member of Axl receptor tyrosine kinase family; its ligand Gas6 can bind Mer, then stimulate the tyrosine kinase activity and downstream cell signal pathway of Mer, and take part in cell inflammation, apoptosis and thrombosis. There are more and more reports on Mer function, while few on its association with malignant diseases. This study was to detect the expression of Mer on T-cell leukemia cell line--Jurkat, and investigate its anti-apoptosis function and the mechanism.

METHODS: Flow cytometry (FCM) was used to detect Mer expression on normal T cells and Jurkat cells. RNA interference (RNAi) was used to block the expression of Mer on Jurkat cells. The effect of Mer on the proliferation of Jurkat cells was assessed by MTT assay, and its effect on serum starvation-induced apoptosis was evaluated by FCM with Annexin V/PI double staining. The expression of apoptosis-associated genes Bcl-2 and Caspase-3 in Jurkat cells was detected by real-time polymerase chain reaction (PCR) after Mer blocking.

RUSELTS: Mer was not expressed in normal T cells both in peripheral blood and bone marrow, but highly expressed in Jurkat cells with a positive rate of 51.1%±5.2%. The inhibition rate of Mer expression in Jurkat cells by RNAi was 86.0%±10.3%. After 48-hour serum starvation, the apoptosis rate was 15.3%±4.3% in Mer-blocking Jurkat cells, and only 1.5%±1.0% in control Jurkat cells(P<0.01). There was no significant difference in Jurkat cell proliferation rate between these 2 groups. After Mer blocking, Bcl-2 expression was decreased by 42.7%±8.6% of control(P<0.01), Caspase-3 expression showed no significant change.

CONCLUSION: Mer is highly expressed in Jurkat cells, and could inhibit cell apoptosis via Bcl-2 signaling pathway.
A Novel Histone Deacetylase Inhibitor MCT-3 Augments Inhibition of Leukemia Cell Growth and Suppresses Induction of MMP-9 Expression by Azacitidine in HL-60 Cells.
Authors: Liu Hongbin , Mayes Penelope A. , Perlmutter Patrick , Mckendrick Joseph J. , Dear Anthony E.
Published: October 2019

Azacitidine (AZA), a demethylating agent, has recently been demonstrated to have efficacy in the treatment of myelodysplasia and acute myeloid leukemia. A potential concern when considering the use of this agent is a recent report demonstrating AZA-mediated re-activation of matrix metalloproteinase-9 (MMP-9) expression facilitating the invasive metastatic phenotype (Sato NM et al J Natl Cancer Inst. 2003 Feb 19;95(4):327–30). Histone deacetylase inhibitors (HDACi) are a recently identified class of agents with considerable in vitro and in vivo activity and early phase clinical efficacy in the treatment of haematological malignancies (Bolden JE Nat Rev Drug Discov. 2006 Sep;5(9):769–84). Pre-clinical in vitro studies suggest that addition of HDACi to 5-aza-2′-deoxycytidine, a derivative of AZA, enhances the efficacy of this agent (Yang H et al Leuk Res. 2005 Jul; 29(7):739–48) whilst early phase clinical trials identify therapeutic activity using a combination of demethylating agents and HDACi (Garcia-Manero G, Blood. 2006 Nov 15;108(10):3271–9). Our current study aimed to determine the in vitro activity and molecular mechanisms of action of the novel HDACi MCT-3, a derivative of Oxamflatin, a hydroxamate analogue, (Dear AE, et al, Org Biomol Chem, 2006, 4, 3778–3784) in the HL-60 cell line alone and combination with AZA. AZA (1.0 microM) and MCT-3 (2.5 microM) alone inhibited HL-60 cell growth over 24hrs by 40%, 30% respectively. The combination of AZA with MCT-3 inhibited HL-60 cell growth up to 50%. Real-time PCR demonstrated that AZA and MCT-3 alone increased p15INK4b and Caspase 3 mRNA expression 2 fold. A Combination of AZA with MCT-3 increased p15INK4b and Caspase 3 mRNA expression up to 2.5 and increased p21WAF1/CIP1 and the orphan nuclear receptor Nur77 expression 2 fold. A combination of AZA and MCT-3 significantly attenuated AZA-induced MMP-9 mRNA expression and proteolytic activity. AZA and MCT-3 alone reduce HL-60 cell growth in vitro. Addition of MCT-3 to AZA increased inhibition of cell growth, suggesting that this HDACi may have the potential for additive activity with demethylating agents. AZA and MCT-3 have similar effects on expression of genes implicated in cell cycle arrest and apoptosis. Increased expression of p21WAF1/CIP1 and the orphan nuclear receptor Nur77 via inhibition of cell cycle progression and enhanced apoptosis may in part be responsible for the enhanced anti-leukaemia activity of the combination of AZA and MCT-3. Importantly MCT-3 is able to inhibit AZA-mediated induction of MMP-9 expression.
Methylation of CpG Islands Was Involved in the Down-Regulation of DAZAP2 in Multiple Meyloma Cells.
Authors: Hu Wei-Xin , Ren Wei , He Le-Qi , Sheng Rong , Shi Yi-Wu
Published: October 2019

Our previous studies showed that DAZAP2 (deleted in azoospermia associated protein 2) was the most profoundly downregulated gene in bone marrow mononuclear cells from multiple meyloma (MM) patients. The results implicated a role for DAZAP2 as a potential tumor suppressor involved in the origin and development of MM. To further understand the molecular mechanism underlying this deregulation, we analyzed epigenetic changes (i.e., DNA methylation) associated with DAZAP2 in MM, given that aberrant promoter methylation contributes to tumorigenesis through inducing transcriptional suppression and tumor suppressor inactivation. Bioinformatics analysis showed that there are three CpG islands in the DAZAP2 promoter region. The fragments of DAZAP2 promoter region with different length were separately PCR-amplified and inserted into the luciferase reporter, pGL2-Basic vector. The reporter constructs were transfected into COS-7 cells and their luciferase activities measured. The results showed that the fragment containing the third CpG island was the most active among the constructs tested. When this fragment was treated with M. Sss I methylase, its luciferase activity was almost lost. On the other hand, after 5-aza-2′-deoxycytidine treatment, the expression of DAZAP2 was restored in KM3 cells, an MM cell line associated with DAZAP2 downregulation. To extend these observations, bone marrow samples from MM patients and normal controls were collected and genomic DNA, RNA and proteins were extracted from each sample for RT-PCR, MSP (methylation-specific PCR) and Western blot analyses. The results were in general agreement with that reported previously, demonstrating that the protein and mRNA of DAZAP2, while detectable in 100% normal controls, were undetectable in 62.5% MM patients. The MSP data further corroborated with RT-PCR and Western blot analysis (94%), which is characterized by a negative correlation between promoter methylation and downregulation of DAZAP2 in multiple myeloma. These results indicate that promoter hypermethylation may play an important role in downregulation of DAZAP2 gene expression in multiple myeloma.
Molecular Mechanisms of NF-kappaB Activation by the HTLV-I Oncoprotein Tax: Role of Post Translational Protein Modification.
Authors: Kfoury Youmna , Nasr Rihab , Favre-Bonvin Arnaud , El-Sabban Marwan , Renault Noemie , Giron Marie-Louise , Hajj Hiba El , Chiari Estelle , Mikati Abdul Ghani , Hermine Olivier , Saib Ali , de Thé Hugues , Pique Claudine , Bazarbachi Ali
Published: October 2019

Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of mature activated T-cells caused by human T cell lymphotropic virus type I (HTLV-I). The Tax viral oncoprotein plays a crucial role in the proliferation and transformation of HTLV-I infected T lymphocytes through various mechanisms, chiefly through activation of the NF-kappaB pathway. We found that ubiquitin binding to Tax C-terminal lysines in the cytoplasm is critical for Tax binding to the IkappaB kinase (IKK) complex and subsequent nuclear translocation of NF-kappaB. Conversely, we demonstrate that the same Tax lysines are modified by SUMO binding in the nucleus, an event required for the formation of Tax nuclear bodies and full NF-kappaB transcriptional activation. We further show that ubiquitylated Tax is not associated with active cytosolic IKK subunits, but binds endogenous IKK subunits and targets them to the centrosome. Indeed, differential ubiquitylation of Tax results in different outcomes: K63-ubiquitylated Tax colocalizes at the centrosome with IKK, while K48-ubiquitylated Tax is degraded by the proteasome. Altogether, these results support a model in which K63-ubiquitylated Tax activates IKK in a centrosome-associated signalosome, leading to the production of Tax-free active cytoplasmic IKK. Thus, ubiquitylation and sumoylation of the same lysine residues of Tax regulate two essential steps controlling NF-kappaB activation, demonstrating how these posttranslational modifications can cooperate to promote Tax-induced transformation. These observations highlight an unsuspected cellular and biochemical complexity in Tax-induced NF-kappaB activation and represents ideal targets for the development of new drugs for ATL therapy.
Gadd45a Behaves as a Tumor Suppressor and Gadd45b Behaves as an Oncogene in myc-Mediated Block in Myeloid Differentiation.
Authors: Mohamed-Hadley Alisha , Gibbs John D. , Liebermann Dan A. , Hoffman Barbara
Published: October 2019

We have observed that deregulated c-Myc expression in M1 myeloid leukemic cells blocks IL-6-induced differentiation at an intermediate stage, the cells fail to exit the cell cycle, proliferating indefinitely, with a portion of the cells always undergoing apoptotic cell death by prematurely recruiting the CD95/Fas apoptotic pathway. Similar results were also observed in normal myeloid cells from bone marrow (Amanullah et al. Oncogene19:2967–77, 2000; Amanullah et al. Oncogene21:1600–1610, 2002). Although there is evidence that c-myc represses the gadd45a promoter, this laboratory has observed elevated gadd45a expression in M1myc cells that are blocked during differentiation. In addition, microarray analysis indicated that gadd45b is a c-myc target gene. Because of these observations and the availability of gadd45a null and gadd45b null mice, the effect of deregulated myc expression in Hoxb8 immortalized macrophage progenitors was ascertained. It was observed that deregulated c-myc expression in gadd45a null cells blocked differentiation at the blast stage, compared to a block at the intermediate stage for wild type progenitors. In contrast, deregulated c-myc expression in gadd45b null progenitors resulted in rapid cell death. These observations suggest that in the context of myc mediated myeloid leukemias Gadd45a behaves as a tumor suppressor and Gadd45b behaves as an oncogene.
Direct and Natural Killer Cell-Mediated Anti-Tumor Effects of Low-Dose Bortezomib in Hepatocellular Carcinoma.
Authors: Krusch Matthias , Armeanu Sorin , Baltz Katrin M. , Lauer Ulrich M. , Steinle Alexander , Kanz Lothar , Bitzer Michael , Salih Helmut R.
Published: October 2019

Background & Aims: Hepatocellular carcinoma (HCC) displays a particular resistance to conventional cytostatic agents. Therefore, alternative treatment strategies focus on novel substances exhibiting anti-neoplastic and/or immunomodulatory activity enhancing for example Natural Killer (NK) cell anti-tumor reactivity. However, the tumor-associated ligands engaging activating NK cell receptors are largely unknown. Exceptions are the NKG2D ligands (NKG2DL) of the MIC and ULBP protein families, which potently stimulate NK cell responses. We studied the consequences of proteasome inhibition with regard to direct and NK cell-mediated effects against HCC.

Methods: Primary human hepatocytes (PHH) from different donors, hepatoma cell lines and NK cells were exposed to Bortezomib. Growth and viability of hepatoma cells and PHH as well as immunomodulatory effects including alterations of NKG2DL expression on hepatoma cells, specific induction of NK cell cytotoxicity and IFN-γ production were investigated.

Results: Bortezomib treatment inhibited hepatoma cell growth with IC50 values between 2.4 and 7.7 nanomol/liter. These low doses increased MICA/B mRNA levels and total and cell surface protein expression in hepatoma cells, which stimulated cytotoxicity and IFN-γ production of cocultured NK cells. Importantly, while IFN-γ production of NK cells was concentration-dependently reduced, low-dose Bortezomib neither induced NKG2DL expression or cell death in PHH nor altered NK cell cytotoxicity.

Conclusions: Low-dose Bortezomib mediates a specific dual anti-tumor effect in HCC by inhibiting tumor cell proliferation and by priming hepatoma cells for NK cell anti-tumor reactivity. Our data suggest the clinical evaluation of Bortezomib treatment in HCC, especially in combination with immunotherapeutic approaches like adoptive NK cell transfer.
Imatinib Mesylate Induced Reversal of Leukemic Gene Phenotype in HL60 Cells Coincides with Stimulation of Oxidative Metabolism.
Authors: Pfeilstocker Michael , Wihlidal Peter , Varga Franz , Pittermann Elisabeth , Karlic Heidrun
Published: October 2019

Besides blockade of tyrosine kinases such as c-kit, Imatinib mesylate (IM) regulates glucose flux through downregulation of GLUT-1 transporters in human leukemia cells. This mechanism has the potential to induce regression of type 2 diabetes and hyperlipidemia as observed in patients with chronic myeloid leukemia or hypereosinophilic syndrome. In addition, there is a stimulatory effect of IM on differentiation of human mesenchymal stem cells. Its synergism with retinoic acid or low dose Ara-C is applied in treatment of acute myeloid leukemia (AML). Thus, the AML-derived c-kit positive cell line HL60 was chosen for studying the effect of IM on expression of genes associated with differentiation and metabolism. We analysed the possible feedback on transcription factors (AML1 and AML3) and consequences regarding differentiation and metabolism - associated genes. Quantitative reverse transcriptase PCR analyses revealed that IM treatment of HL60 cells downregulates mRNA synthesis of AML1 and AML3 by 70% without affecting transcription of the c-abl tyrosine kinase. IM reduces expression of CD34 mRNA from 20% to 6% of the housekeeping gene G6PD. The appearance of differentiated cells was accompanied by a remarkable stimulation of mRNAs from CD11b and CD14 (monocyte markers) reaching 4-fold higher expression levels relative to G6PD. This was associated with an increased proportion of osteocalcin (OCN), which has recently been shown to enhance mitochondrial activity. A 2-fold stimulation of a fat-metabolism associated mitochondrial palmitoyltransferase (CPT1B) and 10-fold stimulation of microsomal carnitine palmitoyltransferase and the carnitine transporter OCTN2 supports previous data indicating an IM-associated stimulation of oxidative metabolism resulting in a regression of type-2 diabetes and hyperlipidemia. Our current investigations show that IM-associated attenuation of cell proliferation inhibited transcription factors AML1 and AML3 and triggered differentiation in the leukemic cell line HL60, as reflected by altered mRNA expression of surface marker genes. The IM - induced stimulation of lipid metabolism in HL60 confirms previous data indicating a reversal of the Warburg effect in K562 cells without cytocidal activity. This indicates a similar mechanism as known for other drugs and strategies targeting glucose or fat metabolism, which are discussed in the context of cancer prevention and treatment.