bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Anti-Human GPVI Autoantibodies Mediating Persistent Target Immnunodepletion over a 15-Year Period.
Authors: Takayama Hiroshi , Hosaka Yoshitaka , Nakayama Kazuyuki , Shirakawa Kamon , Kawahara Tetsushi , Naitoh Katsuki , Shinozaki Mikihiko , Honda Motoyasu , Yamakawa Toru , Furusako Shoji
Published: October 2019

The first reported patient with platelet GPVI deficiency (Blood1987;69:1712) had anti-GPVI antibodies (YA-Abs) in the patient’s serum, which contributed to later progress in identification of GPVI as a novel collagen receptor. Although there is now a concept that anti-GPVI Ab induces immunodepletion of GPVI in vivo and may become a promising therapeutic antithrombotic agent, precise nature of anti-human GPVI Ab mediating immunodepletion of GPVI is unknown. Since the patient’s platelets are still lacking GPVI and unresponsive to collagen stimulation, we purified YA-Abs from the patient’s serum kept in 1988 (YA-Abs-88) and in 2003 (YA-Abs-03) using sequential affinity chromatography on protein A-Sepharose and recombinant human GPVI fused to Fc (rhGPVI-Fc)-Sepharose. The amount of YA-Abs-03 in the serum was estimated to be about 3 μg/mL and twenty times less than that of YA-Abs-88. We also generated 75 mouse monoclonal antibodies (mAbs) against the extracellular part of human GPVI. Among those, several mAbs cross-reacted with YA-Abs for the binding to rhGPVI-Fc, indicating oligoclonal nature of YA-Abs. Two representative mAbs with no cross-reactivity, F1201 and F1232, cross-reacted with YA-Abs-88 and with both YA-Abs-88 and -03, respectively. Previous studies identified two distinct collagen binding sites of human GPVI; the one is a sequence including Lys59 of human GPVI which is recognized by 10B12 Ab, (Blood2004;103:903) and the other is a sequence including Gly30, Val34 and Leu36 which involves the binding to 9O12 Ab (J.Biol. Chem.2004;279:52293). Point mutated rhGPVI-Fc at Lys59 (K59E) was recognized by YA-Abs-88, -03, and F1232 but not by F1201. Another mutated rhGPVI-Fc in which human GPVI sequence from Pro31 to Leu36 was substituted for mouse GPVI counter sequence was recognized by any of those Abs. YA-Abs-88 and F1201but not F1232 evoked surface P-selectin expression or aggregation of human platelets, while F1232 inhibited collagen-induced aggregation. Since both F1201 and F1232 were found to react with nonhuman primate platelets, either mAb was intravenously injected into monkeys at a dose of 0.3 mg/kg that reached its maximum plasma concentration of 10 μg/mL. Within 1 to 2 days after injection, their platelets showed the lack of both GPVI and collagen-induced aggregation. Thus, GPVI-deficiency of the patient’s platelets is now maintained by unexpectedly much decreased amount of specific Abs which contain F1232-like Ab but not F1201-like Ab included in YA-Abs-88. Immunodepletion of GPVI can be safely induced by specific Ab like F1232 recognizing different epitope from previously reported collagen binding sites where their cross-linking causes platelet activation.
Immune Thrombocytopenia Mediated by Anti-GPIbα Antibodies May Occur Via an FcR-Independent Pathway: A Potential Explanation for Refractory Cases to IVIG Therapy.
Authors: Webster Michelle L. , Sayeh Ebrahim , Crow Min , Chen Pingguo , Nieswandt Bernhard , Freedman John , Ni Heyu
Published: October 2019

Intravenous immunoglobulin G (IVIG) is used to treat immune thrombocytopenia (ITP). While benefit has been observed in many cases, for unknown reasons some patients are refractory to this therapy. ITP is characterized by platelet clearance mainly mediated by antibodies against platelet surface glycoproteins (GP) GPIIbIIIa and GPIbα. Approximately 70–80% patients have antibodies against GPIIbIIIa, 20–40% patients have antibodies against GPIbα complex, and some patients have antibodies to both. Phagocytotic macrophages activated via their Fc receptors (FcR) are thought to be responsible for the clearance of opsonized platelets. However, anti-GPIbα antibodies may be able to induce ITP in an FcR-independent manner. Since the prevailing view of the mechanism of action of IVIG is Fc receptor blockade, we hypothesized that IVIG may not be effective at preventing thrombocytopenia induced by anti-GPIbα antibodies. Thrombocytopenia was induced in BALB/c mice using monoclonal antibodies against mouse GPIbα (p0p3, p0p4, p0p5, p0p9, and p0p11). Pretreatment with different doses of IVIG (1, 2, and 4g/kg of mouse) failed to prevent ITP in all anti-GPIbα-treated mice, except for p0p4. These results were repeated with p0p3, p0p4, and p0p5 using 2g/kg IVIG in C57BL/6 mice, and using 2g/kg of a different IVIG preparation. It has been shown that F(ab)2’ fragments of the p0p antibodies can induce thrombocytopenia to the same extent as intact antibodies. To confirm that these anti-GPIbα antibodies act in an FcR-independent manner, we demonstrated that p0p3, p0p4, and p0p5 are able to induce thrombocytopenia in mice lacking activating Fc receptors (FcRγ chain −/−). Interestingly, IVIG was still able to prevent thrombocytopenia induced by p0p4 in these FcRγ chain −/− mice. We demonstrated in vitro that there are no anti-idiotype antibodies against any of the anti-GPIbα antibodies used present in IVIG. Thus, the amelioration of thrombocytopenia in p0p4-treated mice is not due to a neutralization of the antibody by IVIG. In contrast to the anti-GPIbα antibodies, anti-GPIIbIIIa monoclonal antibodies (JON1, JON2, and JON3) that induce thrombocytopenia were sensitive to IVIG treatment, which is consistent with earlier studies using other monoclonal antibodies against GPIIbIIIa. Also, JON2 was unable to induce thrombocytopenia in FcRγ chain −/− mice, confirming that this anti-GPIIbIIIa antibody is FcR-dependent. Our results suggest that ITP induced by anti-GPIbα antibodies may be mediated by FcR-independent mechanisms, many of which may be refractory to IVIG therapy. Thus, IVIG may not be as effective in treating patients with predominantly anti-GPIbα antibodies.
The HPA-15 (Gov) Platelet Alloantigen System: Allele Frequencies in the Canadian Population.
Authors: Chamberlain Kristen , Smith James W. , Moffatt Diana , Moore Jane , Arnold Donald , Kelton John G.
Published: October 2019

Human platelet antigens (HPAs) are genetically defined polymorphisms expressed on plasma membrane proteins. Alloimmunization following exposure to the incompatible antigen during pregnancy, or following transfusion or transplantation, can cause severe thrombocytopenia and bleeding. The Gov-a/Gov-b biallelic system is expressed on CD109, a 175kD glyco sylphosphatidylinositol-anchored glycoprotein found on platelets, endothelial cells, and activated T-cells (Kelton, 1990; Smith, 1995). Alloimmunization to Gov-a/b is implicated in neonatal alloimmune thrombocytopenia (NAT) and post-transfusion purpura (PTP), and anti-Gov antibodies have been found in polytransfused patients. The Gov-a/Gov-b alleles are encoded by an A-C single nucleotide polymorphism (SNP) at position 2108, resulting in a Tyr682Ser substitution in CD109. Sequence analysis determined that CD109 is a member of the alpha 2 macroglobulin/C3, C4, C5 family of thioester-containing proteins (Lin, 2002). The Gov antigen frequencies are expressed almost equally in the population, and European and Asian studies determined that the Gov-b allele was expressed slightly more often than the Gov-a. Based on these studies the Gov-a/Gov-b antigens were assigned to the HPA-15 system (HPA-15a/15b = Gov-b/Gov-a). South American studies found similar gene frequencies in the general population, but significantly higher frequencies for HPA-15a (0.78) in aboriginal populations (Cardone, 2004). No larger North American studies have been reported since the original report of the Gov allele frequencies based on a limited number of platelet donors (Gov-a: 0.532; Gov-b: 0.468, n=33). We used the polymerase chain reaction with sequence-specific primers (PCR-SSP) to investigate the HPA-15 allele frequencies in the Canadian population. DNA from 273 blood donors was tested using PCR-SSP. The frequency of genotypes HPA-15a/15a, 15a/15b, 15b/15b was found to be 27.84, 49.45, and 22.71 respectively. Thus the allele frequencies for the Canadian population were determined to be 0.517 for HPA-15a and 0.483 for HPA-15b. This data is similar to the frequencies reported among some European studies, and supports evidence that the HPA-15a (Gov-b) allele is expressed slightly more frequently in the general population. Initial reports investigating the Gov alloantigens localized the polymorphism to a novel platelet protein, CD109, and lack of sequence data prevented the use of genotyping studies. The original studies of the Gov antigens, which share almost equal distribution, were limited by available alloantisera and complex screening technologies. The results from this report support the relative allele frequencies for the HPA-15a/15b (Gov-b/Gov-a) antigen system, and emphasize the importance of using genotyping studies to determine allele frequencies.
Direct Injection of Allogeneic Multipotent Adult Progenitor Cells Improves Cardiac Function after Myocardial Infarct.
Authors: Hof Wouter van’t , Mal Niladri , Zhang Ming , Forudi Farhad , Deans Robert , Penn Marc
Published: October 2019

Multipotent adult progenitor cells (MAPC) are an early stem cell population identified in the bone marrow of adults. They have been shown to be capable of differentiating into cell populations derived from all 3 germ layers. We hypothesized that delivery of MAPC in the peri-infarct period could lead to improved cardiac function. MAPC were isolated from the bone marrow of Sprague-Dawley rats and stably labeled by using a lentiviral construct encoding green fluorescent protein (GFP). In-vitro differentiation assays confirmed that the GFP-tagged MAPCs had retained the potential for tri-lineage differentiation. Myocardial infarction was induced in Lewis rats by direct LAD ligation. Lewis rats received Sprague-Dawley MAPC either immediately after LAD ligation by directly injecting into the infarct border zone (5 injections of 400,000 cells per injection) or 24 h after MI by intravenous infusion (2 million cells). Immunohistochemistry was performed to identify MAPC (GFP positive cells) within the infarct zone 2 weeks after MI. We identified MAPC in the infarct border zone of all animals that received direct injection of MAPC, 2 weeks after MI, but did not identify any MAPC following intravenous infusion. Echocardiography was performed to determine if the delivery of MAPC to the myocardium at the time of MI led to improvement in cardiac function. At 2 weeks post-MI, we observed a significant increase in shortening fraction in those animals that received direct injection of MAPC compared to control (13.9 ± 2.2% (n=4) vs. 24.0 ± 6.6% (n=7), p=0.018). These data demonstrate that MAPC delivery to acutely infracted myocardium may offer significant benefit. These data further suggest that MAPC are capable of surviving in the myocardial tissue even when used in an allogeneic strategy without inducing a significant increase in inflammation or rejection.
Cobalamins and Cobinamides Inhibit Nitric Oxide Synthase Enzymatic Activity.
Authors: Weinberg J. Brice , Chen Youwei C. , Beasley Bethany E. , Ghosh Dipak K.
Published: October 2019

Cobalamins (Cbl) serve as important co-factors for methionine synthase and methylmalonyl-coA mutase. They also can block the activities of nitric oxide (NO), as the cobalt binds NO and quenches its bioactivity. NO plays very important roles in normal physiology and in various pathologic processes. NO is synthesized by NO synthases (NOS), enzymes that convert L-arginine to L-citrulline and NO. We postulated that various corrin derivatives (e.g., cobalamins or cobinamides) would inhibit the enzymatic activity of NO synthase (NOS) by directly interacting with NOS. We used purified, recombinant neuronal NOS (NOS1) and inducible NOS (NOS2) in in vitro assays measuring conversion of 14-C-L-arginine to 14-C-L-citrulline. Results showed that hydroxocobalamin (OH-Cbl), cobinamide (Cbi), and dicyanocobinamide (CN2Cbi) were potent NOS1 and NOS2 inhibitors, while cyanocobalamin had much less activity. In general, NOS1 was inhibited more actively than was NOS2, but both were inhibited over 80% by the agents. Methylcobalamin and adenosylcobalamin had very little ability to inhibit NOS activity, but light illumination of these two compounds (a process that liberates the methyl and adenosyl groups from cobalamin, generating OH-Cbl) “activated” them so that they had inhibitory activity comparable to native OH-Cbl

Dose (uM) for 50% inhibition of purified NOS activity (ID50) OH-Cbl Cbi CN2Cbi CN-Cbl AdoCbl (dark) Ado-Cbl (light) NOS1 5.7 15.0 0.2 72.0 498.0 6.4 NOS2 21.1 50.0 2.3 1620.0 2148.0 60.0

We examined the interactions of NOS2 with cobalamins and cobinamides by spectroscopy. Under reduced conditions, oxygen binds to the NOS heme iron, a step that is obligatory for the NO synthesis catalytic cycle. Carbon monoxide (CO, which serves as an oxygen mimic) causes a marked change in the NOS spectrum under reduced conditions. CN2Cbi markedly diminished the CO-induced spectral changes in NOS2, indicating that CN2-Cbi likely blocks binding of CO to the NOS2 oxygenase domain under reduced conditions. This suggests that CN2Cbi-mediated inhibition of NOS activity is due to blocking of oxygen binding to heme, a step required for the NOS catalytic cycle. When we incubated OH-Cbl or CN2Cbi with cells of the mouse macrophage cell line J774 activated with interferon-gamma and endotoxin to produce NO, there was a dose-dependent inhibition of NO production. While certain cobalamins and cobinamides have been previously noted to bind and quench/scavenge the actions of preformed NO, we show here for the first time that cobalamins and cobinamides can potently inhibit the enzymatic function of NOS and synthesis of NO. These agents (or their derivatives) might serve as NOS inhibitors for treatment of various conditions. Also, native tissue cobalamins and cobinamides might act as important regulators of NOS activity in normal and pathological conditions in vivo.
Thalidomide-Associated Thromboembolism in Cancer: Reimbursement for Thalidomide’s “Off-Label” Prescribing under the 2004 Medicare Oral Pharmaceutical Demonstration Project Raises Concerns.
Authors: Evens Andrew M. , McKoy June M. , Yarnold Paul R. , McCaffrey Kathyrn , Bennett Charles L.
Published: October 2019

Purpose: In 1999, one year after its approval by the FDA for erythema nodosum leprosum, thalidomide’s effectiveness as an off-label treatment for multiple myeloma was noted. A 28% rate of thromboembolism with thalidomide-doxorubicin therapy for myeloma was reported in 2001. Thalidomide’s inclusion in the 2004 Medicare Oral Pharmaceutical Demonstration Project as treatment for multiple myeloma represents the only off-label use drug covered. FDA regulations prohibit manufacturer dissemination of comprehensive safety information describing thalidomide-associated thromboembolism (TAT) in the off-label oncology setting. Herein, we reviewed FDA and published information for TAT in the oncology setting.

Methods: Adverse event reports contained in FDA databases (n= 190 patients) and 48 prospective clinical trials (n= 2,329 patients) were reviewed for information on thromboembolism occurring among thalidomide-treated cancer patients.

Results: TAT occurred after a median of 52 days of therapy (range, 6 to 469 days), with more than half of these events among persons with multiple myeloma. TAT rates of 30% were noted among patients with newly diagnosed multiple myeloma receiving concomitant doxorubicin. Previously treated myeloma patients receiving thalidomide and doxorubicin and newly diagnosed patients on thalidomide, doxorubicin, and low molecular weight heparin had thromboembolism rates of 15% or lower.

Conclusions: Revision of FDA regulations to allow dissemination of information describing benefits and toxicities of thalidomide as an off-label cancer treatment is important, particularly for multiple myeloma patients receiving thalidomide under the 2004 Medicare Oral Pharmaceutical Demonstration Project.

Cancer drugs covered under the Medicare Replacement Drug Demonstration Project CANCER INDICATION ONCOLOGY DRUG Breast cancer Anastrazole Breast cancer Exemestrane Breast cancer Letrozole Breast cancer Tamoxifen Breast cancer Toremifene Chronic myelogenous leukemia Imatinib mesylate Cutaneous T-cell lymphoma Bexarotene Epithelial ovarian cancer Altretamine GI Stromal Tumor Imatinib mesylate Multiple myeloma Thalidomide Non-small cell lung cancer Gefitinib Non-small cell lung cancer Erlotinib Prophylaxis for ifosfamide-induced hemorrhagic cystitis Mesna
Gene Expression Profile of Nodal vs Extranodal Diffuse Large B-Cell Lymphoma from Saudi Arabia.
Authors: Jehan Zeenath , Siraj Abdul Khalid , Simon Ronald , Sauter Guido , Kuraya Khawla A.l.
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) belongs to the most common malignancies in Saudi Arabia, accounting for 50–60% of adult non-Hodgkin Lymphomas. DLBCL represents a heterogeneous group of tumors with respect to morphology, genetics, and clinical behaviour. The two major subtypes of DLBCL differ in their site of origin, i.e. within (nodal) or outside (extranodal) the lymph nodes, and a separate genetic pathway has been suggested for these subtypes. To detect potential biological differences between these two DLBCL categories, we performed gene expression analysis in 20 nodal and 8 extranodal lymphoma tissues, as well as in 4 normal lymph nodes, using the GeneChip technology (Affymetrix HG-U133_2 array). Data analysis using the D chip software highlighted a total of 215 genes expression patterns of which were significantly different in nodal and extranodal lymphomas. The most striking differences were observed for genes contributing in ATP binding (20 genes), nucleotide binding (12 genes), response to stress (19 genes), RNA metabolism (6 genes), protein translation (4 genes), and inflammatory response (4 genes). Remarkably, also 5 protein tyrosine kinases were found to be differentially expressed. Our data strongly suggests the existence of at least two different genetic pathways for the development of nodal and extranodal DLBCL, which are characterized by these genetic changes. These results prompt for further expression analysis in large cohorts of histomorphologically well defined lymphoma tissues in order to evaluate the clinical relevance of these alterations.
Lack of PRDM1α Expression in Hodgkin/Reed-Sternberg Cells Is Likely Mediated by a Translational or Post-Translational Mechanism.
Authors: Tam Wayne , Tan Leonard , Gomez Mario , Fan Shaoan , Liu Yifang , Wright Kenneth , Chadburn Amy , Knowles Daniel M.
Published: October 2019

Hodgkin/Reed Sternberg (H/RS) cells are the neoplastic cells in classical Hodgkin lymphoma (HL). They are thought to resemble post-germinal center (GC) B cells with expression of markers associated with late stage of B-cell differentiation, for example, interferon regulatory factor -4/multiple myeloma-1 (IRF4/MUM1) and syndecan 1 (CD138). The PR (PRDI-BF1-RIZ) domain zinc finger protein 1 (PRDM1), a transcription repressor with a master regulatory role in plasma cell differentiation, is normally co-expressed with IRF-4/MUM-1 in plasma cells and in a subset of activated GC cells committed to plasma cell fate. We studied expression of PRDM1α, the functional isoform of PRDM1, in 14 classical HL cases [including 3 positive for Epstein-Barr-virus (EBV)] and 4 HL cell lines by immunohistochemistry and Western blotting, respectively. H/RS cells in primary HL cases are negative for PRDM1α, implying a desynchrony in expression between IRF-4/MUM1 and PRDM1. While the myeloma cell line U266 expresses relatively abundant PRDM1α, it was undetectable by Western Blotting in all HL cell lines tested, except for the EBV-positive HL cell line L591 which, unlike in vivo H/RS cells, has a Type III EBV latency pattern. PRDM1α expression in L591 but not in vivo H/RS cells suggests that PRDM1 expression may be modulated by latency type-specific EBV-encoded gene products or the B-cell phenotype exhibited by the cell line. The lack of PRDM1α protein in H/RS cells is not due to impaired gene transcription, since real-time quantitative PCR revealed similarly abundant PRDM1α transcripts in the HL cell lines as U266. In the absence of mutation in the PRDM1 coding region, these results suggest that failure to accumulate PRDM1α protein in H/RS cells is likely due to abnormal translation repression or protein turnover. Loss of functional PRDM1 as a result of translational or post-translational deregulation may represent a novel molecular lesion in HL.
BCR/ABL Post-Translationally Enhances hnRNP E2 Expression and Translation-Inhibitory Function through the Activation of ERK1/2 Mitogen-Activated Protein Kinases.
Authors: Chang Ji-Suk , Notari Mario , Marcucci Guido , Perrotti Danilo
Published: October 2019

Impaired differentiation is a common feature of many hematological malignancies including blast crisis chronic myelogenous leukemia (CML-BC). We previously reported that expression of the RNA binding protein hnRNP E2 is enhanced in CML-BC patient samples by the activity of BCR/ABL oncogenic kinase which uses hnRNP E2 to suppress translation of C/EBP alpha, the main regulator or granulocytic differentiation. Here we show that hnRNP E2 protein but not mRNA expression correlates with BCR/ABL levels in mouse 32Dcl3 myeloid progenitor cell clones expressing different levels of p210 BCR/ABL, and in the BCR/ABL-inducible TonB.210 cell line. Furthermore, exposure of 32D-BCR/ABL cells to imatinib or to the HSP90 inhibitor 17-AAG, which induces BCR/ABL proteasome-dependent degradation, results in downregulation of hnRNP E2 protein levels. Mechanistically, it appears that increased hnRNP E2 expression depends on BCR/ABL-induced post-translational modifications that increase hnRNP E2 protein stability and prevent its proteasome-dependent degradation. Indeed, we have evidence that hnRNP E2 is associated with PRMT5 arginine-methyltransferase and undergoes phosphorylation in K562 cells. Accordingly, inhibition of the mitogen activated ERK1/2 kinases by PD098059 and U0126 reduces hnRNP E2 levels and, in turn, hnRNP E2 binding to the C-rich element contained in the uORF of c/ebp alpha mRNA. Thus, activation of MAPK might be required for the hnRNP E2-dependent suppression of myeloid maturation in CML-BC.
Effect of p53 Silencing on Asenic Tioxide-Induced Apoptotic Pathways in a Myeloma Cell Line Expressing w.t.p53.
Authors: Fatih Kircelli , Cagla Akay , Yair Gazitt
Published: October 2019

Mutations in the p53 gene are the most common genetic abnormality in human cancers. P53 is a tumor suppressor gene, which regulates a wide variety of target genes controlling cell cycle and apoptosis. Arsenic Trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. Our finding in this respect were further supported by our recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (Akay et al.,AACR; Abstract #5344, 2005). We hypothesized that knocking out of p53 or p21 mRNA will result in modifying ATO-induced apoptotic pathways as observed using the p53-Ts conditional mutant of BRK cells. Thus, IM9 cells (expressing w.t.p53) were transfected [AMAXA Nucleoporator, ~60% transfection by green fluorescence protein (GFP) tag] with a GFP-SiRNA expressing plasmid co-expressing anti-sense RNA sequences for p53 (GFP/Sip53) and p21 (GFP/Sip21). As a control, GFP expressing plasmid not containing SiRNA (GFP/cont) was used. We observed in GFP/cont-transfected cells a G1 arrest through activation of p21 and moderate apoptosis, primarily through activation of procaspase 9, with no activation of BID and procaspase 8. In cells transfected with GFP/Sip53 or GFP/Sip21 plasmids we observed rapid mitochondrial membrane depolarization and apoptosis primarily through activation of the extrinsic apoptotic pathway with downregulation of FLIP, activation of procaspase 8 and BID, enhanced release of AIF from mitochondria and rapid apoptosis. Depletion of glutathione was observed with or without silencing of p21 or p53. Furthermore, silencing of p53 or p21 resulted with a decrease in ATO-induced activation of c-jun and Erk and in a decrease in the activation of Akt. These results further substantiate the model we put forward for the role of p53 in ATO-induced apoptosis in myeloma and other cancer cell lines.
BCR/ABL Oncogene Controls MICA Translation.
Authors: Boissel Nicolas , Rea Delphine , Tieng Vannary , Dulphy Nicolas , Brun Manuel , Cayuela Jean-Michel , Rousselot Philippe , Tamouza Ryad , Le Bouteiller Philippe , Mahon François-Xavier , Steinle Alexander , Charron Dominique , Dombret Hervé , Toubert Antoine
Published: October 2019

MHC class I chain-related molecules (MIC) participate in immune surveillance of cancer through the engagement of NKG2D, an activating receptor on NK and T-cells. However, the release of soluble forms of MIC (sMIC) by the tumor may impair this response by decreasing NKG2D surface expression. The molecular basis for MIC expression in tumors is poorly understood. In chronic myeloid leukemia (CML), a malignancy caused by the BCR/ABL fusion oncoprotein., leukemic hematopoietic CD34+ cells expressed MICA at the cell surface, whereas hematopoietic CD34+ cells from healthy donors did not. At diagnosis, CML patients showed increased serum levels of sMICA and a decreased NKG2D expression on NK and CD8+ T-cells. Upon imatinib mesylate (IM) therapy, a specific BCR/ABL inhibitor, serum sMICA and NKG2D expression returned to normal levels. In BCR/ABL positive K562 cell line, IM decreased both surface MICA/B expression and NKG2D-mediated lysis by NK cells. Silencing BCR/ABL gene expression directly evidenced its role in the control of MICA expression. IM did not affect MICA mRNA levels but decreased MICA protein expression and sMICA release. Sucrose density gradient fractionation of cytoplasmic extracts from K562 treated with IM showed a shift in the distribution of MICA mRNA from the polysomal toward the monosomal fractions, favoring a decreased translation. Among the major pathways activated by BCR/ABL that regulate translation, PI3K and mTOR were shown to also control MICA expression. These data provide evidence for a direct control of MICA expression by BCR/ABL oncogene and indicate that post-transcriptional mechanisms may participate to the regulation of MICA expression.
Expression of ERK1/ERK2 MAP Kinase, Protein Kinase C and Akt Kinase by Lymphoid Malignant Cells.
Authors: Murohashi Ikuo , Takahashi Takashi , Ihara Noriko , Wakao Daisuke , Yagasaki Fumiharu , Nakamura Yhuichi , Kawai Nobutaka , Matsuda Akira , Jinnai Itsuro , Bessho Masami
Published: October 2019

ERK1/ERK2 MAP kinase (MAPK), protein kinase C (PKC) and Akt kinase have been shown to inhibit cellular apoptosis. We measured the expression levels of these protein kinases in a total of 102 samples from patients with lymphoid malignancies: 26 with acute lymphoblastic leukemia (ALL), 15 with multiple myeloma (MM), 49 with non-Hodgkin’s lymphoma (NHL), 3 with Hodgkin’s lymphoma (HL) and 6 with chronic lymphocytic leukemia (CLL). Plasma cells were separated by using magnetically labeled CD138, and the purity of the cells were more than 95%. The MAPK and PKC assay systems are based upon the enzyme catalyzed transfer of the [γ-32P] ATP to a specific peptide. Activity of Akt kinase was measured by Western blotting using phospho-specific Akt (Ser 473) antibody. MAPK activity was 0.4±0.0, 4.7±0.7 and 19.0±21.6 p mol/min/106 cells in normal blood mononuclear cells (n=3), normal bone marrow plasma cells (n=10), and patient samples (n=85), respectively. MAPK and PKC activities were 17.1±10.5 and 50.7±58.3 p mol/min/106 cells in ALL, 29.3±39.3 and 29.1±18.9 in MM, 17.3±16.4 and 46.5±46.7 in NHL, 13.6±3.2 and 4.0±3.2 in HL, and 7.3±4.8 and 2.2±0.2 in CLL, respectively. MAPK activity of CLL was significantly low (P<0.05) compared with that of ALL. Phosphorylated Akt was detected in 57 of 59 (97%) patients examined. A significant positive correlation between the activities of PKC and Akt kinase, but not those of MAPK and PKC or MAPK and Akt kinase, was noted. Kaplan-Meier estimates of overall survival (OS) revealed that the OS of the MM patients with lower MAPK was better than those with higher MAPK, although the difference was not significant (P = 0.15). OS was not different by MAPK for ALL or NHL, and by PKC for ALL, MM, or NHL. Effects of various cytokines on the MAPK activity of malignant lymphoid cells in serum free culture were examined. IL-7 significantly enhanced the MAPK activity in 46% of ALL patients, IL-15 in 31% of NHL patients, and IL-15 and IL-6 in 38% and 35% of MM patients, respectively. Taken together, our results suggest that MAPK activity is significantly and markedly elevated and that MAPK may play important roles by various subtype of lymphoid malignancies.
Chromosomal Instability Induced by Overexpression of Telomere Repeats Binding Factor 1.
Authors: Lan Jianping , Huang He , Zhu Yuanyuan , Yu Jian , Sun Jie
Published: October 2019

Telomere dysfunction has been associated with chromosomal stability in process of oncogenesis. Telomere, a special nucleoprotein complex at the termini of linear eukaryotic chromosomes, functions to prevent chromosomes from degradation by endogenous nucleases, fusion and recombination of chromosome ends, triggering DNA damage response and checkpoint-induced cell cycle arrest or apoptosis. Telomere homoeostasis and length are maintained by a set of telomere binding proteins. Among these, telomere repeats binding factor 1(TRF1) serves as a negative regulator of telomere length since TRF1 overexpression would elicit the shortening of telomere length in telomerase-positive cells. To date, the expression level of TRF1 in human cancers remains controversial and its role and mechanism in tumorigenesis are poorly understood. To answer these questions, we introduced EGFP-tagged TRF1 plasmid into HeLa cells to establish a stable cell line with TRF1 overexpression. Positive clones were selected by G418(1mg/ml) and GFP fusion protein was confirmed by immunoblotting analysis with anti-GFP antibodies. Interestingly, these cells demonstrated a higher percentage of mitotic abnormality including chromosome misalignment, anaphase bridging, lagging chromosome and multipolar spindles than controls after over 10 passages(20.0% vs 1.5%, p<0.01). To further clarify if this phenomenon resulted from chromosomal fusion, metaphase chromosome spreads were prepared. As expected, the TRF1 stable cells showed an elevated frequency of dicentric chromosomes compared with untransfected cells, which implicated chromosomal end-to-end fusion in these cells. Our results suggested high expression of TRF1 would induce chromosome missegregation at mitosis, a feature of chromosomal instability which is proposed to be a critical step in carcinogenesis. These findings bring fourth a new hint for our further understanding of carcinogenesis led by telomere dysfunction.
TNFα Stimulates NKG2D-Mediated Lytic Activity of Acute Myeloid Leukemic Cells.
Authors: Laurent Guy , Guilloton Fabien , de Thonel Aurelie , Jean Christine , Demur Cecile , De Mas Veronique , Quillet-Mary Anne
Published: October 2019

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFα, they display cytolytic activity towards various cellular targets including CFU-GM. This mechanism is dependent on stimulation of Granzyme B/Perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patient with acute myeloid leukemia (AML) (FAB M0 to M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by pool >15% of leukemic cells. Futhermore, CD34+ hematopoietic progenitors undergoing granulo-monocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which upon stimulation by TNFα leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFα accumulation, such as AML or myelodisplasic syndromes.
The Effects of Berbamine in Nude Mice Xenograft Model of Human Leukemic Cells.
Authors: Wu Dong , Lin Maofang
Published: October 2019

Background: Berbamine is a kind of bis-benzylisoquinoline extracted from Chinese herb. In our past research, it had been proven that berbamine could obviously inhibit the proliferation and induce apoptosis in different kinds of leukemic cell lines including K562, Jurkat, NB4 in a time-and dosage-dependent manner in vitro. The down-regulation expressions of survivin gene, bcr/abl gene and bcr/abl -related P210 may play an important role in the apoptotic effect of Berbamine in leukemic cells. However, the effects in vivo and its mechanism remain to be identified. In this study, the effects of berbamine in nude mice bearing human leukemic cell xenografts were tested.

Methods: The nude mice (BALB/C-nu/nu) were pre-treated with X ray 400cGy/2min, then 2×107 K562 cells and Jurkat cells were subcutaneously injected into the mice respectively. Rapid growth of solid tumors was observed in nude mice. One week later, the tumor-bearing mice were randomly divided into three treatment groups: untreated controls; berbamine-treated group (a dose of 1.0mg/day × 20 day, subcutaneously injected); Ara-C-treated group(a dose of 0.8mg/day, × 7 day, subcutaneously injected). Four weeks later, all the experimental mice were euthanized.

Results: Mice that received berbamine had significantly slower tumor growth rate than did untreated mice. In K562-bearing mice, the tumor weight of berbamine-treated group was lower than that of the control group(1.46g±0.43g vs 2.90g±0.94g, P<0.01 )and the inhibition rate was 49.66%. In Jurkat-bearing mice, the tumor weight of berbamine-treated group was also lower than that of untreated group(0.96g±0.64g vs 2.28g±0.33g, P<0.01), although it was comparable to that of Ara-C-treated group(0.54g±0.38g, P=0.311). The inhibition rates were 57.89% and 76.32% respectively. Moreover, in Jurkat-bearing mice berbamine inhibited the tumor cells invasion into bone marrow. In K562-bearing mice, berbamine down-regulated the expression level of bcr/abl gene of tumor cells ((berbamine-treated group 1.07±0.05 vs untreated group 1.58±0.24, P<0.05). In addition, no harmful side effects were attributed to berbamine.

Conclusion: In vivo, berbamine could aslo take a better antileukemia effect and the dose of berbamine used was safe with almost no side effects in nude mice. Berbamine extracted from Chinese herb might be a promising candidate of new drugs for clinical anticancer treatment, especially for bcr-abl+ diseases.
Tangeretin Induces Caspase Dependent Apoptosis and G2/M Arrest in the Erythroleukemia Cell Line K562 In Vitro.
Authors: Sofie Lust , Barbara Vanhoecke , Marc Bracke , Fritz Offner
Published: October 2019

Introduction

Tangeretin, a polymethoxyflavon present in citrus peel oil, was found to inhibit Erk-phosphorylation in T47-D breast cancer cells, induce cell death and block invasion in the chick heart invasion assay. Erk-phosphorylation has been implicated in the growth of bcr-abl transformed cell lines as a result of constitutive abl-activation. Here we investigate whether tangeretin can induce apoptosis and growth arrest in the bcr-abl+ erytroleukemia cell line K562, how it affects signal transduction pathways and the balance between pro- and anti-apoptotic proteins.

Methods

Tangeretin was dissolved in DMSO and used at concentrations up to 100 μM. K562 cells were cultured in RPMI 1640–10% FBS in vitro. Proliferation was followed by MTT test. Apoptosis, cell cycle analysis and bcl-2 expression was assessed by flow cytometry. PARP, Erk1/2, p38, Akt, JNK, p70S6K, and P-p38, P-Akt, P-JNK, and P-p70S6K, bcl-XL and mcl-1 were assessed by Western Blot.

Results

Tangeretin shows a time and concentration dependent effect on bcr-abl + K562 cells with a LD50 of 50–70 μM. This effect is accompanied by a G2/M arrest and a significant increase in the percentage of subG0 cells and PARP cleavage at 24 hrs. In short term kinetics (30 minutes) tangeretin inhibits the phosphorylation of Erk. No effect on total Erk1/2, p38, Akt, JNK, p70S6K, and P-p38, P-Akt, P-JNK, and P-p70S6K could be observed. After 24 and 48 hours of treatment, tangeretin is capable of stimulating the phosphorylation of Erk and p70S6K. At the same time activation of procaspase-3 and -9 and downregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL were seen. Bcl-2 expression was analysed by flow cytometry and was also downregulated.

Conclusion

The citrus flavonoid tangeretin is capable of inducing apoptosis and growth arrest in bcr-abl positive K562 cells through activation of caspase-3 and -9 accompanied by a biphasic change in phosphorylation of Erk and p70S6K.
Polymorphisms in Genes of Folate Metabolism and Response to High-Dose Methotrexate in Patients with Primary Central Nervous System Lymphoma.
Authors: Knop Stefan , Loeffler Juergen , Steffens Michael , Guenther Markus , Korfel Agnieszka , Weller Michael , Hebart Holger , Einsele Hermann , Herrlinger Ulrich
Published: October 2019

The antifolate methotrexate (MTX) was shown to be the single most-effective agent in first-line treatment of patients with primary central nervous system lymphoma (PCNSL) when given intravenously in high doses. MTX inhibits 5,10-methylentetrahydrofolate reductase (MTHFR) as well as thymidylate synthase (TS) in target cells what eventually results in decreased DNA synthesis. Intracellular uptake of both folate and MTX is mediated by the reduced folate carrier (RFC). Genetic polymorphisms for all three proteins were described: a C to T base transition at nucleotide 667 (C677T) and an A to C transition at 1298 (A1298C) of MTHFR; a 28-base pair (bp) tandemly repeated sequence polymorphic in the numbers of the repeat (2R/2R; 3R/3R; and 2R/3R) in the 5′-UTR of TS; a 6bp insertion-(+6bp)/deletion(−6bp)-polymorphism in the 3′-UTR of TS; and a polymorphism at nucleotide 80 (G80A) of RFC. We hypothesized that these polymorphisms may be directly linked to response, survival and toxicity in patients with PCNSL who are treated with single high-dose MTX. We prospectively collected blood from 152 subjects who were enrolled into a German multicenter PCNSL trial. They were scheduled to receive HD-MTX 4 g/m2 every 2 weeks. Genomic DNA was extracted from mononuclear cells using QIAGEN DNA Blood Mini Kit®. Genotyping of the two MTHFR (C677T; A1298C) and the RFC (G80A) single nucleotide polymorphisms (SNPs) was performed by melting point analysis with Master Hybridization Probes® using the Light Cycler® technology. Genotyping of the TS 28bp tandem/triplet repeats in the 5′-UTR was performed by conventional PCR followed by high resolution agarose gel electrophoresis. The TS +6bp/−6bp polymorphism in the 3′-UTR was analyzed with PCR, followed by restriction enzyme digest with draI and agarose gel electrophoresis. The fragment sizes were 70 bp and 88 bp for the 6 bp insertion allele and 152 bp for the 6 bp deletion allele. Allele frequencies for heterozygosity and for homozygous mutations of the two MTHFR and the RFC SNPs as well as of the two TS polymorphisms were as expected for a Caucasian population. For the currently 120 evaluable patients logistic regression analysis, Armitage Trend test and Kaplan-Meier survival curves were performed to analyze the role of the polymorphisms as potential predictors for MTX response. Up to now, we only found a trend for an association between response to treatment and survival for the 2 MTHFR mutations. We will analyze haematological and non-haematological toxicities which will be presented at the meeting with updated results for survival and response in the patients who will have completed treatment until then.
Monitoring CML28 mRNA Levels in Patients before and Post HSCT by Real-Time Quantitative RT-PCR.
Authors: Zhang Donghua , Dai Min , Zhou Hongsheng , Wang Yaya , Zhang Lu , Zhang Li , Wang Bin , Cao Wenjing
Published: October 2019

A SYBR Green I real-time quantitative RT-PCR method was established for investigating the correlation between CML28 mRNA expressing levels and relapse of leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitorting of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph+ ALL. The sensitivity of the established method was at 10−4 level, with interassay variation and intraassay variation of standard samples both < 10%. The CML28 was highly expressed in AML and CML-BP or AP. In newly diagnosed group, CML28 was (6.58±2.34)×10−2. In pre-conditioning regimen group was (2.19±0.32)×10−2, in group that 1 month after allo-HSCT was (1.35±1.28)×10−2, in group that 3 months after allo-HSCT was (4.57±6.39)×10−3. CML28 can be detected 3months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2×10−2) survived without relapse, the other 2 patients with high level (>2×10−2) relapsed within one year,1 died and1 received the second time allo-HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2×10−2 and relapsed again. CML28 mRNA level was obviously correlated with the development of diseases. Serial quantification of CML28 mRNA levels were necessary for allo-HSCT recipients, and more informative than a single detection. Use of this assay to evaluate MRD in the patients performed allo-HSCT was helpful for predicition of relapse.
SDF-1-CXCR4 Signaling and Downregulation of CD26/Dipeptidyl-Peptidase IV Are Involved in Skin-Homing of Sezary Cells.
Authors: Russo Giandomenico , Scala Enrico , Bresin Antonella , Caprini Elisabetta , Picchio Maria Cristina , Remotti Daniele , Ragone Gianluca , Nasorri Francesca , Frontani Marina , Cristofoletti Cristina , Arcelli Diego , Lombardo Giuseppe A. , Baliva Giannandrea , Napolitano Monica , Narducci Maria Grazia
Published: October 2019

Sezary Syndrome (SS) is a rare form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving skin and blood. Chemokine and chemokine receptors have been implicated in the spreading process of many cancers including various forms of non-Hodgkin T-cell lymphomas (NHL). In this study we report that chemokine receptor CXCR4 is over-expressed by both circulating and skin-homing neoplastic T-lymphocytes of SS patients and is functionally active as demonstrated by the migration of freshly isolated Sezary (SzS) cells along the chemical gradient of its natural ligand SDF-1. To shed light on the regulation of CXCR4/SDF1 interaction, we also investigated the enzymatic activity of CD26/dipeptidylpeptidase IV (DPPIV) since SDF-1 is efficiently inactivated by CD26, in physiological condition.. This is of particular relevance because one of the hallmark of the circulating SzS cells is the loss of CD26 from the cell surface. We first demonstrated that the CD26 negative phenotype is similarly maintained also in the skin-homing neoplastic T lymphocytes; we then observed that the addition of exogenus soluble CD26 reduces the migratory response of SS cells to SDF-1 whereas the inhibition of the CD26 peptidase activity in Hut78, a CD26-positive CTCL cell line, enhances the SDF-1-induced migration of these cells. We finally showed that SS individuals exhibit a reduced activity of the soluble CD26 as revealed by the measurements performed on the patients derived plasma. Our findings suggest that the SDF-1-CXCR4 axis could play an important role in skin homing of SS through the regulatory activity of CD26.
Impact of RNA Stabilization on Minimal Residual Disease Assessment in Chronic Myeloid Leukemia.
Authors: Thörn Ingrid , Olsson-Strömberg Ulla , Ohlsen Cecilia , Jonsson Anna-Maria , Klangby Ulf , Simonsson Bengt , Barbany Gisela
Published: October 2019

Background and objective: Accurate quantification of BCR-ABL mRNA is of critical importance for managing chronic myeloid leukemia (CML) patients receiving Imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patient samples with long transport times between the hospital and the analyzing laboratory may be subject to RNA degradation with a corresponding loss in sensitivity and possible generation of false negative results. Recently, RNA preservation systems have been developed in order to improve RNA stability.

Design and methods: The aim of the study was to investigate the performance of the PAXgene Blood RNA Kit in CML follow-up peripheral blood samples and compared the results to unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time PCR.

Results: RNA isolated with the PAXgene system gave a superior yield per ml blood than with the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently compared to equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed to decreased sensitivity for MRD detection, resulting in false negatives. The sensitivity was comparable to samples processed routinely 20–30 hours after phlebotomy.

Interpretation and conclusions: We conclude that routinely processed, i.e. unstabilized, peripheral blood that reaches the laboratory and is processed within 30 hours is preferable for MRD detection. Optimal results were achieved with fresh samples processed within 5 hours with the Trizol method. However, RNA stabilization may be useful if sample transit is expected to exceed 30 hours.