The multilevel regulation of CD95 signaling outcome

CD95, also called Fas or APO-1, was the first death receptor (DR) identified and characterized. Studies on CD95 receptor signaling revealed the versatile principles of cell fate regulation via DR. DRs could exert both pro- and anti-apoptotic effects depending on clustering, internalization or signaling thresholds and other extracellular signals. It became clear that molecular network regulating cell death and survival is under the multilevel control. In this Review we focus on the regulation of CD95 signaling and provide brief analysis of molecular switches of its pro- and antiapoptotic functions. At least five levels of life-death cell regulation via CD95 could be tracked: extracellular, membrane, DISC, mitochondrial, and miRNA. The cellular outcome of signaling via DRs depends on other extracellular signals and availability of different intracellular components of signal transduction pathways. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”

Hodgkin’s lymphoma: the role of cell surface receptors in regulation of tumor cell fate

The hallmark of Hodgkin’s lymphoma (HL) are mononucleated Hodgkin’s cells and multinucleated Reed-Sternberg (HRS) cells, which usually account for only about 1% of cells in the tumor tissue. The majority of HRS cells in classical HL are derived from germinal centre B cells that have acquired disadvantageous Ig variable chain gene mutations and escaped from apoptosis

Helicobacter pylori infection of gastric cancercells elevates the level of expression and activation of protein kinase D2

Aim: To test the hypothesis, whether H. pylori infection may affect the level of PKD2 expression and/or activation in gastric cancer cells. Methods: Studies were performed on AGS human gastric adenocarcinoma cell line, gastric tissues samples from 36 cases of different histological variants of gastric cancer. Immunohistochemical, cell and molecular biology, bacteriological and biochemical approaches have been used in this study. Results: H. pylori 16S rRNA gene was detected in 97% cases of gastric tumors, and in 83% of cases cаgA gene was detected. In all tested adenocarcinoma samples cagA+ H. pylori was revealed. These cases were characterized by high level of PKD1/2 expression and autophosphorylation. In adenogenic cancer samples the presence of cagA– H. pylori was identified. Carcinoid and nondifferentiated gastric cancers contain H. pylori, with very low numbers of cagA+ copies. All cases of gastric tumors with cagA– H. pylori had very low levels of PKD1/2 autophosphorylation. AGS cell line infection with cagA– and cagA+ H. рylori resulted in elevation of PKD2 expression levels in 3.29 and 3.66 times respectively (p < 0.001). In cells infected by cag+ H. рylori the level of PKD2 transphosphorylation was 1.39 higher than in cells infected by cagA– H. pylori. For PKD2 autophosphorylation this difference was even higher — 3.27 times (p < 0.001). Conclusion: H. pylori infection enhanced the level of protein kinase D2 expression, trans- and autophosphorylation. The level of PKD2 autophosphorylation/activation was higher in AGS cell line inoculated of with cag+ H. pylori than in AGS cells with cagA– H. pylori. These suggest that H. pylori induces activation of PKD1/2 and could exploit PKD2 mediated signaling pathways that may contribute to the pathogenesis of gastric cancer

CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells

Background: Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150 cell surface expression. From other side we attempted to regulate TFs expression via CD150 and CD180 cell surface receptors. Materials and Methods: Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia (CLL) cells isolated from peripheral blood of 67 primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. Results: Median of PAX5 and EBF1 mRNA expression was higher in cell surface CD150 positive (csCD150⁺) compared to csCD150⁻ CLL cases or normal CD19⁺ and CD19⁺CD5⁺ B-cell subsets. Differences in mRNA expression of IRF8, IRF4 and BLIMP1 between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1 and BCL6 mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6 was uniformly distributed between csCD150⁻ and csCD150⁺ CLL cases. PU.1 protein and CD20 that is direct PU.1 target gene positively correlated with CD150 cell surface expression on CLL cells. Ligation of CD150 and CD180 alone or in combination upregulated IRF8 and PU.1 while downregulated the IRF4 mRNA expression. Signaling via CD150 or CD180 alone elevated the level of BCL6 mRNA. Strong downregulation of IRF4 mRNA was observed after CD150, CD180 or CD150 and CD180 coligation on CLL cells. We found that in CLL cells CD150 is a negative regulator of SPIB while CD180 is involved in upregulation of EBF1 expression level. Moreover, CD180 ligation on CLL cells caused increase of CD150 mRNA level that is a one of the EBF1 target genes. Conclusions: Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 in CLL cells. CD150 and CD180 receptors may modulate transcriptional program in CLL cells by regulating the TFs expression level

Differential expression of PKD1 and PKD2 in gastric cancer and analysis of PKD1 and PKD2 function in the model system

Aim: To study the differential expression of PKD1 and PKD2 in primary gastric cancer samples and to examine the role of PKD1 and PKD2 protein kinases in regulation of gastric tumor cell biology in the model system. Methods: Tumor samples of different histological variants of primary gastric cancer were analyzed. PKD1 and PKD2 expression levels in tumor samples were accessed by Western blot analysis and quantitative polymerase chain reaction (Q-PCR). As a model system we have used gastric adenocarcinoma сell line AGS sublines constitutively transfected by pcDNA3.1 coding PKD1 or PKD2, or empty pcDNA3.1 vector. These cell lines were analyzed by Western blot, Q-PCR, MTT and proliferation assays, in vitro scratch and Transwell assays, clonogenic assay. Results: It was found that primary gastric tumors possess different levels of PKD1 and PKD2 expression on mRNA and protein levels. Low level of PKD1 expression on protein and mRNA level was detected in low differentiated adenocarcinoma and ring cell gastric cancer — disorders with poor clinical prognosis. The high level of PKD2 expression was also found in gastric tumors with poor prognosis: low differentiated adenocarcinoma and adenogen cancer. To find out whether differential expression of PKD1 and PKD2 could affect biology of gastric tumor cells in vitro, we used a model system based on AGS cell line that constitutively expressed PKD1 or overexpressed PKD2. PKD1 transfection led to the inhibition of cell proliferation, migration and colony formation, in the meanwhile, the PKD2 overexpression enhanced proliferation, migration and colony formation capacities of AGS cells. Conclusions: Our data suggest that both downregulation of PKD1 or upregulation of PKD2 expression may determine the behavior of gastric tumor cells, which promotes invasive phenotype and could result in general poor prognosis

Intrinsic defect in B-lymphoblastoid cell lines from patients with X-linked lymphoproliferative disease type 1. II. Receptor-mediated Akt/PKB and ERK1/2 activation and transcription factors expression profile

Background: X-linked lymphoproliferative disease type 1 (XLP1) belongs to genetically determined primary immunodeficiency syndromes with mutations in SH2D1A/DSHP/SAP gene. The dramatic increase of the risk of B-cell lymphoma development in XLP1 patients is linked not only to SAP deficiency of NK, NKT and T cells, but probably to the impairment of B cell differentiation. Aim: To analyze the receptor-mediated Akt/PKB and ERK1/2 phosphorylation and expression of transcription factors that are involved in B cell maturation in EBV-transformed B-lymphoblastoid cell lines (B-LCLs) from XLP1 patients (XLP B-LCLs) in comparison with conventional B-LCLs. Methods: Studies were performed on EBV-transformed XLP B-LCLs IARC 739, SC-XLP and RP-XLP in comparison with SAP-negative B-LCL T5-1 and SAP-positive B-LCL MP-1. Western blot analysis was used for evaluation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204) phosphorylation in response to ligation of CD150, CD40, and IgM cell surface receptors. The expression levels of transcription factors IRF4, IRF8, BCL6, BLIMP1, SPIB, PU.1 and MITF were assessed using quantitative RT-PCR. Results: It was shown that SAP deficiency in XLP B-LCL did not abrogate CD150-mediated Akt and ERK1/2 phosphorylation. At the same time, ligation of CD150 or IgM affects kinetics and amplitude of ERK1/2 activation. In XLP B-LCL the CD150 signaling with IgM coligation play the dominant role in both Akt and ERK1/2 phosphorylation. We found that significantly reduced IRF4, IRF8 and PU.1 expression levels are the key features of XLP B-LCLs. Conclusion: XLP B-LCLs and conventional B-LCLs have differences in kinetics and amplitude of Akt and ERK1/2 phosphorylation. Analysis of transcription factors profile revealed the distinguishing features of XLP B-LCLs with SAP deficiency that may impair B cell differentiation.Key Words: B-lymphoblastoid cell lines, X-linked lymphoproliferative disease type 1, CD150, CD40, BCR, Akt/PKB, ERK1/2, transcription factors. Key Words: B-lymphoblastoid cell lines, X-linked lymphoproliferative disease type 1, CD150, CD40, BCR, Akt/PKB, ERK1/2, transcription factors

Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation

Background: Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts. Materials and Methods: Studies were performed on normal tonsillar B-cell subpopulations, Blymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt’s lymphoma, Hodgkin’s lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expression level of CD150 isoforms was evaluated using qRT-PCR. Results: Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin’s lymphoma cell lines, Burkitt’s lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin’s lymphoma cell lines and Burkitt’s lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi. Conclusion: Malignant Bcell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform

Intrinsic defect in B-lymphoblastoid cell lines from patients with X-linked lymphoproliferative disease type 1. I. Cell surface phenotype and functional studies

Background: Mutations in SH2D1A/DSHP/SAP gene are responsible for the onset of X-linked lymphoproliferative disease type 1 (XLP1) that have increased risk for B-cell lymphoma development. In XLP1 patients SAP deficient NK, NKT and CD8+ cytotoxic T cells are inefficient in eliminating EBV-infected proliferating B cells that may partially contribute to the lymphoma development. However, little is known about impairment of B cell characteristics in XLP1. Aim: To analyze the cell surface phenotype and functional characteristics of EBV-transformed B-lymphoblastoid cell lines from XLP1 patients (XLP B-LCLs) in comparison with conventional B-lymphoblastoid cell lines (B-LCLs). Methods: Studies were performed on SAP-negative B-LCLs T5-1, 6.16, RPMI 1788; SAP-positive B-LCL MP-1 and XLP B-LCLs IARC 739, XLP-D, XLP-8005. Cell surface immunophenotyping was performed using flow cytometry analysis. The level of apoptotic cells (Annexin V-binding), cell viability (MTT assay), and cell proliferation (trypan blue exclusion test) were evaluated in response to ligation of CD40, CD95, CD150 and IgM cell surface receptors. Results: A cell surface phenotype and functional features that distinguish XLP B-LCLs from conventional B-LCLs were revealed. XLP B-LCLs showed the upregulated level of CD20, CD38 and CD86 cell surface expression and downregulation of CD40, CD80 and CD150 expression. The major functional differences of XLP B-LCLs from conventional B-LCLs concern the modulation of CD95 apoptosis via CD40 and CD150 receptors and unresponsiveness to proliferative signals triggered by CD40 or colligation of BCR with CD150. Conclusion: The data suggest that the B-LCL from XLP1 patients have an intrinsic defect that affects cell activation, apoptosis, and proliferation. Key Words: B-lymphoblastoid cell lines, X-linked lymphoproliferative disease type 1, CD150, CD40, CD95, apoptosis

Immunohistochemical studies of protein kinase D (PKD) 2 expression in malignant human lymph

Aim: To study the PKD2 expression, autophosphorylation and localization in reactive lymph nodes and tumors of lymphoid tissues. Materials and Methods: Specific antibodies, which recognize PKD1/2 or PKD2 and autophosphorylated PKD1/2, were used for immunohistochemical and biochemical studies of tonsils, reactive lymph nodes, tumor samples of non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL). Results: Immunohistochemical and biochemical analysis of PKD1 and PKD2 expression showed PKD2 expression in tonsils, reactive lymph nodes and tumor tissues from patients with NHL and HL. Furthermore, we were not able to reveal PKD1 expression in studied lymphoid tissues. In tonsils and reactive lymph nodes the PKD2 expression was detected in T and B cell zones with highest level in germinal centers of lymphoid follicles and the maximum level of autophosphorylation in the light zones of the germinal centers. We found that low level of PKD2 expression and autophosphorylation was characteristic feature for mantle cell lymphomas, Burkitt’s lymphomas, and in 50% of CLL/small lymphocytic lymphomas. Lymphoma cells of germinal center origin and with activated B cell phenotype (diffuse large B cell lymphomas, HL) and anaplastic large cells lymphoma demonstrated the high level of PKD2 expression and autophosphorylation. Conclusions: The level of PKD2 expression and autophosphorylation in neoplastic cells corresponds to the expression pattern of this kinase in their normal analogs, and to the level of cell differentiation and activation.Цель: изучить экспрессию, аутофосфорилирование и локализацию PKD2 в клетках реактивно измененных лимфaтических узлов и опухолях лимфоидной ткани. Материалы и методы: специфические антитела, распознающие PKD1/2 или PKD2 и аутофосфорилированную PKD1/2, были использованы для иммуногистохимического и биохимического анализа клеток небных миндалин, реактивно измененных лимфaтических узлов, биоптатов опухолей у больных неходжкинскими злокачественными лимфомами (НХЛ) и лимфомой Ходжкина (ЛХ). Результаты: при иммуногистохимическом и биохимическом исследовании выявлена экспрессия PKD2 в клетках миндалин, гиперплазированных лимфатических узлов и различных гистологических форм НХЛ и ЛХ. Экспрессия PKD1 в изученных нами лимфоидных тканях выявлена не была. Экспрессия PKD2 была выявлена в T- и B-клеточных зонах миндалин и реактивно измененных лимфатических узлов. Наиболее высокий уровень экспрессии отмечался в клетках зародышевых центров лимфоидных фолликулов, максимальный уровень аутофосфорилирования — в светлых зонах зародышевых центров. Обнаружено, что низкий уровень экспрессии и аутофосфорилирования PKD2 является характерной особенностью лимфом из клеток мантийной зоны, лимфомы Беркитта, в 50% случаях лимфом из малых лимфоцитов/ХЛЛ. Клетки лимфом, возникающих из клеток зародышевых центров с фенотипом активированных B-клеток (диффузная лимфома из крупных B-клеток, ЛХ), а также анапластическая крупноклеточная лимфома характеризовались высоким уровнем экспрессии и аутофосфорилирования PKD2. Выводы: уровень экспрессии и аутофосфорилирования PKD2 в клетках новообразований соответствует особенностям экспрессии этих киназ в соответствующих нормальных аналогах, уровню клеточной дифференцировки клеток и их активации

CD150-mediated Akt signalling pathway in normal and malignant B cells

Aim of this article is to study upstream and downstream events in CD150-mediated Akt signaling pathway in normal human B cells, EBV-transformed lymphoblastoid (LCL) and malignant Hodgkin’s lymphoma (HL) B cell lines