RESUMEN

BACKGROUND: SLAMF1/CD150 receptor is aberrantly expressed in malignant hematopoietic cells compared to ubiquitous expression in their normal analogues. However, the data about CD150 expression and function outside the hematopoietic system are limited. The aim of this pilot study was to examine the profile of mRNA expression of CD150 isoforms and the protein topology in highly and low malignant breast (BC) and prostate cancer (PC) cell lines. MATERIALS AND METHODS: The study was performed on BC T47D, MDA-MB-231, ВСС/Р and BC/ML cell lines and PC LNCap, Du-145 and PC-3 cell lines. The quantitative polymerase chain reaction was applied for study of CD150 isoforms mRNA expression and flow cytometry was used for determination of protein localization. RESULTS: Analyzed BC cell lines did not express CD150 on the cell surface membrane (csCD150-), but more than 45% of cells were positive for CD150 cytoplasmic reaction (cyCD150+). The cyCD150 expression level in T47D cells of luminal BC subtype was higher than in basal BC cell lines MDA-MB-231, ВСС/Р and BC/ML. The PC cell lines expressed CD150 both on the cell surface and in cytoplasm. The highest number of csCD150+ and cyCD150+ cells was revealed in less aggressive androgen responsive, non-metastatic LNCap cell line. All studied BC and PC cell lines expressed mRNA of canonical transmembrane mCD150 and novel nCD150 isoforms but with different pattern of prevalence. Soluble CD150 isoform was revealed at the low level only in BCC/P BC cell line and LNCap, PC-3 PC cell lines. CONCLUSIONS: We have shown that BC and PC cell lines differentially expressed multifunctional receptor CD150 at the mRNA and protein levels that may indicate its association with the degree of their malignancy.

Asunto(s)

Neoplasias de la Mama , Neoplasias de la Próstata/genética , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Células MCF-7 , Masculino , Proyectos Piloto , Isoformas de Proteínas/genética

RESUMEN

UNLABELLED: 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, B-lymphoblastoid 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 B-cell 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.

Asunto(s)

Linfocitos B/patología , Linfoma de Burkitt/genética , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/genética , Mieloma Múltiple/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Linfocitos B/metabolismo , Linfoma de Burkitt/patología , Línea Celular , Línea Celular Tumoral , Enfermedad de Hodgkin/patología , Humanos , Mieloma Múltiple/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , ARN Mensajero/genética , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/análisis

RESUMEN

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.

Asunto(s)

Linfocitos B/patología , Biomarcadores de Tumor/metabolismo , Trastornos Linfoproliferativos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismo , Linfocitos B/metabolismo , Biomarcadores de Tumor/genética , Western Blotting , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Células Tumorales Cultivadas

RESUMEN

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.

Asunto(s)

Antígenos CD/biosíntesis , Apoptosis/genética , Linfocitos B/patología , Proliferación Celular/genética , Trastornos Linfoproliferativos/inmunología , Linfocitos B/inmunología , Línea Celular Transformada , Supervivencia Celular/genética , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Trastornos Linfoproliferativos/patología , Receptores de Superficie Celular/biosíntesis , Linfocitos T Citotóxicos/inmunología

RESUMEN

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".

Asunto(s)

Apoptosis/genética , Transducción de Señal/genética , Receptor fas , Membrana Celular/genética , Membrana Celular/metabolismo , Supervivencia Celular/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Receptor fas/genética , Receptor fas/metabolismo

RESUMEN

AIM: 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. METHODS: To access protein-protein interaction we applied immunoprecipitation, Western blot analysis and surface plasmon resonance (SPR) technique. A novel modification of SPR technique using reduced glutathione bound to golden surface was proposed. Immunostaining and isolation of cytoplasmic fractions and nuclear extracts were performed to detect proteins' localization in cells. Western blot analysis was performed to follow up the phosphorylation of proteins on specific sites and proteins' expression level. RESULTS: It was shown that CD150 ligation induced Akt activation in normal tonsillar B cells (TBC), SH2D1A positive LCL and HL B cell lines. The p85α subunit of PI3K co-precipitated with CD150 cytoplasmic tail. This direct association depends on tyrosine phosphorylation and is mediated by N terminal SH2 domain of p85α. CD150 initiated phosphorylation of FoxO1 transcription factor in normal B cells as well as in LCL MP-1 and HL cell line L1236. At the same time, CD150 ligation triggered GSK-3ß kinase phosphorylation only in immortalized LCL MP-1 and HL cell line L1236. CONCLUSIONS: We have demonstrated that CD150 receptor could trigger PI3K-mediated Akt signaling pathway in normal, EBV-transformed and malignant B cells. CD150-mediated phosphorylation of Akt downstream targets GSK-3ß and FoxO1 in EBV-transformed and HL cells could be one of the mechanisms to avoid apoptosis and support survival program in these immortalized B cells.

Asunto(s)

Antígenos CD/metabolismo , Linfocitos B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Linfocitos B/citología , Linfocitos B/patología , Línea Celular Transformada , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espacio Intracelular/metabolismo , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Dominios Homologos src

RESUMEN

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.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Humanos , Invasividad Neoplásica/genética , Proteína Quinasa D2 , ARN Mensajero/metabolismo

RESUMEN

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.

Asunto(s)

Enfermedad de Hodgkin/enzimología , Linfoma no Hodgkin/enzimología , Proteínas Quinasas/análisis , Enfermedad de Hodgkin/patología , Humanos , Inmunohistoquímica , Ganglios Linfáticos/enzimología , Linfoma no Hodgkin/patología , Tonsila Palatina/enzimología , Fosforilación , Proteína Quinasa C/análisis , Proteína Quinasa D2

RESUMEN

CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negative signals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phosphatase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan's disease SH2-protein (DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome and non-Hodgkin's lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines, where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines from X-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated by CD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosine at Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 are essential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1A may function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptor tyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2 subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin, carcinoembryonic Ag, and leukocyte-inhibitory receptor families.

Asunto(s)

Proteínas Portadoras/fisiología , Glicoproteínas/metabolismo , Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Dominios Homologos src/inmunología , Secuencia de Aminoácidos , Antígenos CD , Linfocitos B/enzimología , Linfocitos B/metabolismo , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/metabolismo , Línea Celular Transformada , Células Cultivadas , Citoplasma/inmunología , Citoplasma/metabolismo , Glicoproteínas/genética , Humanos , Inmunoglobulinas/genética , Células Jurkat , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Unión Proteica/genética , Unión Proteica/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Células Tumorales Cultivadas , Tirosina/genética

RESUMEN

CDw150, a receptor up-regulated on activated T or B lymphocytes, has a key role in regulating B cell proliferation. Patients with X-linked lymphoproliferative disease have mutations in a gene encoding a protein, DSHP/SAP, which interacts with CDw150 and is expressed in B cells. Here we show that CDw150 on B cells associates with two tyrosine-phosphorylated proteins, 59 kDa and 145 kDa in size. The 59-kDa protein was identified as the Src-family kinase Fgr. The 145-kDa protein is the inositol polyphosphate 5'-phosphatase, SH2-containing inositol phosphatase (SHIP). Both Fgr and SHIP interact with phosphorylated tyrosines in CDw150's cytoplasmic tail. Ligation of CDw150 induces the rapid dephosphorylation of both SHIP and CDw150 as well as the association of Lyn and Fgr with SHIP. CD95/Fas-mediated apoptosis is enhanced by signaling via CDw150, and CDw150 ligation can override CD40-induced rescue of CD95-mediated cell death. The ability of CDw150 to regulate cell death does not correlate with serine phosphorylation of the Akt kinase, but does correlate with SHIP tyrosine dephosphorylation. Thus, the CDw150 receptor may function to regulate the fate of activated B cells via SHIP as well as via the DSHP/SAP protein defective in X-linked lymphoproliferative disease patients.

Asunto(s)

Apoptosis , Linfocitos B/metabolismo , Glicoproteínas/metabolismo , Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas , Receptor fas/metabolismo , Antígenos CD , Proteínas Portadoras/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Trastornos Linfoproliferativos/inmunología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Agregación de Receptores , Receptores de Superficie Celular , Transducción de Señal , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Dominios Homologos src , Familia-src Quinasas