Mutations of the tumor suppressor gene SOCS-1 in classical Hodgkin lymphoma are frequent and associated with nuclear phospho-STAT5 accumulation.

The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P. For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here, we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (P < 0.01). Collectively, these findings support the concept that PMBL and cHL share many overlapping features, and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas.

U-HO1, a new cell line derived from a primary refractory classical Hodgkin lymphoma.

The Hodgkin cell line U-HO1 was established from a malignant pleural effusion of a 23-year-old male patient during the end stage of refractory nodular sclerosing classical Hodgkin lymphoma (cHL). Since its establishment in 2005, U-HO1 has maintained stable characteristics in vitro and has a doubling time of about 4 days under standard culture conditions. U-HO1 forms typical Reed-Sternberg cells in suspension, is EBV negative, lacks HLA-A, -B, -C but expresses HLA-D proteins/CD74 and exposes CD15 together with CD30 in the absence of CD19 and CD20 on the cell surface. Karyotype analysis of U-HO1 revealed a hyperdiploid karyotype with multiple clonal aberrations. Most significant is an elongated chromosome 2, der(2)t(2;10)(q35; q16.1)add(2)(p13). CGH analysis revealed the following imbalances: ish cgh dim(1)(p13p31)(p12q21), enh(2)(p13p23), dim(4)(q31.3qter), enh(6)(q22q27), enh(12), enh(18), enh(20) (q13.1pter). FISH analysis showed about six-fold amplification of REL and BCL11A, thus, U-HO1 is prototypical for cHL in every aspect tested so far. As an outstanding feature compared to the existing HL cell lines, U-HO1 has high levels of microRNA transcripts of MIRN216 and MIRN217 located in the amplicon 2p16. Compared to other HL cell lines, U-HO1 proved far less genetically aberrant suggesting that U-HO1's imbalances suffice to cause the full-blown phenotype of primary refractory cHL.

Delineation of the 6p22 amplification unit in urinary bladder carcinoma cell lines.

Eight cell lines from transitional cell carcinoma of the urinary bladder were analyzed by comparative genomic hybridization. All tumor lines exhibited frequent chromosome gains (11.5/cell line) and losses (8.4/cell line). In six cell lines, gain of chromosome 5p was associated with gains of 6p and 20q. In five of these cell lines, amplification of parts of 6p was observed. Cytogenetic investigation combined with fluorescence in situ hybridization analysis revealed typical marker chromosomes with homogeneously staining regions (HSRs) containing material from 6p. By hybridizing individual yeast artificial chromosome probes from a chromosome 6p contig to these HSRs, a contig of three yeast artificial chromosomes common to all 6p HSRs was identified that spans less than 2 Mb. The genes SOX4 and PRL were shown to map to this region and to be coamplified in the cell lines. However, SOX4 was not overexpressed in any cell line and PRL was not expressed at all. Thus, the presumptive 6p oncogene remains to be conclusively identified.

Comparative effects of trypsin, collagenase and mechanical harvesting on cell membrane lipids studied in monolayer-cultured endothelial cells and a green monkey kidney cell line.

Experiments are presented in which membrane lipids of endothelial cells in monolayer culture were labelled with [14C]linoleic acid. Approx. 90% of the radioactive label were incorporated into phospholipids. A comparison of various harvesting methods showed that during the disruption of the labelled endothelial cell monolayer, 0.25% trypsin and 0.125% trypsin (+0.01% EDTA) released 650 and 470% more radioactivity, respectively, than did 0.01% collagenase (+0.01% EDTA). Parallel studies were performed on a green monkey kidney cell line. In this case, 0.25% trypsin released 520% more radioactivity than did 0.1% collagenase (+0.01% EDTA), although 0.125% trypsin in the presence of EDTA (0.01%) was much less traumatic than trypsin alone, the released radioactivity being of the same order of magnitude as that for collagenase. Morphological studies on endothelial cell cultures failed to reveal any distinctive differences in surface morphology following the various enzyme treatments. The results suggest that collagenase treatment of endothelial cell monolayers is the least traumatic harvesting or subculturing method as far as the integrity of the lipids in the cell membrane is concerned.

Cholesterol synthesis in cultured human peripheral lymphocytes. Influence of LDL, HDL, cholesterol/phosphatidylcholine liposomes and complete serum.

Lipoprotein-deficient milieu, freshly isolated human peripheral blood lymphocytes lose about 50% of their membrane cholesterol into the medium within 8 h. The cholesterol loss is counter-regulated by de novo synthesis commencing after a lag phase of 8-12 h, and reaching a steady state within 24 h at a diminished membrane cholesterol level. About 50 micrograms free cholesterol/ml, offered in the form of low-density lipoproteins (LDL) and cholesterol/phosphatidylcholine liposomes, suppressed cholesterol synthesis to about 20% of that controls (lipoprotein-deficient culture). By contrast, pure phosphatidylcholine liposomes enhanced cholesterol synthesis to about 150% of control values. High-density lipoproteins (HDL) exerted a slightly suppressive effect on cholesterol synthesis only at high concentrations (greater than 100 micrograms HDL cholesterol/ml). HDL added to cultures containing fixed concentrations of LDL led to a dose-dependent neutralization of LDL suppression of cholesterol synthesis. Culture medium containing complete serum caused a suppression of cholesterol synthesis to about 50% of the control. The lesser reduction in cholesterol synthesis caused by complete serum compared with LDL or cholesterol/phosphatidylcholine liposomes can be explained by the presence of HDL in the former. Our results support the view that the cholesterol requirement of blood lymphocytes in their lipid-rich milieu is met by cholesterol neosynthesis as well as an exchange mechanism with surrounding lipoproteins. In our system, the cholesterol neosynthesis appears to be controlled by the ratio of LDL to HDL in the surrounding medium.

Specific modifications of the membrane fatty acid composition of human myotubes and their effects on the muscular sodium channels.

The fatty acid (FA) composition of human myotube primary cultures was varied by modifications of the contents of FA in the culture medium. An incubation time of 18 h with a defined FA mixture resulted in the most effective alteration of the original FA pattern of the cells. The increases reached for the relative amounts of palmitic acid (16:0), linoleic acid (18:2) or arachidonic acid (20:4) were 3-5-fold. More than 50% of the extra FA were incorporated in the phospholipid fraction, the remaining share in the triglyceride fraction. Shorter incubation times resulted in less FA incorporation, longer incubation times raised the uptake of FA into the triacylglycerol fraction. For a study of the influence of the membrane modification on the function of the sodium channels, the myotubes were converted into myoballs. The sodium channel properties were then determined using the whole-cell clamp technique. The modified cultures showed no significant alterations in the time constants of activation and inactivation, in the voltage dependence of inactivation (h infinity curves) or in the average amplitudes of the sodium currents.

CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies.

In recent years, some studies on the expression of CD95(Fas/APO-1) ligand (CD95L) in tissues or cells raised concerns about the specificity of the antibodies used. We therefore tested 12 CD95L antibodies for their reliability in immunocyto/histochemistry by (i) staining CD95L-transfected and control CV-1/EBNA cells and (ii) comparing staining patterns in immunohistochemically labeled tissue sections with the localization of CD95L+ cells in in situ hybridization. While G247-4, NOK-1, NOK-2, 4H9, and MIKE-1 stained CD95L-transfected cells and did not significantly bind to controls, G247-4 was the only antibody giving satisfying signals in tissue sections perfectly matching the distribution of CD95L+ cells by in situ hybridization. MAb 33, C-20, and N-20 comparably stained both transfected and control cells and showed considerable background or falsely positive staining in sections. MIKE-2, 8B8, A11, and 4A5 did not or only very faintly bind to either cells and, thus, were not tested on sections. We conclude that G247-4 is the only tested antibody that is recommendable for immunohistochemistry.

Mitochondrial complex I and III mutations and neutral-lipid storage in activated mononuclear macrophages and neutrophils: a case presenting with necrotizing myopathy, poikiloderma atrophicans vasculare, and xanthogranulomatous bursitis.

We report the case of a 57-year-old woman suffering from xanthogranulomatous bursitis, necrotizing myopathy, and poikiloderma atrophicans vasculare, which are associated with marked accumulation of neutral-lipid storage phagocytes. The observed lipid storage was restricted to activated phagocytes independent of the presence of tissue necrosis and was not seen either in circulating blood leukocytes or in muscle fibers. The patient's daughter disclosed xanthomatous inflammatory reaction with profound delay of wound healing secondary to pelviscopy. Examination of the mitochondrial DNAs of the patient, her daughter, and her two grandchildren revealed two homoplasmic mutations at positions 13708 and 15257 of the mitochondrial genome. We discuss the involvement of these mutations in the pathogenesis of xanthomatous and xanthogranulomatous inflammation. Further investigations are required to test whether impairment of aerobic energy production independent from mitochondrial DNA mutations (eg, by hypoxia or microbial toxins) similarly can cause the accumulation of lipid-laden macrophages and explain the persistency of xanthogranulomatous inflammation.

Alterations induced on cytoskeleton by Escherichia coli endotoxin in different types of rat liver cell cultures.

Endotoxins (lipopolysaccharide, LPS) from Gram-negative bacteria are considered as the agents responsible for the induction of endotoxic shock, producing severe cellular metabolic dishomeostasis. Cytotoxic lesions, as well as functional and metabolic disturbances, occur mainly in the liver, which is one of the target organs and exerts an LPS clearance function. In an attempt to approach the molecular basis of endotoxic shock, and to propose an experimental model, we have focused this study on cytoskeleton (microtubules and microfilaments) alterations induced by different doses of endotoxin in different target liver cells. Microfilaments and microtubules were studied by immunofluorescence and different microscopy techniques (optic fluorescence microscopy and confocal laser scanning microscopy) in order to improve the cytoskeleton study resolution. Parenchymal and sinusoidal cell morphology, severely damaged by the LPS action, is related to a disturbance on the cytoskeletal organisation, even more evident in a particular proliferating rat liver cell culture. The most relevant changes are seen in the microtubule patterns in all liver cells tested, which could be related, depending on cell type, either to a direct LPS action or to [Ca+2]i dishomeostasis as well as free radical and cytokine (IL-1beta and TNF-alpha) production. Due to their features, proliferating rat liver cell cultures are used as a sensitive cell model to understand the effect of LPS on cytoskeleton organisation.

Relationships between lymphocyte cholesterol homeostasis and LDL-cholesterol.

The homeostasis of cholesterol was studied in lymphocytes freshly isolated from the blood and cultured with or without low-density lipoprotein (LDL). The content of cholesterol decreased in the lymphocytes cultured without LDL, whereas LDL substituted for cellular cholesterol losses, in spite of almost suppressed LDL-receptor and lymphocyte cholesterol synthesis. Free cholesterol was taken up from LDL mainly via cholesterol exchange and, in contrast to esterified cellular cholesterol, rapidly excreted into the medium. In vitro stimulation of lymphocyte cholesterol synthesis was correlated to the ratio of esterified to free LDL-cholesterol in the blood from which the lymphocytes had been isolated. This result probably reflects the different rates of influx and efflux of esterified or free cholesterol between plasma lipoproteins and lymphocytes. These effects should be taken into account if LDL-cholesterol is determined in plasma for the evaluation of an individual's atherosclerotic risk.

Selective inhibition of human neutrophilic chemotaxis by echo virus, type 9. Virus-induced changes in membrane fluidity.

By means of the excimer-forming lipid technique changes of membrane fluidity of human PMNs exposed to a chemoattractant (FMLP) and/or Echo virus, type 9, strain A. Barty has been investigated. It was shown that preincubation of PMNs with Echo 9 virus results in a viral dose-dependent decrease of membrane fluidity of granulocytes exposed to FMLP. The virus-induced rigidity of cell membrane causes obviously a disturbance in the aggregation of membrane particles to form receptors, and thus can lead to a failure of regular processing of chemotactic signals.

Non-random spatial distribution of intermembraneous particles in red blood cell membrane.

A combined analysis using digital image processing and quantitative evaluation of computed radial distribution of freeze fracture P face particles irradiated and nonirradiated has been carried out. Important quantitative features of the arrangement of particles can be described by computing a statistical measure, the radial distribution function g(r), commonly used to study the structure of liquids. The coordinates for calculating g(r) for each sample were measured automatically by a digital image processing system. The results for the radial distribution function were found to closely resemble g(r) for fluids, indicating the presence of shortrange order. This measure distinguishes three different patterns: i) random distribution, ii) dilute gas like and iii) a liquid like model. The mean number of first nearest neighbours surrounding each particle (coordination number) was found to be of the order of three. The RBC intramembraneous particles are heterogeneous in their behavioural pattern: two thirds random; one fifth, dilute gas; and one tenth, liquid.

Ion transport systems in erythrocytes from paramyotonia patients.

To test the hypothesis of a general defect in the ion transport systems in the cell membranes of patients with paramyotonia congenita, we measured the ouabain-inhibitable and the furosemide-inhibitable K+ influxes in erythrocytes from two patients. In the temperature range examined (15-40 degrees C), the ouabain-inhibitable flux did not differ significantly from control. Also the furosemide-inhibitable flux was well in the rather large control range. The dependence of these fluxes on the external K+ concentration was normal. Energy depletion of the erythrocytes by preincubation with desoxy-glucose led to reduced K+ influxes that were not significantly different in patients and controls. The results do not support the hypothesis of a general defect in the ion transport systems in paramyotonia; they do, however, not rule out the possibility that the ion transport systems in paramyotonic skeletal muscle are defect.

The lipid composition of erythrocyte ghosts from a patient with congenital paramyotonia.

The membrane lipid and fatty acid compositions of red blood cells from a paramyotonia patient were investigated. Cholesterol and total phospholipid contents in paramyotonia were not different from control. Only the sphingomyelin content was lower, and thus the molar ratio of phosphatidylcholine/sphingomyelin was higher than normal. The major abnormality concerned the fatty acid pattern. In all the phospholipid classes saturated fatty acids were increased and unsaturated fatty acids were decreased. The overall ratio of saturated/unsaturated fatty acids was 2.1 vs 1.6 in controls. Similar findings have been reported for the sarcolemma from paramyotonia patients. Thus, the results indicate that the membrane defect in this disease may be generalized.

[Mutations of the tumor suppressor gene SOCS-1 in classical Hodgkin lymphoma are frequent and associated with nuclear phospho-STAT5 accumulation]. / Mutationen des Tumorsuppressorgens SOCS-1 treten im klassichen Hodgkin Lymphom häufig auf und sind assoziiert mit einer Kernakkumulation des phospho-STAT5 Proteins.

AIMS: Suppressors of cytokine signaling (SOCS) negatively regulate Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling involved in proliferation, survival, and apoptosis. We previously showed a loss of SOCS-1 function due to deleterious mutations in a major subset of mediastinal B-cell lymphoma (MBL). In MBL cell lines this leads to retarded JAK2 degradation and sustained phospho-STAT5 action results in enhanced DNA binding of phospho-STAT5. METHODS: To investigate the SOCS-1 gene we laser-microdissected Hodgkin-and Reed-Sternberg (HRS) cells of 19 classical Hodgkin lymphoma (cHL) and performed sequencing analysis. To assess phospho-STAT5 status immunohistochemistry on the corresponding paraffin-embedded cHL tumor tissue was done. RESULTS: We detected mutations of the SOCS-1 gene in HRS cells of 8 of 19 cHL samples and in 3 of 5 cHL-derived cell lines. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells (P <0.01). CONCLUSIONS: In conclusion, these findings support the concept that MBL and cHL share overlapping features and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas.

[Biallelic mutation of SOCS-1 impairs JAK2 degradation and sustains phospho-JAK2 action in MedB-1 mediastinal lymphoma line]. / Die biallelische Mutation von SOCS-1 verhindert den Abbau von JAK2 und prolongiert die Wirkung von phospho-JAK2 in der mediastinalen B-Zell Lymphomlinie MedB-1.

Primary mediastinal B-cell lymphoma (PMBL) is a well-defined subtype of diffuse large B-cell lymphoma. Molecular cytogenetics revealed frequent gains of 9 p24. JAK2, mapping in this region, is presently regarded as a candidate oncogene since expression profiling showed high JAK2 transcript levels and JAK2 was found to be constitutively phosphorylated in mediastinal B-cell lymphomas. We confirm that in the MedB-1 mediastinal B-cell line, harbouring a trisomy 9, JAK2 transcription is elevated and the product is highly phosphorylated. However, JAK2 is not over-expressed at the protein level. On top, JAK2 protein turnover is even delayed. This unexpected finding coincides with a biallelic mutation of the SOCS-1 gene in this cell, which abrogates SOCS box function of the protein. Ectopic expression of wt-SOCS-1 in MedB-1 leads to growth arrest, dramatic reduction of phospho-JAK2 and its downstream partner phospho-STAT5. We conclude that, in MedB-1, action of phospho-JAK2 is sustained due to defective SOCS-1. Hence, SOCS-1 qualifies as a novel tumor suppressor. Of note, the SOCS-1 mutations are also present in the parental tumor of MedB-1 and were detected in 9 of 20 PMBL.

Studies in vitro on the biosynthesis of ceramide and sphingomyelin. A reevaluation of proposed pathways.

The postulated biosynthetic-pathways of ceramide and sphingomyelin were reinvestigated in extensive investigations by means of synthetic stereo- and radio-chemically pure substrates of high specific radioactivity. As a result, the synthesis of ceramides requires the acyl-CoA-mediated acyltransfer to the long chain bases sphingenine and sphinganine. During the biosynthesis of sphingomyelins, phosphocholine is being transferred from the donor CDP-choline to the primary alcohol group of ceramides. Neither can the free long chain sphingosine bases act as acceptor molecule for the phosphocholine group from CDP-choline, nor has a transfer of [N-14CH3]phosphocholine from [N-14CH3]phosphatidyl choline to ceramide by rat liver enzyme preparations been observed. In agreement with previous studies in vivo, the acylation of sphingenylphosphocholine by acyl-CoA or free fatty acid, ATP and CoASH as an alternative pathway in sphingomyelin biosynthesis has been excluded. Other parameters of the CDP-choline:ceramide cholinephosphotransferase reaction (pH-optimum, ion requirement, competitive inhibition by diacyl glycerols, chain length of fatty acids) are reported. Sphingenine-containing ceramide species are preferred as acceptor molecules. Ceramide species with the L-threo (2S,3S)-enantiomeric long-chain bases are better acceptors than the corresponding D-erythro (2S,3R)-isomeric compounds. The meaning of the steric arrangement for the reaction is discussed.

The role of lipoproteins in EBV early antigen induction in Raji cells.

Synthesis of the Epstein-Barr virus (EBV) associated early antigen (EA) can be induced by a variety of agents in Raji cells, a latently EBV-infected Burkitt lymphoma line. We investigated the role of lipoproteins in this EA induction system. Cell growth was not affected by lipoprotein-deficiency, but EA induction by most combinations of the inducers TPA (tetradecanoyl-phorbol-acetate), IdU (iododeoxyuridine), n-BA (n-butyric acid), anti-IgM and EA inducing factor (EIF), was greatly reduced. Only the inducer combination TPA/n-BA was completely independent of the presence of lipoproteins, indicating a different induction pathway. Removing the lipid moieties of the culturing serum did not result in reduced EA induction. Thus, the lowered EA inducibility in lipoprotein-deficiency is due to the absence of the protein moiety (apolipoprotein). Addition of HDL or VLDL partially reconstituted the original EA inducibility, whereas LDL had no effect. Lipoproteins were particularly important during the first 4 hours of induction, the phase where inducers may act on cell membrane structures (e.g., receptors). But lipoproteins were also required throughout the incubation period, even in a late and inducer independent phase.

Alterations in the biophysical properties of the human endothelial cell plasma membrane induced by a chemotactic tripeptide: correlation with enhanced adherence of granulocytes.

Human umbilical vein endothelial cells in monolayer culture were used to study the effects of the chemotactic tripeptide, N-formylmethionylleucylphenylalanine (FMLP), on structure and function of the endothelium. Endothelial cell morphology was unaffected by concentrations of 10(-8)-10(-4)M. No effect on endothelial cell proliferative capacity, as measured by the DNA content of cultures, was seen at the FMLP concentrations studied (10(-8)-10(-6)M). Using fluorescent molecular probes to investigate FMLP-induced alterations in membrane structure, it was shown using the monomer-excimer method with pyrene decanoic acid that FMLP caused a marked restructuring of the plasma membrane. This took the form of a restriction of the surface available to the lipophile reporter molecules, probably caused by a molecular reorganization of the membrane protein component. Experiments with diphenylhexatriene indicated that FMLP did not make the plasma membrane of the endothelial cell more fluid. Concomitant with these changes in the physical properties of the membrane, an FMLP-induced increase in granulocyte adherence to the endothelial cells was observed. A theoretical model is presented correlating granulocyte adherence with the lateral mobility of lipids in the endothelial cell membrane. The significance of the FMLP-induced increase in granulocyte adherence to endothelial cells for the pathogenesis of sepsis is discussed.