Translational efficiency of cMyc mRNA in Burkitt lymphoma cells.

The translational efficiency of cMyc mRNAs was assessed in a variety of cell lines: HeLa cells and Epstein-Barr virus-transformed lymphocytes, both of which contain only the germ line cMyc allele; Daudi, a Burkitt cell line containing a translocated cMyc gene with no apparent alteration; and P3HR-1, a Burkitt line in which the 5' end of the translocated cMyc gene has been altered by the chromosomal translocation. Translational efficiency was inferred by measuring the number of ribosomes associated with the cMyc mRNA, using a procedure by which individual polysomal fractions were analyzed by blot hybridization. Since polysome size is a function of the length of the translated sequence as well as the rate of initiation of protein synthesis, we also determined the number of ribosomes associated with a control mRNA (alpha tubulin) which codes for a protein of similar size to cMyc. We found that the cMyc mRNA was associated with a number of ribosomes comparable to that associated with alpha tubulin mRNA in all the cell lines tested.     

Toll-like receptors expression on Burkitt lymphoma cells

Toll like receptors (TLRs) are an important component of the immune response. They are link between innate and adaptative response. Lymphocytes B express most of the toll-like receptors and they may respond to a broad spectrum of PAMP. Lymphocytes B are one of the major lymphocyte populations in secondary lymphoid tissues, where they represent up to 50% of cells population. These cells are an important element of the defense, largely by using the mechanisms associated with innate response. On the other hand, lymphocytes B are the site of EBV latency, so Burkitt lymphoma cells can may be a convenient model to study the mechanisms associated with EBV infection. The aim of study was to determine the expression of TLRs at the m-RNA level of in Burkitt lymphoma cells treated with ligands for selected TLRs. P3HR, Raji and Namalwa cells were stimulated with Pam3 (10 microg/ml), PolyI:C (25 microg/ml), LPS (10 microg/ml) and measles virus (MeV, moi 0.02). Unstimulated cells and cells treated with PMA (0.5 microg/ml) served as negative and positive controls. After incubation, from stimulated and unstimulated cells mRNA was extracted, RT-PCR reaction was performed and electrophoretic separation was made. The intensity (INT) of bands were determined using the tools for quantitative analysis. In order to analyze the expression of TLR genes, INT values for TLR2, TLR3 and TLR4 in tested cell lines are expressed as %, assuming an average level of GAPDH expression as 100%. The 25% of INT for negative control was accepted as a change in expression level. It was found that the expression of Toll-like receptors in Burkitt lymphoma cells is diverse both in terms of cell type and the type of stimulation.     

Effect of centrifugation on the viability of Burkitt lymphoma cells

This study reports some findings on the effects of centrifugation on the viability of mammalian cells. The authors used Burkitt lymphoma cells cultivated in a synthetic medium containing 10% fetal calf serum for all experiments. Batch centrifugations were conducted in a RC2-B centrifuge (Ivan Sorvall, Incorporated, Norwalk, Connecticut USA) operated at 0 and 25°C. During centrifugation we exposed the cells to gravitational fields ranging from 24,800 to 42.200g. The results showed that at, 0°C and 25,800 or 42,000g no loss in cell viability occurred for up to 90 min exposures in the centrifugal field. However, at 25°C and for gravitational fields of 24,800 and 42,000g, there were appreciable losses in cell viability. Continuous centrifugation studies in the Sharples supercentrifuge (Division of Penn Salt Corporation, Warminister, Pennsylvania USA) were also conducted with bowl speeds up to 28,000 rpm (19,000g) and flow rates ranging from 1.4 to 20 1, hr. Slight, losses in cell viability were noted and postulated as caused by the shear stresses encountered by the cells. Some pumping studies using the lymphoma cells substantiate this conclusion.     

Modulation of apoptotic signalling by carotenoids in cancer cells

There is a growing body of literature on the role of beta-carotene and other carotenoids in human chronic diseases, including cancer. While epidemiological evidence shows that a high dietary intake of fruits and vegetables rich in carotenoids is associated with a reduced risk for cancer, results from intervention trials indicate that supplemental beta-carotene enhances the risk of developing lung cancer incidence and mortality among smokers. A possible mechanism which can explain the dual role of carotenoids as both beneficial and harmful agents in cancer is that their excess or deficiency may bring about changes in molecular pathways involved in apoptotic signalling. Carotenoid ability in inhibiting or in enhancing apoptosis depends on several factors: carotenoid concentration, concerted action of multiple micronutrients, cell type, and redox status. This review summarizes the available evidence for a modulatory action of carotenoids on apoptosis and focuses on the main molecular pathways involved in this process.     

Insulin receptor expression in the Burkitt lymphoma cells Daudi and Raji

The specific binding of insulin to either intact or Triton-solubilized Daudi cells (a Burkitt lymphoma cell line) was reduced by over 95% compared to that to control IM-9 lymphocytes due to a decrease in receptor number without a change in affinity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that 125I-labeled Daudi cells had reduced amounts (approximately 1/20th) of immunoprecipitable binding (alpha) subunit [mol wt (Mr), 130,000] of the receptor and a relatively abundant 210,000 Mr form not seen in IM-9 cells. The transmembranous (beta) subunit (Mr, 90,000) of the receptor, although not detected by 125I surface labeling, could be phosphorylated and, together with the 210,000 Mr form, exhibited the same 2-fold stimulation of phosphorylation by insulin as that in IM-9 cells. Northern blot hybridization revealed a decrease in Daudi cells of all four major species of insulin receptor mRNA. The Raji cell, another Burkitt lymphoma cell line, also exhibited reduced protein and genetic expression of the insulin receptor, indicating that reduced insulin receptor expression may be representative of other Burkitt lymphoma cell lines.     

Cross-talk between different intracellular signalling pathways in the rat hippocampus

The activation of alpha 1-adrenergic receptors in rat hippocampal slices enhances polyphosphoinositide (PPI) breakdown and cyclicAMP (cAMP) accumulation. The latter effect is antagonized by different protein kinase C (PKC) inhibitors and mimicked by a diacylglycerol (DAG) analogue, 1,2-diolein, which activates PKC, suggesting that cAMP synthesis is indirectly affected by alpha 1-adrenoceptors through the stimulated generation of DAG upon PPI hydrolysis. Furthermore the elevation of hippocampal cAMP decreases the ability of alpha 1-receptor agonists to enhance PPI breakdown. It is proposed that the observed effects are part of a complex cross-talk between PPI and AC signalling pathways operating in hippocampal neurons.     

Modulation of phospholipase D stimulation in c-src transfected mesangial cells

Stimulation of rat mesangial cells with platelet-derived growth factor BB (PDGF-BB) enhanced phospholipase D (PLD) activity in a concentration dependent manner. Mesangial cells overexpressing the tyrosine kinase pp60^c-src (c-Src) were used to determine the effect of this non transforming protooncogene on PLD activation. Overexpression of c-Src interfered with PDGF-BB-mediated activation of PLD. This modulation was dependent on the tyrosine kinase activity of c-Src, since overexpression of tyrosine kinase-negative mutants of c-Src did not affect PLD activation. No effect of c-Src overexpression was observed, when PLD was activated by ATP or guanosine 5′-3-O-(thio)triphosphate (GTPγS). The results indicate that the tyrosine kinase c-Src specifically interfered with PDGF-mediated but not with ATP- or GTPγS-mediated PLD activation.     

Roles of Rac and cytosolic phospholipase A2 in the intracellular signalling in response to titanium particles

Titanium (Ti) particle is one of the prosthetic materials commonly used in implantation and has frequently been implicated in pathogenesis such as periprosthetic osteolysis. In the present study, we undertook to understand the intracellular signalling pathway stimulated by exogenous Ti at Rat-2 fibroblasts. By reporter gene analysis following transient transfections, exogenous Ti was shown to stimulate c-fos serum response element (SRE)-dependent luciferase activities in a dose-dependent manner. In addition, Ti-induced SRE activation was shown to be dramatically repressed by RacN17, a dominant negative mutant of Rac1, suggesting that Rac GTPase is essential for the signalling of Ti to c-fos SRE. Furthermore, pretreatment with MAFP, an inhibitor of cytosolic phospholipase A(2) (cPLA(2)), MK886, an inhibitor of 5-lipoxygenase (5-LO), or indomethacin, a general inhibitor of cyclooxygenase (COX), also significantly repressed Ti-induced SRE activation, suggesting mediatory roles of cPLA(2) and subsequent arachidonic acid (AA) metabolisms to leukotrienes (LTs) and prostaglandins (PGs) in the Ti signalling to c-fos SRE. Consistent with these results, intracellular levels of leukotriene B(4) (LTB(4)) and prostaglandin E(2) (PGE(2)) were Rac-dependently elevated in cells exposed to Ti particles.     

EBV interferes with the sensitivity of Burkitt lymphoma Akata cells to etoposide

Burkitt lymphoma (BL) commonly exhibits Epstein-Barr virus (EBV) positivity associated with latent chronic infection. Models of acute EBV infection have been associated with cellular resistance to apoptosis. However, the effect of latent long-term EBV infection on apoptosis induced by drugs is not well defined. To determine this, we have studied the response of the Akata EBV+ cell line (type I latency) to etoposide, before and after downregulating EBV gene expression. We observed that downregulating EBV nuclear antigen-1 (EBNA-1) expression with siRNAs reverted cellular sensitivity to etoposide. In accordance with this finding, Akata EBV+ cells showed increased sensitivity to etoposide, when compared to the Akata EBV- cells. We also observed that Akata EBV+ cells presented increased apoptosis levels and decreased Bcl-xL mRNA and protein levels, when compared to the Akata EBV- cells. In addition, Akata EBV+ cells contained less endoplasmic reticulum (ER) than EBV- cells. Finally, downregulation of EBV with EBNA-1 siRNAs caused an increase in the expression of Bcl-xL indicating that EBV is responsible for the differences found between the Akata EBV+ and EBV- cell lines.     

Aspirin-like drugs prime human T cells. Modulation of intracellular calcium concentrations

Aspirin-like drugs (ALD) enhance T cell proliferation by suppressing PG production in monocytes. Normal human T cells do not produce any eicosanoids. Therefore we studied whether ALD would affect purified T cells directly. We found that ALD enhanced the proliferation and IL-2 production of T cells in the absence of monocytes. This effect did not depend on arachidonic acid metabolism as no lipoxygenase products and only nonsuppressive levels of cyclooxygenase products were detected in T cell cultures. Several possible mechanisms of the ALD effect were ruled out including 1) enhanced mitogen binding, 2) induction of activation markers (IL-2R, transferrin receptor, HLA-DR) on the cell surface, 3) down-regulation of suppressor cells. ALD caused a rise in [Ca2+]i which appeared to reflect an influx of Ca2+ from the extracellular milieu and was more pronounced in CD4+ cells. The rise in intracellular levels of Ca2+, that is considered a necessary second messenger for T cell activation, may prime these cells for an enhanced response to mitogens. In addition, ALD increased T cell membrane fluidity but only at higher concentrations than those found to enhance proliferation. The pharmacologic effect of ALD on T cells presents a possible new immunoenhancing potential of these drugs and may have therapeutic use in immunosuppressed individuals.     

Spiral waves and intracellular calcium signalling.

Confocal imaging of intracellular Ca2+ brings a new level of resolution to the study of hormonal control of intracellular Ca2+ release. This approach has demonstrated the existence of pulsatile circular and spiral waves of Ca+ release induced by receptor activation. The data obtained by confocal imaging support a new framework for understanding intracellular Ca2+ signalling. The goal of this chapter is to review our data on the complexity of intracellular Ca2+ release in Xenopus oocytes, introduce the concept of Ca2+ excitability as a model for Ca2+ release and discuss the implications for encoding intracellular signal information.     

Specific stimulation of phospholipase activities with phosphatidylethanolamine in transformed cells

Phospholipase A1, A2 and C activities with phosphatidylethanolamine were enhanced in C6 cells relative to primary astrocytic cultures. Enhancement was a function of cell density. Phospholipase activities with phosphatidylcholine were unchanged as a function of cell density, while phospholipase C activity with phosphatidylinositol was reduced. All acid phospholipase activities measured were low or essentially absent in the three transformed cell lines examined. These results suggest that arachidonate release upon confluency is mainly from phosphatidylethanolamine.