CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface fas ligand expression and amplifies fas-mediated hepatocyte death during allograft rejection.

We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68(+) macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.

Epstein-Barr virus: LMP1 masquerades as an active receptor.

The Epstein-Barr virus protein LMP1 is essential for transformation of resting B cells by the virus, but how it works is unclear. Recent results suggest that LMP1 acts as a constitutively active receptor that shares certain characteristics with members of the tumour necrosis factor receptor superfamily.

CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily.

CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.

The prognostic significance of Bcl-2 and p53 expression in ovarian carcinoma.

Advanced ovarian cancer is characterized by poor prognosis and the development of resistance to chemotherapy. We have found that Bcl-2 and p53, two proteins implicated in the control of apoptosis, are differently expressed in the ovarian cell line A2780 and its cisplatin-resistant variant 2780CP, with the resistant line overexpressing both proteins. Transfection of the A2780 cells with a Bcl-2- or p53-expressing plasmid increases resistance to various drugs, including cisplatin, suggesting that Bcl-2 and p53 expression may influence the sensitivity of ovarian cancer cell lines to chemotherapy. Expression of these two proteins in vivo was determined by immunohistochemical staining of ovarian tumor biopsies from 70 patients. We found that Bcl-2 and p53 were expressed in 57 and 61% of specimens examined, respectively. Both p53 and Bcl-2 were found to be independent prognostic indicators of survival in ovarian cancer. Survival was poorer in patients with tumors expressing high levels of p53, whereas expression of Bcl-2 was associated with improved survival.

Apolipoprotein A-I inhibits experimental colitis and colitis-propelled carcinogenesis.

In both humans with long-standing ulcerative colitis and mouse models of colitis-associated carcinogenesis (CAC), tumors develop predominantly in the distal part of the large intestine but the biological basis of this intriguing pathology remains unknown. Herein we report intrinsic differences in gene expression between proximal and distal colon in the mouse, which are augmented during dextran sodium sulfate (DSS)/azoxymethane (AOM)-induced CAC. Functional enrichment of differentially expressed genes identified discrete biological pathways operating in proximal vs distal intestine and revealed a cluster of genes involved in lipid metabolism to be associated with the disease-resistant proximal colon. Guided by this finding, we have further interrogated the expression and function of one of these genes, apolipoprotein A-I (ApoA-I), a major component of high-density lipoprotein. We show that ApoA-I is expressed at higher levels in the proximal compared with the distal part of the colon and its ablation in mice results in exaggerated DSS-induced colitis and disruption of epithelial architecture in larger areas of the large intestine. Conversely, treatment with an ApoA-I mimetic peptide ameliorated the phenotypic, histopathological and inflammatory manifestations of the disease. Genetic interference with ApoA-I levels in vivo impacted on the number, size and distribution of AOM/DSS-induced colon tumors. Mechanistically, ApoA-I was found to modulate signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB activation in response to the bacterial product lipopolysaccharide with concomitant impairment in the production of the pathogenic cytokine interleukin-6. Collectively, these data demonstrate a novel protective role for ApoA-I in colitis and CAC and unravel an unprecedented link between lipid metabolic processes and intestinal pathologies.

Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1).

Expression of the oncogenic Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 1 (LMP1) activates signalling on the NF-kappaB axis through two distinct domains in the cytoplasmic C-terminus of the protein, namely CTAR1 (aa 187-231) and CTAR2 (aa 351-386). Whilst this effect is responsible for some of the functional consequences of LMP1 expression, additional LMP1-mediated signalling pathways may exist which contribute to the pleiotropic activities of this protein. In this study we provide evidence of a kinase cascade being activated by LMP1. Thus, we demonstrate that stable or transient expression of the LMP1 prototype from B95.8 in cells of epithelial or B cell origin activates the c-Jun N-terminal kinase (JNK, also known as the stress-activated protein kinase, SAPK) pathway, an effect which was found to be mediated through CTAR2 but not CTAR1. LMP1 from the Cao viral strain or LMP1 homologues from the simian EBV naturally infecting baboons and rhesus monkeys were also able to activate JNK. This phenomenon translates to induction of AP-1, a transcription factor which is readily activated by growth factors and mitogens. Interestingly, an LMP1/ CD40 chimaera comprising of the N-terminus and transmembrane domain of LMP1 and the cytoplasmic tail of CD40 which shares a common TRAF binding motif with CTAR1, effectively induced JNK. As NF-kappaB and JNK are co-activated in LMP1-expressing cells, we investigated whether the two pathways are overlapping or independent. We have found that inhibition of NF-kappaB by metabolic inhibitors or a constitutively active mutated IkappaBalpha does not impair the ability of LMP1 to signal on the JNK axis. Conversely, whilst a dominant negative mutated SEK (JNKK) inhibited LMP1-induced JNK activation, it did not affect NF-kappa-B suggesting that these two LMP1-mediated pathways are divergent.

BHRF1, a viral homologue of the Bcl-2 oncogene, disturbs epithelial cell differentiation.

Epstein-Barr virus (EBV) is associated with tumours of both lymphoid and epithelial origin. Whilst a role for EBV latent genes in the development of these malignancies is accepted, it is also possible that viral proteins involved in EBV replication may influence the oncogenic process. BHRF1 is an immediate early protein which has homology with the Bcl-2 oncogene and can protect B cells from apoptosis. In vivo this protein is most abundantly expressed in the upper layers of oral 'hairy' leukoplakia (HL), a benign hyperparakeratotic tongue lesion which represents a focus EBV replication. We have transfected BHRF1 into the human squamous cell carcinoma line SCC12F which retains several features of normal keratinocytes behaviour in vitro. BHRF1 expression in these epithelial cells is associated with a delay in the commitment of cells to terminal differentiation, increased resistance to the DNA damaging drug, cis-platin and enhanced survival under conditions of serum deprivation. As the differentiation of epithelial cells is an apoptotic process, this data strongly suggests that BHRF1 expression delays the terminal differentiation of epithelial cells through the prevention of apoptosis. This effect of BHRF1, which may normally function to promote productive EBV infection, could contribute to the development of EBV-associated tumours.

Epstein-Barr virus latent membrane protein-1 (LMP1) signalling is distinct from CD40 and involves physical cooperation of its two C-terminus functional regions.

The Epstein-Barr virus (EBV) encoded Latent Membrane Protein-1 (LMP1) mimics a constitutively active receptor molecule, and has been shown to activate NF-kappaB and the MAPK and JNK pathways. Two regions within the cytosolic domain of LMP1 have been found to effect cell signalling. One of these, the carboxy-terminal activation region-1 (CTAR1), binds members of the TRAF family of proteins, and the other (CTAR2) binds TRADD, suggesting that LMP1 transduces signals similarly to the Tumour Necrosis Factor Receptor family of receptors. The ability to bind TRAFs, to activate NF-kappaB and the JNK pathway, to upregulate cellular genes such as CD54 (ICAM-1 adhesion molecule), and to affect cell growth and apoptosis has led to the suggestion that LMP1 signalling is similar to, or even identical to CD40. However, we now show that while ligand-induced CD40 signalling is impaired in the Jurkat T cell line, LMP1 was fully functional; therefore demonstrating that LMP1 and CD40 signalling differ. Mutated LMP1 genes, in which one or other of the CTAR1 and CTAR2 domains was non-functional, behaved more like CD40 in being unable to upregulate the CD54 cell surface marker in Jurkat cells. However, the CTAR1 domain of LMP1, which shared a TRAF-binding sequence motif with CD40, differed from CD40 in being unable to activate NF-kappaB in Jurkat. Cotransfection experiments with LMP1 mutants demonstrated that CTAR1 can cooperative with CTAR2 on separate LMP1 molecules, provided that they exist within the same oligomeric complex.

Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors.

Expression of the Epstein-Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kappaB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187-231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-kappaB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an effect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-kappaB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-kappaB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-kappaB activation and IL6 secretion. The importance of TRAF interactions in mediating these effects was confirmed using dominant negative TRAF2 and TRAF3 mutants which also identified differences in the signalling events mediated by the two NF-kappaB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kappaB activity, also blocked NF-kappaB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.

The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and Bcl-2.

Modulation of apoptosis may influence resistance to chemotherapy and therefore affect the outcome of cancer treatment. Ovarian cancer, one of the most fatal malignancies in women, is often associated with drug resistance but the cellular pathways contributing to this effect remain obscure. We have found that Bcl-2 and p53, two proteins implicated in the control of apoptosis, are frequently expressed in fresh biopsies of primary ovarian carcinoma. Examination of Bcl-2 and p53 protein levels in pairs of cis-platin sensitive and resistant ovarian cell lines demonstrated that the resistant variants over-express Bcl-2 and/or p53, apparently due to progressive expansion of Bcl-2 and/or p53 positive subpopulations during the in vitro development of resistance. Exogenous expression of Bcl-2 or a temperature sensitive mutant p53 (ts p53) in the ovarian cell line A2780 resulted in protection from drug-induced apoptosis and a delay in drug-mediated S-phase arrest. Interestingly, p53 accumulation in response to DNA damage induced by different agents was significantly delayed and reduced in the Bcl-2 transfectants compared to the control A2780 line, suggesting that Bcl-2 may act upstream of the p53 pathway. Similarly, the induction of Bax mRNA and protein was also found to be delayed in the presence of Bcl-2. Overall, our data provide further evidence for cross-talk between Bcl-2, p53 and Bax and suggest that these genes are important determinants of drug-induced apoptosis thereby modulating resistance to chemotherapy.

CD40-induced growth inhibition in epithelial cells is mimicked by Epstein-Barr Virus-encoded LMP1: involvement of TRAF3 as a common mediator.

CD40, a member of the tumour necrosis factor receptor family, is expressed on the surface of B lymphocytes where its ligation provides a potent survival signal. CD40 is also expressed in basal epithelial cells and in a number of different carcinomas where its function remains unknown. We observed that contrary to the studies in normal B cells, CD40 ligation in carcinoma cell lines and in normal primary epithelial cells resulted in growth inhibition and enhanced susceptibility to apoptosis induced by anti-neoplastic drugs, TNF-alpha, Fas and ceramide. This effect was also observed in CD40-transfected Rat-1 fibroblasts. The expression of Bcl-2 did not affect growth inhibition induced by CD40 ligation in epithelial cells but the Epstein - Barr Virus-encoded latent membrane protein 1 (LMP1) blocked the effect. Whilst transient expression of LMP-1 resulted in the inhibition of epithelial cell growth, this effect was not observed with a LMP1 mutant lacking the binding domain for TRAF3, a protein which may mediate signal transduction by interacting with the cytoplasmic domains of both CD40 and LMP1. Transient expression of TRAF3 also inhibited epithelial cell growth, whilst expression of a dominant-negative TRAF3 partially blocked the inhibitory effect of CD40 ligation and of transient LMP1 expression. These results suggest that CD40 regulates epithelial cell growth in a manner mimicked by LMP1 and implicate TRAF3 as a common mediator in the transduction of the growth inhibitory signals generated via the CD40 and LMP1 pathways.

Epstein-Barr virus latent membrane protein 1 (LMP1) upregulates Id1 expression in nasopharyngeal epithelial cells.

Nasopharyngeal carcinoma is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded LMP1 has cell transformation property. It suppresses cellular senescence and enhances cell survival in various cell types. Many of the downstream events of LMP1 expression are mediated through its ability to activate NF-kappaB. In this study, we report a novel function of LMP1 to induce Id1 expression in nasopharyngeal epithelial cells (NP69) and human embryonal kidney cells (HEK293). The Id1 is a basic helix-loop-helix (bHLH) protein and a negative transcriptional regulator of p16(INK4a). Expression of Id1 facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical inhibitors and genetic inhibitors of cell signaling, we showed that induction of Id1 by LMP1 was dependent on its NF-kappaB activation domain at the carboxy-terminal region, CTAR1 and CTAR2. Induction of Id1 by LMP1 may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.

Induction of apoptosis by chemotherapeutic drugs: the role of FADD in activation of caspase-8 and synergy with death receptor ligands in ovarian carcinoma cells.

Although ovarian tumours initially respond to chemotherapy, they gradually acquire drug resistance. The aims of this study were to identify how chemotherapeutic drugs with diverse cellular targets activate apoptotic pathways and to investigate the mechanism by which exposure to a combination of drugs plus death receptor ligands can increase tumour cell kill. The results show that drugs with distinct cellular targets differentially up-regulate TRAIL and TNF as well CD95L, but do not require interaction of these ligands with their receptor partners to induce cell death. Factors that were critical in drug-induced apoptosis were activation of caspases, with caspase-8 being activated by diverse drugs in a FADD-independent manner. Certain drugs also demonstrated some dependence on FADD in the induction of cell death. Caspase-9 was activated more selectively by chemotherapeutic agents. Combining ligation of death receptors with exposure to drugs increased tumour cell kill in both drug resistant cell lines and primary ovarian carcinoma cells, even though these cells were not sensitive to death receptor ligation alone. CD95L was more consistent at combining with drugs than TRAIL or TNF. Investigation of the mechanism by which a combination of drugs plus CD95 ligation can increase cell death showed that caspase-8 was activated in cells exposed to a combination of cisplatin and anti-CD95, but not in cells exposed to either agent alone.

Physical and functional interaction of the TPL2 kinase with nucleophosmin.

Tumor Progression Locus 2 (TPL2) is widely recognized as a cytoplasmic mitogen-activated protein 3 kinase with a prominent role in the regulation of inflammatory and oncogenic signal transduction. Herein we report that TPL2 may also operate in the nucleus as a physical and functional partner of nucleophosmin (NPM/B23), a major nucleolar phosphoprotein with diverse cellular activities linked to malignancy. We demonstrate that TPL2 mediates the phosphorylation of a fraction of NPM at threonine 199, an event required for its proteasomal degradation and maintenance of steady-state NPM levels. Upon exposure to ultraviolet C, Tpl2 is required for the translocation of de-phosphorylated NPM from the nucleolus to the nucleoplasm. NPM is an endogenous inhibitor of HDM2:p53 interaction and knockdown of TPL2 was found to result in reduced binding of NPM to HDM2, with concomitant defects in p53 accumulation following genotoxic or ribosomal stress. These findings expand our understanding of the function of TPL2 as a negative regulator of carcinogenesis by defining a nuclear role for this kinase in the topological sequestration of NPM, linking p53 signaling to the generation of threonine 199-phosphorylated NPM.

Induction of the c-myc but not the cH-ras promoter by platinum compounds.

The recombinant plasmids p324, p330 and p323 carrying the aminoglycoside phosphotransferase (aph) gene and 5' flanking c-myc sequences linked to the reporter gene chloramphenicol acetyl-transferase (cat) were introduced into the mouse erythroleukaemic cell line F412B2TK- and stable transfectants resistant to geneticin G418 were obtained. The effects of cis-platin and two novel platinum compounds, D19466 (lobaplatin) and D17872, on c-myc promoter regions were studied using a large range of drug concentrations and correlated with cytotoxicity data. It was found that cis-platin and D19466 show a similar pattern of cytotoxicity and activation of c-myc promoter-driven cat gene expression with maximum effect (increased expression of 7.4- and 8.1-fold, respectively) at a concentration of 5 x 10(-5) M, while the less cytotoxic D17872 only slightly activates cat expression at the same concentration. However, when the F412B2TK- cell line was transfected with a plasmid carrying 5' flanking sequences of the c-Hras1 gene with promoter/enhancer function, linked to the cat reporter, no similar inductive effect was observed with any of the platinum drugs used. These data suggest that platinum compounds and possibly other DNA-damaging agents may specifically influence the expression of certain genes.

Serum soluble IL-6 receptor concentrations correlate with stages of multiple myeloma defined by serum beta 2-microglobulin and C-reactive protein.

Serum soluble interleukin-6 receptor (sIL-6R) concentrations were measured in 52 patients with multiple myeloma (MM) and 24 normal controls, using a commercially available immunoenzymatic assay kit. Patients were staged according to the Bataille et al. myeloma staging system based on the levels of patients' serum beta 2-microglobulin and C-reactive protein. Twenty-one patients were at stage A of disease, 19 at stage B and 12 at stage C at the time of serum collection for sIL-6R determination. Serum sIL-6R concentrations ranged from 15 to 176 ng/ml with a mean of 64.8 +/- 35.9 ng/ml and a median of 58 ng/ml in the entire group of patients studied. These values were significantly higher than those of 34.4 +/- 13.4 ng/ml found in the controls (P < or = 0.001). Patients of stage C had higher sIL-6R levels (94.8 + 41.2 ng/ml) than patients of stage B (67.7 +/- 31.0 ng/ml) (P < 0.01), and markedly higher than patients of stage A (45.0 +/- 23.1 ng/ml) (P < 0.001). Serum levels of sIL-6R in patients with stage A disease did not differ statistically from those of the controls. A linear positive correlation was observed between serum levels of the receptor and the stage of MM (r = 0.539, P < 0.001). These data strongly suggest that serum sIL-6R concentrations correlate with the stages of MM and may be used as an indicator of the activity of the disease.

Transcriptional activation of the human immunodeficiency virus long terminal repeat sequences by tumor necrosis factor.

The recombinant plasmid pBHIV-1 carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1), linked to the reported chloramphenicol acetyl transferase (CAT) gene, was introduced into human and rat fibroblasts. Stable transfectants were obtained which were resistant to genetecin and expressed CAT-activity from the HIV-1 LTR. The response to TNF alpha was studied. It was found that, at the optimum concentration of 100 IU/ml in human and 1000 IU/ml in rat fibroblasts, the expression of CAT was stimulated by 2.1 and 2.5-fold respectively. Our findings suggest that TNF-alpha in physiological concentrations can transcriptionally activate the HIV-1 LTR sequences and this may play an important role in the pathogenesis of HIV infection.

Adenovirus delivery of human CD40 ligand gene confers direct therapeutic effects on carcinomas.

CD40, a tumor necrosis factor receptor family member, is an emerging target for cancer therapy being best appreciated as an important regulator of the anti-tumor immune response. In this study, we report the development of a replication-defective recombinant adenovirus (RAd) vector expressing human CD40 ligand (RAd-hCD40L) and show that sustained engagement of the CD40 pathway in malignant cells results in direct anti-proliferative and pro-apoptotic effects. Thus, transduction of CD40-positive bladder, cervical and ovarian carcinoma cell lines with RAd-hCD40L potently inhibits their proliferation in vitro, whereas CD40-negative lines remain unresponsive. RAd-hCD40L is also found to be superior to recombinant CD40L in inducing carcinoma cell death and in amplifying the cytotoxic effects of the chemotherapeutic agents 5-fluorouracil, cis-platin and mitomycin C. Soluble CD40L is produced by RAd-hCD40L transduced carcinoma cells but unlike other soluble tumor necrosis factor family ligands, it does not interfere with the death-promoting activity of its membrane-bound form. In a mouse xenograft tumor model bearing a human bladder carcinoma, intratumoral delivery of RAd-hCD40L suppresses cancer growth. These findings highlight the potential of exploiting the CD40 pathway in carcinomas using CD40L gene transfer alone or in combination with other modalities for cancer therapy. Our results have also broader implications in understanding the multifaceted anti-tumor activities of the CD40 pathway in carcinomas, which thus offer an attractive option for future clinical application.

Activated T-lymphocytes with myelosuppressive properties in patients with chronic idiopathic neutropenia.

To characterize the cellular components responsible for the impaired granulopoiesis in chronic idiopathic neutropenia (CIN), we investigated the origin of the proapoptotic cytokine producing cells in the bone marrow (BM) microenvironment of CIN patients. We found that the interferon gamma (IFN gamma) and/or Fas-ligand expressing cells in patient BM mononuclear cells and long-term BM culture stroma cells were the CD3(+) T-lymphocytes but not the CD14(+) monocytes/macrophages. The percentage of activated T-lymphocytes was increased in patients' BM as indicated by the proportions of human leucocyte antigen (HLA)-DR(+), CD25(+), CD38(+), CD69(+) and Fas(+) cells within the CD3(+) fraction. Intracellular IFN gamma expression was higher in the BM than peripheral blood of the patients and was associated with increased BM T-lymphocyte numbers. In crossover experiments, patient CD3(+) T-lymphocytes conferred autologous and allogeneic haemopoietic progenitor cell colony inhibition. Patients' T-cell receptor repertoire and polymerase chain reaction analysis did not reveal any clonal T-lymphocyte expansion, suggesting the absence of a direct, antigen-driven recognition of CD34(+) myeloid progenitor cells by patient T-lymphocytes. We conclude that CIN patients have increased number of activated T-lymphocytes in the BM, probably in the setting of a localized polyclonal immune reaction and that these cells confer an inhibitory effect on myelopoiesis through myelosuppressive cytokines including Fas-ligand and IFN gamma.

LMP1 structure and signal transduction.

The oncogenic Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has structural features and functions reminiscent of a constitutively active TNF family receptor. LMP1 aggregates at the plasma membrane and initiates the activation of signalling pathways, such as NF- kappa B, the mitogen-activated protein kinases JNK and p38, the small GTPase Cdc42 and the JAK/STAT cascade. The constitutive engagement of these signals and the characteristic molecular interactions that regulate them provide the basis for the molecular explanation of the transforming properties of this key EBV protein.