EVI1 acts as an inducible negative-feedback regulator of NF-κB by inhibiting p65 acetylation.

Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense; however, an overactive response is detrimental to the host. Thus, inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in normal development and leukemogenesis. However, its role in regulating NF-κB-dependent inflammation remains unknown. In this article, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae- and TNF-α-induced NF-κB-dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA-binding activity. Moreover, expression of EVI1 itself is induced by nontypeable Haemophilus influenzae and TNF-α in an NF-κB-dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB-dependent inflammation. Thus, our study provides important insights into the novel role for EVI1 in negatively regulating NF-κB-dependent inflammation, and it may also shed light on the future development of novel anti-inflammatory strategies.

The role of resident synovial cells in destructive arthritis.

Infiltration by inflammatory cells, thickening of the lining layer, and destructive invasion into cartilage and bone are pathognomic features of the synovium in rheumatoid arthritis (RA). However, the most common cell types at the sites of invasion are resident cells of the joint, in particular synovial fibroblasts. These cells differ from healthy synovial fibroblasts in their morphology, their expression of proto-oncogenes and antiapoptotic molecules, and in their lack of certain tumor suppressor genes. Through their production of proinflammatory cytokines and chemokines mediated by signaling via Toll-like receptors, they are not only effector cells but also active parts of the innate immune system attracting inflammatory immune cells to the synovium. Most importantly, by producing matrix-degrading molecules they contribute strongly to the destructive mechanisms operative in RA.

Persistent polyclonal binucleated B-cell lymphocytosis and MECOM gene amplification.

BACKGROUND: Persistent Polyclonal Binucleated B-cell Lymphocytosis (PPBL) is characterized by a chronic polyclonal B-cell lymphocytosis with binucleated lymphocytes and a polyclonal increase in serum immunoglobulin-M. Cytogenetic is characterized by the presence of a supernumerary isochromosome +i(3)(q10), premature chromosome condensation and chromosomal instability. Outcome of PPBL patients is mostly benign, but subsequent malignancies could occur. The aim of our study is to provide an update of clinical and cytogenetic characteristics of our large cohort of PPBL patients, to describe subsequent malignancies occurring during the follow-up, and to investigate the role of the long arm of chromosome 3 in PPBL. RESULTS: We analyzed clinical, biological and cytogenetic characteristics (conventional cytogenetic analysis and fluorescent in situ hybridization) of 150 patients diagnosed with PPBL. We performed high-resolution SNP arrays in 10 PPBL patients, comparing CD19(+) versus CD19(-) lymphoid cells. We describe the cytogenetic characteristics in 150 PPBL patients consisting in the presence of supernumerary isochromosome +i(3)(q10) (59%) and chromosomal instability (55%). In CD19(+) B-cells, we observed recurrent copy number aberrations of 143 genes with 129 gains (90%) on 3q and a common minimal amplified genomic region in the MECOM gene. After a median follow-up of 60 months, we observed the occurrence of 12 subsequent malignancies (12%), 6 solid tumors and 6 Non-Hodgkin's Lymphomas, and 6 monoclonal gammopathies of undetermined significance (MGUS), requiring a long-term clinical follow-up. CONCLUSIONS: Our clinical and cytogenetic observations lead us to hypothesize that isochromosome 3q, especially MECOM abnormality, could play a key role in PPBL.

Concordant down-regulation of proto-oncogene PML and major histocompatibility antigen HLA class I expression in high-grade prostate cancer.

Recognition of tumor cells by cytolytic T lymphocytes depends on cell surface MHC class I expression. As a mechanism to evade T cell recognition, many malignant cancer cells, including those of prostate cancer, down-regulate MHC class I. For the majority of human cancers, the molecular mechanism of MHC class I down regulation is unclear, although it is well established that MHC class I down-regulation is often associated with the down-regulation of multiple genes devoted to antigen presentation. Since the promyelocytic leukemia (PML) proto-oncogene controls multiple antigen-presentation genes in some murine cancer cells, we analyzed the expression of proto-oncogene PML and MHC class I in high-grade prostate cancer. We found that 30 of 37 (81%) prostate adenocarcinoma cases with a Gleason grade of 7-8 had more than 50% down-regulation of HLA class I expression. Among these, 22 cases (73.3%) had no detectable PML protein, while 4 cases (13.3%) showed partial PML down-regulation. In contrast, all 7 cases of prostate cancer with high expression of cell surface HLA class I had high levels of PML expression. Concordant down-regulation of HLA and PML was observed in different histological patterns of prostate adenocarcinoma. These results suggest that in high-grade prostate cancer, malfunction of proto-oncogene PML is a major factor in the down-regulation of cell surface HLA class I molecules, the target molecules essential for the direct recognition of cancer cells by cytolytic T lymphocytes.

Complex activation of inducible transcription factors in the brain of normotensive and spontaneously hypertensive rats following central angiotensin II administration.

The effects of intracerebroventricular (i.c.v.) injections of angiotensin II (Ang II) on the expression of inducible transcription factors (ITF) (c-Fos, FosB, c-Jun, JunB, JunD, Krox-20 and Krox-24) in the brain of conscious rats were assessed immunohistochemically using polyclonal antisera. Ang II (1, 10, 100 ng) induced after 90 min a dose-dependent expression of c-Fos, FosB, c-Jun, JunB and Krox-24, which was confined to four specific brain areas, namely the subfornical organ (SFO), median preoptic area (MnPO), paraventricular nucleus (PVN) and supraoptic nucleus (SON). In the above-mentioned regions, JunD exhibited a high basal staining which was not visibly altered by Ang II. Krox 20 was not induced by AnG II. FosB was only induced 4 h after i.c.v. injection of 100 ng Ang II in the MnPO and PVN. The Ang II-AT1 receptor antagonist, losartan, applied i.c.v. 5 min prior to Ang II (100 ng, i.c.v.) prevented the Ang II-induced ITF expression. In spontaneously hypertensive rats (SHR) but not in Wistar rats with nephrogenic hypertension due to aortic banding (WIab), the Ang II-induced expression of c-Fos, and c-Jun was enhanced in all four areas when compared to normotensive Wistar Kyoto (WKY)- and Wistar (WI) rats. The Ang II-induced expression of Krox-24 in the SFO, MnPO and PVN in SHR was also significantly increased when compared to WKY, WI and WIab rats. Our data demonstrate that a stimulation of periventricular Ang II-AT1 receptors induces a temporally and spatially highly differentiated expression pattern of ITFs restricted to four distinct regions of the forebrain involved in blood pressure regulation and body fluid homeostasis. The points to a strictly regulated expression of target genes in the respective regions. The enhanced Ang II-induced expression of ITFs in SHR compared to normotensive controls is not due to elevated blood pressure itself, since it was not observed in secondary hypertensive rats WIab. Thus, the increased sensitivity to Ang II in SHR appears to be genetically determined. The target genes regulated by Ang II-induced ITFs will have to be identified.

Retrovirus-host interactions. The mouse mammary tumor virus model.

Mouse mammary tumor virus (MMTV) is a retrovirus which can induce mammary carcinomas in mice late in life by activation of proto-oncogenes after integration in their vicinity. Surprisingly, it requires a functional immune system to achieve efficient infection of the mammary gland. This requirement became clear when it was discovered that it has developed strategies to exploit the immune response. Instead of escaping immune detection, it induces a vigorous polyclonal T-B interaction which is required to induce a chronic infection. This is achieved by activating and then infecting antigen presenting cells (B cells), expressing a superantigen on their cell surface and triggering unlimited help by the large number of superantigen-specific T cells. The end result of this strong T-B interaction is the proliferation and differentiation of the infected B cells leading to their long term survival.

[Activation of proto-oncogene c-fos in the auditory tract of rats stimulation with wide-band noise]. / La activación del protooncogén c-fos en la vía auditiva en ratas estimuladas con ruido de banda ancha.

The pattern of expression of the proto-oncogene c-fos was mapped in the auditory pathway of Wistar rats kept in three different experimental conditions: a) a dark, soundproofed room; b) with exposure to usual environmental laboratory noise, and c) with exposure to wide-band noise. Under control conditions (a and b), scattered labeled neurons were found in the ventral periolivary nucleus, lateral lemniscus nuclei, inferior colliculus, medial nucleus of the medial geniculate body, and in three divisions of the temporal auditory cortex. Sound stimulation (c) increased the number of fos-like-immunoreactive (FLI) nuclei in all the auditory pathway structures. FLI nuclei were strong in the dorsal cochlear nucleus, anterior and posterior ventral cochlear nuclei, all the superior olivary complex nuclei, lateral lemniscus nuclei, all areas of the inferior colliculus, medial geniculate body, and the three temporal auditory areas, which showed a barrel pattern. Comparison of these results with the literature indicated that fos activation is not merely a sign of transitory neural activation, but a long-term neural processing pathway that is conditioned by factors such as the frequency, intensity, duration, and direction of the auditory stimulus.

[The molecular function of oncogenes]. / Onkogeners molekylaere virkningsmekanismer.

Cancer represents a disruption of the processes in normal cells. To understand cancer, and thereby develop effective treatment, it is necessary to learn about normal reactions in the cells, and their connections. Thus cancer research has been an important source of knowledge about normal cells. Oncogenes are genes that transform normal cells into cancer cells. More than 60 oncogenes have been discovered, and many malignant diseases have been linked to these genes. Human cells contain a normal version of oncogenes, known as protooncogenes, which participate in the normal, cellular processes. This discovery has been of major importance for our understanding of cancer and molecular genetics.

[Surface molecules on the trophoblast cells]. / Overflatemolekyler på trofoblastlaget.

Normal pregnancies depend on successful implantation of the placenta in the uterus. The trophoblast forms the ultimate interface between foetal and maternal tissue. It seems to lack the foreign antigens required to induce immunological rejection reactions in the mother. Therefore a successful pregnancy probably does not depend on the production of blocking antibodies to protect the foetus from maternal cytotoxic T-cells. Non-MHC-molecules may play a role in placentation. Trophoblast cells and cancer cells have several properties in common.

Oncoprotein immunoreactivity in human endocrine tumours.

Abnormally expressed oncogenes are implicated in neoplastic transformation. We have investigated a series of endocrine tumours using immunocytochemistry as a first screening tool to detect oncogene expression. Paraffin sections of 44 pulmonary small cell carcinomas, 15 pulmonary atypical carcinoids, 12 bronchial carcinoids, 28 medullary thyroid carcinomas, 27 phaeochromocytomas, and 17 insulinomas were immunostained with antibodies to c-erbB-2, c-myc, L-myc, and N-myc. Diffuse immunoreactivity was detectable for c-erbB-2 in 8 out of 44 (18 per cent) pulmonary small cell carcinomas, 3 out of 15 (20 per cent) pulmonary atypical carcinoids, and 6 out of 27 (22 per cent) phaeochromocytomas; for c-myc in 18 out of 44 (41 per cent) pulmonary small cell carcinomas and 5 out of 15 (33 per cent) pulmonary atypical carcinoids; for N-myc in 6 out of 28 (21 per cent) medullary thyroid carcinomas; and for L-myc in 4 out of 27 (15 per cent) phaeochromocytomas. There was considerable intratumoral and intertumoral heterogeneity and, in each tumour group, no relationship was found between tumour pattern, mitotic index, and oncoprotein immunoreactivity. These results suggest that oncogene products are present in a proportion of endocrine tumours, and that specific oncoproteins seem to be related to tumour type but not to other histopathological findings. Thus, oncoprotein detection may be a useful tool for identifying subsets of endocrine tumours that are not otherwise recognizable morphologically.

Long-term survival in breast cancer related to overexpression of the c-erbB-2 oncoprotein: an immunohistochemical study using monoclonal antibody NCL-CB11.

In a previous series we have shown poor short-term (3-5 years) survival for patients with tumours overexpressing the c-erbB-2 oncoprotein. In this study we employed archival paraffin-embedded tissue from patients who underwent mastectomy 10-12 years prior to assessment (n = 187). Immunohistochemical staining was carried out by an indirect immunoperoxidase technique using the novel monoclonal antibody NCL-CB11. Tumours were scored according to intensity of membrane staining. Patient and tumour information was obtained by scrutiny of clinical records. Survival analysis was carried out for both time to relapse and time to death, using the log rank test. Patients with tumours demonstrating intense membrane staining had a poor prognosis compared with the rest, with a steeply sloped survival curve over the first 4 years; the survival difference was still evident at 12 years follow-up (P less than 0.001). The survival advantage for c-erbB-2 negative patients was maintained in lymph node negative patients (P less than 0.001). However, c-erbB-2 status did not influence survival in the node positive group, where all patients had a uniformly poor outlook. These results applied to both time to relapse and time to death. In conclusion, c-erbB-2 status, determined using NCL-CB11, is a powerful prognostic indicator, defining in particular node negative patients with a particularly poor prognosis, and for whom alternative therapeutic strategies may be appropriate.

The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance.

Using cells from TCR transgenic mice that do or do not express Fas, we show that there are two mechanistically distinct forms of apoptosis in CD4+ T cells. Naive T cells undergo apoptosis if cultured in the absence of antigen or costimulation. This form of programmed cell death (PCD) is not dependent on Fas, and is prevented by CD28-mediated signals, which lead to the secretion of growth factors and the expression of survival genes, such as bcl-xL. Recently activated T cells undergo apoptotic death upon repeated stimulation. This activation-induced cell death (AICD) is mediated by Fas, but is independent of costimulation and is not prevented by IL-2 or bcl-xL. Finally, we show that peripheral tolerance may be induced in vivo independent of Fas-mediated cell death.

Development of dextran sulphate sodium-induced experimental colitis is suppressed in genetically mast cell-deficient Ws/Ws rats.

Ws/Ws rats have a small deletion of the c-kit gene, and are deficient in both mucosal-type mast cells (MMC) and connective tissue-type mast cells (CTMC). In the present study we investigated the role of intestinal MMC in the development of dextran sulphate sodium (DSS)-induced experimental colitis using Ws/Ws rats. Ws/Ws and control (+/+) rats were given a 3% DSS aqueous solution orally for 10 days, and the subsequent mucosal damage was evaluated macroscopically and histologically. The mucosal myeloperoxidase (MPO) activities and histamine levels were also measured. (i) DSS induced severe oedema and hyperaemia with sporadic erosions in the control (+/+) rats, but these changes were significantly attenuated in the Ws/Ws rats (P < 0.01). (ii) The microscopic mucosal damage score was lower in the Ws/Ws rats than in the control (+/+) rats (P = 0.06). (iii) There were no significant differences in mucosal MPO activity between the Ws/Ws and control (+/+) rats (P = 0.46). (iv) The mucosal histamine levels in the colon were significantly reduced in the Ws/Ws rats compared with the control (+/+) rats (P < 0.05). (v) Significant positive correlations were observed between mucosal histamine levels and the degree of mucosal oedema (calculated as colonic wet weight/protein content) (r = 0.778, P < 0.01), and between histamine levels and the macroscopic damage (r = 0.623, P < 0.05), respectively. (vi) DSS induced a local recruitment of MMC in the colonic mucosa of Ws/Ws rats, and mucosal damage gradually increased in accordance with this MMC recruitment. These results indicate that MMC play an important role in the development of DSS colitis.

Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic microarchitecture, and germinal center reactions.

Chromosomal translocations of bcl-3 are associated with chronic B cell lymphocytic leukemias. Previously, we have shown that Bcl-3, a distinct member of the I kappa B family, may function as a positive regulator of NF-kappa B activity, although its physiologic roles remained unknown. To uncover these roles, we generated Bcl-3-deficient mice. Mutant mice, but not their littermate controls, succumb to T. gondii owing to failure to mount a protective T helper 1 immune response. Bcl-3-deficient mice are also impaired in germinal center reactions and T-dependent antibody responses to influenza virus. The results reveal critical roles for Bcl-3 in antigen-specific priming of T and B cells. Altered microarchitecture of secondary lymphoid organs in mutant mice, including partial loss of B cells, may underlie the immunologic defects. The implied role of Bcl-3 in maintaining B cells in wild-type mice may related to its oncogenic potential.

Regulation of B cell survival in xid mice by the proto-oncogene bcl-2.

CBA/N mice carry an X-linked immunodeficiency (xid) due to a point mutation in the Bruton's tyrosine kinase (btk) gene. xid mice have a smaller peripheral B cell pool than normal animals, lack CD5+ B cells (B1), and are hyporesponsive to mitogenic anti-Igs and thymus-independent type 2 Ags. The proto-oncogene bcl-2 affects B cell homeostasis by suppressing programmed cell death. We hypothesized that reduced bcl-2 expression could enhance programmed cell death in xid B cells, directly causing poor peripheral B cell survival and indirectly affecting Ag responsiveness. We measured and compared levels of endogenous Bcl-2 protein and spontaneous apoptosis in xid and normal B cells, and determined the effect of a human bcl-2/Ig minigene on B cell survival and Ag responsiveness in bcl-2 transgenics. The amount of endogenous Bcl-2 was reduced fivefold in freshly isolated xid B cells compared with that in normal cells, but was equal in xid and normal T cells. Attrition by spontaneous apoptosis was significantly higher in cultured xid B cells. Expression of the bcl-2 transgene suppressed apoptosis equally in normal and xid B cells, prolonged in vitro survival, and markedly expanded in vivo the follicular B cell population normally reduced in xid mice. However, most xid defects persisted; xid/bcl-2 mice remained deficient in B1 cells and hyporesponsive to anti-Igs, thymus-independent type 1 Ags, and thymus-independent type 2 Ags. The data suggest that signal transduction pathways using Btk independently regulate B cell survival and Ag responsiveness.

IL-2R beta agonist P1-30 acts in synergy with IL-2, IL-4, IL-9, and IL-15: biological and molecular effects.

From the sequence of human IL-2 we have recently characterized a peptide (p1-30), which is the first IL-2 mimetic described. P1-30 covers the entire alpha helix A of IL-2 and spontaneously folds into a alpha helical homotetramer mimicking the quaternary structure of a hemopoietin. This neocytokine interacts with a previously undescribed dimeric form of the human IL-2 receptor beta-chain likely to form the p1-30 receptor (p1-30R). P1-30 acts as a specific IL-2Rbeta agonist, selectively inducing activation of CD8 and NK lymphocytes. From human PBMC we have also shown that p1-30 induces the activation of lymphokine-activated killer cells and the production of IFN-gamma. Here we demonstrate the ability of p1-30 to act in synergy with IL-2, -4, -9, and -15. These synergistic effects were analyzed at the functional level by using TS1beta, a murine T cell line endogenously expressing the common cytokine gamma gene and transfected with the human IL-2Rbeta gene. At the receptor level, we show that expression of human IL-2Rbeta is absolutely required to obtain synergistic effects, whereas IL-2Ralpha specifically impedes the synergistic effects obtained with IL-2. The results suggest that overexpression of IL-2Ralpha inhibits p1-30R formation in the presence of IL-2. Finally, concerning the molecular effects, although p1-30 alone induces the antiapoptotic molecule bcl-2, we show that it does not influence mRNA expression of c-myc, c-jun, and c-fos oncogenes. In contrast, p1-30 enhances IL-2-driven expression of these oncogenes. Our data suggest that p1-30R (IL-2Rbeta)(2) and intermediate affinity IL-2R (IL-2Rbetagamma), when simultaneously expressed at the cell surface, may induce complementary signal transduction pathways and act in synergy.

Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene.

During B lymphocyte development, pro-B cells that fail to rearrange an immunoglobulin heavy (IgH) chain allele productively are thought to undergo developmental arrest and death, but because these cells are short-lived in vivo they are not well characterized. Transgenic mice expressing the apoptosis regulatory gene bcl-xL in the B lineage developed large expansions of pro-B cells in bone marrow. V(D)J rearrangements in the expanded populations were nearly all nonproductive, and DJH rearrangements were enriched for joints in DH reading frame 2 and for aberrant joints with extensive DH or JH deletions. Thus, the death of pro-B cells with failed immunoglobulin rearrangements occurs by apoptosis, and bcl-xL can deliver a strong survival signal at the pro-B stage. This analysis also demonstrated that immunoglobulin gene rearrangement is less precise than previously appreciated.

The effect of IL-2 treatment on transcriptional attenuation in proto-oncogenes pim-1 and c-myb in human thymic blast cells.

IL-2 is the major mitogenic cytokine for mature human T cells. This growth factor has been shown previously to induce the expression of a number of genes, including structural proteins, proto-oncogenes, and metabolic enzymes. Multiple mechanisms, including increases in mRNA stability, protein synthesis, and new transcriptional initiation, have been studied to determine how IL-2 induces such a wide variety of genes. The following studies show that a release of transcriptional attenuation is important in IL-2-induced gene expression. A thymic blast cell system was developed and used to demonstrate that IL-2-deprived cells have a marked attenuation of transcription in the 3' ends of the pim-1 and c-myb genes. IL-2 stimulation removes this attenuation and leads to read-through transcription. This effect is gene-specific, as demonstrated by the fact that GAPDH is not attenuated in unstimulated cells. The IL-2-mediated relief of attenuation occurs within 1 h of IL-2 stimulation and is insensitive to the translation inhibitor cycloheximide, suggesting that new protein synthesis is not necessary. Further, the effect is insensitive to the immunosuppressant cyclosporin A, but is sensitive to rapamycin and the tyrosine kinase inhibitor genistein. These studies demonstrate that release of transcription attenuation is a mechanism used to induce gene expression in response to IL-2 treatment.

Regulation of Fas(Apo-1/CD95)- and perforin-mediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2.

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.