Induction of Germinal Centers by MMTV Encoded Superantigen on B Cells

It has not been established whether an endogenous superantigen (SAg) expressed on B cells can induce germinal centers (GCs). An interesting model is that of mammary tumor virus encoded viral SAgs, which induce vigorous T cell proliferation and are predominantly expressed on activated B cells. We have used this model to analyze the possibility that direct stimulation of Mtv7+ DBA/2 B cells by vSAg-responsive (Vβ6+) BALB/c T cells can give rise to GCs. Injection of BALB/c SCID mice iv with 2 × 106 DBA/2 B cells, together with LPS, followed by 2 × 106 BALB/c T cells induces numerous large splenic GCs within 3–5 days. The GCs are still large on day 7, but are very much reduced by day 10. B cell activation with LPS is needed for this effect. These GCs form in spite of the apparent absence of follicular dendritic cells (FDCs) as judged by staining for several FDC surface markers. Control mice receiving either BALB/c T or DBA/2 B cells + LPS alone or DBA/2 T + B cells + LPS fail to exhibit any GCs on days 3–7. Numerous small clusters of PNA+ cells, but few large GCs are observed when TNF-R(p55)-Ig is also injected, whereas LTβR-Ig treatment impeded the formation of aggregations of these cells even further, leaving scattered PNA+ single cells and very small clumps throughout the white pulp of the spleens. Anti-TNFα had no effect. These results suggest that endogenous vSAg mediated GC formation is independent of antigen trapping by FDCs

The Down syndrome critical region protein TTC3 inhibits neuronal differentiation via RhoA and Citron kinase.

The Down syndrome critical region (DSCR) on Chromosome 21 contains many genes whose duplication may lead to the major phenotypic features of Down syndrome and especially the associated mental retardation. However, the functions of DSCR genes are mostly unknown and their possible involvement in key brain developmental events still largely unexplored. In this report we show that the protein TTC3, encoded by one of the main DSCR candidate genes, physically interacts with Citron kinase (CIT-K) and Citron N (CIT-N), two effectors of the RhoA small GTPase that have previously been involved in neuronal proliferation and differentiation. More importantly, we found that TTC3 levels can strongly affect the NGF-induced differentiation of PC12 cells, by a CIT-K-dependent mechanism. Indeed, TTC3 overexpression leads to strong inhibition of neurite extension, which can be reverted by CIT-K RNAi. Conversely, TTC3 knockdown stimulates neurite extension in the same cells. Finally, we find that Rho, but not Rho kinase, is required for TTC3 differentiation-inhibiting activity. Our results suggest that the TTC3–RhoA–CIT-K pathway could be a crucial determinant of in vivo neuronal development, whose hyperactivity may result in detrimental effects on the normal differentiation program

Simple and Rapid In Vivo Generation of Chromosomal Rearrangements using CRISPR/Cas9 Technology

Summary Generation of genetically engineered mouse models (GEMMs) for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs). Intratracheal or intrapulmonary inoculation of lentiviruses induced Eml4-Alk gene rearrangement in lung cells in vivo. Genomic and mRNA sequencing confirmed the genome editing and the production of the Eml4-Alk fusion transcript. All mice developed Eml4-Alk -rearranged lung tumors 2 months after the inoculation, demonstrating that the CRISPR/Cas9 system is a feasible and simple method for the generation of chromosomal rearrangements in vivo

DIFFERENT AQUAPORIN-4 EXPRESSION IN GLIOBLASTOMA MULTIFORME PATIENTS WITH AND WITHOUT SEIZURES

Aquaporin-4 (AQP-4), the most important water channel in the brain, is expressed by astrocyte endfeet abutting microvessels. Altered expression levels of AQP-4 and redistribution of the protein throughout the membranes of cells found in glioblastoma multiforme (GBM) lead to development of the oedema often found surrounding the tumour mass. Dysregulation of AQP-4 also occurs in hippocampal sclerosis and cortical dysplasia in patients with refractory partial epilepsy. This work reports on analysis of the relationship between AQP-4 expression and the incidence of epileptic seizures in patients with GBM. Immunohistochemical and PCR techniques were used to evaluate AQP-4 in biopsy specimens from 19 patients with GBM, 10 of whom had a history of seizures prior to surgery. AQP-4 mRNA levels were identical in the two groups of patients, but AQP-4 expression was more frequently detected on the GBM membranes from specimens of patients with seizures than from those without (10 vs. 2,

The lymphoma-associated NPM-ALK oncogene elicits a p16INKa/pRb-dependent tumour-suppressive pathway

Oncogene induced senescence (OIS) is a barrier for tumour development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumour-suppressive pathways. We report that NPM-ALK, the initiating oncogene of Anaplastic Large Cell Lymphomas (ALCLs), induces DNA-damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is due to activation of the p16INK4a/pRb tumour-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in pre-malignant lesions and decreased tumour latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumours

Inhibition of Rac controls NPM–ALK-dependent lymphoma development and dissemination

Nucleophosmin-anaplastic lymphoma kinase (NPM–ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM–ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM–ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM–ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas