Notch Signaling Activation Suppresses v-Src-Induced Transformation of Neural Cells by Restoring TGF-β-Mediated Differentiation

BACKGROUND: We have been investigating how interruption of differentiation contributes to the oncogenic process and the possibility to reverse the transformed phenotype by restoring differentiation. In a previous report, we correlated the capacity of intracellular Notch (ICN) to suppress v-Src-mediated transformation of quail neuroretina (QNR/v-src(ts)) cells with the acquisition by these undifferentiated cells of glial differentiation markers. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we have identified autocrine TGF-β3 signaling activation as a major effector of Notch-induced phenotypic changes, sufficient to induce transition in differentiation markers expression, suppress morphological transformation and significantly inhibit anchorage-independent growth. We also show that this signaling is constitutive of and contributes to ex-vivo autonomous QNR cell differentiation and that its down-regulation is essential to achieve v-Src-induced transformation. CONCLUSIONS/SIGNIFICANCE: These results support the possibility that Notch signaling induces differentiation and suppresses transformation by a novel mechanism, involving secreted proteins. They also underline the importance of extracellular signals in controlling the balance between normal and transformed phenotypes

Two Novel Variants of the v-srcOncogene Isolated from Low and High Metastatic RSV-Transformed Hamster Cells

AbstractFour different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70–200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-srcprotein in cell lines was not correlated with their metastatic potentialin vivo.Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-srcisoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and v-srcLM: 62 kDa. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-srcsequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the oncoprotein. Both v-srcvariants and chimeric v-srcwith mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretina cells were associated with the mutations in the carboxy-terminal region of the v-srconcogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM

Interférence de la voie de signalisation Notch avec les mécanismes de transformation des cellules de neurorétine aviaires par v-Src

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

ISOLEMENT ET ETUDE FONCTIONNELLE DE MAFA, UN NOUVEAU FACTEUR DE TRANSCRIPTION A B-ZIP DE LA FAMILLE MAF

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

MECANISMES DE DIVISION ET DE DIFFERENCIATION DANS LA NEURORETINE AVIAIRE (ETUDE DE LA REGULATION TRANSCRIPTIONNELLE DU GENE QR1, DES VOIES EN AVAL DE RAS ET DU ROLE DE LA REGULATION DES FACTEURS DE TRANSCRIPTION MAF)

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

TGF-β signaling controls commitment into glial differentiation.

<p>(A) QNR/v-src^ts cells were treated at 37°C with increasing concentrations (0.2–2 ng/ml) of recombinant TGF-β 3 protein during 7 days. Pax6 and Glutamine Synthetase (GS) were detected by western-blot. Protein loading was normalized using Erk antiserum. (B) Pax6 expression was analyzed by immunofluorescence after treatment of QNR/v-Src^ts/ICN cells at 37°C with DMSO (control) or 10 µM SB431542 during 7 days. The majority of control QNR/v-Src^ts/ICN cells did not express nuclear Pax6 but we observed a weak peri-nuclear labeling. Magnification x40.</p

TGF-β3 mRNA is upregulated in QNR/v-src^ts/ICN cells.

<p>QNR/v-src^ts and QNR/v-src^ts/ICN cells were incubated 72 hrs at 37°C or 41°C before extraction of total RNA. After reverse transcription of 1 µg of RNA, TGF-β3 cDNA was amplified by QPCR using specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013572#pone-0013572-t001" target="_blank">Table 1</a>) Results were normalized based on HPRT and TBP transcript levels and presented in relative arbitrary units. Normalized TGF-β3 mRNA levels detected in QNR/v-src^ts cells at 37°C were used as reference equal to 1. Each bar represents the mean -/+ S.E. of experiments realized on 3 different cultures per cell type.</p