Clinical significance of epithelial-mesenchymal transition-related markers expression in the micrometastatic sentinel lymph node of NSCLC.

OBJECTIVES: Metastatic lymph node affectation is the main prognostic factor in localised lung cancer. However, the pathological study of lymph nodes reveals tumour relapse for 20% of patients after oncological curative surgery. Recently, EMT (epithelial-mesenchymal transition) has been established as one of the main factors related to lymphatic dissemination and metastasis. This study evaluated the prognostic value of EMT-related gene expression in micrometastatic sentinel lymph nodes (SLN) of non-small cell lung cancer (NSCLC) patients. METHODS: The presence of genes CDH1, CDH2, VIM, TWIST1, SNAI1, SNAI2, ZEB1, and ZEB2 in mRNA was analysed in tumours and in the SLN of NSCLC patients for whom surgery was planned for treatment. The significant association between the expression level of EMT-related markers and patients' clinicopathological characteristics and relapse was assessed. RESULTS: Of the 96 patients, 56 (58.33%) presented molecular micrometastasis in SLN, which showed higher CDH1, CDH2, and VIM expressions than non-micrometastatic ones. An association linking a low CDH1/CDH2 ratio in SLN with molecular micrometastasis, adenocarcinoma, and non-smoking patients was found. The multivariate Cox regression analysis proved the prognostic accuracy of the CDH1/CDH2 ratio in SLN. CONCLUSIONS: The molecular EMT status of SLN could be used as an independent prognosis predictor in early stage NSLCL patients, and as a new tool to better stratify and predict patient outcomes.

Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells.

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.

GSK3-SCF(FBXW7) targets JunB for degradation in G2 to preserve chromatid cohesion before anaphase.

JunB, an activator protein-1 (AP-1) transcription factor component, acts either as a tumor suppressor or as an oncogene depending on the cell context. In particular, JunB is strongly upregulated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) where it enhances cell proliferation. Although its overexpression is linked to lymphomagenesis, the mechanisms whereby JunB promotes neoplastic growth are still largely obscure. Here, we show that JunB undergoes coordinated phosphorylation-dependent ubiquitylation during the G2 phase of the cell cycle. We characterized a critical consensus phospho-degron that controls JunB turnover and identified GSK3 and SCF(FBXW7) as, respectively, the kinase and the E3 ubiquitin ligase responsible for its degradation in G2. Pharmacological or genetic inactivation of the GSK3-FBXW7-JunB axis induced accumulation of JunB in G2/M and entailed transcriptional repression of the DNA helicase DDX11, leading to premature sister chromatid separation. This abnormal phenotype due to dysregulation of the GSK3ß/JunB/DDX11 pathway is phenocopied in ALK-positive ALCL. Thus, our results reveal a novel mechanism by which mitosis progression and chromatid cohesion are regulated through GSK3/SCF(FBXW7)-mediated proteolysis of JunB, and suggest that JunB proteolysis in G2 is an essential step in maintaining genetic fidelity during mitosis.

Intron-tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells.

Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells. We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium. Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants.

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.

SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase.

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.