Identification and characterization of TNFalpha responsive genes in human breast cancer cells

One of the hallmarks of cancer is the escape of the transformed cells from apoptosis. Therefore, the identification of survival genes, allowing cancer cells to circumvent programmed cell death, could provide new diagnostic markers as well as targets for therapeutic intervention. A well known transcription factor regulating the balance between pro- and anti- apoptotic factors is NF-kappaB, which is strongly induced by tumor necrosis factor alpha (TNFalpha). When cells are stimulated by TNFalpha their response is biphasic with an initial NF-kappaB induction of survival genes which is overridden by the subsequent activation of initiator caspases triggering apoptosis. By combining gene trap mutagenesis with site specific recombination a strategy was developed, which enriches for genes induced by TNFalpha in the human breast cancer cell line MCF-7. The strategy relies on a one way gene expression switch based on Cre/loxP mediated recombination, which uncouples the expression of a marker gene from the trapped cellular promoter thereby enabling the recovery of genes that are only transiently induced by TNFalpha. The marker gene used in these experiments was a dominant negative variant of the TNFalpha-receptor associated protein FADD (dnFADD), which blocks the apoptotic branch of the TNFalpha induced signaling pathway. Initial experiments indicated that MCF-7 cells expressing high levels of dnFADD were insensitive to TNFalpha induced apoptosis and therefore suitable for the installment of a one way gene expression switch susceptible to Cre/loxP mediated recombination. A MCF-7 reporter clone harboring the recombinase dependent gene expression switch was infected with the gene trap retrovirus U3Cre, which inserts the Cre recombinase gene into a large collection of chromosomal sites. Insertion of Cre downstream of an active cellular promoter induces dnFADD expression from the gene expression switch enabling the cells to block TNFalpha triggered apoptosis. From a gene trap integration library containing approximately 2000000 unique proviral integrations, 69 unique TNFalpha inducible gene trap insertion sites were recovered in a two step selection procedure. Sequencing of the genomic regions adjacent to the insertion sites, which were obtained by inverse PCR (gene trap sequence tags, GTSTs), and data base analysis revealed that 42% of the GTSTs belonged to annotated genes, 13% to known cDNAs with open reading frames, 17% to Genscan predicted genes, 9% to ESTs, 9% to repetitive sequences and 10% to unannotated genomic sequence. Overall, 44% of the annotated genes recovered in this screen were directly or indirectly related to cancer, indicating that the gene trap strategy developed here is suitable for the identification of cancer relevant genes. Analysis of the expression patterns of the trapped and annotated genes in wild type cells revealed that 19 out of 24 genes were either up- or down- regulated by a factor of at least 1.45 by TNFalpha. A large fraction of the gene trap insertions were located upstream, in introns or in opposite orientation to annotated transcripts, indicating that the strategy efficiently recovers non-coding RNAs (ncRNAs). While the biological significance of these transcripts still needs to be elucidated, they fall into two main categories. The first category includes gene trap insertions upstream of genes, which could either represent regulatory RNAs interacting with promoter elements or transcripts driven by bidirectional promoters. The second includes inverse orientation gene trap insertions in introns of annotated genes suggesting the presence of natural antisense transcripts (NATs). Interestingly, more than 50% of all antisense integrations are located downstream of transcription start sites predicted by different algorithms supporting the existence of RNAs transcribed from the corresponding genomic regions. Intronic integrations on the coding strand could be derived from cryptic splicing, alternative promoter usage or additional, so far uncharacterized transcripts. Preliminary functional analysis of two genes recovered in this screen encoding the transcription factor ZFP67 and the FLJ14451 protein revealed that FLJ14451 but not ZFP67 inhibited anchorage independent growth in soft agar, suggesting that FLJ14451 might have some tumor suppressor functions. In summary, besides identifying a putative tumor suppressor protein, the present experiments have shown that gene trapping is useful in identifying non-coding transcripts in living cells and may turn out to be the method of choice in characterizing these transcripts whose functions are still largely unknown.Ein zentrales Charakteristikum von Krebs ist die Unterdrückung des zellulären Apoptoseprogramms in den transformierten Zellen. Daher könnte die Identifizierung von Überlebensgenen, welche den Zellen die Umgehung des programmierten Zelltods erlauben, neue diagnostische Marker oder Zielmoleküle für therapeutische Intervention liefern. Die Balance zwischen pro- und anti-apoptotischen Signalen wird durch NF-kappaB, einen gut charakterisierter Transkriptionsfaktor reguliert, welcher wiederum durch Tumornekrosefaktor alpha (TNFalpha) induziert wird. Zellen, die mit TNFalpha stimuliert werden zeigen eine biphasische Antwort, während derer es zu einer initialen Induktion von Überlebensgenen kommt, bevor im folgenden Initiator-Caspasen aktiviert werden, die dann die Apoptose einleiten. Zur Identifizierung TNFalpha induzierbarer Gene in der menschlichen Brustkrebszellinie MCF-7 wurde eine Strategie benutzt, welche auf einer Kombination von Genfallen-Mutagenese und sequenzspezifischer Rekombination beruht. Diese Strategie macht sich einen irreversiblen molekularen Schalter zunutze, der die Expression eines Markergens von dem, die Genfalle aktivierenden, zellulären Promotor entkoppelt. Als Markergen wurde eine dominant negative Variante des TNFalpha-Rezeptor-assozierten Proteins FADD ("Fas-associated death domain protein"; dnFADD) benutzt, die den pro-apoptotische Zweig des TNFalpha-Rezeptor-Signalwegs blockiert. Ein MCF-7 Zellklon mit diesem Rekombinase-aktivierbaren, molekularen Schalter wurde mit dem Genfallen-Retrovirus U3Cre transduziert, welcher das Cre-Rekombinase-Gen in eine große Anzahl unterschiedlicher chromosomaler Loci inserieren kann. Kommt Cre dabei unter die Kontrolle eines aktiven zellulären Promoters, wird die Expression von dnFADD induziert und somit die Apoptose-Induktion durch TNFalpha verhindert. Aus einer Genfallen-Integrationsbank mit ca. 2000000 unabhängigen proviralen Integrationen wurden nach einer zweistufigen Selektion 69 Zellklone mit Genfallen-Integrationen in TNFalpha induzierten Genen erhalten. Die Sequenzierung der benachbart zu den Genfallen-Proviren liegenden, über inverse PCRs amplifizierten, genomischen Regionen und anschließende Datenbankanalysen ergaben folgende Verteilung der Genfallen-Integrationen: 42% lagen in annotierten Genen, 13% in Genen mit offenen Leserahmen unbekannter Funktion, 17% in hypothetischen Genen, 9% in ESTs ("expressed sequence tags"), 9% in repetitiven Elementen und 10% in nicht annotierten genomischen Regionen. 44% der aus diesem Screening-Verfahren erhaltenen, annotierten Gene ließen sich direkt oder indirekt mit Krebserkrankungen korrelieren. Dies ist ein Indiz dafür, dass der hier entwickelte, experimentelle Ansatz zur Identifizierung Krebs-relevanter Gene geeignet ist. Die Expressionanalyse ausgewählter, annotierter Gene in wildtypischen MCF-7 Zellen ergab eine Herauf- bzw. Herunterregulation um einen Faktor von mindestens 1.45 bei 19 von 24 untersuchten Genen. Ein großer Teil der Genfallen-Integrationen befand sich 5'-oberhalb von Genen, in Introns oder in umgekehrter Orientierung zu annotierten Transkripten, was darauf hindeutet, dass mit Hilfe der gewählten Strategie nicht-kodierende RNAs identifiziert werden können. Obwohl der Nachweis dieser Transkripte und ihrer biologischen Relevanz noch aussteht, können sie in zwei Kategorien eingeteilt werden. Die erste umfasst Integrationen 5' zu Genen, die entweder regulatorische RNAs repräsentieren, die mit Promotorelementen interagieren, oder Transkripte, welche unter der Kontrolle von bidirektionalen Promotoren stehen. Die zweite Kategorie, Insertionen auf dem nicht-kodierenden Strang innerhalb von Introns, legen das Vorkommen natürlicher antisense Transkripte (NATs) nahe. Interessanterweise liegen mehr als 50% aller antisense-Integrationen 3' zu potentiellen Transkriptionsstartstellen, die mit verschiedenen Algorithmen vorausgesagt wurden. Dies kann als Hinweis darauf gewertet werden, dass höchstwahrscheinlich RNAs existieren, die Transkripte der entsprechenden genomischen Regionen darstellen. Intronische Integrationen auf dem kodierenden Strang können als Resultat kryptischer Spleißvorgänge oder durch alternative Promotoren transkriptionell aktiviert werden; alternativ könnten es sich bei den durch die Genfalle "abgefangenen" RNAs um zusätzliche, bisher nicht charakterisierte Transkripte handeln. Vorläufige funktionelle Analysen zweier Gene, die für den Tanskriptionsfaktor ZFP67 und das Protein FLJ14451 kodieren, ergaben, dass FLJ14451, aber nicht ZFP67 das Substrat-unabhängige Wachstum von MCF-7 Zellen in Weichagar inhibieren kann. Dies weist auf eine mögliche Tumorsuppresorfunktion des Proteins hin. Die im Rahmen dieser Dissertation durchgeführten Experimente führten nicht nur zur Identifizierung eines potentiellen, neuen Tumorsuppressorgens, sondern zeigten auch, dass Genfallen ein nützliches Werkzeug bei der Suche nach nicht-kodierenden RNAs in lebenden Zellen sein können und ihr Einsatz möglicherweise die Methode der Wahl für die Identifizierung derartiger Transkripte darstellt

Common and Differential Traits of the Membrane Lipidome of Colon Cancer Cell Lines and their Secreted Vesicles: Impact on Studies Using Cell Lines

Colorectal cancer (CRC) is the fourth leading cause of cancer death in the world. Despite the screening programs, its incidence in the population below the 50s is increasing. Therefore, new stratification protocols based on multiparametric approaches are highly needed. In this scenario, the lipidome is emerging as a powerful tool to classify tumors, including CRC, wherein it has proven to be highly sensitive to cell malignization. Hence, the possibility to describe the lipidome at the level of lipid species has renewed the interest to investigate the role of specific lipid species in pathologic mechanisms, being commercial cell lines, a model still heavily used for this purpose. Herein, we characterize the membrane lipidome of five commercial colon cell lines and their extracellular vesicles (EVs). The results demonstrate that both cell and EVs lipidome was able to segregate cells according to their malignancy. Furthermore, all CRC lines shared a specific and strikingly homogenous impact on ether lipid species. Finally, this study also cautions about the need of being aware of the singularities of each cell line at the level of lipid species. Altogether, this study firmly lays the groundwork of using the lipidome as a solid source of tumor biomarkers

El consumo de medios en los jóvenes de Secundaria

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The Novel Anticancer Drug Hydroxytriolein Inhibits Lung Cancer Cell Proliferation via a Protein Kinase Ca- and Extracellular Signal-Regulated Kinase 1/2-Dependent Mechanism

[eng] Membrane lipid therapy is a novel approach to rationally design or discover therapeutic molecules that target membrane lipids. This strategy has been used to design synthetic fatty acid analogs that are currently under study in clinical trials for the treatment of cancer. In this context, and with the aim of controlling tumor cell growth, we have designed and synthesized a hydroxylated analog of triolein, hydroxytriolein (HTO). Both triolein and HTO regulate the biophysical properties of model membranes, and they inhibit the growth of non small-cell lung cancer (NSCLC) cell lines in vitro. The molecular mechanism underlying the antiproliferative effect of HTO involves regulation of the lipid membrane structure, protein kinase C-a and extracellular signal-regulated kinase activation, the production of reactive oxygen species, and autophagy. In vivo studies on a mouse model of NSCLC showed that HTO, but not triolein, impairs tumor growth, which could be associated with the relative resistance of HTO to enzymatic degradation. The data presented explain in part why olive oil (whose main component is the triacylglycerol triolein) is preventive but not therapeutic, and they demonstrate a potent effect of HTO against cancer. HTO shows a good safety profile, it can be administered orally, and it does not induce nontumor cell (fibroblast) death in vitro or side effects in mice, reflecting its specificity for cancer cells. For these reasons, HTO is a good candidate as a drug to combat cancer that acts by regulating lipid structure and function in the cancer cell membrane

Differential effect of 2-hydroxyoleic acid enantiomers on protein (sphingomyelin synthase) and lipid (membrane) targets

The complex dual mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent anti-tumor compound used in membrane lipid therapy (MLT), has yet to be fully elucidated. It has been demonstrated that 2OHOA increases the sphingomyelin (SM) cell content via SM synthase (SGMS) activation. Its presence in membranes provokes changes in the membrane lipid structure that induce the translocation of PKC to the membrane and the subsequent overexpression of CDK inhibitor proteins (e.g., p21Cip1). In addition, 2OHOA also induces the translocation of Ras to the cytoplasm, provoking the silencing of MAPK and its related pathways. These two differential modes of action are triggered by the interactions of 2OHOA with either lipids or proteins. To investigate the molecular basis of the different interactions of 2OHOA with membrane lipids and proteins, we synthesized the R and S enantiomers of this compound. A molecular dynamics study indicated that both enantiomers interact similarly with lipid bilayers, which was further confirmed by X-ray diffraction studies. By contrast, only the S enantiomer was able to activate SMS in human glioma U118 cells. Moreover, the anti-tumor efficacy of the S enantiomer was greater than that of the R enantiomer, as the former can act through both MLT mechanisms. The present study provides additional information on this novel therapeutic approach and on the magnitude of the therapeutic effects of type-1 and type-2 MLT approaches. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy