A novel MCF-10A line allowing conditional oncogene expression in 3D culture

Introduction Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene. Methods MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out. Results MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression. Conclusions Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance

A suicide gene approach using the human pro-apoptotic protein tBid inhibits HIV-1 replication

<p>Abstract</p> <p>Background</p> <p>Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.</p> <p>Results</p> <p>When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)₂R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.</p> <p>Conclusions</p> <p>This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.</p

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Ein neues Regulationssystem für stringent kontrollierte und langzeit-induzierbare Transgenexpression in humanen Zelllinien

The transfer of donor lymphocytes into patients is one way to treat persistent or relapsing malignancies after stem cell transplantation. Safety considerations suggest equipping the cells with conditional suicide switches to stop treatment when adverse effects emerge. The major aim of the present study was to establish a conditional suicide switch based on the expression of human proapoptotic proteins in the human T cell line Jurkat, using the regulatory components of the Tet system. Such an approach could overcome certain limitations posed by the most frequently used and currently clinically tested suicide system which relies on the expression of a viral thymidine kinase. Fundamental requirements for the generation of such a suicide system are i) tight expression control, ii) reliable inducibility of the switch and iii) efficient cell death induction. To achieve tight expression control, the combined activator-repressor strategy was applied to generate Jurkat regulator cell lines. Two mechanistically different acting transsilencers, tTS-KRAB and tTS-PP, harbouring either a KRAB repression domain or a PLDLS motif, were respectively coexpressed with the reverse transactivator rtTA2S-M2. Both approaches conferred excellent regulatory properties to the resulting cell lines. However, when controlling the expression of chromosomally integrated transgenes, tTS-KRAB-mediated repression was shown to silence transgene expression irreversibly, probably due to the recruitment of heterochromatin. Repression mediated by the recently developed transsilencer tTS-PP in the cell line Jr2SM2PP, in contrast, did not interfere with subsequent transactivation, even when repression was maintained for several weeks prior to induction. Because the Tet-inducible promoter can influence the quality of regulation, the performance of four different Tet-inducible promoters was analyzed in Jr2SM2PP cells. The results revealed greatly diverging characteristics concerning basal leakiness and the maximal expression levels achieved and identified TREtight as most approriate for controlling cytotoxic transgenes. Different proapoptotic proteins were tested in transient transfections for doxycycline-dependent cell death induction in Jurkat cells. Using stably integrated, insulator-flanked and TREtight-controlled suicide expression cassettes encoding the recombinant proapoptotic molecules revCasp3 and tBid, efficient suicide switches were generated in the Jurkat regulator cell line Jr2SM2PP. With both switches, up to 99% of all cells died 24 hours after induction. The approach provided stringent expression control which was monitored for several months. After that time-period in the uninduced state, cell death was induced as efficiently as before. The actual target cells of therapeutic safety switch applications are primary human T cells. Episomally persisting herpesvirus saimiri-derived vectors are a promising tool for gene transfer into that cell type. Within the scope of this thesis, different all-in-one Tet-inducible transgene expression cassettes were constructed and cloned into herpesvirus saimiri transfer vectors. They were subsequently tested for doxycycline-dependent regulation of expression in primary human cells in the research group of a collaboration partner. Additionally, the potential of different human bidirectional promoters to drive the coordinated expression of two transgenes was tested in this study. One promoter, pTFP, responsible for the transcription of the mitochondrial trifunctional protein subunits at its endogenous locus, showed promising properties. Stably integrated into HeLa cells it mediated reliable transcription of a dual regulator setup for several months.Die Transfusion von Donorlymphozyten ist eine Möglichkeit, nach erfolgter Stammzelltransplantation persistierende oder wieder aufkeimende bösartige Erkrankungen zu behandeln. Um die Sicherheit solcher Ansätze zu gewährleisten, werden die Donorzellen mit einem konditionalen Suizidschalter ausgestattet, der es erlaubt, die Behandlung beim Auftreten unerwünschter Nebenwirkungen abzubrechen. Hauptziel der vorliegenden Arbeit war die Etablierung eines konditionalen, auf der Expression humaner proapoptotischer Proteine basierenden Suizidschalters in der humanen T-Zelllinie Jurkat, unter Verwendung der regulatorischen Komponenten des Tet-Systems. Solch ein Ansatz könnte gewisse Einschränkungen überwinden, die sich bei der Anwendung des zur Zeit am häufigsten eingesetzten Suizidsystems, das auf der Expression der viralen Thymidinkinase beruht und gegenwärtig klinisch getestet wird, ergeben. Grundlegende Anforderungen an solch ein Suizidsystem sind i) strikte Expressionskontrolle, ii) die konstante Induzierbarkeit des Schalters und iii) effiziente Zelltodinduktion. Zur Gewährleistung strikter Expressionskontrolle wurde die kombinierte Aktivator-Repressor-Strategie angewandt, um Jurkat-Regulatorlinien zu generieren. Zwei mechanistisch unterschiedlich wirkende Transsilencer, tTS-KRAB and tTS-PP, die entweder die KRAB-Repressionsdomäne oder ein PLDLS-Repressionsmotif tragen, wurden jeweils zusammen mit dem reversen Transaktivator rtTA2S-M2 coexprimiert. In beiden Ansätzen wurden hervorragend regulierende Zelllinien erhalten. Bei der Expressionskontrolle chromosomal integrierter Transgene zeigte sich jedoch, dass tTS-KRAB-vermittelte Repression, wahrscheinlich durch Rekrutierung von Heterochromatin, zum Silencing der Transgenexpression führte. Repression durch den kürzlich entwickelten Transsilencer tTS-PP in der Zelllinie Jr2SM2PP dagegen beeinträchtigte die nachfolgende Transaktivierung nicht; und zwar auch dann nicht, wenn die Transgenexpression vor ihrer Induktion schon für mehrere Wochen ausgeschaltet worden war. Da der Tet-regulierbare Promotor die Qualität der Regulation beeinflussen kann, wurde das Verhalten vier verschiedener Promotoren in Jr2SM2PP Zellen analysiert. Die Ergebnisse ließen stark divergierende Eigenschaften in Bezug auf Basalexpression und maximal mögliches Expressionsniveau erkennen. TREtight wurde als am besten geeigneter Promotor für die Kontrolle zytotoxischer Transgene identifiziert. Das Potential verschiedener proapoptotischer Proteine, bei Doxycyclin-abhängiger Überexpression Zelltod auszulösen, wurde zuerst in transienten Transfektionen untersucht. Unter Verwendung stabil integrierter, Isolator-flankierter Suizidgenexpressionskassetten, die unter der Kontrolle von TREtight stehen und für die rekombinanten proapoptotischen Moleküle revCasp3 oder tBid kodieren, wurden effiziente Suizidschalter in der Jurkat-Regulatorlinie Jr2SM2PP generiert. Mit beiden Schaltern wurden bis zu 99% der Zellen innerhalb von 24 Stunden nach Induktion getötet. Mit dem hier verfolgten Ansatz wurde stringente Expressionskontrolle nicht nur erreicht, sondern auch für mehrere Monate aufrecht erhalten. Nach dieser Zeit konnte der Zelltod genauso effizient induziert werden wie vorher. Die eigentlichen Zielzellen therapeutischer Anwendungen solcher Sicherheitsschalter sind primäre humane T-Zellen. Episomal persistierende Herpesvirus saimiri-basierte Vektoren sind ein vielversprechendes Werkzeug für den Gentransfer in diesen Zelltyp. Im Rahmen dieser Arbeit wurden verschiedene all-in-one Tet-regulierbare Transgenexpressionskassetten konstruiert und in Transfervektoren für Herpesvirus saimiri eingebracht. Diese wurden anschließend von einer kooperierenden Arbeitsgruppe auf ihre Fähigkeit überprüft, Doxycyclin-abhängige Regulation von Transgenen in humanen Primärzellen zu vermitteln. Des Weiteren wurde in dieser Arbeit das Potential verschiedener humaner bidirektionaler Promotoren getestet, die simultane Expression zweier Transgene zu vermitteln. Einer der Promotoren, pTFP, der am endogenen Locus für die Transkription der Untereinheiten des mitochondrialen trifunktionalen Proteins zuständig ist, zeigte vielversprechende Eigenschaften. Stabil integriert in HeLa Zellen, vermittelte er zuverlässig die Transkription eines dualen Regulatorsetups über mehrere Monate hinweg

A novel MCF-10A line allowing conditional oncogene expression in 3D culture

Introduction Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene. Methods MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out. Results MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression. Conclusions Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance.</p

tLivin displays flexibility by promoting alternative cell death mechanisms.

Livin is a member of the Inhibitor of Apoptosis (IAP) protein family that inhibits apoptosis triggered by a variety of stimuli. We previously demonstrated that while Livin inhibits caspase activity, caspases can cleave Livin to produce a truncated protein, tLivin and that this newly formed tLivin paradoxically induces cell death. However to date, the mechanism of tLivin-induced cell death is not fully understood. In this study, we set out to characterize the form of cell death mediated by tLivin. Here we demonstrate that, unlike most death-promoting proteins, tLivin is a flexible inducer of cell death capable of promoting necrosis or apoptosis in different cell lines. The unusual flexibility of tLivin is displayed by its ability to activate an alternative form of cell death when apoptosis is inhibited. Thus, tLivin can promote more than one form of cell death in the same cell type. Interestingly, in cells where tLivin induces necrosis, deletion of the caspase binding BIR domain results in tLivin-induced apoptosis, suggesting the BIR domain can potentially hamper the ability of tLivin to induce apoptosis. We further elucidate that tLivin activates the JNK pathway and both tLivin-induced apoptosis and necrosis are partially mediated by JNK activity. Acquired resistance to apoptosis, common in many tumors, impinges on the efficiency of conventional anti-cancer agents that function primarily by inducing apoptosis. The ability of tLivin to induce death of apoptosis-compromised cells makes it an attractive candidate for targeted cancer therapy

tLivin activates the JNK pathway.

<p>(<b>A</b>) 293T cells were either transiently transfected with empty vector (ev), tBID- or tLivin-expressing vectors, treated with 30 µg/ml etoposide for 24 h (etop) or left untreated (con). Cells were harvested at the indicated time points post-transfection and the phosphorylation of JNK, ATF-2 and p38MAPK was analyzed by western blot. (<b>B</b>) Cells of MelA1 clones were harvested at the indicated time points following administration of 2.5 µg/ml dox or following 30 min treatment with 200 nM anisomycin (A), control cells (con) were left untreated. Phosphorylation of JNK and c-JUN was analyzed by western blot. (<b>C</b>) 293T cells were either transiently transfected with empty vector (ev), tLivin- or tLivinΔBIR-expressing vectors, treated with 200 nM anisomycin for 30 min (A) or left untreated (con). Cells were harvested at the indicated time points post-transfection and phosphorylation of JNK and c-JUN was analyzed by western blot.</p

tLivin-induced apoptosis and necrosis are partially mediated by activation of JNK.

<p>(<b>A</b>) Cells of MelA1 clones were treated with 10 µM SP600125 1 h prior to administration of 2.5 µg/ml dox (where indicated) and harvested after 36 h along with untreated cells (con). Cells were stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (<b>B</b>) 293T cells were transiently transfected with the indicated plasmids and harvested 24 h post-transfection along with untransfected cells (con). Cells were stained with PI and the percent of cell death was measured by flow cytometry. (<b>C</b>) 293T cells were transiently transfected with the indicated plasmids, harvested 24 h post-transfection and analyzed by western blot for the protein levels of p-c-JUN, c-JUN, JIP-1, tLivin and GAPDH.</p

tLivin activates an alternative form of cell death when apoptosis is inhibited.

<p>Cells of MelA1 clones were treated with 75 µM zVAD-fmk 1 h prior to addition of 2.5 µg/ml dox or 30 µM cisplatin and harvested after 36 h along with untreated cells (con). (<b>A</b>) Cells were stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (<b>B</b>) Cells from each sample were divided into three aliquots and analyzed for cleavage of caspase 3 by western blot and for the percent of PI positive cells and DNA content by flow cytometry.</p