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.

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.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Adjusting transgene expression levels in lymphocytes with a set of inducible promoters.

BACKGROUND: Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells. METHODS: The performance of the tetracycline-regulated (Tet)-inducible promoters Tet-responsive element (TRE), mouse mammary tumor virus (MMTV)/TRE, TREtight and second generation TRE (SG/TRE) was compared in several B-cell lines and in Jurkat T-cells using transient transfections in combination with Tet-On. To monitor transgene expression in a Jurkat cell line containing a transsilencer and a reverse transactivator, expression cassettes encoding enhanced green fluorescent protein, CD123 or a constitutively active, cytotoxic caspase-3 were flanked with insulators and stably integrated. The performance of TREtight and SG/TRE was furthermore analysed in transiently transfected primary CD4(+) human T-cells. RESULTS: The promoters exhibit greatly diverging characteristics. MMTV/TRE permits moderate, TRE and TREtight permits intermediate and SG/TRE permits very high expression levels. TRE and SG/TRE are leaky, whereas MMTV/TRE and TREtight provide stringent expression control. Tetracycline derivatives add flexibility to transgene expression by introducing intermediate expression levels. CONCLUSIONS: The different expression profiles of the promoters increase the flexibility to adjust transgene expression levels. The promoters provide an additional option to optimize system performance for many applications.

A gene regulation system with four distinct expression levels.

BACKGROUND: The amount of a particular protein, and not just its presence or absence, frequently determines the outcome of a developmental process or disease progression. These dosage effects can be studied by conditionally expressing such proteins at different levels. With typical gene regulation systems like the Tet-On system, intermediate expression levels can be obtained by varying the effector concentration. However, this strategy is limited to situations in which these concentrations can be precisely controlled and, thus, not suited for animal models or gene therapy approaches. Here, we present a Tet transregulator setup that allows establishment of four levels of promoter activity largely independent of effector concentration. METHODS: A newly introduced transsilencer is combined with a reverse transactivator. As the regulators respond differentially to tetracycline derivatives, four expression levels are obtained by adding different effectors. To facilitate integration of the components, we generated versatile all-in-one vectors. Apart from a cassette expressing the transregulators and a selection marker, these vectors encode a bidirectional, regulated promoter driving expression of GFP and the gene of interest. The features of this stepwise regulation system were analyzed by transient and stable transfections of human cell lines. RESULTS: We demonstrate in a variety of experimental settings that coexpression of these transregulators leads to robust stepwise regulation. Depending on the respective effectors, four expression levels are achieved with different responsive promoters, cell lines and target genes. CONCLUSIONS: This system shows that a promoter can be adjusted to different activities and provides an excellent strategy to investigate protein dosage effects.

Transactivator mutants with altered effector specificity allow selective regulation of two genes by tetracycline variants.

A set of Tet repressor (TetR) based eukaryotic transactivators that respond to 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) but no longer to tetracycline (tc) is presented. The novel transactivators exhibit high activation in absence of an effector and a 200-fold reduction of reporter gene activity in the presence of cmt3. The most cmt3-sensitive mutant was coexpressed with a tc-responsive Tet transregulator harbouring an altered DNA recognition specificity. Use of cmt3 and tc yields independent control of expression of two genes in the same cell without crosstalk.