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Dive into the research topics where Lucie Bourget is active.

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Featured researches published by Lucie Bourget.


Molecular and Cellular Biology | 1997

Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.

Dick D. Mosser; Antoine W. Caron; Lucie Bourget; Claude Denis-Larose; Bernard Massie

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.


Molecular and Cellular Biology | 2000

The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

Dick D. Mosser; Antoine W. Caron; Lucie Bourget; Anatoli B. Meriin; Michael Y. Sherman; Richard I. Morimoto; Bernard Massie

ABSTRACT Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochromec-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.


BMC Biotechnology | 2006

The cumate gene-switch: a system for regulated expression in mammalian cells

Alaka Mullick; Yan Xu; René L. Warren; Maria Koutroumanis; Claire Guilbault; Sophie Broussau; Félix Malenfant; Lucie Bourget; Linda Lamoureux; Rita Lo; Antoine W. Caron; Amélie Pilotte; Bernard Massie

BackgroundA number of expression systems have been developed where transgene expression can be regulated. They all have specific characteristics making them more suitable for certain applications than for others. Since some applications require the regulation of several genes, there is a need for a variety of independent yet compatible systems.ResultsWe have used the regulatory mechanisms of bacterial operons (cmt and cym) to regulate gene expression in mammalian cells using three different strategies. In the repressor configuration, regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a strong constitutive promoter. Addition of cumate, a small molecule, relieves the repression. In the transactivator configuration, a chimaeric transactivator (cTA) protein, formed by the fusion of CymR with the activation domain of VP16, is able to activate transcription when bound to multiple copies of CuO, placed upstream of the CMV minimal promoter. Cumate addition abrogates DNA binding and therefore transactivation by cTA. Finally, an adenoviral library of cTA mutants was screened to identify a reverse cumate activator (rcTA), which activates transcription in the presence rather than the absence of cumate.ConclusionWe report the generation of a new versatile inducible expression system.


Infection and Immunity | 2004

Dysregulated inflammatory response to Candida albicans in a C5-deficient mouse strain.

Alaka Mullick; Miria Elias; Serge Picard; Lucie Bourget; Orce Jovcevski; Susan Gauthier; Ashleigh Tuite; Penelope Harakidas; Craig Bihun; Bernard Massie; Philippe Gros

ABSTRACT Experimental infection of inbred mouse strains with Candida albicans provides a good model system to identify host genetic determinants that regulate onset of, response to, and ultimate outcome of disseminated candidiasis. The A/J mouse strain is exquisitely sensitive to infection with C. albicans, while the C57BL/6J strain is relatively resistant, as measured by survival following intravenous injection of Candida blastospores. This differential susceptibility is caused by an A/J-specific loss-of-function mutation in the C5 component of the complement pathway. C5 plays several critical roles in host response to infection, including target lysis and phagocyte recruitment. Therefore, to determine which of its functions were required for host resistance to candidiasis, a detailed comparative analysis of pathophysiology and host response to acute C. albicans infection was conducted in A/J and C57BL/6J mice. C5-sufficient C57BL/6J mice were found to succumb late in infection due to severe kidney pathology, typified by fungal replication and robust neutrophil-based inflammatory response associated with extensive tissue damage. In contrast, A/J mice were moribund within 24 h postinfection but displayed little if any kidney damage despite an inability to mobilize granulocytes and a high fungal load in the kidney. Rather, C5 deficiency in A/J mice was associated with higher levels of circulating cytokines tumor necrosis factor alpha, interleukin-6, monocyte chemotactic protein 1 (MCP-1), MCP-5, and eotaxin in response to C. albicans. Transfer of the C5-defective allele from A/J onto a C57BL/6J genetic background in recombinant congenic strain BcA17 recapitulated the phenotypic aspects of the susceptibility of A/J mice to C. albicans, confirming the causative role of C5 deficiency in the dysregulated cytokine response.


Biotechnology Progress | 1997

Study of adenovirus production in serum-free 293SF suspension culture by GFP-expression monitoring.

Johanne Côté; Lucie Bourget; Alain Garnier; Amine Kamen

The red‐shifted S65T mutant green fluorescent protein (GFP) was used to compare the adenovirus (Ad) production and post‐infection survival of 293SF and 293S cells in serum‐free and serum‐containing flask cultures, respectively. The GFP‐expressing vector permitted the quantification of both the level of GFP expressed by infected cells and the infectious viral content of the cultures by flow cytometry in a simple, fast, sensitive, and reliable way. The GFP has the main advantage of fluorescing without any substrate addition. Infected cultures showed the coexistence of two populations of fluorescent cells, high‐fluorescence cells (HFCs) and low‐fluorescence cells (LFCs), in proportions that varied between 20 and 75 hpi. The gradual increase in the number of LFCs at the expense of HFCs correlated well with the increase in the number of dead cells. This relationship could be used for the continuous measure of a cultures viability with the appropriate on‐line instrumentation. The post‐infection death rate of infected 293SF cells was higher than that of infected 293S cells, but the level of GFP fluorescence in viable, highly fluorescent cells was similar in the two infected cell lines. The number of infectious viral particles (IVPs) was quantified in less than 24 h by an infection assay of 293S cells in wells with viral particles extracted from the culture samples, and the results were more reproducible (±10% variation) than those generally reported for conventional plaque assay titrations or end‐point dilutions. The viable cell‐specific IVP concentrations were for most experiments similar, indicating again that the difference between the two cell lines was their unequal post‐infection viabilities, not the virus production by the infected living cells.


Applied and Environmental Microbiology | 2010

Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains

Young J. Choi; Lyne Morel; Teffanie Le François; Denis Bourque; Lucie Bourget; Denis Groleau; Bernard Massie; Carlos B. Miguez

ABSTRACT A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-β-d-thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTG-induced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.


Infection and Immunity | 2004

Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans

Alaka Mullick; Miria Elias; Penelope Harakidas; Anne Marcil; Malcolm Whiteway; Bing Ge; Thomas J. Hudson; Antoine W. Caron; Lucie Bourget; Serge Picard; Orce Jovcevski; Bernard Massie; David Y. Thomas

ABSTRACT Candida albicans is an opportunistic human pathogen causing both superficial and disseminated diseases. It is a dimorphic fungus, switching between yeast and hyphal forms, depending on cues from its microenvironment. Hyphae play an important role in the pathogenesis of candidiasis. The hosts response to Candida infection is multifaceted and includes the participation of granulocytes as key effector cells. The aim of this investigation was to study host gene expression during granulocyte-Candida interaction. Effector cells were generated by the granulocytic differentiation of HL60 cells. The resulting cell population was shown to be morphologically and functionally equivalent to granulocytes and is therefore referred to as HL60 granulocytoids for the purposes of this study. Gene expression profiles were determined 1 h after hosts were infected with C. albicans. Three Candida-granulocytoid ratios were chosen to reflect different degrees of HL60 granulocytoid inhibition of C. albicans. The data demonstrate that at the high pathogen-host ratio, C. albicans modulated the HL60 granulocytoids response by downregulating the expression of known antimicrobial genes. In addition, looking at the expression of a large number of genes, not all of which have necessarily been implicated in candidastatic or candidacidal mechanisms, it has been possible to describe the physiological response of the HL60 granulocytoid to an infectious challenge with C. albicans. Finally, some of the observed changes in HL60 granulocytoid gene expression were investigated in freshly isolated human polymorphonuclear leukocytes infected with C. albicans. Similar changes were seen in these primary human cells, lending support to the validity of this model.


Journal of Gene Medicine | 2008

A ligand‐pseudoreceptor system based on de novo designed peptides for the generation of adenoviral vectors with altered tropism

Yue Zeng; Maxime Pinard; Jairo Jaime; Lucie Bourget; Phuong Uyen Le; Maureen O'Connor-McCourt; Rénald Gilbert; Bernard Massie

Delivery of transgenes into specific tissues by adenovirus vectors (AdVs) relies on ablations of their natural tropism and on introduction of a new tropism. If the interaction with its natural receptor is ablated, a new packaging cell line is required to produce the AdV. In the present study, we have used two de novo designed peptides (E‐Coil and K‐Coil) that interact with each other with high affinity to establish a new receptor‐ligand system for the propagation of retargeted AdVs.


Cancer Biology & Therapy | 2008

Differential sensitivity of A549 non-small lung carcinoma cell responses to epidermal growth factor receptor pathway inhibitors.

Maria L. Jaramillo; Myriam Banville; Catherine Collins; Beatrice Paul-Roc; Lucie Bourget; Maureen O'Connor-McCourt

It has been demonstrated that A549 non-small cell lung cancer (NSCLC) cells are sensitive to epidermal growth factor receptor (EGFR) inhibitors in in vivo xenograft animal models, but are relatively resistant in conventional in vitro monolayer growth assays. Here, we utilized anchorage-independent cell growth/survival assays as well as motility assays and demonstrated that these tests detect the effects of two EGFR inhibitors, the small molecule inhibitor AG1478 and the ligand-blocking antibody 225 mAb, on A549 cells more sensitively than monolayer growth assays. AG1478 was more effective than 225 mAb at inhibiting EGF-stimulated anchorage-independent cell growth, in part due to its pronounced ability to inhibit cell survival, whereas 225 mAb and AG1478 were both able to inhibit cell motility. In order to determine which EGFR signalling pathway components were most strongly associated with these cell responses, we analyzed in parallel the phosphorylation levels of EGFR itself as well as several downstream pathway elements. We found that the limited ability of 225 mAb to inhibit MAPK, PI3K and STAT3 phosphorylation correlated with its inability to promote anchorage independent apoptosis, but did not correlate with its ability to inhibit motility. Based on our results in A549 cells, we propose that EGF stimulates tumour progression of NSCLC largely through effects on anchorage-independent growth and survival, as well as motility.


Journal of Virological Methods | 2014

Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.

Rénald Gilbert; Claire Guilbault; David Gagnon; Alice Bernier; Lucie Bourget; Seyyed Mehdy Elahi; Amine Kamen; Bernard Massie

E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA). The present study describes the establishment of AdV complementing cells constructed by stable transfection of the minimal E1A and E1B genes into human lung carcinoma (A549). Because some transgene products can be cytotoxic, the cells were engineered to stably express the repressor of the cumate-switch (CymR) to silence transgene transcription during vector growth. For regulatory compliance and to facilitate the scale-up, the resulting complementing cells (SF-BMAdR) were adapted to serum-free suspension culture. The best clone of SF-BMAdR produced AdV carrying an innocuous transgene to the same level as 293 cells, but titers were better for AdV carrying transgene for a cytotoxic product. Elevated titers were maintained for at least two months in suspension culture in the absence of selective agent and the cells did not produce RCA. Because of their advantageous properties, SF-BMAdR cells should become an important tool for developing large-scale production processes of AdV for research and clinical applications.

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Bernard Massie

National Research Council

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Dick D. Mosser

National Research Council

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Alaka Mullick

Université de Montréal

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Rénald Gilbert

National Research Council

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Jairo Jaime

National Research Council

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Yue Zeng

National Research Council

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