Our research program focuses on the molecular and cellular events involved in the initiation, execution and regulation of cell death in mammalian cells. A one page summary of cell death and our research program is available here.
1. Molecular mechanism of apoptosis in amino acid-deprived mammalian cells:
Amino acids are mostly known for their roles as:
1) precursors of protein synthesis
2) metabolic intermediates
4) and antioxydants.
A more recently uncovered and less known role of amino acids is their ability to promote cell survival. This phenomenon is far from being trivial, as it impacts on several biological phenomena. Here are some examples:
1) L-Glutamine supplementation decreases intestinal atrophy observed following partial resection, greatly improving recovery. This effect is attributable to cell death inhibition rather than the promotion of proliferation;
2) Amino acids are essential for the sustained growth of tumor cells;
3) Murine and human cell lines, used in the biotechnological industry to produce monoclonal antibodies and other products, are exquisitely sensitive to amino acid depletion and undergo cell death shortly after exhausting their amino acid supply.
We are involved in the elucidation of the molecular and cellular events responsible for the active pro-survival function of amino acids. We are also studying the molecular pathways underlying the induction of apoptosis following amino acid deprivation.
1) Chartrand, K., Mallory*, M., Deane*, B. and E.R. Gauthier. (2007) Bcl-xL is required in a stress-specific manner for the up-regulation of Gadd153 in Sp2/0-Ag14 hybridoma cells. Submitted.
2) P.J. Guérin*, T. Furtak**, K. Eng** and E.R. Gauthier. (2006). Oxidative stress is not required for the induction of apoptosis upon glutamine starvation of Sp2/0-Ag14 cells. European Journal of Cell Biology . 85: 355-365. Reprint
3) Paquette*, J.C., P. Guérin* and E.R. Gauthier. (2005). Induction of the intrisinc apoptotic pathway by L-glutamine starvation. Journal of Cellular Physiology 202 (3): 912-921. Reprint
2. Control of apoptosis in cell lines of biotechnological interest:
Mammalian cell culture is widely used to produce large amounts of proteins of biotechnological or biomedical interest (e.g. monoclonal antibodies). However, cell death by apoptosis has been shown to severely limit the productivity of these cells, both in batch or perfusion cultures. Apoptotic cell death in long-term culture is attributable to several factors, including the exhaustion of essential nutrients (e.g. amino acids), anoxia and hydrodynamic stress. New means to improve the viability of cultured cell lines are therefore being sought, either by modifying culture conditions or genetically altering the cells themselves.
One aspect of our research program in centred on investigating the role which apoptosis plays in limiting the viability of cell lines in culture. In particular, we are exploring the molecular pathways responsible for apoptosis in long-term cultured cells. Our goal is to use this newly acquired knowledge to improve cell viability using genetic engineering techniques (e.g. ectopic gene expression). We are also exploring the molecular basis of apoptosis induction in hybridoma cells. For these studies, the murine myeloma P3x63Ag8.653 (P3) and the murine hybridoma SP2/O-Ag14 (SP2) are used.
a. Improvement of murine B cell myelomas and hybridomas viability in long-term culture:
We are using genetic engineering procedures in order to increase the resistance of these cell lines to apoptosis. Known proteins inhibiting apoptosis (e.g. Bcl-2, caspase inhibitors) are being modified by mutagenesis to increase their anti-death potency. Techniques used include cell culture, P.C.R., gene cloning and sequencing, mutagenesis, and expression of foreign proteins into mammalian cells.
b. Mechanism of apoptosis induction in long-term cell culture:
We are investigating the molecular events involved in triggering cell death in murine B cell myelomas/hybridomas during cell culture. To achieve this goal, we use biochemical (Western analysis, enzymatic assays), cellular biological (fluorescence microscopy, viability assays), as well as molecular biological approaches (Northern analysis, gene cloning and sequencing, P.C.R., protein expression in bacteria, yeast and mammalian cells).
1) Mallory*, M., K. Chartrand and E.R. Gauthier.(2007).Gadd153 expression does not necessarily correlate with changes in culture behavior of hybridoma cells. Submitted.
2) Charbonneau*, J.R., T. Furtak**, J. Lefebvre** and E.R. Gauthier. (2003). Bcl-xL expression interferes with the effects of L-glutamine supplementation on hybridoma cultures. Biotechnol. Bioeng. 81: 279-290.PDF file
3) Charbonneau*, J. and E.R. Gauthier. (2001). Protection of hybridoma cells against apoptosis by a loop domain-deficient Bcl-xL protein. Cytotechnology. 37: 41-47. PDF file
4) Charbonneau*, J. and E.R. Gauthier. (2000). Prolongation of hybridoma cell viability in stationary batch culture by Bcl-xL expression. Cytotechnology. 34(1-2): 131-39. PDF file