Philippe Gros, Ph.D.
Research in my lab is centred on three main areas:
Resistance to chemotherapy in tumour cells
Multidrug resistance (MDR) is defined by the simultaneous appearance of cellular resistance to structurally and functionally unrelated cytotoxic drugs in tumour cells in vivo and cultured cells in vitro. My laboratory studies the role of two families of membrane transport proteins in the appearance of MDR in tumour cells. These two families of proteins, P-glycoproteins (Pgp) and multidrug resistance associated protein (MRP) are members of the ATP-binding cassette (ABC) families of ATP-driven membrane transporters. My laboratory has previously cloned the genes corresponding to the three members of the Pgp family and has shown that two of the proteins can convey drug resistance and transport unmodified chemotherapeutic drugs, when overexpressed in otherwise drug-sensitive cells. We are currently studying the mechanism of action of these proteins with the aim of identifying novel inhibitors that can reverse MDR and be included in current chemotherapy protocols in the clinic. In the case of MRP, my laboratory has recently cloned and characterized two new members of this family, MRP3 and MRP6. The MRP family of transporters acts as membrane efflux pump on drug molecules that have been modified to glutathione and glucuronide adducts. We are currently trying to elucidate the mechanism of action of these proteins, evaluate their clinical relevance in human tumours and identify potential inhibitors of their action.
In the case of Pgp, the major current research efforts of the lab have been a) to understand the functional role of the two ATP binding sites of the protein in the ATP hydrolysis cycle of the protein, b) to identify the structural determinants involved in the astonishing wide substrate specificity of the protein, c) to identify the protein segments and understand the mechanism involved in signal transduction from the ATP binding sites to the membrane domains to mediate drug efflux, and d) to establish proximity relationships between the different structural domains of the protein, and monitor dynamic changes during ATP hydrolysis and drug transport.
Genetic basis of susceptibility to infectious diseases
My laboratory has used the mouse as an animal model to study and identify genes that affect susceptibility to infectious diseases. The overall approach has consisted in initially identifying differences amongst inbred strains in susceptibility to a specific pathogen, determining if the genetic component of differential susceptibility is simple or complex, and use a positional cloning approach to isolate the genes involved. We have extended this approach to study additional genes that may modulate the expression of a specific susceptibility/resistance factor. Using this approach, we have previously identified the Nramp1 gene as a major determinant of susceptibility to infection with Salmonella, Leishmania and to several species of mycobacteria.
Subsequent studies in humans have shown that Nramp1 is a major predisposing factor to tuberculosis and leprosy. We have also shown that a second member of this family, Nramp2, is expressed at the brush border of the intestine and is dramatically regulated by dietary iron. It is also expressed in the membrane of early endosomes in all cell types. These and other studies show that Nramp2 is the major transferrin-independent iron uptake system of the body, both across the intestinal membrane and across the membrane of transferrin-positive acidified endosomes. Nramp1 is most likely a cation transporter which has been proposed to modulate iron content of the phagosome, thereby affecting microbial replication.
Similarly, we have been carrying out positional cloning of two additional host resistance loci, Lgn1 and Cmv1 (collaboration with S. Vidal, U. Ottawa), that control resistance to infection with Legionella and cytomegalovirus, respectively. Genetic, physical and transcriptional maps of these loci have been assembled and transcription units in these regions are been evaluated (Ly49 family for Cmv1, Naip family for Lgn1). In collaboration with D. Malo (McGill Centre for the Study of Host Resistance), we have also cloned the Lps locus (Tlr4, Toll-like receptor 4), that controls susceptibility to infection with Salmonella and host response to endotoxin. Finally, we have used a genetic approach to study complex traits, in particular susceptibility to malaria infection. Using a quantitative trait linkage analysis, we have identified a major locus on chromosome 8 that controls the level of blood parasitemia during peak of infection. We have also used recombinant congenic strains of mice to identify and map additional single gene effects in the murine model of malaria infection.
Genetic basis of neural tube defects in neurogenesis
Neural tube defects such as spina bifida and anencephaly are the most frequent developmental malformations in humans. These defects in neurogenesis result from failure of the neural tube to close, and failure of neural crest cells to migrate to peripheral structures. Because of their phenotypic similarities with the human condition, the mouse mutants splotch (spina bifida) and loop-tail (craniorachischisis) are accepted models for the study of neural defects in humans. Both mutations map on chromosome 1 (proximal and distal portions, respectively), and behave as semi-dominant traits.
During the past few years, we have used a positional cloning approach to show that the Pax3 gene is mutated in the splotch mouse. Pax3 is a transcription factor that is expressed exclusively during embryogenesis in the dorsal part of the neural tube, where it plays a key role in tissue patterning and development of the neural tube. Subsequently, human Pax3 was also found mutated in Waardenburg syndrome patients, a complex syndrome that shares several phenotypic similarities with the alterations of the splotch mouse (pigmentary defects, craniofacial abnormalities, failure of neural tube to close). Pax3 has two DNA binding domains, a paired domain and a paired-type homeodomain, and analysis of discrete alleles at splotch has shown that the two DNA binding domains strictly cooperate for DNA binding and selection of downstream targets. We have also identified alternatively spliced Pax3 variants with different DNA-binding properties.
The loop-tail mutant has a more profound defect than splotch, with complete failure of the neural tube to initiate closure, resulting in exencephaly and lethality in homozygous embryos. We have completed a high resolution genetic linkage and physical map of the loop tail region, and have cloned the entire region in BAC clones. Transcription mapping by cDNA hybridization and sample sequencing has led to the identification of 11 transcription units in this region, four of which are expressed in neural tissues.