The objective is to improve our understanding of (1) how errors in the segregation of chromosomes occur in mammalian cells, thus leading to aneuploidy, and (2) how segregational errors are caused by chemicals.
The chemicals of interest in this project include: chemotherapeutic agents, such as estramustine and dicarbazine; compounds of environmental relevance, such as vanadium salts and carbamate pesticides; alkylated biomolecules, whose study may shed some light on the mechanisms of action of the vast class of alkylating agents. The capacity of the chemicals to include aneuploidy is studied, when necessary, by in vitro tests, including conventional metaphase chromosome counting, fluorescence in situ hybridization (FISH) with centromeric DNA probes in interphase nuclei, and FISH or indirect immunofluorescence with antikinetochore antibodies in micronuclei. Various approaches are used to monitor the ability of chemicals to interfere with mitotic structures or to disturb specific pre-mitotic or mitotic events. High resolution video microscopy is used to monitor progression through mitosis of mammalian cell lines exposed to the chemicals of interest. This advanced technology allows detection of effects on early mitotic events, such as aster formation and migration and microtubule capturing by kinetochores, on chromosome movement both away from and toward the poles, and on late events, such as chromosome splitting at the onset of anaphase and spindle elongation. Confocal immunofluorescence and electron microscopy are used to clarify or confirm the effect of chemicals on specific steps of mitosis. An in vitro tubulin polymerization assay is used to study the effect of chemicals on the assembly/disassembly of microtubules. Progression through mitosis involves important changes in the state of phosphorylation of a number of cellular proteins. Interference of chemicals with protein phosphorylation is studied by assessing the phosphorylated state of indicator proteins (e.g. ribosomal protein S6) or of microtubule-associated proteins (e.g. MAP1A, associated to kinetochore).
Funding SchemeCSC - Cost-sharing contracts