Investigation into how mitotic spindle checkpoint function is compromised by Sv40 large T antigen
The viral oncoprotein Simian virus 40 large T antigen (LT) efficiently immortalises primary rodent cells and occasionally transforms them to tumourigenicity. It has long been known that LT can cause genomic instability and induce aneuploidy, and that it disrupts the mitotic spindle checkpoint, involved in monitoring the segregation of sister chromatids. LT has recently been shown to interact with the spindle checkpoint protein Bubl. This thesis describes research into the effects of LT on spindle checkpoint function and examines the possibility that interaction of LT with Bubl is responsible. Novel monoclonal antibodies against Bubl were developed to aid this research. The interaction site of Bubl was mapped to amino acids 89-97 of LT using co-immunoprecipitation experiments in several cell lines, each expressing LT with a different mutation in the amino-terminal region. The non-Bub 1-binding (dl89-97) mutant was capable of immortalising rat embryo fibroblasts as efficiently as wild- type LT, but was defective for focus formation, perhaps suggesting a role for Bubl in the mechanism for transformation by LT. In the presence of the microtubule-depolymerising drug nocodazole, the spindle checkpoint is activated and cells arrest in mitosis. LT was shown to compromise spindle checkpoint function, allowing some cells to pass through to the next cell cycle even in the presence of nocodazole. However, without Bubl interaction, LT loses this ability and the spindle checkpoint is robust. An investigation of the checkpoint proteins and complexes present during spindle checkpoint activation in cells expressing wild-type or dl89-97 LT was initiated to analyse possible mechanisms for perturbation of the spindle checkpoint by LT. This study raises the possibility that LT induces genetic instability in mammalian cells by perturbation of spindle checkpoint function, and that this may be a critical component of the mechanism by which LT transforms cells.