Cell context-dependent differences in interaction with Shiga toxin
|Institution:||University of Oslo|
|Full text PDF:||http://urn.nb.no/URN:NBN:no-29100
Cells that grow in a restricted area will eventually become confluent. At that point normal cells will stop dividing, while cancer cells, with their invasive properties, will continue dividing. It has been known for quite some time that certain cellular mechanisms such as rates of endocytosis might be affected by this change in cell density, as cell populations go from being subconfluent to being confluent. In this master thesis cell density-dependent changes in the expression of Gb3 species, and the ability of the cells to bind and internalize Shiga toxin (Stx) was measured. The low density cell populations bind and internalize 3-4 times more toxin per cell than high density cells. The difference in cell density-dependent sensitivity to Stx is even more dramatic, with a 9-fold higher sensitivity in low than in high density cells, suggesting a more efficient sorting of toxin towards the ER in the low density cells. However, this cell density-dependent change does not affect the protein levels of sorting nexin 1, a key component of retrograde transport for Stx. To elucidate if the Stx receptor level was responsible for increased binding and uptake of the toxin in low density cells, the globotriaosylceramide (Gb3) levels were investigated. High performance thin-layer chromatography analysis showed that low density cells have higher Gb3 levels for both C16 and C24 species compared to high density cells, indicating a mechanism for the cell density-dependent change of Stx binding. Butyric acid treatment of the cancer cells HeLa and SW480 resulted in increased levels of Gb3, for both C24 and C16 species. In HeLa cells there was also a shift in species distribution after butyric acid treatment, with a higher increase in C16 compared to C24 species. The implication of these changes for cellular sensitivity to Stx is discussed.