Dania Alkhatib
Queen Mary University of London
Title: The effect of ectopically induced vimentin in vimentin deficient cancer cells
Biography
Biography: Dania Alkhatib
Abstract
Vimentin is a type 3 intermediate filament cytoskeletal protein. It is known to be over expressed in cells that undergo epithelial mesenchymal transition and metastasis. In this project, Neo-pLPC-wild type vimentin DNA and Neo-pLPC-empty-no insert- control DNA were retrovirally transduced into MCF7 cells which showed no gene expression of vimentin in a previous study .
Aim: To test whether transducing vimentin in cells with low/no endogenous levels will induce filament formation. Moreover, to screen the level of vimentin expression in 14 different cell lines. In addition, to test whether when transfecting AcGFP N-terminus tagged vimentin MCF7 cells with wild type vimentin DNA, the aggregates formed will rearrange into filaments or not.
Materials and Methods: Western blotting was done to observe the level of vimentin protein expression in MCF7 cells transduced with wild type vimentin. QPCR was done for different cell lines to investigate the levels of vimentin gene expression. Immunofluorescence was done to demonstrate vimentin expression in MCF7 transduced with vimentin. Transfection of ACGFP N-Terminus tagged vimentin transduced MCF7 cells with non-tagged vimentin DNA was done and proceeded with immunofluorescence.
Results: Immunofluorescence showed an increased expression of vimentin filament when using 5mg/ml of G418 drug compared to 1mg/ml for cell selection. Western blot showed expression of vimentin protein in Neo-pLPC-wild type vimentin MCF7 cells. QPCR showed high level of vimentin expression in Neo-pLPC-wild type vimentin transduced MCF7 cells. In addition, HT1080 expressed vimentin the highest and T47D the lowest. Immunofluorescence after transfection showed that most aggregates didn’t dissolve into filaments.
Conclusion: Vimentin was found to be highly expressed in progressive cancer cell lines. Low endogenous levels of Vimentin were detected in MCF7 cells. When transducing MCF7 cells with a wild type vimentin, all experiments showed that vimentin was expressed in these cells with 5 mg/ml being the G418 drug concentration for selection. Transfecting with a wild type vimentin has a little effect on the vimentin aggregates formed in N- terminus tagged vimentin MCF7 cells, as most aggregates didn’t dissolve into filaments.
Future work: To perform experiments using C tagged vimentin transduced cells to study its role in filament assembly. Invasion and migration assays should also take place on these transduced cells, as if a certain part of vimentin, the head or tail, is proved to play a role in migration and invasion, this could be linked clinically to metastasis. Therefore, this part could be targeted and blocked to stop metastasis and so the tumour could be confined in its place while including in mind other factors causing metastasis. As a result, the tumour could be treated more easily and effectively. This could be used as a potential future cancer treatment.