Abstract

Background
We have previously shown that CRC patients expressing the nuclear envelope protein lamin A/C are twice as likely to die of CRC related causes compared to clinicopathologically identical patients [1]. Furthermore, over-expression of lamin A in CRC cell lines promotes increased cell motility [1]. The aim of my PhD project is to identify cytoskeletal proteins that regulate invasiveness in CRC cells and to assess their potential uses as prognostic biomarkers for CRC.

Method
Biochemical fractionation was used to isolate the nucleo- and cytoskeletons from CRC cells containing either EGFP-lamin A (SW480/lamA) or EGFP as a control (SW480/cntl), in a sequential extraction that produced four insoluble pellets and four soluble supernatants. Immunoblotting was used to detect the presence of nucleo/cytoskeletal proteins in the pellet and supernatant fractions and to assess differences in protein expression at different cell densities.

Results
Immunoblots demonstrated that the nucleo/cytoskeletal proteins keratin 18, α-tubulin, lamin A/C and β-actin were still present in the fourth pellet (P4) and that the membrane-bound protein vinculin had been removed. Pellets were tested for reproducibility in order to carry out downstream proteomic analysis. 2-D gel conditions were optimised and initial expression differences assessed.

Future work: P4 will be used in 2D difference gel electrophoresis (2D DIGE) analysis to compare expression levels of nucleo- and cytoskeletal proteins in cells with high and low levels of lamin A.