Role of cofilin in glioblastoma cell behaviour

Abstract

The actin cytoskeleton is intimately involved in the mechanisms required for cell motility, being able to reorganise into dynamic structures such as cellular protrusions (lamellipodia) at the front edge of crawling cells. Several intracellular proteins associate with the actin cytoskeleton to change its structure, and this is necessary for a cell to respond to motility-inducing signals such as growth factors. Small actin-binding proteins, which include cofilin, actin-depolymerizing factor (ADF) and profilin, are known to synergise in accelerating actin turnover in moving cells.Dysregulation of cell motility plays a pivotal role in conferring invasive behaviour in tumour cells. It is now evident that tumour progression is often associated with abnormal expression of genes that regulate cytoskeletal assembly, as well as genes involved in cytoskeletal turnover. My project focuses on exploring the role of cofilin in glioblastoma tumours of the brain, which frequently infiltrate into adjacent normal tissue. The hypothesis is that altered levels of cofilin expression in glioblastoma cells affect their motility, which may have a significant impact on invasiveness. Cofilin is ubiquitously expressed in eukaryotes and appears to be crucial to the formation of lamellipodia in a variety of motile cells, including metastatic tumour cells. It is likely that the motility observed in tumour cells might arise from disruptions in the activities of cofilin and other proteins that modify actin dynamics.In vitro studies were performed on the human glioblastoma cell line U373 MG, originally derived from a patient. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the actin-binding proteins cofilin, ADF and profilin are expressed by these cells. Using immunochemistry, the distribution of cofilin was investigated in cells cultured under standard conditions and after serum stimulation. To test whether altered levels of cofilin expression in glioblastoma cells affects cell motility, cofilin was overexpressed in the glioblastoma cell line and changes in the motility of transfected cells were analysed and compared to untransfected cells. Overexpression was achieved by transient and stable transfections with a plasmid vector, pCofilin-IRES2-EGFP, which was constructed by subcloning the coding sequence of human cofilin into pIRES2-EGFP. Transfected cells were identified by the expression of EGFP (enhanced green fluorescent protein) in timelapse experiments using confocal microscopy. In order to quantify the relative levels of cofilin overexpression in stable transfectants, cells were immunostained for cofilin using fluorescent detection and analysed by flow cytometry. Western blots confirmed the specificity of the anti-cofilin primary antibody used. The timelapse analyses indicated that overexpression of cofilin increases the motility of glioblastoma cellsThe project was extended to investigate whether variable levels of cofilin overexpression might affect cell motility to different extents. An inducible gene expression system based on the Tet-Off system (Clontech laboratories Inc.) was developed in order to control the level of cofilin overexpression. The coding sequence for cofilin-IRES2-EGFP was subcloned into pTRE, so that cells with inducible expression of cofilin could be identified by green fluorescence. Stable cell lines potentially responsive to the effects of tetracycline or doxycycline antibiotics were cultured and flow sorted to select green fluorescent cells. This system would enable the correlation between cofilin expression and motility to be examined over a wide range of overexpression.