The Localization and Mobility of MSH2 in Cells Undergoing Neoplastic Transformation

Abstract

Genome instability is an underlying factor of all cancers. Defects in DNA repair mechanisms contribute significantly to this factor. Recent reports indicate the mechanical properties of cells change as they advance through different stages of neoplastic transformation. The main goal of this study is to investigate how changes in the intracellular environment and changes in DNA repair protein distribution are altered in cells undergoing neoplastic transformation. For this study we have chosen to use human mammary epithelial (HME) cells from the Weinberg line. Lipofection was used to transiently transfect enhanced green fluorescent protein (egfp) fusions to MSH2 mismatch repair proteins. We have measured the localization and diffusion of these fusions using fluorescence recovery after photobleaching (FRAP) and raster image correlation spectroscopy (RICS) on live cells maintained under appropriate environmental conditions. We have tested normal, immortal, and tumorigenic cells in this study. The acquired data was analyzed using custom written Matlab programs as well as commercially available software. Preliminary results show localization of the fusions to the nucleolus in all cell types tested. The surrounding nucleus generally has the weakest localization signal. We have also observed smaller diffusion coefficients in the nucleus of tumorigenic cells compared to normal cells. This work is supported by NSF Materials and Surface Engineering grant CMMI-1152781.