Kinetics Analyses Of Interactions Between Oncogenic And Tumor Suppressor Proteins By Quantitative High Resolution Microscopy Methods

Abstract

DAB2IP is correlated to the risk of aggressive prostate cancer and loss of DAB2IP not only leads to epithelial-to-mesenchymal transition (EMT), but also makes cells radioresistant. These observations support the notion that improved knowledge of DAB2IP involvement in cellular pathways could be of help to cancer therapy. The goal of this work was to enable the study of how DAB2IP and some of its known key molecular partners interact in the cell by a combination of molecular biology techniques and recently reported quantitative fluorescence microscopy methods. The first part of this work was dedicated on investigating the interaction of DAB2IP with the androgen receptor (AR). AR functions as a transcriptional factor, but how DAB2IP influences AR transcriptional activity remains unclear. In this work, the results revealed that AR transcriptional activity can be inhibited by DAB2IP by preventing AR nuclear translocation. Furthermore, AR nucleocytoplasmic kinetics was shown to be associated with the level of Dihydrotestosterone (DHT) by use of Number & Brightness (N&B) analysis that measures molecular concentration. The second part of this work was dedicated on investigating changes in the kinetics of the DBA2IP protein after exposure to ionizing irradiation (IR). Whether DAB2IP was involved in DNA repair pathways remains unknown. Our results showed that DAB2IP can repress radiation-induced autophagy to prevent cancer cells from death. DAB2IP in combination with NU7441, a DNA-PKcs inhibitor, was verified to enhance the radiation effect by suppressing autophagy. Furthermore, upon radiation treatment co-localization of DAB2IP and ãH2AX, a DSB marker, was not observed. Also, DAB2IP foci in PC-3 and C4-2 D2 cells could not be measured post IR-induction. Although DAB2IP may not directly relocate at DNA DSB sites, post radiation-induction of DAB2IP-EGFP nuclear levels were gradually seen to increase with time. The last part of this work focused on the development of novel quantitative microscopy method to improve quantification of kinetics of proteins related to DAB2IP. The method relies on the use of photoactivatable GFP (PA-GFP) to enable control of the fluorescent protein concentration in the cell to improve the quantification of molecular diffusion coefficients by Raster Image Correlation Spectroscopy (RICS). The results showed that the accuracy of measuring the PA-GFP diffusion coefficient (23.5 µm2/s) was 30% higher than with standard EGFP expression at any time post-transfection. Subsequently, this method was applied to quantifying the kinetics of PA-GFP tagged Skp2, a protein that regulates the degradation of DAB2IP. The free diffusion coefficient of PA-GFP-Skp2 (16.8 µm2/s) could be attained at the estimated value, which was 40-60% higher than the value obtained for EGFP-Skp2 at any time post-transfection, because this method could reduce the contaminating contribution from slowly moving or immobile proteins to the RICS data. The combination of quantitative fluorescence microscopy methods with photoactivation could be used in the future play for the improved quantification of the kinetics of other cellular proteins of interest.