From qualitative to quantitative data in molecular biology: imaging analysis in a breast cancer study

STEVEN REMENAPP; ALEJANDRA C VENTURA; MEI WU; ZHIFEN WU; SOFIA D MERAJVER

Ann Arbor, USA

Simposio; UROP Spring Research Symposium; 2007

University of Michigan, UROP (Undergraduate Research Opportunity Program)

The most lethal form of breast cancer, called inflammatory breast cancer (IBC), has shown a connection between its aggressiveness and increased levels of the RhoC GTPase, a protein that plays a role in the process of metastasis. As with other small GTPases, RhoC has two states, active and inactive, and only in the active state can RhoC recognize target proteins and generate a response (movement, invasion, etc.).  Our lab is working towards a complete and dynamic characterization of the RhoC on-off cycle to understand its deregulation in cancer cells in comparison with normal tissues. Western blot and activation assays in an IBC cell line have been performed, showing a significant increase of RhoC expression and activation quickly after stimulation by lysophosphatidic acid (LPA, a component of serum and a known regulator of RhoC activation).   The aforementioned results are being validated with a different approach: live-cell imaging and quantitative microscopy. A yellow fluorescent tag has been added to RhoC (RhoC-YFP) by transient transfection in order to quantify total RhoC levels. FRET (fluorescence resonance energy transfer) experiments will be done to quantify RhoC activation levels.  The current project focuses on imaging analysis of the data from our live-cell experiments.  Our work in this direction includes focus drift correction of the acquired data, identification and characterization of single cells for analysis, background correction and ratiometric analysis.  The quantitative data generated will later be correlated with mathematical models that mimic RhoC dynamics. A major impact of this work is to quantitatively predict the effects of drugs targeted against RhoC in cancer.