Break

The nuclear shape is a diagnostic and prognostic marker of cancer. The irregular nuclear shape is characterized by blebs, grooves, bulges, concavities in contour, and folds. Currently in our lab, we are conducting a high content screen of chromatin regulators to identify their influence on nuclear shape and lamin texture. 615 genes were silenced using a siRNA library transfected into MCF-10A cells (human breast epithelial cells). Images of the nucleus were captured by using a high content epi-fluorescent microscope. The nuclear shape was measured using an elliptical Fourier analysis and taking the ratio of the size of the first harmonic ellipse to the summation of the next N harmonic ellipses. After conducting the gene screen, we identified multiple genes, that when silenced, increased the irregularity of the nucleus. Conversely, there were genes identified which gave the cells a more circular/ellipsoidal shape which is indicative of increased regularity. This work was supplemented by conducting a high-content screen of epigenetic pharmacological agents to identify other epigenetic modulators effect on nuclear shape. We wanted to know if certain drugs can cross validate results from the gene screen. This was tested by two main questions: can we mimic the phenotype of irregular nuclear shape observed in our MDA-MB-231 (human breast adenocarcinoma) in the MCF-10A cells, and can we “normalize” the irregular nuclear shape in MDA-MB-231 cells? To test this we treated the cells with the 15 different drugs and one control for seven days and imaged them using confocal microscopy. The nuclear shape will be measured using the same method as the gene screen. Based off of current literature we hope to identify drugs which decrease the amount of heterochromatin and/or increase the euchromatin in a cell which will make the nuclear shape more irregular. Conversely, we hope to identify drugs which increase the amount of heterochromatin and/or decrease the euchromatin which “normalizes” the nuclear shape in a cell.