Visualization of nuclear targeting of breast cancer cell lines. [and] Quantification and detection of matrilysin produced by five cancer cell lines

Abstract

The collection of visual evidence of M13 bacteriophage displaying a nuclear localization heptapeptide localizing to the nuclei of breast cancer cells is described in this thesis. The bacteriophages displaying the heptapeptide were conjugated to a fluorochrome prior to incubation with live cells. After the incubation, the cells were fixed and cell nuclei were stained, and a fluorescent microscope was used to visualize and obtain photographs of the cells. Fluorochrome-labeled bacteriophages not displaying the heptapeptide showed no localization to cell nuclei, but fluorochrome-labeled bacteriophages displaying the heptapeptide exhibited peripheral nuclear binding. The second portion of this thesis describes an attempt to measure the amount of the metalloproteinase matrilysin produced by five cancer cell lines by analyzing supernantants taken at regular time points over a 96-hour interval. An ELISA using antibodies specific to matrilysin was performed initially, followed by an assay based on the cleavage activity of the matrilysin enzyme. The ELISA results indicated that the cell lines produced an undetectable level of matrilysin, while the activity assay indicated elevated production by all five of the cell lines. Diagnostic experiments were conducted to determine the cause of the substrate cleavage. It was determined that enzymes present in the samples other than metalloproteinases were cleaving the substrate. Efforts to isolate Cathepsin D, an aspartic protease which was not inhibited during the assays, detected its production by three of the five cell lines.