Core A will provide endpoint analyses for experiments involving apoptosis in both cell culture systems and in animal and human tissues. Apoptosis analyses for cell culture will include bright field examination (LM) using Romanowsky-type dyes or polychrome stains and transmission electron microscopy (TEM). Apoptosis will be quantitated in animal and human colonic specimens using light microscopy. The core will be responsible for maintaining and testing flat mucosa from colon resections and from colonoscopies (from cancer patients, adenoma patients, and """"""""normal"""""""" patients). Tests would include measurements of apoptosis and specific proteins of interest to the project directors. We will use fluorochrome tags on secondary antibodies to detect specific proteins in conjunction with Leica Laser Scanning Confocal Microscopy (LSCM), or produce permanent slides using immunohistochemical procedures. By these approaches we will identify and quantify gene products of significance to the three projects, including proteins which may prove valuable as biomarkers of colon cancer risk (e.g. Hsp70, Cox-2, Gadd153, and others. Quality control studies will be performed to measure expression of potential biomarkers in each of the different regions of the colon of normal individuals as a basis for identifying abnormal expression in high risk individuals. The core will also measure expression of potential biomarkers in the normal appearing mucosa of cancer patients at various distances from the cancer. The core will establish, maintain, subclone and test apoptosis resistant colonic epithelial cell lines for altered gene expression. Cellular effects of chemopreventive agents, of other drugs and of transfections will be evaluated by microscopic methods applicable to the particular experimental situation. Procedures to be made available are ultrastructural cytochemistry (including cryosectioning), fluorescent apoptosis/necrosis assay, bright-field examination of cytospin preparations, semi-thin plastic sectioning, and paraffin embedding. The core will also assist in developing special microscopic procedures to answer specific questions that emerge as experimental data are obtained and analyzed. Two experienced research specialists will be available in the core to provide this assistance.
Showing the most recent 10 out of 88 publications
