STANDARDIZED CHARACTERIZATION OF GENE-EXPRESSION IN HUMAN COLORECTAL EPITHELIUM BY 2-DIMENSIONAL ELECTROPHORESIS

New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein lev el. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed und er altered conditions, and a two-dimensional reference protein map of the normal human colonic epithelium. Normal colonic mucosa, primary tu mors and liver metastases were prepared in the operating room. After w ashing in an ice-cold medium containing protease inhibitors, crypts we re isolated by mechanical preparation without using metalloproteinases . Epithelial cells were then selected using Ber-EP4 Dynabeads. The sam ples were denaturated before processing for immobilized pH gradient tw o-dimensional polyacrylamide gel electrophoresis according to SWISS-2D PAGE standards, The samples contained more than 95% epithelial cells a s confirmed by fluorescence-activated cell sorting using pan-anticytok eratin antibodies. Cell surfaces were not damaged, as assessed by scan ning electronic microscope. A protein reference map of the normal colo nic epithelium was defined. Using gel matching, N-terminal sequencing and/or immunoblotting techniques, 60 polypeptides - including proteins specifically expressed in colorectal epithelium - have now been ident ified, This reproducible method of sample preparation permits the comp arison of protein patterns found in various pathological states with t he present reference map (http://www.expadsy.ch). Some of these patter ns might provide diagnostic or prognostic markers, or even molecular t argets for therapy in the future.