Nonenzymatic extraction of cells from clinical tumor material for analysis of gene expression by two-dimensional polyacrylamide gel electrophoresis.

We have compared different methods of preparation of malignant cells for two-dimensional electrophoresis (2-DE). We found all methods using fresh tissue to be superior compared to methods using frozen tissue. Our results indicate that nonenzymatic methods of preparation of tumor cells, including fine needle aspiration, scraping and squeezing, have advantages over methods using enzymatic extraction of cells. Nonenzymatic methods are rapid, appear to reduce loss of high molecular protein species, and alleviate the necessity of separating viable and nonviable cells by Percoll gradient centrifugation. Using these techniques, high-quality 2-DE maps were derived from tumors of the lung and breast. In the resulting polypeptide patterns, heat shock proteins, non-muscle tropomyosins and intermediate filament were identified. We conclude that nonenzymatic extraction of malignant cells from fresh tumor tissue improves the possibilities that these techniques may be useful in clinical diagnosis.