Detection and characterization of low abundance cellular proteins that specifically increase upon loss of the...

Human epidermoid carcinoma (HEp-3) cells are highly tumorigenic and metastatic in vivo, but their metastatic phenotype is progressively and uniquely lost upon serial passage in vitro. The nonmetastatic phenotype is fully reversible to the highly metastatic state when HEp-3 cells are passaged back in vivo. To study the complex process of metastasis and its possible negative regulation by specific gene products, the expression of specific proteins between the highly metastatic and nonmetastatic HEp-3 cells was investigated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and subsequent computer assisted analyses. Concomitant with the in vitro loss of metastatic potential of HEp-3 cells is the elevated expression of a subset of low abundance proteins detectable in 2D-PAGE but not apparent in high resolution one dimensional PAGE. When the HEp-3 cells revert to the metastatic state, the expression of these proteins declines. The increased cellular abundance of four distinct proteins directly correlates with the loss of the metastatic phenotype: two of the four proteins are associated with isolated cellular membranes (36kD, pI 5.7; 22kDa, pI 5.6), one protein fractionates with the cytoplasm (65kD, pI 6.2), and one protein is enriched in the nuclei fraction (32kD, pI 5.8). These data indicate that computer-assisted analysis of highly sensitive, large-format, 2D-PAGE can be used to identify specific proteins in subcellular compartments that are candidates for negative regulators of the metastatic process.