Two-dimensional gel electrophoretic analysis of the changes after immortalization of human cells: decrease of intracellular alpha-2-macroglobulin fragment.

To study the mechanisms of immortalization of human cells, an early step in cancer development, we compared the cellular proteins of normal and immortalized human fibroblasts. Two-dimensional gel electrophoresis showed that one spot with a molecular mass 20 kDa and an isoelectric point of 6.0, became significantly smaller after immortalization of human cells. Further, the spot was rarely observed in four human liver cancer cell lines. Investigation of the N-terminal amino acids revealed that the spot was a fragment of alpha-2-macroglobulin. Although the 20 kDa fragment contains methionine, the spot was not labeled with [35S]methionine. Thus we concluded that the spot might be derived from the culture medium. These results indicated that intracellular metabolism of a-2-macroglobulin, which is a multifunctional protease inhibitor, changed after the cells were transformed.