CATHEPSIN-D, CATHEPSIN-B, AND CATHEPSIN-L IN TRANSFORMED HUMAN BREASTEPITHELIAL-CELLS

To investigate the regulation of lysosomal enzymes during carcinogenes is, we measured cathepsins (Cats) D, B, and L in MCF-10F, which is a h uman breast epithelial cell line, and cells evolved after treatment wi th carcinogen and transfected with c-Ha-rns oncogene. The clones used in this study, MCF-10FT ras, D3, D3-1, and D3-1Tras, expressed no estr ogen receptors and gradually increased invasive potential, while oncog ene-transfected lines were also tumorigenic in SCID mice [16, 19]. Cat s D, B, and L were determined in the cells and in cell media using enz yme-linked immunosorbent assay (ELISA), specific enzyme activity measu rements, and immunocytochemistry. The major intra- and extracellular l ysosomal proteinase in these cells was Cat D (30-180 pm/mg), followed by Cat B (2-10 pm/mg) and Cat L (1-5 pm/mg). An inverse relationship b etween intracellular Cat D levels and invasive potential of carcinogen -treated and c-Ha-ras oncogene-transfected cell lines was observed. No significant changes in extracellular concentration of Cat D precursor in this series of cell lines was observed. Intracellular levels of Ca ts B and L were unchanged or slightly lower in carcinogen-treated D3 a nd D3-1 cells, as well as in MCF-10FTras. On the other hand, in D3-1Tr as cell line, evolving from c-Ha-ras transfected D3-1 line, 3.5 fold a nd 4.4 fold increases in Cat B and Cat L, respectively, but a 2 fold d ecrease in Cat D, were observed compared to the parental cell line. Im munocytochemical staining showed a granular, polarized perinuclear and cytoplasmic staining of cathepsins in all cell lines. Cysteine protei nases stained more frequently and more intensely in D3-1Tras compared to other lines, confirming the immunochemical assays. We hypothesize t hat several molecular events, caused by a carcinogen and an oncogene s uch as c-Ha-ras, are needed to increase Cat B and Cat L, but not Cat D , expression. Therefore, the cysteine and aspartic lysosomal proteinas es are differentially expressed in the breast cell lines with more inv asive phenotype.