The lysosomal cysteine proteinases cathepsins B and L have been implicated in malignant progression. Increased expression, membrane association and release of cathepsins B and L are observed in transformed fibroblasts and in malignant murine and human tumors, including human breast tumors. The increase in mRNA and the altered trafficking of cathepsins B and L probably reflect modifications in more than one step in the normal pathway that leads to their delivery to lysosomes (i.e., alterations in transcription/translation, posttranslational processing, and/or in intracellular sorting and targeting). Multiple mechanisms appear to be responsible for the secretion of cathepsins B and L since both precursor forms and mature forms are released. An additional factor is that the two enzymes appear to be trafficked in normal cells by at least one similar pathway and one distinct pathway. In the present proposal, we will analyze the steps in the development of a malignant phenotype in human breast epithelium and their link to altered trafficking of cathepsins B and L. For these studies, we will use a recently developed near-diploid human breast epithelial cell line, the MCF-10, and MCF-10 variants that represent some of the initial steps in malignant progression of human breast epithelium (e.g., immortalization, invasiveness after transfection with activated ras).
The specific aims i nclude the analysis of: 1) expression, subcellular distribution and localization of cathepsins B and L; 2) release of precursor and mature forms of cathepsins B and L; 3) processing of cathepsins B and L by pulse-chase studies; 4) components of the normal pathway for trafficking of lysosomal enzymes in order to detect potential changes that could affect delivery of cathepsins B and L to the lysosomes. We will also perform a morphological assessment of the MCF-10 lines including: 5) their surface architecture by scanning electron microscopy and 6) their ultrastructure by transmission electron microscopy. The ultrastructural studies will provide a complement to the pulse-chase studies of the processing of cathepsins B and L by using immunocytochemistry to localize both mature (i.e., fully processed) and precursor forms of cathepsins B and L to specific vesicular populations within the cell. Our overall hypothesis is that alterations in sorting and targeting of lysosomal cysteine proteinases in tumors result in the delivery of cathepsins B and L to small vesicles at the cell periphery and in secretion of these enzymes. During tumor cell invasion, as tumor cells adhere to the basement membrane, local release could be triggered by a variety of mechanisms. Once secreted, the cysteine proteinases can degrade basement membrane directly or indirectly via activation of other proteolytic enzymes, thus facilitating tumor cell invasion.
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