CATHEPSINS B AND L IN MALIGNANT PROGRESSION O MCF-10

溶酶体半胱氨酸蛋白酶组织蛋白B和L被牵连 在恶性进展中。表达增加，膜结合和 在转化的成纤维细胞中观察到组织蛋白酶B和L的释放 在恶性小鼠和人类肿瘤中，包括人类乳腺肿瘤。这个 组织蛋白酶B和L基因表达增加与转运改变的关系 反映正常途径中不止一个步骤的修改 导致它们递送到溶酶体(即， 转录/翻译、翻译后处理和/或在 细胞内分选和定向)。多个机制似乎是 负责组织蛋白酶B的分泌和L既是前体 表格和成熟的表格被发布。另外一个因素是，这两个 在正常细胞中，酶似乎至少由一种类似的 一条路径和一条不同的路径。在本提案中，我们将分析 人类乳腺恶性表型发展的步骤 上皮细胞及其与组织蛋白B和L运输改变的联系。 在这些研究中，我们将使用最近培育的近二倍体人类 乳腺上皮细胞系，MCF-10和MCF-10变异体，代表 人类乳房恶变的一些初始步骤 上皮细胞(如永生化、侵袭性) 激活的RAS)。具体目标包括：1)表情分析， 组织蛋白酶B和L的亚细胞分布和定位；2)释放 组织蛋白酶B和L的前体和成熟形式；3)炮制 组织蛋白酶B和L的脉搏研究；4)正常组分 运输溶酶体酶以检测潜力的途径 可能影响组织蛋白酶B和L向溶酶体输送的变化。 我们还将对MCF-10系列进行形态评估 包括：5)扫描电子显微镜下的表面构型 6)透射电子显微镜观察其超微结构。这个 超微结构研究将为脉冲追逐提供补充 组织蛋白酶B和L的炮制工艺研究 免疫细胞化学定位成熟(即完全加工)和 组织蛋白酶B和L对特定囊泡种群的前体形式 在细胞内。 我们的总体假设是，在排序和定位方面的变化 肿瘤中溶酶体半胱氨酸蛋白酶导致递送 组织蛋白酶B和L对细胞外周和分泌中的小囊泡的作用 这些酶中。在肿瘤细胞侵袭过程中，当肿瘤细胞附着在 基底膜，局部释放可由多种因素触发 机械装置。一旦分泌，半胱氨酸蛋白酶可以降解基底物 通过激活其他蛋白水解物直接或间接地激活细胞膜 酶，从而促进肿瘤细胞的侵袭。

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 include 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.