CATHEPSINS B AND L IN MALIGNANT PROGRESSION O MCF-10

溶酶体半胱氨酸蛋白酶组织蛋白酶B和L与 恶性进展 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， 在恶性鼠和人肿瘤中，包括人乳腺肿瘤。 的 mRNA的增加和组织蛋白酶B和L运输的改变可能是 反映了正常途径中一个以上步骤的修饰， 导致它们被递送到溶酶体（即，的改变 转录/翻译，翻译后加工，和/或 细胞内分选和靶向）。 多种机制似乎 负责组织蛋白酶B和L的分泌， 形式和成熟形式被释放。 另一个因素是， 在正常细胞中，酶似乎通过至少一种类似的 一条路，一条路。 在本报告中，我们将分析 人类乳腺恶性表型发展的步骤 上皮及其与组织蛋白酶B和L的改变的运输的联系。 对于这些研究，我们将使用最近开发的近二倍体人类 乳腺上皮细胞系MCF-10和MCF-10变体， 人类乳腺癌恶性进展的一些最初步骤 上皮（例如，永生化，转染后的侵袭性 激活ras）。 具体目标包括分析：1）表达， 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） 组织蛋白酶B和L的脉冲追踪研究; 4）正常的成分 运输溶酶体酶的途径，以检测潜在的 可能影响组织蛋白酶B和L向溶酶体递送的变化。 我们还将对MCF-10细胞系进行形态学评估 包括：5）通过扫描电子显微镜观察其表面结构 （6）透射电镜观察其超微结构。 的 超微结构研究将为脉冲追踪提供补充 用酶法处理组织蛋白酶B和L的研究 免疫细胞化学定位两个成熟的（即，已完全处理）和 组织蛋白酶B和L的前体形式形成特定的囊泡群 在细胞内。 我们的总体假设是， 肿瘤中的溶酶体半胱氨酸蛋白酶导致 组织蛋白酶B和L在细胞周围和分泌中形成小泡 这些酶。 在肿瘤细胞侵袭过程中，由于肿瘤细胞粘附在 基底膜，局部释放可由多种 机制等 半胱氨酸蛋白酶一旦分泌， 膜直接或间接通过激活其他蛋白水解 酶，从而促进肿瘤细胞侵袭。

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.