REGULATION OF EXTRACELLULAR MATRIX BIOSYNTHESIS

我们的目标是了解调节 细胞外基质蛋白的生物合成。 重大变化 已知基质的组成发生在发育期间， 恶性转化，在激素的影响下，在遗传和 获得性疾病和衰老。 细胞外基质成分，和 特别是纤连蛋白，在细胞粘附和细胞粘附中起主要作用， 迁移 毫无疑问，细胞粘附的问题是 与癌症过程的生物学相关， 调节细胞外基质生物合成的机制将有助于 了解侵袭和转移。 许多意见认为， 恶性细胞的纤连蛋白水平降低， 风景 有大量的信息是关于结构的， 细胞外基质糖蛋白，并取得了迅速进展 最近在克隆和分析它们的结构基因方面取得了进展。 然而，在这方面， 关于调节其生物合成的机制知之甚少。 的 我们提出的方法结合了体细胞遗传学和分子生物学。 我们将分离和表征细胞系变异， 细胞外基质蛋白的合成和/或产生。 这 建议是基于初步结果显示，选择改变 结果显示，HT1080纤维肉瘤变异株产生纤连蛋白， 降低或提高利率。 选择的各种条件改变 将使用隔离器来隔离不同类型的 变体。 直接选择产生改变量的 基质蛋白将在荧光激活的细胞中进行 分拣员。 这些变体将根据它们的附着特性进行表征 分泌的基质蛋白质的性质及其生产率 介质，存在于细胞层和基质附着材料中。 可用的重组DNA克隆将用于测量蓄积 和编码基质蛋白的mRNA的合成速率。 特定基因 将用克隆序列探测编码基质蛋白， 它们的甲基化状态、核酸酶敏感性和 放大 最后，适当选择的控制区域， 基因将被测序。

Our objective is to understand fundamental mechanisms that regulate the biosynthesis of extracellular matrix proteins. Major changes in composition of matrices are known to occur during development, upon malignant transformation, under the influence of hormones, in genetic and acquired diseases and upon aging. Extracellular matrix components, and fibronectin in particular, play a major role in cell adhesion and cell migration. There is little doubt that the problem of cell adhesion is relevant to the biology of the cancer process, and that knowledge of the mechanisms that regulate extracellular matrix biosynthesis will help in understanding invasion and metastasis. Numerous observations that malignant cells have reduced levels of fibronectin strongly support that view. A considerable amount of information is available about the structure of extracellular matrix glycoproteins, and rapid progress has been made recently in the cloning and analysis of their structural genes. However, little is known about the mechanisms that regulate their biosynthesis. The approach we propose combines somatic cell genetics and molecular biology. We will isolate and characterize cell line variants with altered adhesiveness and/or production of extracellular matrix proteins. This proposal is based on preliminary results showing that selection for altered adhesiveness yielded HT1080 fibrosarcoma variants producing fibronectin at reduced or increased rates. Various conditions of selection for altered adhesiveness will be used to favor the isolation of different types of variants. Direct selection for variants producing altered amounts of matrix proteins will be carried out in a fluorescence activated cell sorter. The variants will be characterized in terms of their attachment properties and of their rate of production of matrix proteins secreted in the medium, present in the cell layer and in substrate-attached material. Available recombinant DNA clones will be used to measure the accumulation and rate of synthesis of mRNAs encoding matrix proteins. Specific genes encoding matrix proteins will be probed with cloned sequences to assess their state of methylation, nuclease sensitivity and state of amplification. Eventually, appropriately chosen control regions of these genes will be sequenced.