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。的重大变化 矩阵的组成已知发生在发育过程中，在 恶性转化，在激素的影响下，遗传和 获得性疾病和衰老。细胞外基质成分，以及 尤其是纤维连接蛋白，在细胞黏附和细胞内起主要作用。 迁移。毫无疑问，细胞黏附的问题是 与癌症过程的生物学相关，以及对 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。无数的观察结果表明 恶性细胞的纤维连接蛋白水平降低有力地支持了这一点。 查看。 关于结构的大量信息是可用的 细胞外基质糖蛋白，并取得了快速进展 最近在克隆和分析它们的结构基因。然而， 人们对其生物合成的调控机制知之甚少。这个 我们提出的方法结合了体细胞遗传学和分子生物学。 我们将分离和鉴定具有改变的细胞系变种 细胞外基质蛋白的粘附性和/或产生。这 建议是基于初步结果显示，选择更改 粘附性产生HT1080纤维肉瘤变异体产生纤维连接蛋白 降低或提高税率。选择变更的各种条件 粘附性将被用于有利于分离不同类型的 变种。直接选择产生改变数量的变异体 基质蛋白将在荧光激活的细胞中进行 分拣机。变种将根据它们的依附性来表征 分泌的基质蛋白的性质及其产生率 存在于细胞层和附着在衬底材料中的介质。 可用的重组DNA克隆将被用来测量积累 和编码基质蛋白的mRNAs的合成速度。特定基因 编码基质蛋白将用克隆序列进行探测，以评估 它们的甲基化状态、核酸酶敏感性和 放大。最终，适当选择这些区域的控制区 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.