REGULATION OF EXTRACELLULAR MATRIX BIOSYNTHESIS

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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.