Establishment of A Functional Hgf/C– Met Autocrine Loop in Spontaneous Transformants of Wb–F344 Rat Liver Stem–Like Cells

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Abstract
A model of spontaneous malignant transformation was used to evaluate the molecular changes that take place in WB–F344 rat liver epithelial cells during neoplastic transformation and tumorigenesis. A comparison of wild–type low–passage WB–F344 cells to spontaneously transformed tumor cell lines revealed that the majority of the tumor cell lines have an increased capacity for autonomous proliferation and motility when maintained in serum–free media. In the current study, we show that c–met is expressed at some level in wild–type WB–F344 cells and in all of the spontaneously transformed tumor cell lines, and that 9/16 of the tumor cell lines have acquired hepatocyte growth factor (HGF) expression. In vitro growth of HGF–expressing tumor cell lines is inhibited as much as 68% by the addition of neutralizing antibodies to HGF or antisense HGF oligonucleotides, indicating that the production of HGF by the tumor cells is partially responsible for driving autonomous proliferation in a subset of tumor cell lines. Furthermore, conditioned media collected from HGF–expressing tumor cell lines stimulates DNA synthesis in wild–type WB–F344 cells, and this effect can be abrogated by pre–incubation of the conditioned media with neutralizing antibodies to HGF. Because HGF is a motility–promoting growth factor, all cell lines were evaluated to determine if expression of HGF stimulated motogenesis. All tumor cell lines (regardless of HGF expression) were highly motile in comparison with wild–type WB–F344 cells, with a 3.5–fold to 20–fold greater number of motile cells. The high basal rate of motility characteristic of the tumor cell lines is not a result of the production of HGF, because it is also a property of the cell lines that do not express HGF messenger RNA. Furthermore, tumor cell motility is not inhibited by antisense oligonucleotides or neutralizing antibodies. Establishment of an autocrine HGF/c–met loop in a subset of spontaneously transformed WB–F344 cell lines may influence development and/or expression of the tumorigenic phenotype by driving cellular proliferation.