A MOUSE FIBROBLAST LINE CYCLES BETWEEN MONOLAYER AND SPHEROID FORMS, REGULATES MET AND HGF EXPRESSION, AND RELEASES AN ATTACHMENT AND GROWTH-PROMOTING SUBSTANCE

Authors

  • Douglas O. Halverson,

    1. Intramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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  • James Resau,

    1. ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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  • Donna Faletto,

    1. ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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  • Robert Fisher,

    1. Intramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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  • Miriam Anver,

    1. Pathology Histotechnology Laboratory, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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  • Sing Rong,

    1. ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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  • Jo Ann May,

    1. Pathology Histotechnology Laboratory, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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  • Ilan Tsarfaty,

    1. ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD, 21702-1201, U.S.A.
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    • Tel Aviv University, Sackler School of Medicine, Dept of Human Microbiology, Tel Aviv, Israel.

  • Donald G. Blair

    Corresponding author
    1. Basic Research Laboratory, National Cancer Institute, P.O. Box B, Frederick, MD, 21702-1201, U.S.A.
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To whom correspondence should be addressed: Tel: (301) 846-1318; Fax: (301) 846-6164; blair@ncifcrf.gov

Abstract

A subline of mesoderm-derived mouse NIH3T3 fibroblasts was selected for its ability to proliferate in serum-free media. This cell line (SFDH) grows as a monolayer at low density and spontaneously forms dense, multicellular spheroids at high density. Spheroid formation can also be induced by the addition of dexamethasone, polybrene, or heparin. Spheroids eventually detach from the substrate, but will reattach and re-form monolayers when transferred to fresh culture vessels and media, repeating the cycle again upon reaching high density. Thin section analysis of spheroids shows morphologically-distinct regions of cells, including an attenuated outer surface and a cuboidal interior with occasional lumen-like areas. Over time in culture, spheroids express increasing levels of met, the Met ligand-SF/HGF and cytokeratin, an epithelial marker, in comparison to monolayers. Both monolayer and spheroid-derived cells are rapidly tumorigenic in nude mice. Media conditioned by SFDH cells contain factors that stimulate growth and attachment of a variety of tumorigenic and non-tumorigenic cell lines, inducing cells to divide in serum-free media for up to 14 days when plated on tissue culture-treated and nontreated plastic surfaces pre-coated with SFDH conditional media. The growth-stimulating activity fractionates as a single peak over a sepharose column in the presence of 6m urea, and sediments as a high molecular weight complex. Growth-stimulating activity can be neutralized by several antisera specific for hepatocyte growth factor, and the same sera recognize a novel ∼37kD protein in active supernatants. The cyclic, continuous nature of alternating monolayer and spheroid forms makes this cell line appropriate for studying changing gene expression patterns in progressive cell—cell/cell—matrix interactions.

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