LOSS OF THE TUMORIGENIC PHENOTYPE WITH IN-VITRO, BUT NOT IN-VIVO, PASSAGING OF A NOVEL SERIES OF HUMAN BRONCHIAL EPITHELIAL-CELL LINES - POSSIBLE ROLE OF AN ALPHA-5 BETA-1-INTEGRIN-FIBRONECTIN INTERACTION/

We established an immortalized, nontumorigenic human bronchial epithel ial cell line by transfection with the origin of replication-defective SV40 large T plasmid. This line spontaneously became tumorigenic at p assage 184 (NL20T), although subsequent passages (passages 189, 200, a nd 205) failed to form tumors. The tumorigenic cell line NL20T was rei noculated back into athymic nude mice, and the two subsequently derive d cell lines (NL20T-A and NL20T-B) have been passaged 85 times in vitr o and remain tumorigenic However, late-passage NL20T cells consistentl y lose their tumorigenicity when passaged in vitro on tissue culture p lastic dishes (NL20T-n cells). Thus, two of the cell lines, NL20 and N L20T, reverted to the nontumorigenic phenotype reproducibly and sponta neously following serial passage on plastic tissue culture plates, whe reas cells passaged in mice (NL20T-A and -B) did not. We used these no ntumorigenic (NL20 and NL20T-n) and tumorigenic (early passage NL20T, NL20T-A, and NL20T-B) cells to study the role of the alpha 5/beta 1-in tegrin and attachment to fibronectin in tumorigenicity. The two nontum origenic cell lines (NL20 and NL20T-n) attached slower to fibronectin- coated plates than the two tumorigenic cell lines in a cellular-extrac ellular matrix adhesion assay, Attachment was abrogated by exposure to a blocking antibody to the alpha 5/beta 1-integrin, the fibronectin r eceptor, in the two tumorigenic cell lines, Cell surface expression of the alpha 5/beta 1 cell surface protein by flow cytometry was highest in the tumorigenic NL20T and NL20T-A cells. NL20T-A cells were cultur ed with an antibody to alpha 5/beta 1 and inoculated s.c. into athymic nude mice; tumorigenicity of the NL20T-A cells was inhibited in a dos e-dependent manner, Tumorigenicity was also inhibited partially with m onoclonal antibodies to either alpha 5 or beta 1. A mixture of 10% tum origenic NL20T-A cells and 90% nontumorigenic NL20 cells was cultured on plastic, type IV collagen, laminin, and fibronectin for 9 weeks, On ly cells cultured on fibronectin formed tumors when inoculated s.c. in to athymic nude mice. We conclude that these data are consistent with the hypothesis that neoplastic transformation in our original cell lin e arose from irt vivo selection of a small mutant clone, which had ari sen in culture and was selected subsequently in vivo but was lost with in vitro culture in NL20 cells, and that alpha 5/beta 1-integrin inte raction with the extracellular matrix may play a role in tumorigenicit y in our system.