CHANGES IN RESPONSIVENESS OF RAT TRACHEAL EPITHELIAL-CELLS TO TRANSFORMING GROWTH FACTOR-BETA-1 WITH TIME IN CULTURE

Primary rat tracheal, epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-beta-1 (TGF-beta-1) when treated within 1-2 days after plating. The purpose of the present studies was to examine the effects of TGF-beta-1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGF-beta-1 decreased dramatically as the cultures aged. The IC50 for inhibition of colony forming efficiency was 0.18 pM when TGF-beta-1 was added 24 h after cell plating. When TGF-beta-1 treatment was begun on day 5 of culture, the IC50 was 3-4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGF-beta-1 was begun on day 19, the IC50 was 65 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGF-beta-1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGF-beta-1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGF-beta-1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGF-beta-1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGF-beta-1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-alpha, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGF-beta-1 by about twofold at both early and late times in culture. This indicates that the changes in TGF-beta-1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events.