Figure 1.

v-ras^Ha-transduced TGF-β−/− and AM3 dominant negative TβRII transgenic keratinocytes exhibit increased frequency of in vitro malignant conversion. The indicated cell types were infected with the v-ras^Ha retrovirus on day 3 and allowed to proliferate for 15 days before selection in 0.5 mM calcium as described in Materials and Methods. (A) Calcium-resistant foci per dish generated by TGF-β1+/+, TGF-β1+/−, and TGF-β1−/− keratinocytes. Each bar represents the mean of nine independent experiments each with 7–10 dishes per genotype ±SEM. *, Significantly different from +/+ (P = 0.0027) and +/− (P = 0.0083). (B) Calcium-resistant foci per dish generated by FVB/n control (NT), AM3 keratinocytes (DN), and AM3 keratinocytes cultured in the presence of ZnCl₂ (DN+Zn). Each bar represents the average of six dishes ±SEM. Two independent experiments are shown. ZnCl₂ did not alter the frequency of foci in the NT cultures. *, **, ***, Significantly different from NT (P = 0.024, 0.0009, 0.0001, respectively). (C) The truncated TβRII is induced by ZnCl₂ in the v-ras^Ha-transduced AM3 keratinocytes. ¹²⁵I-labeled TGF-β1 was crosslinked to monolayers of NT or AM3 cells cultured in the presence (+) or absence (−) of 25 mM ZnCl₂ for 24 hr. The crosslinked products were electrophoresed through a precast 4–12% polyacrylamide gel (NOVEX, San Diego). The truncated TβRII runs at a lower molecular mass than the native TβRII. The + in the TGF-β1 row indicates crosslinking in the presence of 100 nM unlabeled TGF-β1. Kd, kilodaltons.