Effect of Rous Sarcoma Virus Transformation of Rat-1 Fibroblasts upon Their Growth Factor and Anchorage Requirements in Serum-free Medium

Abstract

The proliferative response of nontransformed rat embryo (Rat-1) cells and avian sarcoma virus-transformed B31 cells to high-density lipoprotein (HDL), transferrin, insulin, epidermal growth factor (EGF), and fibroblast growth factor has been compared. HDL, added in combination with transferrin, supported the active proliferation of low-density cultures of both Rat-1 and B31 cells. No major difference in the sensitivity of Rat-1 or B31 cells to HDL and transferrin was observed when cells were maintained on dishes coated with an extracellular matrix (ECM) obtained from bovine corneal endothelial cells. The two cell types differed in their response to the other known growth-promoting agents, however. In contrast to Rat-1 cells, transformed B31 cells no longer respond to EGF and fibroblast growth factor and respond only inconsistently to the mitogenic stimulus of insulin.

Nontransformed Rat-1 cells and transformed B31 cells grown in the presence of medium containing, respectively, HDL, transferrin, insulin, EGF, and dexamethasone or HDL, transferrin, and insulin could be subcultured for more than 50 generations in the complete absence of serum without significant alteration in morphology, growth rate, or tumorigenicity (B31 cells). When plastic or collagen-coated dishes were used as the substrate instead of ECM-coated dishes, nontransformed Rat-1 cells grew very slowly in the serum-free medium described above. Dishes coated with collagen were not more efficient than was plastic in supporting growth of Rat-1 cells under these conditions. Coating dishes with fibronectin, however, clearly improved their growth, bringing the final cell density of the cultures up to 50% of that obtained on ECM-coated dishes. In contrast, transformed B31 cells grew significantly in serum-free medium when seeded on plastic or collagen-coated dishes, and the final cell density reached by cells on these substrates was 50% of that of cells maintained on ECM-coated dishes. In addition, B31 cells grew equally well when seeded on fibronectin- or ECM-coated dishes. The transformed cells thus showed less stringent substrate requirements when grown under serum-free conditions than did nontransformed Rat-1 cells. Our data also indicate that HDL, in combination with transferrin, supported efficient anchorage-independent growth of B31 cells. Fibroblast growth factor, but not insulin or EGF, further improved anchorage-independent growth of these cells. The capacity of cells to form colonies in semisolid medium when exposed to HDL and transferrin seems to correlate with high tumorigenic potential.