Enhancement of cell proliferation in low calcium medium by tumor promoters.

The effects of extracellular Ca2+ and phorbol ester tumor promoters on the proliferative capacity of normal and adenovirus-transformed rat embryo (RE) cells has been evaluated. Several early passage normal RE cultures grew 2--6 fold better during a 5 day period in standard Ca2+ (1.25 mM) medium than in low Ca2+ (0.01 mM) medium. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) enhanced growth 2--3 fold in the low Ca2+ medium but produced less than a 1.25 fold enhancement in standard medium. An early passage clone (A18-E) of RE cells transformed by the H5ts125 mutant of adenovirus type 5 grew 3.5 fold better in 1.25 mM than in 0.01 mM Ca2+ medium. With a late passage of the same clone (A18-L) this difference in Ca2+ requirement disappeared. In contrast, both an early passage clone (E11-E) and a late passage clone (E11-L) of carcinogen-pretreated and adenovirus-transformed RE cells grew equally well in low and standard Ca2+ media. TPA caused about a 2 fold enhancement of the growth of all of these adenovirus-transformed clones in low Ca2+ medium, but produced less than a 1.25 fold stimulation in standard medium. A series of diterpene esters with tumor promoting activity, epidermal growth factor (EGF) and melittin also markedly stimulated growth in low Ca2+ medium, whereas phorbol compounds lacking tumor promoting activity did not. Studies on 45Ca uptake indicated that TPA induced a rapid but transient increase in cell associated Ca2+. Thus, adenovirus transformation and in vitro progression decrease the Ca2+ requirement for growth of viral transformed RE cells. Both TPA and EGF can partially overcome the growth restriction of low Ca2+ medium, perhaps by enhancing Ca2+ uptake.