Induced secretion of a Mr 46,000 protein by cultured human hepatoma cells treated with tumor promoters.
Human hepatoma cells, HuH-6 Cl-5, were treated with 12-O-tetradecanoylphorbol-13-acetate at concentrations of 1 ng/ml to 10 micrograms/ml for 6 h in the presence of [35S]methionine. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled proteins secreted into the medium showed that this treatment induced marked secretion of a polypeptide with a molecular weight of about 46,000 (p46). When the labeled proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis, p46 was composed of three isoproteins which had different isoelectric points. The two new classes of tumor promoters, teleocidin and aplysiatoxin, also induced secretion of this protein, although the amount of p46 induced by debromoaplysiatoxin was less than that induced by 12-O-tetradecanoylphorbol-13-acetate, teleocidin, and aplysiatoxin, judging from densitometric scanning of bands on a fluorogram. The nonpromoting phorbol esters, such as 4 beta-phorbol and phorbol-13-monoacetate, did not induce p46. The induction of p46 secretion involved de novo RNA synthesis, since actinomycin D (1 microgram/ml) completely and selectively blocked the incorporation of [35S]-methionine into the protein. "Pulse-chase" experiments indicated that p46 was not a degradation product induced by these potent tumor promoters. In an attempt to identify p46, the total proteins released were treated with two kinds of rabbit anti-human whole serum antisera, but although some proteins were precipitated, p46 was not.