INDUCTION OF c-myc AND c-jun PROTO-ONCOGENE EXPRESSION IN RAT L6 MYOBLASTS BY CADMIUM IS INHIBITED BY ZINC PREINDUCTION OF THE METALLOTHIONEIN GENE

Abstract

Certain proto-oncogenes transfer growth regulatory signals from the cell surface to the nucleus. These genes often show activation soon after cells are exposed to mitogenic stimulation but can also be activated as a nonmitogenic stress response. Cadmium (Cd) is a carcinogenic metal in humans and rodents and, though its mechanism of action is unknown, it could involve activation of such proto-oncogenes. Metallothionein (MT), a metal-inducible protein that binds Cd, can protect against many aspects of Cd toxicity, including genotoxicity and possibly carcinogenesis. Thus, the effects of Cd on expression of c-rnyc and c-jun in rat L6 myoblasts, and the effect of preactivation of the MT gene by Zn treatment on such oncogene expression, were studied. MT protein levels were determined by the Cd-heme assay, and MT, c-myc, and c-jun mRNA levels were measured using oligonucleotide hybridization and standardized to β-actin levels. Cd (5 μM CdC/_2′ 0–30 h) stimulated both c-myc and c-jun mRNA expression. An initial peak of activation of c-myc expression occurred 2 h after initiation of Cd exposure, and levels remained elevated throughout the assessment period. Zn pretreatment markedly reduced the activation of c-myc expression by Cd compared to cells not receiving Zn pretreatment. Cd treatment increased c-jun mRNA levels by up to 3.5-fold. Again, Zn pretreatm ent markedly reduced Cd-induced activation of c-jun expression as minimal increases occurred with Cd exposures of ≤1 h, but otherwise the Zn pretreatment prevented activation of c-jun. The Zn pretreatment elevated MT protein levels >5-fold over control at the point of Cd exposure, but Cd expos ure did not further elevate these Zn-induced MT levels. Similarly, Zn pretreatment did not result in increased relative MT mRNA levels above Cd exposure alone at various time points after Cd exposure. Therefore, Zn pretreatment, possibly by providing elevated MT protein levels at the point of Cd exposure, inhibited the Cd-induced c-myc and c-jun proto-oncogene expression. The extent of Cd-induced proto-oncogene activation thus may be limited by the presence of cellular MT.