REGULATION OF METALLOTHIONEIN MESSENGER-RNA IN HUMAN HEPATOMA (HEP3B) CELLS

Purpose: Metallothionein (MT) has been shown to protect cells from the injurious effects of ionizing radiation. MT is an inducible protein and heavy metals can upregulate transcription of the MT gene. The present study was initiated to investigate regulation of MT mRNA synthesis in a human hepatocellular carcinoma (Hep3B) cell line. Methods and Materials: MT levels in Hep3B cells were measured by the cadmium-hemoglobin assay. Zinc acetate was used as an inducing agent. Levels of the MT mRNA were determined by the slot blot hybridization technique. Cycloheximide was used as an inhibitor of protein synthesis and actinomycin D was used to block transcription. Results: Zinc acetate (0.1 mM) treatment increased the intracellular levels of MT in Hep3B cells. MT levels peaked at 10 h and remained stable for up to 48 h. A time-dependent increase in the MT mRNA was also observed peaking at 16 h and then declining. Addition of cycloheximide and zinc acetate simultaneously, resulted in a decrease in the levels of MT, whereas MT mRNA levels were increased. There was no significant change in the decay rate of MT mRNA when the cells were treated with actinomycin D (7.5 mu g/ml) either in the presence or absence of Zn. Conclusion: These results suggest that neither the increased synthesis of a metal regulatory factor (MRF) nor an increase in half-life of MT mRNA is involved in the mechanism of increased MT biosynthesis upon addition of Zn. These findings support the hypothesis that a preexisting MRF must complex with Zn to initiate increased transcription for MT.