Metallothionein mediates the level and activity of nuclear factor kappa B in murine fibroblasts.

The zinc-binding protein metallothionein (MT) is associated with resistance to apoptosis. We examined whether MT regulates the zinc-dependent antiapoptotic transcription factor nuclear factor KappaB (NF-KappaB), which is up-regulated under many conditions that lead to elevated MT expression. NF-KappaB protein levels and NF-KappaB-dependent reporter gene activity were examined in clonal MT(+) (MT-WT) and MT(-) (MT-KO) fibroblastic cell lines. The amount of cellular NF-KappaB p65 protein in MT-KO was less than 20% of the amount in MT-WT cells, in accord with increased sensitivity of MT-KO cells to apoptosis. NF-KappaB p65 mRNA levels, and NF-KappaB p50 subunit and IKappaBalpha protein levels, were unchanged. NF-KappaB activity assessed by expression of a transfected NF-KappaB reporter construct was less than half that observed in MT-KO cells. Decreased nuclear localization of NF-KappaB p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NF-KappaB p65 than did MT-WT cells, despite a lower cellular NF-KappaB level and function, suggesting that metallothionein mediated the specific activity of NF-KappaB. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NF-KappaB p65 (but not IKappaBalpha or NF-KappaB p50), increased NF-KappaB-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein positively regulates the cellular level and activity of NF-KappaB.

A sensitive time-resolved fluorescent immunoassay for metallothionein protein.

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure MT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of anti-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.