Induction of Metallothionein mRNA in HeLa Cells by Dexamethasone and by Heavy Metals

Abstract

Synthesis of metallothionein, a cysteine-rich heavy metal binding protein, is induced in cultured HeLa cells both by the heavy metals CdZ+, Zn2+ and Cu2+, and by the glucocorticoid hormone dexamethasone. The accumulation of [35S]cysteine-labeled metallothionein and the amount of translatable metallothionein mRNA show identical concentration dependences in response to dexamethasone treatment and in response to zinc exposure. Induction of translatable metallothionein mRNA is rapid in response to both the metal and glucocorticoid inducers. Increased synthesis and accumulation of metallothionein in response to either metal or glucocorticoid exposure is regulated by the level of translatable metallothionein mRNA in HeLa cells.Metallothioneins are low-molecular-weight proteins which bind the heavy metals ZnZf, CdZ+, Hg", Ag2+ and Cu2+. [20].Hepatic metallothioneins are also induced by various types of stress in rats [21]. The stress-related induction of metallothioneins in vivo suggested that this protein might be regulated by hormonal mechanisms. We have recently demonstrated the induction of metallothionein by the synthetic glucocorticoid dexamethasone, both in HeLa cells [22] and in primary hepatocytes [I 51. The induction of metallothionein by glucocorticoids in HeLa cells is mediated through the cbassic glucocorticoid receptor [23]. The metallothioneins induced in HeLa cells by Zn2+ and glucocorticoids are interia [24], although Zn2+ and glucocorticoid hormones appear to induce metallothioneins by mechanisms independent of one another in these cells [25].In rat liver, metallothionein induction by heavy metals is mediated through an increase in translatable metallothionein mRNA [26,27]. Experiments using actinomycin D and cordycepin suggest that metal regulation of metallothionein synthesis in rat liver is probably at the level of available MRNA [28]. Enger et al. [29] MATERIALS AND METHODS ChemicalsTranslation grade [35S]cysteine (> 600 Ci/mmol) was obtained from New England Nuclear; [3H]cysteine (0.5-2 Ci/ mmol) and [3H]leucine (60 Ci/mmol) were from Amersham. Phenol was distilled under N2 and stored at -20°C. lodoacetic acid was recrystallized from light petroleum (b.p. 30-60 "C). Dexamethasone and dithiothreitol were from Sigma.All other chemicals were reagent grade. Guanidine . HCI (ultrapure) was from Schwartz-Mann. Nonidet P40 was from Bethesda Research Labs.Cell Culture in a serum-free hormone-supplemented medium.HeLa cells were grown as described previously [20.22,30] RNA Extraction HeLa cells from three confluent 100-mm plates were scraped into 1.4 ml of ice-cold phosphate-buffered saline and collected by centrifugation in a Beckman microfuge B for 45 s. The pellet was resuspended in 0.35 ml Tris/saline buffer (0.01 M Tris/HCl, pH 7.0; 0.15 M NaCI, 2 mM MgC12) containing 0.5 "/, Nonidet P40 and lysed for 5 min on ice. Nuclei were pelleted by centrifugation for 2 min in a Beckman microfuge and RNA was extracted from the supernatant by one of two different extraction procedures. In the case of the p...

Interleukin 1 regulates human metallothionein gene expression.

“…Maximal levels of MT mRNA were achieved at 12 h postinduction, and the kinetics of the response is similar to MT mRNA induction by glucocorticoids and heavy metals (15,22). In contrast, the response in HL-60 cells is slower, with maximal levels of MT mRNA obtained 24 h after the addition of inducer.…”

Expression and regulation of a human metallothionein gene carried on an autonomously replicating shuttle vector.

“…pBPV69TDNA [ (6). Nuclei were pelleted and used for isolation of either total cellular (20) or episomal DNA (21).…”

“…Nuclei were pelleted and used for isolation of either total cellular (20) or episomal DNA (21). Total cellular RNA was isolated from the cytosols as described (6). DNA was analyzed by electrophoresis on 1% agarose gels and blot hybridization (22).…”

“…In cultured human cells, MTs are induced by exposure to heavy metal ions (2) or glucocorticoids (3). The increase in protein levels is mediated through increased synthesis of MT mRNA (4)(5)(6), which is due to increased transcription rates after treatment with either heavy metals or glucocorticoids (7,8). To gain a better understanding of the molecular mechanisms controlling the expression of human MT genes, we have isolated a cDNA clone of human MT-II mRNA (9) and used it as a hybridization probe to isolate at least 12 different human MT genes from a genomic DNA library (10).…”

Downregulation of metallothionein-IIA expression occurs at immortalization

“…Induction of MT-IIA expression by 5 h exposure to 100 mM CdCl 2 was examined in precrisis and postcrisis IIICF-T/B1 cells; HeLa cells were included as a positive control (Karin et al, 1981). MT-IIA mRNA was induced by cadmium in all cells analysed (Figure 2b), and the relative increase was indistinguishable between precrisis and postcrisis cells.…”