Analysis of cysteine and histidine residues required for zinc response of the transcription factor human MTF-1.

Metal responsive element (MRE)-binding transcription factor-1 (MTF-1) is a zinc finger (ZF) transcription factor that plays a key role in heavy metal homeostasis by regulating relevant genes in response to metals. MTF-1 is known to be activated by heavy metals such as Zn and Cd, but the mechanism of activation remains unclear. In the present study, Cys and His residues of human MTF-1 (hMTF-1), some of which may be involved in interaction with metals or with each other, were screened for their contribution to Zn-dependent transcription. To avoid poor induction ratios of previous transfection assays, we re-examined experimental conditions to establish an assay able to correctly detect Zn-responsive transcription. Using this assay, a series of Cys and/or His substitution mutants were analyzed over the entire hMTF-1 molecule. In five out of the six ZFs (ZF1 to ZF5), Cys mutations that disrupt the ZF structure abolished response to Zn. Of these, ZF5 was shown for the first time to be essential for Zn-responsive transcription, despite it being unnecessary for Zn-induced DNA binding. These results indicate that Zn activation of hMTF-1 involves an additional process besides induction of DNA binding activity. Our assay also confirmed the importance of Cys in the acidic activation domain, as well as those in the C-terminal Cys cluster, implicated in transcription in other studies. The identified Cys residues might contribute to metal response of hMTF-1 through direct metal binding and/or intramolecular interactions, analysis of which will be helpful in understanding the mechanism of metal response.

Cadmium-induced alterations of gene expression in human cells.

We have reported the changes in gene expression in human HeLa cells exposed to a low concentration (5µM) of Cd. In the present study, cells exposed to a higher concentration of Cd were analyzed using a DNA microarray with 9182 human cDNA probes, in an attempt to obtain a comprehensive view on the biological effects of Cd. After a 6h exposure to 50µM Cd, 48 genes were up-regulated 2.5-fold or greater and 14 genes were down-regulated to 40% or less. Marked up-regulation of genes coding for metallothioneins, anti-oxidant proteins, and heat shock proteins was observed. Cd appeared to repress cell proliferation by modulating genes involved in multiple pathways. Cd also affected a number of genes related to apoptosis. Interestingly, it appeared that a series of genes were regulated to accelerate the intrinsic pathway of apoptosis, while others were directed to suppress the extrinsic pathway. Of these, rapid and transient induction of the TR3 gene was noted as a possible key process in Cd-induced apoptosis. Effects on several genes that may reflect mechanistic backgrounds of Cd toxicity were also observed. The present study disclosed a complex pleiotypic response of human cells to Cd, which was composed of a variety of changes in gene expression directed to defense, growth arrest, recovery from damage, apoptosis and so on.

Gene expression profile in human cells exposed to zinc.

Although Zn is an essential trace metal for humans, a comprehensive view of its effects on cellular functions has not been obtained. We used a DNA microarray to assess transcriptional alterations in human HeLa cells after exposure to a moderate concentration of Zn (100 muM ZnSO(4)). Out of 9,182 human genes, expression was increased in 7 genes and decreased in 4 genes twofold or greater. Four of the 7 upregulated genes were those coding for metallothionein isoforms or related proteins. An unexpectedly small extent of changes in gene expression might reflect rapid sequestration of Zn ions by metallothioneins, and the absence of most of the other protective responses indicated the non-toxic nature of Zn at this concentration. Comparison with our previous DNA microarray results for 5 muM CdSO(4)-exposed HeLa cells revealed several genes that are regulated by both metals in parallel, and a gene reciprocally regulated by them.

Ultraviolet irradiation increases the sensitivity of cultured human skin cells to cadmium probably through the inhibition of metallothionein gene expression.

We previously developed an apparatus that can irradiate cultured cells with monochromatic ultraviolet (UV) rays to exactly assess the biological effects of UV components on mammalian cells. Using this device, we studied the effects of UV in and near the UVB region on the general as well as specific protein synthesis of the human skin-derived NB1RGB cells. We found that Cd-induced synthesis of metallothioneins (MTs), which are the proteins involved in the protection against heavy metals and oxidative stress, is inhibited by UV at 280 nm more extensively than total protein synthesis. Such an inhibition was observed when MTs were induced by different inducers such as Cd, Zn, and dexamethasone in three human cell lines, indicating that it is not an event specific to a certain inducer or a certain cell type. By contrast, UV at 300 or 320 nm showed only a marginal effect. UV at 280 nm was likely to block MT gene transcription because Cd-induced increase of MT mRNA was strongly inhibited by irradiation. Cd induction of 70-kDa heat shock protein mRNA was also inhibited by UV irradiation, suggesting that the expression of inducible genes are commonly sensitive to UV. Furthermore, we observed that the irradiation of UV at 280 nm renders NB1RGB cells extremely susceptible to Cd, probably due to the reduced MT synthesis. These observations strongly suggest that UV at 280 nm severely damages cellular inducible protective functions, warning us of a new risk of UV exposure.

DNA microarray analysis of altered gene expression in cadmium-exposed human cells.

Cadmium (Cd) is a heavy metal known to be toxic and carcinogenic, but its mechanism of action remains to be elucidated. Development of the DNA microarray technology has recently made the comprehensive analysis of gene expression possible, and it could be a powerful tool also in toxicological studies. With microarray slides containing 7,000-9,000 genes, we have been studying the gene expression profiles of a human cell line exposed to Cd. By exposure to a non-lethal concentration of Cd, 46 upregulated and 10 downregulated genes whose expression levels changed twofold or greater were observed. The expression of genes related to cellular protection and damage control mechanisms such as those encoding metallothioneins, anti-oxidant proteins and heat shock proteins was simultaneously induced. In addition, altered expression of many genes involved in signaling, metabolism and so on was newly observed. As a whole, a number of genes appear to be coordinately regulated toward survival from Cd toxicity. When cells were exposed to a higher concentration of Cd, more remarkable effects were observed both in the number of affected genes and in the extent of altered expression. These findings will contribute to the understanding of the complicated biological effects of Cd.

Effect of ultraviolet irradiation on the protein synthesis of human skin cells: a study with a monochromatic ultraviolet irradiation apparatus.

Human beings always suffer from serious risk of exposure to ultraviolet (UV) rays in general as well as in work environments but the health effects of UV are not yet fully understood. To enable fine analysis of the biological effects of UV at any given wavelength, we developed an apparatus that can experimentally irradiate cultured mammalian cells with monochromatic UV rays at any given wavelength. With this device, the effects of UV at 260, 280, 300 and 320 nm on the total protein synthesis of human skin-derived NB1RGB cells were examined. An inhibitory effect of UV at 260 and 280 nm was evident, whereas UV at 300 and 320 nm had only a marginal effect. UV at 280 nm also decreased amino acid uptake, which appears, at least partly, to be responsible for reduced protein synthesis. Metallothioneins (MTs), known as proteins defending against heavy metal toxicity, have been reported to be induced after UV irradiation, but UV at 280 nm did not induce MTs in NB1RGB cells. Cadmium pretreatment of cells, that had been assumed to protect cells from UV damage via MT induction, did not increase cell viability after exposure to UV at 280 nm. The present study unambiguously revealed the biological effects of monochromatic UV rays, also showing that the UV-irradiating apparatus can be a powerful tool for studying the health effects of UV.

DNA microarray analysis of human gene expression induced by a non-lethal dose of cadmium.

Cadmium (Cd) is a hazardous heavy metal present in working and living environments. Cd affects many cellular functions, but little is known about the mechanisms of its toxicity and cellular defense against it. Recently, advanced gene expression analysis employing DNA microarrays provided us the means to profile the expression of thousands of genes simultaneously. We describe here a study of Cd-induced gene expression profile. Messenger RNA was prepared from HeLa cells exposed to a non-lethal dose of CdSO4, and analyzed by the use of an array consisting of 7,075 human cDNAs. Many stress response genes including those coding for metallothioneins and heat shock proteins were observed to be induced by Cd. The cellular metabolism inclined toward the synthesis of cysteine and glutathione after Cd exposure. Anti-oxidant genes also appeared to be induced to protect cell components and to quench reactive oxygen species. Ubiquitin pathway was activated as well probably to degrade proteins which might not be renatured. These data suggest that human cells mobilize every genomic resource (induction of some genes and repression of others) to overcome cytotoxicity caused by Cd.