Hirotomo Yamada

DNA Microarray Analysis of Altered Gene Expression in Cadmium- exposed Human Cells

DNA Microarray Analysis of Altered Gene Expression in Cadmium‐exposed Human Cells: Shinji Koizumi, et al. Department of Hazard Assessment, National Institute of Industrial Health—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.

METALLOTHIONEIN INDUCTION IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES BY HEAVY METALS

Human peripheral blood lymphocytes have the capacity to produce metallothioneins (MTs) as a protective response to cadmium exposure. To define the range of metal species inducing lymphocyte MTs, cellular proteins synthesized after exposure to each of 11 heavy metals were analyzed by gel electrophoresis. Toxic metals such as cadmium, mercury and silver were found to induce thioneins (apoproteins of MTs) at relatively low concentrations (maximum at approximately 10 microM), whereas less toxic metals such as zinc, copper and nickel were inductive at relatively high concentrations (maximum at approximately 200 microM). Tin, lead, iron, cobalt, and manganese did not induce thioneins. The heavy metal specificity of MT induction in the lymphocyte resembles that in the liver, and the regulatory mechanism of MT production seems to be similar in both of these tissues. In the cells exposed to highly toxic metals such as cadmium and mercury, expression of cytotoxicity (represented by decline of cysteine uptake) was remarkable at the metal concentrations higher than those saturating thionein induction, supporting the protective role of MTs against heavy metals.

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.

Induction of a 70-kDa protein in human lymphocytes exposed to inorganic heavy metals and toxic organic compounds

Human peripheral blood lymphocytes synthesize a 70-kDa protein (p70) distinct from metallothionein (MT), in response to several inorganic heavy metals. Although Cd and Zn induce both p70 and MTs, Co induces only p70. On the other hand, Cu, Hg, Ni and Ag, which are the inducers of MTs, can not induce p70. This protein is indistinguishable in electrophoretic mobility from the 70-kDa heat shock protein, which is induced by incubation of lymphocytes at 42-45 degrees C. Several environmental pollutants other than the inorganic heavy metals, including organic tin compounds and organic phosphorus compounds, were also found to induce p70. The responsiveness to a variety of environmental stimuli suggests the involvement of p70 in the basic cellular defense mechanism.

Cadmium-induced synthesis of metallothioneins in human T and B cell purified by a fluorescence activated cell sorter

Human peripheral blood lymphocytes were reacted with fluorescein-conjugated antibodies specific to T or B cell surface antigen and fractionated with a fluorescence activated cell sorter. The isolated T and B cells were examined for their capacity to synthesize metallothioneins (MTs). Analysis by gel electrophoresis indicated that both T and B cells were able to produce MTs in a Cd2+-inducible manner, suggesting that both cells types have a mechanism of protection against Cd2+ toxicity.

A nuclear factor that interacts with metal responsive elements of a human metallothionein gene.

Metallothioneins (MTs) are low molecular weight heavy metal-binding proteins which are known to play a major role in heavy metal detoxification and understanding of their regulatory mechanism is toxicologically important. Expression of MT genes is induced by heavy metals and metal responsive elements (MREs) upstream of MT genes are essential for the transcriptional activation. By several types of mobility shift assay with 32P-labeled oligonucleotide probes, we detected HeLa cell nuclear as well as cytoplasmic factors that bind to MRE sequences of human MTIIA (hMTIIA) gene. One of the nuclear factors, which gives stronger signal than others, was further characterized. Competition experiments showed that the nuclear factor (named MREBP) specifically recognizes MREs of hMTIIA gene. EDTA abolished the binding of MREBP to MRE, suggesting that a divalent cation(s) is required for the complex formation. Also in blotting experiments with HeLa nuclear extract and the [32P]MRE probes an EDTA-sensitive 95k protein band, which possibly represents MREBP, was detected.

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.