Seiichiro Himeno

Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: Role of NF-E2-related factor-2†

Thioredoxin reductase (TrxR) is a selenoprotein that catalyzes the reduction of the active site disulfide of thioredoxin (Trx), which regulates the redox status of the cells. In the present study, we found that TrxR1, one of the three TrxR isozymes, was induced by cadmium as well as tumor necrosis factor α (TNFα) in bovine arterial endothelial cells (BAEC), and investigated the mechanism of cadmium‐induced TrxR1 expression. We here showed that cadmium, differently from TNFα, enhanced the promoter activity of the 5′‐flanking region of human TrxR1 gene (nucleotides −1692 to +49). Deletion and site‐directed mutation of antioxidant responsive element (ARE) (nucleotides −62 to −48) in this region abolished the response to cadmium. Overexpression of NF‐E2‐related factor‐2 (Nrf2) augmented the TrxR1 promoter activity. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed cadmium‐induced activation of TrxR1 promoter through the ARE. Chromatin immunoprecipitation (ChIP) assays showed that anti‐Nrf2 antibody precipitated ARE from the chromatin of the cadmium‐treated cells. These results indicated that cadmium‐induced TrxR1 gene expression is mediated by the activation of Nrf2 transcription factor and its binding to ARE in the TrxR1 gene promoter. We further found that in addition to cadmium, the activators of Nrf2, such as diethyl maleate (DEM) and arsenite, induced both TrxR1 and Trx gene expression in BAEC. Nrf2 might play an important role in the regulation of the cellular Trx system consisting of Trx and TrxR.

Modulation of telomerase activity by zinc in human prostatic and renal cancer cells

Because the up-regulation of telomerase in most cancer tissues is considered to be responsible for the unlimited proliferation of cancer cells, suppression of telomerase activity is an attractive potential target for cancer therapy. The mechanism for the activation of telomerase in cancer cells, however, is still unclear. In the present study, we demonstrated that Zn induces an enhancement of telomerase activity in the human renal cell carcinoma (NRC-12) and prostatic cancer (DU145) cell lines. The maximum elevation of the activity was observed 6 hr after treatment with 100 microM Zn; it was diminished by the addition of either metal chelator or cycloheximide. Other metals such as Cd and Cu also enhanced telomerase activity but to a lesser extent, and no correlation between the activation of telomerase and the induction of metallothionein was observed. Our findings provide the first evidence that metals, especially Zn, can modulate telomerase activity in cancer cells.

Overexpression of thioredoxin reductase 1 regulates NF-κB activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C‐terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF‐κB. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor‐α (TNFα) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNFα‐induced DNA‐binding activity of NF‐κB and NF‐κB‐dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF‐κB activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNFα‐induced activation of NF‐κB and the expression of NF‐κB‐targeted proinflammatory genes such as E‐selectin and cyclooxygenase‐2. These results suggest that TrxR1 may act as a positive regulator of NF‐κB and may play an important role in the cellular inflammatory response.

Regulation of glutathione peroxidase mRNA level by dietary selenium manipulation

Glutathione peroxidase (GSH-Px) contains selenium at its active site as a selenocysteine moiety. We have shown that feeding mice a selenium-deficient diet for a long period caused a large decrease in the GSH-Px mRNA level as well as in GSH-Px activity both in the liver and kidneys (Toyoda, H., Himeno, S. and Imura, N. (1989) Biochim. Biophys. Acta 1008, 301-308). In the present study, the transcription rate of the GSH-Px gene was determined by a nuclear run-on assay using liver nuclei of mice fed a selenium-deficient or selenium-adequate diet. The results clearly demonstrate that the transcription rate of the GSH-Px gene was not changed by dietary selenium manipulation, indicating that dietary selenium regulates the level of GSH-Px mRNA in the post-transcriptional step.

The regulation of glutathione peroxidase gene expression relevant to species difference and the effects of dietary selenium manipulation

Glutathione peroxidase (GSH-Px) contains selenium (Se) as selenocysteine in the active site of the enzyme. GSH-Px activities in the cytosol of all guinea-pig tissues examined were extremely low compared with those in mice and rats, while Se concentrations in tissues were almost the same among three animal species. In addition, no GSH-Px mRNA was detectable in any tissues of guinea-pigs, although the guinea-pig had the same copy number (probably a single copy) of the GSH-Px gene in its genomic DNA as that of the mouse and rat, suggesting that the species difference of GSH-Px activity observed in rodents might be due to incapability of gene transcription. On the other hand, feeding of mice with Se-deficient diet for 6 weeks resulted in a remarkable decrease in GSH-Px mRNA as well as GSH-Px activity both in the liver and kidneys. The detailed time-course experiment revealed that the drop in GSH-Px activity preceded the decrease in the mRNA level in Se-depleted mice and the mRNA level recovered rapidly in contrast to the slow rate of increase in the enzyme activity in Se-repleted mice. These results suggested that the alteration in GSH-Px activity in mice subjected to dietary Se manipulation is attributable not only to transcriptional but also to post-transcriptional regulation.

Simian virus 40-transformed metallothionein null cells showed increased sensitivity to cadmium but not to zinc, copper, mercury or nickel.

Primary cultured embryonic cells derived from mice with disrupted metallothionein (MT) I and II genes and from control mice were transformed with a plasmid encoding the simian virus 40 (SV40) large T antigen. The resulting MT-/- and MT+/+ cell strains showed similar cell morphology, cell cycle and no significant differences in glutathione levels or in the activities of glutathione-related enzymes and antioxidant enzymes. The MT-/- cells were more sensitive to Cd than MT+/+ cells, though no increase in the sensitivity to Zn, Cu, Hg or Ni were observed in MT-/- cells. MT+/+ cells accumulated more Cd than MT-/- cells but showed less lesion, suggesting the role of MT induced by Cd in MT+/+ cells as a scavenger of toxic Cd ion. These results suggest a dominant protective role of MT against Cd compared with other metals. SV40-transformed MT-/- cells seem to be a useful tool for the investigation of cellular function of MT.

Role of antioxidant systems in human androgen-independent prostate cancer cells

Most prostate cancer cells respond to initial hormonal therapy; however, some of them eventually acquire resistance to the hormonal therapy. Hormone‐independent prostate cancer usually exhibits resistance to chemotherapy and radiotherapy. Antioxidant systems are known to be involved in the resistance of cancer cells to chemotherapy and radiotherapy. Therefore, it is of significance to examine antioxidant systems of hormone‐independent prostate cancer for enhancing the efficacy of cancer therapy.

Reduced uptake and enhanced release of cadmium in cadmium-resistant metallothionein null fibroblasts.

Metallothionein (MT) is known to play a predominant role in the protection of cells from cadmium (Cd) toxicity. To investigate other factors involved in Cd resistance, we established Cd-resistant cell lines from simian virus 40-transformed MT null fibroblasts. Cd-resistant MT null cells, Cd-rA7 and Cd-rB5, developed approximately 10-fold resistance to Cd compared to parental cells, but showed no cross-resistance to Zn, Cu, Hg, Ni, As, cisplatin or H2O2. Accumulation of Cd in the resistant cells was 13-18% of that of parental cells after treatment with Cd for 24 h. A short-term experiment revealed that the rate of Cd incorporation into the Cd-resistant cells was suppressed, and the rate of Cd release was enhanced in the resistant cells compared with that of parental cells. These results indicate that the altered transport of Cd, slow uptake and rapid release, may confer resistance to Cd on the Cd-resistant cells established from MT null fibroblasts.

Citrate enhances the protective effect of orally administered bismuth subnitrate against the nephrotoxicity of cis-diamminedichloroplatinum.

Attenuation of the renal toxicity of cis-diamminedichloroplatinum (CDDP) is important in the use of this effective but cytotoxic anticancer agent. We have previously shown that the renal toxicity of CDDP can be efficiently reduced by the induction of metallothionein (MT) by preadministration of bismuth compounds in mice. Bismuth subnitrate (BSN) is used as an antigastric ulcer agent and as an antidiarrheic agent, and is suitable for inducing MT in the kidney in cancer patients. However, due to the low absorption rate of Bi from the gastrointestinal tract, the efficacy of BSN in inducing renal MT is low. In the present study, we examined the effects of citrate as a vehicle for oral administration of BSN on the tissue distribution of Bi and induction of MT in the kidneys and tumors in mice inoculated with Meth-A fibrosarcoma. Renal levels of MT and Bi were markedly increased in the mice given BSN dissolved in citrate solution compared with those given BSN suspended in saline. On the other hand, the use of citrate increased Bi accumulation in the tumor only slightly and did not increase tumor MT levels. Administration of BSN with citrate efficiently depressed the renal toxicity of CDDP, but did not affect its antitumor activity. Since both BSN and citrate are used clinically as pharmaceuticals, the combination regimen of BSN and citrate may be readily applicable as a countermeasure against the adverse side effects of CDDP without affecting its antitumor activity.

Application of metallothionein null cells to investigation of cadmium transport

Metallothionein (MT) is an important factor for cadmium resistance in mammalian cells. Most Cd-resistant cell lines thus far established have shown enhanced production of MT protein. However, the presence of high concentrations of MT, which traps cellular cadmium ions efficiently, has hindered the investigation of cadmium transport in Cd-resistant cells. Utilization of MT null mice or cultured cells derived from MT null mice is a useful way to isolate and characterize non-MT factor(s) for Cd resistance. Primary cultured cells derived from embryos of MT-I/II null mouse showed an increased sensitivity to cadmium compared with control cells. Immortalization of these cells by introducing simian virus 40 large T antigen cDNA has enabled the development of Cd-resistant MT null cells. The established Cd-resistant MT null cells exhibited a reduced accumulation of cadmium due to a decreased rate of cellular cadmium uptake. Application of the multitracer technique has demonstrated that the uptake of Mn(II) was also markedly reduced in Cd-resistant MT null cells. Kinetic and competition studies on Cd(II) and Mn(II) uptake in these cells revealed that a high-affinity transport system for Mn(II) is used, at least in part, for cellular Cd(II) uptake. Thus, the utilization of MT null cells has permitted the detection of a novel cadmium transport system.