Carcinogenic nickel compounds alter the program of gene expression in normal cells and induce a pattern of gene expression similar to that found in nickel-induced cancers. Here we have demonstrated that nickel exposure induced hypoxic signaling pathways by inducing hypoxia-inducible transcription factor-1 (HIF-1), which mediated the induction of genes required by cells to survive hypoxia. We also show that a new gene, Cap43, is dependent upon HIF-1 because only HIF-1-proficient cells induced Cap43 when exposed to either hypoxia or nickel. We also show that glyceraldehyde-3-phosphate dehydrogenase, a gene induced by hypoxia through HIF-1, was similar to Cap43 in that it required HIF-1-proficient cells to be induced by either nickel or hypoxia. These data demonstrate that nickel exposure turns on signaling for hypoxic stress, which may be important in its carcinogenesis.
Chronic exposure of workers to inhaled nickel compounds increased the risk of respiratory cancers (1 , 2) . In 1990, the IARC classified nickel compounds as carcinogenic to humans (3) . Numerous studies have confirmed the carcinogenic potency of nickel compounds in animal models (reviewed in Ref. 4 ). Anchorage-independent growth was potentiated by nickel compounds in both human and rodent cells in vitro (5, 6, 7, 8) . We hypothesized that modulation of gene expression may be important in nickel carcinogenicity, and we have identified a number of genes specifically induced or suppressed by this metal (9, 10, 11) . A newly identified human gene, Cap43, has been cloned from human lung carcinoma A549 cells and was of particular interest because this gene was induced by nickel compounds in all of the tested human cell lines (11) .
Nickel treatment produced accumulation of HIF 3 -1α in both A549 and MCF-7 cells (12) . Additionally, nickel-transformed cells had elevated HIF-1 activity relative to p53, suggesting the importance of this transcription factor in nickel carcinogenesis (12) . The HIF-1 transcription factor was isolated from hypoxic cells and was shown to be involved in the regulation of hypoxia-inducible genes (13) . This factor has been shown to be heterodimeric, basic helix-loop-helix protein composed of HIF-1α and HIF-1β subunits (14) . HIF-1α was a short-lived protein that was stabilized under hypoxic conditions and was unique to HIF-1, whereas HIF-1β (ARNT) was less inducible and dimerized with several other basic helix-loop-helix proteins (15) . We also found that the Cap43 gene was induced by hypoxic conditions in three human cell lines with a time course in the A549 cell line similar to nickel-dependent induction. The induction of Cap43 gene expression by hypoxic conditions or nickel was highly dependent on HIF-1 transcription factors because this gene was not induced in fibroblasts that originated from HIF-1α knockout mice. The Cap43 mRNA was increased over 30-fold more than control in cells rendered hypoxic or treated with nickel. The mechanism of this stimulation involved both an increase in the rate of transcription and an increase in mRNA stability. We have shown that stabilization of Cap43 mRNA occurred in cells exposed to hypoxia conditions or Ni,2+ suggesting a similarity of response to both inducers at the transcriptional as well as posttranscriptional levels. Another gene, GAPDH, known to be induced by hypoxia was also induced by nickel treatment in A549 cells. This gene was only induced in HIF-1α-proficient fibroblasts by nickel, cobalt, and hypoxia but not in HIF-1α-deficient fibroblasts. The signaling pathway(s) involved in the transduction of hypoxic signals in mammalian cells are still largely unknown. One hypothetical pathway included an oxygen sensor that was considered to be a heme protein (16) that in turn activated HIF-1 transcription factor, possibly by phosphorylation. However, the protein kinases involved in the hypoxic response in mammalian cells have not been identified. We have shown previously that serine/threonine but not tyrosine phosphorylation was involved in nickel-stimulated Cap43 gene expression (11) . Because hypoxia affected intracellular calcium homeostasis (17) , we tested whether changes in intracellular calcium also affected Cap43 gene expression by hypoxia. The intracellular calcium chelator BAPTA-AM attenuated hypoxia-induced Cap43 gene expression in three different human cell lines. A similar effect was found for Cap43 gene expression induced by nickel (18) . These data suggested that intracellular calcium was essential for signaling gene induction in cells rendered hypoxic or exposed to nickel.
The similarity of gene induction by nickel or hypoxia suggested that exposure of cells to nickel produced a hypoxia-like state of gene expression that may be involved in the progression of normal cells to a neoplastic state.
Materials and Methods
Cell Lines and Culture Conditions.
A549, HCT116, and NCI-H69 cells were purchased from the American Type Culture Collection (Rockville, MD). Human lung bronchoepithelial A549 cells were grown in Ham’s F-12K medium; HCT116 and NCI-H69 were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. HIF-1α+/+ and HIF-1α−/− fibroblasts were obtained from C57 B mice with wild-type, normal, or knockout HIF-1α gene (19) and were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cells were incubated at 37°C in the presence of 5% of CO2 in air. Cells were rendered hypoxic in a chamber with a gas mixture of 0.5% of O2, 5% of CO2 balanced with N2 at 37°C. The level of oxygen in a chamber was verified using a gas monitor (SKC, Inc., Eighty Four, PA).
Northern Blot Analysis.
Total RNA was extracted from cells immediately after exposure using an ULTRASPEC RNA isolation system (Biotecx), and the RNA was electrophoresed (15 μg of total RNA/lane) in 1.0% agarose/formaldehyde gels. Probes were labeled with[α -32P]dCTP using a Random Primed DNA Labeling kit (Boehringer Mannheim).
Western Blot Analysis.
Cells were lysed in TNES buffer [50 mm Tris-HCl (pH 7.5), 2 mm EDTA, 100 mm NaCl, 1 mm sodium orthovanadate, and 1% NP40 containing protease inhibitors (20 μg/ml aprotinin, 20 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). The detection of HIF-1α was as described previously (12) .
Cells Rendered Hypoxic or Exposed to Nickel Exhibit a Similarity in HIF-dependent Gene Expression.
We have reported the cloning of a human gene, Cap43, that was significantly induced by nickel in A549, HTE, Calu-1, WI38, and HOS cells (11) . The basal level of Cap43 expression was very low in these cells, and it was induced by nickel within 4–6 h. Exposure of cells to hypoxia (0.5%) induced Cap43 mRNA within 4 h, and levels continued to increase up to 18 h (Fig. 1) ⇓ . The induction of this gene by hypoxia continued to increase up to 30 h and as with nickel, the induction remained high for at least 60 h (not shown).
We had previously tested numerous stress conditions, such as reducing or oxidative stress, and heat shock; however, these conditions did not induce Cap43 expression (11) . The expression of Cap43 in response to nickel or hypoxia in terms of the kinetics or fold of induction was very similar, suggesting that a common signaling pathway might be activated by both nickel and hypoxia (Fig. 1, A and B ⇓ ; Ref. 11 ). GAPDH, a gene known to be induced by hypoxia, was also induced by 1 mm nickel or 300 μm cobalt chloride in A549 cells (Fig. 1B) ⇓ . The later metal has also been found to induce Cap43. It was conceivable that hypoxia represented a physiological inducer for the Cap43 gene, and nickel stimulated Cap43 expression by imitating hypoxic conditions in cells. The activation of HIF-1 transcription factor is an important part of the molecular response to hypoxia. To determine whether the HIF-1 transcription factor was involved in Cap43 gene expression, we have analyzed the time course of accumulation of HIF-1α in response to hypoxia in A549 cells. HIF-1α was an inducible subunit of HIF-1 transcription factor, and its accumulation correlated with the activation of HIF-1 transcriptional activity (15) . A significant amount of HIF-1α protein was found in A549 cells after an exposure of cells to hypoxia (Fig. 1C) ⇓ . Similarly, accumulation of HIF-1α was observed in nickel-treated A549 cells (12) . To confirm that the HIF-1 transcription factor was involved in the Cap43 gene expression, we compared the Cap43 gene expression in response to hypoxia, as well as nickel and cobalt in mouse fibroblasts that originated from normal or HIF-1α knockout mice. An induction of Cap43 by hypoxia was found in fibroblasts that originated from HIF-1α+/+ mice but not in those from HIF-1α−/− mice (Fig. 2A) ⇓ . Similarly, the induction of Cap43 gene expression by nickel or cobalt was also observed in fibroblasts that originated from HIF-1α+/+ mice but not in those from HIF-1α−/− mice (Fig. 2A) ⇓ . The induction of another gene, GAPDH, by nickel, cobalt, or hypoxia was also found in fibroblasts that originated from HIF-1α+/+ mice and not in cells from HIP-1α−/− mice (Fig. 2B) ⇓ . These data suggested that the induction of gene expression in response to either hypoxia or metals required the HIF-1 transcription factor.
Regulation of Cap43 Gene Expression by Nickel or Hypoxia at the Posttranscriptional Level.
The data presented in Fig. 1C ⇓ showed that the accumulation of HIF-1α protein under hypoxic conditions declined after 6 h and reached baseline levels by 24 h. At the same time, the amount of mRNA for Cap43 continued to increase, suggesting the involvement of posttranscriptional stabilization of mRNA. It was conceivable that the accumulation of large amounts of Cap43 mRNA in response to both hypoxia and nickel resulted from posttranscriptional stabilization of mRNA. To test whether the Cap43 mRNA was stabilized, cells were exposed to hypoxia or nickel for 20 h to induce Cap43, and cells were then transferred to fresh medium under normal oxygen conditions in the presence or absence of 4 μm AD (Fig. 3) ⇓ . In the presence of AD, the half-life of Cap43 mRNA induced by hypoxia or nickel was extended (Fig. 3) ⇓ , whereas in the absence of AD, Cap43 mRNA decayed much faster (Fig. 3) ⇓ . The kinetics of mRNA decay were similar in both nickel or hypoxia-treated cells. Similar stabilization of induced Cap43 mRNA was observed when cells were incubated in the presence of 30μ m cycloheximide (not shown).
A Mobilization of Intracellular Calcium Was Involved in the Induction of Gene Expression by Nickel or Hypoxia.
We have shown previously that the induction of Cap43 gene expression by nickel involved mobilization of intracellular calcium (18) . The calcium ionophore (A23817) was found to be an effective inducer of Cap43 gene expression, and BAPTA-AM attenuated nickel-induced gene expression. Here we tested whether Cap43 induced by hypoxia required changes of intracellular calcium. Three cell lines, A549, HCT116, and NCI-H69, were subjected to hypoxia in the absence or presence of the intracellular calcium chelator BAPTA-AM. In all cases, the addition of BAPTA-AM attenuated the induction of Cap43 gene expression when cells were rendered hypoxic (Fig. 4) ⇓ .
The weak mutagenic activity of nickel compounds suggested that its carcinogenic effects may be exerted at an epigenetic level with a modulation of gene expression by nickel playing a role in the derivation of cells with neoplastic properties. The expression of a number of genes has been shown to be altered in nickel-transformed cells (9 , 20 , 21) . Additionally, we have shown that HIF-1-dependent transcription was significantly activated in nickel-transformed rodent and human cells (12) . The latter observation suggested a sustained activation of a hypoxia-dependent pathway in nickel-transformed cells. The acute exposure of cells to nickel resulted in accumulation of HIF-1 transcription factor (12) , which would promote entry of cells into this pathway.
We have studied the regulation of expression of a human nickel-inducible gene Cap43 to compare signaling pathway(s) activated by hypoxia or NiCl2. We have found that Cap43 gene expression was induced by hypoxia in human cells. This finding will add another gene to the family of hypoxia-inducible genes and further support the idea that exposure of cells to nickel activated a signaling pathway similar to the pathway activated by hypoxia. The time course, the magnitude of gene induction, and the effects of specific activators or inhibitors were found to be similar for nickel and hypoxia.
Nickel induced the expression of a number of genes including GAPDH, VEGF, and Epo (22, 23, 24) . Although the molecular mechanism of induction by nickel was not studied, a common feature of these genes was that they were also induced by hypoxia. A transcription factor (HIF-1) involved in the regulation of VEGF or Epo genes was isolated from hypoxic cells (13 , 14) . We already have shown that nickel significantly induced HIF-1α protein (12) . Here, using fibroblasts that originated from HIF-1α−/− mice, we have shown that the expression of Cap43 and GAPDH by nickel or hypoxia required HIF-1. This finding supported the hypothesis that nickel activated the expression of these genes by mimicking hypoxic conditions.
Because the level of gene expression in mammalian cells can be determined not only by transcriptional activation but also by the rate of mRNA degradation, we compared the degradation rates of Cap43 mRNA induced by nickel or hypoxia. We found that Cap43 mRNA could be stabilized by AD, suggesting the existence of factors that destabilized this mRNA. This stabilization occurred for the induced Cap43 mRNA (i.e., nickel or hypoxia). The basal level of expression, however, was not affected by AD, but basal levels could be induced by cycloheximide (11) . The stabilization of mRNA that was shown for other hypoxia-inducible genes, including VEGF, Epo, and tyrosine hydroxylase (25, 26, 27) , is probably an important part of the cellular response to hypoxic stress. Stabilization of hypoxia-induced VEGF and Epo mRNA by cycloheximide was used to suggest a common mechanism of regulation of these hypoxia-inducible genes (28) . In this study, we found that nickel- or hypoxia-induced Cap43 mRNA was stabilized by AD or cycloheximide, providing additional evidence that nickel or hypoxia exposures produced a similar type of response with respect to these genes.
Previously, we have shown that the induction of Cap43 by nickel involved serine/threonine phosphorylation and mobilization of intracellular calcium (11) , because the calcium ionophore A23187 induced Cap43 gene expression and the intracellular calcium chelator BAPTA-AM attenuated its expression (18) . Although the protein kinases that were activated by calcium in response to nickel exposure were not identified, it was conceivable that the same protein kinase cascade was involved in hypoxia-activated Cap43 gene expression. In this study, we found that the induction of Cap43 gene expression by hypoxia was also attenuated by BAPTA-AM in three human cell lines confirming the involvement of intracellular calcium signaling during hypoxia. This further supported the hypothesis that the same or similar signaling pathway involving calcium was activated by both nickel and hypoxia.
The exposure of cells to nickel-stimulated hypoxia, even under normal oxygen conditions, produced cells that had high levels of HIF-1-dependent transcription and decreased levels of p53-dependent transcription (12) . It was conceivable that selection against p53-dependent cell death occurred during nickel exposure, and cells that survived severe hypoxic stress acquired resistance to diverse physiological and nonphysiological insults including radio- and chemotherapy. It is tempting to speculate that similar changes occur in nickel-transformed cells. Additional experiments are required to understand the acquisition of a hypoxic state in cells exposed to nickel and undergoing cellular transformation.
We thank Tomasz Kluz for excellent technical assistance.
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↵1 This work was supported by Grants ES05512 and ES00260 from the National Institute of Environmental Health Sciences and Grant CA13687 from the National Cancer Institute.
↵2 To whom requests for reprints should be addressed, at New York University School of Medicine, Department of Environmental Medicine, 550 First Avenue, New York, NY 10016. Phone: (914) 731-3516; Fax: (914) 351-2118; E-mail:
↵3 The abbreviations used are: HIF, hypoxia-inducible transcription factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BAPTA-AM, bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester; AD, actinomycin D; VEGF, vascular endothelial growth factor; Epo, erythropoietin.
- Received October 4, 1999.
- Accepted November 11, 1999.
- ©2000 American Association for Cancer Research.