Nickel-induced transformation shifts the balance between HIF-1 and p53 transcription factors
Konstantin Salnikow2,
Won G. An1,
Giovanni Melillo1,
Mikhail V. Blagosklonny1 and
Max Costa
Nelson Institute of Environmental Medicine and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York, NY 10016 and
1 Medicine Branch, National Cancer Institute, National Institute of Health, Bethesda, MD 20892, USA
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Abstract
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Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. In an attempt to unravel the molecular mechanisms of Ni-induced transformation we investigated transcriptional activity of hypoxia-inducible factor (HIF-1) and p53 tumor suppressor protein in Ni-transformed cells. We demonstrated that the activity of HIF-1-responsive promoters was increased in Ni-transformed rodent cells resulting in the increased ratio between HIF-1- and p53-stimulated transcription. To further elucidate the roles of HIF-1 and p53 in Ni-induced transformation we used human osteosarcoma (HOS) cells and a Ni-transformed derivative, SA-8 cells. Since non-functional p53 was expressed in both HOS and SA-8 cells, acute Ni treatment induced HIF-1
protein and HIF-1-dependent transcription without affecting p53. In MCF-7 and A549, human cancer cells with the wild-type p53, both functional p53 and HIF-1
proteins accumulated following exposure to Ni. The induction of HIF-1
and wild-type p53 by Ni was detected after 6 h and was most pronounced by 24 h. These results suggest that acute Ni treatment causes accumulation of HIF-1
protein and simultaneous accumulation of wild-type, but not mutant, p53. We suggest that the induction of hypoxia-like conditions in Ni-treated cells with subsequent selection for increased HIF-1-dependent transcription is involved in Ni-induced carcinogenesis.
Abbreviations: CHE, Chinese hamster embryo fibroblasts; DFX, deferoxamine mesylate; FBS, fetal bovine serum; HIF-1, hypoxia-inducible factor; MOI, multiplicity of infection; VEGF, vascular-endothelial growth factor
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Introduction
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Although Nickel (Ni) compounds are known carcinogens in human and in animal models (14), the mechanisms of Ni-induced cell transformation remain unknown. Insoluble Ni compounds such as Ni subsulfide and Ni oxide have significantly higher carcinogenic potential in vivo compared with soluble Ni sulfate (4,5). The effect is probably associated with the amount of Ni that is delivered into cells when Ni-containing particles are phagocytized and dissolved inside the cell. In vitro, both soluble and insoluble Ni compounds efficiently induce immortalizaton and morphological transformation of human and rodent cells (611). Moreover, at the gene expression level for some genes both soluble and insoluble Ni compounds are equally potent (12).
Ni has low mutagenic potential in many mutational assays (13), hence it may exert carcinogenic activity by modulation of gene expression. We have shown earlier that expression of thrombospondin I was lost in Ni-transformed rodent cells (14,15). This loss was due to the accumulation of transcription factor ATF-1, which is a negative regulator of thrombospondin I gene expression (15). Recently, we cloned a new human gene (Cap43) that is highly induced by both soluble and insoluble Ni compounds (12).
Ni induces expression of a number of genes that are also induced by hypoxia, including glyceraldehyde 3-phosphate dehydrogenase (16), vascular-endothelial growth factor (VEGF) (17) and erythropoietin (18). The same set of genes is under transcriptional control by hypoxia-inducible factor (HIF-1). The inducible subunit (HIF-1
) is stabilized and accumulates in cells following hypoxia or cobalt treatment (19,20). The transcriptional activity of HIF-1 is important for tumor growth and development, particularly for tumor vascularization, since VEGF expression is under HIF-1 transcriptional control (21). It was shown that tumors in which hypoxia cannot induce HIF-1 transcriptional activity remain small and fail to metastasize (22).
The tumor suppressor p53 is another transcription factor which also accumulates following exposure of cultured cells to hypoxia (23). Moreover, in animal models, hypoxia selects against wild-type p53 in tumors (24). It has been demonstrated that wild-type p53 was induced during hypoxia simultaneously with HIF-1
and the binding of these two proteins provides p53 stabilization (25).
p53 protein is involved in the regulation of cell proliferation and apoptosis. Mutations leading to the loss of its transcriptional activity are the most common genetic alterations found in human cancer (26). p53 is known to be induced by DNA damage and some viral oncoproteins (27). Loss of p53 transactivating function due to mutations also results in p53 stabilization, precluding mutant p53 from any further induction following physiological stimuli or inhibition of proteolysis (28). Possible involvement of p53 in Ni-induced transformation was suggested by Maehle et al. (29) since a p53 mutation was found in human kidney epithelial cells transformed with Ni sulfate. On the other hand, human HOS cells have mutant p53 (30), but Ni treatment results in their further transformation. This raises the question that either this p53 is still functional or additional alterations may be involved in Ni-induced transformation.
To further elucidate the role of Ni in the modulation of gene expression we analyzed the effect of acute Ni treatment and Ni-induced transformation on the activity of p53 and HIF-1
. We found that acute Ni treatment resulted in accumulation of wild-type p53, but not mutated and non-functional p53. Acute Ni treatment also induced HIF-1
protein regardless of p53 status. Ni-induced transformation was associated with an increased ratio between HIF-1-dependent transcription and p53-dependent transcription in human and rodent cells.
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Materials and methods
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Chemicals
NiCl2, CoCl2 and CdCl2, were purchased from Alfa Aesar (Ward Hill, MA). Deferoxamine mesylate (DFX) was purchased from Calbiochem (San Diego, CA).
Cell lines
Human lung bronchoepithelial A549 cells were grown in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin; human HOS and Ni-transformed clone SA-8, as well as mouse fibroblast cells BALB/c 3T3 and Ni-transformed B200 cells, were maintained in
-MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Chinese hamster embryo fibroblasts (CHE) and a Ni-transformed clone (Ni2) were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The transformed phenotype (i.e. ability to grow in soft agar, etc.) of SA-8, B200 and Ni2 cells has been previously described (7,10,31). All cells were maintained at 37°C with 5% CO2.
p53 expression vector and p53-responsive promoters
Ad-p53, a wild-type p53-expressing replication-deficient adenovirus, was obtained from Dr B.Vogelstein (Johns Hopkins University). Viral titer was determined as previously described (32). Multiplicity of infection (MOI) is defined as the ratio of total number of viruses used in a particular infection versus number of cancer cells to be infected (i.e. number of viruses per cell). BaxLuc, a Bax promoterluciferase construct, was obtained from Dr K. Vousden (ABL Basic Research Program, NCIFCRDC). WWPLuc, a p21 promoter-luciferase construct, and pG13-Luc, containing a generic p53 response element, were obtained from Dr B.Vogelstein. The control luciferase plasmid, pGL2-Control, driven by the SV40 promoter and enhancer sequences, was purchased from Promega (Madison, WI). PCMV-galactosidase was purchased from Clontech (Palo Alto, CA).
HIF-1-responsive promoters
HRELuc was obtained by subcloning three copies of a double-stranded 21 bp oligonucleotide (5'-AGT GAC TAC GTG CTG CCT-3') in the pGL3 promoter vector (Promega) digested with KpnI and MluI. The oligonucleotide used encompasses the hypoxia-responsive element of the iNOS promoter. The construct was sequenced using Sequenase v.2.0 (US Biochemical Corp., Cleveland, OH). A HIF-responsive, erythropoietin promoter-derived luciferase construct (EpoLuc), inserted into a pGL3-Promoter vector was obtained from Dr H.Bunn and Dr E.Huang (Harvard Medical School, Boston, MA) (18).
Transient transfection assay
Aliquots of 5x104 cells/well were plated into 24-well plates (Costar, Acton, MA). The next day, cells were transfected with plasmids in the presence of Lipofectamine (Gibco BRL, Gaithersburg, MD). If indicated, cells were transfected with TransFast Transfection Reagent (Promega) according to the manufacturer's recommendations. Six hours later, the medium was changed and cells were grown for an additional 24 h, unless indicated otherwise. The cells were lyzed and analyzed for luciferase activity using a TopCount NXT luminescence counter (Packard, Downers Grove, IL).
Western blot analysis
Cells were lyzed in TNES buffer (50 mM TrisHCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 mM sodium orthovanadate, 1% NP40) containing protease inhibitors (20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Proteins were resolved by 8% SDSPAGE for detection of p53, HIF-1
and MDM-2 as previously described (25). Immunoblotting for p53 and MDM-2 was performed using 40 µg of and antibodies were obtained from Oncogene Research (Cambridge, MA).
HIF-1
was detected in 60 µg of nuclear extract using a cocktail of two monoclonal antibodies (OZ12 and OZ15; Lab Vision Corp., Fremont, CA). Signals were detected by chemiluminescence.
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Results
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Expression of HIF-responsive versus p53-responsive constructs in Ni-transformed rodent cells
Acute treatment of cells with carcinogenic metals such as Ni and Co causes the induction of the same sets of genes that are induced by hypoxia (1618). It is conceivable that this induction of gene expression is due to activation of the same transcription factors that are induced by hypoxia, namely HIF-1 and p53. We investigated the activity of these transcription factor in CHE versus Ni-transformed CHE (Ni2) and in 3T3 versus Ni-transformed 3T3 cells (B200). The results were expressed as the ratio of activity of HIF-1-dependent plasmids (EpoLuc) to p53-dependent plasmids (WWPLuc and BaxLuc) in the same cells. The ratio HIF-1:p53 was found to be increased 4- to 6-fold in Ni-transformed CHE Ni2 or mouse B200 cells compared with parental cells (Figure 1A and B
). When expression of the EpoLuc plasmid was normalized to the p53-independent plasmid pGL2, EpoLuc activity was higher in Ni-transformed cells, particularly in B200 cells (not shown). This suggests that the activity of transcription factor HIF-1 was increased in Ni-transformed cells, shifting the ratio between HIF-1- and p53-dependent transcription.

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Fig. 1. HIF-1- and p53-dependent transcription in normal and Ni-transformed rodent cells. Cells were transfected with 1 µg EpoLuc or 1 µg WWPLuc (A) or BaxLuc (B) plasmids. The results presented as the ratio of HIF-1-dependent transcription (EpoLuc) to p53-dependent transcription (WWPLuc or BaxLuc).
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Effect of Ni and Ni-induced transformation in human cells
Next, we investigated human HOS cells and Ni-transformed clone SA-8, taking advantage of the fact that a monoclonal antibody against human HIF-1
was available. HIF-1
protein was not detectable in untreated HOS and was barely detectable in SA-8 cells, but was induced in both parental HOS and in Ni-transformed SA-8 cells following Ni exposure. Induction of HIF-1
protein was more robust in Ni-transformed cells. In contrast, p53 protein was expressed at very high basal levels and was not induced in either HOS or in SA-8 cells (Figure 2A
). Expression of a HIF-1-dependent plasmid (HRELuc) was elevated in Ni-transformed cells (Figure 2B
). The difference between parental HOS and Ni-transformed SA-8 cells was more pronounced if HRELuc expression was normalized to p53-dependent transcription (Figure 2C
). Thus, similarly to rodent cells, the ratio of expression of the HIF-1-dependent to p53-dependent plasmids was shifted to HIF-dependent transcription in SA-8 cells (HREBax) (Figure 2C
). The shift in HIF-1 versus p53 activity was observed for other HIF-1-dependent and p53-dependent plasmids (EpoLucp21, EpoBax and HREp21; data not shown). In agreement with the results demonstrating induction of HIF-1
, but not p53, protein by Ni (Figure 2A
), the ratio of HIF-1
- to p53-dependent transcription was elevated by acute Ni treatment in both cell lines (Figure 2C
). Treatment of cells with DFX, an iron chelator routinely used to mimic hypoxia, similarly demonstrated high HIF-1 inducibility in both HOS and SA-8 cells (Figure 2B and C
).
We found that p53 was not induced by Ni in nuclear extracts of either HOS or SA-8 cells (Figure 2A
), however, HIF-1
could be detected in nuclear extracts. Therefore, we next compared the nuclear and cytoplasmic localization of p53. We found that p53 had a mostly nuclear localization in both HOS and SA-8 cells, which was not changed following Ni treatment (Figure 3A
). We also measured total level of p53 following different times of exposure to Ni and Co and found that neither induced p53; moreover, levels of p53 were slightly decreased following metal exposure (not shown). In addition, neither the DNA-damaging drug adriamycin nor calpain inhibitor I (ALLN) were able to induce p53 (Figure 3B
). In fact, treatment with ALLN decreased levels of p53, which is a specific characteristic of non-functional p53 (33). HOS cells have a p53 mutation at position 156 (30), however, it was not known whether p53 is functional in these cells. To further confirm that p53 was not functional in HOS or SA-8 cells, these cells were infected with an adenovirus expressing wild-type p53 (Ad-p53) and transfected with a p53-dependent (pG13-Luc) construct. Figure 3C
shows a comparison of pG13-Luc reporter activity in HOS and Ni-transformed SA-8 cells infected and not infected with p53 expression vector. A dramatic increase in pG13-Luc activity in cells infected with Ad-p53 suggests that endogenous p53 was not transcriptionally functional in HOS and SA-8 cells.

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Fig. 3. p53 is non-functional in HOS cells. (A) Nuclear and cytoplasmic localization of p53 in HOS and SA-8 cells. Aliquots of 40 µg of nuclear (top) and cytoplasmic (botom) extracts from cells untreated or treated with 1 mM NiCl2 for 16 h were resolved by 8% SDSPAGE and were subjected to western blot analysis. (B) Effect of adriamycin, ALLN and metals on HIF-1 and p53 expression in HOS and Ni-transformed SA-8. Aliquots of 40 µg of nuclear extracts from cells untreated or treated with 1 mM NiCl2, 100 µM ALLN or 200 ng/ml adriamycin (ADR) for 16 h were resolved by 8% SDSPAGE and were subjected to western blot analysis. (C) p53-dependent transcription in HOS and SA-8 cells. Cells were transfected with 1 µg pG13-Luc plasmid and 1 µg pGL2-Control plasmid using TransFast reagent (Promega). If indicated (plus p53), cells were infected with an adenovirus construct expressing wild-type p53 (Ad-p53) at 10 MOI 8 h prior to transfection. If indicated, the day after transfection cells were treated with 1 mM NiCl2 or 260 µM deferoxamine for 16 h. Cells were lysed and luciferase activity was determined as described in Materials and methods.
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Both HIF-1
and wild-type p53 are induced by nickel in human MCF-7 and A549 cancer cells
We next investigated the effects of Ni on human cells which are known to have wild-type p53. MCF-7 and A549 cells were treated with NiCl2 or CoCl2 for different periods of time. The exposure of cells to both NiCl2 and CoCl2 dramatically increased HIF-1
and p53 protein levels (Figure 4A
). Treatment of MCF-7 cells with an equitoxic dose of another metal (CdCl2) that does not produce hypoxic conditions in cells failed to induce either HIF-1
or p53 (Figure 4A
), suggesting that induction of HIF-1
and p53 was specific for NiCl2 and CoCl2 and was not the result of metal toxicity. To study whether p53 and HIF-1
are simultaneously induced by exposure to Ni, protein extracts from A549 cells treated with 1 mM NiCl2 were analyzed by western blot. Time-dependent induction of both proteins was observed (Figure 4B
), suggesting that similar induction of both p53 and HIF-1
could occur in rodent cells. The induction of p53 in MCF-7 cells was accompanied by induction of MDM-2 protein, which is a transcriptional target for p53 (Figure 4C
). Therefore, we conclude that Ni induces transcriptionally active p53 in MCF-7 cells.
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Discussion
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In industrialized countries environmental pollution by heavy metals is potentially hazardous to human health, because many metals are potent carcinogens. The exposure of workers to Ni compounds was shown to correlate with the development of nasal and lung cancers (34,35). The development of malignant tumors was documented in patients at sites of metal implants containing Ni and Co (36). In vitro Ni compounds transform not only rodent, but also human cells that are very resistant to transformation by well-recognized carcinogens (6). In fact, Ni compounds were found to be more potent in immortalization of rodent fibroblasts than ionizing radiation and chemical carcinogens such as benzo[a]pyrene diol epoxide and N-methyl-N-nitrosourea (8).
Treatment of cells with Ni changes expression of several genes, however, the molecular mechanisms remain unclear (14,15). We hypothesized that the carcinogenic activity of Ni compounds was exerted in part by induction of a hypoxia-induced signaling pathway. Ni, Co and the iron chelator DFX have been recognized as important tools in the study of hypoxia-induced signaling pathways since they mimic hypoxic conditions and induce genes that are induced by hypoxia (1618,37). The induction of these genes by hypoxia or Co is mediated by a common mechanism, namely the induction of transcription factor HIF-1
. Here we have demonstrated that acute exposure of cells to Ni strongly induces HIF-1
protein in human cells. Moreover, we found that HIF-1 transcriptional activity was elevated in both Ni-transformed human and rodent cells.
Hypoxia can induce apoptosis (38,39). Accumulation of functional p53 protein followed by p53-dependent apoptosis has been described in cultured cells exposed to hypoxia (24). We found that Ni treatment resulted in accumulation of wild-type p53 in MCF-7 and A549 cells. In HOS cells, where p53 was mutated and non-functional, acute Ni treatment failed to induce p53 or activate transcription of p53-dependent promoters.
The molecular mechanisms of p53 induction by hypoxia were recently investigated, indicating that hypoxic induction of p53 requires concomitant induction of transcription factor HIF-1
and that HIF-1
binds to and stabilizes p53 (25). The role of this factor in apoptotic events was further elaborated in studies which have shown that hypoxia induces p53 and apoptosis only in cells that have HIF-1
, but not in cells deficient in HIF-1
(40). In contrast, p53 inhibits HIF-1-dependent transcription (41), thereby decreasing the chances of normal cells surviving under hypoxia since the expression of most glycolytic enzymes is HIF-1 dependent. This indicates that hypoxia provides conditions to select for cells that have lost their functional p53 as well as their apoptotic program and have acquired a HIF-1-dependent phenotype. It is conceivable that a similar situation exists during Ni-induced transformation. Accordingly, we observed an increased ratio between HIF-1- and p53-dependent transcription in Ni-transformed cells. Involvement of p53 mutation in Ni-induced transformation of human cells was suggested previously (29). However, the parental HOS cells already have a mutant p53 and can be further transformed by Ni compounds (911), indicating that Ni-induced transformation involves additional alterations. We found that the mutated p53 in HOS cells was non-functional and, therefore, mutation of the p53 gene is not sufficient for Ni-induced transformation.
It is important to stress that Ni treatment is cytotoxic and that only a portion of cells survive exposure. The role of p53 in this cytotoxicity is not known. It has been shown that Ni does not induce apoptosis in normal human gingival fibroblasts or in mouse L-929 cells (42). In contrast, Shiao et al. (43) reported a slight induction of p53 and apoptotic DNA fragmentation in CHO cells exposed to Ni acetate for 72 h. The mechanism of p53 induction by Ni in CHO cells was not clear since p53 in these cells was mutated at amino acid 211 (44), was present at high levels and was not induced by X-irradiation (45). Our results demonstrated that Ni induced wild-type p53, but not mutated p53.
In summary, we have found that acute Ni treatment induced both HIF-1
and wild-type p53, but not mutant p53. In Ni-transformed cells, HIF-1-dependent transcription prevailed over p53-dependent transcription, probably as a result of selection for a HIF-1-dependent phenotype.
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Acknowledgments
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This work was supported by grants ES05512 and ES00260 from the National Institute of Environmental Health Sciences and grant CA16037 from the National Cancer Institute.
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Notes
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2 To whom correspondence should be addressed Email: salnikow{at}env.med.nyu.edu 
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Received December 29, 1998;
revised April 28, 1999;
accepted May 12, 1999.