* School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, China;
Department of Hematology, General Hospital of Lanzhou, Gansu 730000, China; and
Institut de Topologie de Dynamique des Systemes, CNRS ESA 7986, Université Paris 7-Denis-Diderot, 1 Rue Guy de la Brosse, 75005 Paris, France
Received February 19, 2003; accepted April 24, 2003
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ABSTRACT |
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Key Words: Nickel; carcinogenesis; histone acetylation; histone acetyltransferase (HAT); histone deacetylase (HDAC); reactive oxygen species (ROS).
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INTRODUCTION |
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Recent studies in this field found that, as human and rodent carcinogens, nickel compounds display a strong potential to inactivate gene expression in spite of their weak mutagenicity. Thus, not gene mutation, but gene silencing is critical in nickel-induced carcinogenesis (Beyersmann, 2002; Cangul et al., 2002
). In the process of gene expression regulation, chromatin remodeling is proved to be crucial because, in eukaryotes, binding of genomic DNA with histones in the nucleosome and folding of the chromatin pose a barrier to transcription, i.e., the structure of chromatin prevents the transcription machinery from interacting with promoter DNA sequences (Fry and Peterson, 2002
). To overcome this, chromatin remodeling is triggered during gene transcription initiation mainly by two classes of enzymes: those that covalently modify nucleosomal histone proteins through acetylation and those that alter chromatin structure through the hydrolysis of adenosine triphosphate (ATP) (Fry and Peterson, 2001
, 2002
; Grunstein, 1997
). Among them, histone acetylation and its related enzymes are continuously needed during gene transcription initiation and elongation. Generally, histone hyperacetylation leads to some genes activation or up-regulation, while histone hypoacetylation leads to inactivation or down-regulation of genes (Archer and Hodin, 1999
; Klochendler-Yeivin and Yaniv, 2001
).
Ni2+ has been found to inactivate the antiangionetic thrombospondin gene and a telomere marker gene by induction of histone H4 hypoacetylation and chromatin condensation (Broday et al., 1999; Salnikow et al., 1997
). Further studies show that Ni2+ inhibits the acetylation of histone H4 through binding with its N-terminal histidine-18, which is located near the lysine residues for histone H4 acetylation (Broday et al., 2000
; Zoroddu et al., 2000
), i.e., histone H4 acetylation is inhibited by the histone H4-bound Ni2+. It is one well-known characteristic of bound-Ni2+ to produce reactive oxygen species (ROS) in vivo, and the generation of ROS by histone-bound Ni2+ has also been proved previously (Bal and Kasprzak, 2002
). Since ROS are very reactive and react easily with surrounding protein, DNA, and other molecules in vivo, their generation likely plays important roles in Ni2+-induced gene expression alterations, carcinogenesis, and especially resistance of mammalian cells to Ni2+ (Denkhaus and Salnikow, 2002
; Qu, 2001; Salnikow et al., 1994
). Hence, we are interested in whether the generation of ROS in vivo, especially those generated by histone-bound Ni2+, can influence the acetylation modification of histones through reacting with histones or molecules around them, such as histone acetyltransferases (HAT) or histone deacetylases (HDAC) (Archer and Hodin, 1999
; Klochendler-Yeivin and Yaniv, 2001
), two classes of enzymes involve in the control of the state of histone acetylation. Our knowledge of the relation between ROS generation and Ni2+-induced histone hypoacetylation still remains unclear. To clarify this will provide a new approach to further understand the role of ROS generation in Ni2+-induced gene silencing, toxicity, and the resistance of mammalian cells to Ni2+.
Here, we suppose that Ni2+-induced generation of ROS may play a role in the carcinogenicity of specific nickel compounds by leading to the inhibition of histone acetylation. To address this hypothesis, effects of Ni2+ on histone acetylation alteration and ROS generation were studied in human hepatoma cells. Ni2+ treatment significantly inhibited histone acetylation through affecting the activity of HAT in cells, and the generation of ROS was induced by Ni2+ and partially involved in the induction of histone hypoacetylation. These results suggested a new approach, diminishing Ni2+-generated ROS, to prevent and cure Ni2+-related histone hypoacetylation, gene suppression, and diseases such as cancer.
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MATERIALS AND METHODS |
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Exposure of cells to Ni2+ and other agents.
NiCl2 (Approx. 98%), H2O2, 2-mercaptoethanol (2-ME) and N-acetyl-cysteine (NAC) were obtained from Sigma Chemical Co. (St. Louis, MO). NiCl2 and NAC stock solution (1 M) in distilled water were filtered through a sterile, pathogen-free nylon filter (pore size: 0.22 µm; MSI Inc., MA), the pH of stock solutions was carefully adjusted to 7.2 using 0.5 N HCl and 1 N NaOH before filtration. A freshly prepared stock solution was mixed with RPMI-1640 at indicated concentrations before use.
Histone purification.
Preparation of histones from Hep3B cells was done according to Cousens et al. (1979) with the following modifications: the washed cells were suspended in lysis buffer (Cousens et al., 1979
) containing trichostatin A (TSA) (100 ng/ml) and PMSF (1 mM). After pipetting up and down for 20 times, the nuclei were washed three times in the lysis buffer and once in 10 mM Tris and 13 mM EDTA (pH 7.4). The histones were extracted from the pellet in 0.4 N H2SO4. After centrifugation, the histones in the supernatant were collected by cold-acetone precipitation, air-dried, then suspended in 4 M urea and stored at -20°C before use.
Western blotting analysis of histone H4 acetylation.
Equal amounts of purified histones (30 mg/lane) were subjected to SDSPAGE on 15% polyacrylamide gels and were electrophoretically transferred to a nitrocellulose membrane. Nitrocellulose blots were blocked with 5% milk in TTBS (Tris-buffered saline plus 0.05% Tween 20, pH 7.5) and incubated overnight at 4°C with a specific antibody for histone H4-acetyl-lysines in TTBS containing 5% milk. After incubation with horseradish peroxidase-conjugated secondary antibody, immunoreactivity was visualized by means of enhanced chemiluminescence (ECL, Amersham, UK).
Histone acetylation assay.
Cells were plated at a density of 2 x 106 cells/ml and exposed to 10 mCi/ml 3H-acetate (5.0 Ci/mmol, Amersham, UK). After incubation for 10 min at 37°C, the cells were stimulated with Ni2+ or/and other agents for the indicated times in the absence or presence of TSA (Sigma, St. Louis, MO, USA). Then histones were purified and 3H-labelled histones were determined by liquid scintillation counting.
HDAC assay.
An HDAC assay was performed as previously reported (Ito et al., 2000). Radiolabeled histones were prepared from Hep3B cells following incubation with TSA (100 ng/ml, 6 h) in the presence of 0.1 µCi/ml 3H-acetate. The histones were purified, dried, and resuspended in 50 mM Tris-HCl buffer (pH 8.0). Crude HDAC preparations were extracted from total cellular homogenates and then incubated with 01000 µM Ni2+ and the 3H-labelled histones in 50 mM Tris-HCl buffer (pH 8.0) for 30 min at 37°C before the reaction was terminated by the addition of 1 N HCl-0.4 N acetic acid. The released 3H-labelled acetic acid was extracted from the reaction mixture and then determined by liquid scintillation counting.
HAT assay.
Crude HAT preparations were extracted from total cellular homogenates of Hep3B cells, and the in vitro acetylation assay was performed as described previously (Chicoine et al., 1987; Ito et al., 2000
). A typical HAT assay was performed using a 50-µl reaction mixture containing: histone protein (20 mg of core histones extracted from Hep3B cells), 20 ml of crude HAT extract (from 1 x 107 cells), 01000 µM Ni2+, 100 ng/ml TSA, 10 mM HEPES (pH 7.8), 4% glycerol and 0.1 µCi 3H-acetyl-CoA (5.6 Ci/mmol, Amersham, UK). Reactions were initiated by the addition of 3H-acetyl-CoA to the mixture, followed by incubation for 1 h at 30°C. After incubation, the reaction mixture was spotted onto Whatman p81 phosphocellulose paper (Whatman), washed extensively with 0.2 M sodium carbonate buffer (pH 9.2), and then briefly washed with acetone. The dried filters were counted by liquid scintillation.
Measurement of intracellular ROS generation.
The level of intracellular ROS was measured by the alteration of fluorescence resulting from oxidation of 29,79-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR) (LeBel, 1992). DCFH-DA was dissolved in DMSO to a final concentration of 20 mM before use. For the measurement of ROS, cells were incubated with 10 µM DCFH-DA at 37°C for 30 min, then the excess DCFH-DA was washed with RPMI-1640 media prior to the treatment with Ni2+ and/or other reagents for a time period indicated in the figure legends. The intensity of fluorescence was recorded using a flow cytometry (Becton Dickenson), with an excitation filter of 485 nm and an emission filter 535 nm. The ROS level was calculated as a ratio: ROS = mean intensity of exposed cells: mean intensity of unexposed cells.
Miscellaneous.
Protein concentrations were determined with the BCA protein assay (Pierce, USA) using bovine serum albumin as a standard. Statistical analysis was performed by analysis of variance (ANOVA post-hoc Bonferroni), and p values less than 0.05 or 0.01 were denoted as * or **, respectively.
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RESULTS |
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DISCUSSION |
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The carcinogenicity of specific insoluble nickel compounds, such as crystalline nickel sulfide (NiS) and subsulfide (Ni3S2), is believed due to the ability of cells to easily phagocytize and dissolve these compounds (Denkhaus et al., 2002; Kargacin et al., 1993). Accurately, it is the accumulation of the Ni2+ ion inside the cell that appears to determine the carcinogenic potencies of different nickel compounds. Although soluble nickel salts enter cells poorly, NiCl2 has been proved to be able to partially enter cells and concentration-dependently increase the Ni2+ ion accumulation inside cells, and hence NiCl2 is often used to study the carcinogenic mechanism of nickel compounds (Broday et al., 2000
; Kargacin et al., 1993
). In this study NiCl2 was chosen as a suitable agent for studying the role of ROS generation in Ni2+-induced histone hypoacetylation.
Exposure of hepatoma cells to Ni2+ resulted in a decreased histone acetylation in cells, as detected by Western blotting and in vivo histone acetylation assays. Two possible mechanisms can be proposed to explain the histone hypoacetylation resulting from Ni2+-exposure. One model postulates that Ni2+ inhibits the acetylation of histones by the direct or indirect down-regulation of overall HAT activity. The other model, on the contrary, involves the promoting of Ni2+ on the deacetylation of acetylated histones through the up-regulation of HDAC activity. The present data show that HDAC activity is not required for the inhibition of Ni2+ on histone acetylation, because TSA, an inhibitor of HDAC (Koyama et al., 2000), had no effect on the inhibition of histone acetylation triggered by Ni2+. Therefore we conclude that the decreased levels of histone acetylation must reflect the direct or indirect repression of HAT activity by Ni2+. This conclusion is confirmed by in vitro HAT and HDAC assays using an hepatoma cell extract, where the activity of HAT, but not that of HDAC, was inhibited by Ni2+ in a dose-dependent manner. Although additional work is needed to clarify how Ni2+ affects HAT activity, we proved that Ni2+ induced histone hypoacetylation through inhibiting HAT activity in hepatoma cells.
Like in other systems (Bal and Kasprzak, 2002; Denkhaus and Salnikow, 2002
; Salnikow et al., 2000
), Ni2+ treatment also led to an increased ROS generation in cells, and the addition of H2O2 in Ni2+-treated cells significantly enhanced the ROS generation, whereas addition of 2-ME or NAC diminished that. A direct linkage between Ni2+-generated ROS and histone hypoacetylation was proved by the results that Ni2+-triggered histone hypoacetylation was efficiently enhanced or suppressed by the enhancement or diminution of ROS generation. This linkage is also confirmed by the HAT assay, where the enhancement or diminution of Ni2+-generated ROS again displayed an efficient enhancing or suppressing effect on the HAT inhibition triggered by Ni2+. The mechanism underlying the inhibition of HAT activity by ROS is unclear; one hypothesis is that the activity of HAT may be weakened by ROS through oxidative modification of critical cysteine or histidine residues in HAT proteins. This hypothesis seems extremely possible in Hep3B cells, since some of HAT proteins such as p300/CBP contain critical cysteine- and histidine-rich domains and have been found expressing in Hep3B cells (Arany et al., 1996
; Newton et al., 2000
). Moreover, this hypothesis is supported by our unpublished data, where the addition of H2O2 inhibited the HAT activity of exogenously introduced p300 in Hep3B cells. Our study also suggests that the generation of ROS is not the whole cause of histone hypoacetylation triggered by Ni2+, since H2O2 at 20 µM, 2-ME at 1 mM, or NAC at 0.5 mM did not enhance or suppress the histone hypoacetylation induced by Ni2+, although they significantly affected the generation of ROS. It suggests that Ni2+ inhibits histone acetylation through a multi-pathway, at least through inhibiting HAT activity by ROS generation (proved by us) and through affecting the access of HAT proteins to histones by the binding of Ni2+ with histones (proved by Broday et al., 2000
). Although there exist other mechanisms, these data herein clearly prove that Ni2+-generated ROS plays a role in the inhibition of Ni2+ on histone acetylation.
The toxicity of Ni2+, especially the carcinogenicity of specific insoluble nickel compounds, is believed mainly resulting from the suppression of gene transcription and expression (Beyersmann, 2002; Cangul et al., 2002
), while the resistance of mammalian cells to Ni2+ is closely allied to their defense capability against the generation of ROS triggered by Ni2+ (Qu et al., 2001
; Salnikow et al., 1994
), i.e., mammalian cells diminish the effects of Ni2+ through diminishing ROS generation. Thus, there must exist a linkage between Ni2+-induced gene suppression and ROS generation. Insofar as we are aware, this study constitutes the first report that Ni2+-induced ROS generation is involved in the inhibition of histone acetylation. The linkage between ROS generation and histone acetylation inhibition opens a new window for us to understand the role of ROS in the toxicity, especially the carcinogenicity of Ni2+ and the resistance of mammalian cells to Ni2+. Presumably, Ni2+ induces carcinogenesis through ROS generation, which inhibits histone acetylating and, hence, suppresses genes expression (Fig. 6
). And whether a mammalian cell is sensitive or resistant to Ni2+ is probably determined by its defense ability against Ni2+-induced gene suppression, while ROS generation plays an important role in this suppression through inhibiting histone acetylation.
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In summary, we conclude that ROS production is increased during exposure of cells to Ni2+, and this production of ROS is involved in the inhibition of histone acetylation. Ni2+ induces histone hypoacetylation through a multi-pathway, at least through affecting the HAT activity by ROS generation and influencing the access of HAT with histones. These data suggest a possibly useful method to prevent nickel-induced histone hypoacetylation, gene silencing, and related diseases (cancer, etc.) through improving the antioxidative potential of people, especially those working in a high nickel-exposed environment.
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ACKNOWLEDGMENTS |
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NOTES |
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