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Interferonβ-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells
www.elsevier.com/locate/issn/10434666 Cytokine 34 (2006) 312–319
Interferonb-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells Takeaki Nagamine a,c,*, Takahiki Kusakabe a,c, Hisashi Takada a,c, Kyoumi Nakazato a, Takuro Sakai b, Masakazu Oikawa b,c, Takahiro Satoh b,c, Kazuo Arakawa b,c a
Gunma University Graduate School of Medicine, Faculty of Medicine, Course of Health Science, 3-39-15 Showa-machi, Maebashi 371-8514, Japan b Advanced Radiation Technology Department, Japan Atomic Energy Agency, 1233 Watanuki-cho, Takasaki 370-1292, Japan c 21st Century COE Program, Gunma University, Japan Received 19 December 2005; received in revised form 10 May 2006; accepted 15 June 2006
Abstract We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonb treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alonetreated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonb in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonb may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei. 2006 Elsevier Ltd. All rights reserved. Keywords: Interferonb; Metallothionein; In-air micro-particle induced X-ray emission; Zinc; Bromine
1. Introduction Interferons (IFNs) are a family of three proteins: IFNa, IFNb and IFNc. While all three have antiproliferative, antiviral, and immunomodulatory effects, IFNa and IFNb predominantly contain the first two effect, whereas IFNc plays predominantly as immunomodulatory mediator [1,2]. IFNa and IFNb share the same cellular receptor and are clinically applied for the main therapy of hepatitis C patients [3]. IFNa and IFNb induce several direct and indirect antiviral mechanisms
*
Corresponding author. Fax: +81 27 220 8923. E-mail address: [email protected] (T. Nagamine).
1043-4666/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2006.06.008
such as intracellular viral RNA degradation, inhibition of viral RNA translation, activation of key components of the cellular immune system important in viral recognition, and prevention of viral infection to susceptible cells [1,2]; however, the predominant in vivo antiviral mechanism of IFNa and IFNb in patients with chronic hepatitis C remains to be clarified. IFNs are a pleiotropic cytokine and exhibits biological activity in relation of selective protein synthesis. MT is one of the proteins induced by IFNa and IFNb [4–7]. MT, a class of small and cysteine-rich proteins, participates in detoxification of heavy metals and in absorption, storage, and homeostasis of essential trace elements [8–11]. Accumulated evidence indicates a significant role for MT in the maintenance of the redox bal-
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ance, scavenging free radicals, and immune responses. A wide range of factors, including metal ions, oxidative stress, hormones and cytokines are known to induce MT synthesis [8–13]. Since putative IFN-stimulated responsive element in the promoter region of MTgene was reported by Friedman and Stark [14], several studies in vivo and in vitro on induction of MT protein and mRNA by IFNa and IFNb have been reported [4–7]. In addition, MT induction by IFN occurs in Zn-sufficient conditions, but not in Zn deficiency [5]. However, relevance of MT to antiviral activity of IFN is largely unknown. The cellular localization of MT changes according to the cell cycle; MT is in the cytoplasm during the G1 phase, and translocates to the nucleus in the S-phase [15–19]. In the nucleus, MT can supply its bound Zn to the zinc-finger proteins and they initiate the transcription process and gene expression [20,21]. By epidermal growth factor or insulin stimulus, MT moves to the intranucleus from cytoplasm; however, the nuclear translocation of MT by IFN is unclear. Because the physiological function of MT on a transcription factor, such as NFjB, alters depending on its localization in the nucleus or in the cytoplasm [19,21,22], it is important to elucidate the cellular induction and subcellular distribution of MT and Zn. In this study, we determined the induction of MT and Zn by IFN in hepatoma cell line. In addition, the subcellular changes of trace elements by IFN were analyzed using in-air micro-particle induced X-ray emission (PIXE). 2. Materials and methods 2.1. Chemicals Natural human IFNb (2 · 108 U/mg protein) was purchased from Cosmo Bio Co. Ltd. (Tokyo, Japan). 5-Brom0-2 0 -deoxyuridine (BrdU) and ZnCl2 were purchased from Wako Chemical Ltd. (Osaka, Japan). PIERCE T-PER Tissue Protein Extraction Reagent and Protein Assay Kit were purchased from Pierce Co. (Rockford, USA) and Bio-Rad Laboratories (Hercules, USA), respectively. Histofine SAB-PO kit was purchased from Nichirei Co. (Tokyo, Japan). Bovine–hemoglobin was purchased from Sigma–Aldrich (Steinheim, Germany). 2.2. Cells and cell culture conditions The human hetatoma cell line (HepG2 cell) was purchased from the Riken Cell Bank (Tukuba, Japan). HepG2 cells were cultured as adherent monolayers in Dulbecco’s modified Eagle’s medium (Nissui Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (JRH Bioscience, Lenexa, KS) and 100 U/mL of penicillin and 100 lg/mL of streptomycin, which contains copper 2 lg/dL, zinc 16 lg/ dL and iron 13 lg/dL. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37 C.
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2.3. MT protein expression following the administration of IFNb and Zn 2.3.1. Immunohistochemistry of MT HepG2 (2 · 105 cells/mL) cells were plated on the culture slide overnight. Cells were treated with Zn100 lM-alone, IFN50 U/mL + Zn100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone derived directly into the medium. No-treated cells were used for control. MT protein in HepG2 cells were determined 9 h after administration of IFN and/or Zn. MT protein was stained immunohistochemically described in a previous our study [23]. In brief, HepG2 cells were incubated in absolute methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase activity, and then treated with normal goat serum for 30 min. They were subsequently incubated with rabbit anti-MT antibody. After being washed with PBS, the sections were reacted with biotinylated goat anti-rabbit IgG and then processed with a Histofine SAB-PO kit, which detects MT. MT polyclonal rabbit antibody used in this study recognizes both MT-I and MT-II isoforms of human, rat, or rabbit MT. 2.3.2. MT concentrations MT concentrations in HepG2 cells were assayed by a modified Cd-hem method described by Onosaka and Cherian [24]. In brief, 1 · 106 cells/mL of HepG2 cells were cultured in plastic dish with a diameter of 10 cm. At 90% confluent, the cells were treated with Zn100 lM-alone, IFN50 U/mL + Zn100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone. No-treated cells were used for control. In 24 h after the treatment, the cells were rinsed with phosphate-buffered saline (PBS), then were harvested and resolved with 1.0 ml of 0.25 M sucrose. The cells were ultra-sonicated, and then centrifuged 20,000g for 30 min. Following the protein concentration assay, the recovered supernatant was used for MT assay. The 0.2-ml supernatant sample was mixed with 1.0 ml of 0.03 M Tris–HCl buffer (pH7.8) containing 1 lg Cd and was incubated for 10 min. For removing non-MT-conjugated Cd, the sample was added 0.1 ml of 5% bovine–hemoglobin and was heated for 90 s at 95 C. After cooling on ice, the sample was centrifuged at 10,000g for 5 min. The addition of bovine– hemoglobin and the heart treatment were repeated three times. MT-conjugated Cd in the supernatant was determined by inductively coupled plasma/mass spectroscopy (ICP-MS) (ELAN6100, Perkinelmer, Japan). Mass-tocharge ratio was 114. MT concentration was calculated by assuming that 1 mole of MT(6600) binds 7 mol Cd [25], and was described per milligram of protein concentration. 2.4. Cellular Zn levels following IFNb and Zn administration HepG2 cells adjusted 1 · 106 cells/mL were cultured in T-25 flask. At 90% confluence, Zn100 lM-alone,
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IFN50 U/mL + Zn100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone were treated into the medium for 9 h. No-treated cells were used for control. After removing medium, cells were rinsed twice with 5 ml PBS. The cells were harvested and resolved with 1.0 ml PIERCE T-PER Tissue Protein Extraction Reagent. The cells were ultra-sonicated, and then centrifuged 3,000 rpm for 30 min at 4 C for removing floating cells. The protein concentration of the recovered supernatant was determined by a Protein Assay Kit. Zn levels of the homogenized solution were determined by atomic absorption spectrophotometry. 2.5. Cell cycle analysis HepG2 cells were plated at a density of 1 · 105 cells/mL in T-25 flask for 24 h. The cells were treated by direct addition of Zn100 lM-alone, IFN50 U/mL + Zn 100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone into the culture medium. No-treated cells were used for control. In 24 h after the treatment, the media were removed and the cells were rinsed with PBS, then released from the dish by isotonic trypsin. The cells were suspended in the medium and centrifuged at 1000 rpm for 5 min at 4 C. After removing supernatant, the cells were added 10% DMSO and stored at 80 C until use. Before analysis the samples were thawed rapidly at 37 C and stained as follows. Tripsin was added to the cell suspension for 15 min, and then RNase solution was added for 15 min. Cells were stained with propidium iodide (PI; 0.0025% in citrate buffer). Cell fluorescence was measured by FACS scan cytometer (Becton Dickinson, Bedford, USA) and analyzed using ModFit software to determine the distribution of cells in various phases of cell cycle. 2.6. Analysis of the cellular elemental distributions in HepG2 cells using in-air micro-PIXE 2.6.1. Preparation of the sample for in-air micro PIXE analysis HepG2 cells adjusted to 5 · 105 cells/ml were cultured with 1 ml of medium on Mylar foil over night. Cells were pretreated with 1 ml of medium containing 100 lM of BrdU, which can be incorporated into the DNA of proliferating cells [26]. In 24 after BrdU treatment, the medium was replaced with IFNb- and Zn-treated medium as follow: Zn100 lM-alone, IFN50 U/mL + Zn100 lM, IFN100 U/ mL + Zn100 lM or IFN100 U/mL-alone. No-treated cells were served as control. After 9 h culture, the cells were rinsed seven times with THAM (tris-hydroxymethylaminomethane) solution and were cryofixed with liquid nitrogen, and dried in a vacuum for 24 h. Finally, the sample was mounted with adhesive onto the sample holder.
distribution in the HepG2 cells sample. Period and dimensions of the scan were set at 30 min and 70 · 70 lm2 areas, respectively. Precise measurement conditions were reported previously [27]. Net count of element yield was calculated by the PC Software program. Because sulfur (S) count is regarded as representative of the whole cell number [28], the net count ratio of Zn to S were evaluated. 2.7. Statistical analysis For comparison for more than two groups, data were analyzed by a one-way ANOVA, followed by Turky test for multiple comparisons. Probabilities of p < 0.05 were regarded as statistically significance. 3. Results 3.1. Immunohistochemical staining of MT induced by IFN and Zn (Fig. 1) We stained MT protein immunohistochemically in the HepG2 cells. MT staining was markedly observed in the cytoplasm and nuclei of hepatocytes treated with IFN and Zn compared to those with the Zn-alone, the IFNalone or the no-treated control. MT expression was similar between the IFN50 U/mL + Zn100 lM-treated cells and the IFN100 U/mL + Zn100 lM-treated ones. The Zn-alone-treated cells showed higher MT expression compared to the control. MT expression was not enhanced in the IFN-alone-treated cells compared to the control. 3.2. Cellular MT concentrations following administration of IFN and Zn (Table 1) Cellular MT concentrations were significantly higher in the IFN- and Zn-treated cells and the Zn–alone-treated cells than in the IFN-alone-treated cells and the control. MT concentrations were significantly higher in the IFN- and Zntreated cells than the Zn-alone-treated cells. MT levels did not increase in the IFN-alone-treated cells. The data are in line with the degree of MT staining shown in Fig. 1. 3.3. Cellular Zn levels following administration of IFN and Zn (Table 2) Cellular Zn levels were significantly higher in the IFNand Zn-treated cells than the IFN-alone-treated cells and the control. The Zn-alone-treated cells showed significant higher Zn levels compared to those of the IFN-alone-treated cells and the control. Cellular Zn levels did not elevate in the IFN-alone-treated ones. 3.4. Cell cycle analysis by flow cytometry (Table 3)
2.6.2. Analysis for cellular elements using in-air micro PIXE A 3.0-MeV proton beam, 1 lm of beam spot size, accelerated by the TIARA single-ended accelerator at JAEATakasaki, was used to analyze the subcellular elemental
For a quantitative evaluation of cell cycle distribution, the percentage of cells in the G0/1-, S-, and G2/M-phase of IFNand/or Zn-treated cultures were determined. The S-phase
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Fig. 1. Immunohistochemical staining of MT in HepG2 cells induced by IFN and Zn. MT protein is mainly observed in the cytoplasm of HepG2 cells. Combination of IFN and Zn markedly increases MT expression in the cytoplasm and nuclei of the cells compared to the Zn-alone- or IFN-alone-treated cells. Table 1 Cellular metallothionein levels following administration of interferon and/ or zinc IFN (U/mL)
Zn (lM)
Metallothionein levels (lg/mg of protein)
0 0 50 100 100
0 100 100 100 0
0.44 ± 0.07 1.78 ± 0.42a,b 2.31 ± 0.20a,b,c 2.21 ± 0.18a,b,c 0.45 ± 0.04
Metallothionein levels were significantly higher in the IFN + Zn-treated cells and the Zn-alone-treated cells than the IFN-alone-treated cells and the control. The IFN + Zn-treated cells showed significant higher metallothionein levels than the Zn-alone-treated ones.Results are mean values ± SD for five different samples per group. a p < 0.01 significant difference compared to the control (IFNO, ZnO). b p < 0.01 significant difference compared to the IFN100 U/mL-alonetreated cells. c p < 0.05 significant difference compared to the Zn 100 l0M-alonetreated cells.
population was higher in the IFN-treated cells than in the Zn-alone-treated cells and the control. A significant S-phase accumulation in the IFN100 U/mL-alone-treated cells was noted accompanied by a decrease in the G2/M phase, sug-
Table 2 Cellular zinc levels following administration of interferon and/or zinc IFN (U/mL)
Zn (lM)
Zinc level (lg/0.01 g of protein)
0 0 50 100 100
0 100 100 100 0
12.9 ± 1.6 25.0 ± 11.1a,b 26.9 ± 8.7a,b 24.3 ± 7.5a,b 15.0 ± 3.3
The Cellular Zn levels were significantly increased in the IFN + Zn-treated cells and the Zn-alone-treated cells than the IFN-alone-treated cells and the control. The cellular Zn levels were unchanged in the IFN-alonetreated cells. Results are mean values ± SD for eight different samples per group. a p < 0.01 significant difference compared to the control (IFNO, ZnO). b p < 0.05 significant difference compared to the IFN100 U/mL-alonetreated cells.
gesting that IFN may delay S-phase of HepG2 cells. Comparing to the IFN100 U/mL-alone, the combination of Zn100 lM and IFN (50 or 100 U/mL) reduced S-phase population and increased M/G2-phase population. The Zn-alone-treated cells showed a very similar pattern of cell-cycle distribution of the control.
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Table 3 Effect of IFN and zinc on cell cycle distribution of HepG2 cells IFN (U/mL)
Zn (lM)
G0/G1 (%)
S (%)
M/G2 (%)
0 0 50 100 100
0 100 100 100 0
56.9 ± 1.3 56.2 ± 2.5 54.5 ± 3.1 51.8 ± 3.2 52.1 ± 2.2
32.5 ± 1.3 32.9 ± 2.5 35.6 ± 2.5 36.7 ± 2.5 39.7 ± 2.5a,b
10.7 ± 1.3 10.9 ± 2.5 10.0 ± 2.5 11.5 ± 2.5c 8.3 ± 2.5
S-phase population was increased in the IFN-treated cells, but was unchanged in the Zn-alone-treated cells. S-phase accumulation in the IFN100 U/mL-treated cells was accompanied by a decrease in the G2/M phase. The IFN100 U/mL + Zn100 lM-treated cells showed reduced Sphase population and increased M/G2 population compared to those with IFN100 U/mL-alone-treated ones. Zn-alone-treated cells showed a similar pattern of cellcycle distribution to the control. Results are mean values ± SD from four different experiments. a p < 0.05 significant difference compared to the control (IFNO, ZnO). b p < 0.05 significant difference compared to the Zn100 mM-alonetreated cells. c p < 0.05 significant difference compared to the IFN100 U/mL-alonetreated cells.
3.5. The cellular elemental distribution analyzed by in-air micro PIXE 3.5.1. The X-ray spectra of HepG2 cells The yield of Zn was increased in the IFN- and Zn-treated cells and the Zn-alone treated cells compared to the
IFN-alone-treated cells or the control (Fig. 2). The net count ratio (%) of Zn to S (Zn/S) were higher in order of the IFN50 U/mL + Zn100 lM, the IFN100 U/ mL + Zn100 lM, the Zn100 lM-alone, the control and the IFN100 U/mL-alone. There was a significant difference between the IFN50 U/mL + Zn100 lM and theIFN100 U/ mL or the control (Table 4). Thus, the Zn/S ratio gave a good consistency with the cellular Zn levels determined by atomic absorption spectrophotometry. 3.5.2. Elemental maps of the HepG2 cells (Fig. 3) The two-dimensional P maps provide a good representation of the physical shape of the cell and highlight the position of the cell nucleus. Since nuclear membrane consists of double layers of phospholipids, it was found by physical correlation that the cell nucleus corresponded to the P-rich region within the cell [29]. In the control sample, Zn map fitted well with P and S maps showed that Zn is assumed to distribute diffusely in the cytoplasm as well as in the nucleus. Focal localizations of Br were found with a spatial correlation with P, suggesting that Br is possibly accumulated within the nucleus. In the Zn100 lM-alone-treated sample, the increased accumulation of Zn was observed, but no enhanced accumulation of Br was found. When IFNb100 U/mL was administered alone, the increased
Fig. 2. The X-ray spectra of HepG2 cells taken by in-air micro-PIXE. Zn yield is higher in order of the IFN50 U/mL + Zn100 lM-treated cells, the IFN100 U/mL + Zn100 lM-treated cells, the Zn100 lM-alone-treated cells, the IFN100 U/mL-alone-treated cells and the control. Representative X-ray spectra from four experiments are shown.
T. Nagamine et al. / Cytokine 34 (2006) 312–319 Table 4 Count ratio of zinc to sulfur calculated by in-air micro-PIXE IFN (U/mL)
Zn (lM)
Zn/S (%)
0 0 50 100 100
0 100 100 100 0
3.0 ± 0.7 6.8 ± 2.0 11.2 ± 1.3a,b 7.0 ± 1.8 2.1 ± 1.4
The mean of Zn/S ratio was significantly higher in the IFN50 + Zn100 mM-treated cells than the IFN100 U/mL-alone-treated cells and the control. Results are mean values ± SD from four different experiments. a p < 0.05 significant difference compared to the control (IFNO, ZnO). b p < 0.05 significant difference compared to the IFN100 U/mL-alonetreated cells.
localization of Br which fitted partially with the Zn map was observed. IFN-alone treatment did not change cellular Zn distribution. The marked accumulation of Zn and Br were observed in the IFN- and Zn-treated sample. The Br and Zn maps showed a good consistency with the P map. Discrete Br accumulation sites were clearly visible with a strong spatial correlation with Zn accumulation. These findings were
observed predominantly in the Zn100 lM-treated samples (Fig. 4).
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IFN50 U/mL-
and
4. Discussion We and others have previously reported hepatic MT induction by IFNa and IFNb in vivo [4–7]. The present in vitro study confirmed previous results; human IFNb directly induced MT expression within the cytoplasm and nuclei of HepG2 cells. Since the promoter region of MTgene have several metal responsive element and the putative IFN-stimulated responsive element [8,14], Zn and IFN may induce MT synthesis in vivo and in vitro. Sato et al. [5] showed that hepatic MT induction by IFN occurred in mice fed with zincsufficient diet, but failed in mice fed Zn-deficient diet. A similar result was obtained from data. When IFNb was in the culture medium, containing such low Zn concentration as 16 lg/dL, MT synthesis was not induced in the cultured hepatocytes. The combination of IFN and Zn markedly expressed hepatic MT compared to those of Zn-alone. At the cellular level, MT is mainly distributed in cytoplasm and to a lesser extent in nuclei [17,30]. In regenerating liver after partial hepatectomy, MTs sift from cytoplasm to nuclei [15,31]. Cellular distribution of MT is
Fig. 3. Elemental maps of HepG2 cells. P maps provide a good representation of the physical shape of the cell and highlight the position of the cell nucleus. The counter plat of P map is drown (upper panels). Zn map is overlapped with the counter plat of P. In the IFN- and Zn-treated samples, Zn distribution fits so well with P map that Zn is assumed to distribute diffusely in the cytoplasm as well as in the nucleus (middle panels). Focal localizations of Br are found with a spatial correlation with P, suggesting that Br is possibly accumulated within the nucleus. Marked accumulation of Br is observed in the IFN- and Zn-treated samples, whereas Br distribution is not enhanced in the Zn-alone-treated samples (lower panels).
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Fig. 4. Accumulation of zinc and bromine by IFN and Zn treatment. HepG2 cells were pre-incubated with 100 lM BrdU for 24 h. Then, the cells were exposed to IFN 50 U/mL and Zn100 lM for 9 h, and intracellular distribution of Zn and Br were determined by micro-PIXE. In the IFN 50 U/mL- and Zn100 lM-treated sample, accumulation of Br and Zn shows a good consistency with the P map. Discrete Br accumulation sites are clearly visible with a strong spatial correlation with Zn accumulation.
also depending on the cell cycle and that MT translocates to nuclei in the S-phase of cells [15–19]. These studies postulated that the nuclear location of MT has been associated to Zn requirements and interactions with nuclear constituents during the cell cycle. The flow cytometry analysis in the present study showed that IFNb100 U/mL-alone delayed the cell cycle at S-phase, which is in line with previous studies [32,33]. Comparing with the cells in IFN100 U/mL-alone, the combination of IFN100 U/mL and Zn100 lM reduced S-phase population and increased of M/G2-phase population, suggesting that Zn may progress cellular proliferation delayed by IFN [34]. Zn-alone-treated cells showed a similar pattern of the cell cycle distribution to the controls. On the other hand, nuclear MT expression was increased by IFNb in combination with Zn compared to those with IFN-alone or Zn-alone. These findings suggest that IFNb in combination with Zn may produce MT protein synergistically and sift MT from cytoplasm to S-phase nuclei of hepatocytes delayed by IFN treatment. To analyze the cellular elements in the HepG2 cells, we applied in-air micro-PIXE technology in the present study, which can determine the subcellular elemental distribution correctly by focusing the beam spot within 1 lm under in-
air scanning [27]. In order to assess the concentration of these trace elements quantitatively, we analyzed the net count of element yield from the PIXE spectrum for every cell. Since the Zn/S ratio (Table 4) indicated a good correlation with the cellular Zn levels determined by atomic absorption spectrophotometry (Table 2), the in-air microPIXE is regarded as a convenient and useful apparatus for examining the trace elements in cultured cells. MicroPIXE spectra showed the enhanced Zn distribution within the Zn-alone-treated sample, which became more predominant with combination of IFN. Zn distribution was not altered by IFN-alone. On the other hand, IFN-alone treatment slightly increased Br distribution, and Zn-alone unchanged Br distribution in HepG2 cells (Fig. 3). As Br is mainly up-taken into the S-phase nucleus [26], IFN-alone is speculated to delay the cell cycle at the S-stage, which is in agreement with the result of flow cytometry. Interestingly, IFN in combination with Zn markedly increased Br accumulation accompanying a spatial correlation with P map that reflects putative nuclear image [29]. Simultaneously, IFN-induced Br accumulation coincides with spatial Zn accumulation. The findings indicate that IFN in combination with Zn may promote cell proliferation of HepG2 cells, accompa-
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nying redistribution of Zn from cytoplasm to S-phase nucleus. Taken together the data from MT induction, flow cytometry and micro-PIXE analysis, the combination of IFNb and Zn induces MT synthesis, which covalently combines Zn, and sifts MT from cytoplasm to IFN-delayed S-phase nuclei. In addition, the enhanced accumulation of Zn into S-phase nucleus by the combination of IFN and Zn leads us a speculation that MT-bound Zn is probably used for zinc-finger protein synthesis [15–17,20]. Further studies are needed to confirm our hypothesis whether IFN-induced MT may promote the activity of transcription factors with a zinc-finger motif by supplying Zn efficiently. Acknowledgment This work was supported in part by the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Ucar R, Sanwo M, Ucar K, Beall G. Interferons: their role in clinical practice. Ann Allerg Asthma Im 1995;75:377–89. [2] Gutterman JU. Cytokine therapeutics; lessons from interferona. Proc Natl Acad Sci USA 1994;91:1198–204. [3] Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Schifman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 2001;358:958–65. [4] Guevara-Ortiz JM, Castellanos VO, Leon-Chavez BA, Achanzar WE, Brambila E. Interferon alpha induction of metallothionein in rat liver is not linked to interleukin-1,interleukin-6, or tumor necrosis factor alpha. Exp Mol Pat 2005;79:33–8. [5] Sato M, Yamaki J, Oguro T, Yoshida T, Nomura N, Nakajima K. Induction of metallothionein synthesis by interferon alpha/beta in response to zinc in mice. Tohoku J Exp Med 1996;178:241–50. [6] Nagamine T, Takagi H, Hashimoto Y, Takayama H, Shimoda R, Nomura N, et al. The possible role of zinc and metallothionein in the liver on the therapeutic effect of interferon-alpha to hepatitis C patients. Biol Trace Elem Res 1997;58:65–76. [7] Sciavolino PJ, Vilcek J. Regulation of metallothionein gene expression by TNF-alpha and IFN-beta in human fibroblasts. Cytokine 1995;7:242–50. [8] Palmiter RD. Metallothionein facts and frustrations. In: Klaassen CD, editor. Metallothionein IV. Basel: Birkhauser Verlag; 1999. p. 215–21. [9] Cherian MG, Chan HM. Biological functions of metallothionein–A review. In: Suzuki KT, Imura N, Kimura M, editors. Metallothionein III. Basel: Birkhauser Verlag; 1993. p. 87–109. [10] Waalkes M, Goering PL. Metallothionein and other cadmiumbinding proteins: recent developments. Chem Res Toxicol 1990;3:281–8. [11] Schroeder JJ, Cousins RJ. Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures. Proc Natl Acad Sci USA 1990;87:3137–41. [12] Kumari MV, Hiramatu M, Ebadi M. Free radical scavenging actions of metallothionein isoforms I and II. Free Radic Res 1998;29:93–101. [13] Crowthers KC, Kline V, Giardina C, Lynes MA. Augmented humoral immune function in metallothionein-null mice. Toxicol Appl Pharmacol 2000;166:161–72.
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[14] Friedman RJ, Stark GR. Alpha-interferon induced transcription of HLA and metallothionein genes containing homologous upstream sequences. Nature 1985;314:637–9. [15] Tohyama C, Suzuki JS, Hemelraad J, Nishimura N, Nishimura H. Induction of metallothionein and its localization in the nucleus of rat hepatocytes after partial hepatectomy. Hepatology 1993;18:1193–201. [16] Nagel WW, Vallee BL. Cell cycle regulation of metallothionein in human colonic cancer cells. Proc Natl Acad Sci USA 1995;92:579–83. [17] Cherian MG, Apostolova MD. Nuclear localization of metallothionein during cell proliferation and differentiation. Cell Mol Biol 2000;46:347–56. [18] Nagano T, Itoh N, Ebisutani C, Takatani T, Miyoshi T, Nakanishi T, et al. The transport mechanism of metallothionein is different from that of classical NLS-bearing protein. J Cell Physiol 2000;185:440–6. [19] Woo ES, Dellapiazza D, Wang A, Lazo J. Energy-dependent nuclear binding dictates metallothionein localization. J Cell Physiol 2000;182:69–76. [20] Zeng J, Heuchel R, Schaffner W, Kagi JH. Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc-finger containing factor Sp1. FEBS Lett 1991;279:310–2. [21] Abdel-Mageed AB, Agrawal KC. Activation of nuclear factor kB: potential role in metallothionein mediated mitogenic response. Cancer Res 1998;58:2335–8. [22] Sakurai A, Hara S, Okano N, Kondo Y, Inoue J, Imura N. Regulatory role of metallothionein in NF-kappa B activation. FEBS Lett 1999;455:55–8. [23] Nagamine T, Suzuki K, Kondo T, Nakazato K, Kakizaki S, Takagi T, et al. Interferon-alpha-induced changes in metallothionein expression in liver biopsies from patients with chronic hepatitis C. Cad J Gastroenterol 2005;19:481–6. [24] Onosaka S, Cherian MG. Comparison of metallothionein determination by polarographic and cadmium-saturation methods. Toxicol Appl Pharmocol 1982;63:270–4. [25] Winge DR, Miklossy KA. Domain nature of metallothionein. J Biol Chem 1982;257:3471–6. [26] Dolbeare F. Bromodeoxyuridine. A diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization. Histochem J 1996;28:531–75. [27] Sakai T, Oikawa M, Sato T, Nagamine T, Moon HD, Nakazato K, et al. New in-air micro-PIXE system for biological applications. Nucl Instr Meth B 2005;231:112–6. [28] Bara M, Moretto P, Razafindrabe L, Llabador Y, Simonoff M, Guiet-Bara A. Nuclear microanalysis of the effect of magnesium and taurine on the ionic distribution in the human amniotic membrane. Cell Mol Biol 1996;42:27–38. [29] Allen GL, Camakaris J, Legge GJF. Elemental microanalysis of fibroblasts by a scanning proton microprobe and application to Menkes’ disease. Biol Trace Elem Res 1994;40:103–26. [30] Nartey NO, Banerjee D, Cherian MG. Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of fetal human liver and kidney and its changes during development. Pathology 1987;19:233–8. [31] Tsujikawa K, Imai T, Kakutani M, Kayamori Y, Mimura T, Otaki N, et al. Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes. FEBS Lett 1991;283:239–42. [32] Yano H, Iemura A, Haramaki M, Ogasawara S, Takayama A, Akiba J, et al. Interferon alpha receptor expression and growth inhibition by interferon alpha in human liver cancer cell lines. Hepatology 1999;29:1708–17. [33] Murphy D, Detjen KM, Welzel M, Wiedenmann B, Rosewicz S. Interferon-alpha delays S-phase progression in human hepatocellular carcinoma cells via inhibition of specific cyclin-dependent kinase. Hepatology 2001;33:346–56. [34] Valko M, Morris H, Cronin MTD. Metals, toxicity and oxidative stress. Curr Med Chem 2005;12:1161–208.
Interferonb-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells Takeaki Nagamine a,c,*, Takahiki Kusakabe a,c, Hisashi Takada a,c, Kyoumi Nakazato a, Takuro Sakai b, Masakazu Oikawa b,c, Takahiro Satoh b,c, Kazuo Arakawa b,c a
Gunma University Graduate School of Medicine, Faculty of Medicine, Course of Health Science, 3-39-15 Showa-machi, Maebashi 371-8514, Japan b Advanced Radiation Technology Department, Japan Atomic Energy Agency, 1233 Watanuki-cho, Takasaki 370-1292, Japan c 21st Century COE Program, Gunma University, Japan Received 19 December 2005; received in revised form 10 May 2006; accepted 15 June 2006
Abstract We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonb treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alonetreated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonb in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonb may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei. 2006 Elsevier Ltd. All rights reserved. Keywords: Interferonb; Metallothionein; In-air micro-particle induced X-ray emission; Zinc; Bromine
1. Introduction Interferons (IFNs) are a family of three proteins: IFNa, IFNb and IFNc. While all three have antiproliferative, antiviral, and immunomodulatory effects, IFNa and IFNb predominantly contain the first two effect, whereas IFNc plays predominantly as immunomodulatory mediator [1,2]. IFNa and IFNb share the same cellular receptor and are clinically applied for the main therapy of hepatitis C patients [3]. IFNa and IFNb induce several direct and indirect antiviral mechanisms
*
Corresponding author. Fax: +81 27 220 8923. E-mail address: [email protected] (T. Nagamine).
1043-4666/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2006.06.008
such as intracellular viral RNA degradation, inhibition of viral RNA translation, activation of key components of the cellular immune system important in viral recognition, and prevention of viral infection to susceptible cells [1,2]; however, the predominant in vivo antiviral mechanism of IFNa and IFNb in patients with chronic hepatitis C remains to be clarified. IFNs are a pleiotropic cytokine and exhibits biological activity in relation of selective protein synthesis. MT is one of the proteins induced by IFNa and IFNb [4–7]. MT, a class of small and cysteine-rich proteins, participates in detoxification of heavy metals and in absorption, storage, and homeostasis of essential trace elements [8–11]. Accumulated evidence indicates a significant role for MT in the maintenance of the redox bal-
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ance, scavenging free radicals, and immune responses. A wide range of factors, including metal ions, oxidative stress, hormones and cytokines are known to induce MT synthesis [8–13]. Since putative IFN-stimulated responsive element in the promoter region of MTgene was reported by Friedman and Stark [14], several studies in vivo and in vitro on induction of MT protein and mRNA by IFNa and IFNb have been reported [4–7]. In addition, MT induction by IFN occurs in Zn-sufficient conditions, but not in Zn deficiency [5]. However, relevance of MT to antiviral activity of IFN is largely unknown. The cellular localization of MT changes according to the cell cycle; MT is in the cytoplasm during the G1 phase, and translocates to the nucleus in the S-phase [15–19]. In the nucleus, MT can supply its bound Zn to the zinc-finger proteins and they initiate the transcription process and gene expression [20,21]. By epidermal growth factor or insulin stimulus, MT moves to the intranucleus from cytoplasm; however, the nuclear translocation of MT by IFN is unclear. Because the physiological function of MT on a transcription factor, such as NFjB, alters depending on its localization in the nucleus or in the cytoplasm [19,21,22], it is important to elucidate the cellular induction and subcellular distribution of MT and Zn. In this study, we determined the induction of MT and Zn by IFN in hepatoma cell line. In addition, the subcellular changes of trace elements by IFN were analyzed using in-air micro-particle induced X-ray emission (PIXE). 2. Materials and methods 2.1. Chemicals Natural human IFNb (2 · 108 U/mg protein) was purchased from Cosmo Bio Co. Ltd. (Tokyo, Japan). 5-Brom0-2 0 -deoxyuridine (BrdU) and ZnCl2 were purchased from Wako Chemical Ltd. (Osaka, Japan). PIERCE T-PER Tissue Protein Extraction Reagent and Protein Assay Kit were purchased from Pierce Co. (Rockford, USA) and Bio-Rad Laboratories (Hercules, USA), respectively. Histofine SAB-PO kit was purchased from Nichirei Co. (Tokyo, Japan). Bovine–hemoglobin was purchased from Sigma–Aldrich (Steinheim, Germany). 2.2. Cells and cell culture conditions The human hetatoma cell line (HepG2 cell) was purchased from the Riken Cell Bank (Tukuba, Japan). HepG2 cells were cultured as adherent monolayers in Dulbecco’s modified Eagle’s medium (Nissui Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (JRH Bioscience, Lenexa, KS) and 100 U/mL of penicillin and 100 lg/mL of streptomycin, which contains copper 2 lg/dL, zinc 16 lg/ dL and iron 13 lg/dL. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37 C.
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2.3. MT protein expression following the administration of IFNb and Zn 2.3.1. Immunohistochemistry of MT HepG2 (2 · 105 cells/mL) cells were plated on the culture slide overnight. Cells were treated with Zn100 lM-alone, IFN50 U/mL + Zn100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone derived directly into the medium. No-treated cells were used for control. MT protein in HepG2 cells were determined 9 h after administration of IFN and/or Zn. MT protein was stained immunohistochemically described in a previous our study [23]. In brief, HepG2 cells were incubated in absolute methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase activity, and then treated with normal goat serum for 30 min. They were subsequently incubated with rabbit anti-MT antibody. After being washed with PBS, the sections were reacted with biotinylated goat anti-rabbit IgG and then processed with a Histofine SAB-PO kit, which detects MT. MT polyclonal rabbit antibody used in this study recognizes both MT-I and MT-II isoforms of human, rat, or rabbit MT. 2.3.2. MT concentrations MT concentrations in HepG2 cells were assayed by a modified Cd-hem method described by Onosaka and Cherian [24]. In brief, 1 · 106 cells/mL of HepG2 cells were cultured in plastic dish with a diameter of 10 cm. At 90% confluent, the cells were treated with Zn100 lM-alone, IFN50 U/mL + Zn100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone. No-treated cells were used for control. In 24 h after the treatment, the cells were rinsed with phosphate-buffered saline (PBS), then were harvested and resolved with 1.0 ml of 0.25 M sucrose. The cells were ultra-sonicated, and then centrifuged 20,000g for 30 min. Following the protein concentration assay, the recovered supernatant was used for MT assay. The 0.2-ml supernatant sample was mixed with 1.0 ml of 0.03 M Tris–HCl buffer (pH7.8) containing 1 lg Cd and was incubated for 10 min. For removing non-MT-conjugated Cd, the sample was added 0.1 ml of 5% bovine–hemoglobin and was heated for 90 s at 95 C. After cooling on ice, the sample was centrifuged at 10,000g for 5 min. The addition of bovine– hemoglobin and the heart treatment were repeated three times. MT-conjugated Cd in the supernatant was determined by inductively coupled plasma/mass spectroscopy (ICP-MS) (ELAN6100, Perkinelmer, Japan). Mass-tocharge ratio was 114. MT concentration was calculated by assuming that 1 mole of MT(6600) binds 7 mol Cd [25], and was described per milligram of protein concentration. 2.4. Cellular Zn levels following IFNb and Zn administration HepG2 cells adjusted 1 · 106 cells/mL were cultured in T-25 flask. At 90% confluence, Zn100 lM-alone,
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IFN50 U/mL + Zn100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone were treated into the medium for 9 h. No-treated cells were used for control. After removing medium, cells were rinsed twice with 5 ml PBS. The cells were harvested and resolved with 1.0 ml PIERCE T-PER Tissue Protein Extraction Reagent. The cells were ultra-sonicated, and then centrifuged 3,000 rpm for 30 min at 4 C for removing floating cells. The protein concentration of the recovered supernatant was determined by a Protein Assay Kit. Zn levels of the homogenized solution were determined by atomic absorption spectrophotometry. 2.5. Cell cycle analysis HepG2 cells were plated at a density of 1 · 105 cells/mL in T-25 flask for 24 h. The cells were treated by direct addition of Zn100 lM-alone, IFN50 U/mL + Zn 100 lM, IFN100 U/mL + Zn100 lM or IFN100 U/mL-alone into the culture medium. No-treated cells were used for control. In 24 h after the treatment, the media were removed and the cells were rinsed with PBS, then released from the dish by isotonic trypsin. The cells were suspended in the medium and centrifuged at 1000 rpm for 5 min at 4 C. After removing supernatant, the cells were added 10% DMSO and stored at 80 C until use. Before analysis the samples were thawed rapidly at 37 C and stained as follows. Tripsin was added to the cell suspension for 15 min, and then RNase solution was added for 15 min. Cells were stained with propidium iodide (PI; 0.0025% in citrate buffer). Cell fluorescence was measured by FACS scan cytometer (Becton Dickinson, Bedford, USA) and analyzed using ModFit software to determine the distribution of cells in various phases of cell cycle. 2.6. Analysis of the cellular elemental distributions in HepG2 cells using in-air micro-PIXE 2.6.1. Preparation of the sample for in-air micro PIXE analysis HepG2 cells adjusted to 5 · 105 cells/ml were cultured with 1 ml of medium on Mylar foil over night. Cells were pretreated with 1 ml of medium containing 100 lM of BrdU, which can be incorporated into the DNA of proliferating cells [26]. In 24 after BrdU treatment, the medium was replaced with IFNb- and Zn-treated medium as follow: Zn100 lM-alone, IFN50 U/mL + Zn100 lM, IFN100 U/ mL + Zn100 lM or IFN100 U/mL-alone. No-treated cells were served as control. After 9 h culture, the cells were rinsed seven times with THAM (tris-hydroxymethylaminomethane) solution and were cryofixed with liquid nitrogen, and dried in a vacuum for 24 h. Finally, the sample was mounted with adhesive onto the sample holder.
distribution in the HepG2 cells sample. Period and dimensions of the scan were set at 30 min and 70 · 70 lm2 areas, respectively. Precise measurement conditions were reported previously [27]. Net count of element yield was calculated by the PC Software program. Because sulfur (S) count is regarded as representative of the whole cell number [28], the net count ratio of Zn to S were evaluated. 2.7. Statistical analysis For comparison for more than two groups, data were analyzed by a one-way ANOVA, followed by Turky test for multiple comparisons. Probabilities of p < 0.05 were regarded as statistically significance. 3. Results 3.1. Immunohistochemical staining of MT induced by IFN and Zn (Fig. 1) We stained MT protein immunohistochemically in the HepG2 cells. MT staining was markedly observed in the cytoplasm and nuclei of hepatocytes treated with IFN and Zn compared to those with the Zn-alone, the IFNalone or the no-treated control. MT expression was similar between the IFN50 U/mL + Zn100 lM-treated cells and the IFN100 U/mL + Zn100 lM-treated ones. The Zn-alone-treated cells showed higher MT expression compared to the control. MT expression was not enhanced in the IFN-alone-treated cells compared to the control. 3.2. Cellular MT concentrations following administration of IFN and Zn (Table 1) Cellular MT concentrations were significantly higher in the IFN- and Zn-treated cells and the Zn–alone-treated cells than in the IFN-alone-treated cells and the control. MT concentrations were significantly higher in the IFN- and Zntreated cells than the Zn-alone-treated cells. MT levels did not increase in the IFN-alone-treated cells. The data are in line with the degree of MT staining shown in Fig. 1. 3.3. Cellular Zn levels following administration of IFN and Zn (Table 2) Cellular Zn levels were significantly higher in the IFNand Zn-treated cells than the IFN-alone-treated cells and the control. The Zn-alone-treated cells showed significant higher Zn levels compared to those of the IFN-alone-treated cells and the control. Cellular Zn levels did not elevate in the IFN-alone-treated ones. 3.4. Cell cycle analysis by flow cytometry (Table 3)
2.6.2. Analysis for cellular elements using in-air micro PIXE A 3.0-MeV proton beam, 1 lm of beam spot size, accelerated by the TIARA single-ended accelerator at JAEATakasaki, was used to analyze the subcellular elemental
For a quantitative evaluation of cell cycle distribution, the percentage of cells in the G0/1-, S-, and G2/M-phase of IFNand/or Zn-treated cultures were determined. The S-phase
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Fig. 1. Immunohistochemical staining of MT in HepG2 cells induced by IFN and Zn. MT protein is mainly observed in the cytoplasm of HepG2 cells. Combination of IFN and Zn markedly increases MT expression in the cytoplasm and nuclei of the cells compared to the Zn-alone- or IFN-alone-treated cells. Table 1 Cellular metallothionein levels following administration of interferon and/ or zinc IFN (U/mL)
Zn (lM)
Metallothionein levels (lg/mg of protein)
0 0 50 100 100
0 100 100 100 0
0.44 ± 0.07 1.78 ± 0.42a,b 2.31 ± 0.20a,b,c 2.21 ± 0.18a,b,c 0.45 ± 0.04
Metallothionein levels were significantly higher in the IFN + Zn-treated cells and the Zn-alone-treated cells than the IFN-alone-treated cells and the control. The IFN + Zn-treated cells showed significant higher metallothionein levels than the Zn-alone-treated ones.Results are mean values ± SD for five different samples per group. a p < 0.01 significant difference compared to the control (IFNO, ZnO). b p < 0.01 significant difference compared to the IFN100 U/mL-alonetreated cells. c p < 0.05 significant difference compared to the Zn 100 l0M-alonetreated cells.
population was higher in the IFN-treated cells than in the Zn-alone-treated cells and the control. A significant S-phase accumulation in the IFN100 U/mL-alone-treated cells was noted accompanied by a decrease in the G2/M phase, sug-
Table 2 Cellular zinc levels following administration of interferon and/or zinc IFN (U/mL)
Zn (lM)
Zinc level (lg/0.01 g of protein)
0 0 50 100 100
0 100 100 100 0
12.9 ± 1.6 25.0 ± 11.1a,b 26.9 ± 8.7a,b 24.3 ± 7.5a,b 15.0 ± 3.3
The Cellular Zn levels were significantly increased in the IFN + Zn-treated cells and the Zn-alone-treated cells than the IFN-alone-treated cells and the control. The cellular Zn levels were unchanged in the IFN-alonetreated cells. Results are mean values ± SD for eight different samples per group. a p < 0.01 significant difference compared to the control (IFNO, ZnO). b p < 0.05 significant difference compared to the IFN100 U/mL-alonetreated cells.
gesting that IFN may delay S-phase of HepG2 cells. Comparing to the IFN100 U/mL-alone, the combination of Zn100 lM and IFN (50 or 100 U/mL) reduced S-phase population and increased M/G2-phase population. The Zn-alone-treated cells showed a very similar pattern of cell-cycle distribution of the control.
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Table 3 Effect of IFN and zinc on cell cycle distribution of HepG2 cells IFN (U/mL)
Zn (lM)
G0/G1 (%)
S (%)
M/G2 (%)
0 0 50 100 100
0 100 100 100 0
56.9 ± 1.3 56.2 ± 2.5 54.5 ± 3.1 51.8 ± 3.2 52.1 ± 2.2
32.5 ± 1.3 32.9 ± 2.5 35.6 ± 2.5 36.7 ± 2.5 39.7 ± 2.5a,b
10.7 ± 1.3 10.9 ± 2.5 10.0 ± 2.5 11.5 ± 2.5c 8.3 ± 2.5
S-phase population was increased in the IFN-treated cells, but was unchanged in the Zn-alone-treated cells. S-phase accumulation in the IFN100 U/mL-treated cells was accompanied by a decrease in the G2/M phase. The IFN100 U/mL + Zn100 lM-treated cells showed reduced Sphase population and increased M/G2 population compared to those with IFN100 U/mL-alone-treated ones. Zn-alone-treated cells showed a similar pattern of cellcycle distribution to the control. Results are mean values ± SD from four different experiments. a p < 0.05 significant difference compared to the control (IFNO, ZnO). b p < 0.05 significant difference compared to the Zn100 mM-alonetreated cells. c p < 0.05 significant difference compared to the IFN100 U/mL-alonetreated cells.
3.5. The cellular elemental distribution analyzed by in-air micro PIXE 3.5.1. The X-ray spectra of HepG2 cells The yield of Zn was increased in the IFN- and Zn-treated cells and the Zn-alone treated cells compared to the
IFN-alone-treated cells or the control (Fig. 2). The net count ratio (%) of Zn to S (Zn/S) were higher in order of the IFN50 U/mL + Zn100 lM, the IFN100 U/ mL + Zn100 lM, the Zn100 lM-alone, the control and the IFN100 U/mL-alone. There was a significant difference between the IFN50 U/mL + Zn100 lM and theIFN100 U/ mL or the control (Table 4). Thus, the Zn/S ratio gave a good consistency with the cellular Zn levels determined by atomic absorption spectrophotometry. 3.5.2. Elemental maps of the HepG2 cells (Fig. 3) The two-dimensional P maps provide a good representation of the physical shape of the cell and highlight the position of the cell nucleus. Since nuclear membrane consists of double layers of phospholipids, it was found by physical correlation that the cell nucleus corresponded to the P-rich region within the cell [29]. In the control sample, Zn map fitted well with P and S maps showed that Zn is assumed to distribute diffusely in the cytoplasm as well as in the nucleus. Focal localizations of Br were found with a spatial correlation with P, suggesting that Br is possibly accumulated within the nucleus. In the Zn100 lM-alone-treated sample, the increased accumulation of Zn was observed, but no enhanced accumulation of Br was found. When IFNb100 U/mL was administered alone, the increased
Fig. 2. The X-ray spectra of HepG2 cells taken by in-air micro-PIXE. Zn yield is higher in order of the IFN50 U/mL + Zn100 lM-treated cells, the IFN100 U/mL + Zn100 lM-treated cells, the Zn100 lM-alone-treated cells, the IFN100 U/mL-alone-treated cells and the control. Representative X-ray spectra from four experiments are shown.
T. Nagamine et al. / Cytokine 34 (2006) 312–319 Table 4 Count ratio of zinc to sulfur calculated by in-air micro-PIXE IFN (U/mL)
Zn (lM)
Zn/S (%)
0 0 50 100 100
0 100 100 100 0
3.0 ± 0.7 6.8 ± 2.0 11.2 ± 1.3a,b 7.0 ± 1.8 2.1 ± 1.4
The mean of Zn/S ratio was significantly higher in the IFN50 + Zn100 mM-treated cells than the IFN100 U/mL-alone-treated cells and the control. Results are mean values ± SD from four different experiments. a p < 0.05 significant difference compared to the control (IFNO, ZnO). b p < 0.05 significant difference compared to the IFN100 U/mL-alonetreated cells.
localization of Br which fitted partially with the Zn map was observed. IFN-alone treatment did not change cellular Zn distribution. The marked accumulation of Zn and Br were observed in the IFN- and Zn-treated sample. The Br and Zn maps showed a good consistency with the P map. Discrete Br accumulation sites were clearly visible with a strong spatial correlation with Zn accumulation. These findings were
observed predominantly in the Zn100 lM-treated samples (Fig. 4).
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IFN50 U/mL-
and
4. Discussion We and others have previously reported hepatic MT induction by IFNa and IFNb in vivo [4–7]. The present in vitro study confirmed previous results; human IFNb directly induced MT expression within the cytoplasm and nuclei of HepG2 cells. Since the promoter region of MTgene have several metal responsive element and the putative IFN-stimulated responsive element [8,14], Zn and IFN may induce MT synthesis in vivo and in vitro. Sato et al. [5] showed that hepatic MT induction by IFN occurred in mice fed with zincsufficient diet, but failed in mice fed Zn-deficient diet. A similar result was obtained from data. When IFNb was in the culture medium, containing such low Zn concentration as 16 lg/dL, MT synthesis was not induced in the cultured hepatocytes. The combination of IFN and Zn markedly expressed hepatic MT compared to those of Zn-alone. At the cellular level, MT is mainly distributed in cytoplasm and to a lesser extent in nuclei [17,30]. In regenerating liver after partial hepatectomy, MTs sift from cytoplasm to nuclei [15,31]. Cellular distribution of MT is
Fig. 3. Elemental maps of HepG2 cells. P maps provide a good representation of the physical shape of the cell and highlight the position of the cell nucleus. The counter plat of P map is drown (upper panels). Zn map is overlapped with the counter plat of P. In the IFN- and Zn-treated samples, Zn distribution fits so well with P map that Zn is assumed to distribute diffusely in the cytoplasm as well as in the nucleus (middle panels). Focal localizations of Br are found with a spatial correlation with P, suggesting that Br is possibly accumulated within the nucleus. Marked accumulation of Br is observed in the IFN- and Zn-treated samples, whereas Br distribution is not enhanced in the Zn-alone-treated samples (lower panels).
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Fig. 4. Accumulation of zinc and bromine by IFN and Zn treatment. HepG2 cells were pre-incubated with 100 lM BrdU for 24 h. Then, the cells were exposed to IFN 50 U/mL and Zn100 lM for 9 h, and intracellular distribution of Zn and Br were determined by micro-PIXE. In the IFN 50 U/mL- and Zn100 lM-treated sample, accumulation of Br and Zn shows a good consistency with the P map. Discrete Br accumulation sites are clearly visible with a strong spatial correlation with Zn accumulation.
also depending on the cell cycle and that MT translocates to nuclei in the S-phase of cells [15–19]. These studies postulated that the nuclear location of MT has been associated to Zn requirements and interactions with nuclear constituents during the cell cycle. The flow cytometry analysis in the present study showed that IFNb100 U/mL-alone delayed the cell cycle at S-phase, which is in line with previous studies [32,33]. Comparing with the cells in IFN100 U/mL-alone, the combination of IFN100 U/mL and Zn100 lM reduced S-phase population and increased of M/G2-phase population, suggesting that Zn may progress cellular proliferation delayed by IFN [34]. Zn-alone-treated cells showed a similar pattern of the cell cycle distribution to the controls. On the other hand, nuclear MT expression was increased by IFNb in combination with Zn compared to those with IFN-alone or Zn-alone. These findings suggest that IFNb in combination with Zn may produce MT protein synergistically and sift MT from cytoplasm to S-phase nuclei of hepatocytes delayed by IFN treatment. To analyze the cellular elements in the HepG2 cells, we applied in-air micro-PIXE technology in the present study, which can determine the subcellular elemental distribution correctly by focusing the beam spot within 1 lm under in-
air scanning [27]. In order to assess the concentration of these trace elements quantitatively, we analyzed the net count of element yield from the PIXE spectrum for every cell. Since the Zn/S ratio (Table 4) indicated a good correlation with the cellular Zn levels determined by atomic absorption spectrophotometry (Table 2), the in-air microPIXE is regarded as a convenient and useful apparatus for examining the trace elements in cultured cells. MicroPIXE spectra showed the enhanced Zn distribution within the Zn-alone-treated sample, which became more predominant with combination of IFN. Zn distribution was not altered by IFN-alone. On the other hand, IFN-alone treatment slightly increased Br distribution, and Zn-alone unchanged Br distribution in HepG2 cells (Fig. 3). As Br is mainly up-taken into the S-phase nucleus [26], IFN-alone is speculated to delay the cell cycle at the S-stage, which is in agreement with the result of flow cytometry. Interestingly, IFN in combination with Zn markedly increased Br accumulation accompanying a spatial correlation with P map that reflects putative nuclear image [29]. Simultaneously, IFN-induced Br accumulation coincides with spatial Zn accumulation. The findings indicate that IFN in combination with Zn may promote cell proliferation of HepG2 cells, accompa-
T. Nagamine et al. / Cytokine 34 (2006) 312–319
nying redistribution of Zn from cytoplasm to S-phase nucleus. Taken together the data from MT induction, flow cytometry and micro-PIXE analysis, the combination of IFNb and Zn induces MT synthesis, which covalently combines Zn, and sifts MT from cytoplasm to IFN-delayed S-phase nuclei. In addition, the enhanced accumulation of Zn into S-phase nucleus by the combination of IFN and Zn leads us a speculation that MT-bound Zn is probably used for zinc-finger protein synthesis [15–17,20]. Further studies are needed to confirm our hypothesis whether IFN-induced MT may promote the activity of transcription factors with a zinc-finger motif by supplying Zn efficiently. Acknowledgment This work was supported in part by the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Ucar R, Sanwo M, Ucar K, Beall G. Interferons: their role in clinical practice. Ann Allerg Asthma Im 1995;75:377–89. [2] Gutterman JU. Cytokine therapeutics; lessons from interferona. Proc Natl Acad Sci USA 1994;91:1198–204. [3] Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Schifman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 2001;358:958–65. [4] Guevara-Ortiz JM, Castellanos VO, Leon-Chavez BA, Achanzar WE, Brambila E. Interferon alpha induction of metallothionein in rat liver is not linked to interleukin-1,interleukin-6, or tumor necrosis factor alpha. Exp Mol Pat 2005;79:33–8. [5] Sato M, Yamaki J, Oguro T, Yoshida T, Nomura N, Nakajima K. Induction of metallothionein synthesis by interferon alpha/beta in response to zinc in mice. Tohoku J Exp Med 1996;178:241–50. [6] Nagamine T, Takagi H, Hashimoto Y, Takayama H, Shimoda R, Nomura N, et al. The possible role of zinc and metallothionein in the liver on the therapeutic effect of interferon-alpha to hepatitis C patients. Biol Trace Elem Res 1997;58:65–76. [7] Sciavolino PJ, Vilcek J. Regulation of metallothionein gene expression by TNF-alpha and IFN-beta in human fibroblasts. Cytokine 1995;7:242–50. [8] Palmiter RD. Metallothionein facts and frustrations. In: Klaassen CD, editor. Metallothionein IV. Basel: Birkhauser Verlag; 1999. p. 215–21. [9] Cherian MG, Chan HM. Biological functions of metallothionein–A review. In: Suzuki KT, Imura N, Kimura M, editors. Metallothionein III. Basel: Birkhauser Verlag; 1993. p. 87–109. [10] Waalkes M, Goering PL. Metallothionein and other cadmiumbinding proteins: recent developments. Chem Res Toxicol 1990;3:281–8. [11] Schroeder JJ, Cousins RJ. Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures. Proc Natl Acad Sci USA 1990;87:3137–41. [12] Kumari MV, Hiramatu M, Ebadi M. Free radical scavenging actions of metallothionein isoforms I and II. Free Radic Res 1998;29:93–101. [13] Crowthers KC, Kline V, Giardina C, Lynes MA. Augmented humoral immune function in metallothionein-null mice. Toxicol Appl Pharmacol 2000;166:161–72.
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