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Articles

Tumor Suppressor Protein p53 mRNA and Subcellular Localization Are Altered by Changes in Cellular Copper in Human Hep G2 Cells^{1 ,2}

Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306-4340

³To whom correspondence should be addressed. E-mail: levenson{at}neuro.fsu.edu.

	ABSTRACT

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Copper toxicity causes hepatic damage that can lead to the development of hepatocarcinoma. Similarly, copper deficiency has been reported to increase hepatocyte tumorigenesis. Thus, the objective of this work was to explore the role of copper toxicity and deficiency in the regulation of the tumor suppressor protein p53. Using Northern analysis, Western analysis, immunocytochemistry and the human hepatoma cell line Hep G2, this work showed that elevations in hepatocyte copper consistent with Wilson’s disease (5.7-fold increase) induced p53 mRNA and confirmed that copper toxicity is correlated with apoptotic cell death. However, Western analysis and immunocytochemistry showed that post-transcriptional mechanisms are a significant part of the process, with p53 translocation from the cytosol into the nucleus of copper-treated cells. Treatment of Hep G2 cells with increasing concentrations of the copper chelator tetraethylenepentamine (TEPA, 0–50 µmol/L, 48 h) reduced cellular copper and increased mean p53 mRNA abundance by over fourfold with nuclear translocation of the wild-type protein. However, TEPA treatment did not result in a loss of cell viability or appear to induce apoptosis.

KEY WORDS: • p53 • apoptosis • Hep G2 • liver

	INTRODUCTION

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The trace element copper is required for the activity of many important enzymes (1) including superoxide dismutase, which scavenges free radicals (2) , cytochrome c oxidase, an integral component of the mitochondrial electron transport chain (3 ,4) , and dopamine ß-monooxygenase, which is required for the synthesis of the catecholamine norepinephrine (5) . The serum protein ceruloplasmin, which has ferrioxidase activity and is essential for normal iron metabolism, is also dependent on the presence of copper (6) . Because of the requirement for copper in these and other enzymes, deficiency symptoms including anemia, neutropenia, cardiovascular abnormalities and neurodegeneration have been reported (7 8 9) .

Although the essentiality of copper has been well documented, copper is also known to be a toxic metal. Copper toxicity can be caused by a mutation in the gene that codes for a copper-transporting P-type ATPase (10 11 12) . This mutation leads to Wilson’s disease (WD)⁴ in humans (13 ,14) or, in rats, produces the Long-Evans Cinnamon rat (LEC) (12) , both of which are characterized by severe copper accumulation. In humans with WD, hepatic copper can be between 5 and 8 times greater than normal (15 ,16) . Copper poisoning can lead to copper toxicity (17) . The resulting tissue damage is particularly pronounced in the liver where copper accumulation can lead to hepatitis, cholagiofibrosis, cirrhosis and hepatocellular carcinoma (13 ,17 18 19 20) .

Copper overload can result in apoptotic cell death (21 22 23) . It has previously been shown that the addition of 250 µmol/L copper to cultured hepatocytes induces apoptosis (22) that appears to be dependent on the tumor-suppressor protein p53 (22 ,23) . The mechanisms responsible for copper regulation of p53-mediated death are not known, but it would be reasonable to hypothesize that they would include an increase in p53 mRNA because other hepatotoxic treatments have been shown to significantly increase p53 gene transcription (24 ,25) . However, only a small (twofold) increase in p53 mRNA in LEC rat liver relative to normal Sprague-Dawley control rats has been reported (26) , suggesting that other mechanisms may be involved. Thus, this study was designed to test the hypothesis that copper-induced hepatocyte apoptosis is mediated largely by post-transcriptional mechanisms involving p53. Furthermore, given that copper deficiency has been associated with tumorigenesis, this study also examined the possible regulation of hepatocyte apoptosis and p53 regulation by copper deficiency.

	MATERIALS AND METHODS

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Cell culture.

Human hepatoma cells (Hep G2), initially isolated from a liver biopsy in a 15-y-old Caucasian male (27) , were obtained from the American Type Culture Collection (ATCC, Rockville, MD). This established cell line was grown in a humidified incubator containing 5% CO₂ and 95% air at 37°C, and maintained in Minimum Essential Medium (MEM, Sigma Chemical, St.Louis, MO) supplemented with 10% calf serum (Cosmic Calf Serum, Hyclone Laboratories, Logan, UT), 500 µg/L gentamicin (Life Technologies, GIBCO BRL, Rockville, MD) and antibiotic-antimycotic solution (Sigma Chemical) containing penicillin (1 x 10⁵ U/L), streptomycin (100 mg/L) and amphotericin B (250 µg/L). At 50–75% confluence (169 ± 24 x 10⁴ cells/well), cells were treated with 200 µmol/L copper as cupric sulfate (18 or 48 h) or the copper chelator tetraethylenepentamine (TEPA, 50 µmol/L, 48 h) (Sigma Chemical) and harvested for cell counting using the vital dye neutral red (n = 6 in two separate experiments). Additional wells were used for measurement of total cellular copper (n = 3) by graphite furnace atomic absorption spectroscopy (Zeeman 5100, Perkin Elmer, Norwalk CT). Copper concentration data were expressed as nmol Cu/mg total protein as measured by the Lowry protein assay (28) . Statistical significance of differences was determined by ANOVA and a post-hoc Dunnett’s test using the statistical program GraphPad Prism (version 3.0, San Diego, CA).

Northern analysis.

Using acid guanidinium thiocyanate-phenol-chloroform extraction (29) , total cellular RNA was collected from cells treated with 200 µmol/L copper (0–24 h, n = 9 in three separate experiments) and 0, 10, 25 and 50 µmol/L TEPA (48 h, n = 6 in two separate experiments) and subjected to Northern analysis as previously described (30) . Briefly, RNA was fractionated by size on a 10 g/L agarose gel containing 0.66 mol/L formaldehyde and transferred to a nylon membrane (GeneScreen, NEN, Boston, MA) by capillary blotting. RNA transfer was confirmed by visualization of ethidium bromide–stained RNA under UV light. Blots were UV cross-linked and stored at 4°C until hybridization overnight at 65°C with random primed ³²P-labeled cDNA probes for p53 (ATCC) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ATCC) as a control (RadPrime DNA Labeling System, Life Technologies, GIBCO BRL). Blots were exposed to Kodak X-OMAT AR film at -80°C. Relative amounts of bound cDNA probe were determined by computer evaluated densitometry (Quantity One Quantification) created by Protein and DNA Imaging (PDI, Boston, MA) and expressed as a function of GAPDH mRNA abundance. Statistical significance of differences was determined by ANOVA and a post-hoc Dunnett’s test using the statistical program GraphPad Prism (version 3.0).

Cellular and nuclear morphology.

Cells were first grown on glass coverslips to 50% confluence. After treatment with 200 µmol/L copper (18 h) or 50 µmol/L TEPA (48 h), live Hep G2 cells were observed microscopically for the formation of cytosolic blebbing associated with apoptosis (n = 6 in two separate experiments). Nuclear staining of cells fixed to coverslips was used to examine the nuclei of cells (31) . Cells were washed 3 times in PBS (pH 7.4) and fixed with 3.7% formaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in PBS for 10 min. Cells were then permeabilized with 0.2% Triton X-100 in PBS. After three additional PBS washes, coverslips were incubated in 4',6-diamidino-2-phenylindole (DAPI, Sigma Chemical) for 10 min, washed in PBS and mounted onto microscope slides with a commercially prepared antifading mounting medium (FluorSave Reagent, Calbiochem-Novabiochem, La Jolla, CA). Nuclei were examined and photographed using a Nikon Microphot-FX equipped with epifluorescence.

Immunocytochemistry.

Cells (n = 6 in two separate experiments) were grown on glass coverslips to permit immunocytochemical localization of p53 during copper toxicity or chelation (32) . After treatment with 200 µmol/L copper (24 h) or TEPA (48 h), cells were washed with PBS (pH 7.4) and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100 in PBS. After three additional PBS washes, coverslips were incubated for 2 h at 37°C with a commercially prepared goat polyclonal immunoglobulin (Ig)G raised to human p53 (DO-1, Santa Cruz Biotechnologies, Santa Cruz, CA) at a concentration of 1:1000 in 100 g/L bovine serum albumin. This antibody was designed to permit detection of both wild-type and mutant p53. An additional antibody, designed to detect the presence of p53 in the mutant conformation, was also used (Pab240, Santa Cruz Biotechnologies) in separate dishes. After three washes in PBS, cells were incubated with a donkey anti-goat IgG (fc fragment specific) antibody conjugated to the fluorescent dye cyanine 3 (Cy3) (Jackson ImmunoResearch Laboratories, Westgrove, PA) for 2 h at 37°C. The secondary antibody was designed to have minimal cross-reactivity with human serum proteins produced by Hep G2 cells. Cells were then rinsed in PBS and mounted onto microscope slides as previously described. For photomicrographs, exposure times were held constant to permit comparisons among treatment groups.

Isolation of nuclear extracts.

Cytoplasmic and nuclear proteins were separated following a modification of previously described procedures (33) . In two separate experiments, cells were grown in 75 mm² flasks. After treatment with copper (200 µmol/L, 24 h) or TEPA (50 µmol/L, 48 h), cells were mechanically dislodged and collected in serum-free media at 4°C. Untreated cells served as controls. After centrifugation at 1500 x g for 5 min at 4°C, the pellet was resuspended in ice-cold buffer A [10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT)] with freshly added protease inhibitor cocktail (Sigma Chemical). After 15 min of incubation at 4°C, 312 µL of 10% NP-40 solution was added per 5 mL of buffer A. Samples were vortexed and centrifuged at 16000 x g for 1 min at 4°C. The supernatant fraction containing cytoplasmic proteins was collected and concentrated. The pellets containing nuclei were resuspended in ice-cold buffer C (20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1mmol/L EDTA, 1 mmol/L DTT) with freshly added protease inhibitor cocktail. Samples were continuously agitated at 4°C for 15 min and centrifuged at 16000 x g for 15 min at 4°C. The supernatant containing nuclear proteins was collected and concentrated. Total protein concentration for both cytosolic and nuclear fractions was determined using the bicinchoninic acid method (Pierce, Rockford, IL).

Western blot analysis.

Cytoplasmic or nuclear proteins (50 µg) were added to an equal volume of sample buffer (0.2 mol/L Tris, pH 6.8, 1% SDS, 30% glycerol, 7.5% mercaptoethanol, 0.1% bromophenol blue), heated at 95°C for 5 min and subjected to SDS-PAGE using a 10% polyacrylamide gel (33) . Samples were transferred to a nitrocellulose membrane on ice and the membrane blocked (Tris buffered saline with 5% nonfat dry milk, 0.1% Tween 20) for 15 min at room temperature. Antibodies to wild-type and mutant p53 (previously described) were diluted 1:250 in blocking solution and incubated overnight with continuous agitation at 4°C. After washing, chemiluminescence was used to detect p53 according to the manufacturer’s protocol (ECL Western Blotting Analysis System, Amersham Pharmacia Biotech, Piscataway, NJ). Autoradiography detected a single band with an approximate molecular weight of 45 kDa. Densitometry was used as previously described to determine relative abundance of p53 in cytosolic and nuclear fractions. Values in the text are means ± SD.

	RESULTS

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Cellular copper concentrations.

Copper treatment increased cellular copper from 0.15 ± 0.03 in untreated control cells to 0.86 ± 0.09 nmol Cu/mg protein (n = 3, P 0.001). Figure 1 shows that increasing concentrations of media TEPA for 48 h resulted in a consistent trend toward a decrease in cellular copper concentrations that reached 50% of control at 50 µmol/L TEPA (0.15 ± 0.03 vs. 0.8 ± 0.02, P 0.05). TEPA treatment did not alter cellular zinc concentrations. The control zinc concentration was 3.36 ± 0.26 nmol Zn/mg protein, whereas TEPA-treated cells contained 3.30 ± 0.14 nmol Zn/mg protein. Normal media, supplemented with 10% serum contained 1.25 µmol Cu/L (by analysis).

View larger version (36K): [in this window] [in a new window]   Figure 1. Effect of tetraethylenepentamine (TEPA) treatment on Hep G2 cellular copper. Cells were incubated in media containing 0–50 µmol/L of the copper chelator TEPA for 48 h. Bars are mean ± SD, n = 3. *Significantly different from control (0 µmol/L TEPA), P 0.05.

There were no significant changes in cell viability after 18 or 48 h of TEPA treatment (Fig. 2 ). Longer treatment times (up to 96 h) with TEPA also did not significantly reduce the number of live cells (data not shown). Additionally, 18 h of copper treatment significantly reduced the number of viable cells that were able to incorporate the vital dye neutral red (P 0.001, Fig. 2 ), with a further reduction in viability at 48 h. Consistent with apoptosis, blebbing of the plasma membrane was observed beginning 18 h after the addition of copper to the media. Examination of nuclear morphology by DAPI staining supported the conclusion that copper induced apoptosis in these cells. Copper treatment resulted in condensation of nuclear material, nuclear fragmentation, dispersion of nuclear aggregates to the edges of the nuclear membranes and nuclear blebbing (Fig. 3 ).

View larger version (31K): [in this window] [in a new window]   Figure 2. Effect of alterations in copper on Hep G2 cell viability. Cells were treated with media containing; 200 µmol/L copper (as cupric sulfate) or 50 µmol/L of the copper chelator tetraethylenepentamine (TEPA) for 18 and 48 h. Viability was determined by counting of cells able to incorporate the vital dye neutral red. Bars are mean ± SD, n = 6 dishes. *Significantly different from controls, P 0.001.

View larger version (54K): [in this window] [in a new window]   Figure 3. Effect of copper treatment on cultured Hep G2 nuclei. Cells (n = 3 dishes) were treated with 200 µmol/L copper for 18 h, fixed with 3.7% formaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI). Photos show representative cells at X300 magnification. Normal control cells had uniformly stained nuclei, whereas copper-treated cells consistently developed chromatin aggregation and budding of nuclear material as shown.

Northern analysis showed that copper treatment significantly increased p53 mRNA abundance compared with untreated cells at 18 (P < 0.05) and 24 h (P < 0.001) (Fig. 4 ). Additionally, 48 h of increasing concentrations of the copper chelator TEPA resulted in a consistent trend toward an increase in p53 mRNA that peaked at 50 µmol/L TEPA (P < 0.01) (Fig. 5 ).

View larger version (49K): [in this window] [in a new window]   Figure 4. Effect of copper on relative p53 mRNA abundance in Hep G2 cells. Cells were treated with 200 µmol/L copper as cupric sulfate for 0–24 h. Total cellular RNA was then collected for Northern analysis. Inset shows representative Northern. Bars represent p53 mRNA abundance expressed as a function of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA abundance; values are means ± SD, n = 9. *Significantly different from control (t = 0), P 0.05. **Significantly different from control (t = 0), P 0.001.

View larger version (34K): [in this window] [in a new window]   Figure 5. Effect of copper chelation on relative p53 mRNA abundance in Hep G2 cells. Cells were treated with 0–50 µmol/L tetraethylenepentamine (TEPA) for 48 h; then, total cellular RNA was collected for Northern analysis. Bars represent p53 mRNA abundance expressed as a function of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA abundance; values are means ± SD, n = 6. *Significantly different from control (0 µmol/L TEPA), P 0.01.

Immunocytochemistry of p53 showed that untreated control Hep G2 cells synthesized p53 that was evenly distributed throughout the cell (cytosol and nucleus) (Fig. 6 ). After TEPA treatment, there was movement of p53 into the nucleus of cells without a significant increase in overall immunodectectable p53. Copper treatment increased the p53 staining intensity approximately twofold (P < 0.01) in copper-treated cells. Immunocytochemistry also showed that p53 was almost exclusively localized to the nucleus of copper-treated cells (Fig. 6) . Western analysis of cytosolic and nuclear p53 showed that most of the p53 was localized to the nucleus of Hep G2 cells (Fig. 7 ). Treatment of Hep G2 cells resulted in a change in the ratio of cytosolic to nuclear p53 with a shift away from cytosolic localization in both TEPA- and copper-treated cells (Fig. 7) .

View larger version (60K): [in this window] [in a new window]   Figure 6. Immunocytochemical localization of p53 in control, tetraethylenepentamine (TEPA)-treated (50 µmol/L, 48 h) and copper-treated (200 µmol/L, 24 h) Hep G2 cells. Photomicrographs are representative of images from dishes at X300 magnification.

View larger version (41K): [in this window] [in a new window]   Figure 7. Western analysis of p53 in the cytosol and nuclei of tetraethylenepentamine (TEPA)-treated (50 µmol/L, 48 h) and copper-treated (200 µmol/L, 24 h) Hep G2 cells. Inset shows representative Western analyses. Bars represent the relative abundance of cytosolic and nuclear p53 for each treatment condition (means ± SD) in two separate experiments.

	DISCUSSION

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The current report confirms previous work showing that copper toxicity induces hepatocyte apoptosis (22) . Hep G2 cells underwent morphological changes and a pattern of nuclear condensation consistent with apoptosis. Specifically, the nuclear blebbing seen in this study after copper treatment occurs only in apoptosis, and not in necrosis (35) . Furthermore, the dispersal of aggregated nuclear material to the inside of the nuclear membrane seen here is indicative of apoptosis, whereas a random pattern would be suggestive of necrosis (35) . The cellular damage appeared to be due at least in part to copper-induced DNA damage (36 37 38) . Compared with other metals such as nickel, iron, cobalt, lead and chromium, copper ions are the most mutagenic (38) . Although much of this damage has been attributed to oxidant injury (38 ,39) , the ability of copper to damage DNA is not due solely to the production of hydroxyl radicals because free radical scavengers appear to be unable to completely eliminate the damage. Also, Cu⁺¹ and Cu⁺² ions are equally effective at causing DNA damage (38) . The DNA strand-breaks caused by copper do not appear to be random (36 37 38) . Rather, there is evidence of high affinity copper-binding sites on double-stranded DNA that show cooperative, saturable copper binding (37) and are susceptible to copper-mediated damage.

The use of the human hepatoma cell line Hep G2 as a model to study the regulation of human p53 by alterations in copper availability is a good one because these cells carry wild-type copies of the p53 gene that codes for a zinc-finger protein. Previous work suggested that the ability of copper to induce hepatocyte apoptosis is dependent on p53 because Huh7 cells with mutant p53 and Hep3b cells without p53 did not undergo apoptosis (22) . The present study shows that copper-induced apoptosis not only increases p53 mRNA, but that essentially all of the p53 is translocated into the nucleus of copper-treated Hep G2 cells after translation. This is consistent with the role of p53 as a nuclear transcription factor that acts to regulate downstream genes involved in apoptosis (40) .

Removal of copper from the media by chelation also resulted in consistent increases in p53 mRNA that were inversely proportional to cellular copper concentrations. As seen in copper treatment, TEPA treatment reduced cytosolic p53 levels. Inexplicably, however, increases in p53 in the nucleus (in both treatment groups) were more easily observed using immunocytochemistry than by Western analysis of nuclear proteins.

In a recent report examining the regulation of p53 by zinc, p53 mRNA abundance in zinc-deficient Hep G2 cells was shown to be elevated by approximately twofold relative to controls (33) . The increases in p53 mRNA that we report here are not the result of zinc chelation or antagonism of zinc by high levels of copper because no changes in cellular zinc accompanied the changes in cellular copper. Furthermore, a significant reduction in cellular zinc was required to produce an increase in p53 and p53 mRNA (33) . However, copper displaces zinc from a variety of metal-binding sites and has been shown to bind to p53 in vitro (41) . Thus, caution is warranted when interpreting the present results because the activity of p53 (DNA binding) may have been compromised by treatment (41) . This is particularly true in the case of TEPA treatment in which there is no cell death although p53 is clearly localized to the nucleus. Thus, the possibility exists that p53 DNA-binding activity has been compromised. This possibility warrants continued investigation, given the role of p53 as a tumor-suppressor protein (42 43 44) . It has been shown repeatedly that the inability to synthesize normal DNA-binding p53 is a significant risk factor for the development of cancerous neoplasms (45 46 47) . Furthermore, the possibility that copper deficiency inhibits the ability of hepatic p53 to bind DNA may also help to explain a previous report showing the induction of hepatic tumorigenicity after copper restriction (48) .

	ACKNOWLEDGMENTS

 
The authors thank Kimberly A. Riddle at the Biological Science Imaging Resource Facility, Florida State University, for her continued microscopy assistance, Charles Badland, also at Florida State University, for his expertise in photography, and Joan Hare, director of the Core Cell Culture Facility at FSU.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 99, April 17–20, Washington, DC [Narayanan, V. S., Martin, A. E. & Levenson, C. W. (1999) Copper-induced apoptosis in Hep G2 cells is preceded by an increase in hsp 70, but not p53, mRNA. FASEB J. 13: A372 (abs.)].

² Supported by National Institutes of Health grant DK50472 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

⁴ Abbreviations used: DAPI, 4',6-diamidino-2-phenylindole; DTT, dithiothreitol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Ig, immunoglobulin; LEC, Long-Evans Cinnamon rat; TEPA, tetraethylenepentamine; WD, Wilson’s disease.

Manuscript received August 21, 2000. Initial review completed September 18, 2000. Revision accepted February 19, 2001.

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