Zinc status affects p53, gadd45, and c-fos expression and caspase-3 activity in human bronchial epithelial cells

Jessica C. Fanzo1, Scott K. Reaves1, Libin Cui2, Lei Zhu1, John Y. J. Wu1, Yi Ran Wang1, and K. Y. Lei2

1 Department of Nutritional Sciences, University of Arizona, Tucson, Arizona 85721; and 2 Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was designed to examine the influence of zinc depletion and supplementation on the expression of p53 gene, target genes of p53, and caspase-3 activity in normal human bronchial epithelial (NHBE) cells. A serum-free, low-zinc medium containing 0.4 µmol/l of zinc [zinc deficient (ZD)] was used to deplete cellular zinc over one passage. In addition, cells were cultured for one passage in media containing 4.0 µmol/l of zinc [zinc normal (ZN)], which represents normal culture concentrations (Clonetics); 16 µmol/l of zinc [zinc adequate (ZA)], which represents normal human plasma zinc levels; or 32 µmol/l of zinc [zinc supplemented (ZS)], which represents the high end of plasma zinc levels attainable by oral supplementation in humans. Compared with ZN cells, cellular zinc levels were 76% lower in ZD cells but 3.5-fold and 6-fold higher in ZA and ZS cells, respectively. Abundances of p53 mRNA and nuclear p53 protein were elevated in treatment groups compared with controls (ZN). For p53 mRNA abundance, the highest increase (3-fold) was observed in ZD cells. In contrast, the highest increase (17-fold) in p53 nuclear protein levels was detected in ZS cells. Moreover, gadd45 mRNA abundance was moderately elevated in ZD and ZA cells and was not altered in ZS cells compared with ZN cells. Furthermore, the only alteration in c-fos mRNA and caspase-3 activity was the twofold increase and the 25% reduction, respectively, detected in ZS compared with ZN cells. Thus p53, gadd45, and c-fos and caspase-3 activity appeared to be modulated by cellular zinc status in NHBE cells.

apoptosis; zinc-deficient cells; zinc-supplemented cells; lung cancer


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE P53 TUMOR SUPPRESSOR GENE has been termed the guardian of the genome (21) because of its importance in the regulation of cell proliferation, DNA repair, and cell death and in the prevention of genomic alterations (16). p53 is a 393-amino acid transcription factor that regulates many downstream genes involved in DNA repair, cell cycle arrest, and apoptosis (7). p53 protein binds to the promoter region of p21, a cyclin-dependent kinase inhibitor (9) that is able to arrest cells in the G1 phase of the cell cycle. This arrest allows time for cells to repair DNA damage induced by cytotoxic stresses. p53 upregulates growth arrest DNA damage protein (Gadd45), which binds to proliferating cell nuclear antigen (PCNA) and inhibits DNA synthesis (31), allowing for DNA repair. p53 can also induce apoptosis by upregulating Bax (26), an apoptotic potentiating factor, and downregulating Bcl-2, an antiapoptotic protein. p53 has also been shown to repress regulators of cell proliferation, such as c-fos and c-jun (19), both of which are early-response nuclear oncogenes.

p53 is mutated in over 50% of human cancers (15), and many of these mutations occur in the DNA-binding domain of the p53 protein. The DNA-binding domain consists of a loop-sheet-helix structure and two beta -sheets containing two loops. These loops are connected by a zinc atom bound to cysteine and histidine amino acids (5). This zinc atom is important in stabilizing DNA-binding activity of p53 (35). When wild-type p53 is exposed to membrane-permeable zinc chelators or a divalent cation resin, the wild-type protein adopts a mutant conformation and exhibits a decreased ability to bind DNA (13, 35). Furthermore, in zinc-depleted HepG2 cells, p53 mRNA and p53 nuclear protein were increased (29), indicating that zinc influences the expression of p53.

One of the main functions of p53 is to induce apoptosis. Zinc has been shown to influence apoptosis both in vivo and in vitro and can modulate the activity of apoptotic regulators in the cell death pathway (4). Zinc supplementation has been shown to inhibit caspase-3 activity, a major implementer of apoptosis, as well as other caspases (10, 27). Zinc has also been shown to increase the bcl-2/bax ratio in U937 cells pretreated with hydrogen peroxide. This increased ratio is indicative of enhanced cell survival (10). Overall, zinc plays a role as a cytoprotectant by stimulating cell proliferation and inhibiting apoptosis and, ultimately, acts as a regulator of mitosis and cell death in monitoring tissue growth (4). Marginal or subclinical zinc deficiency in certain segments of the U.S. population has been shown to occur (30), and these alterations in a dietary factor, such as zinc, may have an impact on p53 function and expression, as well as overall protection.

The objective of this work was to examine the effects of cellular zinc status on the expression of p53 and p53-related genes in normal human bronchial epithelial (NHBE) cells. Our data suggest that cellular zinc levels in NHBE cells are readily responsive and that p53 expression may be sensitive to these alterations in zinc status. Optimal cellular zinc concentrations in the bronchial epithelial cells may be crucial in order for p53 to perform its protective role in maintaining genomic stability.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and treatment media. The NHBE cell line was purchased from Clonetics (San Diego, CA) and was used as a representative of human lung epithelial cell behavior in response to different levels of zinc. All reagents used in cell culture were obtained from Clonetics and Life Technologies (Grand Island, NY). NHBE cells were maintained in Clonetics' recommended bronchial epithelial growth media (BEGM), which included supplements of 13 g/l bovine pituitary extract, 0.5 g/l hydrocortisone, 0.5 mg/l human recombinant epidermal growth factor, 0.5 g/l epinephrine, 10 g/l transferrin, 5 g/l insulin, 0.1 mg/l retinoic acid, 6.5 mg/l 3,3',5-triiodo-L-thryonine, 50 g/l gentamicin, and 50 mg/l amphotericin B (Clonetics). Media were replaced every other day. Cells reached confluency in approximately 9 to 9.5 days, and nearly confluent cells were subcultured using trypsin-EDTA at a ratio of 1:8 at passage 3 for experimental treatment.

Zinc-free BEGM baseline media in which Clonetics omitted the addition of ZnSO4 was used as the zinc-depleted media. This media consisted of bronchial epithelial basal media with the added growth components and contained residual amounts of zinc (0.4 µmol/l), as detected by flame atomic absorption spectrophotometry. The zinc-free basal medium of 0.4 µmol/l ZnSO4 was suitable without affecting overall growth in NHBE cells and was used as the zinc-depleted medium (ZD). For the other three treatment groups, zinc was added to the media in the form of ZnSO4 so that the only difference between these media was the zinc concentration. For zinc-normal (ZN) media, 4.0 µmol/l ZnSO4 was added to the zinc-free basal media; the zinc-adequate (ZA) media contained 16 µmol/l ZnSO4; and the zinc-supplemented (ZS) media contained 32 µmol/l ZnSO4. The ZN media was used as a comparison to standard culture media and was used as the control group for experiments. The ZA treatment was used as a representative of human plasma zinc levels, and the ZS group was used to represent plasma zinc levels attainable by oral supplementation in humans. After NHBE cells were subcultured into one of the four assigned groups, the cells were cultured overnight in ZN media before changing to their respective media. Cells were then grown in ZD, ZN, ZA, or ZS media for 9 days (1 passage). The cells were harvested for cellular zinc and DNA content, total cellular RNA isolation, nuclear protein extraction, and cell lysate isolation.

Cellular zinc and DNA content determination. Both cells and media were collected by scraping from 100-mm tissue culture plates. Cell suspensions were then centrifuged at 500 g for 5 min at 4°C, and cell pellets were washed twice with phosphate-buffered saline (PBS). Cells were resuspended into 1.5 ml PBS and sonicated for two 30-s intervals. An aliquot of the sonicated cell suspension was used to measure cellular zinc content by flame atomic absorption spectrophotometry (Hitachi, San Jose, CA) as previously described (29). Zinc standard solutions (Fisher) ranging from 0.05 to 1.0 parts per million were used to generate a linear standard curve. The zinc content of the cells was determined based on these zinc reference solutions. In addition, the certified zinc solutions were compared with bovine liver standard reference (U.S. Department of Commerce, National Institute of Standards and Technology, Gaithersburg, MD). Appropriate blanks were employed for all measurements. From the same sample, a small aliquot of the sonicated cell suspension was used to measure cellular DNA content using diphenylamine (37). Data were expressed as cellular zinc per microgram of DNA because a linear relationship between cellular DNA and cell number was previously established (37).

RNase protection assays. Total cellular RNA was isolated from NHBE cells using the RNAqueous kit (Ambion, Austin, TX) according to manufacturer's instructions, and the integrity of the RNA was verified by electrophoresis. The mRNA abundance of human apoptotic genes including p53, gadd45, p21, c-fos, bax, bcl-x, bcl-2, and mcl-1 was measured by RNase protection assay (RPA) using a human-stress 1 multiprobe (Pharmingen, San Diego, CA). The human L32 and glyceraldehyde-3-phosphate dehydrogenase probes were also included in the multiprobe and were used as internal references for normalization. Labeled riboprobes were synthesized using an in vitro transcription system kit with T7 RNA polymerase (Pharmingen) and [alpha -32P]UTP (3,000 Ci/mmol; NEN, Boston, MA).

RPA were performed using the Pharmingen RPA kit. Each sample contained 5 µg total RNA from NHBE cells and 2.9 × 105 counts · min-1 · µl-1 of the multiprobe. The RNA and labeled probe were coprecipitated with ammonium acetate and ethanol and then resuspended in hybridization buffer at 56°C for 13 h. The RNase digestions were performed at 30°C for 45 min, followed by inactivation using a proteinase K cocktail and subsequent precipitation. Protected fragments were separated by PAGE using Quickpoint nucleic acid polyacrylamide minigel (Novex, San Diego, CA). In controls without digestion, only full-length probes were observed. No protected bands appeared in controls in which yeast RNA replaced NHBE RNA, indicating that digestions were complete. RPA gels were dried and exposed to film. Band intensities of protected signals were quantified by a laser densitometer (Molecular Dynamics). The relative mRNA abundance in each sample was expressed as the arbitrary units of the apoptotic gene bands per arbitrary unit of L32 in the same RPA reaction.

Western blot analysis. Nuclear extracts were prepared as previously described by Reaves et al. (29). Western blots were performed as previously described by Gadbois and Lehnert (11) with slight modifications. Nuclear extract (10 µg) was combined with an equal volume of sample loading buffer (20% glycerol, 10% 2-mercaptoethanol, 5% SDS, 200 mmol/l Tris · HCl, pH 6.7, 0.01% bromphenol blue), boiled for 3 min, and then subjected to 10% SDS-PAGE (BioRad, Hercules, CA). After electrophoresis, gels were briefly equilibrated in transfer buffer (20% methanol, 192 mmol/l glycine, 25 mmol/l Tris, 0.05% SDS) before transfer onto nitrocellullose membranes. Transfer was performed at 30 V overnight at 4°C. Equal loading of samples was verified by staining a duplicate gel with Coomassie brilliant blue R-250 and scanning with a laser densitometer to compare optical density units between lanes. After blocking (5% dried, nonfat milk, 10 mmol/l Tris · HCl, pH 8.0, 150 mmol/l NaCl, 0.05% Tween 20) for at least 1 h, blots were then incubated with anti-p53 antibody DO-1 (Santa Cruz Biotech, Santa Cruz, CA), diluted to 0.2 µg/ml in Tris-buffered saline-Tween 20 (TBST) with 5% dried, non-fat milk at 4°C overnight followed by four 10-min washes in TBST. A 1-h incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Santa Cruz Biotechnology), diluted to 0.25 mg/l in TBST with 5% dried, nonfat milk, was followed by four 10-min washes in TBST. Autoradiography was performed utilizing enhanced chemiluminescence according to the manufacturer's instructions (Amersham, Arlington Heights, IL). p53 bands were verified by running human p53-GST fusion protein (Oncogene Research Products, Cambridge, MA) on a lane in each gel. Blots were also stained with amido black and photographed to document equivalent protein loading. Laser densitometry (Molecular Dynamics, Sunnyvale, CA) was used to quantify p53 bands after establishing linearity curves.

Caspase-3 activity assay in Zn-treated NHBE cells. Caspase-3 activity was measured with the ApoAlert CPP32 colorimetric assay kit (Clontech Laboratories, Palo Alto, CA). This method uses a colorimetric assay to monitor cleavage of an acetyl-Asp-Glu-Val-Asp-7-amino-4-p-nitroanilide (Ac-DEVD- p-nitroanilide) substrate, which resembles the caspase-3 cleavage site (18). The cells were cultured in their respective zinc treatments for one passage. Briefly, 2 × 106 cells were lysed in lysis buffer on ice for 10 min. The cell lysates were then centrifuged at 200 g for 3 min at 4°C to precipitate cellular debris. After centrifugation, 50 µl of 2× reaction buffer (Clontech), containing 10 mmol/l dithiothreitol (DTT), and 50 µmol/l conjugated Ac-DEVD-p-nitroanilide substrate were added to the supernatant fractions. The supernatant fractions were then incubated at 37°C for 2 h in a water bath and measured in a colorimetric spectrometer at 405 nm. A negative control using 0.5 µl Ac-DEVD-fluoromethyl ketone, a caspase-3 irreversible inhibitor, was used to indicate specificity. Samples from Jurkat cells induced with 0.25 mg/l anti-human Fas (clone CH11, Upstate Biotech, Lake Placid, NY) for 16 h were used as a positive control for caspase-3 activity. A parallel control reaction that did not contain conjugated substrate was also used, as was a sample from uninduced Jurkat cells. A calibration curve using concentrations ranging from 0 to 20 nmol of p-nitroanilide was established. Values were expressed as the concentration of Ac-DEVD-p-nitroanilide cleaved over the course of a 2-h incubation interval.

Statistical analysis. Statistical analyses were performed using SAS software [general linear models (GLM) release, version 6; SAS Institute, Cary, NC]. The data were analyzed using one-way ANOVA, and the means were further analyzed by least significant differences (LSD). Values were expressed as means ± SE with a statistical probability of P < 0.05 being considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Zinc-deficient medium depleted cellular zinc. NHBE cells were cultured for one passage in zinc treatment media. We purchased basal epithelial media in which zinc was not added (Clonetics). The additive growth factors contained residual amounts of zinc, as determined by atomic absorption (data not shown), and increased the zinc concentration of the media to 0.4 µmol/l. When the cells were cultured directly in this basal medium containing 0.4 µmol/l of zinc, they were able to reach a confluent state that was comparable to cells cultured in the regular medium of 4.0 µmol/l of zinc. This indicates that cell growth was not affected by the low-zinc status of the basal medium, yet the cell is able to maintain a zinc-depleted state. The suitable levels of zinc for bronchial epithelial cells were determined by a dosage curve, with the DNA content per plate as an index of cell growth. From the zinc-depleted levels of zinc, to the higher dosage of zinc supplementation, the level of DNA content was essentially the same (Fig. 1A). In contrast, the cellular zinc content exhibited a dose-dependent increase as the zinc concentration in the media increased (Fig. 1A). Furthermore, cells were cultured for up to two passages in the various zinc treatments, and growth was not significantly different among groups (data not shown).


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Fig. 1.   Cellular zinc levels and DNA content in zinc-depleted (ZD, 0.4 µmol/l zinc), zinc-normal (ZN, 4.0 µmol/l zinc), zinc-adequate (ZA, 16.0 µmol/l zinc), and zinc-supplemented (ZS, 32.0 µmol/l zinc) normal human bronchial epithelial (NHBE) cells. Cells were cultured for 1 passage in basal epithelial growth media (BEGM) with zinc added as a supplement to the ZD medium. Cellular zinc was measured by atomic absorption spectrophotometry, and DNA was measured by the diphenylamine method. Cellular zinc levels and DNA content (A) as well as zinc concentration per microgram DNA (B) were expressed as a percentage of ZN controls. Values are means ± SE from 3 experiments. Different letters indicate significantly different means, P < 0.05. Treatments with the same letters indicate no significant difference.

Culture in zinc-depleted media resulted in significant reductions in cellular zinc. In the ZD cells, cellular zinc concentration was reduced to 34% of their ZN controls (Fig. 1B). Cells cultured in medium containing 16 µmol/l of zinc (ZA) had significantly higher cellular zinc concentrations than both ZN control and ZD cells (Fig. 1B). Cells cultured in 32 µmol/l of zinc (ZS) had nearly 450% higher cellular zinc levels compared with ZN controls. The zinc concentrations of ZA and ZS were significantly different from each other as well (340.0 ± 55.0 vs. 546.4 ± 61.4%). Cellular zinc levels were expressed per cellular DNA to correct for any differences in cell numbers between plates. Growth did not appear to be affected by the medium zinc concentration because no significant differences in DNA were observed among treatment groups (Fig. 1A).

Zinc treatment altered p53 mRNA abundance and p53 nuclear protein levels. p53 mRNA abundance in the ZD cells was nearly 170% higher than the level found in ZN control cells (Fig. 2) and was also significantly higher than both ZA and ZS cells. Interestingly, the p53 mRNA abundance in the ZA group was also increased (184.0 ± 15.7%) compared with the ZN control group but was not different from the ZS group (Fig. 2). Nuclear p53 protein in ZD cells, as measured by Western blot analysis, was significantly higher than the ZN controls (586.9 ± 65.9 vs. 100 ± 13.9%) (Fig. 3). The ZA cells also had increased p53 protein levels (778.3 ± 39.1%) compared with ZN control, but these levels were not significantly different from that of ZD cells (Fig. 3). Interestingly, the ZS group exhibited a drastic increase in the level of nuclear p53 protein (1,712 ± 116.7%) compared with ZN controls and was also significantly different from the ZD and ZA groups as well.


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Fig. 2.   Relative p53 mRNA abundance in ZD (0.4 µmol/l zinc), ZN (4.0 µmol/l zinc), ZA (16.0 µmol/l zinc), and ZS (32.0 µmol/l zinc) NHBE cells. Cells were cultured for 1 passage in BEGM with zinc added as a supplement to the ZD medium. RNase protection products were separated on a polyacrylamide gel and quantitated by laser densitometry. L32 was used as an internal reference, and values are expressed as a percentage of ZN controls. Representative samples from each treatment group are shown at bottom. Values are means ± SE from 4 (ZD and ZA) and 3 (ZN and ZS) experiments. Different letters indicate significantly different means, P < 0.05. Treatments with the same letters indicate no significant difference.



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Fig. 3.   Relative nuclear p53 protein levels in ZD (0.4 µmol/l zinc), ZN (4.0 µmol/l zinc), ZA (16.0 µmol/l zinc), and ZS (32.0 µmol/l zinc) NHBE cells. Cells were cultured for 1 passage in BEGM with zinc added as a supplement to the ZD medium. Nuclear protein extracts were separated on 10% polyacrylamide-SDS gels, transferred onto nitrocellulose membranes, and incubated with anti-p53 antibody. Autoradiography was performed using enhanced chemiluminescence and quantitated by laser densitometry. Values are expressed as a percentage of ZN controls. Representative samples from each treatment group are shown at bottom. Values are means ± SE from 3 (ZD and ZN) and 4 (ZA and ZS) experiments. Different letters indicate significantly different means, P < 0.05. Treatments with the same letters indicate no significant difference.

Zinc treatment affects gadd45 and c-fos mRNA abundance. gadd45 mRNA abundance closely resembled the trend of the p53 mRNA effects, with the ZD group being nearly 100% higher than the ZN control group (Fig. 4). The ZA group was also significantly higher (160.9 ± 19.3%) than the ZN control but was not statistically different from the ZD group (Fig. 4). The ZS cells were not different from the ZN or ZA cells but were significantly lower than the ZD cells (Fig. 4). For c-fos mRNA, only ZS cells had increased mRNA abundance compared with the other groups (ZD, 77.3 ± 23.6%; ZN, 100 ± 17.9%; ZA, 131.3 ± 26.1%; ZS, 180.5 ± 58.7%). The mRNA abundances of bcl-x, bcl-2, mcl-1, bax, and p21 were not significantly different among treatment groups. The lack of difference in p21 (ZD, 87.4 ± 25.5%; ZN, 100 ± 20.9%; ZA, 83.7 ± 32.6%; ZS, 80.6 ± 8.8%) mRNA among treatments may account for the lack of effect of zinc on NHBE cell growth.


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Fig. 4.   Relative gadd45 mRNA abundance in ZD (0.4 µmol/l zinc), ZN (4.0 µmol/l zinc), ZA (16.0 µmol/l zinc), and ZS (32.0 µmol/l zinc) NHBE cells. Cells were cultured for 1 passage in BEGM with zinc added as a supplement to the ZD medium. RNase protection products were separated on a polyacrylamide gel and quantitated by laser densitometry. L32 was used as an internal reference, and values are expressed as a percentage of ZN controls. Representative samples from each treatment group are shown at bottom. Values are means ± SE from 4 (ZD and ZA) and 3 (ZN and ZS) experiments. Different letters indicate significantly different means, P < 0.05. Treatments with the same letters indicate no significant difference.

Caspase-3 activity was depressed by zinc supplementation. Caspase-3 activity was measured in bronchial epithelial cells treated with different zinc concentrations for one passage, and values are shown in Fig. 5. No differences in caspase-3 activity were observed among the ZD, ZN, or ZA cells. However, in the ZS group, caspase-3 activity was significantly reduced compared with the other three groups. These data suggest that supplementation of the media with 32 µmol/l of zinc may exert a protective effect of zinc against apoptosis.


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Fig. 5.   Caspase-3 activity in ZD (0.4 µmol/l zinc), ZN (4.0 µmol/l zinc), ZA (16.0 µmol/l zinc), and ZS (32.0 µmol/l zinc) NHBE cells. Cells were cultured for 1 passage in BEGM with zinc added as a supplement. Caspase-3 activity was measured using a colorimetric enzymatic assay as described in MATERIALS AND METHODS. Values are expressed as the concentration of the Ac-DEVD-p-nitroanilide substrate cleaved after a 2-h interval and represent means ± SE from 4 experiments. Different letters indicate different means, P < 0.05. Treatments with the same letters indicate no significant difference.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was designed to determine the influence of intracellular zinc concentrations on the expression of p53 and p53-regulated genes in human bronchial epithelial cells. We chose this particular cell line as our model for several reasons. Bronchial epithelial cells play an important role in airway defense mechanisms and in the pathogenesis of airway disorders (34) and are also considered to be the progenitors of human bronchial cancer (2). They are more susceptible to developing cancerous tumors because of their direct contact with toxins, DNA-damaging agents, and carcinogens, such as cigarette smoke. With exposure to cigarette smoke, increased proliferation of atypical cells and a loss of cilia often occur in bronchial epithelial cells (2). In addition, with constant stress and exposure to cytotoxins, cell proliferation and renewal rates are high, squamous metaplasia occurs, and the risk of genetic instability increases (36). Thus NHBE cells serve as a good model for studying target genes in human lung carcinogenesis (23), as well as transformation by modulation in gene expression. The use of NHBE cells may establish how p53, a major regulator in cell growth and genetic stability, functions in normal epithelial cells and provide information on how nutrients, such as zinc, play a role in p53 activity and expression.

Lung cancer is the most prominent cancer death among both men and women in the United States (3), and the vast majority of the lung cancers arise from the bronchial epithelium (36). Often in lung cancer a loss of heterozygosity occurs on 17p (20), and p53, which is found on 17p13.1, is often mutated in lung cancer (32). p53 mutations in lung cancer consist of homozygous deletions and point mutations, and p53 mRNA expression is also often low in lung tumors (33). A high incidence of p53 mutations have also been shown to occur in tobacco-related cancers, such as squamous cell carcinomas (22), and in 50-80% of lung cancer cases from cigarette smokers, p53 is mutated (12). In non-small cell lung cancer, the most common mutation in p53 consists of a GC-to-TA point mutation, usually due to a carcinogen found in tobacco, benzo(a)pyrene, from cigarette smoke exposure (6). Because zinc is such an important component in p53 protein structure, DNA binding, and overall function, and because low serum zinc levels are found in patients with bronchogenic carcinoma (8), the effect of zinc status on p53 gene expression and p53 target genes was determined in this study.

To deplete zinc from the cells, cell-permeable chelators or chelating resins were not used in the present studies. Instead, zinc was omitted in the preparation of the basal media, which provided an effective, yet noninvasive, technique to deplete zinc from the cells. Interestingly, the zinc-depleted cells did not exhibit a decrease in cell growth, and morphologically, the cells did not appear to be different among the treatment groups. This depletion over one passage, without altered growth, may be representative of conditions that occur in a marginally zinc-deficient state found in certain subpopulations of the United States (30). The cells treated with a zinc level of 16 µmol/l were used for comparison to normal human plasma zinc levels. The supplementation group of 32 µmol/l of zinc represents a level of zinc that may be attainable for humans without pharmacological concentrations being present (32). NHBE cells in the present study were very responsive to the different zinc concentrations in the media. The zinc-depleted cells had a much lower concentration of zinc than controls, and with higher zinc levels, intracellular zinc concentrations increased dramatically. The intracellular zinc concentration in each treatment group was significantly distinct from each other, indicating that in vitro, epithelial cells are extremely sensitive to slight variations in zinc homeostasis.

Our data suggest that zinc-depleted cells demonstrated a significant increase in p53 mRNA abundance. gadd45 mRNA abundance was also higher in the ZD cells; however, p21, bax, bcl-2, mcl-1, bcl-x, and c-fos mRNA levels were not affected by the zinc depletion. p53 nuclear protein was also increased almost 500% in the ZD group compared with the control group. Reaves et al. (29) also found that in zinc-depleted HepG2 cells, using a low-serum media, p53 mRNA abundance and p53 nuclear protein were increased. However, the mechanism of how zinc deficiency upregulates p53 gene expression remains unclear. One possible mechanism is that NHBE cells may be undergoing oxidative stress, and the oxidation of critical cysteines that regulate DNA binding within the p53 protein may be altered (35). Altered conformation of p53 to a mutant form and depressed ability of p53 to bind DNA at critical cysteine residues have been observed in cells depleted of zinc by culturing with chelators (25). However, this may not be the case in the present studies in view of the gadd45 data. The gadd45 promoter contains p53 DNA-binding response elements in its regulatory regions, and once activated, it can bind to PCNA and promote DNA excision repair as well as G2 arrest (31). With zinc depletion, p53 mRNA is upregulated, and there is a concomitant increase of p53 protein in the nucleus. p53 protein may then activate Gadd45 (growth arrest and DNA damage-inducible protein) at the transcriptional level.

With zinc depletion, p53 gene expression may be increased to induce DNA repair, but not to the extent that it depresses growth rate of these cells. In this study, the increase in p53 and gadd45 genes may be a prerepair event, and the cells may be able to be repaired before decreasing growth. Interestingly, bronchial epithelial cells have been shown to be less sensitive to effects of DNA damage compared with other cell types and have a short-lived G1 and G2 cell cycle arrest response to insult (11). This may explain why cell growth was not affected in the ZD cells. Changes in zinc levels may have also induced a mutant conformational change in p53 that subsequently decreases the ability of p53 to bind to DNA and act as a transcription factor in activating downstream targets, such as p21. In our findings, p21 remained unaltered. Degradation and posttranslational modification of p53 protein may have been altered with zinc treatments as well, which ultimately has an affect on downstream targets in repair and apoptosis.

The ZA group, which was used for comparison to normal zinc levels found in the human plasma, had significantly higher p53 mRNA abundance compared with ZN control levels. This increase was accompanied by a dramatic increase (778% compared with control) in p53 protein, which may have been responsible for the increased gadd45 mRNA in these cells. These results are surprising because the 16 µmol/l of zinc is considered to be the normal plasma zinc level in humans. However, these cells are cultured in an in vitro system and may be preconditioned to lower zinc levels than are found in normal media. Mimicking in vivo conditions is difficult, and bronchial epithelial cells in vitro have demonstrated a sensitive response to zinc. Whether this sensitivity may occur in vivo is uncertain. Perhaps the level of zinc used in the ZA group is considered stressful to the cells, and induction of p53 and gadd45 genes is necessary for repair. With zinc supplementation, DNA synthesis is increased (28). In ZA cells, increased gadd45 may possibly induce DNA repair. This balance between repair and synthesis may not have changed cell growth in these NHBE cells.

The zinc-supplemented cells also had an increase in both p53 mRNA and p53 nuclear protein. The increase in p53 protein is almost 20-fold, which indicates that the cells are accumulating large amounts of p53 in the nucleus, perhaps as part of a stress-response mechanism. This large accumulation of p53 protein in the nucleus may be due to posttranslational modification of p53 protein, such as stabilization of the protein through NH2-terminal phosphorylation, decreased Mdm-2 binding, COOH-terminal acetylation, and nuclear localization signaling. An enhanced phosphorylation, acetylation, or localization of the p53 protein may have played a role in the increased accumulation within the nucleus. Zinc supplementation may have a negative effect on Mdm-2, a protein that degrades p53; however, no studies have examined the effects of zinc on mdm-2 gene expression. Further studies examining p53 conformation and DNA-binding activity will have to be performed to understand the role of zinc in p53 function and activation.

Zinc may be signaling another factor that interacts with the p53 promoter to upregulate transcription and translation. A complex called activator protein-1 (AP-1) is a transcription factor that regulates cell growth and differentiation. It is formed from the dimerization of c-Fos and c-Jun. Once these dimerize, AP-1 can bind and activate the p53 promoter (17). AP-1 has been shown to be responsive to zinc (14), and our supplementation may be inducing c-Fos to dimerize with c-Jun and activate p53 promoter activity. c-fos mRNA was increased in the ZS group, as was p53 mRNA and p53 protein. At present, how zinc affects the relationship of c-fos and p53 is uncertain, and future research examining stress induced by zinc and its effect on these genes will help elucidate their interaction.

In the zinc-supplemented cells, caspase-3 activity was depressed compared with the other three zinc treatment groups. To the best of our knowledge this is the first report of zinc supplementation affecting caspase-3 activity in NHBE cells. Zinc has been shown to inhibit caspase-3 and other caspases in models other than NHBE cells by others as well (1, 24). Perhaps zinc may be acting as a protectant against cell death to promote cell survival and integrity with increased repair.

In these studies, we have examined the effect of both zinc depletion and zinc supplementation on the expression of p53 and p53-regulated genes. Using NHBE cells, we have shown that in both zinc depletion and zinc supplementation, p53 gene expression was increased. In zinc depletion, gadd45 mRNA abundance was higher, whereas in zinc-supplemented cells, c-fos mRNA abundance was increased and caspase-3 activity was depressed. Based on these results, p53 gene expression and downstream targets of p53 may be responsive to cellular zinc status. The mechanism(s) of how zinc influences p53 function and activity is still unclear. These data suggest that suboptimal and physiological levels of zinc in NHBE cells may not only play an integral part in p53 gene expression but also in the expression of downstream target genes.


    ACKNOWLEDGEMENTS

This work was supported by the Arizona Disease Control Research Commission (S. K. Reeves), U.S. Department of Agriculture National Research Initiative Competitive Grant 96-35200-3248, and funds from the University of Arizona Agricultural Experiment Station (K. Y. Lei).


    FOOTNOTES

This research was presented in part at Experimental Biology 2000 and was published in abstract form (9a).

Address for reprint requests and other correspondence: K. Y. Lei, Dept. of Nutrition and Food Science, Univ. of Maryland, 3304 Marie Mount Hall, College Park, MD 20742 (E-mail: dl165{at}umail.umd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 October 2000; accepted in final form 23 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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