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Department of Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR 97331
* To whom correspondence should be addressed. E-mail: Emily.Ho{at}oregonstate.edu.
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ABSTRACT |
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Introduction |
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B (NFB), and AP-1 transcription factors in rat glioma C6 cells (7). These studies suggest that a decrease in cellular zinc alone causes DNA damage and impairs DNA damage response mechanisms, resulting in a loss of DNA integrity and potential for increased cancer risk. The prostate contains the highest concentrations of zinc compared with other soft tissues in the body (8). Interestingly, as prostate cells develop cancer, zinc concentrations decrease by 60–70% (9). Moreover, the main region of zinc accumulation in the prostate, the peripheral zone, is also the main region of prostate cancer development (9). Although the connections between prostate zinc concentrations and prostate cancer have been well established, the precise function of zinc in the prostate remains unknown. We hypothesized that zinc deficiency would disrupt the function of critical zinc dependent proteins that maintain DNA integrity in prostate cells, ultimately resulting in increased DNA damage.
To further understand the role of zinc in maintaining DNA integrity in the prostate, we used genomic approaches to examine the molecular changes that occur with zinc deficiency. The goal of this study was to investigate the impact of zinc deficiency on DNA damage and subsequent alterations in gene expression and protein function by examining global gene expression changes and alterations in transcription factor binding activities using microarray and transcription factor array technologies in normal prostate epithelial cells (PrEC). These studies will offer insight into the function of zinc in the maintenance of DNA integrity as well as help identify zinc-regulated genes and zinc-dependent transcription factors in the prostate.
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Methods |
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Inductively coupled plasma-optical emission spectroscopy. Zinc levels in ZnDF and ZnAD cells and media were determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cell pellets containing 5 million cells, or 1 mL media, were vortexed with 1 mL 70% ultrapure nitric acid (EMD Omnitrace) overnight. Following incubation, samples were diluted with chelex-treated nanopure water to a 7% acid solution, centrifuged, and analyzed by the Prodigy High Dispersion ICP-OES instrument (Teledyne Leeman Labs) against known standards.
Comet assay. Detection of single-strand DNA breaks were determined by alkali single-cell gel electrophoresis as described by Singh (10). Cells were suspended in 0.5% agarose and applied to microscope slides. Cells were subsequently lysed in Comet lysis buffer (Trevigen) for 1 h, subjected to alkali buffer for 20 min, and underwent electrophoresis. Nuclear material was stained with Sybr-green (Molecular Probes). Fifty cells from 4 independent samples were scored without knowledge of the treatments on a scale of 0–4 for tail migration intensity.
RNA isolation and microarray. Total RNA was isolated from ZnDF and ZnAD PrEC cells using the Qiagen RNeasy Mini kit. RNA integrity was determined using OD260/280 ratios and agarose gel electrophoresis with ethidium bromide staining. Global alterations in RNA transcripts from ZnDF and ZnAD cells were determined using the Affymetrix human genome HG-U133A GeneChip (Affymetrix). Three replicates per treatment group were performed. For microarray analysis, RNA samples were sent to the Center for Genome Research and Biocomputing Core Laboratories at Oregon State University, Corvallis, OR for RNA integrity screening, probe synthesis, hybridization, and scanning according to the GeneChip Expression Analysis technical manual (701021 Rev. 5).
Array data analysis.
GeneSifter software (VizX Labs) was used for all analysis of microarray data. Array data were normalized to the all median signal intensity value for each experiment (each gene chip) and signal values were log base 2 transformed. Genes that did not have present calls for all 3 replicates in at least 1 treatment group were omitted. Fold-change was calculated and genes with 
2- or 
2-fold change in the ZnDF group compared with ZnAD group and a P-value <0.05 after the t test was applied were identified as differentially expressed by zinc deficiency. Genesifter grouped differentially expressed genes according to gene function using Gene Ontology and KEGG databases.
Real-time quantitative PCR.
P53, Calpain 6, tyrosine 3-monooxygenase, nuclear factor of light polypeptide gene enhancer in B-cells 2 (NFB2), ribosomal P2, myeloid cell leukemia sequence 1 (BCL-2 related), metallothionein 1 (MT-1), cyclin-dependent kinase inhibitor 1A (p21), BCL2-associated X protein (BAX), transformed 3T3 cell double minute 2, p53 binding protein (mouse) (Mdm2), growth arrest and DNA-damage-inducible, 
(GADD45), and insulin-like growth factor binding protein 3 (IGF-BP3) were analyzed by quantitative PCR (qPCR). Five micrograms total RNA was reverse transcribed to cDNA using SuperScript First Strand Synthesis system for RT-PCR (Invitrogen). Primer sequences and annealing temperatures are listed in Supplemental Table 1. Presence of double-stranded PCR product was monitored using DyNAmo SYBR Green qPCR kit (New England Biolabs) using the Chromo4 Real Time PCR detections system (MJ Research). Melting curve analysis and agarose gel electrophoresis with ethidium bromide staining was conducted to ensure single PCR product of correct amplicon length. Each sample was run in triplicate. Normalized intensity was determined using the standard curve method. Fold-change for each gene was assessed after normalization of intensity value to β-actin (ACTB).
Western blot. Nuclear extracts were isolated from ZnDF and ZnAD PrEC cells using NE-PER Nuclear Cytoplasmic Extraction Reagents (Pierce). Standard Western blot procedure was preformed using 20 µg nuclear extract loaded and separated on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and transferred onto nitrocellulose membrane (BioRad Laboratories). Antibody dilutions were as follows: p53, 1:1000 [p53 (DO-1), Santa Cruz Biotechnologies]; ACTB, 1:4000 (A5441, Sigma-Aldrich); and secondary antibody concentrations were 1:20,000 (goat anti-mouse IgG-horseradish peroxidase, Santa Cruz). Detection was by SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) with image analysis on an AlphaInnotech photodocumentation system. Quantification of signal intensity was determined using Image J 1.37v (NIH) software. Triplicate samples for each treatment group were analyzed.
Transcription factor array. Transcription factor binding activity was analyzed using Panomics Protein/DNA Array I. Nuclear extracts were prepared as described previously and hybridized to the Protein/DNA Array I membrane as directed by the manufacturer. The membrane was spotted with 54 different consensus-binding sequences, each corresponding to a different transcription factor or family of transcription factors. Chemiluminescence detection of consensus sequence binding was obtained with Hyperfilm ECL. Quantification of signal intensity was determined using Image J software. Triplicate Arrays were conducted per treatment group. Fold-change was calculated as the ratio of the signal intensity as ZnDF:ZnAD for each spot on the membrane.
Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) for p53 was analyzed with the Odyssey Infrared Imaging System (LI-COR Biosciences) using the p53 IRDye 700 Infrared Dye Labeled Oligonucleotides as directed by the manufacturer. Briefly, 5 µg nuclear extract, 50 fmol p53 oligo IRDye 700 Infrared Dye, 1 µg poly [poly(2'-deoxyinosinic-2'-deoxycytidylic acid)], 2 µL 10x binding buffer (1 mol/L Tris, 1 mol/L KCl, 1 mol/L dithiothreitol, and 500 mmol/L EDTA), 2 µL 25mmol/L dithiothreitol plus 2.5% Tween-20, and 2 µL loading dye was incubated at room temperature in a total reaction volume of 20 µL for 30 min. For specific competitor reaction, the sample was incubated for 10 min with 5 pmol of unlabeled p53 oligo before addition of 50 fmol of labeled probe. Reaction mixture was separated on a 6% acrylamide gel at 150 V for 2 h and then imaged and quantified using the Odyssey Infrared Imaging System.
Statistics.
Statistical analysis of microarray data were performed using GeneSifter software as described above. Western blot and EMSA were analyzed by unpaired t test using GraphPad Prism Version 4.01. For the transcription factor array, T-ratio was calculated using the mean fold-change and was compared with 1, representing no change in transcription factor binding ability. The critical 2-tailed P-value was determined using T-distribution table with = 0.05. Values in the text are means ± SEM.
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Results |
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Microarray and qPCR.
A total of 286 of 
22,000 genes represented on the Affymetrix HG-U133A gene chip were significantly differentially expressed at least 2-fold with zinc deficiency. Of these genes, 146 genes were down-regulated (Supplemental Table 2) by zinc deficiency and 140 genes were up-regulated by zinc deficiency (Supplemental Table 3). Further analysis using Genesifter showed that differentially expressed genes were involved in a variety of biological processes, such as cellular metabolism, regulation of physiological processes, transport, cell organization and biogenesis, cell growth, cell homeostasis, and response to extracellular stimuli (Supplemental Fig. 1). Microarray data revealed none of the well-characterized genes involved in regulating zinc homeostasis were differentially expressed. Transcription of metallothionein, which is known to be largely controlled by zinc levels (11), was not differentially expressed. qPCR analysis showed that MT-1 expression tended to be lower in ZnDF than ZnAD cells (P = 0.06; Table 1).
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-1-A, receptor, BCL2-associated athanogene 5, and nucleolar protein 3, which were upregulated; and complement component 7, FOS-like antigen 2, Harakiri, BCL2-interacting protein, p73, and myeloid cell leukemia sequence 1, which were down-regulated. Genes such as p53, p73, BRCA2, and MRE11A have overlapping functions as they are also involved in DNA damage response and repair. Additionally, the gene, X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) was also downregulated. XRCC4 is also involved in DNA repair.
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B2, Fos-like antigen 2, and TP73 were downregulated, whereas p53, metal responsive element binding factor 2, and Kruppel-like factor 6 were upregulated with zinc deficiency. Furthermore, genes directly involved in transcription such as the AT-rich interactive domain 1A (SWI-like), which encodes a protein that is part of the chromatin remodeling complex (SNF/SWI), and polymerase (RNA) II (DNA) directed polypeptide A, which encodes for the protein that makes up the largest subunit of RNA polymerase II, were also upregulated with zinc deficiency. Overall, several key genes that regulate cell cycle progression, apoptosis, DNA damage response and repair, as well as genes that function in transcription and as transcriptional regulators were influenced by zinc deficiency.
Gene expression changes of p53, Calpain 6, tyrosine 3-monooxygenase, NFB2, and myeloid cell leukemia sequence 1 (BCL-2 related) were confirmed by qPCR. ACTB was chosen to normalize copy numbers before assessment of fold-change, because microarray and qPCR analysis of ACTB showed no change in mRNA expression with zinc deficiency in PrEC cells. The direction and magnitude of the fold-changes found with qPCR for these genes were similar to microarray results, with the exception of ribosomal P2 [p53 and Calpain 6, P < 0.05; tyrosine 3-monooxygenase, P = 0.08; NFB2, P = 0.06; myeloid cell leukemia sequence 1 (BCL-2 related), P = 0.16; and ribosomal P2, P = 0.23] (Table 1).
Nuclear p53 protein expression. Western blot analysis revealed a significant increase in nuclear p53 expression in ZnDF PrEC compared with ZnAD PrEC. After normalization to ACTB, the OD for ZnDF cells (0.8606 ± 0.0932) was higher than that for ZnAD cells (0.4386 ± 0.0406; P < 0.05) (Fig. 1A). Importantly, the expression of targets downstream of p53, including p21, BAX, GADD45, Mdm2, and IGF-BP3, showed no significant change in expression with zinc deficiency by qPCR analysis (Table 3).
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Discussion |
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Prostate epithelial cells have a unique ability to accumulate high levels of zinc and have the highest concentration of zinc compared with other soft tissues in the body. It has been hypothesized that zinc accumulation is required for the inhibition of m-aconitase so citrate can accumulate for secretion in prostatic fluid (12,13). Moreover, during malignancy, the ability of the prostate cells to accumulate zinc is lost (14). The mechanisms leading to loss of zinc during prostate cancer progression is unclear but have been linked to aberrant expression of the zinc transporters Zip1,2 and 3 in cancer cells (15,16). A loss of cellular zinc through dietary zinc deficiency may exacerbate or accelerate this process leading to malignancy. Likewise, alterations in a variety of genes, mostly involved in signal transduction, stress response, and metabolism, have also been observed other cell and tissue types (17–19). Ho et al. (4) reported that zinc deficiency in IMR 90 cells, a primary human lung fibroblast cell line, resulted in altered expression of genes involved in stress response, cell signaling, protein degradation, and DNA damage/repair. Studies of zinc deficiency in rats have also shown differential expression of genes involved in growth and metabolism, stress response, and transcription/translation (20,21). Together, these data support the diverse biological role of zinc and indicate regulation of genes involved in DNA damage/repair and transcription by zinc.
Unexpectedly, none of the well-described genes involved in regulating zinc homeostasis, such as metallothionein and zinc transporters, were differentially expressed during zinc deficiency in prostate cells. Metallothionein functions to regulate zinc levels, acts as an antioxidant (due to multiple thiol groups on the molecule), and serves to protect against heavy metal toxicity. Zinc, other heavy metals, and oxidative stress can induce metallothionein expression. Transcription of metallothionein is partly controlled through interactions between the metal-responsive transcription factor 1 and metal response elements (MRE) located in the promoter region (11,22,23). However, the presence of other promoter elements also controls metallothionein expression due to cadmium overload or oxidative stress (24). Increased expression of metallothionein and metal-responsive transcription factor 1 with zinc treatment has been reported in several different prostate cancer cell lines (25,26). Decreased MT-1 expression due to zinc deficiency has been observed in other cell and tissue types (4,20). In most cell types, zinc is often sequestered through binding to metallothionein, keeping free zinc concentrations fairly low. In contrast, in the prostate, there is considerable free zinc available in the cell, much of it bound to citrate. Thus, the control, regulation, and synthesis of metallothionein may be different in prostate cells compared with other cell types. Indeed, variable and low metallothionein expression intensity and localization patterns in normal human prostate samples and normal RWPE prostate epithelial cells have been observed (27). Thus, it is possible there are unique mechanisms to control zinc homeostasis in the prostate that could account for the lack of change in typical zinc-sensitive genes. In this study, zinc deficiency in normal PrEC resulted in a nonsignificant decrease in MT-1 gene expression, a 1.6-fold increased binding of metal response factors to MRE, and no change in zinc transporter expression. These data highlight the unique metabolic responses to zinc depletion that are specific to the prostate and are an important area of future research.
In addition to the direct increases in DNA damage, our data suggest that zinc deficiency in the prostate impaired p53 binding activity, leading to an inability to signal downstream targets responsible for carrying out essential mechanisms involved in DNA damage signaling and repair. Increased gene and/or nuclear p53 protein expression due to zinc deficiency has also been reported in other cell types both in vitro and in vivo (4,7,28–30). Studies have shown that removal of zinc from the DNA binding region of p53 results in a nonfunctional protein that has lost its site-specific DNA-binding activity (3,31). Increased p53 expression without a concomitant increase in downstream targets has also been seen in other cell types such as aortic endothelial cells (30,31). In vivo studies by Fong et al. (32–34) have shown dietary zinc deficiency results in increased esophageal cell proliferation, increased expression of p53, mutations in p53, and the Ha-ras oncogene as well as increased tumor development in rats. However, these effects were modified with zinc replenishment (35). These data further support the essential role of zinc in DNA damage response/repair mechanisms and protection against cancer development.
Many other transcription factors, in addition to p53, contain zinc finger DNA-binding motifs (36). Several studies have indicated that loss of zinc from zinc-dependent enzymes, or mutations in the zinc-finger domain, can result in loss of protein function (2,3,37,38). In this study, 4 of the 16 transcription factors that showed significant increases in DNA binding activity with zinc deficiency were zinc ion binding. In addition to p53, Sp1, RXR, and GATA also contain zinc finger motifs in their DNA binding region (39,40). A hierarchy may exist of proteins/transcription factors in which proteins acquire or lose zinc depending on the tissue type and extent of zinc deficiency. Determining this hierarchy could reveal novel biomarkers for zinc deficiency and identify specific mechanisms by which zinc deficiency may increase prostate cancer risk.
Several other genes involved in DNA damage response were also affected with zinc deficiency, including downregulation of p73, XRCC4, MRE11A, and BRCA2. Recent studies suggest that p73 may also play a role in regulating p53 transcription (41). The BRCA2 gene, which is involved in repair of double-strand DNA breaks through homologous recombination (42), and mutations in BRCA2 have been associated with increased risk of developing breast and prostate cancer (43–46). Finally, protein phosphatase 1A was also upregulated. Protein phosphatase 1A has been shown to be involved in activation of p53, possibly via its dephosphorylation activity (47).
In conclusion, low cellular zinc levels in PrEC cells resulted in DNA damage and altered expression of genes involved in cell cycle, apoptosis, DNA damage, and repair and transcription. Interestingly, zinc deficiency increased both the transcript and nuclear p53 protein expression but did not significantly upregulate the DNA binding activity of p53. In addition, there was no change in gene expression of the p53 downstream targets, p21, BAX, and Mdm2. Although epidemiological studies are often inconsistent (48–52), there is experimental evidence that supports the protective role of zinc against prostate cancer (9). This study confirms the role of zinc in protecting DNA integrity and demonstrates that zinc deficiency may compromise DNA integrity by impairing the function of zinc-dependent proteins involved in the DNA damage response. These data suggest that zinc deficiency may impair cellular mechanisms that respond to and repair DNA damage that could result in an accumulation of DNA mutations and increased cancer risk.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: M. Yan, Y. Song, C. P. Wong, K. Hardin, and E. Ho, no conflicts of interest.
3 Supplemental Tables 1–3 and Supplemental Figures 1 and 2 are available with the online posting of this paper at jn.nutrition.org.
4 Abbreviations used: ACTB, β-actin; BAX, BCL2-associated X protein; BRCA2, breast cancer 2, early onset; Brn-3, POU domain, class 4, transcription; CREB, cAMP responsive element-binding protein; EMSA, electrophoretic mobility shift assay; FAST-1, forkhead box H1; GADD45, growth arrest and DNA-damage-inducible, ; GATA, GATA binding protein; IGF-BP3, insulin-like growth factor binding protein 3; Mdm2, transformed 3T3 cell double minute, p53 binding protein (mouse); MRE, metal response elements; MRE11A, MRE11 meiotic recombination 11 homolog A; MT-1, metallothionein 1; NFB, nuclear factor B; NFB2, nuclear factor of light polypeptide gene enhancer in B-cells 2; p21, cyclin-dependent kinase inhibitor A1; PrEC, prostate epithelial cells; PrEGM, Prostate Epithelial Cell medium; qPCR, quantitative PCR; RXR, retinoid X receptor; Smad 3/4, MAD, mothers against decapentaplegic homolog 3/4; Stat4, signal transducer and activator of transcription 4; TP53 (p53), tumor protein p53; TP73 (p73), tumor protein p73; XRCC4, X-ray repair complementing defective repair in Chinese hamster cells 4; ZnAD, zinc adequate; ZnDF, zinc deficient.
Manuscript received 19 June 2007. Initial review completed 30 July 2007. Revision accepted 3 January 2008.
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LITERATURE CITED |
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