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Am J Physiol Cell Physiol 292: C2175-C2184, 2007. First published February 15, 2007; doi:10.1152/ajpcell.00256.2006
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CELLULAR METABOLISM

Zinc deficiency depresses p21 gene expression: inhibition of cell cycle progression is independent of the decrease in p21 protein level in HepG2 cells

Stephen H. K. Wong,1 Yangqing Zhao,1 Norberta W. Schoene,2 Chung-Ting Han,1 Rita S. M. Shih,1 and Kai Y. Lei1

1Department of Nutrition and Food Science, University of Maryland, College Park; and 2Nutrient Requirements and Functions Laboratory, Beltsville Human Nutrition, Research Center, Agricultural Research Service/United States Department of Agriculture, Beltsville, Maryland

Submitted 10 May 2006 ; accepted in final form 7 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The influence of zinc status on p21 gene expression was examined in human hepatoblastoma (HepG2) cells. Cells were cultured for one passage in a basal medium depleted of zinc to induce severely zinc-deficient (ZD) cells or in basal medium supplemented with 0.4, 4.0, 16, or 32 µM zinc to represent mild zinc deficiency (ZD0.4), the amount of zinc in most normal media (ZN), the normal human plasma zinc level (zinc-adequate; ZA), or the high end of plasma zinc attainable by oral supplementation (ZS), respectively. In ZD and ZD0.4 cells, the nuclear p21 protein level, mRNA abundance, and promoter activity were reduced to 40, 70, and 65%, respectively, of ZN cells. However, p21 protein and mRNA levels, as well as p21 promoter activity, were not altered in ZA and ZS cells compared with ZN cells. Moreover, the amounts of acetylated histone-4 associated with the proximal and distal p21 promoter regions, as a measure of p21 promoter accessibility, were decreased in ZD (73 and 64%, respectively) and ZD0.4 (82 and 77%, respectively) cells compared with ZN cells (100 and 100%, respectively). Thus multiple lines of evidence indicate that the transcriptional process of p21 is downregulated by depressed zinc status in HepG2 cells. Furthermore, the transfection of 5 µg of plasmid cytomegalovirus-p21 plasmid, which constitutively expressed p21, was able to normalize the reduction in p21 protein level and cyclin D1-cdk4 complex activity but not the inhibition of cell growth and G1/S cell cycle progression in ZD cells.

p21 mRNA; p21 promoter accessibility; zinc status; human hepatoblastoma cells

CELL PROLIFERATION IS INDUCED by active cyclin/cyclin-dependence kinase (cdk) complexes. Cell cycle progression is impaired by the inhibition of cyclin/cdk complexes by cyclin kinase inhibitors (CKIs). p21WAF1/Cip1, a member of the Cip/Kip family of CKIs (9, 37), is capable of inhibiting the activity of cyclin/cdk complexes. DNA damage induces p21 overexpression, which leads to growth arrest at the G1 and G2 phases (27) or S phase (29). Cellular p21 level is modulated by various mechanisms: transcriptional regulation, epigenetic silencing, and mRNA stability, as well as ubiquitin-dependent and -independent protein degradation. p21 is a short-lived protein, and proteasome inhibition leads to an increase in p21 protein levels and in p21 half-life (26, 33). Basal turnover of p21 controlled by the proteosome does not appear to require ubiquitination (3), although a role for NH2-terminal ubiquitination of p21 has been proposed (5). Thus p21 expression may be regulated posttranslationally by both ubiquitin-dependent and -independent proteasome-mediated degradation (26, 34). Moreover, p21 protein levels have been reported to be regulated by posttranslational modifications such as phosphorylation (23).

As for the transcriptional modulation of p21 expression, both p53-dependent and -independent mechanisms are involved. Two conserved p53-binding sites are located in the p21 promoter. After DNA damage, at least one of these is required for p53 responsiveness (10). The p53-independent activation of p21 transcription involves a variety of transcription factors, including Sp1, Sp3, Ap2, STATs, C/EBP{alpha}, C/EBPbeta, and the bHLH proteins BETA2 and MyoD, which are induced by a number of different signaling pathways (14). Six conserved Sp1-binding sites are located in the proximal promoter of the human p21 gene. Within the p21 promoter, the Sp1 site between –87 and –72 from the start site appeared to be essential for the activation of the p21 promoter by histone deacetylase activity complex (HDAC) inhibitors (16).

Paradoxically, p21 stabilizes cyclin D1-cdk4/cdk6 assembly into active complexes that modulate cell cycle positively (21). The role of p21 as an assembly factor for cyclin D1-cdk4/cdk6 is completely opposite to its function as a cdk inhibitor. Repression of p21 depresses cyclin D1-cdk4/cdk6 complex formation and results in impaired cell cycle progression. In smooth muscle cells, Sp1 repression of p21 transcription mediated by Sp1-binding sites in the proximal p21 promoter reduced formation of the cyclin D1-cdk4/cdk6-p21 complex, whose integrity is essential for G1/S transition, and subsequently inhibited cell growth (19, 20). Moreover, in endothelial cells, the Notch pathway mediated p21 repression and decreased cyclin D1-cdk4 formation and nuclear targeting, which led to reduced retinoblastoma protein phosphorylation, as well as depressed S phase entry and cell cycle progression (28). Thus p21 repression may also lead to depressed cell cycle progression.

Reductions of zinc status may promote apoptosis in certain cell types (13) and depress G1/S cell cycle progression in human hepatoblastoma (HepG2) cells (7). In studies using a membrane-permeable chelator to induce severe zinc deficiency, zinc has been shown to play a structural role in p53 and is essential for its DNA-binding activity, as well as its stability (15). However, membrane-permeable chelators may induce unknown side effects, as well as cause an extremely severe deficiency not achievable by dietary depletion. Thus we have elected to induce zinc deficiency by using low-zinc or zinc-depleted media, without culturing cells with membrane-permeable chelators, to examine the influence of zinc status on gene expression. Cells were cultured for one passage in a basal medium depleted of zinc to induce severely zinc-deficient (ZD) cells or in basal medium supplemented with 0.4, 4.0, 16, or 32 µM zinc to represent mild zinc deficiency (ZD0.4), the amount of zinc in most normal media (ZN), the normal human plasma zinc level (zinc-adequate; ZA), or the high end of plasma zinc attainable by oral supplementation (ZS), respectively. Reductions in cellular zinc are at the same magnitude as those observed in tissue zinc from animals fed zinc-deficient diets and are much less severe than when using membrane-permeable chelators. Compared with ZN cells, both p53 mRNA and nuclear protein abundance were first reported to be increased twofold in ZD cells and normalized by culturing of ZD cells in ZA medium in the last 24 h in HepG2 cells (32). By use of a low-zinc growth medium (ZD), nuclear p53 protein levels were increased about fivefold and twofold in ZD cells compared with ZN cells of normal human bronchial endothelial (NHBE) and normal human aortic endothelial cells (HAECs), respectively. p53 mRNA level was increased 2.5-fold in ZD cells compared with ZN NHBE cells, and it was too low to be quantitated in HAECs (11, 12). Our first observation, that p21 mRNA was not upregulated in the presence of p53 nuclear accumulation, was seen in the ZD NHBE cells. Moreover, in HAECs, although there was no significant difference for p21 mRNA abundance between ZD and ZN cells, there was a trend of 20% reduction in ZD cells.

The present study was designed to test the hypothesis that zinc deficiency would repress the expression of p21 mRNA and nuclear protein, as well as the p21 promoter accessibility and activity, in HepG2 cells. In addition, the hypothesis that, in zinc-deficient HepG2 cells, the impaired cell growth and G1/S cell cycle progression is independent of the depressed p21 expression, was also examined. The reasons for using HepG2 hepatoblastoma cells for the present study are as follows. 1) Normal human hepatocytes do not divide, and the cost for their usage would be financially impractical. 2) HepG2 hepatoblastoma cells will grow for many passages in serum-containing media of variable zinc status to provide sufficient quantities of cells for needed assays. 3) HepG2 cells are known to have normal p21 and p53 genes, and they have been used for studying p21, p53, and signal transduction pathways. 4) HepG2 cells respond well in serum-containing culture systems of variable zinc status, and they do not require the expensive serum-free medium required for normal human cells in primary culture. And 5) the zinc-depleted medium for HepG2 cell culture is lower in zinc than those used for HAECs and NHBE cells and can induce a more severe zinc-deficient state.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and treatment. The human hepatoblastoma cell line, HepG2, was purchased from the American Type Culture Collection (Manassas, VA). Chelex100 ion exchange resin (Bio-Rad, Hercules, CA) was used to deplete zinc from FBS before FBS was added to DMEM (32). The resin was first neutralized to physiological pH with 0.25 M HEPES, pH 7.4, and then mixed with FBS in a 1:4 ratio at 4°C for 2 h. The Chelex100 resin was separated from FBS by centrifugation, followed by filtration through a 0.4-µm filter for sterilization and removal of residual Chelex100 resin. The zinc concentration in this chelexed FBS was <1.0 µM, as determined by flame atomic absorption spectrophotometry (Hitachi, San Jose, CA). The basal medium consisted of DMEM with 10% chelexed FBS, containing <0.1 µM zinc, and was termed the ZD medium. For the other 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. The ZD0.4 medium was prepared by addition of 0.4 µM ZnSO4 to ZD medium. Previous studies have shown that addition of at least 0.4 µM ZnSO4 to ZD medium is required to prevent the marked depression of HepG2 cell growth observed in severe zinc deficiency. The zinc-normal (ZN) and the zinc-adequate (ZA) media were prepared by adding 4.0 and 16.0 µM ZnSO4 to ZD medium to mimic the zinc level observed in normal culture medium and in human plasma, respectively. The zinc-supplemented (ZS) medium consisted of the ZD medium plus 32 µM ZnSO4 and was used to represent the plasma zinc level attainable by oral zinc supplementation in humans. The HepG2 cells were cultured overnight in ZN medium before being changed to their respective medium. Cells were then cultured in ZD, ZD0.4, ZN, ZA, or ZS medium for the indicated time period. In experiments with the rescue treatment, cells were cultured in ZD medium for 4 days and then switched over to ZS medium for the last 2 days of culture before being harvested as ZD+32µM cells.

Determination of cell number and viability, as well as cellular zinc levels. Cells and media were collected from tissue culture plates. Cell number was measured by using a hemocytometer. Cell viability was assessed by the traditional Trypan blue assay. In addition, nonviable cell counts were also determined by NucleoCounter using the NucleoCassette Kit and NucleoView software (ChemoMetec, Aller µd, Denmark), according to the manufacturer's protocol. The system features an integrated fluorescence microscope designed to detect signals from a fluorescent dye, propidium iodide, that intercalate to DNA in the nuclei of nonviable cells. Moreover, cell pellets, resulting from centrifugation at 200 g for 2 min at 4°C, were washed twice with phosphate-buffered saline (PBS), resuspended in 1.5 ml PBS, and sonicated. Cellular zinc content was measured by flame atomic absorption spectrophotometry (model no. 5000; Perkin Elmer, Norwalk, CT) by using standard curves of 0.05–1.0 ppm generated with certified zinc reference solutions (Fisher Scientific, Fair Lawn, NJ), as previously described (32). Furthermore, the certified zinc solutions were compared with Bovine Liver Standard Reference (United States Department of Commerce, National Institute of Standards, Gaithersburg, MD). Appropriate blanks were employed for all measurements. Cellular zinc was calculated as nanograms per million cells and presented as percentage of ZN cells.

Nuclear and cytoplasmic extract preparation. The NE-PER Nuclear and Cytoplasmic Extraction Reagents and the Halt Protease Inhibitor Cocktail Kits (Pierce Biotechnology, Rockford, IL) were used for nuclear and cytoplasmic extract preparation, according to the manufacturer's instructions, based on the method of Smirnova (34). Nuclear and cytoplasmic extracts were then stored in aliquots at –80°C. Protein concentrations were determined by using the BCA Protein Assay Reagent Kit (Pierce). Contamination of nuclear extracts by cytoplasmic proteins or contamination of cytoplasmic extracts by nuclear proteins, detected by Western blot analysis of heat shock protein-90 (Hsp90) or organic cationic transporter, respectively, was routinely found to be <5% in our lab.

Western blot analysis. Forty micrograms of nuclear and cytoplasmic protein were resolved on 10% SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) by using a mini-transfer system (Bio-Rad, Hercules, CA). Membranes were blocked with 5% nonfat dry milk in PBS-T (10 mM phosphate buffer, pH 7.3, 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20) for 1 h at room temperature, before incubation with 1 µg/ml rabbit anti-p21 polyclonal antibody (C-19) from Santa Cruz Biotechnology (Santa Cruz, CA) in PBS-T containing 5% nonfat milk at 4°C overnight. Membranes were then washed three times with PBS-T and blotted with a secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) at room temperature for 1 h, followed by three washes in PBS-T. The protein was visualized by use of Western Blot Luminol Reagent (Santa Cruz Biotechnology) and exposed to film. The optical densities of the protein bands were quantified by the Alpha Innotech Imaging System (San Leandro, CA).

RNase protection assays. Total cellular RNA was isolated from HepG2 cells, using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer's instructions. The integrity of the RNA was verified by RNA gel electrophoresis. The mRNA abundance of p21 was measured by the nonradioactive RNase Protection Assay System (BD Biosciences, San Diego, CA), used according to the manufacturer's instructions. The human GAPDH probe was used as an internal reference for normalization. Biotin-labeled riboprobes were synthesized using Non-Rad In Vitro Transcription Kit with T7 RNA polymerase (BD Biosciences) according to the manufacturer's instructions.

p21 promoter activity. The influence of zinc status on human p21 gene promoter activity was studied by transient transfection of a p21-promoter-luciferase gene into HepG2 cells. This approach was used to provide further evidence that the transcription process is depressed by a reduction in p21 gene promoter activity in zinc-deficient HepG2 cells.

Preparation of luciferase construct. The p21 promoter construct was a kind gift provided by Dr. Lieberman (Columbia University, New York, NY) (38) that consisted of a 2,337-bp fragment of the human p21 promoter (10) containing two p53-binding consensus sites, and with HindIII and XhoI sites added on the ends. It was isolated and inserted into the plasmid pGL3-basic (Promega, Madison, WI) to generate construct pGL3-p21-Luci plasmid. This pGL3-p21-Luci plasmid was transformed into Escherichia coli DH5{alpha} competent cells (Invitrogen) by standard protocol for mass production. The plasmid was prepared by using the Wizard PureFection Plasmid DNA Purification System from Promega.

Transient transfection and luciferase assay. HepG2 cells were transfected by using Tfx-20 reagent, according to the protocol provided by the manufacturer (Promega). HepG2 cells, in DMEM with 10% FBS without antibiotics, were seeded at a density of 2 x 105 cells/well in 24-well plates and cultured for 4 days in DMEM containing 10% chelexed FBS plus 0, 0.4, 4.0, 16.0, or 32.0 µM zinc. Just before transfection, the medium was removed. Transfections were performed in triplicate with 500 ng of the plasmid DNA containing the wild-type p21 promoter luciferase reporter construct and 10 ng of an internal control plasmid, pRL-SV40 (Promega). One hour after transfection, the transfection medium was changed to the respective media and cultured for 2 more days. Luciferase activity was measured in the Luminometer TD-20/20 (Turner Designs, Sunnyvale, CA) by using the Dual-Luciferase Reporter Kit according to recommendations by the manufacturer (Promega). Changes in firefly luciferase activity were calculated and plotted after normalization with changes in Renilla luciferase activity in the same sample.

p21 promoter accessibility. The chromatin immunoprecipitation (ChIP) assay of Lagger et al. (22) was performed using the ChIP Assay Kit (Upstate Biotechnology) according to the manufacturer's recommended protocol for the determination of the association of acetylated histone with the proximal and distal regions of the p21 promoter. This approach was used to verify our contention that depression of the p21 transcription process by zinc deficiency is partially due to depressed p21 promoter accessibility of the proximal and distal (with p53 responsive element) regions of the p21 promoter (see GoGoGoGoFig. 5A). Formaldehyde-cross-linked chromatin was prepared from HepG2 cells, and immunoprecipitation was performed using the ChIP Assay Kit. One-tenth of the DNA from each immunoprecipitation was used in each PCR reaction. All PCRs were performed on a Perkin Elmer GeneAmp PCR System 2400 with Applied Biosystems' Golden Taq polymerase. PCR primers were the same as those published by other researchers (22), which amplified the proximal (5'-GGT GTC TAG GTG CTC CAG GT-3' and 5'-GCA CTC TCC AGG AGG ACA CA-3') and distal (5'-GGT CTG CTA CTG TGT CCT CC-3' and 5'-CAT CTG AAC AGA AAT CCC AC-3') regions of the p21 promoter. Primers for GAPDH promoter (5'-AAA AGC GGG GAG AAA GTA GG-3' and 5'-CTA GCC TCC CGG GTT TCT CT-3') were used as a control. PCR condition was as follows: 95°C for 5 min, once; 95°C for 1 min, 50°C for 1 min, 72°C for 2 min, 35 cycles; and 72°C for 7 min, once. The amplified DNA was separated on 2.0% agarose gel and visualized with ethidium bromide. The optical densities of the DNA bands were quantified by scanning densitometry (Alpha Innotech) and normalized to that of ZN cells.


Figure 1
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  		Fig. 1. Zinc-deficient (ZD) medium depletes cellular zinc. A: human hepatoblastoma (HepG2) cells were cultured for one passage in a basal medium depleted of zinc to induce severe ZD cells or in basal medium supplemented with 0.4, 4.0, 16, or 32 µM zinc to represent mild zinc deficiency (ZD0.4), the amount of zinc in most normal media (ZN), the normal human plasma zinc level (zinc-adequate; ZA), or the high end of plasma zinc attainable by oral supplementation (ZS), respectively. For the rescue treatment, cells were cultured in ZD medium for 4 days and then switched over to ZS medium for the last 2 days of culture before being harvested as ZD+32 µM cells. Cells were counted by use of hemacytometer. Cell no. was expressed as a percentage of ZN controls. Values are means ± SE from 3 experiments. Data were analyzed by 1-way ANOVA. Different letters indicate significantly different means (P < 0.05), while means with the same letter are not significantly different. B: cellular zinc was measured by atomic absorption spectrophotometry. Cellular zinc levels were expressed as a percentage of ZN controls. Values are means ± SE from 3 experiments. Different letters indicate significantly different means (P < 0.05).

 

Figure 2
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  		Fig. 2. Low intracellular zinc status reduces p21 protein level. Nuclear and cytoplasmic proteins (40 µg) prepared from HepG2 cells were separated on SDS-PAGE, Western blotted, and probed with antibody against p21 (C-19). The nuclear or cytoplasmic protein blots were also probed with antibody against Oct-1 (H-65) or actin (I-19), respectively. Optical densities of the protein bands were quantified by the Alpha Innotech Imaging System (San Leandro, CA). The nuclear (A) or cytoplasmic (B) p21 protein band was normalized to the corresponding Oct-1 or actin value, respectively, and expressed as a percentage of ZN cells. Values shown in bar graphs represent means ± SE from 3 separate experiments. Data were analyzed by 1-way ANOVA. Different letters indicate significantly different means (P < 0.05), while means with the same letter are not significantly different.

 

Figure 3
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  		Fig. 3. Low intracellular zinc status reduces p21 mRNA level. Total RNA was isolated from cells by use of RNeasy Kit (Qiagen, Valencia, CA). The abundance of p21 mRNA was measured using a nonradioactive RNase protection assay (RPA) kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. The RPA probe set, which included p21 and GAPDH probe, was from the same manufacturer. Optical densities of the protected p21 bands were quantified by the Alpha Innotech Imaging System and then normalized to GAPDH. Values shown in bar graphs represent means ± SE from 3 separate experiments. Data were analyzed by 1-way ANOVA. Different letters indicate significantly different means (P < 0.05), while means with the same letter are not significantly different.

 

Figure 4
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  		Fig. 4. Low intracellular zinc status depresses p21 promoter activity. HepG2 cells were cultured in media containing different zinc concentrations (ZD, <0.1 µM; ZD0.4, 0.4 µM; ZN, 4 µM; ZA, 16 µM; and ZS, 32 µM zinc supplemented to the basal medium) for 4 days. Thereafter, cells were transiently transfected in serum-free DMEM media using Tfx-20 with 500 ng of luciferase reporter construct (PGL3-p21) containing the p21 promoter, together with 10 ng of pRL-SV40 as an internal control. PGL3 basal vector without carrying any promoter was used as the vector control. After transfection, cells were cultured in corresponding media for 2 more days. For the rescue treatment, cells were cultured in ZD medium for 4 days, transfected, and then switched over to ZS medium for the last 2 days of culture before being harvested as ZD+32 µM cells. Cell extracts were then assayed by the dual-luciferase reporter system, and signals were measured by a luminometer. Values represent means ± SE from 3 experiments. Data were analyzed by 1-way ANOVA. Different letters indicate significantly different means (P < 0.05), while means with the same letter are not significantly different.

 

Figure 5
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  		Fig. 5. Acetylation of histones on the proximal and distal p21 promoter is depressed in ZD HepG2 cells. A: relative positions of PCR primers for the amplification of proximal or distal p21 promoter fragments, shown in a schematic drawing. B: chromatin immunoprecipitation (ChIP) analysis of the p21 proximal promoter was performed with antibodies to acetylated histone-H4 and unspecific antibodies (control), using chromatin isolated from HepG2 cells. C: same as for B but with ChIP analysis for the p21 distal promoter. D: primers for the human GAPDH gene were used as a control. Data shown are representative of 3 independent experiments. AcH4, acetylated histone-H4; No Ab, without antibody.

 
Transient transfection of p21 overexpression plasmid PCMV-p21. HepG2 cells were cultured in media with respective zinc concentrations (ZD = ≤0.1 µM; ZN = 4 µM) for 4 days. Thereafter, cells were transiently transfected in serum-free DMEM media by using Tfx-20 with graded amounts of PCMV-p21 plasmids (1, 3, 5, 7, and 10 µg). PCMV plasmid without carrying p21 gene (PCMV-control) was used as a control. ZD and ZN cells transfected with PCMV control are indicated as ZD + 0 and ZN + 0. The PCMV-p21 plasmid was a kind gift provided by Dr. Ariga (30), which consisted of a constitutive promoter followed by the full-length p21 coding sequence. Both PCMV-p21 and PCMV control plasmids were individually transformed into E. coli DH5{alpha} competent cells (Invitrogen) by standard protocol for mass production. The plasmids were prepared by using Wizard PureFection Plasmid DNA Purification System from Promega. After transfection, cells were cultured in corresponding media for 2 more days. Total proteins were then extracted and subjected to protein expression analysis by Western blot analysis.

Cdk4 immunoprecipitation kinase assay. Cdk4 binds with cyclin D1 to form an active complex that can phosphorylate Rb protein at Ser795 (31, 37). The established method of Ding et al. (8), which measures the phosphorylation of Rb at Ser795, was used. Cells were lysed by using radioimmunoprecipitation assay (RIPA) lysis buffer according to manufacturer's protocol (Santa Cruz Biotechnologies). Total cell lysate was immunoprecipitated overnight at 4°C with mAb anti-cdk4, mAb anti-cyclin D1, or mouse IgG. Immunoprecipitates were then washed twice with the Rb kinase buffer [50 mM HEPES (pH 7.5), 1 mM EGTA, 10 mM KCl, 10 mM MgCl2, and 1 mM dithiothreitol], resuspended in the Rb kinase buffer with the addition of 10 mM ATP and 0.5 µg of recombinant Rb protein (QED Bioscience, San Diego, CA), and incubated with gentle shaking for 30 min at 30°C. The kinase assay was stopped by the addition of SDS sample buffer. The reactants were subjected to 12% SDS-PAGE, immunoblotted with rabbit anti-phosphospecific Rb [pSer795] IgG, stripped, and reprobed with rabbit anti-Rb IgG.

Cell cycle analysis. DNA contents of cells were assayed by fluorescence-activated cell sorting (FACS) by using a FACScalibur cytometer (Becton Dickinson, San Jose, CA). HepG2 cells were cultured in respective zinc media for one passage, trypsinized, washed in PBS (Ca2+, Mg2+ free), and fixed in 70% ethanol. Cells were then stained with propidium iodide. Flow cytometry and FACS analysis (FACScanner, Becton Dickson) were used to quantify the distribution of DNA fluorescence and intensity. Flow cytometric data files were collected and analyzed by use of the CELLQuest program (Becton Dickinson). A total of 10,000 cell events were collected for DNA analyses. Cell cycle distribution percentages of stained nuclei were calculated by using Modfit LT software (Verity Software House, Topsham, ME). The calibration standard LinearFlow green and the DNA QC Particle Kit, for verification of instrument performance, were purchased from Molecular Probes (Eugene, OR) and Becton Dickinson, respectively.

Statistical analysis. The data were analyzed with one-way ANOVA by using SPSS 14.0 Windows software (SPSS, Chicago, IL). The means were further analyzed by least significant differences. Values were expressed as means ± SE, with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Zinc-deficient medium depletes cellular zinc. HepG2 cells were cultured for one passage in the zinc-depleted and supplemented media. Growth as measured by cell number was significantly depressed in ZD and ZD0.4 cells to 63 and 65%, respectively, of ZN cells (Fig. 1A). In addition, no differences in cell number were observed among the ZN, ZA, ZS, and ZD+32 µM cells. Cell viability was very high, at the optimal level, and no difference was detected among all treatments (98 and 99% or higher, as determined by the NucleoCounter method and the Trypan blue exclusion assay, respectively). Cellular zinc levels were expressed per million cells to correct for any differences in cell numbers between plates. In ZD and ZD0.4 cells, cellular zinc concentration was reduced to 43 and 50%, respectively, of ZN controls (Fig. 1B). Moreover, cellular zinc levels in ZA, ZS, and ZD+32 µM cells were 52, 77, and 85% higher, respectively, than in ZN cells. Furthermore, cellular zinc levels in ZS and ZD+32 µM cells were higher than in ZA. Thus the supplementation of ZD cells with ZS medium (ZD+32 µM) for the last 2 days of culture was found to normalize cell growth and cellular zinc level to that of ZN cells. This rescue approach demonstrated the specificity of zinc treatment and the speed of normalization of cell growth and cellular zinc level by zinc replenishment.

Zinc depletion decreases nuclear and cytoplasmic p21 protein as well as p21 mRNA levels. Nuclear p21 protein levels in the ZD and ZD0.4 cells were significantly reduced to 40 and 43%, respectively, of that in ZN cells (Fig. 2A). Cytoplasmic p21 protein levels in ZD and ZD0.4 cells were similarly reduced (Fig. 2B). The p21 mRNA levels in ZD and ZD0.4 cells were almost 30% lower than in ZN cells (Fig. 3). In contrast, no significant differences were detected in nuclear and cytoplasmic p21 protein levels, as well as p21 mRNA abundance among the ZN, ZA, ZS, and ZD+32 µM cells. Most importantly, the rescue approach demonstrated the specificity of zinc treatment and the rapid normalization of both p21 protein and mRNA levels by zinc replenishment.

p21 promoter activity is decreased in zinc-depleted HepG2 cells. Transient transfection of a p21-promoter-luciferase reporter gene into HepG2 cells indicated that the p21 promoter activity of ZD and ZD0.4 cells was significantly reduced to 64 and 66% of that of ZN cells, respectively (Fig. 4). In contrast, no differences were observed among the ZA, ZS, ZD+32 µM, and ZN cells. Thus the supplementation of ZD cells with ZS medium (ZD+32 µM) for the last 2 days of culture was found to normalize the p21 promoter activity to that of the ZN cells. This rescue approach demonstrated the specificity of zinc treatment and the speed of normalization of p21 promoter activity by zinc replenishment.

Zinc depletion reduces the p21 promoter accessibility. To test whether cellular zinc status affects local histone acetylation pattern on the p21 promoter, we performed ChIP experiments with chromatin isolated from HepG2 cells. After cross-linking with formaldehyde, chromatin was sonicated and immunoprecipitated with antibodies against the acetylated form of histone-H4. In addition, ChIP assays were performed in parallel without antibodies to control the specificity of the reaction. Moreover, primers specific for the human GAPDH gene were used as a control. And PCRs were performed with standards consisting of fixed amounts of genomic DNA as a template to assure that amplification reactions were within the linear range. As shown in Fig. 5, the amounts of acetylated histone-H4 associated with the proximal and distal p21 promoter regions were significantly decreased in ZD (73 ± 5 and 64 ± 5%, respectively) and ZD0.4 cells (82 ± 1 and 77 ± 6%, respectively) compared with ZN cells (100 ± 3 and 100 ± 3%, respectively). Although there appeared to be a trend for the amounts of acetylated histone-H4 associated with the proximal or distal p21 promoter region to be lower in ZD than in ZD0.4 cells, the differences were not significant. No significant changes in acetylation at the GAPDH locus were observed among the treatments. In the absence of specific antibodies, no specific PCR products were amplified. Our data demonstrate that, in zinc-deficient HepG2 cells, the reductions in p21 protein and mRNA expression correlate well with the depressed p21 promoter activity and accessibility.

Transfection of the PCMV-p21 plasmid, at 5 µg/plate, normalizes p21 protein level in ZD cells to that in ZN cells. The method of Ariga and colleagues (30), to overexpress p21 by transient transfection of the PCMV-p21 plasmid, was used to normalize the decrease in p21 protein level induced by zinc deficiency. By transfecting graded amounts of PCMV-p21 plasmid (1, 3, 5, 7, and 10 µg/plate), which constitutively expressed p21, the amount of PCMV-p21 at 5 µg/transfection was established to be the right dosage capable of restoring the p21 expression level in the PCMV-p21-transfected ZD cells (ZD + 5) back to the level of ZN control cells (ZN + 0) (Fig. 6). In addition, ZN cells transfected with 5 µg of PCMV-p21 (ZN + 5) demonstrated a 48% increase in p21 protein level compared with ZN control cells (ZN + 0).


Figure 6
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  		Fig. 6. Transfection of p21 expression plasmid PCMV-p21, at 5 µg/plate, restores the p21 protein level in ZD cells back to that of ZN cells. HepG2 cells were cultured in media with respective zinc concentrations (ZD, ≤0.1 µM; ZN, 4 µM) for 4 days. Thereafter, cells were transiently transfected in serum-free DMEM media, using Tfx-20 with graded amounts of PCMV-p21 plasmids [0 (+0), 1 (+1), 3 (+3), 5 (+5), 7 (+7), and 10 µg (+10)]. PCMV plasmid without carrying p21 gene was used as control (PCMV-control). ZD or ZN cells transfected with PCMV-control plasmid are indicated as ZD + 0 or ZN + 0 cells, respectively. After transfection, cells were cultured in corresponding media for 2 more days. Total proteins were then extracted and subjected to protein expression analysis by Western blot analysis. Values are means ± SE from 3 experiments. Data were analyzed by 1-way ANOVA. Different letters indicate significantly different means (P ≤ 0.05), while means with the same letter are not significantly different.

 
Normalization of p21 protein level in ZD-PCMV-p21 cells restores the cyclin D1-cdk4 complex activity to the level of ZN cells. The role of p21 as an assembly factor, for the formation of the active cyclin D1-cdk4 complex, which modulates cell cycle positively, has been reported in several cell models (19, 20, 28). Thus studies were designed to examine whether the repressed p21 is responsible for the reduction in cell growth observed in ZD HepG2 cells. First, cyclin D1-cdk4 complex activity was determined by the standard nonradioactive method of Ding et. al. (8). The experimental design involved the usage of ZN and ZD cells transfected with PCMV-p21 or PCMV-control plasmids. As a measure of cyclin D1-cdk4 complex activity, the level of phosphorylation at Ser795 of a recombinant truncated Rb protein (31, 35) was determined and found to be significantly lower by only 20% in ZD vs. ZN cells (Fig. 7). Most interestingly, after the transfection of 5 µg of PCMV-p21, cyclin D1-cdk4 complex activity was normalized in ZD cells and remained unaltered in ZN cells compared with ZN cells transfected with control plasmid (Fig. 7). In addition, as a control to assess the quality of the lysate, equivalent amounts of the lysate from both ZD and ZN cells transfected with PCMV-p21 or PCMV-control plasmids were electrophoresed by SDS-PAGE and immunoblotted with mAb anti-GAPDH (Fig. 7C) and found to be optimal among all treatments. Thus the results indicated that the small reduction in cyclin D1-cdk4 complex activity appeared to be the result of the decreased p21 protein level in ZD cells.


Figure 7
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  		Fig. 7. Transfection of p21 expression plasmid PCMV-p21, at 5 µg/plate, restores the cyclin D1-cdk4 complex activity in ZD cells back to that of ZN cells. An equivalent amount of lysate (900 µg) was immunoprecipitated (IP) with mAb anti-cdk4 or mouse IgG and washed, and the immunoprecipitated protein was subjected to the cdk4 kinase assay, followed by 12% SDS-PAGE and immunoblotting with anti-Rb [pSer795] IgG (A); it was then stripped and reprobed with anti-total Rb IgG (B). C: an equivalent amount of lysate (100 µg) was subjected to SDS-PAGE and immunoblotted with mAb anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control for the quality of the lysate. Values are means ± SE from 3 experiments. Data were analyzed by 1-way ANOVA. Different letters indicate significantly different means (P ≤ 0.05), while means with the same letter are not significantly different.

 
Inhibition of cell growth and G1/S cell cycle progression in ZD HepG2 cells is independent of the decrease in p21 protein level. Cell growth and cell cycle progression analyses were also determined in the same batch of cells used for the measurement of cyclin D1-cdk4 complex activity after the rescue of p21 level in ZD cells. The result indicated that cell growth remained repressed in ZD-PCMV-p21 cells, with normalized p21 expression, compared with ZD-PCMV control cells. (Fig. 8). Most importantly, in the ZN-PCMV-p21 cells transfected with the same amount of PCMV-p21 plasmid, the marked increase in p21 expression was accompanied by a 30% reduction in cell growth, which demonstrated the growth inhibitory effect of p21 expression at a high level. In addition, consistent with our previous report (7), cell cycle progression was impaired at the G1/S phase in ZD cells (Fig. 9). However, the impaired G1/S cell cycle progression remained unchanged in the ZD-PCMV-p21 cells, with normalized p21 level and cyclin D1-cdk4 complex activity (Fig. 9). Moreover, the G1/S progression was mildly impaired in the ZN-PCMV-p21 cells, with markedly elevated p21 protein level, which indicated that the overexpressed p21 is functionally effective in inhibiting cell cycle progression and growth. Thus these findings suggest that, in ZD HepG2 cells, the decreased p21 protein level is not responsible for the associated inhibition of cell growth and cell cycle progression.


Figure 8
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  		Fig. 8. Zinc deficiency-induced inhibition of cell growth remains unchanged after normalization of p21 protein level. HepG2 cells were cultured in media with respective zinc concentrations (ZD, ≤0.1 µM; ZN, 4 µM) for 4 days. Thereafter, cells were transiently transfected in serum-free DMEM media by using Tfx-20 with either 5 µg of PCMV-p21 or control plasmid. After transfection, the cells were cultured in corresponding media for 2 more days. Cell nos. were counted and expressed as %ZN control. Values are means ± SE from 3 experiments. Data were analyzed by 1-way ANOVA. Different letters indicate significantly different means (P ≤ 0.05), while means with the same letter are not significantly different.

 

Figure 9
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  		Fig. 9. Zinc deficiency-induced inhibition of G1/S cell cycle progression remains unchanged after normalization of p21 protein level. In cell cycle analyses, DNA content of HepG2 cells was assayed by flow cytometry using a FACScalibur cytometer. Cells were cultured in respective zinc concentration (ZD, ≤0.1 µM; ZN, 4 µM) for 4 days. Thereafter, cells were transiently transfected in serum-free DMEM media using Tfx-20 with either 5 µg of PCMV-p21 or control plasmid. After transfection, cells were cultured in corresponding media for 2 more days. Washed cells were fixed in ethanol and stained for DNA content. Flow cytometric data files were collected and analyzed using the CELLQuest program. A total of 10,000 cell events were collected for DNA analyses. Cell cycle distribution percentages of stained nuclei were calculated using Modfit LT software. The calibration standard LinearFlow green and the DNA QC Particle Kit were used for verification of instrument performance. Histograms are representative of 3 independent experiments.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In previous studies (7), we have reported reductions in cell growth and cellular zinc status in ZD HepG2 cells. In addition, cellular zinc status was highly responsive to the zinc content of the culture medium among treatments. Moreover, cell cycle analysis indicated that progression from G1 to S phase was depressed in ZD cells. And no DNA fragments were detected in region M1 of flow cytometric examinations for all treatments, which indicates the presence of few or no apoptotic cells. In view of available data indicating that p21 repression and overexpression may lead to depressed cell cycle progression, we designed the present study to examine the influence of zinc status on the transcriptional process of the p21 gene. In the present study, similar reductions in cell growth for ZD cells and cellular zinc status among zinc-deficient cells were observed, as in our previous studies. Thus the changes in cell growth in ZD cells and cellular zinc status among treatments were similarly replicated in the present studies.

In the present report, we have presented multiple lines of evidence, demonstrating, for the first time, that the transcriptional process and expression of p21 are depressed in zinc-depleted HepG2 cells. The observed reductions of 60% in nuclear p21 protein and almost 30% in p21 mRNA in zinc-depleted cells compared with control ZN cells (Figs. 2 and 3) suggest that the transcriptional process might be compromised in zinc-depleted cells. Since a similar magnitude of reduction for cytoplasmic p21 protein was also observed in zinc-depleted cells, it appears unlikely that the observed reduction in nuclear p21 protein resulted from a decreased transport into the nucleus or an enhanced turnover in the nucleus. One possible explanation for the larger magnitude of reduction in protein level vs. mRNA abundance in ZD cells is that an impaired translational process or enhanced degradation of p21 protein or both may have contributed to the higher magnitude of reduction in p21 protein level in ZD HepG2 cells. Nevertheless, in the present study, we have elected to concentrate our efforts on the transcriptional process by determining the influence of zinc status on the p21 promoter activity and accessibility.

In the present study, the p21 promoter activity was measured by transient transfection of a p21 promoter-reporter gene into HepG2 cells. Marked reductions of 36 and 34% in p21 promoter activity in ZD and ZD0.4 cells, respectively, compared with ZN cells indicate that the transcription process was compromised by zinc deficiency. In addition, the supplementation of ZD cells with ZS medium for the last day of culture was able to normalize the p21 promoter activity to that of ZN cells. This approach substantiates the specificity of zinc supplementation and the speed for normalization of p21 promoter activity by zinc replenishment. Moreover, the similar magnitude of reduction for p21 mRNA level, to almost 30% lower in ZD cells than in controls, compared with the 34–36% reduction for p21 promoter activity, may suggest that depressed promoter activity is mainly responsible for the reduced mRNA abundance.

DNA exists in the form of highly organized chromatin in the eukaryotic nucleus. The nucleosome is the basic unit, with DNA wrapped around an octamer of histones composed of a pair of H2A, H2B, H3, and H4. To activate gene transcription, the highly dense chromatin structure must be disrupted by chromatin remodeling complexes to enhance the accessibility of the promoter of a gene to transcription factors. Histone-modifying enzymes and ATP-dependent remodeling complexes are two major groups of chromatin-modifying complexes capable of disrupting chromatin structure. Among the well-characterized histone-modifying enzymes, histone acetyltransferase is one that acetylates the histone tails, reduces the interaction between DNA and histone, enhances the accessibility of DNA to transcriptional factors, and promotes transcriptional activation (34). The ChIP assay, which employs antibodies against epigenetic protein markers, such as acetylated histones, has been used to determine the chromatin state of individual genes.

Recent ChIP experiments performed by Lagger et al. (22) demonstrated that, on induction by actinomycin D, p53 can replace HDAC at the p21 promoter, and the resultant increase in histone acetylation on the p21 promoter correlates well with the activation of the p21 gene. Similar ChIP studies by Liu et al. (24) reported that the amount of acetylated histones on the p21 promoter, especially the proximal promoter, directly correlates with the extent of p21 expression.

In our ChIP study, we have selected a region proximal to the TATA box as the proximal p21 promoter region. The proximal promoter is the region to which the basal transcription machinery would bind. In addition, we have selected a region containing the first downstream p53-responsive element as the distal p21 promoter region. Both promoter regions are the same as those used by Lagger et al. (22) and are similar to those used by Liu et al. (24). Data from our ChIP experiments indicate that, in ZD and ZD0.4 cells, the amounts of acetylated histone-H4 on the proximal region of the p21 promoter were decreased to 73 and 82% of ZN cells, respectively. Similarly, the amounts of acetylated histone-H4 on the distal region of the p21 promoter were markedly reduced in ZD and ZD0.4 cells to 64 and 77% of ZN cells, respectively. These reductions in acetylated histone-H4 on the proximal and distal regions of the p21 promoter suggest that the p21 promoter in zinc-depleted cells may be less accessible to the transcription machinery. Thus the present ChIP data provide another line of evidence supporting the promoter activity data, indicating that the transcriptional process is depressed in zinc-depleted cells. Furthermore, the magnitude of reductions in histone acetylation on the p21 promoter correlates well with the magnitude of depressed p21 promoter activity and p21 mRNA abundance in zinc-depleted cells.

p21 has been commonly reported as a negative cell cycle regulator by acting as an inhibitor of cyclin/cdk. In this context, a repression of p21 enhances cell cycle progression. However, p21 may also function as a positive cell cycle regulator by functioning as an assembly factor for the cyclin D1-cdk4 complex (21). In this context, repression of p21 impairs cell cycle progression. Recently, impairment of cell proliferation induced by repressed p21 expression has been demonstrated in several cell models, including smooth muscle cells (19, 20), endothelial cells (28), and LoVo colon cancer cells (2). Therefore, the ability of the depressed p21 level induced by zinc deficiency to downregulate cyclin D1-cdk4 activity, which, in turn, contributes to cell growth depression, was investigated. The result indicated that cyclin D1-cdk4 complex kinase activity was significantly lower, by 20%, in ZD cells compared with ZN cells. By restoring the p21 protein level in ZD cells back to the level in ZN cells, with transient transfection of a PCMV-p21 plasmid that constitutively expressed p21, the cyclin D1-cdk4 complex activity in ZD-PCMV-p21 cells was normalized to the same level as in ZN cells. Thus the decrease in p21 protein level in ZD cells appeared to be responsible for the small reduction in cyclin D1-cdk4 complex activity. However, data from both cell growth and cell cycle analyses indicated that impaired cell growth and G1/S cell cycle progression remained unaltered in ZD-PCMV-p21 cells, with restored p21 protein level and cyclin D-cdk4 complex activity. Thus, in ZD HepG2 cells, the decrease in p21 level appeared not to be responsible for the inhibition of cell growth and G1/S progression. Therefore, in ZD HepG2 cells, other effects of zinc deficiency not related to p21 may be responsible for the inhibition of cell cycle progression and growth. Recently, MacDonald (25) reviewed past reports on the role of zinc in growth and cell proliferation. However, the exact mechanism responsible for the inhibition of cell growth induced by zinc deficiency has not been fully established. The author postulated that metalloenzymes (6, 36) such as DNA polymerase, involved in DNA synthesis, may be responsible for the reduction in DNA synthesis and cell growth.

In summary, the present data suggest that the reduction in acetylated histone-H4 on the p21 promoter resulted in a depressed p21 promoter accessibility, which contributed to the decrease in p21 promoter activity and the downregulation of p21 mRNA and protein expression in zinc-depleted HepG2 cells. Moreover, the decreased level of p21 protein appeared not to be responsible for the impaired cell growth and G1/S cell cycle progression in zinc-depleted HepG2 cells. Recent studies in our laboratory (1) have established, in ZD HepG2 cells, a marked reduction in the level of nuclear p300 protein, which is known to be the main coactivator recruited by p53 to responsive elements of a target gene. Apart from the scaffolding role of p300, its function in chromatin remodeling has been identified through the intrinsic histone acetyltransferase (HAT) activity of p300 (18). Thus the marked reduction in nuclear p300, accompanied by an expected reduction in its intrinsic HAT activity, may have contributed to the observed decreased amounts of acetylated histone-H4 associated with the proximal and distal promoter regions of the p21 gene in the ZD cells. Future studies can be designed to establish the influence of cellular zinc status on the interactions and functions of transcription factors, including p300, on the p21 promoter.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the United States Department of Agriculture National Research Initiative Grant 2002-35200-12241 (K. Y. Lei).


   ACKNOWLEDGMENTS
 
The assistance provided by Noella A. Bryden in the analyses of trace minerals in Dr. Richard A. Anderson's laboratory at the Agricultural Research Service/United States Department of Agriculture is gratefully acknowledged. We also acknowledge the initial exploratory efforts on p21 in HepG2 cells provided by Drs. Libin Cui and Ali Alshatwi while in K. Y. Lei's laboratory before April 2003.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Y. Lei, Dept. of Nutrition and Food Science, Univ. of Maryland, 0121 Skinner Bldg., College Park, MD 20742 (e-mail: dlei{at}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.


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