Children's Cancer Institute Australia for Medical Research, Iron Metabolism and Chelation Program, PO Box 81, High Street, Randwick, Sydney, New South Wales 2031, Australia and The Heart Research Institute, Iron Metabolism and Chelation Group, 145 Missenden Road, Camperdown, Sydney, New South Wales 2050, Australia
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Abstract |
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Abbreviations: Act D, actinomycin D; BSA, bovine serum albumin; BSS, Hank's balanced salt solution; cdks, cyclin-dependent kinases; CHX, cycloheximide; DFO, desferrioxamine; EMSA, electrophoretic mobility shift assays; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HIF-1, hypoxia-inducible factor-1
; MEM, Eagle's modified minimum essential medium; PBS, phosphate-buffered saline; PIH, pyridoxal isonicotinoyl hydrazone; TBS, Tris-buffered saline; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; 1,10-P, 1,10-phenanthroline; 311, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone
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Introduction |
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The importance of Fe in tumor cell growth is demonstrated by many in vitro studies in cell culture and in clinical trials (1). These investigations showed that the clinically used Fe chelator desferrioxamine (DFO) (Figure 1) can markedly inhibit the growth of a variety of tumors (1). The ability of DFO and other chelators to prevent tumor growth is probably due to the high Fe requirement of neoplastic cells that is necessary for DNA synthesis (2). Up to the present, most studies in vivo have examined rapidly growing or aggressive neoplasms (e.g. leukemia and neuroblastoma) that could be expected to be affected by Fe deprivation (1,2). The effect of chelators on more slowly growing tumors, e.g. some carcinomas or sarcomas, has not yet been extensively assessed. The fact that studies with DFO and other chelators have demonstrated selective antitumor activity in animal models and clinical trials suggests an exploitable therapeutic window does exist (1,2). However, Fe chelation using DFO suffers from its requirement for long infusions and poor membrane permeability that moderate its efficacy as an antitumor agent (2).
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Previous studies showed that 311 and other chelators markedly increased the mRNA levels of the p53-transactivatable genes WAF1 and GADD45 (7,11). This was interesting, since WAF1 encodes p21CIP1/WAF1, which is a universal inhibitor of cyclin-dependent kinases (cdks) and induces a G1/S arrest (12). The GADD45 gene encodes a molecule critical for DNA repair and cell cycle arrest at G1/S (12). The ability of 311 and DFO to inhibit proliferation and increase WAF1 expression was dependent on their ability to bind Fe, as their Fe complexes had no effect (6). Moreover, the effects of Fe chelators on increasing WAF1 mRNA levels was reversible after Fe supplementation (11). Considering these results in cells with wild-type p53, chelators may increase p53 DNA-binding activity to transactivate its target genes. However, WAF1 may also be transactivated by AP2, Sp1 or the p53 homologs p63 and p73 (13,14). Furthermore, the transactivation of WAF1 after Fe chelation has been demonstrated to occur in cell types with (7,15) or without p53 (11,16). These latter experiments suggest, but do not prove, that a p53-independent mechanism was involved in increasing WAF1 expression. Thus, it is important to assess the role of p53 in transactivation of WAF1 after exposure of cells with wild-type or mutant p53 to Fe chelators.
Very few studies have investigated the effect of potent Fe chelators on the DNA-binding activity of transcription factors. Like many cysteine-containing transcription factors, such as AP1, AP2, Sp1 and NFB (17,18), p53 was found to be subject to redox regulation (19). It is known that cysteines and zinc(II) are essential for p53 DNA-binding activity (19) and disruption of these by reductants/oxidants or metal chelators can abolish DNA-binding activity. In fact, in vitro, the chelator 1,10-phenanthroline (1,10-P) and oxidants result in a decrease in p53 DNA-binding activity (20,21). In contrast, using intact cells, 1,10-P increased p53 transcriptional activity without increasing p53 protein, while DFO was shown to increase p53 levels via activating the hypoxia-inducible factor-1
(HIF-1
) (15).
In the current investigation, we examined the mechanism involved in the chelator-mediated increase in WAF1 expression in cells with mutant or wild-type p53. Our work suggests that a p53-independent pathway responds to Fe chelation to induce WAF1 mRNA levels by a transcriptional and post-transcriptional mechanism. The identification of this p53-independent pathway could be important for discovering specific molecular targets that inhibit cancer cell growth by chelators and other drugs.
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Materials and methods |
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Cell culture
The human SH-SY-5Y neuroblastoma cell line was provided by Mr Hiroki Nishimura (Queensland Pharmaceutical Research Institute, Brisbane, Australia). Human SK-N-MC neuroepithelioma cells, human MCF-7 breast cancer cells and human MRC-5 fibroblasts were obtained from the American Type Culture Collection (Rockville, MD). All cell lines were cultured in Eagle's modified minimum essential medium (MEM) (Gibco, Rockville, MD) containing 10% fetal calf serum (FCS) (CSL, Melbourne, Australia), 2 mM L-glutamine (Sigma), 1% (v/v) non-essential amino acids (Gibco), 100 U/ml penicillin (Gibco), 100 µg/ml streptomycin (Gibco) and 0.28 µg/ml fungizone (Squibb Pharmaceuticals, Montréal, Canada) at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were grown and viability assessed using standard techniques described previously (23).
Preparation of 59Fe-transferrin
Human apotransferrin (Sigma) was labeled with 59Fe (Dupont NEN, Boston, MA) to produce 59Fe2-transferrin, as previously described (23). Unbound 59Fe was removed by exhaustive vacuum dialysis against 0.15 M NaCl buffered to pH 7.4 using 1.4% NaHCO3 (23). Fully saturated diferric transferrin was used in all experiments.
Iron efflux experiments
To assess the ability of the chelator to permeate cell membranes and bind intracellular Fe pools, cells were prelabeled with 59Fe-transferrin (0.75 µM) for 3 h at 37°C using established methods (6). The cells were then placed on ice and washed four times with ice-cold Hank's balanced salt solution (BSS) and then reincubated for 3 h at 37°C in the presence of medium alone (control) or medium containing DFO (550 µM) or 311 (550 µM). The cells were then reincubated with MEM for incubation periods of 3 h at 37°C. The overlying medium was then removed and placed into -counting tubes. The cells were removed from the Petri dishes in 1 ml of BSS using a plastic spatula and transferred to a separate set of
-counting tubes. The radioactivity in the samples were then quantitated using a
-scintillation counter (Wallace 1282 Compugamma; LKB, Turku, Finland).
Antibodies used in western blot analysis
The mouse monoclonal anti-human ß-actin antibodies were from Sigma (clone AC-15) and were used at 1/5000. The rabbit polyclonal anti-human p53 (catalog no. sc-6243) was used at 0.0025 µg/ml, respectively, and was from Santa Cruz Biotechnology (Santa Cruz, CA).
Western blot analysis
Cells were collected and incubated for 20 min at 4°C with the lysis buffer [20 mM HEPES, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 10 mM NaCl, CompleteTM protease inhibitors (Roche; Mannheim, Germany)] and centrifuged at 5000 r.p.m. for 5 min at 4°C. For nuclear extracts, the pellet containing nuclei was vigorously resuspended in lysis solution containing 500 mM NaCl and gently rocked for 1.5 h at 4°C. The lysates were then centrifuged at 10 000 r.p.m. for 15 min at 4°C and the supernatant containing the nuclear proteins collected. The protein concentration of the lysate was assessed with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).
The lysates were then mixed with loading buffer containing 20% ß-mecaptoethanol and loaded at 100 µg per sample onto an SDSPAGE gel consisting of a 4% stacking and 15% resolving gel. After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ) overnight at 4°C. These membranes were then soaked in methanol and immediately blocked with 5% skimmed milk in Tris-buffered saline (TBS) for 2 h at room temperature. Following blocking, the membranes were incubated with the primary antibodies diluted to working concentrations in 5% skimmed milk in TBS for 3 h at room temperature. Membranes were then washed four times in TBS containing 0.1% Tween-20 (Sigma) for 10 min each. After washing, anti-mouse (0.030.1 mg/ml; Sigma) or anti-rabbit (0.050.1 mg/ml; Zymed) antibodies conjugated with horseradish peroxidase were incubated with the membranes for 1 h at room temperature. After washing, the membranes were developed using the ECL PlusTM western blot detection reagent (Amersham Biosciences). The films were scanned and montages assembled in Microsoft Word®. To ensure even loading of proteins, membranes were probed for ß-actin. All densitometric data were normalized to ß-actin.
Indirect immunofluorescence
Cells were plated on a 1 cm2 sterile glass slide in a Petri dish and then incubated at 37°C with control medium or medium containing DFO (150 µM), 311 (25 µM) or Act D (9 nM). The treated cells were then washed three times with phosphate-buffered saline (PBS) and fixed in 4% (w/v) formaldehyde, freshly obtained from paraformaldehyde, in PBS for 20 min at room temperature. After fixation, the cells were extensively washed three times with PBS and the membranes permeabilized at room temperature with 0.2% Triton X-100 in PBS (v/v) for 5 min. The samples were then washed three times with PBS and blocked for 2 h in 1% (w/v) bovine serum albumin (BSA) in PBS at room temperature. Following blocking, the cells were incubated with rabbit anti-human p53 polyclonal antibody (6 µg/ml) in blocking solution for 2 h at room temperature. The cells were then washed four times in PBS for 10 min and the anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC) (20 µg/ml) (Sigma) was added. After the secondary antibody incubation, the cells were again thoroughly washed with PBS and mounted onto a glass slide with ProlongTM antifade mounting medium (Molecular Probes, Eugene, OR). For each experiment, 10 fields were assessed containing a total of more than 500 cells. The experiment was then repeated three times.
Electrophoretic mobility shift assays (EMSA)
Nuclear protein extracts of all cell types were prepared by previously described methods (24). Protein- and DNA-binding reactions were incubated at 4°C overnight and contained 15 µg of nuclear extract in a volume of 20 µl containing 2 mM Tris (pH 7.5), 10 mM NaCl, 1 µg poly(dI·dC), 0.2 mM dithiothreitol, 0.2 mM EDTA and 1% glycerol. All double-stranded oligonucleotides (p53, AP2 and Sp1) were end-labeled with [32P]ATP and 105 c.p.m. was used per reaction. Sequences of all the oligonucleotides are shown in Table I. The double-stranded oligonucleotide for detecting p53 corresponded to the p53 cis-element of the ribosomal gene cluster promoter (25,26). For the analysis of p53 DNA-binding, 100 ng of the p53-specific polyclonal antibody (FL-393; Santa Cruz) were added to each reaction that contained the p53 oligonucleotide (26). Specificity of the anti-p53 antibody was shown by the addition of a 50-fold excess of a p53 oligonucleotide competitor that prevented the interaction. DNAprotein complexes were then resolved on a 4% polyacrylamide gel, run for 2 h at 4°C at 200 V, dried and exposed to X-ray film.
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For transient transfections, SK-N-MC, MRC-5, SH-SY-5Y and MCF-7 cells were used. The SK-N-MC cell line has a mutant p53 gene (29) and was a relevant control to the other cell types that have native p53 (30,31). The plasmid WAF1 12280 was introduced using Lipofectamine® reagent (Gibco BRL, Rockville, MD) according to the manufacturer's instructions. As a relevant negative control, only the empty vector (pGL2-Basic; Promega Corp) without the insert was transfected.
Cells were seeded in 35 mm dishes with MEM containing 10% FCS. When cells reached 70% confluence, the medium was removed and replaced by serum-free MEM. Then 4 µg of WAF1 (12280) and 2 µg of ß-galactosidase plasmid DNA (ß-Galactosidase Enzyme Assay System, Promega catalog no. E2000) were added to the cells and incubated at 37°C for 5 h. The reaction mixture containing plasmid DNA was then replaced with MEM containing 10% FCS and the cells reincubated at 37°C for 20 h. All cell types were then treated with DFO (150 µM), 311 (5, 10 and 25 µM) or Act D (9 nM) for 24 h at 37°C. It should be noted that Act D (9 nM) was used as a relevant positive control as at this concentration it is known to induce WAF1 expression via the classical p53 pathway (30). Luciferase assays (Luciferase Assay System, Promega catalog no. E4030) were performed according to the manufacturer's instructions. The activity of luciferase was normalized to ß-galactosidase (27).
Northern blot analysis
Northern blot analysis was performed by isolating total RNA using the Total RNA Isolation Reagent from Advanced Biotechnologies Ltd (Epsom, UK), as described previously (11). The membranes were hybridized with probes specific for human WAF1 and ß-actin. The WAF1 probe consisted of a 1 kb fragment from pSXV (ATCC catalog no. 79928). The ß-actin probe consisted of a 1.4 kb fragment from human ß-actin cDNA cloned into pBluescript SK- (ATCC catalog no. 37997).
Nuclear run-on transcription assays
In vitro nuclear transcription reactions were performed according to the methods of Greenberg and Ziff (32) and Groudine et al. (33). In brief, isolated nuclei (25 x 107) were incubated in a total volume of 200 µl in buffer composed of 35% glycerol, 10 mM Tris (pH 7.5), 5 mM MgCl2, 80 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 1 mM ATP, GTP and CTP and 150 µCi of [-32P]UTP at 37°C for 45 min. Nuclei were digested with 10 µl DNase I (10 mg/ml; Gibco) at 30°C for 15 min. The reaction mixture was further treated with 2 µl proteinase K (10 mg/ml) and 5 µl yeast tRNA (10 mg/ml) at 37°C for 30 min and the RNA extracted three times with a 1:1 mixture of phenol/chloroform. The labeled RNA was precipitated using isopropanol. The 32P-labeled RNA pellet was resuspended in 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) (pH 7.4), 0.2% SDS, 10 mM EDTA at 5 x 106107 c.p.m./ml. This RNA solution was mixed with an equal volume of 10 mM TES (pH 7.4), 0.2% SDS, 10 mM EDTA, 600 mM NaCl and 2 ml of the RNA solution was hybridized at 65°C for 50 h to DNA immobilized on Hybond-N+ filter (Amersham Biosciences). In a given experiment, each filter was hybridized with the same number of c.p.m. of 32P-labeled RNA.
Five micrograms of DNA in 200 µl of 0.2 N NaOH was heated at 95°C for 5 min, cooled on ice and immobilized onto Hybond-N+ membranes using a slot blot apparatus (Bio-Rad). In the current study, membranes were spotted with human WAF1 cDNA (linearized in pSXV; ATCC catalog no. 79928) and human GADD45 cDNA (linearized in pHu145B2; kindly provided by Dr Albert Fornance, National Cancer Institute, Bethesda, MD). The membrane was also spotted with the pSXV vector alone (non-specific binding control), the loading control human ß-actin (linearized in pBluescript SK-; ATCC catalog no. 37997) and human transferrin receptor 1 (transferrin R1) cDNA (linearized in pCD-TR1; kindly provided by Dr Lucas Kühn, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland). It should be noted that transferrin R1 is post-transcriptionally up-regulated by Fe depletion and was an appropriate negative control in this study using Fe chelators (for a review see 2). After hybridization, the membranes were washed three times with solutions of 2 x SSC (1 x SSC = 0.15 M NaCl and 15 mM sodium citrate, pH 7.0) containing 0.1% SDS at 65°C for 60 min and then 0.1 x SSC and 0.1 x SDS at 65°C for 60 min. The membranes were then exposed to X-ray film (Kodak X-Omat AR) for 27 days at -70°C.
Statistics
Experimental data were compared using Student's t-test. Results were considered statistically significant at P < 0.05.
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Results |
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To investigate the ability of the chelators to bind intracellular Fe pools, cells were labeled with 59Fe-transferrin (0.75 µM) for 3 h at 37°C. The cells were then washed and reincubated for 3 h at 37°C in medium containing DFO or 311 (550 µM). For both cell types, 311 was more effective than DFO at mobilizing internalized 59Fe at all chelator concentrations (Figure 2). Comparing the ability of the chelators to mobilize 59Fe from the two cell types, DFO was less effective at mobilizing 59Fe from SK-N-MC than from MRC-5 cells (Figure 2). The low activity of DFO at removing 59Fe from SK-N-MC cells has been noted previously when comparing these cells with other neoplastic cell types (e.g. BE-2 neuroblastoma cells; 11). Hence, the effect observed for DFO in MRC-5 fibroblasts was not unique. Further, it is important to note that we cannot claim any differences in the activity of the chelators between normal and neoplastic cells using such a small number of cell types. In contrast to the results found for DFO, 311 was more effective at mobilizing 59Fe from SK-N-MC cells than MRC-5 fibroblasts (Figure 2). The high activity of 311 as a function of concentration probably reflects its considerable lipophilicity and marked membrane permeability (6,11). In contrast, DFO is quite hydrophilic and does not readily permeate the plasma membrane (11; for a review see 2).
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The effect of the chelators on p53 protein expression
Western analysis was used to assess nuclear p53 levels following incubation of MRC-5, MCF-7, SH-SY-5Y and SK-N-MC cells with DFO (150 µM) or 311 (25 µM) for 24 h at 37°C (Figure 3AD). As a positive control, cells were incubated with the DNA-damaging agent Act D (9 nM). At this concentration Act D does not prevent transcription and increases p53 protein levels (30). Both DFO and 311 increased p53 2- to 3-fold in MRC-5 cells, while Act D resulted in a marked 6-fold increase (Figure 3A). Similar results were found when treating MCF-7 cells with DFO, 311 or Act D, which resulted in 2-, 5- and 9-fold increases in p53 protein levels, respectively (Figure 3B). For the SH-SY-5Y cell type (Figure 3C), 311 and Act D markedly increased p53 levels to 7- and 15-fold that of the control, while DFO was less effective, increasing it to four times the control value. Hence, in both normal and neoplastic cell types with wild-type p53, chelators could increase p53 protein expression in a similar way. In contrast, when examining SK-N-MC cells, DFO or 311 only slightly increased nuclear p53 compared with the control, while Act D markedly increased it to 4-fold that of the control (Figure 3D). Considering this, it is of interest to note that SK-N-MC cells have mutant p53 (29) while the other cell types all have wild-type protein (30,31). The mutation of p53 in SK-N-MC cells results in a shorter transcript (7,29), indicating a relatively large scale disruption in the gene. In fact, the deletion encompasses a 200300 bp fragment that includes exons 2 and 3 (29). Collectively, our data indicate that DFO or 311 could only increase nuclear p53 protein levels in cells with wild-type but not the mutant gene.
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The effect of DFO and 311 on the DNA-binding activity of other redox-sensitive transcription factors in normal and neoplastic cells
Considering the results showing an increase in DNA-binding activity for p53 after exposure to the Fe chelators (Figure 5), we thought it prudent to examine other redox-sensitive transcription factors that could be affected by intracellular Fe levels and play roles in cellular proliferation. Examining the literature, AP2 and Sp1 are cysteine-containing transcription factors that are subject to redox regulation (17,18) and these molecules play critically important roles in differentiation, proliferation and cell cycle control. Furthermore, the WAF1 promoter has one AP2 and six Sp1 DNA-binding sites (13), and these transcription factors have previously been shown to play important roles in the expression of WAF1. Hence, an increase in DNA-binding activity of AP2 and/or Sp1 to the WAF1 promoter could possibly explain the p53-independent increase in WAF1 mRNA expression seen in SK-N-MC and K562 cells after exposure to chelators (11).
AP2 DNA-binding activity
Incubation of control cell lysates with the AP2 oligonucleotide (Table I) resulted in three major bands (Figure 6A, lane 2) that were not present when the probe alone was run (Figure 6A, lane 1). Addition of a 50-fold excess of specific competitor (i.e. non-labeled AP2 oligonucleotide) totally ablated the top two bands and slightly reduced the intensity of the bottom band (Figure 6A, lane 3). A non-specific competitor oligonucleotide (i.e. Sp1) at a 50-fold excess had little effect on AP2 DNA-binding (Figure 6A, lane 4) when compared with the control (Figure 6A, lane 2). The mutant AP2 probe (Table I) showed that the bottom two bands became bound to this oligonucleotide (Figure 6A, lane 7), indicating that the top band represented specific AP2 DNA-binding activity.
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Sp1 and Sp3 DNA-binding activity
Addition of the Sp1 oligonucleotide (Table I) to control lysates resulted in two bands (Figure 6B, lane 2) that were consistent with binding of the transcription factors Sp1 and Sp3 (13). These latter two bands were not found when the probe alone was run (Figure 6B, lane 1). An excess of specific competitive oligonucleotide (unlabeled Sp1) totally inhibited DNA binding (Figure 6B, lane 3), while the non-specific competitor (unlabeled AP2) had no significant (P > 0.05) effect (Figure 6B, lane 4) over three experiments. Mutant probe (Table I) did not result in any DNA-binding activity (Figure 6B, lane 7).
The incubation of SK-N-MC cells with DFO or 311 resulted in a marked increase in Sp1 DNA-binding activity to 328 and 439% of the control, respectively, while the increase in Sp3 DNA-binding activity was less marked but still significant (P < 0.005) compared with the control (Figure 6B). In the other neoplastic cell types, Sp1 and Sp3 DNA-binding activity in the presence of DFO or 311 was also significantly (P < 0.005) increased above the control and ranged from 190 to 258% of the control (Figure 6B). In contrast to neoplastic cells, DFO had no significant effect on either Sp1 or Sp3 DNA-binding activity in MRC-5 fibroblasts, while 311 caused significant (P < 0.005) 219 and 231% increases, respectively, compared with the control (Figure 6B). Collectively, the DNA-binding results from neoplastic cells suggested that AP2, Sp1 or Sp3 may play a role in the p53-independent increase in WAF1 expression.
The effect of chelators on WAF1 promoter activity
To determine the possible role of p53, AP2, Sp1 or Sp3 in the chelator-mediated increase in WAF1 expression, experiments were performed using a WAF1 promoter construct containing functional p53, AP2 and Sp1 DNA-binding sites linked to the luciferase reporter gene (Figure 7, closed bars) (27). As a negative control, cells were also transfected with the same vector that did not contain the WAF1 promoter (Figure 7, open bars). These constructs were transiently transfected into SK-N-MC cells that have mutant p53 (negative control) (29), or MCF-7, SH-SY-5Y or MRC-5 cells that have wild-type p53 (positive controls) (30,31) (Figure 7AD).
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Chelators increase WAF1 mRNA levels via a transcriptional mechanism
Our current studies using northern blotting demonstrate that 311 markedly increases WAF1 mRNA levels in MCF-7 and SK-N-MC cells (Figure 8A). These experiments confirmed our previous investigations using five different cell types (7,8,11,35). While we showed that p53, Sp1, Sp3 or AP2 were not involved in the transactivation of WAF1 (Figure 7), it was essential to determine whether the increase in WAF1 mRNA levels was due to a transcriptional mechanism. To examine this, MCF-7 and SK-N-MC cells with wild-type or mutant p53, respectively, were incubated with either control medium or 311 and nuclear run-on assays performed (Figure 8A and B). The 311 ligand was chosen for these assays due to its marked effect on the expression of WAF1 (11).
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The role of transcriptional and post-transcriptional mechanisms in the expression of WAF1 mRNA
Additional studies were performed to determine the role of transcriptional and post-transcriptional processes in the increase in WAF1 mRNA levels after incubation with 311. In these experiments, SK-N-MC cells were incubated for 5 h at 37°C with the protein synthesis inhibitor cycloheximide (CHX) (5 µg/ml) and the transcription inhibitor Act D (0.4 µM) (Figure 9). These concentrations of CHX and Act D are widely used to inhibit protein synthesis and transcription, and in this study and previous investigations (11), they were shown to markedly inhibit [3H]leucine and [3H]uridine incorporation, respectively (data not shown).
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Further studies were performed in SK-N-MC cells to determine the half-life of WAF1 mRNA in the presence of Act D alone or Act D and 311 (Figure 10). In the presence of Act D alone, the half-life of WAF1 mRNA was determined to be 6 h, and this increased to 10 h in the presence of 311 as well as Act D (Figure 10).
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Discussion |
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As part of our research program to understand the influence of Fe chelation on tumor cell growth, it was important to examine the effect of chelators on molecules that play a role in the expression of genes vital for proliferation. In particular, the G1/S arrest observed after chelation (34,36) suggests a role for p53 and its downstream target WAF1, which regulates S phase entry (12). Previous studies have suggested that p53 can act as a metabolic sensor (38). In fact, when nucleotide pools were disturbed by inhibitors this resulted in a reversible G0/G1 cell cycle arrest associated with induction of p53 and WAF1 (38). Since 311 and DFO inhibit ribonucleotide reductase and decrease [3H]thymidine incorporation (34,42), this could act as a signal to induce p53 transcriptional activity. Certainly, our previous studies examining a variety of chelators demonstrated that high antiproliferative activity was well correlated with the mRNA levels of the downstream p53 targets WAF1 and GADD45 (8,11,35).
The present work shows that incubation of DFO or 311 with normal or neoplastic cells possessing wild-type p53 increased p53 protein levels and p53 DNA-binding activity. Moreover, DFO and 311 were effective at inducing the nuclear accumulation of wild-type p53. These studies were in contrast to those of Sun et al. (19), in which treatment of cells with the chelator 1,10-P was shown to increase p53 transcriptional activity, but have no effect on p53 protein levels. In addition, these observations were different to the results found in vitro using isolated p53 protein, where 1,10-P inactivated p53 DNA-binding activity (19). However, this latter study used very high ligand concentrations (0.6251.25 mM), which may be necessary to directly remove Zn(II) from p53. Indeed, considering that Zn(II) plays a role in the tertiary structure of this protein, the removal of this ion could disrupt its function. The reason for the difference in the results obtained with 1,10-P compared with DFO or 311 could be related to their different specificities for metal ions. The 1,10-P ligand is a strong divalent metal ion chelator, with high affinity for Fe(II) and Zn(II) (41). In contrast, for DFO and the aroylhydrazone class of chelators, such as 311, the specificity for Fe(III) is extremely high, with much lower affinity for Fe(II) and Zn(II) (9,10). Moreover, 311 or DFO may not be able to gain access to the Zn(II)-binding site of p53 due to steric constraints. It should also be noted that the changes in p53 DNA-binding activity observed could be due to other effects of Fe chelation within the cell. For instance, we cannot exclude the possibility that Fe chelation changes intracellular redox status that may then indirectly alter the DNA-binding activity of p53 and other redox-sensitive transcription factors.
Several studies have examined the effect of DFO on the levels of p53 protein or the transactivation of its target genes after exposure to chelators (7,11,15,16,30). However, this is the first work to assess whether there was increased p53 DNA-binding activity that is directly involved in the chelator-mediated up-regulation of WAF1 (11). This was important to assess since p53 activation has been shown to depend on an increase in protein levels and a shift from the latent to the DNA-binding form of the protein (37). We demonstrated that DFO and 311 were less effective than Act D at increasing p53 protein levels and its DNA-binding activity. The increase in p53 DNA-binding activity in mortal MRC-5 fibroblasts after incubation with DFO or 311 was similar to that found for two neoplastic cell types (i.e. MCF-7 and SH-SY-5Y; Fig. 5C). In addition, our immunofluorescence studies clearly showed nuclear targeting of p53 after incubation with DFO or 311 in MRC-5 and MCF-7 cells (Figure 4). These experiments suggest that the response of p53 to Fe chelators was similar in these normal and neoplastic cells and that the protein can be targeted to the nucleus. However, the response of p53 to chelators was less marked than that observed with Act D.
In contrast to our investigation, Ashcroft et al. (30) reported that DFO only partially activated stabilized but latent p53 in MRC-5 fibroblasts and this response was lost in tumor cell lines. While these latter authors did not perform p53 DNA-binding studies to support this suggestion, they did show that a loss of p53 transcriptional activity correlated with its cytosolic accumulation, indicating a defect in pathways allowing nuclear targeting after chelation (30). We have not been able to confirm these latter results in tumor cell types and we did not observe accumulation of cytosolic p53 even when using higher DFO concentrations (i.e. 250 µM; data not shown). In fact, Fe chelators induced similar changes in p53 protein levels, nuclear localization and p53 DNA-binding activity in mortal MRC-5 cells and the two neoplastic cell lines with wild-type p53 (i.e. MCF-7 and SH-SY-5Y).
The precise molecular mechanism(s) responsible for the p53-independent increase in WAF1 and GADD45 expression after exposure to chelators remains unknown. However, our investigation indicates that transcriptional up-regulation is at least partially responsible (Figure 8). It is of interest that recent studies suggest that the p53 homologs, p63 and p73, may play important roles in transactivating p53 target genes when p53 is mutated (14). Indeed, both these molecules bind p53 consensus sequences in the WAF1 and GADD45 promoters (14). It is clear from our reporter construct studies that the regulation appears to be occurring upstream of the p53, AP2 and Sp1 consensus binding elements that are within the 2280 bp of the WAF1 promoter (Figure 8). Further studies are essential in order to determine the nature of the transcriptional regulation. However, irrespective of the precise molecular mechanism involved, the ability of chelators to potentially inhibit tumor cell growth by a p53-independent pathway is of significance, as p53 is the most frequently mutated gene in human cancer (12). These results may explain why cells with wild-type or mutant p53 are similarly sensitive to the antiproliferative effects of Fe chelators (42). Indeed, it appears that another pathway is vital for the cell cycle arrest observed after Fe chelation. The discovery of this p53-independent pathway could be important for the identification of specific molecular targets that inhibit cancer cell growth by chelators and other drugs.
Apart from transcriptional induction of WAF1 expression, we also obtained evidence for a post-transcriptional mechanism that increased the half-life of WAF1 mRNA (Figure 10). Previous studies have demonstrated that post-transcriptional mechanisms can also be involved in increasing WAF1 mRNA levels (4245). Of interest to the present study, Esposito et al. (42) showed that during oxidative stress, p21 can be induced via a p53-independent pathway that involves a post-transcriptional mechanism. Obviously, the chelation of Fe from cells could perturb the intracellular redox equilibrium and, hence, a similar mechanism could be responsible for the increased stability of WAF1 mRNA observed in the current investigation.
It is paradoxical that increased p53 protein levels, DNA-binding activity and its targeting to the nucleus did not lead to the observed increase in the mRNA levels of a potential p53 target gene, i.e. WAF1. This is surprising considering that DFO and 311 induce a G1/S arrest and that WAF1 encodes p21CIP1/WAF1, the universal inhibitor of cyclin-dependent kinases which prevents cell cycle progression (12). However, it is well known that after exposure to 311 and, to a lesser extent, DFO there is a marked decrease in DNA synthesis due to the inhibition of ribonucleotide reductase (1,34,46), which prevents entrance into S phase. Moreover, exposure of cells to chelators leads to a decrease in the expression of molecules that play key roles in G1 progression, e.g. cyclins D1, D2 and D3 and cdk2 (7). Furthermore, other cell cycle regulators could also be involved in mediating the G1/S arrest after incubation with Fe chelators, e.g. p27Kip1 (47), alleviating the requirement for p21CIP1/WAF1.
Recently, we have shown that while WAF1 mRNA is markedly increased after chelation, the protein levels of p21 are decreased (48). Considering this, it is clear that the effect of these ligands on the regulation of WAF1 is complex, with a p53-independent mechanism being involved in the transcriptional and post-transcriptional regulation of the molecule, which results in increased mRNA levels. Paradoxically, Fe chelation then subsequently prevents translation, an effect that can be readily reversed by the addition of Fe (48).
Apart from our studies, others have shown that the transcription of p21 was inhibited after incubating HL-60 cells with DFO and a phorbol ester (49). These results are clearly quite different to those found in the current study and may reflect the specific cell type used and the non-physiological differentiating agent. It is also of interest that the protein levels of p21 can be regulated by proteasomal degradation (50,51). Considering this, Fukuchi et al. (52) demonstrated that p21 accumulation after incubation with an Fe chelator or DNA-damaging agent was dependent on inhibition of the proteasome. However, our previous studies (48) demonstrated that the proteasome played a small role in the chelator-mediated effects on WAF1 expression.
The question remains, what is the function of increased p53 expression and DNA-binding activity observed after incubation with chelators? Recent studies in our laboratory using gene arrays showed that other p53-transactivatable genes were up-regulated after incubation with 311, including mdm2 and GADD45 (N.T.V.Le and D.R.Richardson, unpublished results). Additional studies determining whether these changes in gene expression lead to alterations in protein levels have been initiated using proteomic analysis and are the subject of on-going investigation.
The high Fe chelation activity of 311 in the current study and its ability to markedly induce WAF1 expression is probably at least partially attributed to its high membrane permeability (11). On the other hand, far higher concentrations of DFO were necessary to achieve appropriate Fe chelation efficacy (Figure 2). The low activity of DFO is probably related to the fact that the molecule shows low efficacy at permeating cell membranes (53) and may be internalized by non-specific pinocytosis (54,55).
In summary, we examined the complex mechanisms involved in the chelator-mediated increase in WAF1 expression. The potent chelator 311 and more so the DNA-damaging agent Act D caused an increase in nuclear p53 protein and p53 DNA-binding activity in cells with wild-type or mutant p53. However, reporter construct studies showed that p53 played no role in the 311-mediated increase in WAF1 transcription. Nonetheless, our experiments demonstrated that the elevated WAF1 mRNA expression was at least partially due to increased transcription and also to a post-transcription mechanism that was sensitive to CHX. Our studies indicate that a p53-independent pathway responds to Fe chelation to induce WAF1 transcription.
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