Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21CIP1/WAF1, but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation

Nghia T.V. Le and Des R. Richardson1

The Heart Research Institute, Iron Metabolism and Chelation Group, 145 Missenden Road, Camperdown, Sydney, New South Wales 2050, Australia and Children's Cancer Institute Australia for Medical Research, Iron Metabolism and Chelation Program, High Street (PO Box 81), Randwick, Sydney, New South Wales 2031, Australia

1 To whom correspondence should be addressed at: Iron Metabolism and Chelation Program, Children's Cancer Institute Australia for Medical Research, High Street (PO Box 81), Randwick, Sydney, New South Wales 2031, Australia Email: d.richardson{at}ccia.org.au


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Iron (Fe) chelators are potential antitumor agents. Cellular Fe depletion results in a G1/S arrest but the precise molecular mechanisms involved remain unclear. Recent studies have shown that this process is complex with multiple cell cycle molecules being involved. We previously showed that Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were far more potent antitumor agents than the clinically used ligand, desferrioxamine (DFO). To further characterize the effects of chelators on cell cycle arrest, we compared their activity with the DNA-damaging agents actinomycin D (Act D) and cisplatin (CP). These latter two compounds increase the expression of p53 and its target genes such as the universal cyclin-dependent kinase inhibitor, p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased nuclear p53, the effect being pronounced for Act D and CP. As expected, both Act D and CP also markedly increased nuclear p21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (P<0.0004) decrease. This latter effect was surprising, as these chelators markedly increased mRNA levels of this molecule. Immunofluorescence studies showed that Act D and CP caused nuclear localization of p21CIP1/WAF1. In contrast, the chelators prevented translation of p21CIP1/WAF1. This did not appear to be due to a general effect of the chelators on preventing translation, as transferrin receptor 1 was markedly up-regulated 15- to 21-fold by DFO and 311. Combination of 311 with Act D or CP prevented translation of p21CIP1/WAF1 and its nuclear localization observed with these DNA-damaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 was reversed by the Fe donor ferric ammonium citrate, indicating that p21CIP1/WAF1 translation was dependent on intracellular Fe levels. This study demonstrates that while Fe chelators markedly up-regulate the mRNA levels of p21CIP1/WAF1 they paradoxically inhibit translation.

Abbreviations: Act D, actinomycin D; BSA, bovine serum albumin; cdks, cyclin-dependent kinases; CP, cisplatin; DFO, desferrioxamine; FAC, ferric ammonium citrate; Fe-Tf, Fe2-transferrin; FITC, fluorescein isothiocyanate; HBSS, Hank's balanced salt solution; mAb, monoclonal antibody; PBS, phosphate-buffered saline; pRb, retinoblastoma protein; TBS, Tris-buffered saline; TBST, TBS containing 0.1% Tween-20; TfR1, transferrin receptor 1; 311, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Iron (Fe) is a critical element for many cellular processes, including DNA synthesis, respiration, oxygen sensing and energy production (for a review see 1). For many years it has been known that Fe deprivation by chelators typically results in a G1/S arrest (2,3). This was thought to be primarily due to inhibition of the Fe-containing enzyme ribonucleotide reductase, which is the rate-limiting step for DNA synthesis (4). However, recent studies have demonstrated that Fe depletion results in a more complex response that affects not only ribonucleotide reductase, but also a range of cell cycle control molecules (511).

At present, knowledge regarding the role of Fe in cell cycle control remains in its infancy. The most well-studied molecules include the cyclins and cyclin-dependent kinases (cdks), which associate to form active cyclin–cdk complexes (11). These latter molecules phosphorylate substrates such as retinoblastoma protein (pRb) to facilitate cell cycle progression. The treatment of cells with Fe chelators was shown to cause a marked decrease in the expression of cyclins D1, D2 and D3 (7,8,10), cdk2 (5,7), cdk1 (3) and the hypophosphorylation of pRb (7,8,12). Changes in the expression of cdk2 and cyclins D1, D2 and D3 may result in the G1/S arrest observed after Fe deprivation.

During the G1/S transition, cyclins D and E are bound to cdk4 and cdk2, respectively, to phosphorylate pRb (for a review see 11). This latter molecule regulates the progression of cells from G1 to S phase of the cell cycle. The phosphorylation of pRb results in the release of the E2F family of transcription factors that are critical for the transactivation of genes required for proliferation (11). This process is further amplified once cyclin E is expressed to bind to cdk2 and commit the cell to progress from G1 to S (11). After incubation with Fe chelators there was a significant decrease in cyclin D and cdk2 expression, resulting in a reduction in phosphorylated pRb that may contribute to G1/S arrest (7,8,10).

The activities of the cyclin–cdk complexes can be modulated by a number of cyclin-dependent kinase inhibitors (for a review see 13). One of the most important is p21CIP1/WAF1 (hereafter called p21), which is a universal cdk inhibitor (1416). The WAF1 gene which encodes p21 can be transactivated by the tumor suppressor protein p53 (14) or by other transcription factors such as AP2, Sp1 or Sp3 (17). Marked transactivation of WAF1 after Fe chelation has been demonstrated to occur in several cell types with native p53 (7,18,19) and mutant p53 (6,7,20). This finding suggests a potential role for p21 in the G1/S arrest observed after Fe chelation (3,9).

The over-expression of p21 affects many cellular processes to result in cell cycle arrest. For example, p21 causes a G1/S arrest by binding cdk2–cyclin E to prevent pRb phosphorylation (13). This arrest may be reinforced by the ability of p21 to directly inhibit the production and activation of caspase 3 to prevent apoptosis (21). In addition, p21 can also stop DNA replication by binding to proliferating cell nuclear antigen (22,23). Other proteins which are affected by p21 binding include transcription factors involved in cell cycle progression such as E2F and c-myc (24,25). The fact that p21 can inhibit many pathways involved in the cell cycle and apoptosis makes this molecule a critical regulator and potential therapeutic target.

Besides cell cycle arrest, when expressed at very low levels p21 is important for cell cycle progression, to stabilize and mediate the nuclear accumulation of cyclin D–cdk4 or cyclin D–cdk6 complexes (26,27). To achieve this level of regulation, p21 is readily exported into the cytoplasm for ubiquitination and proteasomal degradation (28).

A number of Fe chelators have been shown to possess potent antitumor activity (1,29,30). We have previously identified a novel group of aroylhydrazone Fe chelators known as the 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone analogs (31). These ligands are members of the pyridoxal isonicotinoyl hydrazone class and show high selectivity and affinity for Fe(III) (32,33). Subsequent studies using one of the most effective of these ligands, namely 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) (Figure 1A), showed that the anti-proliferative and Fe chelation activity of this compound was much greater than the clinically used chelator desferrioxamine (DFO) (Figure 1B) (6,9,31). Furthermore, 311 was shown to be a potent ribonucleotide reductase inhibitor via its ability to chelate Fe (20). 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 (6,31,34). Moreover, the effects of Fe chelators on increasing WAF1 mRNA levels was reversible after Fe supplementation (6).



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Fig. 1. Schematic illustration showing the structures of the chelators (A) 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) and (B) desferrioxamine (DFO).

 
The present study examines the expression of p21 at the protein level following Fe chelation. We demonstrate that nuclear p21 protein decreases after incubation of cells with DFO or 311. This was surprising, since we previously showed that WAF1 mRNA was markedly up-regulated 20–30 h after incubation with Fe chelators that exhibit antiproliferative activity (6,7,35). Immunofluorescence studies demonstrated that chelators did not increase the levels of cytoplasmic or nuclear p21 protein, while the DNA-damaging agents actinomycin D (Act D) and cisplatin (CP) (3638) caused a robust increase in nuclear p21. In contrast, when cells were treated with Act D or CP together with our most potent Fe chelator, 311, p21 protein expression was inhibited. Studies examining cellular localization showed that the lack of p21 expression following Fe chelation was reversed after Fe supplementation, resulting in nuclear accumulation of the protein. Moreover, Fe chelators prevented nuclear accumulation of p21 in the presence of DNA-damaging agents, suggesting a possible reason for the greater antitumor activity of these compounds when used in combination.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell treatments and reagents
The aroylhydrazone chelator 311 and its Fe complex in a 2:1 ligand:Fe(III) ratio was synthesized and characterized as described previously (34). DFO was from Novartis Pharmaceutical Co. (Basel, Switzerland). Act D, human apotransferrin, ferric ammonium citrate (FAC), MG132 and lactacystin were purchased from Sigma Chemical Co. (St Louis, MO). CP was obtained from Pharmacia and Upjohn Inc. (Sydney, Australia).

Cell culture
Human MRC-5 fibroblasts, WI-38 fibroblasts, MCF-7 breast cancer cells and U2OS osteosarcoma cells were obtained from the American Type Culture Collection (Rockville, MD). The SH-SY5Y neuroblastoma cell line was obtained from Mr Hiroki Nishimura (Queensland Pharmaceutical Research Institute, Brisbane, Queensland, Australia). All cell types were grown in MEM containing 10% fetal calf serum (Gibco, Sydney, Australia), 1 mM sodium pyruvate (Trace Scientific, Melbourne, Australia), 1 mM L-glutamine (Trace Scientific), 1% (v/v) non-essential amino acids (Gibco), 100 µg/ml streptomycin (Gibco), 100 U/ml penicillin (Gibco) and 0.28 µg/ml fungizone (Squibb Pharmaceuticals, Montréal, Canada). Cells were grown in an incubator (Forma Scientific, Marietta, OH) at 37°C in a humidified atmosphere of 5% CO2/95% air and sub-cultured as described previously (39,40). Cell growth and viability were assessed by phase contrast microscopy, Trypan blue (Sigma) staining and cell adherence to the culture substratum.

Protein preparation and labeling
Apotransferrin (Sigma) was labeled with 59Fe (Dupont NEN, Boston, MA) to produce Fe2-transferrin (Fe-Tf) using established procedures (39,40).

Effect of chelators on cellular proliferation
The effect of the chelators and other cytototoxic drugs on the proliferation of normal and neoplastic cells was examined using the MTT assay by the same method as described previously (28,34). For all cell types, the proliferation data obtained using the MTT assay correlated well with cell numbers estimated via microscopy (31).

Flow cytometric analysis
The frequencies of cells in the phases of the cell cycle were examined by enumerating the distribution of nuclei containing double-stranded DNA using propidium iodide staining and flow cytometric analysis as described in our previous studies (9).

Iron uptake and iron efflux experiments
The effect of chelators on 59Fe uptake from 59Fe-Tf and 59Fe release from prelabeled cells was studied using standard procedures (31). Briefly, the ability of the chelators to prevent 59Fe uptake from 59Fe-Tf was examined by incubating the cells for 0.5–24 h at 37°C in medium containing 59Fe-Tf (0.75 µM) and either DFO (250 µM) or 311 (25 µM). The cells were then placed on ice and washed four times with ice-cold Hank's balanced salt solution (HBSS) (Gibco) to remove non-specifically bound 59Fe-Tf. The amount of 59Fe internalized by cells was assessed by established methods via incubation with the general protease pronase (1 mg/ml) for 30 min at 4°C to remove membrane-bound 59Fe and Tf (40,41). The cells were then removed from the Petri dishes using a plastic spatula and the supernatant and pellet separated into {gamma}-counting tubes. Radioactivity was measured using a {gamma}-scintillation counter (LKB Wallace, Turku, Finland).

To assess the ability of the chelator to permeate cell membranes and bind intracellular Fe pools, standard procedures were followed (31). Briefly, cells were prelabeled with 59Fe-Tf (0.75 µM) for 3 h at 37°C. The cells were then placed on ice and washed four times with ice-cold HBSS and then reincubated for 0.5–24 h at 37°C in the presence of medium alone (control) or medium containing DFO (250 µM) or 311 (25 µM). The overlying medium was then decanted and placed in {gamma}-counting tubes. The cells were removed from the Petri dishes in 1 ml of HBSS using a plastic spatula and transferred to a separate set of {gamma}-counting tubes.

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 (6). 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).

Antibodies used in western blot analysis
The mouse monoclonal anti-human ß-actin antibody was from Sigma (clone AC-15) and was used at a dilution of 1/5000. The mouse anti-human transferrin receptor antibody was from Zymed Laboratories (catalog no. 13-6890; San Francisco, CA) and was used at 0.16 µg/ml. The mouse monoclonal anti-human p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 µg/ml, respectively.

Western blot analysis
For cytoplasmic extracts, cells were collected and incubated for 20 min at 4°C with 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 dithiotheitol, 10 mM NaCl, CompleteTM protease inhibitors (Roche, Mannheim, Germany)], centrifuged at 3210 g for 5 min at 4°C and the supernatant retained. For nuclear extracts, the pellets were resuspended in lysis buffer containing 500 mM NaCl and gently rocked for 1.5 h at 4°C (7). The lysates were then centrifuged at 25 200 g for 15 min at 4°C and the supernatant containing the nuclear proteins collected. Protein concentrations of both nuclear and cytoplasmic lysates were assessed with a Bio-Rad protein assay kit (Hercules, CA).

The lysates were then mixed with loading buffer containing 20% ß-mecaptoethanol (Sigma) and loaded at 100 µg per sample onto a SDS–PAGE gel consisting of a 4% stacking and 15% resolving gel. After electrophoresis, the proteins were transferred onto PVDF 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 with 5% skimmed milk in TBS for 3 h at room temperature. Membranes were subsequently washed four times in TBS containing 0.1% Tween-20 (TBST) (Sigma) for 10 min each. After washing, anti-mouse (0.03–0.1 mg/ml) (Sigma) or anti-rabbit (0.05–0.1 mg/ml) (Zymed) antibodies conjugated with horseradish peroxidase were incubated with the membranes for 1 h at room temperature. The membranes were then washed with TBST and developed using the ECL PlusTM Western blot detection reagents (Amersham Biosciences). 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 the ß-actin loading controls.

Direct and indirect immunofluorescence
Cells were seeded and treated on dishes containing sterile glass coverslips. The treated cells were then washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (w/v) 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. After blocking, the cells were incubated with either mouse anti-human p21 directly conjugated with fluorescein isothiocyanate (FITC) (10 µg/ml) or rabbit anti-human p53 antibodies (6 µg/ml) in blocking solution for 2 h at room temperature. As a control for indirect immunofluorescence (i.e. for p53), slides were incubated with PBS and 1% BSA and no first antibody and were always found to be negative. The cells were then washed four times in PBS for 10 min each. For the detection of p53, the respective secondary anti-rabbit antibody containing a FITC conjugate (20 µg/ml) (Sigma) was then incubated with the cells for 1 h at room temperature and the cells again washed with PBS. The coverslip was mounted onto a glass slide with ProlongTM anti-fade mounting medium (Molecular Probes, Eugene, OR). Slides were then examined using a Zeiss Axioplan2 fluorescence microscope (Children's Cancer Institute Australia for Medical Research).

Statistics
Experimental data were compared using Student's t-test. Results were considered statistically significant when P<0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chelator 311 is more active than DFO at inhibiting Fe uptake from transferrin and increasing Fe mobilization from neoplastic and normal cells
To determine the effects of the chelators on binding cellular Fe, we examined their ability to reduce 59Fe uptake from the serum Fe-binding protein Tf and increase 59Fe mobilization from prelabeled MCF-7 cells. Both of these indices are critical for determining the ability of a chelator to induce cellular Fe depletion (31,42). Our previous studies have shown that in comparison with 311, DFO at an equimolar concentration has low efficacy at chelating intracellular Fe pools (6,9). Considering this, we examined the effect of DFO at 250 µM compared with 311 at 25 µM (Figure 2A and B).



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Fig. 2. The chelators 311 and DFO (A) decrease 59Fe uptake from 59Fe-Tf, (B) increase 59Fe mobilization from prelabeled MCF-7 breast cancer cells, (C) increase TfR1 expression and (D) increase WAF1 mRNA levels. (A) MCF-7 cells were incubated for 30 min–24 h at 37°C with 59Fe-Tf (0.75 µM) and either DFO (250 µM) or 311 (25 µM). After this incubation the cells were washed and then incubated with Pronase (1 mg/ml) for 30 min at 4°C to separate internalized from membrane-bound 59Fe (see Materials and methods for details). The results are means ± SD from three determinations in a typical experiment. (B) MCF-7 cells were labeled with 59Fe-Tf (0.75 µM) for 3 h at 37°C. The cells were then washed and reincubated with DFO (250 µM) or 311 (25 µM) for 30 min–24 h at 37°C. At the end of this reincubation period the efflux medium and cells were separated (see Materials and methods for details). The results are means ± SD from three determinations in a typical experiment. (C) MCF-7 cells were incubated with control medium (CON) or medium containing FAC (100 µg/ml), 311 (25 µM) or DFO (250 µM) for 24 h and cytoplasmic lysates were then prepared for western blotting using anti-TfR1 antibody (see Materials and methods for details). (D) MCF-7 cells were incubated with control medium (CON) or medium containing 311 (25 µM), DFO (250 µM), Act D (9 nM) or CP (20 µM) and the RNA prepared for northern blotting (see Materials and methods for details). The results are a typical experiment from three performed.

 
Examining the effect of chelators at inhibiting 59Fe uptake from 59Fe-Tf (0.75 µM) by MCF-7 cells, our results showed that 311 (25 µM) was more effective than DFO (250 µM) at all time points up to 24 h (Figure 2A). For instance, after a 6 h incubation, DFO and 311 reduced 59Fe uptake to 38 and 7% of the control, respectively (Figure 2A). These results demonstrating the greater efficacy of 311 over DFO at chelating cellular Fe pools in MCF-7 breast cancer cells was in agreement with our previous studies using a number of neoplastic cell types (6,9,31).

In experiments examining the efficacy of DFO (250 µM) and 311 (25 µM) at mobilizing 59Fe from cells prelabeled with 59Fe-Tf (0.75 µM), 311 was again more active at a 10-fold lower concentration (Figure 2B). These data again confirm our previous investigations examining the Fe chelation efficacy of 311 compared with DFO in other cell types (6,9,31). Our results from the 59Fe uptake (Figure 2A) and 59Fe mobilization studies (Figure 2B) suggested that DFO and 311 caused marked depletion of cellular Fe.

Studies examining the antiproliferative effects of DFO and 311 showed that as found in many tumor cell types (9,43), 311 had significantly (P<0.001) greater activity than DFO in MCF-7 cells. In fact, the relative IC50 values after a 24 h incubation were 0.4 and 27 µM for 311 and DFO, respectively. As shown previously in a variety of cell types using flow cytometry (8), DFO (250 µM) and 311 (25 µM) increased the proportion of MCF-7 and MRC-5 cells in the G1 phase of the cell cycle (data not shown) consistent with a G1 arrest (2,8).

Transferrin receptor 1 (TfR1) plays an important role in Fe uptake from Tf (1). The up-regulation of TfR1 expression after Fe chelation with DFO or 311 has been well characterized in previous studies and was an appropriate positive control for cellular Fe depletion (6,44). Indeed, the ability of chelators to induce this effect was due to their efficacy at reducing Fe uptake from Tf (Figure 2A) and increasing Fe mobilization from cells (Figure 2B) (6). Incubation of MCF-7 cells with 311 (25 µM) or DFO (250 µM) resulted in a marked increase in TfR1 expression (Figure 2C, lanes 3 and 4) compared with the control (Figure 2C, lane 1). As can be expected from the relative abilities of DFO and 311 to chelate Fe (Figure 2A and B), TfR1 levels were far greater in cells treated with 311 than DFO, namely a 21- and 15-fold increase compared with the relevant control, respectively (Figure 2C, cf. lanes 3 and 4). Collectively, our experiments demonstrated that under the present experimental conditions DFO, and particularly 311, caused marked intracellular Fe depletion.

To confirm previous studies showing that treatment of a variety of cell types with Fe chelators increased WAF1 mRNA levels (6,7,35), we conducted northern blot experiments on MCF-7 cells following incubation with control medium, DFO (250 µM), 311 (25 µM), Act D (9 nM) or CP (20 µM) (Figure 2D). The concentrations of these agents implemented were within the range used previously to increase WAF1 mRNA and p21 protein levels (6,7,35,36,45,46). Compared with cells incubated with medium alone, both chelators increased WAF1 mRNA levels to a comparable level to that found with the DNA-damaging agents Act D and CP (Figure 2D).

The chelators DFO and 311 increase nuclear p53 but decrease the expression of p21
In Figure 2D and in previous studies we showed that incubation of cells with DFO or 311 resulted in a marked increase in the expression of WAF1 mRNA after a 20–30 h incubation (6,7,35). Considering that p21 is important in the p53-mediated G1 arrest of tumor cells (47), we examined the effect of Fe chelators on the protein levels of p53 and p21. In addition, we wanted to evaluate the chelator-mediated p53 response compared with the well-characterized effects of DNA-damaging agents such as Act D and CP (3638,48,49).

Experiments were initially conducted to compare the expression of p53 and p21 in neoplastic and normal cells after a 24 h incubation with Fe chelators and DNA-damaging agents (Figure 3A and B). As a neoplastic cell type we used MCF-7 breast cancer cells that are immortal and compared the response with MRC-5 fibroblasts that are mortal (45). Incubation of both MCF-7 breast cancer cells and MRC-5 fibroblasts with Act D and CP resulted in a marked and significant (P<0.001) increase in the expression of nuclear p53 and p21 (Figure 3A and B, lanes 4 and 5) compared with the control (Figure 3A and B, lane 1). In contrast, despite the significant (P<0.01) increase in nuclear p53 expression after incubation with DFO and more so 311, surprisingly there was a significant (P<0.0004) decrease in nuclear p21 (Figure 3A and B, lanes 2 and 3) compared with the control (Figure 3A and B, lane 1). Examination of p21 in total cell lysates after incubation with 311 also demonstrated very similar results to those obtained for nuclear lysates (data not shown). Experiments also examined the expression of nuclear p21 as a function of time. Using incubation times of 3–9 h with 311 (25 µM) there was no change in nuclear p21 levels between control and chelator-treated cells, while at 24 h a decrease was observed. To validate these data using the anti-p21 monoclonal antibody (mAb) from Santa Cruz Inc., the experiments above were repeated using an anti-p21 mAb from Zymed Laboratories Inc. (catalog no. 33-7000), with identical results being obtained.



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Fig. 3. Western blot analysis demonstrating that DFO or 311 decreased nuclear p21 expression while the DNA-damaging agents Act D and CP increased the expression of this protein in (A) MCF-7 breast cancer cells and (B) MRC-5 fibroblasts. Cells were incubated for 24 h at 37°C with control medium (CON) or medium containing DFO (250 µM), 311 (25 µM), Act D (9 nM) or CP (20 µM). Nuclear lysates were then prepared for western blot analysis (see Materials and methods for details). Results from densitometry are tabulated below each figure and are normalized to the ß-actin loading control in each case. Results are a typical experiment from four performed.

 
The proteasomal degradation pathway is not responsible for the 311-mediated decrease in nuclear p21
It has been previously demonstrated that the protein level of p21 can be regulated by proteasomal activity (28,50). Furthermore, the treatment of cells with proteasomal inhibitors can result in the accumulation of p53 and p21 (28,51,52). We therefore examined whether the decrease in nuclear p21 levels after exposure to Fe chelators was due to increased proteasome-mediated degradation (Figure 4). To assess this, we incubated MCF-7 cells with two chemically distinct proteasome inhibitors, namely lactacystin (53) and MG132 (54). The data obtained with lactacystin were very similar to those with MG132 and, thus, only results using this latter agent are shown (Figure 4).



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Fig. 4. Western blot analysis demonstrating that inhibition of the proteasome using MG132 does not prevent the decrease in nuclear p21 found after treatment with the chelator 311. MCF-7 cells were initially incubated for 24 h at 37°C with control medium (CON) or medium containing either DFO (250 µM), 311 (25 µM) or Act D (9 nM). To inhibit proteasomal activity, MG132 (50 µM) was added to the respective treatments and the incubation allowed to continue for a further 3 h at 37°C. Nuclear lysates were then prepared for western blot analysis (see Materials and methods for details). Results from densitometry are tabulated below each figure and are normalized to ß-actin in each case. Results are a typical experiment from four performed.

 
In agreement with previous studies (28,50,51), the incubation of cells with MG132 resulted in an increase in the nuclear levels of p53 and p21 (Figure 4, lane 2) compared with the control (Figure 4, lane 1). This indicates that MG132 was able to prevent the activity of the proteasome and provided an appropriate internal positive control. Incubation of cells with DFO and MG132 increased nuclear p21 and p53 levels relative to DFO alone (Figure 4, cf. lanes 3 and 4). Hence, these data indicate that for DFO-treated cells, the slight decrease in nuclear p21 in the presence of chelator was at least partially due to increased proteasome activity.

In contrast to the data found for DFO, the incubation of 311-treated cells with MG132 did not result in nuclear accumulation of p21 compared with 311 alone (Figure 4, cf. lanes 5 and 6). This suggested that proteasomal degradation was not responsible for the decreased nuclear p21 levels following Fe chelation by 311. The fact that MG132 (Figure 4, lane 6) and lactacystin (data not shown) did not increase p21 levels in the presence of 311 may suggest a proteasome-independent mechanism for decreasing nuclear p21. When compared with 311 alone, no significant decrease in p53 levels was observed when MG132 was added with 311 (Figure 4, cf. lanes 5 and 6). These results may indicate that 311-treated cells exhibited inhibition of proteasomal activity leading to nuclear p53 accumulation. A possible reason for the contrasting results after proteasomal inhibition between DFO and 311 may be related to their different mechanisms of transport into cells and their ability to access intracellular compartments. While the very lipophilic nature of 311 readily allows diffusion through cellular membranes, the more hydrophilic DFO is far less effective at accessing intracellular Fe pools (6,9). Indeed, DFO has been suggested to be internalized into cells by fluid phase endocytosis (5557), although limited passive diffusion of DFO has never been ruled out. Thus, the efficiency with which 311 enters cells may enhance and/or contribute to the ability of the ligand to fully inhibit the proteasome compared with DFO. Using cells incubated with the DNA-damaging agent Act D, addition of MG132 had no significant effect on p53 or p21 compared with Act D alone (Figure 4, cf. lanes 7 and 8). This is because DNA damage is known to inhibit proteasomal activity (52).

Immunofluorescence studies demonstrate that iron chelation increased nuclear p53 but did not increase nuclear or cytoplasmic p21
Further studies then assessed the cellular localization of p21 and p53 in MCF-7 cells by immunofluorescence after exposure to DFO, 311 or Act D (Figure 5). Examination of control cells revealed weak nuclear and cytoplasmic p21 staining (Figure 5A). However, upon incubation with DFO or 311, there was little change in p21 expression compared with the control (Figure 5B and C). Similar staining of p21 after exposure to chelators was also found in a variety of other human cell types, namely U2OS osteosarcoma cells, SH-SY-5Y neuroblastoma cells, WI-38 fibroblasts and MRC-5 fibroblasts (data not shown). This suggested that the effects observed with the Fe chelators were not specific to MCF-7 cells. In contrast to the effects of Fe chelation, p21 was confined to the nucleus in cells incubated with Act D (Figure 5D).



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Fig. 5. Immunofluorescence studies demonstrating that DFO and 311 do not increase p21 expression but result in nuclear accumulation of p53 in MCF-7 breast cancer cells. Cells were incubated for 24 h at 37°C with control medium (CON), DFO (250 µM), 311 (25 µM) or Act D (9 nM) and then prepared for immunofluorescence to assess p21 or p53 distribution (see Materials and methods for details). The magnification used was 100x. The exposure period used for photography was equivalent for each picture. Results are a typical experiment from five performed.

 
The distribution of p53 was quite different to that observed for p21 after Fe chelation (cf. Figure 5B and C with F and G). In control cells, p53 was confined to the cytoplasm and largely excluded from the nucleus (Figure 5E), whereas treatment with either DFO, 311 or Act D resulted in nuclear localization of p53 (Figure 5F–H). The results with Act D for both p21 and p53 were in agreement with previous studies (45), while the chelator-mediated effects have not been previously reported. Taken together with the western blot analysis (Figure 3), our experiments using immunofluorescence suggested that DFO and 311 inhibited p21 translation (Figure 5B and C) but increased p53 localization in the nucleus.

Iron chelation inhibits p21 protein expression and its nuclear accumulation in DNA-damaged cells
The nuclear accumulation of p21 and p53 after DNA damage has been well characterized in many studies (5861). Having established that the translation of p21 was impaired following Fe chelation (Figures 3A and B and 5B and C), we then examined whether incubation with DFO or 311 could inhibit the nuclear accumulation of p21 in cells simultaneously treated with the DNA-damaging agents CP and Act D (Figures 6 and 7).



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Fig. 6. Western blot analysis demonstrating that 311 prevents nuclear accumulation of p21 in MCF-7 cells treated with the DNA-damaging agent CP, but has no effect on nuclear p53 localization. Cells were incubated for 24 h with either control medium (CON), DFO (250 µM), DFO (250 µM) and CP (20 µM), 311 (25 µM), 311 and CP (20 µM) or CP (20 µM). Nuclear lysates were then prepared for western blot analysis (see Materials and methods for details). Results from densitometry are tabulated below each figure and are normalized to ß-actin in each case. Results are a typical experiment from three performed.

 


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Fig. 7. Immunofluorescence studies demonstrating that DFO and 311 prevent nuclear accumulation of p21 in MCF-7 cells treated with the DNA-damaging agent Act D, but have no effect on nuclear p53 localization. Cells were incubated for 24 h at 37°C with either control medium (CON) or medium containing either DFO (250 µM), DFO (250 µM) and Act D (9 nM), 311 (25 µM), 311 and Act D (9 nM) or Act D (9 nM). After this incubation, cells were then prepared for immunofluorescence to assess p21 or p53 distribution (see Materials and methods for details). The magnification used was 100x. The exposure period used for photography was equivalent for each picture. The results are a typical experiment of two performed.

 
In these experiments, MCF-7 breast cancer cells were co-incubated for 24 h with CP (20 µM) or Act D (9 nM) and either DFO (250 µM) or 311 (25 µM) (Figures 6 and 7). As illustrated in Figure 3, the increase in p53 expression was more marked in cells exposed to CP than after incubation with Fe chelators (Figure 6, cf. lane 6 with lanes 2 and 4). The treatment of cells with CP and either DFO or 311 (Figure 6, lanes 3 and 5) resulted in a marked increase in the expression of nuclear p53 protein relative to untreated cells (Figure 6, lane 1). This increase in nuclear p53 protein in the co-incubated samples was comparable with that found in cells treated with CP alone (Figure 6, cf. lanes 3 and 5 with lane 6). As shown in Figure 3A and B, incubation of cells with CP resulted in the increased expression of nuclear p21 compared with the relevant control (Figure 6, cf. lanes 1 and 6). However, when CP was co-incubated with DFO (Figure 6, lane 3), the nuclear p21 accumulation was slightly inhibited compared with CP alone (Figure 6, lane 6). In samples co-incubated with 311 and CP (Figure 6, lane 5), the inhibition of p21 nuclear accumulation was significantly (P<0.001) more marked than that found for DFO and CP (Figure 6, lane 3). Very comparable results to those observed with CP were also obtained using Act D (data not shown). Taken together, our results demonstrate that 311 prevented nuclear accumulation of p21 in cells co-incubated with DNA-damaging agents.

To further confirm the observations reported above using western analysis, we conducted immunofluorescence studies examining p21 and p53 localization in cells co-incubated with Fe chelators and Act D (Figure 7). As shown in Figure 5, DFO or 311 had little effect on p21 protein expression (Figure 7B and D) compared with the control (Figure 7A). Upon the addition of Act D to 311, no nuclear localization of p21 was observed (Figure 7E), whereas DFO did not totally prevent nuclear p21 staining in the presence of Act D (Figure 7C). In contrast, incubation with Act D alone resulted in a marked accumulation of nuclear p21 (Figure 7F). The co-incubation of chelators and Act D did not prevent nuclear accumulation of p53 (for example for DFO cf. Figure 7H and I). Identical results in immunofluorescence studies were obtained when CP was used instead of Act D (data not shown). Collectively, these results confirm our western blot data (Figure 6) showing that Fe chelators prevent nuclear accumulation of p21 in cells incubated with DNA-damaging agents.

The supplementation of iron into cells restores nuclear p21 accumulation after Fe chelation
In order to further assess whether the translation of p21 was dependent on Fe, we conducted experiments aimed at repleting intracellular Fe levels following Fe chelation with 311. In these studies, the expression of p21 and p53 were examined using both western blot analysis (Figure 8) and immunofluorescence (Figure 9). Iron was donated to cells by incubation with FAC (100 µg/ml) or the 311-Fe complex (25 µM) for 24 h after the initial 24 h preincubation with control medium (MEM) or 311. Many previous studies have shown that incubation of cells with FAC or aroylhydrazone-Fe complexes (e.g. 311-Fe) results in cellular Fe accumulation that reverses the effects of Fe depletion (40,6265). Indeed, reincubation of 311-treated cells with FAC (Figure 8, lane 5) or the 311-Fe complex (Figure 8, lane 6) was shown to decrease TfR1 levels when compared with cells incubated with 311 alone (Figure 8, lane 3). These data demonstrated effective Fe donation to the cell.



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Fig. 8. Western blot analysis demonstrating that supplementation of Fe to cells pre-treated with chelators restores nuclear p21 accumulation. MCF-7 cells were initially incubated for 24 h at 37°C with either control medium alone (MEM) or 311 (25 µM). This medium was then removed and the cells incubated for a further 24 h at 37°C with either MEM, FAC (100 µg/ml), 311 (25 µM) or the Fe complex of 311 (311-Fe) (25 µM). Nuclear lysates were then made for western analysis for p21 and p53, while cytoplasmic lysates were prepared for TfR1 (see Materials and methods for details). Data from densitometry are tabulated below each figure and are normalized to ß-actin in each case. Results are a typical experiment from three performed.

 


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Fig. 9. Immunofluorescence studies demonstrating that supplementation of iron to cells pre-treated with 311 restores nuclear p21. MCF-7 cells were initially incubated for 24 h at 37°C with either control medium alone (MEM), 311 (25 µM) or Act D (9 nM). This medium was then removed and replaced with MEM only or MEM containing the Fe donor FAC (100 µg/ml), 311 (25 µM), the Fe complex of 311 (311-Fe) (25 µM) or Act D (9 nM). After this incubation, cells were then prepared for immunofluorescence to assess p21 distribution (see Materials and methods for details). The magnification used was 100x. The exposure period used for photography was equivalent for each picture. Results are a typical experiment from two performed.

 
The incubation of MCF-7 cells with FAC after the initial incubation with MEM resulted in no significant (P>0.05) change in nuclear p53 (Figure 8, lane 2) compared with cells incubated with MEM only (Figure 8, lane 1). In contrast, incubation of cells for 24 h with 311 after the initial incubation with this chelator resulted in a significant (P<0.001) 3.1-fold increase in p53 (Figure 8, lane 3) compared with cells incubated with MEM alone (Figure 8, lane 1). Incubation of cells with MEM, FAC or the 311-Fe complex (311-Fe) after the initial incubation with 311 (Figure 8, lanes 4–6) did not significantly (P>0.05) alter the 311-mediated p53 response compared with cells treated with 311 alone (Figure 8, lane 3). This demonstrated that p53 induction was maintained even in the absence of 311 in the reincubation medium. As expected, using immunofluoresence studies, cells initially treated with 311 followed by reincubation with FAC, MEM or 311-Fe showed nuclear p53 staining that was very similar to that observed in cells incubated with 311 alone (data not shown).

Incubation of MCF-7 cells with FAC after the initial incubation with MEM did not significantly (P>0.05) affect nuclear p21 expression (Figure 8, lane 2) compared with cells incubated with MEM only (Figure 8, lane 1). Incubation of cells with MEM after the initial incubation with 311 (Figure 8, lane 4) resulted in no change in nuclear p21 relative to cells treated with 311 alone (Figure 8, lane 3). However, the addition of FAC after the initial incubation with 311 resulted in a significant increase (P<0.001) in nuclear p21 (Figure 8, lane 5) compared with cells incubated with 311 only (Figure 8, lane 3).

Incubation of cells with the 311-Fe complex after the initial incubation with 311 resulted in a significant (P<0.001) increase in nuclear p21 (Figure 8, lane 6) compared with cells incubated with 311 only (Figure 8, lane 3). The 311-Fe complex was slightly more effective than FAC at increasing p21 levels in cells initially treated with 311 (Figure 8, cf. lanes 5 and 6). The reason for this may be because of the high efficiency of lipophilic aroylhydrazone-Fe complexes (e.g. 311-Fe complex) at donating Fe to cells (63,64). In accordance with this, in 311-treated cells TfR1 levels decreased to a greater extent when cells were incubated with 311-Fe (Figure 8, lane 6) than FAC (Figure 8, lane 5). Collectively, the results in Figures 5, 7 and 8 suggest that Fe chelation by 311 prevents translation and the nuclear accumulation of p21 and that Fe repletion then increases nuclear p21 levels to above the control level (Figure 8).

To further characterize the ability of FAC to restore p21 nuclear accumulation in cells treated with chelators, we conducted the same type of experiment as in Figure 8 using immunofluorescence (Figure 9). As shown in Figures 5C and 7D, in MCF-7 cells treated with 311 there was little change in p21 expression (Figure 9C) when compared with the control (Figure 9A). The addition of MEM following the initial incubation with 311 (Figure 9D) resulted in no change in p21 staining when compared with 311 alone (Figure 9C). The addition of FAC or the 311-Fe complex following the initial incubation with 311 resulted in marked nuclear p21 staining (Figure 9E and F). In contrast to Fe chelators, reincubation of Act D-treated cells with MEM or FAC (Figure 9H and I) did not alter the nuclear accumulation of p21 compared with Act D alone (Figure 9G).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Iron is fundamental for cellular proliferation and DNA synthesis. Recently, Fe chelators have been demonstrated to have potential in the treatment of a number of tumors in vitro using cell culture and in vivo in animals and humans (1,3,30,31, 6069). In particular, some chelators of the aroylhydrazone class (e.g. 311) show much promise as antiproliferative agents suitable for the treatment of cancer (6,9,31,43). Although it is well known that Fe depletion results in a G1/S arrest (3), further work is required to understand the precise molecular mechanisms involved.

The present study demonstrated that p21 protein expression was decreased after incubation of cells with DFO or 311 (Figure 3). This was unexpected, since we previously showed that WAF1 mRNA was markedly up-regulated 20–30 h after incubation with Fe chelators that exhibit antiproliferative activity (6,7,35) (Figure 2D). This investigation is the first to demonstrate that Fe chelation using DFO and 311 prevents translation of the universal cdk inhibitor p21 in a variety of cell types. Moreover, the effect of the chelators on p21 expression and localization was markedly different to the classical DNA-damaging agents CP and Act D. The ability of the chelators to directly or indirectly inhibit p21 translation is important, since nuclear import of p21 is vital for cell cycle arrest and DNA repair (1416,70,71). Indeed, the cellular localization of p21 has been proposed to be critical in regulating its function (72,73). The failure in p21 expression after incubation with chelators could have important consequences in terms of cellular proliferation (72). The present investigation clearly demonstrates that a pharmacological intervention (i.e. Fe chelation) can affect p21 protein levels. Further, our results may be important in understanding the dysregulation of the cell cycle following Fe depletion.

It could be suggested that the decrease in p21 protein expression could be due to the ability of the chelators to generally inhibit the translational apparatus of the cell. In fact, previous studies have indicated that Fe chelation using DFO can inhibit the synthesis of hypusine, a component of translation initiation factor 5A (74). However, a general inhibition of translation does not appear to be occurring, as TfR1 protein levels are markedly up-regulated after incubation with DFO or 311 (Figures 3C and 8). The increase in TfR1 levels after incubation with both these chelators is well characterized and is due to Fe depletion that stabilizes TfR1 mRNA via the iron-regulatory protein–iron responsive element mechanism (6; for review see 44). Moreover, we previously showed that cyclin E protein levels are up-regulated after incubation with chelators, an effect that is mediated by increased translation of the protein (75).

It is paradoxical that Fe chelation using DFO and 311 caused cell cycle arrest (3,9), whereas this treatment does not result in the nuclear localization of the universal cell cycle inhibitor p21 (Figures 3 and 5). Cell cycle arrest should theoretically be associated with nuclear accumulation of p21 (14,15). Hence, cellular arrest observed after treatment with Fe chelators may be independent of p21. For instance, Fe chelation is known to decrease the expression of a number of proteins which play vital roles in G1/S progression, including cyclin D and cdk2 (7,8). Interestingly, a lack of p21 expression may prevent nuclear importation of cyclin D (26,27) that is required for pRb phosphorylation and cell cycle progression which could contribute to G1/S arrest. Moreover, the ability of DFO and 311 to inhibit ribonucleotide reductase (20,76) and reduce dNTP levels probably acts as another mechanism to prevent entrance into S phase. Considering this, it is known from studies using p21-null mice that a G1 arrest can occur in these cells after incubation with cytotoxic agents, suggesting that other regulators can be involved (77). Indeed, there are many cdk inhibitors, such as the INK family (13) and p27Kip1 (78), that may be important in the G1/S arrest observed after incubation with chelators.

In agreement with previous studies using DFO (18), we have demonstrated that Fe chelation by DFO or, particularly, 311 can increase p53 protein expression (Figure 3A and B). The mechanism of this effect has been suggested to be due to the direct interaction of hypoxia-inducible factor 1{alpha} at stabilizing p53 protein (18). Further studies are essential to determine whether the elevated p53 levels play any role in mediating the increase in WAF1 mRNA levels observed in the present investigation. However, it is important to note that our previous experiments have shown that the increase in WAF1 mRNA levels can occur in cells with mutant p53, indicating a p53-independent mechanism (6). Studies by investigators using other chelators have also suggested that while p53 expression is increased it does not appear to be involved in the transactivation of WAF1 (66). The precise molecular mechanism involved in the increase in WAF1 mRNA levels remains unclear at present and was outside the scope of this investigation.

It is uncertain if the lack of nuclear p21 accumulation observed in this study plays a role in apoptosis or the marked antiproliferative effects observed after Fe chelation (this study; 9,31). However, it is known that cells with a defective p21 response undergo apoptosis upon treatment with cytotoxic agents (7982). Under these conditions the cells become hypersensitive to DNA damage, resulting in the accelerated onset of apoptosis (79). It has been shown that in the absence of p21, DNA-damaging agents dysregulate the cell cycle leading to death (82). Certainly, in the present study (data not shown) and in previous investigations (83) greater cytotoxicity of Fe chelators was observed when they were combined with DNA-damaging agents than when either agent was used alone. It is possible to speculate that the lack of p21 protein expression and the dysregulation of its function could be important in enhancing the induction of apoptosis after incubation with chelators.

The expression of p21 can be regulated by transcriptional and/or post-transcriptional mechanisms. Indeed, several studies have investigated the transcriptional regulation of p21 following treatment with DFO (5,45). Boldt and colleagues showed that the transcription of p21 was inhibited after incubating HL-60 cells with DFO and a phorbol ester (5). The changes in p21 expression after exposure to Fe chelators could be affected by alterations in the p53 pathway (45,66). In fact, Vousden et al. (45) showed that the p53 transcription factor was stabilized, but may be functionally inactive following Fe chelation with DFO. This increase in p53 protein levels did not result in a robust induction of p21 mRNA after DFO treatment and, as a consequence, there was no increase in p21 protein expression (45). It should also be noted that the protein levels of p21 can be regulated by proteasomal degradation (28,50). Considering this, Fukuchi et al. (19) demonstrated that p21 accumulation after incubation with an Fe chelator or DNA-damaging agent was dependent on inhibition of the proteasome. These investigators showed that despite a marked increase in p21 mRNA after Fe chelation, there was no detectable expression of the protein (19). This process was reversed following the addition of a potent proteasomal inhibitor to allow p21 to accumulate beyond basal levels after DFO treatment.

In contrast to the studies described above, we demonstrated that p21 protein levels decreased despite the marked increase in its mRNA following incubation with DFO or 311. In addition to this, the decrease in p21 protein expression was not caused by the proteasomal degradation pathway after incubation with 311 (Figure 4). The supplementation of Fe to Fe-deprived cells reversed the inhibitory effects of the Fe chelators resulting in a marked increase in p21 protein (Figures 8 and 9). This demonstrates that Fe was required for p21 translation. In addition, previous studies showed that incubation of cells with the Fe complex of DFO or 311 could not induce WAF1 expression (6). Hence, Fe chelation increased WAF1 mRNA expression (6,7) but prevented p21 translation. To date, the precise molecular mechanism responsible for this inhibition is unknown and requires further investigation.

In summary, we demonstrate that p21 protein expression decreased after incubation of cells with DFO or 311. In contrast, the classical DNA-damaging agents Act D and CP caused a marked increase in nuclear p21 protein. When cells were treated with these DNA-damaging agents together with our most potent Fe chelator, 311, this prevented the elevation of nuclear p21 levels in a variety of cell types. This observation suggested a reason for the greater antitumor activity of these agents when used in combination. Studies examining p21 localization showed that the failure of p21 to be translated and be targeted to the nucleus following chelation was reversed following Fe supplementation. Taken together, our experiments reveal that Fe was required for the translation and, hence, nuclear accumulation of p21.


    Acknowledgments
 
The authors thank Dr Richard Lock and members of the Iron Metabolism and Chelation Program for their comments on the manuscript prior to submission. We very much appreciate the great assistance provided by Dr Maria Kavallaris in performing the immunofluorescence studies, Dr Simon Liang for help with northern blots and Ms Claudia Flemming for advice on flow cytometry. Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital. This project was supported by a fellowship and project grant from the National Health and Medical Research Council of Australia.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 18, 2002; revised March 4, 2003; accepted March 6, 2003.