Oncogene regulation of tumor suppressor genes in tumorigenesis

Jimmy Sung, Joel Turner, Susan McCarthy, Steve Enkemann, Chan Gong Li, Perally Yan, Timothy Huang and Timothy J. Yeatman*

Department of Surgery and Interdisciplinary Oncology, H.Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612, USA and Department of Molecular Virology, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA

* To whom correspondence should be addressed Email: yeatman{at}moffitt.usf.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
We attempted to demonstrate whether there is an epigenetic link between oncogenes and tumor suppression genes in tumorigenesis. We designed a high throughput model to identify a candidate group of tumor suppressor genes potentially regulated by oncogenes. Gene expression profiling of mock-transfected versus v-src-transfected 3Y1 rat fibroblasts identified significant overexpression of DNA methyltransferase 1, the enzyme responsible for aberrant genome methylation, in v-src-transfected fibroblasts. Secondary microarray analyses identified a number of candidate tumor suppressor genes that were down-regulated by v-src but were also re-expressed following treatment with 5-aza-2'-deoxycytidine, a potent demethylating agent. This candidate group included both tumor suppressor genes that are known to be silenced by DNA hypermethylation and those that have not been previously identified with promoter hypermethylation. To further validate our model, we identified tsg, a tumor suppressor gene that was shown to be down-regulated by v-src and found to harbor dense promoter hypermethylation. Our model demonstrates a cooperative relationship between oncogenes and tumor suppressor genes mediated through promoter hypermethylation.

Abbreviations: DAC, 5-aza-2'-deoxycytidine; DNMT1, DNA-cytosine 5-methyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
It is well known that cancer cells are often the product of multiple genetic alterations that cause cellular transformation. To date, numerous specific genetic alterations have been identified that activate proto-oncogenes genes or inactivate tumor suppressor genes. In fact, the sine qua non of a cancer gene is one that is affected by a mutational event with a significant prevalence. In addition, a ‘third’ pathway to tumorigenesis has been identified whereby the expression of key genes is regulated through promoter hypermethylation and silencing. Tumor suppressor genes, in particular, may be subject to this mechanism of inactivation, in addition to mutational events (1).

Oncogenes and tumor suppressor genes have classically been assigned distinct, independent roles in cancer development and progression. The interrelationships, structural and temporal, between these tumor-promoting processes, however, are still poorly understood. We have hypothesized that the inhibition of tumor suppressor genes that occurs via promoter hypermethylation may be initiated and regulated by the activation of oncogenes, explaining in part how a single oncogene can result in cellular transformation. To test this hypothesis we designed a high throughput model to identify tumor suppressor genes potentially regulated by oncogenes through methylation events. We used the well-defined cellular model of v-src-mediated cellular transformation to demonstrate these relationships (2). We have previously examined the genes that were principally up-regulated by v-src and, in fact, we were able to develop a ‘Src fingerprint’ of genes commonly regulated by Src that was detected in human colon cancer specimens well known to harbor high levels of Src activity (3). In the current study we chose to examine genes down-regulated by v-src as a means of understanding the relationship of tumor suppressor genes to the v-src oncogene. We demonstrated that a single oncogene, v-src, can inhibit the expression of a large number of genes during the process of cellular transformation. Using a gene expression profiling approach, a comparison of v-src-transfected with mock-transfected rat fibroblasts identified a number of candidate tumor suppressor genes that are down-regulated by v-src and then re-expressed to baseline levels following treatment with 5-aza-2'-deoxycytidine (DAC) (46). The regulation of one of these candidate genes, tsg, was the principal subject of this study in which we demonstrated that a tumor suppressor gene is silenced by an oncogene through promoter hypermethylation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cell culture
Pools of stably transfected 3Y1 rat fibroblasts were a kind gift from Richard Jove (H.Lee Moffitt Cancer Center and Research Institute). Mock-transfected and v-src-transfected 3Y1 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies) with 10% fetal bovine serum at 37°C, 5% CO2.

Microarray analysis
The methods for preparation and subsequent handling of RNA up through hybridization followed the recommendations of the Affymetrix GeneChip® Expression analysis system. Rat U34 GeneChips, containing ~8000 elements per array, were used in all experiments. Scanned output files were visually inspected for hybridization artifacts and then analyzed using Affymetrix Microarray 5.1 software.

Real-time quantitative PCR (real-time qPCR)
A quantitative primer/probe set was designed to evaluate and to quantitative rat tsg (accession no. S72637), rat vhl (accession no. U14746), rat dnmt1 (accession no. NM_053354) and gapdh in 3Y1 v-src-transfected cell lines. The primers were as follows. Rat tsg: forward primer 5'-CACGCAGATTGTGGGCC-3'; reverse primer 5'-TCCTAGGCACGCCCTGTTC-3'; probe 5'-AGCGGCTGTGAGGCCAAGTCTATCC-3'. Rat dnmt1: forward primer 5'-GGAAGGTGAGCATCGACGAA-3'; reverse primer 5'-GATCATCCGGAATGACCGAG-3'; probe 5'-ACTCTGGAGGTGGGCGACTG-CG-3'. Rat vhl: forward primer 5'-GCTGCCTTTGTGGCTCAAC-3'; reverse primer 5'-GGTGCCCGGTGGTAAGGT-3'; probe 5'-TTGATGGTGAGCCTCAGCCCTACCC-3'. gapdh: forward primer 5'-TGAAGGTCGGAGTCAACGG-3'; reverse primer 5'-AGAGTTAAAAGCAGCCCTGGTG-3'; probe 5'-TTTGGTCGTATTGGGCGCCTGG-3'. Total RNA was extracted by the guanidine isothiocyanate, phenol/chloroform method (Trizol; Gibco) with the addition of 20 µg glycogen as a carrier for the RNA. Reverse transcription of RNA was performed using 15 U omniscript reverse transcriptase (Qiagen), 1x cDNA synthesis buffer, 40 U RNase inhibitor (Gibco), 5 mM DTT, 1 mM dNTP mixture, 0.5 µg oligo(dT) primer and 2 µg RNA. Primers and probes for real-time PCR were designed using Primer Express software (Applied Biosystems).

Methylation-specific PCR (MS-PCR)
Genomic DNA was isolated from cells by digestion with 100 g/ml proteinase K followed by standard phenol/chloroform (1:1) extraction and ethanol precipitation. DNA was treated with sodium bisulfite as follows. A sample of 1 g genomic DNA was denatured by incubation with 0.2 M NaOH for 10 min at 37°C. Then 10 mM hydroquinone (Sigma Chemical Co., St Louis, MO) and 3 M sodium bisulfite (pH 5.0) (Sigma Chemical Co.) were added and the solution was incubated at 50°C for 16 h. Treated DNA was purified using the Wizard DNA Purification System (Promega Corp., Madison, WI), desulfonated with 0.3 M NaOH, precipitated with ethanol and resuspended in water. Modified DNA was stored at –80°C until used. MS-PCR was performed with primers specific for the methylation reaction. The primers were as follows. Methylated tsg promoter: forward 5'-TGGCCCCGGCACCCCTGCTCGTCGCGCG-3'; reverse 5'-CGCGGCCGCCGCGCGCGGTAAGTCTC-3'. Unmethylated tsg promoter: forward 5'-TGGCCCTGGCACCCCTGCTTGTTGTGTG 3'; reverse 5' TGTGGCTGCTGTGTGTGGTAAGTCTC-3'. Negative control samples without DNA were included for each set of PCR. PCR products were analyzed on 2% agarose gels containing ethidium bromide.

Bisulfite sequencing and combined bisulfite restriction analysis (COBRA) assay
Sodium bisulfite modification of genomic DNA was conducted using a CpGenome DNA Kit according to the manufacturer's instructions (Intergen). Bisulfite-treated DNA (~1 ng) was used as a template for PCR with specific primers flanking the BstUI sites located within the CpG island regions of interest. Primers used for amplification were as follows: tsg_121U23, 5'-TTAGAATTTAATTTTTTGAGGGA-3'; tsg_294L22, 5'-TTACTCCRCCTAACTTTTCTAA-3'. After amplification, PCR products incorporating 32P were digested with BstUI (New England Biolabs), which recognizes sequences unique to the methylated alleles. The undigested control and digested DNA samples were separated in parallel on 8% polyacrylamide gels and subjected to autoradiography using a PhosphorImager (Amersham-Pharmacia). The region of interest within the tsg promoter site was amplified from bisulfite-treated genomic DNA by PCR using the same primer pairs as described earlier. Amplified products were subcloned using the TOPO-TA Cloning System (Invitrogen). Plasmid DNA of 10–15 insert-positive clones were isolated using a QIAprep Spin Miniprep kit (Qiagen) and sequenced using the ABI sequencing system (Applied Biosystems).

Western analysis
DNA-cytosine 5-methyltransferase (DNMT1) protein levels were assessed using goat anti-rat antibody purchased from Santa Cruz Inc. (Santa Cruz, CA) under the following conditions. The phosphotyrosine level in v-src-transfected 3Y1 cells was dectected using anti-phosphotyrosine-specfic antibody clone 4G10 (Upstate Biotechnology, Lake Placid, NY). Cells were lysed in 50 mM Pipes–KOH (pH 6.5), 2 mM EDTA, 0.1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid, 5 mM DTT, 20 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml apoprotinin and 2 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged at 4°C for 30 min at 20 000 g and the supernatant fraction recovered. Protein extracts (100 g) were fractionated through a 10% criterion precast gel (Bio-Rad, Hercules, CA) and blotted onto pure nitrocellulose membranes (Bio-Rad).

Final protein detection used horseradish peroxidase-conjugated mouse anti-goat IgG secondary antibodies purchased from Biosource (Camarillo, CA) and Santa Cruz Biotech (Santa Cruz, CA) and SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

tsg transfection
Full-length rat tsg was cloned and inserted into the PCDNA3.1 expression vector (Invitrogen) and transiently transfected (48 h) using 2 µl of SuperFect (Qiagen) into v-src-transformed 3Y1 rat fibroblasts to assess effects on tumorigenicity. Photographs were obtained 3 days after transfection.

Antisense and 5-aza-2'-deoxycytidine treatments of cells in culture
DAC (200 nm) was dissolved in fresh phosphate-buffered saline each day and added to fresh medium for 6 days.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Verification of v-src transfection
We first verified the quality of our 3Y1 v-src transfection by comparing Src protein activity between the mock- and v-src-transfected 3Y1 cells with a phosphotyrosine western blot (Figure 1). These data demonstrate a high degree of Src-specific activity in v-src-transfected cells versus mock-transfected controls and is consistent with the transformation phenotype observed.



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Fig. 1. Phosphotyrosine western blot demonstrating high levels of phosphotyrosine in v-src-transfected rat fibroblasts when compared with mock-transfected controls.

 
Identification of genes down-regulated by v-src
In order to identify candidate tumor suppressor genes regulated by v-src we performed a genome-wide microarray analysis (in triplicate) of 3Y1 rat fibroblasts stably transfected (and transformed) with v-src and compared them with mock-transfected controls. After standard normalization and scaling procedures, we compared the expression of these two cell lines, gene by gene, to identify the genes that were significantly (P < 0.001) down-regulated by v-src. A large number of significantly down-regulated genes were identified. A representative sample of these genes is shown in Table I. A complete list of all down-regulated genes is shown in Supplementary material, Table SI.


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Table I. Genes down-regulated by v-src

 
Identification of v-src-regulated methyltransferases
Because we hypothesized that v-src might regulate a number of candidate tumor suppressor genes though promoter hypermethylation and silencing, we examined the expression of all evaluable methyltransferases on the Affymetrix GeneChip. Of numerous methyltransferases examined, DNMT1 was prominently overexpressed in v-src-transformed fibroblasts when compared with mock controls (Figure 2). DNMT1 expression was confirmed at both the RNA level by real time qPCR (Figure 3A) and at the protein level by western analysis (Figure 3B), supporting its potential in the regulation of promoter activity.



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Fig. 2. Gene expression profile of all methyltransferases on a U34A GeneChip shows prominent up-regulation of dnmt1 in v-src-transfected rat 3Y1 fibroblasts compared with mock-transfected controls.

 


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Fig. 3. Confirmation of induction of dnmt1 expression in v-src-transformed fibroblasts. (A) Real-time quantitative PCR analysis identified a 5-fold increase in expression when compared with mock-transfected cells. (B) Protein expression determined by western analysis confirms the 5-fold increase in expression.

 
Experimental screening for candidate tumor suppressor genes regulated by promoter hypermethylation: identification of tsg
In order to identify genes that were regulated by promoter hypermethylation we combined the gene expression profiling approach with a demethylating treatment of the same cells using DAC to find genes that were down-regulated by v-src but then re-expressed following demethylation. By comparing the gene expression of cells transformed by v-src with those same cells treated with DAC we were able to identify a number of candidate tumor suppressor genes potentially regulated by promoter hypermethylation. A heat map identifying these candidate genes and demonstrating their expression consequent to DAC treatment is shown in Figure 4. Prominent among a number of genes on this list was the von Hippel-Lindau tumor suppressor gene (vhl), previously shown to be regulated by promoter hypermethylation, validating our approach to this screening process (7). In addition, a new gene called tumor suppressor gene (tsg) was identified. We chose tsg as the principal focus of this study because it has been previously identified as a strong tumor suppressor gene candidate with the potential to reverse the transformation of neoplastic cells (8). However, tsg was not previously know to be regulated by methylation. We have confirmed the previously reported tumor suppressive properties of tsg by demonstrating that transient transfection of tsg into v-src-transformed cells can reverse the transformation phenotype of v-src-transected cells (Figure 5).



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Fig. 4. Gene expression profile of genes significantly down-regulated by v-Src and re-expressed on treatment with 5-azacytidine. Data identify tumor suppressor gene candidates regulated by promoter methylation. Prominent amongst these genes were tsg and vhl.

 


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Fig. 5. Effect of tsg transfection on cellular transformation. (A) Mock-transfected rat 3Y1 fibroblasts show normal density inhibition of growth. (B) 3Y1 fibroblasts transfected with v-src take on the transformed phenotype with evidence of focus formation and loss of intercellular adhesion. (C) Reversion of the transformed phenotype by tsg transfection shows the potential tumor suppressive properties of tsg.

 
Expression of both vhl and tsg was subsequently confirmed by real-time qPCR to be down-regulated by v-src transfection and then re-expressed by demethylation following treatment with DAC (Figure 6).



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Fig. 6. Real-time qPCR analysis of gene expression. (A) tsg re-expression following demethylation with 5-aza-2'-deoxycytidine. (B) vhl re-expression after treatment with 5-aza-2'-deoxycytidine (DAC).

 
Validation of tsg promoter methylation by bisulfite sequencing
While data demonstrating re-expression of down-regulated genes after DAC treatment is compelling, definitive evidence of promoter hypermethylation is required since it has been demonstrated that not all genes re-expressed following DAC treatment are controlled via promoter hypermethylation (4). We first compared the tsg promoter site in mock- and v-src-transfected 3Y1 cells using MS-PCR and found evidence of promoter hypermethylation (Figure 7A). To further confirm this finding, we then selected a region of the tsg promoter site (region 1) for more definitive evaluation by combined bisulfite restriction analysis (COBRA) assay and bisulfate sequencing. We found that v-src-transfected 3Y1 cells contained a specific region of high density methylation (Figure 7B and C). Finally, we sequenced the remaining region of the promoter site (region 2) and observed generalized hypermethylation of the tsg promoter site in v-src-transfected 3Y1 cells (Figure 7D).



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Fig. 7. Analysis of the methylation state of tsg in v-src-transformed 3Y1 fibroblasts confirmed hypermethylation of the tsg promoter. (A) Methylation-specific PCR analysis of tsg promoter suggesting hypermethylation. (B) Methylation analysis by COBRA of the tsg promoter site. (C) Bisulfite sequencing confirmed hypermethylation of the tsg promoter. Seven clones of v-src-transformed and five clones of mock-transfected 3Y1 fibroblasts were analyzed for methylation status. Methylated and unmethylated CpG sites are represented by closed and open circles, respectively. (D) Bisulfite sequencing of the remaining portion of the tsg promoter. Methylated and unmethylated CpG sites are represented by closed and open squares, respectively.

 
Using this approach we have demonstrated that the promoter region of tsg contains CpG islands and, once transfected with v-src, these CpG islands increased in methylation density and were noted to contain specific regions of high density methylation.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
To identify a potential relationship between oncogenes and tumor suppressor genes, the classic model of v-src fibroblast transformation was selected. We and others have previously demonstrated that v-src is a potent inducer of transformation that is characterized by a signature of genes whose expression is both up-regulated as well as down-regulated relative to mock-transfected controls (3). Much attention has been devoted to the induced genes, however, those genes suppressed by v-src that are potential tumor suppressors have been less well examined. First, we sought to identify a number of genes that were down-regulated by v-src and then re-expressed following demethylation. Based on gene expression profiles of v-src-transformed fibroblasts, we hypothesized that v-src might inhibit the expression and function of a number of tumor suppressor genes through the process of promoter hypermethylation mediated by DNA methyltransferases, in particular DNMT1. Both mock-transfected and v-src-transformed 3Y1 rat fibroblasts were then treated with DAC, a demethylating agent (9). The Affymetrix U34 rat GeneChip was used to identify genes that were down-regulated in v-src-transformed as compared with mock-transfected cells but re-expressed after treatment with DAC. A comparison of mock-transfected versus v-src-transfected rat fibroblasts (3Y1) found that DNMT1 was prominently overexpressed in v-src- compared with mock-transfected cells (Figure 2). Further analysis using MAS 5.0 software (Affymetrix) identified 912 probe sets significantly down-regulated (P < 0.001) by v-src (Table I). Of these probe sets, 141 were up-regulated (P < 0.001) by treatment with DAC and, within these probe sets, at least six of these were annotated genes with a putative tumor suppressor function and were rich in mathematically defined CpG islands (vhl, tsg, cdkn1a, par-4, p53 and st13) (10).

Among the genes meeting our criteria, we first selected vhl, Von Hippel-Lindeu tumor-suppressor gene, for further validation. We were encouraged by the identification of vhl with our model because previous studies have definitively demonstrated that vhl is silenced by promoter hypermethylation in renal carcinoma and can be reactivated by DAC (7). However, the involvement of v-src in the down-regulation of vhl has not been reported. On the other hand, our model identified a second gene that is a known v-src-regulated putative tumor suppressor gene, the 3Y1 tumor suppressor gene. Unlike vhl, the relationship between tsg and promoter site hypermethylation has not been reported. tsg, also known as N03, was first identified as a novel gene that is down-regulated in v-src, v-mos and SV-40 transformed 3Y1 cells (11). It was named the "3Y1 v-src tumor suppressor gene" because subsequent studies confirmed tsg's role in the suppression of colony formation in soft agar and tumorigenicity in nude mice (8). In accordance with these previous findings we also observed the reversal of phenotype by transfecting 3Y1 v-src transformed fibroblasts with tsg (Figure 5)

To see whether tsg was also silenced by CpG island promoter hypermethylation, as in the case of vhl, we first validated our microarray findings with real-time quantitative polymerase chain reaction (qPCR). We demonstrated a significant inhibition of both tsg and vhl and expression in v-src-transformed cells but a dramatic induction and re-expression of vhl and tsg following treatment with DAC (Figure 6).

The main epigenetic modification in human beings is methylation of the cytosine bases that are located 5' to a guanosine in a CpG dinucleotide. Short regions of the genome that are rich in CpG dinucleotides are called CpG ‘islands.’ Hypermethylation of these CpG islands of a promoter leads to silencing of the gene (12). Using computational analysis to survey the promoter region of tsg, we found the region to be rich in CpG islands. We first evaluated the tsg promoter site methylation status with methylation specific polymerase chain reaction (MS-PCR) and found hypermethylation of the tsg promoter site in 3Y1 v-src cells compared to 3Y1 mock (Figure 7A). This finding was confirmed by combined bisulfate restriction analysis (COBRA) (Figure 7B) and bisulfite sequencing (Figure 7C) which showed a specific region of the tsg promoter site, designated as region 1, to be hypermethylated in all 7 clones of the v-src transformed cells examined, whereas no mock-transfected cells (5 clones) were methylated. Furthermore, a complete bisulfate sequencing of the remaining promoter site, designated as region 2, showed no methylation in the mock transfected clone and hypermethylation in all of the v-src transfected clones (Figure 7D). The issue as to what region of methylation regulates suppression of gene expression is controversial. That is, suppression of gene expression may be the result of a total increase in methylation density or a result of hypermethylation of a particular region of the promoter site (13–16). In our analysis, we found both hypermethylation of a particular region (region 1) in the 5' end of the tsg promoter site and a generalized total increase of methylation density of the entire promoter site (regions 1 and 2). Most importantly, we found that methylation status of the promoter site correlated with tsg expression (Figure 6A).

Currently, several DNA methyltransferase have been associated with hypermethylation, including DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3b3 (1721). Of these, the role of DNMT1 in hypermethlation of tumor suppressor genes is best established (22,23). Furthermore, previous studies have shown that DNMT1 is necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells (24,25). Therefore, we attempted to link v-src to hypermethylation of tumor suppressor genes to DNMT1 expression. Previous studies by others showed up-regulation of dnmt1 by Fos and Ras (26,27). In addition, analysis of the dnmt1 promoter site linking DNMT1 to the c-Jun pathway provided an explanation to the responsiveness of DNMT1 to oncogenic signals (28). In our real-time PCR analysis we found significant up-regulation of DNMT1 in v-src-transformed compared with mock-transfected 3Y1 cells (Figure 3A). Furthermore, our western blot analysis demonstrated that DNMT1 protein is indeed up-regulated in v-src-transformed 3Y1 cells (Figure 3B). These findings further support our hypothesis that promoter methylation is a mechanism linking the activities of oncogenes and tumor suppressor genes.

Both oncogenes and tumor suppressor genes have been implicated in carcinogenesis and in cancer progression. Long regarded as independent, we hypothesized that these distinct genetic elements might be related through epigenetic mechanisms. Our model provides evidence that v-src, an oncogene, silenced the vhl and tsg tumor suppressor genes through promoter hypermethylation in transformed rat fibroblasts. Our data suggest that this same approach can be used with other transformed cell models (i.e. with activated Ras and Myc transformation models) to identify additional tumor suppressor gene candidates that are epigenetically controlled by oncogenes. Moreover, this process can be evaluated in different species and different tissues of origin. The presence of an epigenetic linkage between oncogenes and tumor suppressor genes suggests that tumorigenesis occurs by cooperative mechanisms.


    Supplementary material
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Supplementary material is available online at: http://www.carcin.oupjournals.org.


    Acknowledgments
 
The authors would like to thank Bernard W. Futscher, Ph.D. of the Arizona Cancer Center for his helpful discussions regarding the preparation of this manuscript.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received August 18, 2004; revised October 8, 2004; accepted October 12, 2004.





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