Inactivation of DNA repair gene O6-methylguanine–DNA methyltransferase by promoter hypermethylation and its relation to p53 mutations in esophageal squamous cell carcinoma

Lei Zhang*, Wenfu Lu*, Xiaoping Miao, Deyin Xing, Wen Tan and Dongxin Lin1

Department of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

1 To whom correspondence should be addressed Email: dlin{at}public.bta.net.cn


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of esophageal squamous cell carcinoma (ESCC) has been linked to exposure to carcinogens such as nitrosamines that cause various alkyl DNA damages and O6-methylguanine–DNA methyltransferase (MGMT) is a primary defence against alkylation-induced mutagenesis and carcinogenesis. This study was to investigate the role of inactivation of MGMT by promoter hypermethylation and its relation to p53 mutations in ESCC. Methylation of MGMT promoter was determined by methylation-specific polymerase chain reaction in 119 ESCC specimens, 22 corresponding tissue samples adjacent to the tumors, and 21 normal epithelial specimens of the esophagus. The levels of MGMT protein in ESCC with methylated or unmethylated MGMT were analyzed by quantitative immunohistochemistry. Mutations of p53 in 119 ESCC were detected by denaturing high-performance liquid chromatography and sequencing. We found that all 21 normal esophageal tissues had unmethylated MGMT; however, among 119 ESCC, 46 (38.7%) had hypermethylated MGMT. This epigenetic change also occurred in some normal tissues adjacent to the tumors. The level of MGMT protein in MGMT-methylated ESCC was significantly lower than that in MGMT-unmethylated ESCC, whereas great inter-individual variation and poor expression was also observed among MGMT-unmethylated ESCC. Fifty-one percent (61/119) ESCC showed p53 mutations but the distribution of the mutations did not differ significantly between MGMT-methylated ESCC (44%) and MGMT-unmethylated ESCC (56.2%; P = 0.18). MGMT promoter hypermethylation was neither associated with overall G:C to A:T mutations nor associated with this type of mutations in non-CpG dinucleotides in p53. Our results demonstrate that inactivation of MGMT by aberrant promoter methylation is a frequent molecular event in ESCC. This epigenetic alteration is an important, but may not be the sole, mechanism leading to the impaired expression of MGMT. Aberrant MGMT methylation seemed not to be associated with overall frequency and spectrum of p53 mutations in ESCC.

Abbreviations: DHPLC, denaturing high-performance liquid chromatography; ESCC, esophageal squamous cell carcinoma; MGMT, O6-methylguanine–DNA methyltransferase; MSP, methylation-specific polymerase chain reaction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in certain parts of China, with about 250 000 cases diagnosed every year. Epidemiological and ecological studies of esophageal cancer in China have suggested some environmental factors are involved in the etiology of this disease. Among them, exposure to nitrosamines has received particular attention (13). Nitrosamines are potent alkylating agents that can produce various alkyl DNA adducts, particularly O6-methylguanine. If not removed, O6-methylguanine can preferentially mispair with thymine rather than cytosine during replication, thus causing G:C to A:T mutations, which may initiate carcinogenesis if the mutations occur in the tumor-related genes (4). A previous study has demonstrated the existence of relatively high levels of O6-methylguanine in esophageal DNA from individuals at high-risk of esophageal cancer in China (5), suggesting that O6-methylguanine formed by alkylating agents such as nitrosamines, may play an important role in mutagenesis and subsequent carcinogenesis of the esophagus. Molecular studies of Chinese ESCC have revealed somatic mutations in oncogenes and tumor suppressor genes (6). Among them, the p53 gene has been shown to be the most frequently mutated, with a mutational spectrum showing that G:C to A:T transitions are common (6). The etiology of p53 mutations in ESCC is unclear, although it has been suggested that these mutations might be linked to exposure to dietary or environmental carcinogens such as nitrosamines (6,7).

The DNA repair protein, O6-methylguanine–DNA methyltransferase (MGMT), specifically transfers alkyl groups at the O6 position of guanine to a cysteine residue within its own sequence in an auto inactivating reaction (8). Therefore, avoidance of the mutagenic/carcinogenic effect of such DNA damage is directly associated with the level and de novo synthesis rate of MGMT within the target cells. All normal human tissues express MGMT protein, although the levels vary significantly among tissue types; however, a proportion of human tumors lack MGMT activity (911). Previous studies have found that hypermethylation of the CpG islands within the MGMT promoter is associated with loss or reduction of MGMT expression in a variety of primary human carcinomas (1214), indicating that inactivation of this DNA repair mechanism may be involved in the development of these different types of cancer. In addition, it has been shown that promoter hypermethylation of MGMT is associated with G:C to A:T mutations in K-ras and p53 in colorectal cancer (15,16) and in p53 in lung cancer and astrocytomas (17,18). However, little or nothing is known so far about the status of MGMT hypermethylation and inactivation and its association with mutations in cancer-related genes in ESCC.

On the basis of the potential association between ESCC and exposure to environmental carcinogens, such as nitrosamines, and the role played by MGMT in protecting cells against DNA lesions caused by these carcinogens, we speculated that inactivation of MGMT, by promoter hypermethylation, may be an important host factor in carcinogenesis of the esophagus. In the present study, we analyzed the MGMT promoter hypermethylation in ESCC, paired normal tissues adjacent to the tumors and normal esophageal epithelium. We also examined the correlation between this epigenetic change in MGMT and the levels of MGMT expression and the status of p53 mutations in ESCC.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue samples and DNA extraction
119 ESCC and 22 corresponding normal tissues adjacent to the tumors were obtained from surgically removed specimens of individual patients who underwent an esophagectomy at the Cancer Hospital, Chinese Academy of Medical Science (Beijing). No patients received any antitumor treatment before the operation and the diagnosis as ESCC was histologically confirmed. The paired normal tissues adjacent to the tumor were sampled at least 5 cm away from the center of the tumor. The adjacent tissue was divided into two pieces: one was analyzed for methylation status of MGMT and the other was examined for invasiveness of tumor cells. The adjacent tissues showing tumor cell invasion were excluded in the study. Twenty-one samples of normal esophageal mucosa were collected from healthy individuals by biopsy. All the samples were frozen in liquid nitrogen immediately after resection or biopsy and stored at -80°C until processing. DNA was extracted using a standard method as described previously (19). This study was approved by the Institutional Review Board of the Chinese Academy of Medical Science Cancer Institute.

Bisulfite treatment of DNA and methylation-specific PCR
Conversion of unmethylated, but not methylated, cytosines to uracils in DNA samples by bisulfite treatment was carried out essentially according to the procedure of Herman et al. (20). Briefly, 1 mg DNA was resuspended in 50 ml H2O and denatured for 10 min in boiling water. The denatured DNA was then diluted in 550 ml freshly prepared solution containing 10 mM hydroquinone and 3 M sodium bisulfite, pH 5.0. The DNA solution was incubated at 50°C for 16 h and then desalted through a Wizard DNA Clean-Up System (Promega, Madison, WI, USA), treated with 0.3 M NaOH for 5 min at room temperature, precipitated with ethanol and resuspended in 30 ml of H2O for immediate use.

The methylation status of the MGMT gene was analyzed by a two-step methylation-specific PCR (MSP) approach, as described by Palmisano et al. (21). The bisulfite-treated DNA was used as a template in the first PCR to amplify a 289 bp fragment of the gene with the primers 5'-GGA TAT GTT GGG ATA GTT/5'-CCA AAA ACC CCA AAC CC. Amplification was achieved with initial denaturing at 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 52°C for 30 s and 72°C for 30 s, and then a final extension at 72°C for 7 min. The amplified products were diluted and subjected to the second PCR, with the primers 5'-TTT CGA CGT TCG TAG GTT TTC GC/5'GCA CTC TTC CGA AAA CGA AAC G-3' for the unmethylated reaction and 5'-TTT GTG TTT TGA TGA AAG TAG GTT TTT GT/AAC TCC ACA CTC TTC CAA AAA CAA AAC A for the methylated reaction. The reaction mixture (25 µl) contained the first PCR product (~20 ng), each primer (1.0 µM), dNTP (1.25 mM), Taq DNA polymerase (1.5 U) with the 1x reaction buffer (16.6 mM ammonium sulfite, 67 mM Tris, pH 8.8, 6.7 mM MgCl2 and 10 mM 2-mercaptoethanol). PCR was performed under the following conditions: an initial melting step of 3 min at 95°C; followed by 40 cycles of 15 s at 95°C, 15 s at 66°C and 15 s at 72°C; and a final elongation step of 7 min at 72°C. DNA from normal lymphocytes was used as negative control for methylated alleles of MGMT, and placental DNA treated in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) was used as positive control for methylated alleles of MGMT. PCR products were analyzed by electrophoresis in 4% agarose gel containing ethidium bromide.

The PCR products amplified with primers specific either for the methylated or for the unmethylated DNA were purified and ligated into the pMD18-T vector (Takara Biotech Inc., Dalian, China). Recombinants were then transformed into Escherichia coli. The plasmid DNA isolated from E.coli were analyzed by ABI 377 autosequencing system (Applied Biosystems).

Immunohistochemical analysis of MGMT
A representative panel of 21 tumors selected on the basis of availability of sections and known MGMT methylation status was evaluated for MGMT expression by immunohistochemistry. Immunostaining was essentially based on the procedures described previously (22) but using the HistostainTM-SP immunochemistry kit and DAB kit (Zymed, South San Francisco, CA, USA) with mouse monoclonal anti-human MGMT antibody (Neomarkers, Fremont, CA, USA; MT 3.1; 200 µg/ml PBS). Briefly, sections of formalin-fixed, paraffin-embedded tissues (4 µm thick) were deparaffinized with xylenes, dehydrated by using graded ethanol and endogenous peroxidase activity was blocked by 0.3% H2O2 in methanol. To unmask antigens, sections were treated with 10 mM citrate buffer (pH 6.0) at 100°C in a pressure vessel. After cooling at room temperature, sections were washed three times with water and brought to PBS for 5 min followed by incubation with 5% goat sera for 10 min. The sections were then incubated overnight with the monoclonal antibody against MGMT (1:50 dilution) at 4°C according to manufacturers' protocol. After washing with PBS, secondary antibody was added and incubated for 30 min at 37°C followed by the addition of streptavdin–peroxidase and incubated for 20 min at 37°C. The peroxidase reaction was finally developed with diaminobenzidine for 10 min. After the sections were washed several times, they were counterstained with hematoxylin, dehydrated with ethanol, rinsed in xylene and the sections were mounted with gum and used for microscopic examination and quantitative immunohistochemistry. As a control, isotyping to the primary antibody and omission of the primary antibody were done, and both gave no staining. Normal esophageal tissues were used as positive control samples.

Stained sections were quantitatively analyzed with MPIAS-500 Multimedia Color Pathologic Image Analysis System (Qing Ping Imaging Inc., Wuhan, China). All measurements were done with a stabilized light source with the same condenser and threshold settings and by the same operator. For accurate and consistent results, the intensity of the light was checked by capturing a blank field and shade correction was done by computer before reading each slide. All samples were analyzed 2 h after switching on the microscope as the light density stabilized in 2 h. Five randomly selected fields (865 µm2 each) were examined for the density of the immunostain for each sample. The level of immunohistochemical staining of MGMT was expressed as mean integrated gray.

Detection of p53 mutations
Mutations in exons 5–9 of the p53 gene were detected by PCR based denaturing high-performance liquid chromatography (DHPLC) analysis and sequencing. The published primers (23) were used for amplification of exons 5–8 of the gene. The exon 9 was amplified with primers 5'-GGGAGCACTAAGCGAGGTAA-3' and 5'-CCACTTGATAAGAGGTCCCAAG-3'. The lengths of the amplified fragments containing exons 5–9 were 229, 160, 156, 185 and 213 bp, respectively. PCR was accomplished with a 25 µl reaction mixture containing ~100 ng of DNA, 0.25 µM each primer, 0.2 mM dNTP, 2.5 mM MgCl2, 1.0 U Taq DNA polymerase with 1x reaction buffer (Promega). The reaction was carried out under the following conditions: an initial melting step of 2 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 60°C for exons 5–7 and 9 and 55°C for exon 8, 45 s at 72°C and a final elongation of 7 min at 72°C.

DHPLC analysis was performed on a Transgenomic WAVE System (Transgenomic Inc., Omaha, NE, USA) essentially identical with that described previously (24). Briefly, each PCR product was denatured for 3 min at 94°C, and then gradually re-annealed by decreasing sample temperature from 94 to 45°C over a period of 30 min. The PCR product was applied to the DHPLC column and eluted with a linear acetonitrile gradient at a flow rate of 0.9 ml/min. The mobile phase temperatures were 66, 64, 61, 60 and 61°C for the PCR products of exons 5–9, respectively. The mutations revealed by DHPLC analyses were confirmed by direct DNA sequencing of the PCR products with an ABI 377 autosequencing system.

Statistical analysis
Statistical analyses were performed using the SigmaStat (Jandel Scientific, San Rafael, CA, USA). The association between the MGMT methylation status and protein expression was assessed by Mann–Whitney Rank Sum Test. Pearson's {chi}2 test was used to examine differences in distribution of p53 mutations between ESCC with methylated MGMT and ESCC without methylated MGMT. Differences were considered statistically significant for P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MGMT promoter hypermethylation
To confirm the reliability of the nested, MSP method, PCR products amplified with primers specific for either the methylated or the unmethylated DNA were cloned and sequenced. DNA sequencing verified that both sequences are in the promoter of the MGMT gene, and the marked differences in these two sequences validate this approach. All the cytosines in the unmethylated product were converted to thymines after bisulfite treatment and amplification, suggesting that the MGMT gene is unmethylated. However, the cytosines in the CpG dinucleotides of methylated product remained unchanged, as methylated cytosines cannot be modified by bisulfite, which indicated that the CpG islands of the gene are methylated. DNA samples obtained from 21 normal esophageal mucosa, 119 ESCC and 22 paired normal tissues adjacent to ESCC were subjected to MGMT promoter hypermethylation study using MSP. Figure 1 shows representative examples of the MSP products analyzed on an agarose gel for the MGMT gene. It was found that 46 (38.7%) ESCC had aberrant MGMT promoter hypermethylation, whereas this epigenetic change of the gene did not occur in all specimens of normal esophageal mucosa. Among 22 paired ESCC and their adjacent normal tissues, nine ESCC exhibited MGMT promoter hypermethylation, of which 5 (55.6%) also had this change in their adjacent normal tissues. Interestingly, among the remaining 13 ESCC without MGMT promoter hypermethylation, two had this epigenetic alteration in their adjacent normal tissues.



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Fig. 1. MSP of MGMT. The presence of a visible PCR product in lane U indicates the presence of unmethylated genes of MGMT. The presence of product in lane M indicates the presence of methylated genes. Corresponding lanes are: ESCC (1,2), in vitro methylated DNA (IVD) as positive control for methylation, normal lymphocytes (NL) as negative control for methylation, and water control for PCR reaction.

 
Association between MGMT promoter methylation and its expression
Because tumor tissues are often contaminated with normal cells, such as infiltrating lymphocytes and endothelial cells, which express MGMT, western blot analysis may not be helpful in quantification of MGMT expression. Immunohistochemistry was therefore used to determine 21 ESCC tissues with known methylation status of the MGMT promoter. MGMT protein was detected primarily in the nuclei, while it was also seen in the cytoplasm. All sections examined had the staining of MGMT in normal cells adjacent to or within tumors, which provided an internal positive control. The representative immunohistochemical staining of the expression or loss of expression of MGMT protein is shown in Figure 2. Quantitative analyses of stained sections showed that the levels of MGMT protein observed in ESCC varied greatly among individuals. However, as shown in Table I, when the levels of MGMT expression were compared among individuals according to the status of MGMT promoter hypermethylation, the median value for ESCC with hypermethylated MGMT (n = 9) was significantly lower than that for ESCC with unmethylated MGMT (n = 12) (0.19 versus 2.66, P = 0.019). Furthermore, it should be noted that even among the unmethylated ESCC, MGMT expression showed a considerable inter-individual variation, ranging from 0.07 ± 0.02 to 11.66 ± 2.06.



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Fig. 2. Immunohistochemistry of MGMT in ESCC. (A) ESCC unmethylated at MGMT shows expression of the protein in tumor cells. (B) ESCC hypermethylated at MGMT shows complete lack of expression in tumor cells, but expression in lymphocytes.

 

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Table I. MGMT levels in ESCC as a function of MGMT methylation status

 
Relationship between MGMT promoter hypermethylation and p53 mutations
Among 119 ESCC analyzed, 61 (51.3%) demonstrated mutant p53 and of them, three had two mutations in different exons. It was found that 43.5% (20/46) of the p53 mutations were presented in MGMT methylated ESCC, which was not significantly different from that (56.2%; 41/73) in MGMT unmethylated ESCC (P = 0.18). Analysis of mutational spectrum showed that most of the p53 mutations were transitional mutations (59.4%), with G:C to A:T, C:G to T:A, and other types being 29.7, 23.4 and 6.3%, respectively. Transversions and insertions/deletions accounted for 29.7 and 10.9%. The presence of MGMT promoter hypermethylation was neither associated with overall G:C to A:T mutations nor associated with this type of mutations in non-CpG dinucleotides in p53 (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The promoter hypermethylation of the MGMT gene has been shown to be a common epigenetic event in certain human cancers, including brain cancer, colon cancer, lung cancer, head and neck cancer, gastric cancer, lymphomas, cervical cancer and retinoblastoma (1214,21,2527). Aberrant methylation of this DNA repair gene has also been observed in esophageal adenocarcinoma (28). In the present study, we have extended the finding to ESCC, showing for the first time that 38.7% of ESCC had promoter hypermethylation of the MGMT locus. Furthermore, we have demonstrated that loss or poor expression of MGMT was associated with MGMT CpG island methylation. These results suggest that inactivation by the promoter hypermethylation of MGMT is a common molecular event in ESCC and, thus, it may be involved in the development of this cancer. Another interesting finding in the present study was the observation that the promoter hypermethylation of MGMT also occurred in some normal tissues adjacent to the tumors, but not in normal esophageal epithelium obtained from cancer-free individuals. Furthermore, the promoter hypermethylation of MGMT was also detected in two normal tissues adjacent to the tumors without this epigenetic change. These results may implicate a field methylation of MGMT. Parallel findings have been reported for p53 mutations and p53 protein accumulation in ESCC and the corresponding adjacent mucosa, showing that among tumors without p53 accumulation, portions of adjacent mucosa were p53 positive (2931). Our results along with those of p53 are consistent with the observation of field cancerization in the esophagus (32,33) and indicate that the promoter hypermethylation of MGMT may be an early molecular event in esophageal carcinogenesis.

Because MGMT plays a central role in the repair of O6-alkylguanine DNA adducts which can be formed by exposure to nitrosamine carcinogens, its involvement in esophageal carcinogenesis is biologically plausible. Previous studies have shown that dietary exposure to nitrosamines and the host capability to form these carcinogens are associated with an increased risk of ESCC (13). Furthermore, a higher amount of O6-methylguanine was actually found in the esophageal epithelium (5). It could be possible that among individuals exposed to carcinogenic nitrosamines that target the esophagus, inactivation of MGMT by the epigenetic alteration in the target tissue may allow the persistence of O6-methylguanine in genomic DNA and accelerate the accumulation of mutations in cancer-related genes, which may lead to an increased risk for developing the cancer. The importance of MGMT in nitrosamine-induced carcinogenesis has been evident in animal models. It has been demonstrated that transgenic mice overexpressing MGMT are protected against nitrosamine-induced O6-methylguanine formation and risk of tumor development (3436), whereas MGMT knockout mice are sensitive to the lethal effects and carcinogenesis caused by nitrosamines and other alkylating agents (37,38). The data together with our results suggest that nitrosamine exposure and impaired MGMT function may be important environmental and host factors in the etiology of ESCC.

Previous studies have revealed that loss of MGMT function is most frequently due to epigenetic changes, especially hypermethylation of the promoter region. Hypermethylation of the MGMT CpG islands as the cause of the gene transcriptional silencing in cell lines defective in O6-methylguanine repair capacity has been well-documented (3942). Recently, Esteller et al. (12) reported in their study on the relationship between MGMT methylation and MGMT expression in human tumors that 92% tumors that lack MGMT expression showed MGMT promoter hypermethylation, whereas 94% tumors that retained expression of MGMT were unmethylated at the MGMT CpG island. Our results in the present study are generally in agreement with theirs, showing that MGMT levels in ESCC with MGMT promoter hypermethylation were significantly lower than those in the unmethylated tumors, and in some methylated cases, the expression was almost absent. However, contrary to their findings, we found that even among unmethylated tumor tissues, inter-individual variation in MGMT expression was great (over a ~160-fold range) and poor expression was also observed. This observation suggested that factors other than promoter hypermethylation might also be responsible for the suppression of MGMT expression. It has been shown that loss of MGMT expression occurs rarely due to deletion, mutation or rearrangement of the gene (43,44), or due to mRNA instability (45). Therefore, other genetic or epigenetic alterations such as single nucleotide polymorphisms should be considered as a potential mechanism as germ line polymorphisms within the promoter region or coding region may have impact on the transcription or the protein stability (46,47). Further investigations are needed to elucidate this important issue.

Recent studies have shown that in certain cancers including colorectal cancer, non-small cell lung cancer and astrocytomas, the presence of mutations, especially G:C to A:T transitions in p53 are associated with MGMT promoter hypermethylation (1618). Because p53 mutations have been shown to be common in ESCC and the origin is thought to be attributable to environmental exposure (6,7), we therefore hypothesized that these mutations might be associated with aberrant MGMT promoter methylation. However, our results in the present study did not support this speculation as no association was found between aberrant MGMT methylation and frequency of overall p53 mutations or the presence of G:C to A:T transitions in this suppressor gene. This inconsistency with the previous reports on other types of cancer (1618) may reflect the difference in etiology and pathogenesis of these different cancers. For example, Esteller et al. (16) showed that 60% of p53 mutations in colorectal cancers were G:C to A:T transitions. In contrast, we found that this type of p53 mutation was only 29.7% in ESCC. Another possibility to explain our observation is that p53 mutations may correlate to the levels of MGMT expression or activity but not only MGMT promoter hypermethylation, because we found that MGMT expression showed a considerable inter-individual variation even among the unmethylated ESCC and the levels in some unmethylated cancers were similar to that in methylated cancers. Finally, it is also possible that in esophageal carcinogenesis, p53 mutations may occur earlier than does aberrant methylation of MGMT so that these two molecular events are entirely unrelated. However, it should be noted that as this analysis was based on a relatively small sample set and the mutational spectrum of p53 is complicated, the failure to find correlation might also be due to limited statistical power. In addition, although we determined p53 mutations at the hot spots (i.e. exons 5–9), we did not determine mutations in other regions of the gene, which could have the potential to bias the results. The finding should hence be interpreted with caution before this preliminary result is confirmed in further studies.


    Notes
 
* Lei Zhang and Wenfu Lu contributed equally to this work. Back


    Acknowledgments
 
This work was supported by grants 39825122 and 30128020 from National Natural Science Foundation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 17, 2002; revised April 3, 2003; accepted April 4, 2003.