Telomerase activation and p53 mutations in urethane-induced A/J mouse lung tumor development

Joji Ohno, Yoshitsugu Horio4,, Yoshitaka Sekido1,, Yoshinori Hasegawa, Masahide Takahashi2,, Jun-ichi Nishizawa3,, Hidehiko Saito, Fuyuki Ishikawa3, and Kaoru Shimokata1,

First Department of Internal Medicine,
1 Department of Clinical Preventive Medicine and
2 Department of Pathology II, Nagoya University School of Medicine, Nagoya 466-8550, Japan and
3 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mouse telomerase holoenzyme, which synthesizes telomeric DNA de novo, is a ribonucleoprotein complex that includes the mouse telomerase RNA component (mTERC), mouse telomerase-associated protein (mTEP1) and mouse telomerase reverse transcriptase (mTERT). To determine the role of telomerase in urethane-induced lung tumorigenesis in A/J mice we examined telomerase activity and the expression of each telomerase subunit in 20 tumor samples, harvested at 16, 28, 40 and 50 weeks after urethane treatment. The telomeric repeat amplification protocol assay showed that statistically significant telomerase activation occurred both early and late in tumorigenesis. Semi-quantitative reverse transcription–polymerase chain reaction analysis revealed that mRNA expression levels of mTEP1 and mTERT were up-regulated during tumor progression, while mTERC expression was not significantly different between tumors and normal lung. We further examined mTEP1 protein expression in normal lung tissue and lung tumors; western blot analysis showed preferential expression of mTEP1 protein in lung tumors compared with normal lung and immunohistochemistry revealed that a majority of the adenoma cells were positively stained in the nucleus, whereas only a few of the adjacent normal alveolar cells were immunoreactive. In addition, we investigated DNAs of the 20 tumor samples by single strand conformation polymorphism and sequencing analyses to examine whether alterations of the p53 gene in exons 5–8 were associated with telomerase activity. Although we found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six late stage tumors, there was no apparent correlation between telomerase activity and p53 mutations. Collectively, these results suggest that mTEP1 as well as mTERT may be involved in the regulation of telomerase activity and that telomerase activation may contribute to lung tumorigenesis in A/J mice independently of p53 gene alterations.

Abbreviations: ITAS, internal telomerase assay standard; mTEP1, mouse telomerase-associated protein; mTERC, mouse telomerase RNA component; mTERT, mouse telomerase reverse transcriptase; RT–PCR, reverse transcription–polymerase chain reaction; SSCP, single strand conformation polymorphism; TRAP, telomeric repeat amplification protocol.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lung cancer is the leading cause of cancer mortality in the USA and Japan (1). It comprises four major histological subtypes, including adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell carcinoma. The incidence of adenocarcinoma of the lung has been gradually increasing for the past two decades. However, the peripheral location and late symptomatic manifestation of adenocarcinoma of the lung prevent its detection at an early stage as well as comprehensive analysis of the early molecular events associated with it. To resolve them we need multiple approaches against lung cancer that encompass epidemiological and clinical data as well as adequate in vitro and/or in vivo experimental systems.

Among many animal models available for the analysis of human lung adenocarcinoma, urethane-induced lung tumorigenesis in A/J mice is thought to be one of the most useful because of its faithful reproducibility, histological similarity and time-dependent progression from hyperplasia through adenoma and eventually to adenocarcinoma (2,3). This model is also important because atypical adenomatous hyperplasia, which is histologically similar to hyperplasia/adenoma in the A/J mouse model, has been categorized as a premalignant lesion of bronchioloalveolar carcinoma in a new histological classification of human lung cancer (4). Furthermore, mouse lung epithelial cells have been shown to accumulate a number of genetic changes sequentially, involving oncogenes and tumor suppressor genes, to develop overt lung cancer during the multistage process of carcinogenesis and human lung cancer arises through a similar process (3,5). Among such genetic changes examined so far, K-ras and p53 are frequent targets for developing lung adenocarcinomas in urethane-treated A/J mice as well as in human (5,6). Based on such histological and molecular similarities, further elucidation of the genetic and/or biochemical aspects of mouse lung carcinogenesis would enable us to understand the pathogenesis of human lung cancer and to utilize this model to evaluate molecular and pharmacological interventions, which may prove efficacious in treatment and possible prevention.

It is becoming clearer that one of the key processes of neoplasia is characterized by activation of telomerase, a ribonucleoprotein enzyme complex that adds telomeric repeats to the ends of replicating chromosomes, the telomere (7). However, the role of telomerase in human lung carcinogenesis has not been fully clarified to date. Thus, using a well-established A/J mouse model we examined telomerase activity and expression of the three major subunits of telomerase, mouse telomerase reverse transcriptase (mTERT), mouse telomerase-associated protein (mTEP1) and mouse telomerase RNA component (mTERC). Furthermore, to determine whether genetic alterations of the p53 gene were associated with telomerase activity, we performed mutation analysis of the p53 gene exons 5–8. We report herein that telomerase activity is activated in both the early and late stages of tumorigenesis, which might contribute to carcinogenesis independently of the p53 alteration, and that mTEP1 as well as mTERT may play an important role in the regulation of telomerase activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lung tumor induction
A/J inbred mice at 5–7 weeks of age were given a single i.p. injection of urethane in 0.9% saline at a dose of 1 mg/g body wt. Groups of mice were killed at 16, 28, 40 and 50 weeks after urethane administration. The lungs were removed and placed in ice-cold phosphate-buffered saline. Lung tumors were dissected from the surrounding non-tumor lung tissue, frozen in liquid nitrogen and stored quickly at –80°C until use. Since tumors at 16 and 28 weeks after urethane administration were small, between two and four tumors were pooled and assayed as a single sample.

Telomeric repeat amplification protocol (TRAP) assay
Extraction of cell lysates and assays of their telomerase activity were done with a TRAPeze kit (Oncor, Gaithersburg, MD) according to the modified version of the manufacturer's instructions. Briefly, frozen tumor samples were homogenized in an appropriate amount of CHAPS lysis buffer and incubated for 30 min on ice. After centrifugation at 16 000 g for 20 min at 4°C the concentration of the supernatant was measured with a BCA protein assay kit (Pierce Chemical Co., Rockford, IL). An aliquot of the cell lysates containing 2 µg protein was used for each TRAP assay unless otherwise indicated. Each telomerase reaction product labeled with [{gamma}-32P]ATP was purified by phenol/chloroform extraction to avoid PCR inhibitory proteins, then amplified in the presence of an internal telomerase assay standard (ITAS, 36 bp product), which works as an internal PCR control for each TRAP assay. TRAP assay products were separated by 12.5% PAGE, dried and autoradiographed. The intensity of each TRAP assay product and ITAS was calculated by measuring the generated bands using NIH Image v.1.61, which is a powerful image processing application and can be downloaded from the NIH Image ftp site (rsbweb.nih.gov). This application can simultaneously measure density and spatial data from a properly calibrated image. The quantitation of telomerase activity was estimated by the following formula: relative specific activity of telomerase = the intensity of each TRAP assay product ÷ the intensity of each ITAS. As a positive control, 0.25 and 0.5 µg cell extract from human lung cancer cell line NIH H1299 were used; as a negative control, 2 µl of lysis buffer without any cell extract was used.

Reverse transcription–polymerase chain reaction (RT–PCR)
Total cellular RNAs were isolated from the normal lung of untreated mice, pooled small tumor samples and middle sized individual tumors of urethane-treated mice by a single-step guanidine isothiocyanate method, using Isogen (Nippongene, Tokyo, Japan), and were treated with DNase I (Takara Shuzo Co., Japan) for 60 min at 37°C in the presence of RNase inhibitor (Takara Shuzo Co.) to eliminate genomic DNA contamination. Random primed, first strand cDNAs were synthesized from 3 µg total RNAs using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions.

PCR amplification was carried out using Amplitaq Gold (Roche Molecular Systems, Branchburg, NJ) in the presence of 10% (v/v) dimethyl sulfoxide. PCR cycles consisted of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C. The number of cycles used was in the linear range and varied from 28 to 40 cycles in different PCRs. Primers used were as follows: mTEP1, 765S (5'-gaagaaagcacagaagtccc-3') and 1073AS (5'-agaactgtattggcgatgtc-3'); mTERC, 121S (5'-ctggtcttttgttctccgc-3') and 446AS (5'-tgcacttcccacagctca-3'); mTERT, 2674S (5'-atggcgttcctgagtatg-3') and 2931AS (5'-ttcaaccgcaagaccgacag-3'); mouse ß-actin, 409S (5'-tgaaccctaaggccaaccgtg-3') and 804AS (5'-gctcatagctcttctccaggg-3'). To normalize the amount of RNA, amplification of the mouse ß-actin gene was used as a control.

Western blot analysis
Fifteen micrograms of cell lysates prepared from normal lungs of untreated mice and lung tumors of urethane-treated mice were electrophoresed and blotted as described previously (8). Immunoblots were detected using an ECL chemiluminescence system (Amersham, Little Chalfont, UK) according to the manufacturer's instructions and exposed on Amersham Hyperfilm. The rabbit polyclonal antibody specific for mTEP1 was generated against recombinant mTEP1 protein.

Immunohistological staining
Frozen sections (4 µm thick) of normal lung and lung tumor tissues were fixed with 4% paraformaldehyde in 0.1 M NaH2PO4 with 8% sucrose (pH 7.4) for 30 min on ice and immunohistological staining was performed by the avidin–biotin–peroxidase complex method as described previously (9). Nuclear staining was done with methyl green.

Single strand conformation polymorphism (SSCP) analysis and DNA sequencing analysis
DNA was prepared from each CHAPS cell lysate using a standard phenol/chloroform extraction method (10) and subjected to nested or hemi-nested PCR amplification to detect p53 mutations between exons 5 and 8, including the exon–intron boundaries, as previously described (5). The PCR products labeled with [{alpha}-32P]dCTP were diluted with formamide dye solution and electrophoresed on 6% polyacrylamide gels. No PCR products were detected in an all CHAPS control after two rounds of amplification. Aberrant bands were reamplified with the same primer set and the purified PCR products were sequenced using an Applied Biosystems model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, CT) as described previously (11).

Statistical analysis
Associations between telomerase activities and tumor development were evaluated using a non-parametric statistical method. All statistical tests were conducted at the two-sided 0.05 level of significance.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activation of telomerase in urethane-induced lung tumorigenesis in A/J mouse
Since urethane-induced lung tumorigenesis in A/J mice follows a time-dependent progression, we collected lung tumors at 16, 28, 40 and 50 weeks after treatment, which have been shown to correspond to very small adenoma, small adenoma, middle sized adenoma and large adenoma/adenocarcinoma, respectively (2,5). Because of the small sizes of the lung tumors at both 16 and 28 weeks following urethane administration, between two and four tumors were pooled at each time and assayed as a sample. To determine the relationship between telomerase activity and tumor development we measured telomerase activities in 20 samples at the four different time points and in four normal lung tissues of untreated mice at 20, 33, 45 and 55 weeks of age, whose ages almost corresponded to those of mice examined after urethane treatment (Figure 1Go and Table IGo). The level of telomerase activity per 2 µg cell extract protein was very low in normal lung tissues compared with the high activity of either 0.25 or 0.5 µg extract derived from NCI-H1299, a human lung carcinoma cell line previously shown to express high telomerase activity. Although there were variations among tumors, the average levels of telomerase activity of lung tumors at the four different time points were significantly elevated compared with that of normal lung tissues (Table IGo; P < 0.05, normal lung versus lung tumors at 16, 28, 40 or 50 weeks). Among all tumors examined the average level of telomerase activity of the 50 week tumors was significantly higher than that of the other tumors (P < 0.05, 50 week tumors versus 16–40 week tumors), indicating that telomerase activities might be elevated in both the early and late stages of tumorigenesis. Heat inactivation or RNase treatment of each sample abolished telomerase activity, indicating that TRAP products were attributable to telomerase (data not shown).



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Fig. 1. Telomerase activity in normal lung tissues and urethane-induced lung tumors. For each mouse sample an aliquot of the extract containing 2 µg protein/reaction was used in a TRAP assay. An extract derived from human lung carcinoma cell line H1299 having telomerase activity was used as a positive control. The levels of telomerase activity in lung tumor samples were increased during the course of progression while those of normal lung tissues revealed very low activity. The case numbers are given at the top of the lanes. NC, lysis buffer used as a negative control; NL, normal lung.

 

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Table I. Telomerase activity and p53 mutation in normal lung tissue and lung tumor samples at different timepoints after urethane administration
 
mTERC, mTERT and mTEP1 mRNA expression in tumorigenesis
To determine the molecular mechanisms through which mouse telomerase is activated in tumors we examined expression of the three major subunits, including mTEP1, mTERT and mTERC. We collected another set of tumor samples at 16, 28, 40 and 50 weeks after urethane treatment and normal lung tissues of untreated mice at 20 and 55 weeks of age, extracted RNAs and performed semi-quantitative RT–PCR (Figure 2Go). Expression of mTEP1 mRNA was weak in normal lung tissue but increased in all lung tumors tested, indicating that mTEP1 is up-regulated at an early stage of tumorigenesis. Up-regulation of mTERT mRNA expression was also observed in tumors, suggesting that mTEP1 as well as mTERT might be involved in the regulation of telomerase activity. In contrast, the expression level of mTERC were almost the same in normal lung tissue and lung tumors at different time points, suggesting that telomerase activity may not be regulated by expression of mTERC in the urethane-induced lung tumor model. RT–PCR of ß-actin mRNA was done as a control to normalize the amount of RNAs among these samples.



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Fig. 2. Semi-quantitative RT–PCR analyses for mTERC, mTERT and mTEP1 mRNA expression. Expression of mTEP1 mRNA is very weak in normal lung tissue of untreated mice but relatively high in lung tumors. mTERT mRNA expression was up-regulated in tumors during the course of development. The expression levels of mTERC were almost the same in normal lung tissue and lung tumors at different time points. The case numbers are given at the top of the lanes.

 
mTEP1 protein expression in normal lung and lung tumors
To confirm preferential expression of mTEP1 in lung tumors compared with normal lung tissue we performed western blot analyses using a polyclonal antibody specific for mTEP1 protein. As shown in Figure 3AGo, mTEP1 protein expression was up-regulated at a similar level in both 16 and 40 week lung tumors compared with normal lung tissue, which was consistent with their mRNA expression levels. Further investigations of the expression and location of mTEP1 protein by immunohistochemical analysis indicated that only a few normal alveolar cells were immunoreactive, whereas the majority of the 40 week adenoma cells showed positive nuclear staining (Figure 3B and CGo). The same result was obtained in the 16 week adenomas (data not shown). Association of up-regulation of mTEP1 expression with telomerase activation at an early stage of tumor progression suggested that mTEP1 might be one of the key regulators of telomerase activity in this model.



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Fig. 3. mTEP1 protein expression in normal lung and lung tumors. (A) Western blot analysis using polyclonal antibody specific for murine TEP1 protein. mTEP1 protein expression was up-regulated in both 16 and 40 week lung tumors compared with normal lung tissue. (B and C) Immunohistochemical analysis of mTEP1 expression. A majority of 40 week adenoma cells were positively stained in the nucleus while a few normal alveolar cells were immunoreactive. Large arrows point to adenoma cells. Small arrows point to normal alveolar cells. Magnification: (B) 100x; (C) 450x.

 
Association of telomerase activation and p53 gene alteration
Since we previously found that p53 mutations between exons 5 and 8 occurred ~40 weeks after urethane treatment in this model, we hypothesized that p53 mutations might affect activation of telomerase at a late stage of tumor progression. To test this we isolated DNAs from 20 cell lysates used for TRAP assay and performed PCR–SSCP analysis for p53 mutations (Figure 4Go). Six tumors (two at 40 weeks and four at 50 weeks after treatment) showed six altered bands with different mobilities from those of normal lung in exons 5 and 8, but no alterations were observed in exons 6 and 7. These six tumors were further analyzed by sequencing (Table IGo). We found one nonsense, two missense and two silent and one double mutation at different codons. We did not distinguish whether these mutations accompanied wild-type allele loss, due to inevitable contamination by normal cell DNAs. Although p53 mutations occurred in the late stage during tumor progression, no apparent correlation was found between the level of telomerase activity and the p53 gene alteration, suggesting that p53 alterations might not have an effect on telomerase activation.



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Fig. 4. Representative PCR–SSCP analyses of the p53 gene mutations. Five p53 defects were observed in exon 5. The case numbers are given at the top of the lanes. The arrowheads indicate aberrant band shifts.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we found that telomerase activity was elevated at the early stage and further increased at the late stage during the progression of tumorigenesis with statistical significance, suggesting that activation of telomerase might occur both early and late in urethane-induced A/J mouse lung tumorigenesis. Two previous studies indicated that telomerase activity was up-regulated in late stage tumors using three transgenic mouse models for breast, pancreatic and skin cancers (12,13) and a study of chemically induced skin carcinogenesis revealed that telomerase activity was increased early in premalignant stages of tumor progression (14). The different timing of telomerase activation during tumor progression may depend on the tissue analyzed and the mouse model used.

Although we observed statistically higher levels of telomerase activity in lung tumors than in normal lung tissue, it is unclear how telomerase activation contributes to tumor development in our model. Indeed, telomerase activation does not seem to be essential for tumor development, since tumors were formed in telomerase-defective mTERC knockout mice (15). In this regard it is noteworthy that in double knockout mice for both mTERC and the tumor suppressor genes p16/p19ARF, tumors developed with ~50% incidence compared with that in p16/p19ARF knockout mouse (16). Moreover, restoration of mTERC into MEF cells significantly restored oncogenic potential (16), suggesting that activation of telomerase may play an important role in promoting tumor progression.

TEP1, the mammalian homolog of Tetrahymena p80 telomerase protein, has been shown to interact specifically with TERC and TERT. TEP1 contains repeated sequences in the C-terminus, referred to as the WD-40 motif, which has been suggested to function in protein–protein interactions (17,18). Indeed, it has been reported that TEP1 is a shared component of the telomerase complex and the vaults (19). The vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Furthermore, it has also been reported that the region close to the N-terminus of hTEP1 and the C-terminal region of p53 interact in vitro (20). However, the function of TEP1 has not yet been elucidated clearly, even in telomere regulation. In our study we found that both mTEP1 up-regulation and telomerase activation occurred simultaneously in an early stage of tumorigenesis. This implies that mTEP1 may participate in the regulatory mechanisms that control telomerase activity. Moreover, the role of TEP1 in the regulation of telomerase may be different between human and mouse, since hTEP1 mRNA has been found to be constitutively expressed in normal lung and lung cancer (21).

In human lung cancer the hTERT expression level is well correlated with telomerase activity and is considered as a surrogate marker for it (21,22). In the A/J mouse lung tumor model the mTERT mRNA expression level was also up-regulated in tumors compared with normal lung tissue, suggesting that mTERT might be involved in the regulation of telomerase activity. However, there was not so much difference in mTERT mRNA expression among tumors during progression, while telomerase activity in the late stage of tumorigenesis was further increased. This suggests that other regulatory mechanisms, such as loss of telomerase inhibitors, may be involved in telomerase activation.

mTERC has been shown to be a possible regulator of telomerase activity in mouse skin and pancreatic cancer models, mTERC being up-regulated in the hyperproliferative stages of progression before telomerase activity is increased (12). However, mTERC might not affect telomerase activity in the murine lung, since we found that mTERC was expressed at the same level in normal lung and lung tumors. Rather, as hTERC and hTERT were shown to be able to reconstitute telomerase activity in vitro with properties similar to those of native telomerase (23), expression of mTERC and mTERT in normal lung tissue may merely reflect the basal level of intrinsic telomerase activity.

The tissue distribution of hTERC has been examined using the RT–PCR, northern blot analysis and in situ hybridization methods. RT–PCR and northern blot analysis showed hTERC to be expressed in most somatic tissues, although its expression level was much lower than in cancer and gonads (24). Another study, using RT–PCR, has reported that hTERC was expressed in most adjacent non-neoplastic lung tissues and all lung cancer tissues examined (21). However, hTERC expression detected by a radioactive in situ hybridization method has been reported to be in the basal cells of bronchi, in bronchioles and in all lung cancer tissues examined, but not in alveolar cells and areas of atypical adenomatous hyperplasia (25). Another study found infrequent hTERC expression in pulmonary adenocarcinoma by in situ hybridization (26). Thus, the tissue distribution of hTERC detected by in situ hybridization appeared more restricted than that detected by RT–PCR or northern blot analysis. On the other hand, the tissue distribution of mTERC has been analysed by northern blot analysis and RT–PCR but not by in situ hybridization. This latter technique would be necessary to examine the precise tissue distribution and localization of mTERC in murine tissues. Furthermore, a comparative study by in situ hybridization or immunohistoligical analysis of all three telomerase components and the in situ version of the TRAP assay (27) may determine which is the critical component for regulation of telomerase activity.

Since we previously reported that p53 mutation frequently occurs as a late event in urethane-induced lung tumorigenesis (5) and because the p53 and/or Rb tumor suppressor pathways have been implicated as crucial for the checkpoint responses of telomere length (28), we hypothesized that inactivation of the p53 gene might affect telomerase activity. To test this we examined p53 alterations in exons 5–8, where the vast majority of p53 mutations occur (6). We found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six tumors at the late stage, but found no apparent correlation between the p53 mutations and the level of telomerase activity, although there may be some p53 mutations outside exons 5–8. However, since it has been reported that murine fibroblast cell lines established from p53+/+ and p53–/– mice expressed telomerase regardless of the p53 status of their tissue of origin (29), telomerase activation and p53 inactivation in the A/J mouse lung tumor model may be independent and cooperating molecular events that occur during the multistage process of carcinogenesis.

In human non-small cell lung cancer several groups have shown statistically significant associations between mutant p53 expression and telomerase activity (3032), while another study has not found any significant association (33). Although additional studies will be needed to draw a definitive conclusion, the p53 gene may have different roles in the regulation of telomerase activity in human and mouse.

In the present study we have demonstrated the significance of telomerase activation during lung tumor progression in the A/J mouse and the involvement of mTEP1 and mTERT in the regulation of telomerase activity. With further studies of telomerase activation and telomere regulation in murine lung tumorigenesis, the commonalities and distinctions between human and mouse may provide an integrated understanding of the complex genetic events underlying human lung cancer development.


    Notes
 
4 To whom correspondence should be addressed Email: yhorio{at}med.nagoya-u.ac.jp Back


    Acknowledgments
 
We are grateful to Ms K.Shimamoto for her technical assistance. This work was supported by a Grant-in-Aid for COE Research from the Ministry of Education, Science, Sports and Culture of Japan and a research grant from Aichi Cancer Center Research Foundation.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 23, 2000; revised January 15, 2001; accepted January 16, 2001.