Affiliations of authors: C. Kitanaka, K. Sakurada, A. Tomiyama, K. Noguchi, Y. Kuchino, Biophysics Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan; K. Kato, R. Ijiri, Y. Tanaka (Division of Pathology), Y. Toyoda (Division of Oncology), H. Kigasawa (Division of Hematology), T. Nishi (Division of Surgery), Kanagawa Children's Medical Center, Minami-ku, Yokohama, Japan; Y. Nagashima, Second Department of Pathology, School of Medicine, Yokohama City University, Kanazawa-ku, Yokohama; A. Nakagawara, Division of Biochemistry, Chiba Cancer Center Research Institute, Chuoh-ku, Chiba, Japan; T. Momoi, Division of Development and Differentiation, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo; M. Shirouzu, S. Yokoyama, Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi, Yokohama; S. Yokoyama, Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo; Y. Kuchino, Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama, Japan.
Correspondence to: C. Kitanaka, M.D., or Y. Kuchino, Ph.D., Biophysics Division, National Cancer Center Research Institute, 51-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan (e-mail: ckitanak{at}ncc.go.jp).
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ABSTRACT |
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INTRODUCTION |
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On the other hand, molecular biologic markers such as H-Ras, TrkA, and N-Myc have been associated with the prognosis of neuroblastoma. The amplification and/or overexpression of the N-myc gene have been associated with poor prognosis (10). In contrast, the overexpression of H-Ras as well as TrkA is a favorable prognostic factor of neuroblastoma (1115), suggesting that H-Ras and/or TrkA overexpression might contribute to the elimination of tumor cells through an as yet unknown mechanism. Given our recent observation (9,16) that Ras causes autophagic degeneration in a cell type-dependent manner, these lines of evidence together give rise to the intriguing possibility that increased expression of Ras may contribute to neuroblastoma regression through the activation of a nonapoptotic cell suicide program. In this study, we examine this possibilitythrough in vivo analyses using neuroblastoma tissues and through in vitro analyses using neuroblastoma cell linesfor the purpose of understanding the mechanism involved in spontaneous neuroblastoma regression.
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MATERIALS AND METHODS |
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Tumor samples were obtained from neuroblastoma patients identified through the mass screening program (2,17) (mass screened patients) and, for comparison, from patients (1 year old) clinically detected and at stage 3 or 4 according to the International Neuroblastoma Staging System (INSS) (18) (clinically detected, advanced-stage patients over 1 year of age), who underwent tumor resection at Kanagawa Children's Medical Center from 1972 through 1999. Tumor samples obtained from patients who received preoperative chemotherapy or radiotherapy and those tumors less than 1 cm in diameter or judged to be not suitable for immunohistochemistry because of poor sample preparation (this was checked by a pilot immunohistochemical analysis using antibodies such as anti-neurofilament and anti-CD68) were excluded. As a result, a total of 87 tumors from mass screened patients and 24 tumors from clinically detected, advanced-stage patients over 1 year of age were available for the following analyses in this study.
For immunohistochemical analyses, sections from formalin-fixed, paraffin-embedded tumor samples were used. After xylene deparaffinization and ethanol rehydration, the samples were treated with 0.3% hydrogen peroxide in methanol to inactivate endogenous peroxidase and then subjected to microwave treatment in 0.01 M citrate buffer (pH 6.0) for antigen retrieval. To detect the H-Ras protein, the tissue sections were first treated with 5% normal swine serum to block nonspecific staining, followed by incubation first with anti-c-H-Ras mouse monoclonal antibody (Ab-1, 1 : 100 dilution; Calbiochem, San Diego, CA) overnight at 4 °C and then with a peroxidase-conjugated anti-mouse immunoglobin G rat antibody (1 : 50 dilution) for 1 hour at room temperature (MBL, Nagoya, Japan). Bound antibody was visualized using diaminobenzidine as a chromogen, and the sections were lightly counterstained with hematoxylin. The specificity of H-Ras immunostaining was confirmed by omission of the primary antibody and the use of multiple irrelevant primary antibodies and by the immunoadsorption method (preincubation of the anti-c-H-Ras antibody with recombinant human c-H-Ras protein fused to glutathione S-transferase [Calbiochem]). Parenchymatous cells of the adrenal gland and connective tissues served as positive and negative controls, respectively, in H-Ras immunostaining of tumor samples (19). For evaluation of Ras immunoreactivity, a simple two-staged grading was adopted; i.e., positive or negative. Only cells unequivocally positive for Ras were judged to be Ras positive, and the others were judged to be Ras negative. A focal area of degeneration associated with Ras expression was defined as a cluster (20 or more cells or cell fragments) of Ras-positive, degenerating tumor cells without intervening Ras-negative, nondegenerating tumor cells. For the semiquantitative assessment of the appearance of focal areas of degeneration associated with Ras expression for each tumor, we examined one set of tumor sections stained with hematoxylineosin (H&E) and their adjacent sections stained with the anti-Ras antibody; a tumor sample was judged to be negative if the set analyzed contained no focal areas of degeneration associated with Ras expression and to be positive if the set contained one or more such focal areas. Immunostaining with antibody against active caspase-3 fragments (p20/p17) and TUNEL analysis of paraffin sections have been described (20,21).
For electron microscopy, tumor samples were fixed with 2.5% glutaraldehyde, postfixed with 2% OsO4, and embedded in Epon. Semithin sections (1 µm) were stained by the periodic acid-Schiff (PAS) method (oxidation in 1% aqueous periodic acid for 10 min, followed by treating for 10 min with Schiff's reagent and rinsing in a sulfurous acid solution). After localization of PAS-positive, degenerating cells in semithin sections, the contiguous tumor samples were trimmed under a microscope, and serial adjacent ultrathin sections (0.04 µm) encompassing PAS-positive, degenerating cells were cut. The ultrathin sections were then examined by transmission electron microscopy after staining with uranyl acetate and lead citrate.
Plasmids and Reagents
pcDNA3wtRas and pcDNA3RasV12 express wild-type and constitutively activated oncogenic H-Ras proteins, respectively, under the control of the cytomegalovirus immediate early promoter of the pcDNA3 expression vector (Invitrogen, Groningen, The Netherlands). cDNAs coding for enhanced green fluorescent protein (GFP), baculovirus p35 protein, and mouse Bcl-xL was subcloned into pcDNA3 to create pcDNA3EGFP, pcDNA3p35, and pcDNA3mbclxL, respectively. The entire coding region of human TrkA cDNA was subcloned into pFLAG CMV2 (Kodak, New Haven, CT) in-frame to the FLAG epitope to create pTrkA. Plasmids for inducible expression, pTA-Hyg, pT2-GN, pT2-wtRas, and pT2-RasV12, have been described (16). Staurosporine was purchased from Sigma Chemical Co. (St. Louis, MO). zVAD-fmk and zAsp-CH2-DCB were obtained from Peptide Institute (Osaka, Japan). Boc-Asp-fmk and -nerve growth factor (NGF) were purchased from Calbiochem.
Cells, Transfection, and Colony Formation Assay
Human neuroblastoma cell lines SH-SY5Y and GAMB were maintained on collagen Icoated dishes (Biocoat; BD Biosciences, Franklin Lakes, NJ) in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Multiser; Cytosystems, Castle Hill, Australia). For transfection, neuroblastoma cells (2 x 105) were seeded into 60-mm dishes 2 days before transfection, and transfection was done using Effectene transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instruction. The total amount of transfected DNA was always kept constant by use of an empty vector plasmid. Colony formation assay was done essentially as described (16). In brief, neuroblastoma cells transfected with 1 µg of pcDNA3-based expression plasmids were cultured in the presence of a selection drug (800 µg/mL Geneticin (GIBCO BRL, Rockville, MD) for approximately 2 weeks, and the number of colonies formed after drug selection was counted under a phase contrast microscope.
Immunoblot Analyses
Both adherent and detached cells were collected and were subjected to lysis in the lysis buffer (25 mM TrisHCl pH 7.6, 50 mM NaCl, 2% Nonidet P-40, 0.5% deoxycholate, 0.2% sodium dodecyl sulfate). After determination of protein concentrations, equal amounts of cell lysates were separated by SDSpolyacrylamide gel electrophoresis (15% for Ras and 8% for poly[ADP-ribose] polymerase [PARP]), transferred to a nitrocellulose membrane, and blotted first with a primary antibody against c-H-Ras (Ab-1; Calbiochem, or C-20; Santa Cruz Biotechnology, Santa Cruz, CA) or PARP (rabbit polyclonal; Boehringer Mannheim, Mannheim, Germany) and subsequently with an appropriate horseradish peroxidase-conjugated secondary antibody (Zymed, South San Francisco, CA). Blots were visualized by enhanced chemiluminescence (ECL; Amersham-Pharmacia, Buckinghamshire, England).
Generation and Analyses of Cell Lines Inducible with Tetracycline Withdrawal
SH-SY5Y-TA-GN, SH-SY5Y-TA-wtRas, and SH-SY5Y-TA-RasV12 cells were established by cotransfection of SH-SY5Y cells with pT2-GN, pT2-wtRas, and pT2-RasV12, respectively, together with pTA-Hyg followed by drug selection with Hygromycin B (Calbiochem) and Geneticin (GIBCO BRL). Inducible expression of LacZ (in SH-SY5Y-TA-GN cells), wt-Ras, and RasV12 was verified by immunoblot analyses. The stable transfectants were maintained in the presence of 400 U/mL Hygromycin B, 200 µg/mL Geneticin, and 0.5 µg/mL tetracycline (Sigma). The percentage of cell death was defined as 100 x (number of dead cells)/(number of dead cells + number of live cells). Dead cells were detected by their loss of ability to exclude trypan blue dye, but small fragments of dead cells with a diameter apparently shorter than half that of live cells were not counted. Transmission electron microscopic analysis was carried out as described (16). TUNEL assay was done using an in situ apoptosis detection kit (Wako, Osaka, Japan) according to the manufacturer's instructions. In brief, cells seeded on a collagen Icoated glass coverslip and treated as described in the figure legends were fixed in phosphate-buffered saline with 4% formaldehyde. After treatment with the permeabilization buffer (0.1% sodium citrate0.1% Triton X-100), cells were incubated first with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate (FITC)-labeled dUTP and then with peroxidase-conjugated anti-FITC antibody. A positive signal was visualized by the addition of diaminobenzidine. For propidium iodide staining, cells were instead incubated with a 25 µg/mL propidium iodide (Sigma) solution after the permeabilization step. Analyses of inducible cell lines were done using multiple independent clones for each cell line, and essentially identical results were obtained from different clones of each. The results of representative clones are presented.
Statistical Analysis
The chi-square test was used to determine distribution differences in the frequency of the appearance of focal areas of degeneration associated with Ras expression between tumors from mass screened patients and from clinically detected, advanced-stage patients over 1 year of age; a P value of less than .05 was considered to be statistically significant. The results of quantitative in vitro analyses were presented as the mean and 95% confidence intervals. All statistical analyses were two-sided.
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RESULTS |
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To test the idea that Ras expression mediates neuroblastoma cell death, we first examined the morphology of neuroblastoma cells expressing Ras protein using tumor samples from neuroblastoma patients. It has been well documented that H-Ras protein is expressed at high levels in neuroblastomas with a favorable prognosisincluding those detected through mass screening (11,12,14,22). Consistent with such reports, pilot immunohistochemical analysis of mass screened tumors detected definite H-Ras immunoreactivity in these tumors. We then went on, using serial tumor sections, to compare corresponding areas stained with anti-H-Ras antibody and with H&E. Strikingly, we found that intense H-Ras staining clearly colocalizes with cells that are undergoing or have undergone degeneration in H&E sections (Fig. 1, compare A-a with A-b, B-a with B-b, C-a with C-b, and D-a with D-b). Closer analysis at a higher magnification revealed that the degenerating nuclei do not show apparent chromatin condensation and undergo extensive fragmentation (Fig. 1
, D-e; the double arrows indicate fragmented nuclei, whereas the single arrows indicate unfragmented ones). Fragmentation of the degenerating cells was also evident from the marked irregularity in size of cellular debris and of immunoreactive signals for Ras (Fig. 1
, A-b, B-b, C-b, D-b, and E). Such Ras-positive, degenerating cells were found either scattered among viable cells (Fig. 1
, E) or as focal areas of cellular degeneration (Fig. 1
, AD) and either distant from or close to fibrovascular stroma. Similar findings were observed also in tumors from clinically detected, advanced-stage patients over 1 year of age known to have a poor prognosis (10). However, consistent with previous observations that H-Ras overexpression occurs more frequently in neuroblastomas with a favorable prognosis (11,12,14,22), focal areas of degeneration associated with H-Ras expression were found statistically significantly more frequently in tumors from mass screened patients than in those from clinically detected, advanced-stage patients over 1 year of age (53 of 87 versus 7 of 24, respectively; P = .006) (Table 1
).
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The above in vivo observations using tumor samples from neuroblastoma patients clearly indicated that Ras overexpression in human neuroblastoma is closely associated with caspase-independent, nonapoptotic tumor cell death. We then examined in vitrousing human neuroblastoma cell lineswhether Ras expression is the cause of such cell death. In human neuroblastoma, ras gene mutations frequently found in other human cancers are quite rare, suggesting that mutational activation of Ras does not have a role in the genesis and development of human neuroblastoma (25,26). Nevertheless, a constitutively activated H-Ras with oncogenic mutation (RasV12) was used in this study in addition to wild-type H-Ras (wt-Ras) for the purpose of efficient activation of the Ras signaling pathway. We tested a series of human neuroblastoma cell lines (SH-SY5Y, GAMB, IMR32, LA-N-5, SMS-KCN, TGW, and RTBM-1) to examine the effect of wt-Ras and RasV12 expression. Of these neuroblastoma cell lines, we could achieve Ras expression levels comparable to those expressed in tumor samples on immunoblot analysis in SH-SY5Y and GAMB cells (data not shown), and therefore subjected these cell lines to subsequent analyses. As shown in Fig. 3, A, when wt-Ras or RasV12 was coexpressed with GFP in SH-SY5Y and GAMB cells, GFP-positive cells showed morphologic degeneration characterized by rounding up and fragmentation into irregular sizes in both cell lines. Cytoplasmic vacuolation was only rarely observed, in contrast to glioma cells, in which Ras induced a cell death characterized by prominent vacuolation (9,16). This morphologic feature of Ras-induced neuroblastoma cell degeneration appeared distinct from that of apoptosis induced by staurosporine treatment or serum deprivation, in which cell fragmentation was not prominent and fragmented cells, if any, were rather of uniform size (Fig. 3
, A, and data not shown). Quantitative analysis revealed that, although both wt-Ras and RasV12 induced morphologic degeneration of neuroblastoma cells, RasV12 produced more degenerating cells than wt-Ras (Fig. 3
, B). Given that RasV12 is expressed at a level comparable to or lower than wt-Ras (Fig. 3
, C), the results indicate that the ability of Ras to induce morphologic degeneration is associated with its activity as an intracellular signaling molecule. We further asked whether the morphologic alteration induced by Ras expression is actually a manifestation of cellular demise. The results of colony formation assays demonstrated that Ras expression suppresses (RasV12 more potently than wt-Ras) clonogenic survival of both neuroblastoma cell lines (Fig. 3
, D). Consistent with the idea that the induction of cell death by Ras depends on its activity, RasV12/D38Nhaving an inactivating point mutation in the Ras effector loop region in a RasV12 background (27)failed to induce neuroblastoma cell death in either morphologic or colony formation assays (data not shown). These results indicate that Ras expression induces neuroblastoma cell death in vitro, most likely through the activation of the downstream signaling pathway(s).
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To further characterize Ras-induced neuroblastoma cell death in the absence of secondary effects of transfection, we established stable transfectants of SH-SY5Y cells that can be induced to express wt-Ras or RasV12 following tetracycline withdrawal (Fig. 4, A). Induction of Ras expression increased the proportion of dead cells (Fig. 4
, B), and the dying cells were characterized by extensive fragmentation into irregular sizes (Fig. 4
, C), as observed in the transient transfection assays (Fig. 3
, A). Nuclear staining using propidium iodide revealed that, in contrast to apoptotic cells showing nuclear condensation, fragmented dead cells produced by Ras expression contained nuclear fragments without apparent condensation (Fig. 5
, A), which was identical to what occurred in vivo in neuroblastoma samples (compare with the fragmented, noncondensed nuclei indicated by double arrows in Fig. 1
, D-e). TUNEL analysis indicated that Ras-induced cell death is negative for apoptotic DNA fragmentation (Fig. 5
, B). Electron microscopic analysis demonstrated increased lysosomal structures and lack of nuclear changes in cells undergoing Ras-induced death (Fig. 5
, C), just as observed in vivo in tumor samples (Fig. 2
). Collectively, these results indicate that Ras expression in neuroblastoma cells triggers nonapoptotic PCD with morphologic features of autophagic degeneration.
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Interestingly, coexpression of Ras and TrkA is associated with a better prognosis in neuroblastoma patients than the expression of either alone (12). We hypothesized that TrkA augments the activity of Ras to induce neuroblastoma cell death through its demonstrated ability to activate Ras (3032), thereby increasing the chance of tumor cell eradication. As reported previously (33), transfection-mediated expression of TrkA (in the absence of Ras overexpression) induced differentiation of SH-SY5Y cells, and NGF treatment alone failed to induce differentiation but could enhance differentiation induced by TrkA expression (Fig. 7, A). We then assessed the effect of TrkA signaling on Ras-induced neuroblastoma cell death. As shown in Fig. 7
, B, the expression of TrkA in the presence of different levels of Ras expression revealed that TrkA expression increases the proportion of degenerating cells in the presence of Ras overexpression much more efficiently than in its absence. Importantly, NGF treatment increased the proportion of degenerating cells in the presence of TrkA expression but not in its absence, providing unequivocal evidence that the augmentation of Ras-induced cell death is mediated by the TrkA signaling pathway. TrkA expression and/or NGF treatment did not affect Ras protein expression itself (Fig. 7
, C), indicating that TrkA-mediated augmentation of Ras-induced cell death signaling occurred downstream of Ras expression. These results indicate that TrkA can augment Ras-mediated death signaling, and this synergy of TrkA and Ras in neuroblastoma cell death may provide an explanation for the clinical observation that coexpression of TrkA and Ras predicts quite a favorable prognosis (12).
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DISCUSSION |
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Our immunohistochemical analyses of neuroblastoma tissues also revealed that focal areas of degeneration overexpressing Ras are occasionally accompanied by "gaps" or "clefts," suggesting that the degenerating areas are actively shrinking (see Fig. 1, C, for example). This implies that Ras-mediated neuroblastoma cell degeneration can contribute, at least locally, to volume reduction (regression) of tumor. We therefore asked whether such a local mechanism of regression is indeed associated with overall regression of neuroblastoma. Among subgroups of neuroblastomas, at least one third of mass screened neuroblastomas have been reported to undergo spontaneous regression (2,17), while clinically detected, advanced-stage neuroblastomas from patients over 1 year of age are presumed to have a very low chance of regression. The immunohistochemical analysis of tumor samples from these subgroups of neuroblastoma indicated that degenerating tumor cells overexpressing Ras are found much more frequently in mass screened neuroblastomas than in clinically detected, advanced-stage neuroblastomas from patients over 1 year of age, establishing a clear correlation between Ras-associated degeneration and the propensity to undergo spontaneous regression. Although it may not necessarily exclude the existence of other mechanisms, this clear correlation strongly supports the idea that Ras-mediated nonapoptotic tumor cell death plays an important role in spontaneous regression of neuroblastoma. This idea explains why previous observations failed to establish a definite correlation between apoptosis and factors associated with spontaneous regression (57), and our results thus provide a novel and significant insight into the mechanism underlying spontaneous neuroblastoma regression. It may also be extrapolated that, even in neuroblastomas unlikely to regress spontaneously, Ras expression contributes to a favorable prognosis by facilitating nonapoptotic tumor cell death in conjunction with anticancer treatments. In support of this idea, recent observations indicate that autophagic changes are prominent not only in untreated but also in treated neuroblastomas (8) and that an anticancer drug (fenretinide) can induce nonapoptotic cell death in neuroblastoma cell lines in a caspase-independent manner (35). While our results thus suggest that increased Ras expression likely contributes to a favorable prognosis through induction of nonapoptotic tumor cell death, it is also possible that Ras does so through other mechanisms. Indeed, we observed modest H-Ras immunoreactivity in tumor cells showing ganglionic maturation in some cases (Kato, K, unpublished observation). It would therefore be interesting to speculate that Ras negatively regulates the growth of neuroblastoma by inducing cell death when expressed at higher levels and differentiation when expressed at lower levels.
At present, it remains unknown what triggers Ras expression in neuroblastoma. Our observations that Ras-positive cells are located both distant from and close to fibrovascular stroma suggest that it is unlikely to be local environmental factors, including ischemic conditions. Rather, inherent genetic control seems more plausible. Presumably, in neuroblastomas that ultimately regress, tumor cells are programmed to express Ras, and the proportion of Ras-overexpressing tumor cell subpopulations may be increasing during the regression phase, though it would not be ethically acceptable to obtain surgical specimens from actively regressing neuroblastomas to examine this. In neuroblastomas that do not ultimately regress, however, tumor cell subpopulations that are programmed to express Ras may undergo local regression within the tumor; but they will eventually be outgrown by other subpopulations that are not programmed to express Ras. The molecular mechanism by which Ras protein expression is regulated in neuroblastoma also remains unknown, but the mechanism appears to be either transcriptional or posttranscriptional, as H-Ras overexpression in neuroblastoma is not accompanied by H-ras gene amplification (11). In this respect, it would be interesting to note that the expression of N-Myc, a well-established predictor of a poor prognosis of neuroblastoma (10), is inversely correlated with H-Ras expression in neuroblastomas (14). It is, therefore, possible that N-Myc may contribute to poor prognosis through inhibition of H-Ras expression. At any rate, elucidation of the molecular mechanism involved in the regulation of Ras expression may enable us to artificially manipulate Ras expression in neuroblastomas and thus open a novel avenue to neuroblastoma therapy.
Finally, this study for the first time to our knowledge reveals the role of caspase cascade-independent, nonapoptotic cell death in human diseases or healthy conditions. Nonapoptotic PCD now appears to be involved in more diverse in vivo processes than has been previously assumed (9,36). In this sense, this study sets the first good example that exploration of the roles and mechanisms of nonapoptotic PCD under physiological and pathological conditions could bring about important progress in biomedical science that would not be possible by apoptosis research alone. Our study also indicates for the first time the significance of caspase cascade-independent, nonapoptotic PCD in the growth control of cancer. Elucidation of the molecular mechanisms of nonapoptotic PCD is expected to contribute to the development of novel therapeutic strategies against human diseases, including cancer, which often acquires resistance against apoptotic cell death during development (37).
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NOTES |
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Manuscript received August 23, 2001; revised November 21, 2001; accepted January 22, 2002.
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