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Original Articles |
Departments of Obstetrics and Gynecology (H.U., K.I., R.K., S.S., K.O.) and Pathology (T.S., C.K., J.T., H.S.), Tohoku University School of Medicine, Sendai 980-8574; Research Institute Life Science (J.N., K.K., M.Y.), Snow Brand Company Ltd., Tochigi 329-05; and Department of Biochemistry (N.H.), Fujita Health University School of Medicine, Aichi 470-1192, Japan
Address correspondence and requests for reprints to: Hiroki Utsunomiya M.D., Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. E-mail: H-Utsu{at}ob-gy.med.tohoku.ac.jp
Abstract
Intratumoral metabolism and synthesis of estrogens are considered to play very important roles in the pathogenesis and development of human endometrial adenocarcinoma. The 17ß-hydroxysteroid dehydrogenase (17ß-HSD) isozymes catalyze the interconversion of estradiol (E2) and estrone and thereby serve to modulate the tissue levels of bioactive E2. To elucidate the possible involvement of this enzyme in human endometrial carcinoma, we first examined the expression of 17ß-HSD type 1 and type 2 in 20 normal cycling human endometria, 36 endometrial hyperplasia, and 46 endometrial endometrioid adenocarcinoma using immunohistochemistry, and we then studied immunoreactivity of 17ß-HSD type 2 using immunoblotting analyses, the activity of 17ß-HSD type 1 and type 2 using thin-layer chromatography and their expression using RT-PCR in endometrial endometrioid adenocarcinoma. We correlated these findings with various clinicopathological parameters to examine the biological significance of 17ß-HSDs in human endometrial disorders. 17ß-HSD type 2 immunoreactivity in normal endometrium was present in all cases of secretory phase (n = 14), but not in any endometrial mucosa of proliferative phase (n = 6). In addition, 17ß-HSD type 2 immunoreactivity was detected in 27 of 36 (75%) endometrial hyperplasia and 17 of 46 (37%) carcinoma cases. 17ß-HSD type 1 immunoreactivity was not detected in all the cases examined. In both endometrial hyperplasia and carcinoma cases there were significant positive correlations between 17ß-HSD type 2 and progesterone receptor labeling index (LI). In carcinoma cases, a significant inverse correlation was detected between 17ß-HSD type 2 immunoreactivity and age. In addition, 17ß-HSD type 2 immunoreactivity was also correlated with 17ß-HSD type 2 enzymatic activity, and semiquantitative analyses of 17ß-HSD type 2 messenger RNA. No significant correlations were detected between 17ß-HSD type 2 and estrogen receptor LI, Ki67 LI, amount of aromatase messenger RNA or histological grade. These data indicated that the expression of 17ß-HSD type 2 in hyperplastic and/or neoplastic endometrium may represent altered cellular features through hyperplastic and neoplastic transformation. However, 17ß-HSD type 2 may also play some protective and/or suppressive roles toward unopposed estrogenic effects through inactivating E2 in situ, especially in premenopausal patients.
ENDOMETRIAL CARCINOMA IS one of the most common malignancies in developed countries, and its incidence has recently increased (1, 2). In situ estrogen metabolism, including its synthesis and degradation, has been recently considered to play a very important role in the development and progression of various human estrogen-dependent neoplasms, including endometrial endometrioid adenocarcinoma (3, 4). In endometrial endometrioid adenocarcinoma, in situ 17ß-estradiol (E2) availability has been demonstrated to be closely related to the pathogenesis and development of endometrial proliferative disorders, including endometrial hyperplasia and carcinoma, especially of the endometrioid type (5). The enzyme 17ß-hydroxysteroid dehydrogenase (17ß-HSD) catalyzes the reversible interconversion of estrone (E1) and E2. Recently, isozymes of 17ß-HSD have been characterized. 17ß-HSD type 1 catalyzes the 17ß reduction of biologically inactive E1 to E2 (6, 7, 8), whereas 17ß-HSD type 2 preferentially catalyzes the oxidation of E2 to E1 (9). Both 17ß-HSD type 1 and type 2 regulate the tissue level of E2 and modulate estrogenic actions in estrogen target tissues such as the endometrium (10, 11). Therefore, the analyses of the expression and activity of 17ß-HSD type 1 and type 2 in the human endometrium and its disorders are considered as very important in understanding estrogen-related biological phenomenon.
Recently, immunohistochemical studies of 17ß-HSD isozymes have been reported in human breast carcinoma (12, 13). In addition, the presence of 17ß-HSD isozymes has been reported in normal human endometrium (14, 15). In these studies, 17ß-HSD type 2 was reported to be present in the endometrial glandular epithelium of secretory phase, suggesting inactivation of E2 and subsequent diminished estrogenic actions by this enzyme. However, the distribution and possible biological roles of these 17ß-HSD isozymes in the endometrial hyperplasia and carcinoma have not been examined. Therefore, in this study, 17ß-HSD type 1 and type 2 were immunolocalized in the human endometrial hyperplasia and carcinoma as well as normal cycling human endometrium. These findings were then correlated with various clinicopathological parameters to study biological significance of the in situ regulatory mechanisms of estrogens conferred by 17ß-HSDs in endometrial disorders. In human endometrial endometrioid adenocarcinoma, we also performed immunoblotting analyses for 17ß-HSD type 2, enzymatic assay for 17ß-HSD type 1 and type 2 using thin-layer chromatography (TLC) and semiquantitative analyses of messenger RNA (mRNA) by RT-PCR method to further characterize 17ß-HSD type 1 and type 2 in endometrial malignancies.
Materials and Methods
Endometrium
Twenty normal cycling human endometria (6 proliferative phase and 14 secretory phase), 36 endometrial hyperplasias (18 hyperplasia simple, 11 hyperplasia complex, and 7 atypical hyperplasia complex), and 46 endometrial endometrioid adenocarcinomas (23 well differentiated, 14 moderately differentiated, and 9 poorly differentiated; 28 stage I, 8 stage II, 9 stage III, and 1 stage IV) were obtained from surgical pathology files of Tohoku University Hospital (Sendai, Japan). This study was approved by the Ethical Committee of the Tohoku University School of Medicine. We obtained nonpathological endometria from hysterectomy specimens performed due to carcinoma in situ of the uterine cervix. Endometrial hyperplasia and carcinoma specimens were obtained from total dilatation and curretage and hysterectomy, respectively. All the patients examined had not received irradiation or chemotherapy before surgery. The histopathological classification in each specimen was evaluated according to FIGO histologic grading system for endometrial adenocarcinoma in 1988 (16).
The specimens were all routinely processed (i.e.10% formalin fixed for 24–48 h) paraffin embedded, and thin sectioned (3 µm).
Antibodies
The characteristics of the primary antibodies used in this study
are summarized in Table 1
. 17ß-HSD type
1 antibody was a rabbit polyclonal antibody against the enzyme purified
from human placenta (12) and was kindly provided by Dr.
M. H. Poutanen (University of Oulu, Oulu, Finland). The monoclonal
antibody of 17ß-HSD type 2, mAB-C2-12, was produced by immunizing
mice with a synthetic carboxyl-terminal peptide corresponding to amino
acids 375–387 of 17ß-HSD type 2 (17) and was kindly
provided by Dr. S. Andersson (University of Texas Southwestern Medical
Center, Dallas, TX). Utilization of these antibodies for
immunohistochemistry has been reported previously
(18).
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Immunohistochemical analyses were performed using the streptavidin-biotin amplification method using a Histofine Kit (Nichirei, Tokyo, Japan) and have been previously described in detail (19). The antigen-antibody complex was visualized with 3.3'-diaminobenzidine solution [1 mM 3.3'-diaminobenzidine, 50 mM Tris-HCl buffer (pH 7.6), and 0.006% H2O2], and counterstained with hematoxylin. Tissue sections of full-term placenta were used as positive controls for 17ß-HSD type 1 and type 2 (18). As negative controls, normal rabbit or mouse IgG was used instead of the primary antibodies. No specific immunoreactivity was detected in these tissue sections.
Scoring of immunoreactivity
For evaluation of 17ß-HSD type 1 and type 2 immunoreactivity, we determined the labeling index (LI; i.e. the percentage of positive cells) according to the report by Sasano et al. (13). After completely reviewing the immunostained sections of each lesion, two of the authors (H.U. and T.S.) independently divided the cases into the following three groups: 2+, more than 50% positive cells, +, 0–50% positive cells; and -, no immunoreactivity. Scoring of estrogen receptor (ER), progesterone receptor (PR), and Ki67 in gland or carcinoma cells was performed on high-power fields (x400) using a standard light microscope. In all cases, the total of more than 500 glandular or carcinoma cells from three different representative fields were counted independently by the same two authors above, and the percentage of immunoreactivity (i.e. LI) was determined. These three fields were simultaneously determined by two of the authors before counting using a double-headed light microscope. Cases with disconcordant results (interobserver differences with >5%) were simultaneously reevaluated by the same two authors using double-headed light microscope. Consequently, interobserver differences were less than 5% in this study.
17ß-HSD enzymatic assay
Forty-six carcinoma cases of fresh frozen tissues (i.e. the cases immediately frozen in liquid nitrogen and stored at -80 C) were available for examination of enzymatic assay.
The samples were homogenized at 4 C in phosphate buffer [100 mM KCl, 10 mM KH2PO4, 10 mM Na2HPO4, and 1 mM EDTA (pH 7.5)] and incubated at 37 C with the phosphate buffer containing [3H]E1 or [3H]E2 with cofactor (NADPH or NADP+). After the separation of steroids on TLC plates, the rates of product formation were measured by GS-250 Molecular Imager (Bio-Rad Laboratories, Inc., Hercules, CA). Protein amount of each sample was measured by the method of Lowry et al. (20).
Immunoblot analyses
Immunoblot analyses were performed in seven specimens of the endometrial endometrioid adenocarcinoma. The samples were homogenized at 4 C in phosphate buffer [100 mM KCl, 10 mM KH2PO4, 10 mM Na2HPO4, and 1 mM EDTA (pH 7.5)]. After centrifugation, 30 µg of the supernatant proteins (whole cell extracts) were subjected to SDS-PAGE (10% acrylamide gel). After SDS-PAGE, proteins were transferred to polyvinylidene difluoride (Immobilon P; Millipore Corp., Bedford, MA) in 20 mM Tris/150 mM glycine/3.5mMSDS/20% (vol/vol) methanol for 3 h at 75 mA constant current. Briefly, the blots were blocked in 5% nonfat dry milk/phosphate-buffered saline/0.1% Tween 20 (Difco, Detroit, MI) for 1 h at room temperature and then incubated with diluted antibody for 17ß-HSD type 2 at 4 C overnight. After incubation with horseradish peroxidase-linked sheep antimouse Ig (dilution 1:3000 for 17ß-HSD type 2) for 1 h. At room temperature, antibody/protein complexes on the blots were detected using enhanced chemiluminescence plus detection reagents (Amersham International, Buckinghamshire, UK).
Semiquantitative analyses of mRNA
Semiquantitative analyses of 17ß-HSD type 1, type 2,
aromatase, and ß-actin mRNAs were carried out using total RNA
fractions from tissues by RT-PCR using a specific pair of fluorescent
dye-labeled primers and their standard RNAs, as described previously
(21, 22). Oligonucleotide primers for RT-PCR were
synthesized as summarized in Table 2. The
coding sequence between the two PCR primer sites is located 5' upstream
of the RT primer site in each transcript of 17ß-HSD type 1, type 2,
aromatase, and ß-actin, which is interrupted by at least 2 introns in
the genes. The standard RNAs for 17ß-HSD type 1, type 2, aromatase,
and ß-actin were synthesized in vitro with T7 RNA
polymerase using their complementary DNA as templates, purified, and
then spectrophotometrically quantitated (21, 22). Total
RNA (0.2–2 µg) and standard RNAs (0.02–2 amol) were subjected to RT
with SUPERSCRIPT II reverse transcript (Life Technologies, Inc., Grand Island, NY) and a specific antisense primer at 47 C
for 45 min. The resulting complementary DNA was amplified by PCR using
a specific pair of fluorescent dye-labeled sense primers and antisense
primers for 23–28 cycles. The fluorescent PCR products were analyzed
on a 2% agarose gel with a Gene Scanner 362 Fluorescent Fragment
Analyzer (Perkin-Elmer Corp. Co., Foster City, CA). Each
amount of 17ß-HSD type 1, type 2, aromatase, and ß-actin mRNAs in
the tissue RNA was calculated from the peak areas of the fluorescent
PCR products of a specific mRNA and its standard RNA. The
proportionality between the amounts of their RNAs and the peak areas of
their PCR products was detected over a wide range of 0.002–20
attomoles in the assays. All semiquantitated data of 17ß-HSD type 1,
type 2, and aromatase mRNA were expressed as a ratio of ß-actin mRNA
levels.
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Values for patient age and LIs of ER, PR, and Ki67 were
presented as mean ± 95% confidence interval (CI). Association
between 17ß-HSD immunoreactivity and these parameters were evaluated
using Bonferroni test. We also evaluated the statistical differences
between patients for 17ß-HSD immunoreactivity and stage or
histological grade in a cross-table using the
2 test. P values less than 0.05
were considered as significant.
Results
Normal cycling endometrium
17ß-HSD type 2 immunoreactive protein was detected only in the
cytoplasm of glandular cells in the secretory phase (Fig. 1A). 17ß-HSD type 2 immunoreactivity
was not detected in the stromal cells of secretory phase endometrium
and neither epithelium nor stromal cells of proliferative phase
endometrium (Fig. 1B). 17ß-HSD type 1 immunoreactivity was not
detected in any of the cases examined. ER and PR were detected in the
nuclei of epithelial and stromal cells through all phases.
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Results are summarized in Table 3.
17ß-HSD type 2 immunoreactivity was detected only in epithelial
cells, but not in stromal cells (Fig. 2).
17ß-HSD type 1 immunoreactivity was not detected in any of the cases
examined. There was a significantly positive correlation between
17ß-HSD type 2 immunoreactivity and PR LI [P <
0.02; +(47.4 ± 5.8) vs. -(39.4 ± 6.9)]. No
significant correlation was detected between 17ß-HSD type 2
immunoreactivity and ER LI, Ki67 LI, age, or histological
classification including simple, complex, and atypical endometrial
hyperplasia.
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Immunoreactivity. 17ß-HSD type 2 immunoreactivity was
detected in the cytoplasm of carcinoma cells (Fig. 3A), but not in stromal cells. 17ß-HSD
type 1 immunoreactivity was not detected in any of the carcinoma cases
examined. There was a statistically significant positive correlation
between 17ß-HSD type 2 immunoreactivity and PR LI [P
< 0.02; 2+(48.5 ± 25.6) vs. -(26.3 ± 21.6)].
PR immunoreactivity was detected in the nuclei of carcinoma cells (Fig. 3B), adjacent to the area where 17ß-HSD type 2 immunoreactivity was
expressed. A statistically significant inverse correlation was detected
between 17ß-HSD type 2 immunoreactivity and age [P
< 0.01; 2+(46.2 ± 9.9) vs. -(60.3 ± 13.8)].
There was no significant correlation between 17ß-HSD type 2
immunoreactivity and ER LI, Ki67 LI, grade, or the relative amount of
aromatase mRNA expression determined by RT-PCR analyses.
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Immunoblot analyses for 17ß-HSD type 2. 17ß-HSD type 2 was
detected as a single band (approximately 45 kDa for 17ß-HSD type 2)
corresponding to the expected molecular weight (Fig. 4). Results of immunoblotting were
consistent with those of immunohistochemical, enzymatic, and mRNA
semiquantitative studies of 17ß-HSD type 2 in seven cases of
endometrioid endometrial adenocarcinoma (data not shown).
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Results (examined by 17ß-HSD type 2 immunoreactivity) are summarized
in Table 4.
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In the great majority of human breast and endometrial endometrioid adenocarcinoma, estrogens, especially E2, which is a biologically potent estrogen, have been demonstrated to contribute greatly to the growth and development of these neoplasms (23, 24). Therefore, numerous studies have been performed to evaluate the detailed metabolism of serum estrogens in patients diagnosed with endometrial carcinoma, but there has been no consistent evidence of increased serum estrogen concentrations or other systemic estrogen abnormalities reported in women with endometrial endometrioid adenocarcinoma (25, 26, 27). However Tseng et al. (28) and Yamaki et al. (29) demonstrated the possibility of in situ estrogen production through increased aromatization activity, which results in the conversion of serum androgens to estrogens . In addition, Watanabe et al. (30) reported marked aromatase immunoreactivity and mRNA in endometrial carcinoma but not in normal or hyperplastic endometrium, including atypical hyperplasia. Therefore, in patients with endometrial carcinoma, especially of the endometrioid type, intratumoral estrogens derived from in situ aromatization could function as an autocrine steroid, such as in the stimulation of growth and proliferation in cancer cells. E2 can, therefore, promote the growth of these carcinoma cells, regardless of serum concentration of this steroid (25). However, it is also true that aromatase is primarily involved in the production of E1, a relatively weak estrogen, but it is E2 that is considered to play a more important role in the development and biological behavior of human endometrial disorders as a biologically potent sex steroid. Therefore, it is very important to examine 17ß-HSDs, which are involved in the interconversion of E1 and E2 in the human endometrium, and its disorders to study their intratumoral estrogen synthesis and metabolism in detail.
In our present study, 17ß-HSD type 2 immunoreactivity was significantly correlated with the relative amount of 17ß-HSD type 2 mRNA expression determined by semiquantitative analyses using RT-PCR and the enzymatic activity of 17ß-HSD type 2 examined by TLC assay. 17ß-HSD immunoreactive protein examined by immunohistochemistry may therefore represent potential enzymatic activity of this enzyme in human endometrium.
In human normal endometrium, 17ß-HSD type 2 immunoreactive protein was detected in the secretory phase, but not in the proliferative phase. In support of our findings, a study by Mustonen et al. (14) found that 17ß-HSD type 2 mRNA by in situ hybridization was localized to the glandular epithelial cells of human endometrium during the secretory phase of the menstrual cycle, but not during the proliferative phase. In the normal cycling human endometrium, the estrogen receptor content of the endometrium is highest in the proliferative phase and markedly decreased in the secretory phase (31). In addition, it is well known that less estrogenic actions are required in the endometrium following ovulation. Casey et al. (15) reported that 17ß-HSD type 2 mRNA was markedly expressed in endometrial glandular epithelial cells of the secretory phase, but 17ß-HSD type 1 mRNA was not detected in any phase. These findings indicate that 17ß-HSD type 2 is likely to play a very important role in the maintenance of hormonal environments to make preparation for implantation during the secretory phase of the human menstrual cycle by inactivating E2, which results in diminished local estrogenic actions together with a decrease in the number of estrogen receptors, as described above. This increased 17ß-HSD type 2 expression during the secretory phase may be caused by serum progesterone levels that increase following ovulation, but it awaits further investigation for clarification. We could not detect 17ß-HSD type 1 immunoreactivity in normal cycling human endometrium. However, Zeitoun et al. (32) reported that 17ß-HSD type 1 mRNA was expressed in proliferative phase by Northern analyses. 17ß-HSD type 1 antibody that was used in this study has been widely used in various immunohistochemical studies (12, 13, 18). Therefore, it is difficult to compare the results of 17ß-HSD type 1 protein expression with those of 17ß-HSD type 1 mRNA expression. It awaits further investigations to clarify these differences on 17ß-HSD type 1 expression.
Estrogens are also considered to play very important roles in the development of endometrial hyperplasia. Some endometrial hyperplasia, especially atypical hyperplasia, are likely precursors of endometrial endometrioid adenocarcinoma. In our study, 17ß-HSD type 2 immunoreactivity was detected in hyperplastic glandular epithelium of endometrial hyperplasia, in contrast to normal proliferative endometrial glands in which 17ß-HSD type 2 immunoreactivity was not detected (14). Endometrial hyperplasia was, in general, considered to be caused by continuous estrogenic stimulation such as ovulatory dysfunction, which results in abnormal cell proliferation with altered patterns of several gene expression (33, 34).
Expression of 17ß-HSD type 2 in hyperplastic endometrium, in contrast to the absence of the enzyme in normal proliferative endometrium, may be also considered within the spectrum of these alterations occurring through the development of hyperplasia. On the other hand, the expression of 17ß-HSD type 2 in proliferative glandular cells of endometrial hyperplasia may also represent one of the in situ defensive mechanisms of modulating unopposed estrogenic effects. However, further investigations, including the analyses of endometrial mucosa with estrogen breakthrough bleeding, are required to clarify this hypothesis. The positive correlation between 17ß-HSD type 2 and PR LI in endometrial hyperplasia also indicates that progesterone may be involved in the regulation of 17ß-HSD type 2 expression in endometrial hyperplasia, as in secretory phase of normal cycling endometrium.
In endometrial adenocarcinoma, 17ß-HSD type 1 expression and activity were not detected in our study. This is in contrast to the study of 17ß-HSD type 1 in breast cancer in which nearly half of the cases demonstrated 17ß-HSD type 1 immunoreactivity in carcinoma cells, and 17ß-HSD type 2 was not expressed (12, 13). These results indicate that intratumoral estrogen metabolism is different between human breast and endometrial carcinoma, although both of them are sex steroid-dependent malignancies. In endometrial endometrioid adenocarcinoma, the amount of aromatase expression and the number of ER-positive cells are, in general, higher than breast carcinoma (25). However, the presence of 17ß-HSD type 2 and the absence of 17ß-HSD type 1 in tumor tissue suggests the presence of in situ degradation of E2 in endometrial carcinoma, which may be postulated inconsistent considering the importance of in situ availability of E2 in the development of endometrial endometrioid adenocarcinoma. This expression of 17ß-HSD type 2 in neoplastic endometrial cells may also represent the findings associated with neoplastic transformation of human endometrium. However, this in situ expression of 17ß-HSD type 2 in carcinoma is also considered to play some protective or suppressive roles in neoplastic proliferation through degrading E2 in situ, especially in premenopausal women who have relatively higher serum concentration of E2, because the significantly inverse correlation was detected between 17ß-HSD type 2 and age of the patients. It then becomes important to study the correlation between the status of 17ß-HSD type 2 expression and biological behavior and/or clinical outcome of the patients. However, the cases examined in this study were relatively recent cases and sufficient data were not available for these studies above.
In contrast to premenopausal women in endometrial carcinoma, postmenopausal women in endometrial carcinoma was associated with markedly low levels of serum E2 (35). In addition, E1 is relatively available in carcinoma tissue because of marked aromatase expression and the absence of 17ß-HSD type 1 expression. Therefore, 17ß-HSD type 2 expression in postmenopausal patients may not play important roles compared with premenopausal patients because of little in situ E2 availability in endometrial carcinoma tissue of postmenopausal women, although aromatase can catalyze the synthesis of active E2 from testosterone. It is also well known that the frequency of ER-positive endometrial carcinoma in postmenopausal patients was significantly smaller than that in premenopausal patients (36). These results are likely to indicate that the factors such as phosphatase and tensin homolog deleted on chromosome 10 (PTEN) mutation, K-Ras mutation, microsatellite instability and others play more important roles in postmenopausal patients than in premenopausal patients (37, 38, 39).
To obtain a better understanding of the local regulation of E2 in human endometrial endometrioid adenocarcinoma, it is very important to examine the relationship between 17ß-HSD type 2 and ER LI, PR LI, Ki67 LI, amount of aromatase mRNA, or histological grade. In this study, a significant linear correlation was detected between 17ß-HSD type 2 immunoreactivity and PR LI (P < 0.02). Medroxyprogesterone acetate (MPA) has been used for medical treatment of endometrial proliferative disease, especially in premenopausal patients. MPA is occasionally administrated to patients with endometrial endometrioid adenocarcinoma of the well differentiated type. A relatively favorable response has been occasionally reported in some cases (40, 41, 42, 43). MPA is considered to act directly on carcinoma cells, resulting in the suppression of tumor cell proliferation (44, 45). However, the positive correlation between the expression of 17ß-HSD type 2 and PR may also suggest that MPA acts not only directly on tumor cell proliferation but also on the induction of 17ß-HSD type 2 which may result in decreased in situ availability of biologically active estrogen, but it awaits further investigations for clarification.
In conclusion, 17ß-HSD type 2 mainly regulates the tissue levels of E2 and modulates estrogenic actions in both hyperplastic and/or neoplastic endometrium. 17ß-HSD type 2 may play some protective and/or suppressive roles toward unopposed estrogenic effects through inactivating E2 in situ especially in premenopausal patients.
Acknowledgments
We appreciate Andrew David Darnel (Department of Pathology, Tohoku University School of Medicine, Sendai, Japan) for editing this manuscript.
Footnotes
1 Supported in part by The Grant-in-aid for Cancer Research 7-1 from
The Ministry of Health and Welfare, Japan; a grant-in-aid for
scientific research area on priority area (A-11137301) from The
Ministry of Education, Science and Culture, Japan; a grant-in-aid for
Scientific Research (B-11470047) from Japan Society for the Promotion
of Science; and a grant from The Naitou Foundation and Suzukenn
Memorial Foundation. 
Received September 14, 2000.
Revised February 26, 2001.
Accepted March 14, 2001.
References
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