Roles of p38 and c-jun in the differentiation, proliferation and immortalization of normal human endometrial cells

In-Sun Hong1, Seok-Hyun Kim2, Mi Kyoung Koong3, Jin Hyun Jun3, Sung-Hoon Kim4, Yong-Soon Lee1,* and Kyung-Sun Kang1,*

1 Laboratory of Stem Cell and Tumor Biology, Department of Veterinary Public Health, College of Veterinary Medicine and 2 Laboratory of Reproductive Biology and Infertility, College of Medicine, Seoul National University, Seoul 151-742, 3 Department of Biology and Infertility, Samsung Cheil Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul 100-380 and 4 Graduate School of East–West Medical Science, Kyunghee University, Yongin 449-701, Korea

* To whom correspondence should be addressed. orEmail: leeys{at}snu.ac.kr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It has been reported that p38 and c-jun operate as mediators of cell proliferation and differentiation. Therefore, by studying the roles of c-jun and p38 in the proliferation and differentiation of normal human endometrial cells, we can better understand the mechanism of these processes in endometrial cells. METHODS: Separation of glandular and stromal components was based on a modification of the work of Satyaswaroop et al. To confirm the purification of the endometrial cells and the expression of the transfected SV40 large T antigen, immunocytochemical analysis and western blot analysis were performed. RESULTS: There were polygonal shapes in the stromal cells in the early passage 1–2, while the aged endometrial stromal cells were spindle shaped. To investigate passage-dependent molecular events in endometrial cells, the c-jun and pp38 levels were examined. Both c-jun and pp38 were significantly reduced with cellular aging and passages. To understand the role of c-jun, endometrial stromal cells were treated with SP600125 which is a specific inhibitor of c-jun. SP600125 induced morphological changes of young endometrial stromal cells with polygonal shape; the young cells appeared as aged endometrial cells with spindle shape. In addition, an immortalized endometrial cell line was established and shown to express activated c-jun, similiar to normal endometrial cells. CONCLUSIONS: These results suggest that the modulation of p38 and c-jun may play an important role in the differentiation and proliferation of human endometrial cells.

Key words: c-jun/differentiation/endometrial epithelial cells/endometrial stromal cells/p38


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In vitro cell culture has been developed to study the physiology, biology and pharmacology of several organs such as the breast, kidney, liver, prostate, bone and thyroid, either normal or malignant. Endometrial tissue is an interesting model for understanding the role of intrinsic and extrinsic factors, i.e. hormones and growth factors, involved in its normal and pathologic development, as well as its cyclic growth (Merviel et al., 1995Go). The endometrium is one of the most rapidly renewing tissues of the body, and the proliferation of all endometrial cell types is believed to be mainly dependent on estrogen. The endometrium is composed of epithelial and stromal cells containing fibroblast and myofibroblast. In particular, stroma can promote epithelial development or differentiation in the female reproductive tract (Cooke et al., 1986Go, 1997Go; Donjacour et al., 1991Go).

The proto-oncogene c-jun is one of the immediate early genes, and its activation is related to cellular proliferation, differentiation and apoptosis, depending on the tissue studied (Colotta et al., 1992Go; Landers et al., 1992Go; Schuchard et al., 1993Go). The c-jun proto-oncogene,which encodes a central component of AP-1 transcription factors, is a major regulator of the proliferative and stress responses of mammalian cells, and a contributor to oncogenesis (Derijard et al., 1994Go; Kyriakis et al., 1994Go; Wisdom et al., 1999Go). The p38 group kinases are involved in inflammation, cell growth, cell differentiation, the cell cycle, and cell death (New et al., 1998Go; Ono et al., 2000Go). Furthermore, the p38 pathway plays a regulatory role in the proliferation and differentiation of cells (Pietersma et al., 1997Go; Craxton et al., 1998Go; Cho et al., 2002Go) and the involvement of p38 in cell growth became apparent when it was noticed that an over expression of p38 in yeast led to a significant slowing of proliferation (Ziegler-Heitbrock et al., 1992Go).

Given these roles of p38 and c-jun in cellular proliferation and differentiation, we hypothesized that p38 and c-jun might serve as modulators of proliferation and differentiation of normal human endometrial cells. Subsequently, the primary objective of the present work was to demonstrate that p38 and c-jun play an important role in the proliferation and differentiation of normal human endometrial cells. To investigate their roles for these cell processes in normal human endometrial cells, we isolated two types of normal endometrial cells and then examined the levels of c-jun and pp38. Our results suggest that the modulation of c-jun might be closely related to regulating the proliferation and differentiation of normal endometrial cells in humans.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Tissue specimens
Human endometrial tissues were obtained from normal cycling patients who had undergone a hysterectomy. Tissue samples (2–3 g) were obtained from women who were in the proliferative (days 5–14) and secretory phase (days 15–26), and who had not received hormonal therapy within 30 days prior to tissue extraction. Institutional Review Board approval was obtained and patients gave informed consent. Endometrial tissue was transported to the laboratory in isolation medium consisting of M199/F12 culture medium (Gibco Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum (Gibco Life Technologies) plus antibiotic and antimycotic agents to yield the final concentrations of 200 U of penicillin, 0.2 mg/ml of streptomycin and 0.5 µg/ml of amphotericin B (antibiotic/antimycotic solution, Sigma, St Louis, MO).

Isolation of endometrial epithelial and stromal cells
Endometrial tissue was rinsed in HBSS to remove blood and debris. Separation of glandular and stromal components was based on a modification of the work of Satyaswaroop et al. (1979)Go and Arnold et al. (2001)Go. After gentle centrifugation (600 g for min), the supernatant was removed, and the tissue was then placed on a 100 mm plastic tissue culture dish (Corning-Costar, Cambridge, MA). The entire procedure was performed under a sterile laminar flow hood. The tissue was minced with sterile scalpels into 1–2 mm fragments and digested with collagenase (2 mg/ml; Sigma) in M199/F12 (as above) for 2.5 h at 37°C on a shaking incubator. The tissue digest was vigorously pipetted and added to a stacked sterile wire sieve assembly with a number 100 wire cloth sieve (140 µm size; Newark Wire Co., Newark, NJ), followed by a number 400 wire cloth sieve (37 µm). After the endometrial digest was added to the top of the sieve assembly, the epithelial glands were retained in the number 100 and 400 sieves while the stromal cells passed through to the receptacle below. The glands were back-flushed out of the sieves onto a 100 mm sterile dish using the same medium. Any stromal cells remaining with the glands were further separated by selective adherence to plastic tissue culture dishes for 1 h. The stromal cells were collected from the lower receptacle, pelleted by centrifugation at g for min and resuspended in isolation medium. Red blood cells were removed by careful washing after the stromal cells had attached to a 100 mm sterile dish.

Cell culture medium
Stromal cells were maintained in medium consisting of a 1:1 mixture of M199:Ham's F12 medium (Gibco Life Technologies) supplemented with 10% FBS (Gibco Life Technologies), ITS+[containing insulin (0.62 µg/ml), transferrin (0.62 µg/ml), selenium (0.62 ng/ml), bovine serum albumin (125 µg/ml) and linoleic acid (52.6 µg/ml) (Sigma] plus 100 U of penicillin, 0.1 mg/ml of streptomycin and 0.25 µg/ml of amphotericin B (antibiotic/antimycotic solution, Sigma) according to the method of Arnold et al. (2001)Go. The culture medium was routinely changed every 2–3 days. The epithelial gland cells required little or no serum and were cultured in medium consisting of M199 and F12 (1:1) with added bovine pituitary extract (BPE; 2 ml/l; Collaborative Biomedical Products, Bedford, MA), ITS+(concentrations as above) and antibiotic and antimycotic agents as described above.

Immunocytochemistry
To confirm the purification of the stromal cells and the expression of the transfected SV40 LTag, immunocytochemical analysis was performed using cytokeratin 8/18 (Santa Cruz Biotechnology, CA) as a marker of epithelial cells, vimentin (Chemicon, Temecula, CA) as a marker of stromal cells, and a monoclonal antibody against SV40 LTag (Santa Cruz). For immunocytochemistry, cultures were rinsed with PBS and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. They were then treated with ice-cold 100% methanol for 10 min, then 100% acetone for 5 min, then 0.4% Triton X-100 in PBS for 10 min, with triple PBS rinses in between each treatment. Samples were treated with 2% horse serum and 2% goat serum in PBS containing 4% BSA (PBS/BSA) for 100 min at 37°C to block non-specific binding of primary antibodies. Antibodies were diluted in PBS/BSA plus 2% horse and 2% goat sera at 1:100 for cytokeratin, 1:100 for vimentin, and 1:100 for the antibody against SV40 LTag. The primary antibodies were incubated on cells for 1 h at 37°C. Samples were rinsed three times with PBS. Fluorescent secondary antibodies were added concurrently: FITC anti-mouse IgG diluted to 1:100 (Zymed Laboratories Inc., San Francisco, CA) in PBS/BSA plus 2% horse and 2% goat sera for 45 min at 37°C. Slides were rinsed with PBS and coverslips were added. Fluorescence was visualized using a fluorescence microscope.

Western blot analysis
Western blot analysis was performed as described previously (Hayashi et al., 1997Go; Sai et al., 1998Go). Cells were grown in a 100 mm tissue culture dish (Nunc, Rochester, NY) to the same confluency and were then treated with test compounds. Proteins were extracted with a 20% SDS solution containing 1 mM phenylmethylsulfonyl fluoride (a protease inhibitor), 10 mM iodoacetoamide, 1 mM leupeptin, 1 mM antipain, 0.1 mM sodium orthovanadate and 5 mM sodium fluoride. Protein content was determined using the DC assay kit (Bio-Rad, Hercules, CA) and separated on 12.5% SDS–PAGE according to the method using leupeptin (Laemmli et al., 1970). The proteins were then transferred to nitrocellulose membranes at 100 V and 350 mA for 1 h. All antibodies were used according to the manufacturers' instructions and protein bands were detected using an ECL detection kit (Amersham, Piscataway, NJ).

Immortalization of endometrial cells
Plasmid pMK 16, containing the SV40 replication origin defective gene (SV40 ori–), was used in this study. Normal cells were transfected on the 12th day in culture until they became subconfluent. This purified plasmid DNA was transferred into the cells using DNA superfect (Qiagen, Valencia, CA) according to the manufacturer's instructions. Briefly, 1.5 µl of plasmid and 40µl of DNA superfect in 3 ml of serum-free medium were added to the culture which had been washed twice with serum-free medium. After 24 h of incubation at 37°C, fresh medium was added to the culture. A successful transfection was confirmed after 5 or 6 days by a sharp increase of cell doubling time and large T antigen immunostaining. Growth curves of normal or transfected endometrial stromal cells were evaluated: 2 x 105 cells were plated in a 75 cm3 flask and cultured as reported above. The medium was changed every 2 days. The proliferation potential of transformed clones was determined by their total CPDL (cumulative population-doubling levels) using the formula cpdl = ln(Nf/Ni)/ln 2, where Ni and Nf are initial and final cell numbers, respectively, and ln is the natural log. The initial cell number was 2 x 105 for each propagation.

Bioassay of GIIC
The scrape loading/dye transfer (SL/DT) technique was adapted from the method of EI-Fouly et al. (1987)Go. Following incubation, the cells were washed twice with 2 ml of PBS. Lucifer yellow was added to the cells and three scrapes were made with a surgical steel blade scalpel under low light intensities. The purpose of three scrapes was to ensure that the scrape traversed a large group of confluent cells. After an additional 3 min incubation period, the cells were washed with 10 ml of PBS and then fixed with 2 ml of 4% formalin solution. The distance that the dye travelled perpendicular to the scrape was observed with an inverted fluorescence microscope (Olympus Ix 70, Tokyo, Japan).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Isolation and immortalization of normal endometrial cells
We isolated endometrial epithelial and stromal cells. Epithelial cells were polyhedral and grew as islands in a whirl-like pattern around a glandular fragment; whereas stromal cells were spindle-shaped, more long-lived and grew rapidly to form parallel bundles of cells as shown in Figure 1. Stromal cells typically appeared spindle-shaped and reached confluence within a short period of time. Epithelial cells derived from the glandular epithelium initially presented a whirled morphology, but within 2 weeks cell integrity deteriorated. The intermediate filaments, vimentin and cytokeratin were immunocytochemically detected to prove the epithelial or stromal origin of the cultivated cells. Cytokeratin 8/18 (the marker of epithelial cells) was positive for the epithelial cells, whereas vimentin (the marker of stromal cells) was positive for stromal cells as shown in Figure 2.



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Figure 1. Photomicrographs of endometrial epithelial and stromal cells. The different cultures were observed with a phase-contrast inverted microscope. Stromal cells typically appeared spindle-shaped and reached confluence within a short period of time (A). Epithelial cells derived from the glandular epithelium initially presented a whorled morphology, but within 2 weeks cell integrity deteriorated (B).

 


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Figure 2. Immunofluorescent staining of endometrial epithelial and stromal cells. Stromal cells were immunostained for vimentin (A), but stromal cells were not stained for cytokeratin as shown (B). However, epithelial cells were immunostained for cytokerain (C), but epithelial cells were not stained for vimentin (D).

 
Next, endometrial stromal cells were immortalized by SV40 large T antigen. One to two weeks after transfection (with SV40 large T antigen), rapidly growing colonies were observed. After confluence, these cells lost contact inhibition and grew as an overlayer. Transfected endometrial stromal cells were tested for the expression of several specific antigens previously observed in normal endometrial cells. The expression of SV40 large T antigen was tested by immunocytochemistry and showed a strong nuclear staining in 100% of the transfected endometrial stromal cells as shown in Figure 3. The immortalized endometrial stromal cells had an extended life span (CPDL60) as shown in Figure 4. Transfected endometrial stromal cells were revealed by a 100% vimentin positive and 100% negative expression of cytokeratin 8/18 expression as shown in Figure 5.



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Figure 3. The SV40 expression of normal endometrial stromal cells and immortalized endometrial stromal cells. Both in immunostaining (A) and western blot analysis (B), SV40 large T antigens strongly expressed in immortalized endometrial stromal cells, but, in normal endometrial stromal cells, SV40 large T antigens were not expressed.

 


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Figure 4. Cumulative growth curve of transformed cells at immortalization. This study observed the cumulative population-doubling levels (CPDLs) of immortalized endometrial stromal cells to study its growth characteristics and production. This result suggests that immortalized endometrial stromal cells had an extended life span.

 


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Figure 5. Immunochemical trait of immortalized endometrial stromal cells and normal endometrial stromal cells. Phenotypic characteristics of transfected cells were similar to those of their normal corresponding endometrial cells. Transfected endometrial stromal cells were revealed by a 100% positive vimentin expression and negative cytokeratin 8/18 expression.

 
To quantitate the connexin expression level for normal and immortal endometrial stromal cells, we isolated proteins and analyzed them using western blot analysis. The results show that normal and transfected endometrial stromal cells had expressed connexin 32 and 43. Connexin 26 was not expressed in both normal and transfected endometrial stromal cells as shown in Figure 6.



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Figure 6. SL/DT (A) and western blot analysis (B) were conducted to quantify the connexin expression level for normal and immortalized endometrial stromal cells. The normal and transfected endometrial stromal cells have the same expression level of connexin 32 and 43. Connexin 26 was not expressed in both the normal and transfected endometrial stromal cells. These results suggest that SV40 large T antigens did not affect GJIC in endometrial stromal cells.

 
Roles of p38 and c-jun in differentiation and proliferation of endometrial stromal cells
Cellular and molecular events in early (passage 1–2) and aged (passage 10–15) human endometrial stromal cells were investigated. There were morphological alterations following these passages. The early cells from passage 1–2 were polygonal, while the aged cells from passage 10–15 were spindle-shaped as shown in Figure 7. To examine the mechanisms involved in this morphological alteration, western blot analysis was performed. The results show that the c-jun and pp38 levels were significantly reduced in aged cells as shown in Figure 8. To clarify the role of c-jun, SP600125, which is an inhibitor of JNK kinase, was administered onto the cells. As a result, the polygonal early passage stromal cells changed morphologically into aged endometrial spindle-shaped cells as shown in Figure 9.



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Figure 7. Photomicrographs of endometrial stromal cells. The different cultures were observed with a phase-contrast inverted microscope. Younger (passage 1–2) endometrial stromal cells had more morphological alterations from a polygonal morphology. (A) As the cells aged (passage 5), the endometrial stromal cells became more spindle-shaped than younger endometrial stromal cells (B). The more aged endometrial stromal cells (passage 10–15) became completely elongated and spindle-shaped (C).

 


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Figure 8. Expression of c-jun and p38 is essential for differentiation of endometrial stromal cells. The c-jun and pp38 levels were significantly reduced with an increase of in vitro aged cells. But the total Erk and p38 expressions were not changed.

 


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Figure 9. The inhibition of the c-jun expression induced differentiation of endometrial stromal cells. Treatment with SP600125, an inhibitor of JNK kinase (C), induced morphological changes of young endometrial stromal cells (A) to aged endometrial stromal cells (B) as shown.

 
In addition, this study was also performed to examine the roles of c-jun and p38 during cell proliferation of endometrial stromal cells using immortalized cells. Western blotting methods were also used to investigate the expression pattern of several specific proteins in normal and transfected endometrial stromal cells. The results show that normal and transfected endometrial stromal cells had the same expression level of total Erk, p53, p38 and pp38. But c-jun expression levels were significantly increased in immortalized endometrial stromal cells as shown in Figure 10.



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Figure 10. Normal and immortalized endometrial stromal cells have the same expression level of total Erk, p53, p38 and pp38. But the c-jun and c-myc expression levels are significantly increased in immortalized endometrial stromal cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The aim of this work was to understand the roles of c-jun and p38 for the differentiation and proliferation of normal human endometrial cells. In the present study, we isolated two types of normal endometrial cells and then examined the level of c-jun and pp38 during differentiation and immortalization.

Normal endometrial stromal cells were spindle-shaped, more long-lived and grew rapidly to form parallel bundles of cells. There were polygonal shapes in the stromal cells in the early passage 1–2, while the aged endometrial stromal cells were spindle-shaped as shown in Figure 7. To investigate passage-dependent molecular events in endometrial cells, the c-jun and pp38 levels were examined. Both c-jun and pp38 were significantly reduced with cellular aging and passages. In this study, we did not demonstrate all the mechanisms of differentiation in endometrial cells. However, certain findings may be drawn concerning how p38 and c-jun operate as mediators of cell proliferation and differentiation.

It has been reported that the p38 signaling transduction pathway, a mitogen-activated protein (MAP) kinase pathway, plays an essential role in regulating many cellular processes including inflammation, cell differentiation, cell growth and death (New et al., 1998Go; Ono et al., 2000Go). In addition to these, p38 was found to play an important role in cell differentiation for several different cell types. For example, the differentiation of 3T3-L1 cells into adipocytes and the differentiation of PC12 cells into neurons both require p38 (Engelman et al., 1998Go; Takaya et al., 1998). The p38 pathway was found to be necessary and sufficient for SKT6 differentiation into hemoglobinized cells as well as C2C12 differentiation into myotubes (Anna et al., 1999). The involvement of p38 in cell growth became apparent when it was noticed that an over expression of p38 in yeast led to a significant slowing of proliferation. While, as a central component of AP-1 transcription factors, c-jun is also a major regulator of proliferation and the stress responses of mammalian cells, and a contributor to oncogenesis (Derijard et al., 1994Go; Kyriakis et al., 1994Go; Wisdom et al., 1999Go). The transcription factor AP-1 was first defined as a DNA binding activity specific for positive regulatory elements in the SV40 early promoter. Because the jun genes were initially brought into view by virtue of their presence in the genomes of oncogenic retroviruses, the connection of AP-1 proteins to cancer has been apparent since their discovery. The transcription factor AP-1 is activated in response to an incredible array of stimuli, including mitogenic growth factors, inflammatory cytokines, growth factors of the TGF-{beta} family, UV and ionizing irradiation, cellular stress, antigen binding and neoplastic transformation (Wisdom et al., 1999Go). The major findings of this study are that c-jun was inactivated during cell differentiation, while immortalized cell lines were shown to activate c-jun, more than normal cells. To confirm the role of c-jun, cells were treated with SP600125 (inhibitor of JNK kinase). SP600125 induced morphological changes of young endometrial cells to appear as aged primary endometrial cells as shown in Figure 9. Furthermore, in this study, the pure endometrial stromal cells were immortalized by SV40 large T antigen using a similar protocol as previously described by Kang et al. (1997)Go. These findings demonstrate that the mechanism of SV40 large T antigen-mediated proliferation is associated with an increased expression of c-jun in human endometrial cells (Endo et al., 1992Go; Lu et al., 1998Go; Kim et al., 2001Go).

In our study, SV40-induced immortalization occurred at a rare frequency. There are two implications of immortalization. The first is the traditional view that suggests immortalization occurs as would a mutation in a critical gene—to revert a normal mortal cell to an immortal cell that could then be neoplastically transformed. The opposite interpretation is that SV40 transfects all of the primary cells in the population, which include mostly progenitor or mortal cells. These cells would eventually senesce and die (‘crisis’ event). However, in that primary population, there exist a few stem cells which are already, naturally, ‘immortal’. SV40 would not immortalize these cells but would prevent them from terminally differentiating. Therefore, comparing the genes (c-jun; p38) in the primary normal cells with the ‘immortalized’ clones may be a false comparison. Because the primary cells contain mostly mortal progenitor cells, their gene expression would be very different from the few primary adult stem cells in that population. Therefore, the true comparison would be to compare the SV40 immortalized cells to the few normal adult stem cells. Therefore, we are planning to isolate adult stem cells from the human uterus for the comparison of these stem cells to immortalized endometrial stromal cells in the near future.

It has been reported that poor gap junctional intercellular communication (GJIC) is associated with uncontrolled cell growth and immortalization with SV40 large T antigen (Khoo et al., 1998Go; Saito et al., 2001Go). However, our dye transfer and western blot analysis data show that there was gap junction in both normal and immortal endometrial cells as shown in Figure 6. An inadequate culture condition, or the fact that the low level of dye transfer might be caused by other connexins that were not examined in our western blot analysis are possible limitations to this study. Taken together, these assessments suggest that the modulation of p38 and c-jun might play an important role for the differentiation and proliferation of normal human endometrial cells.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by a grant (01-PJ10-PG8-01EC01-0007) from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. This work was also supported by the Research Institute for Veterinary Science, Seoul National University.


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 Discussion
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 References
 
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Submitted on December 16, 2003; resubmitted on June 2, 2004; accepted on June 24, 2004.





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