- Email: [email protected]
Dipeptidyl peptidase IV expression in endometrial endometrioid adenocarcinoma and its inverse correlation with tumor grade
Dipeptidyl peptidase IV expression in endometrial endometrioid adenocarcinoma and its inverse correlation with tumor grade Ei Ei Khin, MD,a Fumitaka Kikkawa, MD, PhD,a Kazuhiko Ino, MD, PhD,a Hiroaki Kajiyama, MD,a Takahiro Suzuki, MD,a Kiyosumi Shibata, MD, PhD,a Koji Tamakoshi, MD, PhD,b Tetsuro Nagasaka, MD, PhD,c and Shigehiko Mizutani, MD, PhDa Nagoya, Japan OBJECTIVES: Dipeptidyl peptidase IV (DPPIV)/CD26 is a cell surface aminopeptidase. This study investigated the expression and localization of DPPIV in endometrial endometrioid adenocarcinomas of different grades. STUDY DESIGN: Immunohistochemical analysis was performed by using DPPIV and regulated on activation, normal T-cell expressed and secreted (RANTES) specific monoclonal antibodies. Cell proliferation was evaluated by bromodeoxyuridine (BrdU) uptake assay. RESULTS: Immunohistochemical analyses showed that DPPIV was strongly or moderately stained in glandular cells of the normal secretory phase. In endometrial adenocarcinoma, the DPPIV expression decreased with advancing grade (P < .01). Furthermore, RANTES, one of the possible DPPIV substrates, was highly expressed in all grades of endometrial adenocarcinoma cells. The addition of RANTES to endometrial adenocarcinoma cells increased proliferation in a concentration-dependent manner. CONCLUSION: DPPIV is expressed in normal endometrial glandular cells, but its expression in endometrial adenocarcinoma is down-regulated with increasing grade. Our data also suggest a regulatory role of this ectoenzyme in neoplastic transformation and progression of endometrial adenocarcinomas possibly by degrading certain bioactive peptides such as RANTES. (Am J Obstet Gynecol 2003;188:670-6.)
Key words: Dipeptidyl peptidase IV, RANTES, immunohistochemistry, endometrial endometrioid adenocarcinoma
Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a cell surface peptidase that cleaves N-terminal dipeptides from polypeptides with a proline or alanine at the penultimate position. This 110-kd type II membrane glycoprotein has been identified as the cluster differentiation 26 (CD26) and has multiple functions, including serine protease activity,1 the ability to bind adenosine deaminase, which is associated with T-cell activation and proliferation,2 and adhesion to extracellular matrix components such as fibronectin and collagens.3 Enzymologically, DPPIV can cleave specific peptide substrates such as substance P,
From the Departments of Obstetrics and Gynecology,a and Public Health,b Nagoya University Graduate School of Medicine, and Division of Pathology, Clinical Laboratory, Nagoya University Hospital.c This work was supported in part by Grants-in-Aid from the Ministry of Posts and Telecommunications for specific medical research (collaboration with Nagoya Teishin Hospital) and Education, and the fund of Showakai and Ogya. Received for publication February 7, 2002; revised September 3, 2002; accepted November 5, 2002. Reprint requests: Dr Fumitaka. Kikkawa, Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Showa-ku, Tsurumai-cho 65, Nagoya 466-8550, Japan. E-mail: [email protected] © 2003, Mosby, Inc. All rights reserved. 0002-9378/2003 $30.00 + 0 doi:10.1067/mob.2003.169
670
growth hormone–releasing factor, glucagon-like peptides, and some chemokines, including regulated on activation, normal T-cell expressed and secreted (RANTES),4 and reduces cellular responses to these bioactive peptides. This ectoenzyme is widely distributed in activated T cells, epithelial cells of the small intestine, liver, prostate, renal proximal tubules, and also in melanocytes.5 In addition, DPPIV is expressed in the female reproductive organs such as the placenta,6 ovary,7 and endometrium.8 In addition to its expression in normal tissues, DPPIV expression and its roles in human tumors have been reported not only in hematologic malignancies but also in certain solid tumors, including melanoma, thyroid carcinoma, prostate carcinoma, and colon carcinoma.9-12 Recent studies have shown that cell surface peptidases, including DPPIV, play key roles in the control of growth, differentiation, and signal transduction of many cellular systems by modulating the activity of peptide factors.13 Thus, abnormalities in the expression pattern and/or catalytic function of these peptidases result in altered peptide activation or inactivation. These cause disruption of normal cellular homeostasis and may contribute to neoplastic transformation or tumor progression. In human endometrium, we have recently demonstrated that neutral endopeptidase (NEP)/CD10 is
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highly expressed in stromal cells of the normal endometrium and grade 1 endometrial adenocarcinoma but its expression is decreased or lost with advancing tumor grade.14 In contrast to NEP expression in endometrial stroma, DPPIV is known to be expressed in endometrial glandular cells.8 However, DPPIV expression and function in endometrial adenocarcinoma have not yet been studied. The purpose of this study was to investigate DPPIV expression and localization by immunohistochemical analysis in normal endometrium and different grades of endometrial adenocarcinomas and also to determine whether its expression pattern is related to neoplastic transformation, differentiation, and disease progression. Furthermore, we also examined immunohistochemical expression of the chemokine RANTES, which is one of the DPPIV substrates, in the same tissue materials, and discuss the possible role of DPPIV in differentiation and progression of endometrial carcinoma through the regulation of this chemokine. Material and methods Cell line and cell culture. Endometrial endometrioid adenocarcinoma cell line HEC1A was purchased from the American Type Culture Collection (ATCC), and ISHIKAWA was provided by Dr Nishida (University of Tsukuba, Tsukuba, Japan). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St Louis, Mo) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 U/mL) at 37°C in a humidified 5% carbon dioxide atmosphere. Tissues. Formalin-fixed, paraffin-embedded tissues were obtained surgically from 41 patients who underwent hysterectomy at Nagoya University Hospital from August 1994 to December 2000. Normal endometrial tissues (n = 12) were obtained from premenopausal women who underwent hysterectomy for benign ovarian tumor or uterine myoma. Tumor tissues were obtained from 29 patients with endometrial endometrioid adenocarcinoma with different tumor grades. Tumor histologic features and grade were determined according to the criteria of the World Health Organization (grade 1: well differentiated; grade 2: moderately differentiated; grade 3: poorly differentiated). Clinical staging was determined according to the Federation of International Gynecology and Obstetrics (FIGO). Tumors consisted of 16 stage I, 5 stage II, and 8 stage III (10 grade 1, 9 grade 2, and 10 grade 3). Informed consent for the use of tissues was obtained from each patient. Flow cytometric analysis. Fluorescence-activated cell sorting (FACS) analysis was performed to quantify DPPIV expression on the endometrial adenocarcinoma cell surface. Cells were stained with phycoerythrin (PE)-conjugated anti-DPPIV monoclonal antibodies (mAb), MA-261 (Pharmingen, San Diego, Calif), and TS-145 (kindly pro-
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vided by Dr Ryuzo Ueda, Nagoya City University),15 or isotype-matched control mouse immunoglobulin G (IgG) (Coulter, Hialeah, Fla) for 1 hour at 4°C. After three washes, cells were resuspended in phosphatebuffered saline solution (PBS). FACS data were acquired on the FACS calibur system (Becton Dickinson, San Jose, Calif), and analyzed with CELLQuest software (Becton Dickinson). Each assay was performed three times. Western blotting. Tissues were homogenized for 10 minutes on ice in lysis buffer composed of PBS solution, 1% Triton X-100, and protease inhibitor cocktail tablet (Roche Diagnosis GmbH, Mannheim, Germany). Tissue extracts and cell lysates were cleared by centrifugation at 14,000g for 20 minutes at 4°C and stored at –80°C. Protein concentrations were determined with a protein assay kit (Bio-Rad Laboratories, Hercules, Calif). Extract (30 µg) in each lane was separated by SDS/7.5% polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and immunoblotted with mouse monoclonal anti-DPPIV antibody (TS-145, 1:40 dilution). The biotinylated secondary antibody (Vector Laboratories, Burlingame, Calif) was used at 1:200 dilution. Immunoreactive proteins were detected by ECL Western blotting detection systems (Amersham Biosciences, Tokyo, Japan). In the negative control, the primary antibody was replaced with normal mouse IgG. Immunohistochemistry. Immunohistochemical staining was performed with the avidin-biotin immunoperoxidase technique (Histofine SAB-PO kit, Nichirei, Tokyo, Japan). Sections were cut at a thickness of 4 µm and were deparaffinized and rehydrated. For heat-induced epitope retrieval, deparaffinized sections were treated three times for 5 minutes at 90°C at 750 W in a microwave oven. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide, and nonspecific immunoglobulin binding was blocked by incubation with 10% normal rabbit serum. Incubation with primary mAb for DPPIV (TS-145) at a dilution of 1:100 and for RANTES (PeproTech EC, London, UK) at a dilution of 1:400 was performed overnight at 4°C. After a washing with PBS solution, secondary biotinylated antibody was applied for 10 minutes. After washing, the streptavidinperoxidase conjugate was applied for 5 minutes. After washing, sections were incubated with 3-amino-9-ethylcarbazole (AEC, Nichirei, Japan) to develop a red reaction product and then counterstained with Mayer’s hematoxylin. For the negative control, mouse IgG was replaced with primary antibody. Sections from normal kidney proximal tubules were used as a positive control staining for DPPIV. Staining intensity was scored semiquantitatively on a four-titered scale (negative: –; weakly positive: +; moderately positive: ++; strongly positive staining: +++) relative to the known positive and negative controls. Each specimen was scored twice, independently by two investigators.
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Fig 1. Cell surface expression of DPPIV on endometrial adenocarcinoma cell line (HEC1A) by FACS. Cells were stained with two different PE-labeled DPPIV mouse mAb (shaded area) or PE-labeled isotype-matched mouse IgG (open area). A, Stained with MA-261. B, Stained with TS-145.
Fig 2. Immunoblot analysis of DPPIV using mouse anti-CD26 monoclonal antibody (TS-145). Protein (30 µg) extracted from normal endometrial tissue, endometrial adenocarcinoma tissue, and endometrial adenocarcinoma cell lines (ISHIKAWA and HEC1A) was separated on SDS/7.5% polyacrylamide gel and electroblotted onto nitrocellulose membranes. Lane 1, Grade 1 endometrial adenocarcinoma tissue; lane 2, grade 2; lane 3, grade 3; lane 4, proliferative phase normal endometrium; lane 5, secretory phase; lane 6, ISHIKAWA cells; lane 7, HEC1A cells.
Cell proliferation assay. Cells were cultured in 96-well microplates in serum-free medium for 4 hours. After incubation with various concentrations of RANTES for 24 hours, cell proliferation was evaluated by bromodeoxyuridine (BrdU) uptake assay by using a cell proliferation enzyme-linked immunosorbent assay (ELISA) system (Amersham Biosciences). Reverse transcriptase–polymerase chain reaction experiments. After informed consent was obtained from patients, messenger RNA (mRNA) was extracted from cell lines (ISHIKAWA and HEC1A) and endometrial adenocarcinoma tissues by using a Quick Prep mRNA Purification Kit (Amersham Biosciences). Reverse transcription–polymerase chain reaction (RT-PCR) was performed with an RNA PCR kit (Perkin-Elmer Corp, Norwalk, Conn) according to the manufacturer’s protocol. The sense and an-
tisense specific primers for human RANTES were 5´CTACTCGGGAGGCTAAGGCAGGAA-3´ and 5´GAGGGGTTGAGACGGCGGAAGC-3´, respectively. These primers yield a 318-bp fragment. Statistics. The nonparametric Kruskal-Wallis test was performed to compare the staining scores among all groups. In addition, the staining score were compared between normal endometrium and neoplastic tissues by using the Mann-Whitney test with Bonferroni correction. Analysis of variance was used to determine the significance of the effect of RANTES on endometrial adenocarcinoma cell proliferation. P < .05 was considered significant. Results To evaluate the immunoreactivity and specificity of the anti-DPPIV mAb, TS-145, that was used for subsequent Western blotting and immunohistochemical studies, FACS analysis was performed using the endometrial carcinoma cell line (HEC1A). As shown in Fig 1, strong DPPIV expression on HEC1A cells was detected by FACS analysis by using the TS-145 mAb. A similar expression level and specificity were also observed by the commercially available DPPIV-specific mAb (MA-261). Western blot analysis for DPPIV protein expression was performed with TS-145 (Fig 2). A single immunoreactive band was detected as approximately 110 kd in both the proliferative and secretory phases of normal endometrial tissues, although the DPPIV expression in the proliferative phase was lower than that in the secretory phase. A 110-kd band of immunoreactive DPPIV protein was also detected in each grade of endometrial adenocarcinoma tissue as well as in HEC1A.
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Fig 3. DPPIV immunostaining of normal premenopausal endometrium in different phases of menstrual cycle and endometrial adenocarcinoma specimens. (Original magnification, 200.) A, Normal premenopausal endometrium (proliferative phase). B, Normal premenopausal endometrium (secretory phase). C, Grade 1 adenocarcinoma tissue. D, Grade 1. E, Grade 2. F, Grade 3.
To evaluate the immunoreactivity of TS-145 mAb on paraffin-embedded tissue sections, we first immunostained the normal renal proximal tubular tissue as a positive control. Strong staining intensity was observed in the renal proximal tubular cells (data not shown). DPPIV immunoreactivity was only localized in endometrial glandular cells but not detected in stromal cells. DPPIV expression was weakly or moderately detected in the proliferative phase of normal endometrium (Fig 3, A), whereas in the secretory phase of normal endometrium its expression was strongly detected in glandular cells (Fig 3, B). These findings are consistent with those of the previous report.8
In endometrial adenocarcinoma, DPPIV was also localized in adenocarcinoma cells but not in stromal cells. In grade 1, DPPIV immunoreactivity was strongly or moderately detected (Fig 3, C and D). However, weak or no expression of DPPIV was found in grades 2 and 3 (Fig 3, E and F). There was an inverse correlation between DPPIV immunoreactivity and tumor grading, and comparison of the five groups showed a significant difference (P < .0005, Fig 4). However, there was no correlation with the clinical stage of endometrial adenocarcinoma (data not shown). The results of immunohistochemical staining for RANTES in normal endometrium and endometrial adenocarcinomas were summarized in the Table. RANTES
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Fig 4. Staining intensity score of DPPIV immunoreactivity in glandular cells of normal endometrium and grades 1, 2, and 3 endometrial adenocarcinoma. Score decreased with advancing tumor grade.
Table. Intensity of RANTES expression in sections of patients at normal and different grades of endometrial adenocarcinomas Intensity of RANTES expression Normal (n = 8) Grade 1 (n = 10) Grade 2 (n = 9) Grade 3 (n = 10)
–
+
++
+++
0 (0%) 0 (0%) 0 (0%) 0 (0%)
1 (13%) 2 (20%) 1 (12%) 3 (30%)
3 (37%) 3 (30%) 5 (55%) 5 (50%)
4 (50%) 5 (50%) 3 (33%) 2 (20%)
immunoreactivity was localized in the glandular cells of normal endometrium and was strongly or moderately detected in the secretory phase of normal endometrium (Fig 5, A). Most endometrial adenocarcinomas also highly or moderately expressed RANTES (Fig 5, B). Similar moderate expression of RANTES was found in breast carcinoma tissues as a positive control (data not shown). However, there was no significant correlation between RANTES expression and tumor grade. We studied the effect of RANTES on endometrial adenocarcinoma cell proliferation. Fig 6 shows that RANTES induced cell proliferation in both HEC1A and ISHIKAWA cells in a concentration-dependent manner. RT-PCR demonstrated that mRNA of RANTES was present in endometrial adenocarcinoma tissues as well as in ISHIKAWA and HEC1A cells as shown in Fig 6, C. Comment In this study, we have demonstrated the expression and localization of DPPIV in normal endometrium and endometrial adenocarcinoma by immunohistochemical analysis. DPPIV immunostaining was more strongly positive in the secretory phase of normal endometrium than in the proliferative phase. These findings were highly
consistent with the previous report,8 and it is suggested that DPPIV plays a role in the differentiation of endometrial glandular cells and also possibly in the maintenance of homeostasis of the normal endometrium. Strong or moderate DPPIV expression was still observed in most grade 1 adenocarcinomas, but weak or no expression was detected in grades 2 and 3. These findings suggest that down-regulation of DPPIV expression is related to neoplastic transformation and tumor progression. Similar findings were reported in melanoma.9,16 However, increased expression of DPPIV was reported in thyroid, skin, and prostate carcinoma.10,17,18 Our recent studies demonstrated that the expression of aminopeptidase A, one of the cell surface aminopeptidases, was upregulated in choriocarcinoma and invasive cervical carcinoma.19,20 Iwata and Morimoto21 suggested that upregulation or down-regulation of these ectopeptidases appear to be tissue specific, and even cell type specific, in a variety of malignancies in their review. This hypothesis was supported by Papandreou et al,22 who proposed two basic mechanisms of cell surface peptidase involvement in the malignant process. One is loss of function resulting in an inability of the tumor cells to inactivate a stimulatory peptide. The other is gain of function resulting in
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Fig 5. RANTES immunostaining in normal premenopausal endometrium and endometrial adenocarcinoma specimens. (Original magnification, 200.) A, Normal premenopausal endometrium. B, Grade 3 adenocarcinoma.
the activation processing of a stimulatory peptide or the inactivation of an inhibitory peptide. In this study, the intensity of DPPIV staining in endometrial adenocarcinoma was inversely correlated with the degree of tumor differentiation and was down-regulated with advancing tumor grade. Similarly, we recently reported that another cell-surface aminopeptidase, NEP/CD10, was expressed in normal endometrial stromal cells, and its stromal expression was down-regulated with increasing tumor grade in endometrial carcinoma, whereas stromal endothelin-1, one of the NEP substrates, was up-regulated.14 On the basis of these two findings, it is suggested that these two ectoenzymes are differentially expressed in endometrial glandular cells and stromal cells and may play a regulatory role not only in the maintenance of normal endometrial function but also in the neoplastic transformation and tumor progression through modulating their specific peptide substrates. Enzymologically, DPPIV is a serine-type protease that preferentially cleaves Xaa-Pro and Xaa-Ala dipeptides from the NH2 terminus of peptides and proteins. The optimal size and features of substrates for this enzyme have not been fully characterized. Potential DPPIV substrates
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Fig 6. After incubation with various concentrations of RANTES for 24 hours, cell proliferation was evaluated by BrdU uptake assay by using a cell proliferation ELISA system. A, HEC1A. B, ISHIKAWA. C, RT-PCR was performed by using primers of RANTES. Lanes 1 and 7, 100-bp ladder; lane 2, grade 1 endometrial adenocarcinoma tissue; lane 3, grade 2; lane 4, grade 3; lane 5, ISHIKAWA cells; lane 6, HEC1A cells.
include neuropeptides and some chemokine families such as RANTES.4 Among these various DPPIV substrates, several chemokines were shown to be involved in tumor cell proliferation and progression. Recently, Luboshits et al23 have reported that the expression of RANTES is directly correlated with the advance of breast carcinoma, indicating that RANTES may be involved in breast carcinoma progression. Our results showed that significant levels of RANTES expression was observed in both normal endometrial glandular cells and endometrial adenocarcinoma cells. Hornung et al24 previously showed RANTES expression in human endometrial and endometriosis tissues. To our knowledge, our current study is the first report showing the expression of RANTES in endometrial carcinomas. Furthermore, RANTES stimulated the proliferation of HEC1A endometrial carcinoma cells in vitro (Fig 6). Mrowietz et al25 also found that RANTES is secreted by human melanoma cells and is associated with enhanced tumor formation in vivo. It is still unclear whether these
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chemokines could be involved in the progression of endometrial carcinomas in vivo. However, one can speculate that the loss of DPPIV in advanced endometrial carcinoma may result in the inability to degrade peptide growth factors such as RANTES. These factors may be secreted from carcinoma or stromal cells as an autocrine/paracrine growth factor and may contribute to tumor progression. In conclusion, this report has demonstrated the expression and localization of DPPIV in normal endometrium and endometrial adenocarcinoma. Because DPPIV expression decreased with the advancing tumor grade, reducing this enzyme may have a beneficial effect on carcinoma progression caused by the loss of degrading activity of bioactive factors such as RANTES in endometrial adenocarcinoma. REFERENCES
1. Misumi Y, Hayashi Y, Arakawa F, Ikehara Y. Genome, nucleotide, OMIM, protein molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface. Biochim Biophys Acta 1992;1131:333-6. 2. Dong RP, Kameoka J, Hegen M, Tanaka T, Xu Y, Schlossman SF, et al. Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response. J Immunol 1996;156:1349-55. 3. Loster K, Zeilinger K, Schuppan D, Reutter W. The cysteine-rich region of dipeptidyl peptidase IV (CD 26) is the collagen-binding site. Biochem Biophys Res Commun 1995;217:341-8. 4. Proost P, De Meester I, Schols D, Struyf S, Lambeir AM, Wuyts A, et al. Amino-terminal truncation of chemokines by CD26/dipeptidyl-peptidase IV: conversion of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection. J Biol Chem 1998;273:7222-7. 5. Hartel S, Gossrau R, Hanski C, Reutter W. Dipeptidyl peptidase (DPP) IV in rat organs: comparison of immunohistochemistry and activity histochemistry. Histochemistry 1988;89:151-61. 6. Mizutani S, Sumi S, Narita O, Tomoda Y. Purification and properties of human placental dipeptidyl peptidase IV. Nippon Sanka Fujinka Gakkai Zasshi 1985;37:769-75. 7. Fujiwara H, Maeda M, Imai K, Fukuoka M, Yasuda K, Takakura K, et al. Human luteal cells express dipeptidyl peptidase IV on the cell surface. J Clin Endocrinol Metab 1992;75:1352-7. 8. Imai K, Maeda M, Fujiwara H, Kariya M, Takakura K, Kanzaki H, et al. Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells. Hum Reprod 1992;7:1189-94. 9. Morrison ME, Vijayasaradhi S, Engelstein D, Albino AP, Houghton AN. A marker for neoplastic progession of human melanocyte is a cell surface ectopeptidase. J Exp Med 1993;177:1135-43. 10. Tanaka T, Umeki K, Yamamoto I, Sakamoto F, Noguchi S, Ohtaki S. CD26 (dipeptidyl peptidase IV/DPP IV) as a novel molecular
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marker for differentiated thyroid carcinoma. Int J Cancer 1995;64:326-31. Bogenrieder T, Finstad CL, Freeman RH, Papandrou CN, Scher HI, Albino AP, et al. Expression and localization of aminopeptidase A, aminopeptidase N and DPPIV in benign and malignant human prostate tissue. Prostate 1997;33:225-32. Ten Kate J, van den Ingh HF, Khan PM, Bosman FT. Adenosine deaminase complexing protein (ADCP) immunoreactivity in colorectal adenocarcinoma. Int J Cancer 1986;37:479-85. Kenny AJ, O’Hare MJ, Gusterson BA. Cell-surface peptidases as modulators of growth and differentiation. Lancet 1989;2:785-7. Suzuki T, Kikkawa F, Ino K, Nagasaka T, Tamakoshi K, Mizutani S. Imbalance between neutral endopeptidase 24.11 and endothelin-1 expression in human endometrial carcinoma. Oncology 2001;60:258-67. Tsuge I, Ueda R, Nishida K, Namikawa R, Seto M, Maruyama T, et al. Five antigens on human T cells detected by mouse monoclonal antibodies. Clin Exp Immunol 1984;58:444-52. Wesley UV, Albino AP, Tiwari S, Houghton AN. A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells. J Exp Med 1999;190:311-22. Moehrle MC, Schlagenhauff BE, Klessen C, Rassner G. Aminopeptidase M and dipeptidyl peptidase IV activity in epithelial skin tumors: a histochemical study. J Cutan Pathol 1995;22:241-7. Wilson MJ, Ruhland AR, Quast BJ, Reddy PK, Ewing SL, Sinha AA. Dipeptidylpeptidase IV activities are elevated in prostate cancers and adjacent benign hyperplastic glands. J Androl 2000;21:220-6. Ino K, Nagasaka T, Okamoto T, Uehara C, Nakazato H, Nakashima N, et al. Expression of aminopeptidase A in human gestational choriocarcinoma cell lines and tissues. Placenta 2000;21:63-72. Fujimura H, Ino K, Nagasaka T, Nakashima N, Nakazato H, Kikkawa F, et al. Aminopeptidase A expression in cervical neoplasia and its relationship to neoplastic transformation and progression. Oncology 2000;58:342-52. Iwata S, Morimoto C. CD26/dipeptidyl peptidase IV in context: the different roles of a multifunctional ectoenzyme in malignant transformation. J Exp Med 1999;190:301-6. Papandreou CN, Usmani B, Geng Y, Bogenrieder T, Freeman R, Wilk S, et al. Neutral endopeptidase 24.11 loss in metastatic human prostate cancer contributes to androgen-independent progression. Nat Med 1998;4:50-7. Luboshits G, Shina S, Kaplan O, Engelberg S, Nass D, LifshitzMercer B, et al. Elevated expression of the CC chemokine regulated on activation, normal T cell expressed and secreted (RANTES) in advanced breast carcinoma. Cancer Res 1999;59:4681-7. Hornung D, Ryan IP, Chao VA, Vigne JL, Schriock ED, Taylor RN. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab 1997;82:1621-8. Mrowietz U, Schwenk U, Maune S, Bartels J, Kupper M, Fichtner I, et al. The chemokine RANTES is secreted by human melanoma cells and is associated with enhanced tumour formation in nude mice. Br J Cancer 1999;79:1025-31.
Key words: Dipeptidyl peptidase IV, RANTES, immunohistochemistry, endometrial endometrioid adenocarcinoma
Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a cell surface peptidase that cleaves N-terminal dipeptides from polypeptides with a proline or alanine at the penultimate position. This 110-kd type II membrane glycoprotein has been identified as the cluster differentiation 26 (CD26) and has multiple functions, including serine protease activity,1 the ability to bind adenosine deaminase, which is associated with T-cell activation and proliferation,2 and adhesion to extracellular matrix components such as fibronectin and collagens.3 Enzymologically, DPPIV can cleave specific peptide substrates such as substance P,
From the Departments of Obstetrics and Gynecology,a and Public Health,b Nagoya University Graduate School of Medicine, and Division of Pathology, Clinical Laboratory, Nagoya University Hospital.c This work was supported in part by Grants-in-Aid from the Ministry of Posts and Telecommunications for specific medical research (collaboration with Nagoya Teishin Hospital) and Education, and the fund of Showakai and Ogya. Received for publication February 7, 2002; revised September 3, 2002; accepted November 5, 2002. Reprint requests: Dr Fumitaka. Kikkawa, Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Showa-ku, Tsurumai-cho 65, Nagoya 466-8550, Japan. E-mail: [email protected] © 2003, Mosby, Inc. All rights reserved. 0002-9378/2003 $30.00 + 0 doi:10.1067/mob.2003.169
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growth hormone–releasing factor, glucagon-like peptides, and some chemokines, including regulated on activation, normal T-cell expressed and secreted (RANTES),4 and reduces cellular responses to these bioactive peptides. This ectoenzyme is widely distributed in activated T cells, epithelial cells of the small intestine, liver, prostate, renal proximal tubules, and also in melanocytes.5 In addition, DPPIV is expressed in the female reproductive organs such as the placenta,6 ovary,7 and endometrium.8 In addition to its expression in normal tissues, DPPIV expression and its roles in human tumors have been reported not only in hematologic malignancies but also in certain solid tumors, including melanoma, thyroid carcinoma, prostate carcinoma, and colon carcinoma.9-12 Recent studies have shown that cell surface peptidases, including DPPIV, play key roles in the control of growth, differentiation, and signal transduction of many cellular systems by modulating the activity of peptide factors.13 Thus, abnormalities in the expression pattern and/or catalytic function of these peptidases result in altered peptide activation or inactivation. These cause disruption of normal cellular homeostasis and may contribute to neoplastic transformation or tumor progression. In human endometrium, we have recently demonstrated that neutral endopeptidase (NEP)/CD10 is
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highly expressed in stromal cells of the normal endometrium and grade 1 endometrial adenocarcinoma but its expression is decreased or lost with advancing tumor grade.14 In contrast to NEP expression in endometrial stroma, DPPIV is known to be expressed in endometrial glandular cells.8 However, DPPIV expression and function in endometrial adenocarcinoma have not yet been studied. The purpose of this study was to investigate DPPIV expression and localization by immunohistochemical analysis in normal endometrium and different grades of endometrial adenocarcinomas and also to determine whether its expression pattern is related to neoplastic transformation, differentiation, and disease progression. Furthermore, we also examined immunohistochemical expression of the chemokine RANTES, which is one of the DPPIV substrates, in the same tissue materials, and discuss the possible role of DPPIV in differentiation and progression of endometrial carcinoma through the regulation of this chemokine. Material and methods Cell line and cell culture. Endometrial endometrioid adenocarcinoma cell line HEC1A was purchased from the American Type Culture Collection (ATCC), and ISHIKAWA was provided by Dr Nishida (University of Tsukuba, Tsukuba, Japan). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St Louis, Mo) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 U/mL) at 37°C in a humidified 5% carbon dioxide atmosphere. Tissues. Formalin-fixed, paraffin-embedded tissues were obtained surgically from 41 patients who underwent hysterectomy at Nagoya University Hospital from August 1994 to December 2000. Normal endometrial tissues (n = 12) were obtained from premenopausal women who underwent hysterectomy for benign ovarian tumor or uterine myoma. Tumor tissues were obtained from 29 patients with endometrial endometrioid adenocarcinoma with different tumor grades. Tumor histologic features and grade were determined according to the criteria of the World Health Organization (grade 1: well differentiated; grade 2: moderately differentiated; grade 3: poorly differentiated). Clinical staging was determined according to the Federation of International Gynecology and Obstetrics (FIGO). Tumors consisted of 16 stage I, 5 stage II, and 8 stage III (10 grade 1, 9 grade 2, and 10 grade 3). Informed consent for the use of tissues was obtained from each patient. Flow cytometric analysis. Fluorescence-activated cell sorting (FACS) analysis was performed to quantify DPPIV expression on the endometrial adenocarcinoma cell surface. Cells were stained with phycoerythrin (PE)-conjugated anti-DPPIV monoclonal antibodies (mAb), MA-261 (Pharmingen, San Diego, Calif), and TS-145 (kindly pro-
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vided by Dr Ryuzo Ueda, Nagoya City University),15 or isotype-matched control mouse immunoglobulin G (IgG) (Coulter, Hialeah, Fla) for 1 hour at 4°C. After three washes, cells were resuspended in phosphatebuffered saline solution (PBS). FACS data were acquired on the FACS calibur system (Becton Dickinson, San Jose, Calif), and analyzed with CELLQuest software (Becton Dickinson). Each assay was performed three times. Western blotting. Tissues were homogenized for 10 minutes on ice in lysis buffer composed of PBS solution, 1% Triton X-100, and protease inhibitor cocktail tablet (Roche Diagnosis GmbH, Mannheim, Germany). Tissue extracts and cell lysates were cleared by centrifugation at 14,000g for 20 minutes at 4°C and stored at –80°C. Protein concentrations were determined with a protein assay kit (Bio-Rad Laboratories, Hercules, Calif). Extract (30 µg) in each lane was separated by SDS/7.5% polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and immunoblotted with mouse monoclonal anti-DPPIV antibody (TS-145, 1:40 dilution). The biotinylated secondary antibody (Vector Laboratories, Burlingame, Calif) was used at 1:200 dilution. Immunoreactive proteins were detected by ECL Western blotting detection systems (Amersham Biosciences, Tokyo, Japan). In the negative control, the primary antibody was replaced with normal mouse IgG. Immunohistochemistry. Immunohistochemical staining was performed with the avidin-biotin immunoperoxidase technique (Histofine SAB-PO kit, Nichirei, Tokyo, Japan). Sections were cut at a thickness of 4 µm and were deparaffinized and rehydrated. For heat-induced epitope retrieval, deparaffinized sections were treated three times for 5 minutes at 90°C at 750 W in a microwave oven. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide, and nonspecific immunoglobulin binding was blocked by incubation with 10% normal rabbit serum. Incubation with primary mAb for DPPIV (TS-145) at a dilution of 1:100 and for RANTES (PeproTech EC, London, UK) at a dilution of 1:400 was performed overnight at 4°C. After a washing with PBS solution, secondary biotinylated antibody was applied for 10 minutes. After washing, the streptavidinperoxidase conjugate was applied for 5 minutes. After washing, sections were incubated with 3-amino-9-ethylcarbazole (AEC, Nichirei, Japan) to develop a red reaction product and then counterstained with Mayer’s hematoxylin. For the negative control, mouse IgG was replaced with primary antibody. Sections from normal kidney proximal tubules were used as a positive control staining for DPPIV. Staining intensity was scored semiquantitatively on a four-titered scale (negative: –; weakly positive: +; moderately positive: ++; strongly positive staining: +++) relative to the known positive and negative controls. Each specimen was scored twice, independently by two investigators.
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Fig 1. Cell surface expression of DPPIV on endometrial adenocarcinoma cell line (HEC1A) by FACS. Cells were stained with two different PE-labeled DPPIV mouse mAb (shaded area) or PE-labeled isotype-matched mouse IgG (open area). A, Stained with MA-261. B, Stained with TS-145.
Fig 2. Immunoblot analysis of DPPIV using mouse anti-CD26 monoclonal antibody (TS-145). Protein (30 µg) extracted from normal endometrial tissue, endometrial adenocarcinoma tissue, and endometrial adenocarcinoma cell lines (ISHIKAWA and HEC1A) was separated on SDS/7.5% polyacrylamide gel and electroblotted onto nitrocellulose membranes. Lane 1, Grade 1 endometrial adenocarcinoma tissue; lane 2, grade 2; lane 3, grade 3; lane 4, proliferative phase normal endometrium; lane 5, secretory phase; lane 6, ISHIKAWA cells; lane 7, HEC1A cells.
Cell proliferation assay. Cells were cultured in 96-well microplates in serum-free medium for 4 hours. After incubation with various concentrations of RANTES for 24 hours, cell proliferation was evaluated by bromodeoxyuridine (BrdU) uptake assay by using a cell proliferation enzyme-linked immunosorbent assay (ELISA) system (Amersham Biosciences). Reverse transcriptase–polymerase chain reaction experiments. After informed consent was obtained from patients, messenger RNA (mRNA) was extracted from cell lines (ISHIKAWA and HEC1A) and endometrial adenocarcinoma tissues by using a Quick Prep mRNA Purification Kit (Amersham Biosciences). Reverse transcription–polymerase chain reaction (RT-PCR) was performed with an RNA PCR kit (Perkin-Elmer Corp, Norwalk, Conn) according to the manufacturer’s protocol. The sense and an-
tisense specific primers for human RANTES were 5´CTACTCGGGAGGCTAAGGCAGGAA-3´ and 5´GAGGGGTTGAGACGGCGGAAGC-3´, respectively. These primers yield a 318-bp fragment. Statistics. The nonparametric Kruskal-Wallis test was performed to compare the staining scores among all groups. In addition, the staining score were compared between normal endometrium and neoplastic tissues by using the Mann-Whitney test with Bonferroni correction. Analysis of variance was used to determine the significance of the effect of RANTES on endometrial adenocarcinoma cell proliferation. P < .05 was considered significant. Results To evaluate the immunoreactivity and specificity of the anti-DPPIV mAb, TS-145, that was used for subsequent Western blotting and immunohistochemical studies, FACS analysis was performed using the endometrial carcinoma cell line (HEC1A). As shown in Fig 1, strong DPPIV expression on HEC1A cells was detected by FACS analysis by using the TS-145 mAb. A similar expression level and specificity were also observed by the commercially available DPPIV-specific mAb (MA-261). Western blot analysis for DPPIV protein expression was performed with TS-145 (Fig 2). A single immunoreactive band was detected as approximately 110 kd in both the proliferative and secretory phases of normal endometrial tissues, although the DPPIV expression in the proliferative phase was lower than that in the secretory phase. A 110-kd band of immunoreactive DPPIV protein was also detected in each grade of endometrial adenocarcinoma tissue as well as in HEC1A.
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Fig 3. DPPIV immunostaining of normal premenopausal endometrium in different phases of menstrual cycle and endometrial adenocarcinoma specimens. (Original magnification, 200.) A, Normal premenopausal endometrium (proliferative phase). B, Normal premenopausal endometrium (secretory phase). C, Grade 1 adenocarcinoma tissue. D, Grade 1. E, Grade 2. F, Grade 3.
To evaluate the immunoreactivity of TS-145 mAb on paraffin-embedded tissue sections, we first immunostained the normal renal proximal tubular tissue as a positive control. Strong staining intensity was observed in the renal proximal tubular cells (data not shown). DPPIV immunoreactivity was only localized in endometrial glandular cells but not detected in stromal cells. DPPIV expression was weakly or moderately detected in the proliferative phase of normal endometrium (Fig 3, A), whereas in the secretory phase of normal endometrium its expression was strongly detected in glandular cells (Fig 3, B). These findings are consistent with those of the previous report.8
In endometrial adenocarcinoma, DPPIV was also localized in adenocarcinoma cells but not in stromal cells. In grade 1, DPPIV immunoreactivity was strongly or moderately detected (Fig 3, C and D). However, weak or no expression of DPPIV was found in grades 2 and 3 (Fig 3, E and F). There was an inverse correlation between DPPIV immunoreactivity and tumor grading, and comparison of the five groups showed a significant difference (P < .0005, Fig 4). However, there was no correlation with the clinical stage of endometrial adenocarcinoma (data not shown). The results of immunohistochemical staining for RANTES in normal endometrium and endometrial adenocarcinomas were summarized in the Table. RANTES
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Fig 4. Staining intensity score of DPPIV immunoreactivity in glandular cells of normal endometrium and grades 1, 2, and 3 endometrial adenocarcinoma. Score decreased with advancing tumor grade.
Table. Intensity of RANTES expression in sections of patients at normal and different grades of endometrial adenocarcinomas Intensity of RANTES expression Normal (n = 8) Grade 1 (n = 10) Grade 2 (n = 9) Grade 3 (n = 10)
–
+
++
+++
0 (0%) 0 (0%) 0 (0%) 0 (0%)
1 (13%) 2 (20%) 1 (12%) 3 (30%)
3 (37%) 3 (30%) 5 (55%) 5 (50%)
4 (50%) 5 (50%) 3 (33%) 2 (20%)
immunoreactivity was localized in the glandular cells of normal endometrium and was strongly or moderately detected in the secretory phase of normal endometrium (Fig 5, A). Most endometrial adenocarcinomas also highly or moderately expressed RANTES (Fig 5, B). Similar moderate expression of RANTES was found in breast carcinoma tissues as a positive control (data not shown). However, there was no significant correlation between RANTES expression and tumor grade. We studied the effect of RANTES on endometrial adenocarcinoma cell proliferation. Fig 6 shows that RANTES induced cell proliferation in both HEC1A and ISHIKAWA cells in a concentration-dependent manner. RT-PCR demonstrated that mRNA of RANTES was present in endometrial adenocarcinoma tissues as well as in ISHIKAWA and HEC1A cells as shown in Fig 6, C. Comment In this study, we have demonstrated the expression and localization of DPPIV in normal endometrium and endometrial adenocarcinoma by immunohistochemical analysis. DPPIV immunostaining was more strongly positive in the secretory phase of normal endometrium than in the proliferative phase. These findings were highly
consistent with the previous report,8 and it is suggested that DPPIV plays a role in the differentiation of endometrial glandular cells and also possibly in the maintenance of homeostasis of the normal endometrium. Strong or moderate DPPIV expression was still observed in most grade 1 adenocarcinomas, but weak or no expression was detected in grades 2 and 3. These findings suggest that down-regulation of DPPIV expression is related to neoplastic transformation and tumor progression. Similar findings were reported in melanoma.9,16 However, increased expression of DPPIV was reported in thyroid, skin, and prostate carcinoma.10,17,18 Our recent studies demonstrated that the expression of aminopeptidase A, one of the cell surface aminopeptidases, was upregulated in choriocarcinoma and invasive cervical carcinoma.19,20 Iwata and Morimoto21 suggested that upregulation or down-regulation of these ectopeptidases appear to be tissue specific, and even cell type specific, in a variety of malignancies in their review. This hypothesis was supported by Papandreou et al,22 who proposed two basic mechanisms of cell surface peptidase involvement in the malignant process. One is loss of function resulting in an inability of the tumor cells to inactivate a stimulatory peptide. The other is gain of function resulting in
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Fig 5. RANTES immunostaining in normal premenopausal endometrium and endometrial adenocarcinoma specimens. (Original magnification, 200.) A, Normal premenopausal endometrium. B, Grade 3 adenocarcinoma.
the activation processing of a stimulatory peptide or the inactivation of an inhibitory peptide. In this study, the intensity of DPPIV staining in endometrial adenocarcinoma was inversely correlated with the degree of tumor differentiation and was down-regulated with advancing tumor grade. Similarly, we recently reported that another cell-surface aminopeptidase, NEP/CD10, was expressed in normal endometrial stromal cells, and its stromal expression was down-regulated with increasing tumor grade in endometrial carcinoma, whereas stromal endothelin-1, one of the NEP substrates, was up-regulated.14 On the basis of these two findings, it is suggested that these two ectoenzymes are differentially expressed in endometrial glandular cells and stromal cells and may play a regulatory role not only in the maintenance of normal endometrial function but also in the neoplastic transformation and tumor progression through modulating their specific peptide substrates. Enzymologically, DPPIV is a serine-type protease that preferentially cleaves Xaa-Pro and Xaa-Ala dipeptides from the NH2 terminus of peptides and proteins. The optimal size and features of substrates for this enzyme have not been fully characterized. Potential DPPIV substrates
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Fig 6. After incubation with various concentrations of RANTES for 24 hours, cell proliferation was evaluated by BrdU uptake assay by using a cell proliferation ELISA system. A, HEC1A. B, ISHIKAWA. C, RT-PCR was performed by using primers of RANTES. Lanes 1 and 7, 100-bp ladder; lane 2, grade 1 endometrial adenocarcinoma tissue; lane 3, grade 2; lane 4, grade 3; lane 5, ISHIKAWA cells; lane 6, HEC1A cells.
include neuropeptides and some chemokine families such as RANTES.4 Among these various DPPIV substrates, several chemokines were shown to be involved in tumor cell proliferation and progression. Recently, Luboshits et al23 have reported that the expression of RANTES is directly correlated with the advance of breast carcinoma, indicating that RANTES may be involved in breast carcinoma progression. Our results showed that significant levels of RANTES expression was observed in both normal endometrial glandular cells and endometrial adenocarcinoma cells. Hornung et al24 previously showed RANTES expression in human endometrial and endometriosis tissues. To our knowledge, our current study is the first report showing the expression of RANTES in endometrial carcinomas. Furthermore, RANTES stimulated the proliferation of HEC1A endometrial carcinoma cells in vitro (Fig 6). Mrowietz et al25 also found that RANTES is secreted by human melanoma cells and is associated with enhanced tumor formation in vivo. It is still unclear whether these
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chemokines could be involved in the progression of endometrial carcinomas in vivo. However, one can speculate that the loss of DPPIV in advanced endometrial carcinoma may result in the inability to degrade peptide growth factors such as RANTES. These factors may be secreted from carcinoma or stromal cells as an autocrine/paracrine growth factor and may contribute to tumor progression. In conclusion, this report has demonstrated the expression and localization of DPPIV in normal endometrium and endometrial adenocarcinoma. Because DPPIV expression decreased with the advancing tumor grade, reducing this enzyme may have a beneficial effect on carcinoma progression caused by the loss of degrading activity of bioactive factors such as RANTES in endometrial adenocarcinoma. REFERENCES
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