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Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a cellsurface peptidase that cleaves N-terminal dipeptides frompolypeptides with a proline or alanine at the penultimateposition. This 110-kd type II membrane glycoprotein hasbeen identified as the cluster differentiation 26 (CD26)and has multiple functions, including serine protease ac-tivity,1 the ability to bind adenosine deaminase, which isassociated with T-cell activation and proliferation,2 andadhesion to extracellular matrix components such as fib-ronectin and collagens.3 Enzymologically, DPPIV cancleave specific peptide substrates such as substance P,

growth hormone–releasing factor, glucagon-like pep-tides, and some chemokines, including regulated on acti-vation, normal T-cell expressed and secreted (RANTES),4

and reduces cellular responses to these bioactive pep-tides. This ectoenzyme is widely distributed in activated Tcells, epithelial cells of the small intestine, liver, prostate,renal proximal tubules, and also in melanocytes.5 In ad-dition, DPPIV is expressed in the female reproductive or-gans such as the placenta,6 ovary,7 and endometrium.8 Inaddition to its expression in normal tissues, DPPIV ex-pression and its roles in human tumors have been re-ported not only in hematologic malignancies but also incertain solid tumors, including melanoma, thyroid carci-noma, 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 cellularsystems by modulating the activity of peptide factors.13

Thus, abnormalities in the expression pattern and/orcatalytic function of these peptidases result in alteredpeptide activation or inactivation. These cause disruptionof normal cellular homeostasis and may contribute toneoplastic transformation or tumor progression.

In human endometrium, we have recently demon-strated that neutral endopeptidase (NEP)/CD10 is

From the Departments of Obstetrics and Gynecology,a and PublicHealth,b Nagoya University Graduate School of Medicine, and Divisionof Pathology, Clinical Laboratory, Nagoya University Hospital.cThis work was supported in part by Grants-in-Aid from the Ministry of

Posts and Telecommunications for specific medical research (collabora-tion with Nagoya Teishin Hospital) and Education, and the fund ofShowakai and Ogya. Received for publication February 7, 2002; revised September 3, 2002;accepted November 5, 2002.Reprint requests: Dr Fumitaka. Kikkawa, Department of Obstetrics andGynecology, Nagoya University School of Medicine, Showa-ku, Tsuru-mai-cho 65, Nagoya 466-8550, Japan. E-mail: kikkawaf@med.nagoya-u.ac.jp© 2003, Mosby, Inc. All rights reserved.0002-9378/2003 $30.00 + 0doi:10.1067/mob.2003.169

Dipeptidyl peptidase IV expression in endometrial endometrioidadenocarcinoma 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 investi-gated the expression and localization of DPPIV in endometrial endometrioid adenocarcinomas of differentgrades.STUDY DESIGN: Immunohistochemical analysis was performed by using DPPIV and regulated on activa-tion, normal T-cell expressed and secreted (RANTES) specific monoclonal antibodies. Cell proliferation wasevaluated by bromodeoxyuridine (BrdU) uptake assay.RESULTS: Immunohistochemical analyses showed that DPPIV was strongly or moderately stained in glan-dular cells of the normal secretory phase. In endometrial adenocarcinoma, the DPPIV expression decreasedwith advancing grade (P < .01). Furthermore, RANTES, one of the possible DPPIV substrates, was highly ex-pressed in all grades of endometrial adenocarcinoma cells. The addition of RANTES to endometrial adeno-carcinoma cells increased proliferation in a concentration-dependent manner.CONCLUSION: DPPIV is expressed in normal endometrial glandular cells, but its expression in endometrialadenocarcinoma is down-regulated with increasing grade. Our data also suggest a regulatory role of this ec-toenzyme in neoplastic transformation and progression of endometrial adenocarcinomas possibly by degrad-ing certain bioactive peptides such as RANTES. (Am J Obstet Gynecol 2003;188:670-6.)

Key words: Dipeptidyl peptidase IV, RANTES, immunohistochemistry, endometrial endometrioidadenocarcinoma

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highly expressed in stromal cells of the normal en-dometrium and grade 1 endometrial adenocarcinomabut its expression is decreased or lost with advancingtumor grade.14 In contrast to NEP expression in en-dometrial stroma, DPPIV is known to be expressed in en-dometrial glandular cells.8 However, DPPIV expressionand function in endometrial adenocarcinoma have notyet been studied. The purpose of this study was to investi-gate DPPIV expression and localization by immunohisto-chemical analysis in normal endometrium and differentgrades of endometrial adenocarcinomas and also to de-termine whether its expression pattern is related to neo-plastic transformation, differentiation, and diseaseprogression. Furthermore, we also examined immuno-histochemical expression of the chemokine RANTES,which is one of the DPPIV substrates, in the same tissuematerials, and discuss the possible role of DPPIV in dif-ferentiation and progression of endometrial carcinomathrough the regulation of this chemokine.

Material and methods

Cell line and cell culture. Endometrial endometrioidadenocarcinoma cell line HEC1A was purchased fromthe American Type Culture Collection (ATCC), andISHIKAWA was provided by Dr Nishida (University ofTsukuba, Tsukuba, Japan). Both cell lines were culturedin Dulbecco’s modified Eagle’s medium (DMEM, Sigma,St Louis, Mo) supplemented with 10% heat-inactivatedfetal calf serum, penicillin (100 U/mL), and strepto-mycin (100 U/mL) at 37°C in a humidified 5% carbondioxide atmosphere.

Tissues. Formalin-fixed, paraffin-embedded tissueswere obtained surgically from 41 patients who underwenthysterectomy at Nagoya University Hospital from August1994 to December 2000. Normal endometrial tissues (n =12) were obtained from premenopausal women who un-derwent hysterectomy for benign ovarian tumor or uter-ine myoma. Tumor tissues were obtained from 29patients with endometrial endometrioid adenocarci-noma with different tumor grades. Tumor histologic fea-tures and grade were determined according to thecriteria of the World Health Organization (grade 1: welldifferentiated; grade 2: moderately differentiated; grade3: poorly differentiated). Clinical staging was determinedaccording to the Federation of International Gynecologyand Obstetrics (FIGO). Tumors consisted of 16 stage I, 5stage II, and 8 stage III (10 grade 1, 9 grade 2, and 10grade 3). Informed consent for the use of tissues was ob-tained from each patient.

Flow cytometric analysis. Fluorescence-activated cellsorting (FACS) analysis was performed to quantify DPPIVexpression on the endometrial adenocarcinoma cell sur-face. Cells were stained with phycoerythrin (PE)-conju-gated anti-DPPIV monoclonal antibodies (mAb), MA-261(Pharmingen, San Diego, Calif), and TS-145 (kindly pro-

vided by Dr Ryuzo Ueda, Nagoya City University),15 orisotype-matched control mouse immunoglobulin G(IgG) (Coulter, Hialeah, Fla) for 1 hour at 4°C. Afterthree washes, cells were resuspended in phosphate-buffered saline solution (PBS). FACS data were acquiredon the FACS calibur system (Becton Dickinson, San Jose,Calif), and analyzed with CELLQuest software (BectonDickinson). Each assay was performed three times.

Western blotting. Tissues were homogenized for 10minutes on ice in lysis buffer composed of PBS solution,1% Triton X-100, and protease inhibitor cocktail tablet(Roche Diagnosis GmbH, Mannheim, Germany). Tissueextracts and cell lysates were cleared by centrifugation at14,000g for 20 minutes at 4°C and stored at –80°C. Pro-tein concentrations were determined with a protein assaykit (Bio-Rad Laboratories, Hercules, Calif). Extract (30µg) in each lane was separated by SDS/7.5% polyacry-lamide gel electrophoresis, transferred onto nitrocellu-lose membranes, and immunoblotted with mousemonoclonal anti-DPPIV antibody (TS-145, 1:40 dilution).The biotinylated secondary antibody (Vector Laborato-ries, Burlingame, Calif) was used at 1:200 dilution.

Immunoreactive proteins were detected by ECL West-ern blotting detection systems (Amersham Biosciences,Tokyo, Japan). In the negative control, the primary anti-body was replaced with normal mouse IgG.

Immunohistochemistry. Immunohistochemical stain-ing was performed with the avidin-biotin immunoperoxi-dase technique (Histofine SAB-PO kit, Nichirei, Tokyo,Japan). Sections were cut at a thickness of 4 µm and weredeparaffinized and rehydrated. For heat-induced epitoperetrieval, deparaffinized sections were treated three timesfor 5 minutes at 90°C at 750 W in a microwave oven. En-dogenous peroxidase activity was blocked by incubationwith 3% hydrogen peroxide, and nonspecific im-munoglobulin binding was blocked by incubation with10% normal rabbit serum. Incubation with primary mAbfor DPPIV (TS-145) at a dilution of 1:100 and forRANTES (PeproTech EC, London, UK) at a dilution of1:400 was performed overnight at 4°C. After a washingwith PBS solution, secondary biotinylated antibody wasapplied for 10 minutes. After washing, the streptavidin-peroxidase conjugate was applied for 5 minutes. Afterwashing, sections were incubated with 3-amino-9-ethyl-carbazole (AEC, Nichirei, Japan) to develop a red reac-tion product and then counterstained with Mayer’shematoxylin. For the negative control, mouse IgG was re-placed with primary antibody. Sections from normal kid-ney proximal tubules were used as a positive controlstaining for DPPIV. Staining intensity was scored semi-quantitatively on a four-titered scale (negative: –; weaklypositive: +; moderately positive: ++; strongly positive stain-ing: +++) relative to the known positive and negative con-trols. Each specimen was scored twice, independently bytwo investigators.

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Cell proliferation assay. Cells were cultured in 96-wellmicroplates in serum-free medium for 4 hours. After in-cubation with various concentrations of RANTES for 24hours, cell proliferation was evaluated by bromod-eoxyuridine (BrdU) uptake assay by using a cell prolifer-ation enzyme-linked immunosorbent assay (ELISA)system (Amersham Biosciences).

Reverse transcriptase–polymerase chain reaction experi-ments. After informed consent was obtained from patients,messenger RNA (mRNA) was extracted from cell lines(ISHIKAWA and HEC1A) and endometrial adenocarci-noma tissues by using a Quick Prep mRNA Purification Kit(Amersham Biosciences). Reverse transcription–poly-merase chain reaction (RT-PCR) was performed with anRNA PCR kit (Perkin-Elmer Corp, Norwalk, Conn) ac-cording to the manufacturer’s protocol. The sense and an-

tisense specific primers for human RANTES were 5´-CTACTCGGGAGGCTAAGGCAGGAA-3´ and 5´-GAGGGGTTGAGACGGCGGAAGC-3´, respectively. Theseprimers yield a 318-bp fragment.

Statistics. The nonparametric Kruskal-Wallis test wasperformed to compare the staining scores among allgroups. In addition, the staining score were compared be-tween normal endometrium and neoplastic tissues byusing the Mann-Whitney test with Bonferroni correction.Analysis of variance was used to determine the signifi-cance of the effect of RANTES on endometrial adenocar-cinoma cell proliferation. P < .05 was consideredsignificant.

Results

To evaluate the immunoreactivity and specificity of theanti-DPPIV mAb, TS-145, that was used for subsequentWestern blotting and immunohistochemical studies,FACS analysis was performed using the endometrial car-cinoma cell line (HEC1A). As shown in Fig 1, strongDPPIV expression on HEC1A cells was detected by FACSanalysis by using the TS-145 mAb. A similar expressionlevel and specificity were also observed by the commer-cially available DPPIV-specific mAb (MA-261).

Western blot analysis for DPPIV protein expression wasperformed with TS-145 (Fig 2). A single immunoreactiveband was detected as approximately 110 kd in both theproliferative and secretory phases of normal endometrialtissues, although the DPPIV expression in the prolifera-tive phase was lower than that in the secretory phase. A110-kd band of immunoreactive DPPIV protein was alsodetected in each grade of endometrial adenocarcinomatissue as well as in HEC1A.

Fig 1. Cell surface expression of DPPIV on endometrial adenocarcinoma cell line (HEC1A) by FACS. Cells werestained with two different PE-labeled DPPIV mouse mAb (shaded area) or PE-labeled isotype-matched mouse IgG (openarea). A, Stained with MA-261. B, Stained with TS-145.

Fig 2. Immunoblot analysis of DPPIV using mouse anti-CD26monoclonal antibody (TS-145). Protein (30 µg) extracted fromnormal endometrial tissue, endometrial adenocarcinoma tissue,and endometrial adenocarcinoma cell lines (ISHIKAWA andHEC1A) was separated on SDS/7.5% polyacrylamide gel andelectroblotted onto nitrocellulose membranes. Lane 1, Grade 1endometrial 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.

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To evaluate the immunoreactivity of TS-145 mAb onparaffin-embedded tissue sections, we first immunos-tained the normal renal proximal tubular tissue as a posi-tive control. Strong staining intensity was observed in therenal proximal tubular cells (data not shown). DPPIV im-munoreactivity was only localized in endometrial glandu-lar cells but not detected in stromal cells. DPPIVexpression was weakly or moderately detected in the pro-liferative phase of normal endometrium (Fig 3, A),whereas in the secretory phase of normal endometriumits expression was strongly detected in glandular cells (Fig3, B). These findings are consistent with those of the pre-vious report.8

In endometrial adenocarcinoma, DPPIV was also local-ized in adenocarcinoma cells but not in stromal cells. Ingrade 1, DPPIV immunoreactivity was strongly or moder-ately detected (Fig 3, C and D). However, weak or no ex-pression of DPPIV was found in grades 2 and 3 (Fig 3, Eand F). There was an inverse correlation between DPPIVimmunoreactivity and tumor grading, and comparison ofthe five groups showed a significant difference (P < .0005,Fig 4). However, there was no correlation with the clinicalstage of endometrial adenocarcinoma (data not shown).

The results of immunohistochemical staining forRANTES in normal endometrium and endometrial ade-nocarcinomas were summarized in the Table. RANTES

Fig 3. DPPIV immunostaining of normal premenopausal endometrium in different phases of menstrual cycle and en-dometrial adenocarcinoma specimens. (Original magnification, �200.) A, Normal premenopausal endometrium (pro-liferative phase). B, Normal premenopausal endometrium (secretory phase). C, Grade 1 adenocarcinoma tissue. D,Grade 1. E, Grade 2. F, Grade 3.

674 Khin et al March 2003Am J Obstet Gynecol

immunoreactivity was localized in the glandular cells ofnormal endometrium and was strongly or moderately de-tected in the secretory phase of normal endometrium(Fig 5, A). Most endometrial adenocarcinomas alsohighly or moderately expressed RANTES (Fig 5, B). Simi-lar moderate expression of RANTES was found in breastcarcinoma tissues as a positive control (data not shown).However, there was no significant correlation betweenRANTES expression and tumor grade.

We studied the effect of RANTES on endometrial ade-nocarcinoma cell proliferation. Fig 6 shows that RANTESinduced cell proliferation in both HEC1A andISHIKAWA cells in a concentration-dependent manner.RT-PCR demonstrated that mRNA of RANTES was pre-sent in endometrial adenocarcinoma tissues as well as inISHIKAWA and HEC1A cells as shown in Fig 6, C.

Comment

In this study, we have demonstrated the expression andlocalization of DPPIV in normal endometrium and en-dometrial adenocarcinoma by immunohistochemicalanalysis. DPPIV immunostaining was more strongly posi-tive in the secretory phase of normal endometrium thanin the proliferative phase. These findings were highly

consistent with the previous report,8 and it is suggestedthat DPPIV plays a role in the differentiation of endome-trial glandular cells and also possibly in the maintenanceof homeostasis of the normal endometrium.

Strong or moderate DPPIV expression was still ob-served in most grade 1 adenocarcinomas, but weak or noexpression was detected in grades 2 and 3. These findingssuggest that down-regulation of DPPIV expression is re-lated to neoplastic transformation and tumor progres-sion. Similar findings were reported in melanoma.9,16

However, increased expression of DPPIV was reported inthyroid, skin, and prostate carcinoma.10,17,18 Our recentstudies demonstrated that the expression of aminopepti-dase A, one of the cell surface aminopeptidases, was up-regulated in choriocarcinoma and invasive cervicalcarcinoma.19,20 Iwata and Morimoto21 suggested that up-regulation or down-regulation of these ectopeptidases ap-pear to be tissue specific, and even cell type specific, in avariety of malignancies in their review. This hypothesiswas supported by Papandreou et al,22 who proposed twobasic mechanisms of cell surface peptidase involvementin the malignant process. One is loss of function resultingin an inability of the tumor cells to inactivate a stimula-tory peptide. The other is gain of function resulting in

Fig 4. Staining intensity score of DPPIV immunoreactivity in glandular cells of normal endometrium and grades 1, 2, and3 endometrial adenocarcinoma. Score decreased with advancing tumor grade.

Table. Intensity of RANTES expression in sections of patients at normal and different grades of endometrial adenocar-cinomas

Intensity of RANTES expression – + + + + + +

Normal (n = 8) 0 (0%) 1 (13%) 3 (37%) 4 (50%)Grade 1 (n = 10) 0 (0%) 2 (20%) 3 (30%) 5 (50%)Grade 2 (n = 9) 0 (0%) 1 (12%) 5 (55%) 3 (33%)Grade 3 (n = 10) 0 (0%) 3 (30%) 5 (50%) 2 (20%)

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the activation processing of a stimulatory peptide or theinactivation of an inhibitory peptide.

In this study, the intensity of DPPIV staining in en-dometrial adenocarcinoma was inversely correlated withthe degree of tumor differentiation and was down-regu-lated with advancing tumor grade. Similarly, we recentlyreported that another cell-surface aminopeptidase,NEP/CD10, was expressed in normal endometrial stro-mal cells, and its stromal expression was down-regulatedwith 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, itis suggested that these two ectoenzymes are differentiallyexpressed in endometrial glandular cells and stromalcells and may play a regulatory role not only in the main-tenance of normal endometrial function but also in theneoplastic transformation and tumor progressionthrough modulating their specific peptide substrates.

Enzymologically, DPPIV is a serine-type protease thatpreferentially cleaves Xaa-Pro and Xaa-Ala dipeptidesfrom the NH2 terminus of peptides and proteins. The op-timal size and features of substrates for this enzyme havenot been fully characterized. Potential DPPIV substrates

include neuropeptides and some chemokine familiessuch as RANTES.4 Among these various DPPIV sub-strates, several chemokines were shown to be involved intumor cell proliferation and progression. Recently, Lu-boshits et al23 have reported that the expression ofRANTES is directly correlated with the advance of breastcarcinoma, indicating that RANTES may be involved inbreast carcinoma progression. Our results showed thatsignificant levels of RANTES expression was observed inboth normal endometrial glandular cells and endome-trial adenocarcinoma cells. Hornung et al24 previouslyshowed RANTES expression in human endometrial andendometriosis tissues. To our knowledge, our currentstudy is the first report showing the expression ofRANTES in endometrial carcinomas. Furthermore,RANTES stimulated the proliferation of HEC1A en-dometrial carcinoma cells in vitro (Fig 6). Mrowietz etal25 also found that RANTES is secreted by humanmelanoma cells and is associated with enhanced tumorformation in vivo. It is still unclear whether these

Fig 5. RANTES immunostaining in normal premenopausal en-dometrium and endometrial adenocarcinoma specimens. (Orig-inal magnification, �200.) A, Normal premenopausalendometrium. B, Grade 3 adenocarcinoma.

Fig 6. After incubation with various concentrations of RANTESfor 24 hours, cell proliferation was evaluated by BrdU uptakeassay by using a cell proliferation ELISA system. A, HEC1A. B,ISHIKAWA. C, RT-PCR was performed by using primers ofRANTES. Lanes 1 and 7, 100-bp ladder; lane 2, grade 1 endome-trial adenocarcinoma tissue; lane 3, grade 2; lane 4, grade 3; lane5, ISHIKAWA cells; lane 6, HEC1A cells.

676 Khin et al March 2003Am J Obstet Gynecol

chemokines could be involved in the progression of en-dometrial carcinomas in vivo. However, one can specu-late that the loss of DPPIV in advanced endometrialcarcinoma may result in the inability to degrade peptidegrowth factors such as RANTES. These factors may be se-creted from carcinoma or stromal cells as an au-tocrine/paracrine growth factor and may contribute totumor progression.

In conclusion, this report has demonstrated the ex-pression and localization of DPPIV in normal en-dometrium and endometrial adenocarcinoma. BecauseDPPIV expression decreased with the advancing tumorgrade, reducing this enzyme may have a beneficial effecton carcinoma progression caused by the loss of degrad-ing activity of bioactive factors such as RANTES in en-dometrial adenocarcinoma.

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