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[Cancer Research 65, 6950-6956, August 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

CD26 Regulates p38 Mitogen-Activated Protein Kinase–Dependent Phosphorylation of Integrin ß1, Adhesion to Extracellular Matrix, and Tumorigenicity of T-Anaplastic Large Cell Lymphoma Karpas 299

Tsutomu Sato1, Tadanori Yamochi1, Toshiko Yamochi1, Ugur Aytac1, Kei Ohnuma2, Kathryn S. McKee1, Chikao Morimoto2 and Nam H. Dang1

1 Department of Lymphoma/Myeloma, University of Texas M.D. Anderson Cancer Center, Houston, Texas and 2 Department of Clinical Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Requests for reprints: Nam H. Dang, Department of Hematologic Oncology, Nevada Cancer Institute, 10000 West Charleston Boulevard, Suite 260, Las Vegas, NV 89135. Phone: 702-821-0000; Fax: 702-821-0021; E-mail: ndang{at}nvcancer.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD26 is an antigen with key role in T-cell biology and is expressed on selected subsets of aggressive T-cell malignancies. To elucidate the role of CD26 in tumor behavior, we examine the effect of CD26 depletion by small interfering RNA transfection of T-anaplastic large cell lymphoma Karpas 299. We show that the resultant CD26-depleted clones lose the ability to adhere to fibronectin and collagen I. Because anti–integrin ß1 blocking antibodies also prevent binding of Karpas 299 to fibronectin and collagen I, we then evaluate the CD26-integrin ß1 association. CD26 depletion does not decrease integrin ß1 expression but leads to dephosphorylation of both integrin ß1 and p38 mitogen-activated protein kinase (MAPK). Moreover, our data showing that the p38MAPK inhibitor SB203580 dephosphorylates integrin ß1 and that binding of the anti-CD26 antibody 202.36 dephosphorylates both p38MAPK and integrin ß1 on Karpas 299, leading to loss of cell adhesion to the extracellular matrix, indicate that CD26 mediates cell adhesion through p38MAPK-dependent phosphorylation of integrin ß1. Finally, in vivo experiments show that depletion of CD26 is associated with loss of tumorigenicity and greater survival. Our findings hence suggest that CD26 plays an important role in tumor development and may be a novel therapeutic target for selected neoplasms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD26/dipeptidyl peptidase IV is a 110-kDa cell surface glycoprotein that belongs to the serine protease family and is expressed on a variety of tissues, including T lymphocytes, endothelial cells, and epithelial cells. It is composed of a short cytoplasmic domain, a transmembrane region, and an extracellular domain with dipeptidyl peptidase IV activity, which selectively removes the NH2-terminal dipeptide from polypeptides containing either a proline or an alanine at the penultimate residue. This enzymatic activity seems to regulate the effect of several crucial cytokines and chemokines (1). Work over the past decade has shown CD26 to have an important role in T-cell biology both as a marker of T-cell activation and as a structure associated with key molecules and signaling pathways (2–5).

Although the role of CD26 in the regulation of normal T-cell physiology has been well characterized, its involvement in tumor biology is still unclear, although early data suggested that it may have a role in the development of selected neoplasms. Studies of patient samples showed that CD26 is highly expressed on lung adenocarcinoma, thyroid carcinoma, and B-cell chronic lymphocytic leukemia (6–8). Meanwhile, CD26 is a marker of aggressive disease for selected subsets of T-cell non-Hodgkin's lymphomas/leukemias. Carbone et al. showed that CD26 expression is restricted to such aggressive types of T-cell malignancies as T-lymphoblastic lymphomas (T-LBL), T-acute lymphoblastic leukemias (T-ALL), and T-anaplastic large cell lymphomas (T-ALCL). Furthermore, multivariate analysis indicated that the expression of CD26 on T-LBL/T-ALL tumor cells is associated with a worse outcome compared with CD26-negative T-LBL/T-ALL tumors (9). Similarly, we showed recently that CD26 expression is a marker of poor prognosis for T-large granular lymphocyte lymphoproliferative disorder (10). Although these findings suggested that CD26 may regulate the malignant behavior of selected tumors, the exact mechanisms involved in the role of CD26 in tumor biology remain to be elucidated.

In this article, we evaluate the role of CD26 in tumor biology by depleting the expression of CD26 on the T-ALCL cell line Karpas 299 with the small interfering RNA (siRNA) technique. We show that CD26 mediates cell adhesion to the extracellular matrix (ECM) proteins fibronectin and collagen I through p38 mitogen-activated protein kinase (MAPK)–dependent phosphorylation of integrin ß1. We also show that CD26 expression regulates topoisomerase II{alpha} level and tumor sensitivity to the topoisomerase II inhibitor doxorubicin. Importantly, loss of CD26 expression on Karpas 299 cells results in decreased tumorigenicity and improved survival in a severe combined immunodeficient (SCID) mouse animal model, hence suggesting that targeting CD26 may be an effective therapeutic approach for selected CD26-positive T-cell malignancies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. All culture plates and dishes were purchased from Falcon Plastics (Oxnard, CA). Mouse anti–integrin ß1 antibody (P4C10) was from Chemicon (Temecula, CA). Mouse anti–integrin ß1 antibody (4B4) and isotypic control mouse IgG were from Beckman Coulter (Miami, FL). Mouse anti-CD26 antibody (202.36) was from Santa Cruz (Santa Cruz, CA). Mouse anti-CD26 antibodies (1F7 and 5F8) were prepared as described previously (11). Specific inhibitors against p38MAPK SB203580 was from Biomol (Plymouth, Meeting, PA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma (St. Louis, MO). Doxorubicin was from Calbiochem (La Jolla, CA).

Cell culture. The human T-ALCL cell line Karpas 299 was supplied by American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Sigma) with 10% heat-inactivated FCS (Sigma) and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin) at 37°C in a humidified atmosphere containing 5% CO2.

Depletion of CD26. The expression of CD26 was suppressed by Knockout RNAi System (Clontech, Palo Alto, CA) as described previously (12). Briefly, the target sequence ATCATGCATGCAATCAAC, which corresponds to the nucleotide sequence from 1,768 to 1,785 of CD26 cDNA (accession no. NM_001935), was first determined using siDESIGN program at Dharmacon siDESIGN Center (http://design.dharmacon.com/). Complementary oligonucleotides encoding siRNA were then designed and ligated into vector RNAi-Ready pSIREN-RetroQ (Clontech) according to the manufacturer's instructions. Oligonucleotides encoding missense siRNA, in which the target sequence was replaced with the missense sequence ATCTTGCAAGCAAACAAC, were also ligated into the vector as controls. For the retroviral packaging, these constructs were cotransfected with p10A1 (Clontech) into GP2-293 cells (Clontech) using LipofectAMINE reagent (Invitrogen, Carlsbad, CA). The supernatants containing retrovirus were collected 72 hours after the transfection. Then, this supernatant was added to culture medium of Karpas 299 cells, which was supplemented with 8 µg/mL polybrene (Sigma). After 3 days of incubation, the cells were cultured with 0.4 µg/mL puromycin (Clontech) to eliminate nontransfected cells. Isolated clones were obtained by the standard limiting dilution method.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideassay. The cells were cultured with 100 or 300 µL medium in each well of a 96- or 24-well plate, respectively. To quantify the total number of cells, a quarter volume of the MTT solution, PBS containing 5 mg/mL MTT, was added to the culture medium. To quantify the number of adhesive cells, the culture medium, in which nonadhesive cells were floating after the plates were shaken orbitally several times, was discarded. Then, the same volume of fresh medium with a quarter volume of MTT solution was added to the remaining adhesive cells. In both quantifications, the cells were cultured for 2 hours and added with the same volume of lysis buffer as described previously (13). After overnight incubation at 37°C, the absorbance of dissolved blue formazan was measured at 570 nm in spectrophotometer µQuant (Bio-Tek, Winooski, VT) with KC junior software.

Flow cytometry. The cells were collected, washed twice with PBS, and resuspended in 0.5 mL fluorescence-activated cell sorting buffer, PBS containing 0.5 mmol/L EDTA and 1% (w/v) bovine serum albumin (Sigma). Then, the cells were incubated on ice for 30 minutes with anti-CD26 (Caltag, Burlingame, CA) or anti–integrin ß1 (4B4) mouse monoclonal antibody. Isotypic mouse IgG was used as a control. Surface antigens detected by these antibodies were visualized with FITC-conjugated anti-mouse IgG antibody (Pierce, Rockford, IL), followed by the use of FACSCalibur (Becton Dickinson, San Jose, CA) with CellQuest software.

Western blotting. Cells (1 x 107) were collected, washed twice with PBS, and resuspended in 0.3 mL lysis buffer [1% SDS, 10 mmol/L Tris-HCl (pH 7.4), 10 µg/mL leupeptin, 10 µg/mL aprotinin, 2 mmol/L phenylmethylsulfonyl fluoride] and then boiled for 5 minutes. After passage through a 20-gauge needle 10 times and centrifugation at 15,000 rpm for 20 minutes, the aliquot was boiled again with a standard 4x SDS loading buffer containing 15% (v/v) 2-mercaptoethanol for 5 minutes; 10 µL of which were then subjected to SDS-polyacrylamide gel for electrophoresis followed by the transfer to Immobilon membrane (Millipore, Bedford, MA). The membrane was hybridized with goat anti–CD26/dipeptidyl peptidase IV (R&D, Minneapolis, MN), mouse anti–topoisomerase II{alpha} (Ki-S1, Chemicon), mouse anti–phosphotopoisomerase II{alpha} Thr1342 (3D4, MBL, Nagoya Japan), goat anti–integrin ß1 L-16 (Santa Cruz), rabbit anti–integrin ß1 pSer785 (Biosource, Camarillo, CA), rabbit anti-p38MAPK (Cell Signaling, Beverly, MA), or rabbit anti–phospho-p38MAPK Thr180/Tyr182 (Cell Signaling) antibodies. Proteins detected by these antibodies were visualized with horseradish peroxidase–conjugated anti-mouse (DAKOCytomation, Kyoto, Japan), rabbit (Bio-Rad, Hercules, CA), or goat (DAKOCytomation) antibody followed by the use of SuperSignal West Pico Stable Peroxidase Solution (Pierce).

In vivo experiments. Three-week-old female CB-17 SCID mice were purchased from Taconic Farms (Germantown, NY). The mice were kept in laminar flow rooms at constant temperature and humidity. They had free access to food and water. Experimental protocols were approved by the Institutional Ethics Committee for Animal Experimentation. Briefly, every mouse has received i.p. injection of 0.2 mL rabbit anti–asialo-GM1 antisera (Wako Pure Chemical, Osaka, Japan) on day –1. The mice were subsequently divided into three groups, each of which included five animals. Every mouse in each group was then injected i.p. with 0.3 x 106 cells of mock or CD26-depleted clones (dep-1 and dep-2) on day 0. The growth of the palpable tumor in the inguinal regions was followed by measurements with a caliper and its volume was calculated according to the following formula: MD x TL2 x 1/2, where MD and TL are the maximum diameter and transverse length, respectively. The mice were sacrificed before the volume of the tumor mass reached 3,000 mm3 for ethical reason.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transfection of small interfering RNA against CD26 depletes its expression on Karpas 299 cells. Because CD26 surface expression is associated with aggressive disease in certain types of T-cell malignancies, including T-ALCL (9, 14), we evaluated the role of CD26 in the T-ALCL cell line Karpas 299, which expresses high level of CD26. To deplete CD26 expression, Karpas 299 cells (parent) were retrovirally transfected with siRNA against CD26. Two permanent transfectants were subsequently obtained and designated as CD26-depleted clone 1 (dep-1) and clone 2 (dep-2). The cells transfected with missense siRNA were used as controls (mock). Flow cytometry studies (Fig. 1A) and Western blotting analyses (Fig. 1B) indicate that CD26 expression is almost completely abrogated on CD26-depleted clones.



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  		Figure 1. Effect of CD26 depletion on topoisomerase II{alpha} expression and sensitivity to doxorubicin. Karpas 299 cells (parent) were retrovirally transfected with siRNA against CD26. The clones selected by the standard limiting dilution method were designated CD26-depleted clone 1 (dep-1) and clone 2 (dep-2). The control was a clone transfected with missense siRNA (mock). The expression of CD26 was examined by flow cytometry (A) and Western blotting (B). A,solid lines or broken lines, cells treated with anti-CD26 antibody or isotype control IgG, respectively. B, equal amount of proteins was loaded in each lane, with ß-actin as control. C, Karpas 299 cells (parent, {circ}), mock ({square}), and CD26-depleted clones (dep-1, {blacktriangleup}; dep-2, {blacklozenge}) were plated onto 96-well culture plate (2 x 104 cells per well) and cultured with the indicated concentrations of doxorubicin for 3 days. Following the incubation period, the number of total cell was quantified by MTT assay as described in Materials and Methods. Points, mean of five separate experiments (n = 5); bars, SD. D, topoisomerase II{alpha} (Topo) and phosphorylated topoisomerase II{alpha} at Thr1342 (p-Topo) of Karpas 299 cells (parent), mock, and CD26-depleted clones (dep-1 and dep-2) were evaluated by Western blotting as described in Materials and Methods, with each lane being loaded with equal amount of proteins and with ß-actin as control.

 
CD26 depletion is associated with decreased topoisomerase II{alpha} level and decreased sensitivity to doxorubicin. We showed previously that transfectional overexpression of CD26 results in the up-regulation of topoisomerase II{alpha} expression, associated with enhanced sensitivity to the topoisomerase II inhibitor doxorubicin (12, 15–17). Consistent with our earlier work, our present studies clearly show that CD26 depletion results in decreased doxorubicin sensitivity (Fig. 1C). Furthermore, we found that the expression of topoisomerase II{alpha} as well as its phosphorylated active form (18) is down-regulated in CD26-depleted clones (Fig. 1D). In contrast, the expression of other structures associated with doxorubicin sensitivity, such as lung resistance protein, multidrug resistance protein-1, and P-glycoprotein, is not influenced by CD26 depletion (data not shown).

CD26 regulates cell adhesion to extracellular matrix through phosphorylation of integrin ß1. CD26 has been described previously to play a role in cell adhesion to the ECM under certain experimental conditions (1, 19). Evaluating the potential effect of CD26 on tumor binding to the ECM, we found that parental Karpas 299 cells and mock clone display greater adhesion to the ECM proteins fibronectin and type I collagen I than CD26-depleted clones (Fig. 2A). Because integrins have a well-established role in cell adhesion to ECM proteins, we evaluated the potential association between CD26 and integrin ß1, which is involved in cell adhesion to both fibronectin and collagen I (20). We found that anti–integrin ß1 antibodies significantly block the adhesion of Karpas 299 cells to both fibronectin and collagen I as shown in Fig. 2B. Examining further the association between CD26 expression and integrin ß1–dependent cell adhesion, we found no difference in the expression level of integrin ß1 among parental Karpas 299 cells, mock cells, and CD26-depleted clones by flow cytometry (data not shown) and Western blotting (Fig. 2C). We next focused on the level of phosphorylated integrin ß1 at residue Ser785, which is necessary for integrin ß1 to function as an adhesion molecule (21). Our findings showed that there is lower level of phosphorylation at integrin ß1 Ser785 in CD26-depleted clones than in parental Karpas 299 and mock cells (Fig. 2C).



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  		Figure 2. Effect of CD26 depletion on cell adhesion to ECM and integrin ß1 phosphorylation. A, Karpas 299 cells (parent; open columns), mock (shaded columns), and CD26-depleted clones (dep-1 and dep-2; closed columns) were plated onto 60 mm dishes (3 x 106 cells per dish) coated with fibronectin (FN), collagen I (CL), or laminin (LN) and cultured for 3 hours. Then, nonadhesive and adhesive cells were separately collected and counted using a hemocytometer. Adhesive cells (%): adhesive cells / adhesive cells + nonadhesive cells. Columns, mean of five separate experiments (n = 5); bars, SD. B, Karpas 299 cells were preincubated with 0 (open columns), 0.4 (shaded columns), or 2 (hatched columns) µg/mL blocking antibodies against integrin ß1 (P4C10 or 4B4) for 30 minutes at room temperature. The cells were then plated onto 24-well plates (3 x 105 cells per well) coated with fibronectin or collagen I and placed at room temperature for 1 hour. Following washing of plates to discard nonadhesive cells, the number of adhesive cells remaining was then quantified by MTT assay as described in Materials and Methods. Cells preincubated with isotype control IgG were used as control. Columns, mean of five separate experiments (n = 5); bars, SD. C, expression of integrin ß1 (ß1) and phosphorylated integrin ß1 at Ser785 (p-ß1) on Karpas 299 cells (parent), mock, and CD26-depleted clones (dep-1 and dep-2) was examined by Western blotting as described in Materials and Methods, with equal amount of proteins being loaded in each lane and with ß-actin as control.

 
p38MAPK intermediates between CD26 and integrin ß1. We reported recently an association between CD26 and p38MAPK, with overexpression of CD26 resulting in increased p38 phosphorylation at Thr180/Tyr182 and depletion of CD26 level leading to decreased p38 phosphorylation at the same residues (12). We therefore examined the possible relationship between p38MAPK and integrin ß1 using the p38MAPK-specific inhibitor SB203580. Although incubation of Karpas 299 cells with SB203580 for 6 hours up to a concentration of 40 µmol/L (Fig. 3A) does not lead to decreased cell viability, there is a dose-dependent inhibition of cell adhesion (Fig. 3B). In addition, treatment with SB203580 is accompanied by a gradual dephosphorylation of integrin ß1 at Ser785 as well as p38MAPK at Thr180/Tyr182 (Fig. 3C).



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  		Figure 3. Effect of the p38MAPK inhibitor SB203580 on integrin ß1 phosphorylation. Karpas 299 cells were initially cultured in 24-well plates (3 x 105 cells per well; A and B) or 10 cm dish (1 x 107 cells per dish; C) coated with fibronectin for 6 hours. The cells were then incubated with the indicated concentrations of the p38MAPK inhibitor SB203580 dissolved in DMSO. A final DMSO concentration of 0.4% (v/v) was achieved equally in all samples. A, following incubation with SB203580 at 6 ({blacksquare}) or 12 ({bullet}) hours, MTT assays were then done as described in Materials and Methods to determine cell viability. Points, mean of five separate experiments (n = 5); bars, SD. B, following incubation with SB203580 at 6 hours, plates were washed to discard nonadhesive cells and the number of adhesive cells remaining was quantified by MTT assay as described in Materials and Methods. Columns, mean of five separate experiments (n = 5); bars, SD. C, following incubation with SB203580 at 6 hours, levels of p38MAPK (p38), phosphorylated p38MAPK at Thr180/Tyr182 (p-p38), integrin ß1, and phosphorylated integrin ß1 at Ser785 were evaluated by Western blotting as described in Materials and Methods, with each lane being loaded with equal amount of proteins and with ß-actin as control.

 
Anti-CD26 antibody 202.36 inhibits cell adhesion with dephosphorylation of p38MAPK and integrin ß1. To further delineate CD26 association with p38MAPK and integrin ß1, we evaluated the effect of anti-CD26 binding on Karpas 299 cell adhesion to the ECM and p38MAPK and integrin ß1 phosphorylation. We found that the anti-CD26 antibody 202.36 suppresses adhesion of Karpas 299 cells, whereas antibodies recognizing other CD26 epitopes display no inhibitory effect on cell adhesion (Fig. 4A). Of note is the fact that 202.36 does not affect cell viability at the dose and time tested (Fig. 4B). Furthermore, the inhibitory effect is in a dose-dependent manner (Fig. 4C). Importantly, we observed decreased phosphorylation of p38MAPK and integrin ß1 at the Thr180/Tyr182 and Ser785 residues, respectively, following treatment with the anti-CD26 antibody 202.36 (Fig. 5).



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  		Figure 4. Effect of the anti-CD26 antibody 202.36 on adhesion of Karpas 299 cells to ECM. A, Karpas 299 cells were plated onto 24-well plates (3 x 105 cells per well) coated with fibronectin. Shortly thereafter, each antibody (10 µg/mL) was added and the cells were incubated for the indicated times. Open, shaded, hatched, or closed columns, isotype control IgG or anti-CD26 antibody clone 1F7, 5F8, or 202.36, respectively. After the incubation period, plates were washed to discard nonadhesive cells and the number of adhesive cells remaining was then quantified by MTT assay as described in Materials and Methods. B, cells were treated with 10 µg/mL IgG or 202.36 and incubated for the indicated times. After the incubation period, MTT assays were then done as described in Materials and Methods to determine cell viability. C, cells were treated with the indicated concentrations of IgG or 202.36 and incubated for 60 minutes. After the incubation time, plates were washed to discard nonadhesive cells and the number of adhesive cells remaining was then quantified by MTT assay as described in Materials and Methods. Columns, mean of five separate experiments (n = 5); bars, SD (A-C).

 


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  		Figure 5. Effect of the anti-CD26 antibody 202.36 on phosphorylation of p38MAPK and integrin ß1. Karpas 299 cells were cultured with 10 µg/mL anti-CD26 antibody 202.36 or isotype control IgG for the indicated times. Levels of p38MAPK, phosphorylated p38MAPK at Thr180/Tyr182, integrin ß1, and phosphorylated integrin ß1 at Ser785 were evaluated by Western blotting as described in Materials and Methods, with each lane being loaded with equal amount of proteins and with ß-actin as control.

 
CD26 depletion is associated with decreased tumorigenicity and increased survival in a severe combined immunodeficient mouse model. To investigate the effect of depletion of CD26 expression in an in vivo animal model, we inoculated mock and CD26-depleted clones into SCID mice i.p. and then monitored for tumor development and overall survival (Fig. 6). All of the mice inoculated with mock Karpas 299 clones developed tumor masses in the inguinal areas. The largest palpable tumor mass was monitored by serial measurements, and all the mice with tumors were eventually sacrificed per protocol. Importantly, none of the mice inoculated with CD26-depleted clones developed tumor masses over the observed time. Similar studies done with CD26-depleted clone 2 also showed no tumor development over similar times tested (data not shown).



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  		Figure 6. Effect of CD26 depletion on tumorigenicity in an in vivo SCID mouse model. SCID mice pretreated with anti–asialo-GM1 antibody were injected i.p. with mock or a CD26-depleted clone dep-1. The maximum diameter (MD) and transverse length (TL) of the largest inguinal mass were measured with a caliper, with tumor volume being calculated by the formula: MD x TL2 x 1/2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this article, we provide evidence linking CD26 to structures with important biological roles. We extend our previous studies by demonstrating the association between CD26 and topoisomerase II{alpha}, with implications for the treatment of selected neoplasms with topoisomerase II inhibitors, such as doxorubicin. Importantly, our study is the first to report a linkage among CD26, p38MAPK, and integrin ß1. Our data indicate that CD26 is associated with integrin-dependent adhesion of Karpas 299 cells to the ECM by regulating p38MAPK-dependent phosphorylation of integrin ß1 at Ser785. In addition, we show that down-regulation of CD26 expression decreases tumorigenicity of Karpas 299 cells in an in vivo SCID mouse xenograft model, hence suggesting that targeting CD26 may be an effective therapeutic strategy for selected neoplasms, specifically aggressive hematologic malignancies associated with high level of CD26 expression.

Previous work has suggested that CD26 may have a role in cell adhesion to ECM in selected experimental conditions (19, 22–24), although the mechanism involved with CD26 role in cell adhesion has not been clearly elucidated. Our present findings indicate that the T-ALCL Karpas 299 cells bind to the ECM proteins fibronectin and collagen I and that this binding is regulated by CD26. Importantly, we show that CD26 affects ECM binding through integrin ß1 as supported by our data demonstrating that treatment with anti-integrin antibody inhibits binding of parental Karpas 299 to ECM and that the anti-CD26 antibody 202.36 also inhibits cell adhesion by suppressing phosphorylation of p38MAPK and integrin ß1 of Karpas 299. Furthermore, depletion of CD26 expression by siRNA transfection results in decreased integrin ß1 phosphorylation and inhibition of cell adhesion to ECM. Our data also suggest that the specific epitope recognized by the anti-CD26 antibody 202.36, which is also the HIV-1 gp120-binding domain of CD26 (25), is responsible for CD26 regulation of cell adhesion, as other anti-CD26 antibodies recognizing other epitopes do not affect ECM binding.

Previous studies showed the importance of phosphorylation of the Ser785 residue of integrin ß1 in cell adhesion, as substitution of serine to methionine or aspartate at position 785 results in loss of binding (21). Our work extends these data by demonstrating specifically that the phosphorylation status of integrin ß1 at Ser785 regulates integrin ß1–dependent cell adhesion. Of note is the fact that CD26 depletion does not have an effect on the phosphorylation status of integrin ß1 at Thr788/Thr789 (data not shown), which has also been reported to regulate integrin ß1–dependent cell adhesion (26, 27). We also note that we consistently observe two bands on Western blotting with the particular antibody used to detect integrin ß1 phosphorylated at Ser785. Although the exact reason for this observation is presently unclear, several potential explanations can be offered. It is possible that particular breakdown products are detected along with the full-length protein. Alternatively, the level of phosphorylation at Ser785 residues may differ for different integrin ß1 molecules, hence slightly altering the molecular weights of individual integrin ß1 molecule and resulting in the heterogeneous bands detected. It is also possible that variants of integrin ß1 exist in Karpas 299 cells that are phosphorylated at Ser785, leading to the observation of multiple protein bands.

Our data show that CD26 regulates integrin ß1 phosphorylation of Karpas 299 cells through its effect on p38MAPK. However, the mechanism involved in p38MAPK regulation of the phosphorylation status of integrin ß1 remains to be elucidated. Of note is the fact that transfectional overexpression of CD26 on the human Burkitt B-cell lymphoma cell line Jiyoye results in increased phosphorylation of p38MAPK (12) but does not lead to an accompanying enhancement in phosphorylation of integrin ß1 or induction of cell adhesion (data not shown). It is presently unclear how p38MAPK is able to induce phosphorylation of integrin ß1 in the T-cell lymphoma line Karpas 299 but not in the B-cell lymphoma line Jiyoye cells. It is likely that p38MAPK regulation of integrin ß1 phosphorylation is dependent on other factors that exist in the T-cell line Karpas 299 but not in the B-cell line Jiyoye. Likewise, it is presently unclear as to how CD26 regulates p38 phosphorylation. Given the fact that CD26 is a serine protease capable of cleaving selected biological factors, it is possible that CD26 indirectly regulates p38 phosphorylation pathway via the activity of its cleaved substrates. In addition, because CD26 physically and functionally associates with molecules with key roles in signal transduction, including the tyrosine phosphatase CD45 (5), CD26 may have an effect on p38 phosphorylation through its associated molecules.

An important aim of our present study is to clarify the role of CD26 in regulating the malignant behavior of tumors as a marker of aggressive disease for selected subsets for T-cell malignancies (9, 10, 14), which may allow for the rational selection of CD26 as a potential target for novel therapy. Our in vivo data with SCID mouse xenografts conclusively show that depletion of CD26 expression on Karpas 299 cells results in loss of tumorigenicity and enhanced survival. It is likely that the inability to bind to the ECM as a result of CD26 depletion prevents tumor development in the animal model, given the fact that there is no difference in the rate of proliferation or the level of spontaneous cell death between CD26-positive and CD26-depleted Karpas 299 cells (data not shown). Given the fact that Karpas 299 is a T-ALCL cell line, it is interesting to note that in a study involving 8 cases of patients with T-ALCL, 5 of 8 (63%) cases were positive for CD26 expression (9). Taken together, our findings suggest that CD26 ability to regulate cell adhesion through p38MAPK-dependent phosphorylation of integrin ß1 plays a key role in tumorigenicity of the T-ALCL cells Karpas 299 and that treatment strategies targeting CD26 may be an effective therapeutic approach for selected CD26-bearing tumors, including aggressive T-cell malignancies.


   Acknowledgments
 
Grant support: Japan Society for the Promotion of Science (T. Sato), Kanae Foundation for Life and Socio-Medical Science (T. Sato), M.D. Anderson Cancer Center Physician-Scientist Award (N.H. Dang), Gillson Longenbaugh Foundation (N.H. Dang), and Goodwin Funds (N.H. Dang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/28/05. Revised 5/ 2/05. Accepted 5/18/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pro B, Dang NH. CD26/dipeptidyl peptidase IV and its role in cancer. Histol Histopathol 2004;19:1345–51.[Medline]
  2. Fox DA, Hussey RE, Fitzgerald KA, et al. Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody. J Immunol 1984;133:1250–6.[Abstract]
  3. Dang NH, Torimoto Y, Deusch K, Schlossman SF, Morimoto C. Comitogenic effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathways. J Immunol 1990;144:4092–100.[Abstract]
  4. Dang NH, Torimoto Y, Sugita K, et al. Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. Analysis of surface expression and human T cell activation. J Immunol 1990;145:3963–71.[Abstract]
  5. Torimoto Y, Dang NH, Vivier E, Tanaka T, Schlossman SF, Morimoto C. Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes. J Immunol 1991;147:2514–7.[Abstract/Free Full Text]
  6. Asada Y, Aratake Y, Kotani T, et al. Expression of dipeptidyl aminopeptidase IV activity in human lung carcinoma. Histopathology 1993;23:265–70.[Medline]
  7. Aratake Y, Kotani T, Tamura K, et al. Dipeptidyl aminopeptidase IV staining of cytologic preparations to distinguish benign from malignant thyroid diseases. Am J Clin Pathol 1991;96:306–10.[Medline]
  8. Bauvois B, De Meester I, Dumont J, Rouillard D, Zhao HX, Bosmans E. Constitutive expression of CD26/dipeptidylpeptidase IV on peripheral blood B lymphocytes of patients with B chronic lymphocytic leukaemia. Br J Cancer 1999;79:1042–8.[CrossRef][Medline]
  9. Carbone A, Gloghini A, Zagonel V, et al. The expression of CD26 and CD40 ligand is mutually exclusive in human T-cell non-Hodgkin's lymphomas/leukemias. Blood 1995;86:4617–26.[Abstract/Free Full Text]
  10. Dang NH, Aytac U, Sato K, et al. T-large granular lymphocyte lymphoproliferative disorder: expression of CD26 as a marker of clinically aggressive disease and characterization of marrow inhibition. Br J Haematol 2003;121:857–65.[CrossRef][Medline]
  11. Torimoto Y, Dang NH, Tanaka T, Prado C, Schlossman SF, Morimoto C. Biochemical characterization of CD26 (dipeptidyl peptidase IV): functional comparison of distinct epitopes recognized by various anti-CD26 monoclonal antibodies. Mol Immunol 1992;29:183–92.[CrossRef][Medline]
  12. Yamochi T, Yamochi T, Aytac U, et al. Regulation of p38 phosphorylation and topoisomerase II{alpha} expression in the B-cell lymphoma line Jiyoye by CD26/dipeptidyl peptidase IV (DPPIV), associated with enhanced in vitro and in vivo sensitivity to doxorubicin. Cancer Res 2005;65:1973–83.[Abstract/Free Full Text]
  13. Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 1989;119:203–10.[CrossRef][Medline]
  14. Carbone A, Cozzi M, Gloghini A, Pinto A. CD26/dipeptidyl peptidase IV expression in human lymphomas is restricted to CD30-positive anaplastic large cell and a subset of T-cell non-Hodgkin's lymphomas. Hum Pathol 1994;25:1360–5.[CrossRef][Medline]
  15. Sato K, Aytac U, Yamochi T, et al. CD26/dipeptidyl peptidase IV enhances expression of topoisomerase II{alpha} and sensitivity to apoptosis induced by topoisomerase II inhibitors. Br J Cancer 2003;89:1366–74.[CrossRef][Medline]
  16. Aytac U, Sato K, Yamochi T, et al. Effect of CD26/dipeptidyl peptidase IV on Jurkat sensitivity to G2-M arrest induced by topoisomerase II inhibitors. Br J Cancer 2003;88:455–62.[CrossRef][Medline]
  17. Aytac U, Claret FX, Ho L, et al. Expression of CD26 and its associated DPPIV enzyme activity enhances sensitivity to doxorubicin-induced cell cycle arrest at G2-M checkpoint. Cancer Res 2001;61:7204–10.[Abstract/Free Full Text]
  18. Ishida R, Iwai M, Marsh KL, et al. Threonine 1342 in human topoisomerase II{alpha} is phosphorylated throughout the cell cycle. J Biol Chem 1996;271:30077–82.[Abstract/Free Full Text]
  19. Dang NH, Torimoto Y, Schlossman SF, et al. Human CD4 helper T cell activation: functional involvement of two distinct collagen receptors, 1F7 and VLA integrin family. J Exp Med 1990;172:649–52.[Abstract/Free Full Text]
  20. Elangbam CS, Qualls CW Jr, Dahlgren RR. Cell adhesion molecules—update. Vet Pathol 1997;34:61–73.[Abstract]
  21. Mulrooney JP, Hong T, Grabel LB. Serine 785 phosphorylation of the ß1 cytoplasmic domain modulates ß1A-integrin-dependent functions. J Cell Sci 2001;114:2525–33.[Abstract/Free Full Text]
  22. Cheng HC, Abdel-Ghany M, Pauli BU. A novel consensus motif in fibronectin mediates dipeptidyl peptidase IV adhesion and metastasis. J Biol Chem 2003;278:24600–7.[Abstract/Free Full Text]
  23. Loster K, Zeilinger K, Schuppan D, Reutter W. The cysteine-rich region of dipeptidyl peptidase IV (CD26) is the collagen-binding site. Biochem Biophys Res Commun 1995;217:341–8.[CrossRef][Medline]
  24. Kikkawa F, Kajiyama H, Ino K, Shibata K, Mizutani S. Increased adhesion potency of ovarian carcinoma cells to mesothelial cells by overexpression of dipeptidyl peptidase IV. Int J Cancer 2003;105:779–83.[CrossRef][Medline]
  25. Herrera C, Morimoto C, Blanco J, et al. Comodulation of CXCR4 and CD26 in human lymphocytes. J Biol Chem 2001;276:19532–9.[Abstract/Free Full Text]
  26. Wennerberg K, Fassler R, Warmegard B, Johansson S. Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin ß1A. Requirement for threonines 788–789 in receptor activation. J Cell Sci 1998;111:1117–26.[Abstract]
  27. Stroeken PJ, van Rijthoven EA, Boer E, Geerts D, Roos E. Cytoplasmic domain mutants of ß1 integrin, expressed in ß1-knockout lymphoma cells, have distinct effects on adhesion, invasion and metastasis. Oncogene 2000;19:1232–8.[CrossRef][Medline]



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