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[Cancer Research 63, 8500-8506, December 1, 2003]
© 2003 American Association for Cancer Research

Endocrinology

Biological Significance of Aminopeptidase N/CD13 in Thyroid Carcinomas

Astrid Kehlen1, Uwe Lendeckel3, Henning Dralle2, Jürgen Langner1 and Cuong Hoang-Vu2

1 Institute of Medical Immunology and
2 Department of Surgery, University of Halle-Wittenberg, Halle, Germany, and
3 Institute of Experimental Internal Medicine, University Hospital Magdeburg, Magdeburg, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aminopeptidase N (APN)/CD13 is a transmembrane ectopeptidase expressed on a wide variety of cells. However, the precise function of APN/CD13 in tumor cells and the relationship of APN/CD13 to thyroid cancer remain unclear. In our study, we quantified the expression of APN/CD13 and additionally dipeptidyl peptidase IV (DPIV)/CD26 in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. Undifferentiated anaplastic thyroid carcinomas expressed more APN/CD13 than differentiated thyroid carcinomas. DPIV/CD26 showed an opposite expression pattern. We detected higher levels of DPIV/CD26 in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas than in undifferentiated anaplastic thyroid carcinomas. In the undifferentiated thyroid carcinoma cell line 1736, APN/CD13 mRNA expression could be increased by epidermal growth factor, basic fibroblast growth factor, interleukin-6, and tumor necrosis factor {alpha}. FTC-133 cells stably transfected with an expression vector for APN-enhanced green fluorescent protein showed a higher migration rate than FTC-133 cells transfected with the enhanced green fluorescent protein-control plasmid. Overexpression of APN/CD13 in stably transfected cells is associated with down-regulation of N-myc down-regulated gene (NDRG)-1, melanoma-associated antigen ME491/CD63, and DPIV/CD26 gene expression. Inhibition of APN/CD13 mRNA expression by small interfering RNA induced NDRG-1, ME491/CD63, and DPIV/CD26 mRNA expression in cells of the undifferentiated thyroid carcinoma cell line C643. We conclude that APN/CD13-associated down-regulation of NDRG-1, ME491/CD63, and DPIV/CD26 in thyroid carcinoma cells is an important step of tumor progression to more malignant phenotypes, and we underline the important role of APN/CD13 as mediator in a multimolecular process regulating cell migration.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Four types of thyroid carcinomas comprise >98% of all thyroid malignancies, including PTC,4 FTC, UTC, and medullary thyroid carcinoma (Ref. 1 ). PTC may have a very benign course, whereas UTC belongs to the most aggressive human malignancies. All steps of tumor growth such as local invasion, intravasation, extravasation, angiogenesis, and metastasis require the activity of proteolytic enzymes (2 , 3) . The involvement in these processes of peptidases from the classes of metallo- and serine peptidases has been shown, but individual contribution of each enzyme in thyroid carcinoma is not clear. This work deals with the role of the Zn-dependent ectopeptidase APN and the serine-type ectopeptidase DPIV in thyroid carcinoma. APN/CD13 (EC 3.4.11.2) catalyzes the removal from small peptides of NH2-terminal, preferentially neutral, amino acid residues (for review see Refs. 4 and 5 ). This 150-kDa metalloprotease has been found in many tissues, with especially high expression in brush border membranes of kidney proximal tubules, intestine, and placenta. It has been suggested that APN/CD13 plays a role in antigen processing, neuropeptide and cytokine degradation, cell-cell contact, tumor invasion, and extracellular matrix degradation (6, 7, 8, 9, 10) . Recent reports have shown that APN/CD13 is also important in angiogenesis and is activated by angiogenic signals (6 , 10 , 11) . Investigations identified the APN/CD13 cell surface antigen as the principal receptor for peptides with the Asn-Gly-Arg (NGR) motif and demonstrated that coupling of TNF-{alpha} to NGR peptides enhanced the immunotherapeutic properties of TNF-{alpha} (6 , 11, 12, 13) .

DPIV/CD26 (E.C. 3.4.14.5.) is a 110-kDa transmembrane glycoprotein with ubiquitous expression (for review see Refs. 14 and 15 ). DPIV/CD26 has a variety of functions, not only in protease activity, which liberates NH2-terminal X-proline from peptides, but also in various cellular processes such as regulation of immune response, signal transduction, and interaction with molecules of extracellular matrix (16, 17, 18, 19) . Recent studies have provided evidence that DPIV/CD26 may play a role in tumor progression, especially in cell adhesion and invasion (20) . Other studies have reported that DPIV/CD26 expression has a suppressing effect on the malignant phenotype, has anti-invasive function, and is linked with a prolonged survival (21, 22, 23) . High DPIV/CD26 expression was found in follicular cell-derived thyroid carcinomas (PTC, Hürthle cell carcinoma, and FTC), whereas thyroid glands with benign conditions are exclusively negative for CD26 expression (24, 25, 26, 27) .

To further clarify the role of the ectopeptidase APN/CD13 in the process of motility and invasion of thyroid carcinomas, we (a) studied the expression level of APN/CD13 in comparison with the DPIV/CD26 level in thyroid carcinomas, (b) investigated the regulation of APN/CD13 in cultured thyroid carcinoma cells, and (c) examined the effect of APN/CD13 overexpression on motility and gene expression in stably transfected thyroid carcinoma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues and Cell Lines.
A total of 40 thyroid tissues, including 10 FTCs, 10 PTCs, 10, UTCs, and 10 goiters, were obtained from patients of the Department of Surgery of the University of Halle by surgical resection and stored at -80°C until used. The local committee of medical ethics approved the use of human tissues, and all patients gave their written informed consent. Each tumor was scored based on the TNM (tumor-node-metastasis) classification (Table 1Citation ; Ref. 28 ). Seven human thyroid cancer cell lines (C643, 1736, Hth74, FTC-133, FTC-236, FTC-238, and B-CPAP) were cultured in the recommended growth medium. For stimulation experiments, 1736 cells were serum-starved for 3 days (29) and treated with the following recombinant cytokines: TNF-{alpha} (1 ng/ml); TGF-ß1 (10 ng/ml); IL-1ß (10 ng/ml); IL-4 (100 units/ml); and IL-6 (100 units/ml; Strathmann Biotech, Hamburg, Germany). The following growth factors were used: EGF (10–100 ng/ml); and bFGF (50–100 ng/ml; Cell Concepts, Umkirch, Germany).


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  		Table 1 List of patients with goiter and thyroid carcinomas

 
Flow Cytometry Analysis.
Cells were detached from 6-well plates using Accutase (PAA, Linz, Austria). After two cold washes, standard surface membrane immunofluorescence techniques were used. Cells were stained with either the APN-specific mAb Leu-M7, the DPIV-specific mAb M-A262, or an IgG1 isotype control (Becton Dickinson, Heidelberg, Germany) at 4°C for 40 min. After two cold washes with PBS containing 0.1% sodium azide, cells were labeled with the phycoerythrin-conjugated secondary antibody goat antimouse IgG (Dianova, Hamburg, Germany) at 4°C for 30 min and washed and fixed using 1% paraformaldehyde. Fluorescence was analyzed in a Becton Dickinson FACSCalibur using Cellquest software. Ten thousand cells per sample were counted. MFI was calculated as sample MFI - control antibody MFI.

RNA Isolation and cDNA Synthesis.
Total cellular RNA was isolated according to Chomczynski and Sacchi (30) . The first strand of DNA was synthesized (after a 10-min incubation at 20°C) at 42°C for 50 min using 500 ng of total RNA in 5.5 µl of diethyl pyrocarbonate water, 2 µl of 5x first-strand buffer [250 mM Tris/HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2], 0.5 µl of deoxynucleoside triphosphate mix (10 mM each of dATP, dCTP, dGTP, and dTTP), 1 µl of 0.1 M DTT, 0.5 µl (50 pmol) of random primer (Roche, Mannheim, Germany), and 0.5 µl (200 units/µl) of Superscript II-RT (Invitrogen, Karlsruhe, Germany). For quantitative RT-PCR, six dilutions of standard RNA and total RNA, respectively, were converted into cDNA in separate tubes.

Construction of RNA Standards.
The standards were constructed by procedures described previously (31) . Briefly, for the construction of standard RNA, a composite primer was synthesized (see Table 2Citation for primer sequences). This primer 1 contained a sequence for the SP6 RNA polymerase (underlined) and also one of the specific sequences of the appropriate gene. The product of the PCR amplification with primer 1 and 2 was gel purified (QIAquick Gel Extraction kit; Qiagen, Hilden, Germany) followed by in vitro transcription from the SP6 promoter using the transcription system of Roche. The recombinant RNA was quantified at 260 nm and used as the standard in the quantitative RT-PCR reaction.


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  		Table 2 Sequences of primers used for standard construction and PCR amplification

 
Quantitative PCR Analysis.
For quantification, 1 µl of the reverse transcriptase reaction mixture was added to 25 µl of reaction mixture consisting of 1x reaction buffer, 1.5 units of Taq polymerase (Qiagen), 1.8 mM MgCl2, 0.1x SYBR Green (Biozym, Hessisch Oldendorf, Germany), 200 µM each deoxynucleoside triphosphate, and 0.5 µM each of primers 3 and 2 (Table 2)Citation . A negative control without template was included. Assays of both the standard cDNA and the sample cDNA, respectively, were run in triplicates in a Rotor-Gene 2000 (LTF, Wasserburg, Germany). Initial denaturation at 95°C for 300 s was followed by 40 cycles with denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and elongation at 72°C for 20 s. The fluorescence intensity of the double-strand-specific SYBR Green, reflecting the amount of formed PCR product, was read after each elongation step at 82°C. To verify the PCR products, melting curves were generated. Absolute RNA amounts were determined with the software Rotor-Gene version 4.4 in quantitation mode. Relative quantification of NDRG1 and ME491/CD63 gene expression, respectively, was performed with the software Rotor-Gene version 4.6 in comparative quantitation mode. This mode allows the user to compare samples treated differently relative to a control sample. The second derivative of the raw data is taken to calculate the take off point. On the basis of the take off point and the reaction efficiency, it calculates the relative concentration of each sample compared with the control sample.

Transient Transfection and Luciferase Assay.
The 1736 cells were washed with OptiMem medium and resuspended in OptiMem medium at a concentration of 5 x 105 cells/ml. The appropriate construct containing the epithelial APN promoter (1.5 µg; kindly provided by J. Olsen, Copenhagen, Denmark) and 0.5 µg of the internal control plasmid pRL-TK (Renilla luciferase gene; Promega) were cotransfected into the cells using 2 µl of LipofectAMINE 2000 (Invitrogen) reagent according to the instructions of the manufacturer. The preparation of cell extracts and the measurement of luciferase activities were carried out using the Dual-Luciferase Reporter Assay System (Promega) according to the recommendations of the manufacturer. The assays for firefly luciferase activity and Renilla luciferase activity were performed sequentially using one reaction tube in a luminometer with two injectors (Lumat LB9507; EG&G Berthold, Bad Wildbad, Germany). Changes in firefly luciferase activity were calculated and plotted after normalization with changes in Renilla luciferase activity in the same sample.

Transfection and Selection of Clones.
FTC-133 cells (5 x 105 cells) were transfected with 1.5 µg of either the pAPN-EGFP construct (kindly provided by J. Topsch, Mainz, Germany) or the pEGFP-C3 host vector (BD Clonetech, Heidelberg, Germany) using 3 µl of LipofectAMINE 2000 (Invitrogen) according the manufacturer’s instructions. Selection was initiated 48 h after transfection by adding 1 mg/ml G418 (Invitrogen). Selection medium was changed every 4 days for 6 weeks until all untransformed cells died. Resistant clones were isolated and expanded for further characterization.

Motility.
Cellular motility of transfected cell clones was evaluated in 24-well Transwell chambers (Costar, Bodenheim, Germany). The upper and lower culture compartments were separated by polycarbonate filters with a pore size of 8 µm. In motility assays, 2 x 105 cells/well were seeded in RPMI 1640 containing 0.1% BSA on the filters for 18 h. Then cells on the upper surface of membrane were removed by wiping with cotton swabs, and cells that had migrated through the membrane and stuck to the lower surface of the membrane were measured using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay (Roche) as described by the manufacturer. Formazan production was measured as the absorbance of 492–690 nm. Results are expressed as a percentage of the absorbance of cells that migrated through the polycarbonate membrane into the lower Transwell chamber. All quantifications were performed in triplicate. The inhibitor bestatin (10-4 M; Sigma-Aldrich, Taufkirchen, Germany) and the mouse antihuman APN/CD13 antibody WM15 (5 µg/ml; Dunn, Asbach, Germany) were added to the cells 30 min before the migration assay.

siRNA and Transfection of RNA Oligonucleotides.
Double-stranded siRNA was designed to APN/CD13 (5'-AAAGCGTGGAATCGTTACCGC-3') and synthesized by Qiagen. GFP-22 siRNA Rhodamine (Qiagen) was used as control. Approximately, 1.5 x 105 cells/well were plated in a 12-well plate in media containing 10% FCS to give 80–90% confluence, and transfection of the RNA oligonucleotides was performed using GeneEraser (4 µl/well; Stratagene, Amsterdam, the Netherlands) to result in a final RNA concentration of 100 nM. After 48 h, the cells were lysed, and RNA was isolated as described above.

Statistical Analysis.
Data are expressed as mean ± SE. Wilcoxon’s rank-sum test was used to determine whether two experimental values were significantly different (P < 0.05, *; P < 0.01, **).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
High Expression of APN/CD13 in Undifferentiated Thyroid Carcinomas.
To investigate the ectopeptidase mRNA expression of seven different thyroid carcinoma cells, we developed a quantitative RT-PCR assay using RNA standards and specific APN/CD13 as well as DPIV/CD26 primers (see Table 2Citation ) as described by the method of Bustin (32) . Quantitative RT-PCR revealed higher absolute levels of APN/CD13 mRNA in the undifferentiated thyroid carcinomas cell lines 1736, C643, and Hth74 as compared with the differentiated cell lines FTC-133, FTC-236, FTC-238, and B-CPAP (Fig. 1)Citation . The highest APN/CD13 mRNA amount was found in C643 cells with 880 fg/µg total RNA. DPIV/CD26 showed an opposite expression pattern. A higher expression of DPIV/CD26 mRNA was found in follicular and PTC cell lines as compared with undifferentiated cells. FTC-238 cells showed the highest DPIV/CD26 mRNA expression with 2.3 fg/µg total RNA. As given in Fig. 2Citation , flow cytometry analysis showed similar results for the expression of APN/CD13 and DPIV/CD26 at the protein level; high APN/CD13 protein expression was seen in UTC cell lines versus a very low expression level in differentiated cells. In contrast to the APN/CD13 expression, the DPIV/CD26 expression in differentiated cells is higher than that in undifferentiated cells.



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  		Fig. 1. Comparison of the absolute amounts of APN/CD13 and DPIV/CD26 mRNA in thyroid carcinoma cell lines (each in triplicate experiments). Results of quantitative RT-PCR are as described in "Materials and Methods."

 


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  		Fig. 2. Surface expression of APN/CD13 and DPIV/CD26 on thyroid carcinoma cell lines. After staining with the anti-APN/CD13 antibody Leu-M7 and the anti-DPIV/CD26 antibody M-A262, respectively, cells were analyzed using flow cytometry. Results are given as relative mean fluorescence.

 
Furthermore, quantification of the mRNA expression of both ectopeptidases, APN/CD13 and DPIV/CD26, in tissue samples of patients with thyroid carcinomas and in control tissues of patients with goiter was done. The mean APN/CD13 mRNA level was 47 fg/µg total RNA (range, 3–149 fg/µg total RNA) in tissues of patients with UTC, 32 fg/µg total RNA in PTC (range, 0.1–190 fg/µg total RNA), 6 fg/µg total RNA in FTC (range, 0.01–18 fg/µg total RNA), and 2 fg/µg total RNA in goiter (range, 0.8–5 fg/µg total RNA), respectively (Fig. 3A)Citation . The mean amount of DPIV/CD26 mRNA was 0.1 fg/µg total RNA in UTC (range, 0.001–0.5 fg/µg total RNA), 8.7 fg/µg total RNA in PTC (range, 0.002–57 fg/µg total RNA), 0.8 fg/µg total RNA in FTC (range, 0.002–3.6 fg/µg total RNA), and 0.6 fg/µg total RNA in goiter (range, 0.008–2.2 fg/µg total RNA), respectively (Fig. 3B)Citation .



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  		Fig. 3. Comparison of the absolute amounts of APN/CD13 mRNA and DPIV/CD26 mRNA in tissues of patients with thyroid carcinoma and in goiter tissue, respectively (each group, n = 10 in triplicate experiments). Results of quantitative RT-PCR are as described in "Materials and Methods."

 
EGF and IL-6 Augment APN/CD13 Expression in 1736 Cells.
An addition of 10% FCS to the culture medium resulted in a decrease of the APN/CD13 mRNA level (Fig. 4)Citation . Therefore, 72 h before modulation, the undifferentiated thyroid carcinoma cell line 1736 was cultured in serum-free medium. After treatment of 1736 cells with IL-1ß and TGF-ß1 for 24 h, no significant change in the APN/CD13 mRNA level was found when compared with the unstimulated control. However, the cytokines IL-6, TNF-{alpha}, and IL-4 induced an increase in APN/CD13 mRNA expression (Fig. 4)Citation . The APN/CD13 mRNA expression was 5.2-fold higher in IL-6-treated 1736 cells, 4.7-fold higher in TNF-{alpha}-treated 1736 cells, and 2-fold higher in IL-4-stimulated 1736 cells (Fig. 4)Citation . Furthermore, EGF and bFGF were able to stimulate the APN/CD13 mRNA levels, and addition of 10% FCS down-regulated the APN/CD13 mRNA expression. The stimulation of the APN/CD13 mRNA amounts by EGF was concentration dependent. Addition of 100 ng/ml EGF resulted in a 6-fold higher expression of APN/CD13 mRNA compared with the unstimulated control. bFGF stimulated the APN/CD13 mRNA expression 3-fold (Fig. 4)Citation .



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  		Fig. 4. Regulation of APN/CD13 mRNA expression in UTC-1736 cells by different cytokines and growth factors. Serum-starved 1736 cells (each in triplicate experiments) were cultured for 24 h with the appropriate mediator. Results of quantitative RT-PCR are given as a percentage of the basal control (culture without cytokine = 100%; * denotes statistically significant differences from control at the P < 0.05 level).

 
To further investigate the effect of IL-6 and EGF on APN/CD13 mRNA levels, the epithelial APN/CD13 promoter activity was measured by using transfected 1736 cells and quantification of the reporter gene activity with the Dual Luciferase assay system. 1736 cells transfected with the APN/CD13 promoter construct showed a 2.5-fold increase of reporter gene activity after stimulation with 100 ng/ml EGF. A 1.8-fold increase of APN/CD13 promoter activity was detectable after addition of IL-6.

APN/CD13 Is Involved in Motility of Thyroid Carcinoma Cells.
Because of their origin from a primary tumor and their low basal APN/CD13 expression as shown in Figs. 1Citation and 2Citation , the FTC-133 cell line was used. FTC-133 cells were transfected with an expression vector (pEGFP-C3) containing the full-length APN/CD13 coding sequence. FTC-133 cells transfected with the vector alone served as control. Cell clones that have stably integrated the vector constructs were selected for G418 resistance within 6 weeks. Using flow cytometry, we quantified the APN/CD13 expression of the stably transfected clones. More than 90% of the FTC-133 cells (MFI = 1280) expressing the APN/CD13 construct (FTC-133 APN-GFP) were positive for APN/CD13 using the Leu-M7 mAb specific for APN/CD13. The control cells (FTC-133 GFP) were stained very weakly (<5% of the cells were positive for APN/CD13, MFI = 10; Fig. 5Citation ). Additionally, by measuring [3H]thymidine incorporation, no significant difference was found in the proliferation behavior of both clones, the FTC-133 GFP cells and the FTC-133 APN-GFP cells. To determine cell motility, the migrating ability of FTC-133 GFP and FTC-133 APN-GFP cells was tested. As shown in Fig. 6Citation , the APN-GFP-transfected cells (136 ± 15%; P < 0.05) passed the filter barrier faster than did the GFP-transfected cells (control = 100%). The migration of FTC-133 APN-GFP cells could be inhibited with bestatin (10-4 M) and with an enzyme activity inhibiting mAb against APN/CD13 (WM15; 5 µg/ml). The WM15 antibody did not affect the migration of FTC-133 GFP cells, whereas bestatin was able to inhibit it weakly.



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  		Fig. 5. Surface expression of APN/CD13 on stably transfected FTC-133 cells. Open histogram from flow cytometry represents APN/CD13 expression of FTC-133 GFP cells, and shaded histogram corresponds to the FTC-133 APN-GFP cells.

 


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  		Fig. 6. Effect of APN/CD13 and inhibitors of APN/CD13 on the migration of FTC-133 cells. FTC-133 GFP cells or FTC-133 APN-GFP cells (2 x 105 cells/well) were seeded into the upper compartment of a Transwell chamber, and the chamber was incubated for 18 h. The inhibitor bestatin (10-4 M) and the anti-APN/CD13 antibody WM15 (5 µg/ml) were added to the cells 30 min before the migration assay. After incubation for 18 h, the migrated cells were measured using a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. Formazan production was measured as the absorbance. Results are expressed as a percentage of the absorbance of cells that migrated through the polycarbonate membrane into the lower Transwell chamber (absorbance of FTC-133 GFP = 100%). The mean ± SD of triplicate determinations is shown.

 
APN/CD13 Changes Gene Expression of DPIV/CD26, NDRG1, and ME491/CD63.
To study the effect of APN/CD13 overexpression on the DPIV/CD26 expression, the transcript level of DPIV/CD26 was measured in stably transfected FTC-133 cells. DPIV/CD26 mRNA levels were strongly down-regulated in APN/CD13-transfected cells (0.30-fold) as compared with the nontransfected FTC-133 cells (1.0-fold; Fig. 7Citation ). Furthermore, differential expression of N-myc downstream-regulated gene 1 (NDRG-1/Drg-1) and melanoma-associated antigen ME491/CD63 was investigated using real-time RT-PCR. These genes are associated with promoting/inhibiting proliferation and differentiation in carcinomas. The gene expression of NDGR1 (0.37-fold) and ME491/CD63 (0.68-fold), respectively, was down-regulated in APN-GFP FTC-133 cells compared with FTC-133 cells (1.0-fold; Fig. 7Citation ). RNA interference was used in an attempt to decrease APN/CD13 in C643 cells expressing high levels of APN/CD13 to address whether its overexpression is involved in the gene expression of NDRG-1, ME491/CD63, and DPIV/CD26. siRNA directed against APN/CD13 reduced the levels of APN/CD13 (0.38-fold; Fig. 8Citation ) compared with the mock-transfected cells but did not affect the levels of actin (data not shown). Interestingly, siRNA directed against APN/CD13 increased the mRNA levels of NDRG-1, ME491/CD63, and DPIV/CD26 (1.92-, 1.8-, and 2.2-fold, respectively), whereas siRNA directed against GFP did not.



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  		Fig. 7. Effect of APN/CD13 on the gene expression of DPIV/CD26, NDRG1, and ME491/CD63 in APN/CD13-overexpressing FTC-133 cells. Results of quantitative RT-PCR are given as a fold change of the control (FTC-133 = 1.0; n = 4; * denotes statistically significant differences from control at the P < 0.05 level; **, P < 0.01).

 


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  		Fig. 8. Effect of inhibition of APN/CD13 mRNA expression by siRNA on the gene expression of DPIV/CD26, NDRG1, and ME491/CD63 in C643 cells. Results of quantitative RT-PCR are given as a fold change of the control (mock-transfected C643 cells = 1.0; n = 4; * denotes statistically significant differences from control at the P < 0.05 level).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using cultured thyroid carcinoma cells and thyroid tissues of patients suffering from thyroid carcinomas, we report for the first time differences in the absolute levels of the ectopeptidases APN/CD13 and DPIV/CD26. UTCs expressed more APN/CD13 than differentiated thyroid carcinomas. DPIV/CD26 showed an opposite expression pattern, i.e., higher levels of DPIV/CD26 in FTCs and PTCs than in UTCs. Furthermore, in APN/CD13-transfected FTC-133 cells, the DPIV/CD26 mRNA expression is down-regulated. It is known that follicular cell-derived thyroid carcinomas (PTC, Hürthle cell carcinoma, and FTC) express high DPV/CD26 amounts (24, 25, 26, 27) . Recently, Aratake et al. (33) demonstrated that galectin-3 and DPIV/CD26 were consistently coexpressed at protein and mRNA levels in differentiated thyroid carcinomas and could be used as preoperative diagnostic markers. Here, we showed that the expression levels for the ectopeptidases APN/CD13 (UTC > PTC, FTC) and DPIV/CD26 (PTC, FTC > UTC) could be used for characterization of undifferentiated anaplastic and differentiated thyroid carcinomas.

Furthermore, we examined the modulation of APN/CD13 expression in the undifferentiated thyroid carcinoma cell line 1736 by cytokines and growth factors. After addition of the cytokines IL-6, TNF-{alpha}, or IL-4 to 1736 cells, we detected an increased APN/CD13 mRNA expression. In renal cell carcinoma cells, TNF-{alpha} and IL-4 were also able to induce the APN/CD13 mRNA (34) , and in osteosarcoma cells, IL-6 enhanced the APN/CD13 expression and the invasive potential (35) . Interestingly, Basolo et al. (36) reported a reduced expression of IL-6 in UTCs, whereas levels of IL-8, TGF-{alpha}, and TGF-ß1 were similar to those produced by normal cells. EGF is able to stimulate the proliferation of normal and thyroid cells; it enhanced migration and inhibited differentiation (37, 38, 39, 40) . EGF induced an increased APN/CD13 steady-state mRNA level and a higher APN/CD13 promoter activity in 1736 cells in a concentration-dependent manner. EGF may have divergent effects on the expression of peptidases: on one hand, EGF augments APN/CD13 expression in the undifferentiated thyroid carcinoma cell line 1736; and on the other hand, it decreases DPIV/CD26 expression in FTC-133 cells.5 In lung alveolar epithelial cells, treatment with EGF and dexamethasone resulted in an increase of APN/CD13 mRNA amounts (41) . bFGF (fibroblast growth factor 2) expression is elevated in thyroid carcinomas. bFGF may act as mitogen to thyrocytes producing hyperplasia and could play a role as an angiogenic agent (42, 43, 44) . As in endothelial cells, bFGF was able to stimulate APN/CD13 expression in 1736 cells. Addition of 10% FCS to endothelial cells increased the APN/CD13 expression (6) ; however, FCS treatment decreased the APN/CD13 mRNA level in the anaplastic thyroid carcinoma cell line 1736. Information regarding the specific pathways regulated by IL-6, EGF, or bFGF that induce APN/CD13 transcription will be important for understanding the mechanism of APN/CD13 expression on anaplastic thyroid carcinomas.

To study the function of APN/CD13 in thyroid carcinoma, we stably transfected FTC-133 cells with an expression vector for APN/CD13. APN/CD13-overexpressing FTC-133 cells showed an increased motility. The motility of FTC-133 APN-GFP cells could be inhibited by the antibody WM15 (enzyme activity inhibiting) and by the competitive inhibitor bestatin. Bestatin (Ubenimex) is an antibiotic with inhibitory activity of some but not all aminopeptidases (45) ; i.e., it has immunomodulatory properties and can act as an antitumor agent (46, 47, 48, 49) . The inhibition of other aminopeptidases could be the reason for the inhibitory effect of bestatin on the motility of the FTC-GFP control cells, whereas the anti-APN/CD13 antibody WM15 did not affect the motility of FTC-133 GFP cells. The WM15 antibody has neutralizing activity for APN/CD13, showing that the epitope might be involved at the active site of the metal-chelating domain of the enzyme (50 , 51) . In melanoma, APN/CD13 plays a role in invasion of basement membranes that could be inhibited by a specific antibody and the inhibitors of APN/CD13, bestatin, and amastatin, and APN/CD13 is involved in degradation of collagen IV (9 , 52 , 53) . Ishii et al. (54) reported a high expression of APN/CD13 in cancerous prostate and suggested a function in invasion and metastasis. In colon cancer, APN/CD13 may be an indicator of a poor prognosis for node-positive patients, and it also participates in cell motility and angiogenesis (10) . Investigations by Pasqualini et al. (11) and Curnis et al. (13) demonstrated the involvement of APN/CD13 in angiogenesis and the capability of this peptidase to function as a target for delivering drugs into tumors and for inhibiting angiogenesis.

For the first time, we report that overexpression of APN/CD13 in thyroid carcinomas is associated with down-regulation of both NDRG1 and ME491/CD63 gene expression. N-myc downstream-regulated gene-1 (NDRG1/Drg-1) was originally identified as a gene up-regulated during cellular differentiation (55 , 56) . The transcription factors N-myc and c-myc were shown to repress the expression of the mouse orthologue (57) . In tumors, NDRG1 expression is reduced, restoration of NDRG1 expression results in a growth-inhibitory effect, and its overexpression causes morphological changes in metastatic colon carcinoma cells suggesting increased differentiation (58, 59, 60) . NDGR1 may be involved in the formation of the E-cadherin/catenin complex (58 , 61) . ME491/CD63 tetraspanin, a member of the transmembrane-4 superfamily, has been reported to suppress tumor progression and metastasis (62, 63, 64) . The transmembrane tetraspanins can form complexes with various integrins, and assembly of these complexes could modulate the function of cell surface receptors in migration and tumor invasion (65, 66, 67, 68) . Thus, our results support the assumption that APN/CD13-associated down-regulation of NDRG1 and ME491/CD63 in thyroid carcinoma cells is an important step of tumor progression to more malignant phenotypes. These results also underline the important role of APN/CD13 as mediator in a multimolecular process regulating cell migration. The pathways by which down-regulation of these two genes contributes to tumorigenesis are still unclear. No functional link is known between APN/CD13 and NDRG1 or ME491/CD63. As shown schematically in Fig. 9Citation , APN/CD13 could signal via cleavage or modulation of peptide substrates or via their localization in membrane microdomains (69) . Membrane microdomains are discussed as platforms for assembly of signaling complexes and play an important role in signaling regulation. Many of the known physiological substrates of APN/CD13 act via G-protein-coupled receptors known to be microdomain associated. However, APN/CD13 might act as a negative regulator by cleavage or modulation of substrate peptides. Our group could report that APN/CD13 is linked directly to signal transduction pathways in monocytes (70) . Pathways that link microdomains and NDRG-1 with two other proteins known to be involved in thyroid carcinomas, i.e., PPAR{gamma} and PTEN, are clearer. Both PPAR{gamma} and PTEN are dysregulated in thyroid carcinomas, can be found in microdomains, and are able to regulate the expression of NDRG-1 (58 , 71) . Whether direct links exist between APN/CD13 and PTEN or PPAR{gamma}, respectively, is a part of our ongoing investigation.



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  		Fig. 9. Model of APN/CD13 signaling showing putative pathways involved in down-regulation of NDRG1 and ME491/CD63. See text for details.

 
In summary, we could show that APN/CD13 is involved in cell motility of thyroid carcinoma cells, and it seems to be a marker for dedifferentiation. Additional studies of the function of APN/CD13 may open new insights in the processes of tumor invasion, metastasis, and angiogenesis.


   ACKNOWLEDGMENTS
 
We thank S. Fuhrmann, K. Hammje, I. Peters, and C. Sauer for excellent technical support; B. Korant (Wilmington, DE) for critical reading of the manuscript; and O. Gimm (Halle, Germany) for discussions.


   FOOTNOTES
 
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.

Requests for reprints: Astrid Kehlen, Martin Luther University Halle-Wittenberg, Institute of Medical Immunology, Magdeburger Strasse 2, D-06097 Halle, Germany. Phone: 49-345-5574736; Fax: 49-345-5574055; E-mail: astrid.kehlen{at}medizin.uni-halle.de

4 The abbreviations used are: PTC, papillary thyroid carcinoma; APN, aminopeptidase N; DPIV, dipeptidyl peptidase IV; UTC, undifferentiated anaplastic thyroid carcinoma; FTC, follicular thyroid carcinoma; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; IL, interleukin; TNF, tumor necrosis factor; EGFP, enhanced green fluorescent protein; siRNA, small interfering RNA; TGF, transforming growth factor; mAb, monoclonal antibody; MFI, mean fluorescence intensity; RT-PCR, reverse transcription-PCR; GFP, green fluorescent protein; PPAR, peroxisome proliferator-activated receptor; PTEN, phosphatase and tensin homologue deleted from chromosome 10. Back

5 Astrid Kehlen, unpublished results. Back

Received 6/16/03. Revised 8/28/03. Accepted 9/ 9/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gimm O. Thyroid cancer. Cancer Lett., 163: 143-156, 2001.[Medline]
  2. Liotta L. A., Tryggvason K., Garbisa S., Hart I., Foltz C. M., Shafie S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature (Lond.), 284: 67-68, 1980.[Medline]
  3. Iwata S., Morimoto C. CD26/dipeptidyl peptidase IV in context. The different roles of a multifunctional ectoenzyme in malignant transformation. J. Exp. Med., 190: 301-306, 1999.[Free Full Text]
  4. Shipp M. A., Look A. T. Hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key!. Blood, 82: 1052-1070, 1993.[Free Full Text]
  5. Riemann D., Kehlen A., Langner J. CD13: not just a marker in leukemia typing. Immunol. Today, 20: 83-88, 1999.[Medline]
  6. Bhagwat S. V., Lahdenranta J., Giordano R., Arap W., Pasqualini R., Shapiro L. H. CD13/APN is activated by angiogenic signals and is essential for capillary tube formation. Blood, 97: 652-659, 2001.[Abstract/Free Full Text]
  7. Thiele K., Riemann D., Navarrete S. A., Langner J., Kehlen A. Cell-cell contact of human T cells with fibroblasts changes lymphocytic mRNA expression: increased mRNA expression of interleukin-17 and interleukin-17 receptor. Eur. Cytokine Netw., 11: 53-58, 2000.[Medline]
  8. Weber M., Uguccioni M., Ochensberger B., Baggiolini M., Clark-Lewis I., Dahinden C. A. Monocyte chemotactic protein MCP-2 activates human basophil and eosinophil leukocytes similar to MCP-3. J. Immunol., 154: 4166-4172, 1995.[Abstract]
  9. Saiki I., Fujii H., Yoneda J., Abe F., Nakajima M., Tsuruo T., Azuma I. Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation. Int. J. Cancer, 54: 137-143, 1993.[Medline]
  10. Hashida H., Takabayashi A., Kanai M., Adachi M., Kondo K., Kohno N., Yamaoka Y., Miyake M. Aminopeptidase N is involved in cell motility and angiogenesis: its clinical significance in human colon cancer. Gastroenterology, 122: 376-386, 2002.[Medline]
  11. Pasqualini R., Koivunen E., Kain R., Lahdenranta J., Sakamoto M., Stryhn A., Ashmun R. A., Shapiro L. H., Arap W., Ruoslahti E. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res., 60: 722-727, 2000.[Abstract/Free Full Text]
  12. Curnis F., Arrigoni G., Sacchi A., Fischetti L., Arap W., Pasqualini R., Corti A. Differential binding of drugs containing the NGR motif to CD13 isoforms in tumor vessels, epithelia, and myeloid cells. Cancer Res., 62: 867-874, 2002.[Abstract/Free Full Text]
  13. Curnis F., Sacchi A., Borgna L., Magni F., Gasparri A., Corti A. Enhancement of tumor necrosis factor {alpha} antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nat. Biotechnol., 18: 1185-1190, 2000.[Medline]
  14. De Meesters I., Korom S., Van Damme J., Scharpe S. CD26, let it cut or cut it down. Immunol. Today, 20: 367-375, 1999.[Medline]
  15. Morimoto C., Schlossman S. F. The structure and function of CD26 in the T-cell immune response. Immunol. Rev., 161: 55-70, 1998.[Medline]
  16. Nemoto E., Sugawara S., Takada H., Shoji S., Horiuch H. Increase of CD26/dipeptidyl peptidase IV expression on human gingival fibroblasts upon stimulation with cytokines and bacterial components. Infect. Immun., 67: 6225-6233, 1999.[Abstract/Free Full Text]
  17. Gaetaniello L., Fiore M., de Filippo S., Pozzi N., Tamasi S., Pignata C. Occupancy of dipeptidyl peptidase IV activates an associated tyrosine kinase and triggers an apoptotic signal in human hepatocarcinoma cells. Hepatology, 27: 934-942, 1998.[Medline]
  18. Hanski C., Huhle T., Reutter W. Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen. Biol. Chem. Hoppe-Seyler, 366: 1169-1176, 1985.[Medline]
  19. 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., 217: 341-348, 1995.[Medline]
  20. Cheng H. C., Abedl-Ghany M., Elble R. C., Pauli B. U. Lung endothelial dipeptidyl peptidase IV promotes adhesion and metastasis of rat breast cancer cells via tumor cell surface-associated fibronectin. J. Biol. Chem., 273: 24207-24215, 1998.[Abstract/Free Full Text]
  21. Wesley U. V., Albino A. P., Tiwari S., Houghton A. N. A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells. J. Exp. Med., 190: 311-322, 1999.[Abstract/Free Full Text]
  22. Pethiyagoda C. L., Welch D. R., Fleming T. P. Dipeptidyl peptidase IV (DPPIV) inhibits cellular invasion of melanoma cells. Clin. Exp. Metastasis, 18: 391-400, 2000.[Medline]
  23. Kajiyama H., Kikkawa F., Suzuki T., Shibata K., Ino K., Mizutani S. Prolonged survival and decreased invasive activity attributable to dipeptidyl peptidase IV overexpression in ovarian carcinoma. Cancer Res., 62: 2753-2757, 2002.[Abstract/Free Full Text]
  24. Aratake Y., Kotani T., Tamura K., Araki Y., Kuribayashi T., Konoe K., Ohtaki S. Dipeptidyl aminopeptidase IV staining of cytologic preparations to distinguish benign from malignant thyroid diseases. Am. J. Clin. Pathol., 96: 306-310, 1991.[Medline]
  25. Kotani T., Aratake Y., Ogata Y., Umeki K., Araki Y., Hirai K., Kuma K., Ohtaki S. Expression of dipeptidyl aminopeptidase IV activity in thyroid carcinoma. Cancer Lett., 57: 203-208, 1991.[Medline]
  26. Kotani T., Kawano J., Suganuma T., Hirai K., Umeki K., Aratake Y., Konoe K., Ohtaki S. Immunohistochemical localization of dipeptidyl aminopeptidase IV in thyroid papillary carcinoma. Int. J. Exp. Pathol., 73: 215-222, 1992.[Medline]
  27. Iwabuchi H., Toriya K., Mimura T., Tamai S., Ito K., Kato H. Staining for dipeptidyl aminopeptidase IV activity in nodular thyroid diseases. Acta Cytol., 40: 158-163, 1996.[Medline]
  28. Sobin L. H., Fleming I. D. TNM Classification of Malignant Tumors, 5th ed. (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer (Phila.), 80: 1803-1804, 1997.
  29. Hoelting T., Zielke A., Siperstein A. E., Clark O. H., Duh Q. Y. Aberrations of growth factor control in metastatic follicular thyroid cancer in vitro. Clin. Exp. Metastasis, 12: 315-323, 1994.[Medline]
  30. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159, 1987.[Medline]
  31. Kehlen A., Thiele K., Riemann D., Langner J. Expression, modulation and signalling of IL-17 receptor in fibroblast-like synoviocytes of patients with rheumatoid arthritis. Clin. Exp. Immunol., 127: 539-546, 2002.[Medline]
  32. Bustin S. A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol., 25: 169-193, 2000.[Abstract]
  33. Aratake Y., Umeki K., Kiyoyama K., Hinoura Y., Sato S., Ohno A., Kuribayashi T., Hirai K., Nabeshima K., Kotani T. Diagnostic utility of galectin-3 and CD26/DPPIV as preoperative diagnostic markers for thyroid nodules. Diagn. Cytopathol., 26: 366-372, 2002.[Medline]
  34. Kehlen A., Gohring B., Langner J., Riemann D. Regulation of the expression of aminopeptidase A, aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 in renal carcinoma cells and renal tubular epithelial cells by cytokines and cAMP-increasing mediators. Clin. Exp. Immunol., 111: 435-441, 1998.[Medline]
  35. Kido A., Krueger S., Haeckel C., Roessner A. Possible contribution of aminopeptidase N (APN/CD13) to invasive potential enhanced by interleukin-6 and soluble interleukin-6 receptor in human osteosarcoma cell lines. Clin. Exp. Metastasis, 17: 857-863, 1999.[Medline]
  36. Basolo F., Fiore L., Pollina L., Fontanini G., Conaldi P. G., Toniolo A. Reduced expression of interleukin 6 in undifferentiated thyroid carcinoma: in vitro and in vivo studies. Clin. Cancer Res., 4: 381-387, 1998.[Abstract]
  37. Hoelting T., Goretzki P. E., Duh Q. Y. Follicular thyroid cancer cells: a model of metastatic tumor in vitro. Oncol. Rep., 8: 3-8, 2001.[Medline]
  38. Hoelting T., Siperstein A. E., Clark O. H., Duh Q. Y. Epidermal growth factor enhances proliferation, migration, and invasion of follicular and papillary thyroid cancer in vitro and in vivo. J. Clin. Endocrinol. Metab., 79: 401-408, 1994.[Abstract]
  39. Duh Q. Y., Clark O. H. Factors influencing the growth of normal and neoplastic thyroid tissue. Surg. Clin. N. Am., 67: 281-298, 1987.
  40. Westermark K., Westermark B., Karlsson F. A., Ericson L. E. Location of epidermal growth factor receptors on porcine thyroid follicle cells and receptor regulation by thyrotropin. Endocrinology, 118: 1040-1046, 1986.[Abstract]
  41. Tangada S. D., Peterson R. D., Funkhouser J. D. Regulation of expression of aminopeptidase N in fetal rat lung by dexamethasone and epidermal growth factor. Biochim. Biophys. Acta, 1268: 191-199, 1995.[Medline]
  42. Thompson S. D., Franklyn J. A., Watkinson J. C., Verhaeg J. M., Sheppard M. C., Eggo M. C. Fibroblast growth factors 1 and 2 and fibroblast growth factor receptor 1 are elevated in thyroid hyperplasia. J. Clin. Endocrinol. Metab., 83: 1336-1341, 1998.[Abstract/Free Full Text]
  43. Eggo M. C., Hopkins J. M., Franklyn J. A., Johnson G. D., Sanders D. S., Sheppard M. C. Expression of fibroblast growth factors in thyroid cancer. J. Clin. Endocrinol. Metab., 80: 1006-1011, 1995.[Abstract]
  44. Shingu K., Sugenoya A., Itoh N., Kato R. Expression of basic fibroblast growth factor in thyroid disorders. World J. Surg., 18: 500-505, 1994.[Medline]
  45. Scornik O. A., Botbol V. Bestatin as an experimental tool in mammals. Curr. Drug Metab., 2: 67-85, 2001.[Medline]
  46. Mathe G. Bestatin, an aminopeptidase inhibitor with a multi-pharmacological function. Biomed. Pharmacother., 45: 49-54, 1991.
  47. Yoneda J., Saiki I., Fujii H., Abe F., Kojima Y., Azuma I. Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin). Clin. Exp. Metastasis, 10: 49-59, 1992.[Medline]
  48. Furuse K., Fukuoka M., Genka K., Kato H., Tsubura E., Ohta M., Nakai H., Kinuwaki E., Nakano M., Ishikawa S., et al Double blind controlled study of ubenimex (Bestatin) against squamous cell lung cancer-a multicenter cooperative study. Gan To Kagaku Ryoho, 20: 1187-1194, 1993.[Medline]
  49. Ezawa K., Minato K., Dobashi K. Induction of apoptosis by ubenimex (Bestatin) in human non-small-cell lung cancer cell lines. Biomed. Pharmacother., 50: 283-289, 1996.[Medline]
  50. Helene A., Beaumont A., Roques B. P. Functional residues at the active site of aminopeptidase N. Eur. J. Biochem., 196: 385-393, 1991.[Medline]
  51. O’Connell P. J., Gerkis V., d’Apice A. J. Variable O-glycosylation of CD13 (aminopeptidase N). J. Biol. Chem., 266: 4593-4597, 1991.[Abstract/Free Full Text]
  52. Fujii H., Nakajima M., Saiki I., Yoneda J., Azuma I., Tsuruo T. Human melanoma invasion and metastasis enhancement by high expression of aminopeptidase N/CD13. Clin. Exp. Metastasis, 13: 337-344, 1995.[Medline]
  53. Menrad A., Speicher D., Wacker J., Herlyn M. Biochemical and functional characterization of aminopeptidase N expressed by human melanoma cells. Cancer Res., 53: 1450-1455, 1993.[Abstract/Free Full Text]
  54. Ishii K., Usui S., Sugimura Y., Yoshida S., Hioki T., Tatematsu M., Yamamoto H., Hirano K. Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion. Int. J. Cancer, 92: 49-54, 2001.[Medline]
  55. van Belzen N., Dinjens W. N., Diesveld M. P., Groen N. A., van der Made A. C., Nozawa Y., Vlietstra R., Trapman J., Bosman F. T. A novel gene which is up-regulated during colon epithelial cell differentiation and down-regulated in colorectal neoplasms. Lab. Investig., 77: 85-92, 1997.[Medline]
  56. Piquemal D., Joulia D., Balaguer P., Basset A., Marti J., Commes T. Differential expression of the RTP/Drg1/Ndr1 gene product in proliferating and growth arrested cells. Biochim. Biophys. Acta, 1450: 364-373, 1999.[Medline]
  57. Shimono A., Okuda T., Kondoh H. N-myc-dependent repression of ndr1, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant. Mech. Dev., 83: 39-52, 1999.[Medline]
  58. Guan R. J., Ford H. L., Fu Y., Li Y., Shaw L. M., Pardee A. B. Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer. Cancer Res., 60: 749-755, 2000.[Abstract/Free Full Text]
  59. van Belzen N., Dinjens W. N., Eussen B. H., Bosman F. T. Expression of differentiation-related genes in colorectal cancer: possible implications for prognosis. Histol. Histopathol., 13: 1233-1242, 1998.[Medline]
  60. Kurdistani S. K., Arizti P., Reimer C. L., Sugrue M. M., Aaronson S. A., Lee S. W. Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage. Cancer Res., 58: 4439-4444, 1998.[Abstract/Free Full Text]
  61. Lachat P., Shaw P., Gebhard S., van Belzen N., Chaubert P., Bosman F. T. Expression of NDRG1, a differentiation-related gene, in human tissues. Histochem. Cell Biol., 118: 399-408, 2002.[Medline]
  62. Hotta H., Hara I., Miyamoto H., Homma M. Overexpression of the human melanoma-associated antigen ME491 partially suppresses in vivo malignant phenotypes of H-ras-transformed NIH3T3 cells in athymic nude mice. Melanoma Res., 1: 125-132, 1991.[Medline]
  63. Kondoh M., Ueda M., Ichihashi M., Mishima Y. Decreased expression of human melanoma-associated antigen ME491 along the progression of melanoma pre-canceroses to invasive and metastatic melanomas. Melanoma Res., 3: 241-245, 1993.[Medline]
  64. Radford K. J., Mallesch J., Hersey P. Suppression of human melanoma cell growth and metastasis by the melanoma-associated antigen CD63 (ME491). Int. J. Cancer, 62: 631-635, 1995.[Medline]
  65. Sordat I., Decraene C., Silvestre T., Petermann O., Auffray C., Pietu G., Sordat B. Complementary DNA arrays identify CD63 tetraspanin and {alpha}3 integrin chain as differentially expressed in low and high metastatic human colon carcinoma cells. Lab. Investig., 82: 1715-1724, 2002.[Medline]
  66. Boucheix C., Rubinstein E. Tetraspanins. Cell. Mol. Life Sci., 58: 1189-1205, 2001.[Medline]
  67. Sauer G., Kurzeder C., Grundmann R., Kreienberg R., Zeillinger R., Deissler H. Expression of tetraspanin adaptor proteins below defined threshold values is associated with in vitro invasiveness of mammary carcinoma cells. Oncol. Rep., 10: 405-410, 2003.[Medline]
  68. Berditchevski F., Odintsova E. Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling. J. Cell Biol., 146: 477-492, 1999.[Abstract/Free Full Text]
  69. Nilsson M., Ekholm R., Fayet G., Maroux S., Ericson L. E. Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles. Cell Tissue Res., 250: 295-303, 1987.[Medline]
  70. Santos A. N., Langner J., Herrmann M., Riemann D. Aminopeptidase N/CD13 is directly linked to signal transduction pathways in monocytes. Cell Immunol., 201: 22-32, 2000.[Medline]
  71. Unoki M., Nakamura Y. Growth-suppressive effects of BPOZ and EGR2, two genes involved in the PTEN signaling pathway. Oncogene, 20: 4457-4465, 2001.[Medline]



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