Gastroenterology

Gastroenterology

Volume 122, Issue 2, February 2002, Pages 376-386
Gastroenterology

Basic Research
Aminopeptidase N is involved in cell motility and angiogenesis: Its clinical significance in human colon cancer,☆☆

https://doi.org/10.1053/gast.2002.31095Get rights and content

Abstract

Background & Aims: The molecular basis of cell motility is highly complex and is controlled by a number of molecular systems, whereas angiogenesis is an important biological component of tumor progression. The aims of this study were to investigate the possible involvement of proteins at the cell surface in controlling cell motility and angiogenesis, and to identify the cell surface molecules involved in gastrointestinal tumors. Methods: We addressed these issues using functional monoclonal antibodies, which inhibit cell motility, endothelial cell migration, and tube formation. Furthermore, we investigated the relationship between this antigen and colon cancer, and showed the prognostic significance in human colon cancer. Results: We established a murine monoclonal antibody MH8-11, which inhibits cell motility and in vitro angiogenesis. This epitope was a 165-kilodalton protein, and the sequencing analysis revealed that it was almost identical to aminopeptidase N (APN)/cluster of differentiation (CD) 13. APN/CD13 expression was associated with tumor status (P = 0.025). The disease-free and overall survival rate for patients with positive APN/CD13 expression tumors was significantly lower than that for patients with negative APN/CD13 expression tumors (P = 0.014, 0.033, respectively). Among 47 node-positive patients, the survival rate of patients with negative APN/CD13 expression was better than that of those with positive APN/CD13 expression. Conclusions: Our data suggest that APN/CD13 is involved in cell motility and angiogenesis, and APN/CD13 expression may be a useful indicator of a poor prognosis for node-positive patients with colon cancer.

GASTROENTEROLOGY 2002;122:376-386

Section snippets

Immunization and antigen

HT1080 human fibrosarcoma cells were used as the antigen. Approximately 4 × 105 cells were injected 3 times intraperitoneally into a Balb/c mouse, which was injected 14 days later with an adjuvant antigen of about 4 × 105 cells. Hybridomas were produced 3 days after the last injection. Cell suspensions were prepared from the spleen of the mouse, and SP2 myeloma cells were fused with the aid of polyethylene glycol and hypoxanthine-aminopterin-thymidine selection as previously described.14

Selection of mAb and inhibition of cell motility

Cell

Selection of mAb MH8-11 and inhibition of HT1080 motility

About 5000 hybridoma supernatants were screened in the penetration assay. Five hybridomas were found to have a strong inhibitory effect on HT1080 motility, one of which was mAb MH8-11 with a dose-dependent effect resulting in constant inhibition at 5 μg/mL (Figure 1).

. Dose-dependent effect of mAb MH8-11 on cell penetration. Cell penetration was determined with HT1080-penetrating cells into the lower chamber of a Transwell. A constant inhibitory effect was produced by an mAb MH8-11 concentration

Discussion

We have established that functional mAb MH8-11 regulated cell motility and in vitro angiogenesis, and cloned the cDNA recognized by this mAb. Its sequence was almost identical to that of APN, which is produced by myeloid and other cells.23, 24 APN/CD13 is a zinc-dependent metalloprotease that binds to the membrane through an N-terminal segment and is encoded by a gene located on the long arm of chromosome 15 at bands q25-26. The cDNA sequence predicts 967 amino acids that are integral type II

Acknowledgements

The authors thank Drs. Tadashi Ohbayashi and Tomoko Okuno for the histologic examination of the tumor samples, and Mitsuko Shirata for her assistance in preparation of the manuscript.

References (37)

  • J Haier et al.

    Cell surface molecules and their prognostic values in assessing colorectal carcinomas

    Ann Surg

    (2000)
  • CR Boland et al.

    Colorectal cancer prevention and treatment

    Gastroenterology

    (2000)
  • M Miyake et al.

    A specific cell surface glycoconjugate controlling cell motility: evidence by functional monoclonal antibodies that inhibit cell motility and tumor cell metastasis

    Biochemistry

    (1991)
  • LA Liotta et al.

    Metastatic potential correlates with enzymatic degradation of basement membrane collagen

    Nature

    (1980)
  • I Saiki et al.

    Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation

    Int J Cancer

    (1993)
  • LA Liotta et al.

    Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane

    Cancer Res

    (1981)
  • BF Sloane et al.

    Cathepsin B activity in B16 melanoma cells: a possible marker for metastatic potential

    Cancer Res

    (1987)
  • R Pasqualini et al.

    Aminopeptidase N is a receptor for tumor-forming peptides and a target for inhibiting angiogenesis

    Cancer Res

    (2000)
  • Cited by (0)

    Address requests for reprints to: Masayuki Miyake M.D., Ph.D., Department V of Oncology and Department of Thoracic Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, 2-4-20, Ohgimachi, Kita-ku, Osaka 530-8480, Japan. e-mail: [email protected]; fax: (81) 6-6312-8816.

    ☆☆

    Supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (to M.M.; No. 12470280) and the Vehicle Racing Commemorative Foundation (to M.M.).

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