Basic ResearchAminopeptidase N is involved in cell motility and angiogenesis: Its clinical significance in human colon cancer☆,☆☆
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).
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.
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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.
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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.).