Shedding of membrane type 1 matrix metalloproteinase in a human breast carcinoma cell line

Takeshi Harayama, Eiko Ohuchi, Takanori Aoki, Hiroshi Sato, Motoharu Seiki, Yasunori Okada

研究成果: Article査読

25 被引用数 (Scopus)

抄録

Membrane type 1 matrix metalloproteinase (MT1-MMP) with a transmembrane domain is a new member of the MMP gene family and is expressed on the cell surfaces of many carcinoma cells to activate the zymogen of MMP-2 (gelatinase A). We have previously reported that MT1-MMP is released into culture media in a complex form with tissue inhibitor of metalloproteinases 2 (TIMP-2) from a human breast carcinoma cell line, MDA-MB-231, treated with concanavalin A (Con A). In the present study, we further studied the release mechanism of MT1-MMP. Immunoblot analysis indicated that the amounts of MT1-MMP in culture media increase with the time of exposure and the concentration of Con A, and those in cell lysates conversely decrease in a similar way. Time- and dose-dependent release of MT1-MMP into the media was confirmed by a sandwich enzyme immunoassay specific to MT1-MMP. The molecular weight of the immunoreactive MT1-MMP in the media was M(r) 56,000, which was 4000-M(r) smaller than that in the cell lysates. Northern blot analysis demonstrated that the mRNA expression level of MT1-MMP is about 3-fold enhanced after a 24 h-exposure to Con A and this is maintained up to 72-h exposure. The release of MT1-MMP from the Con A-treated cells was inhibited by metalloproteinase inhibitors such as EDTA and o-phenanthroline, but not by MMP inhibitors including TIMP-1, TIMP-2 and BB94 or other proteinase inhibitors of serine, cysteine and aspartic proteinases. During the Con A treatment of the cells, cell viability decreased time- and dose-dependently and dead cells reacted positively in the TdT-mediated dUTP Nick-End Labeling (TUNEL) method. Con A-treated MDA cells showed apoptotic morphology when stained with Hoechst dye and hematoxylin and eosin. DNA ladder formation was detected by electrophoresis of the DNA from Con A-treated MDA cells. These results suggest that MT1-MMP release from Con A-treated cells is due to shedding mediated by metalloproteinase(s) other than MMPs, and is associated with apoptosis.

本文言語English
ページ(範囲)942-950
ページ数9
ジャーナルJapanese Journal of Cancer Research
90
9
DOI
出版ステータスPublished - 1999 9

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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