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A Functional Proteomics Screen of Proteases In Colorectal Carcinoma

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Abstract

Background

Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout.

Materials and Methods

An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity.

Results

The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), ca-thepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metallopro-teases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by im-munohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor.

Conclusions

This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.

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Acknowledgments

The authors thank William Stetler-Stevenson, Bonnie Sloane, and Ellen Kick for reagents; the UCSF Liver Center Clinical Core for normal human liver samples; Mark Segal, Division of Biostatistics, UCSF, for consultation on statistical analysis; and Ramona Soto for preparation of the manuscript. This work was supported by a National Cancer Institute Program Project Grant #CA72006-02.

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Authors and Affiliations

  1. Department of Pathology, UCSF/VA Medical Center, 4150 Clement Street - 113B, San Francisco, CA, 94121, USA

    James H. McKerrow Ph.D., M.D., Vivek Bhargava, Elizabeth Hansell, Thomas Kuwahara, Mary Matley & Lisa Coussens

  2. Department of Surgery, University of California, San Francisco, CA, USA

    Robert Warren

  3. The Liver Center, University of California, San Francisco, CA, USA

    Sandra Huling

  4. The Hormone Research Laboratory, University of California, San Francisco, CA, USA

    Lisa Coussens

Authors
  1. James H. McKerrow Ph.D., M.D.
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  2. Vivek Bhargava
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  3. Elizabeth Hansell
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Correspondence to James H. McKerrow Ph.D., M.D..

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Communicated by F. E. Cohen.

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McKerrow, J.H., Bhargava, V., Hansell, E. et al. A Functional Proteomics Screen of Proteases In Colorectal Carcinoma. Mol Med 6, 450–460 (2000). https://doi.org/10.1007/BF03401787

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  • DOI: https://doi.org/10.1007/BF03401787

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