No conflicts of interest were declared.
Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation†
Article first published online: 27 FEB 2008
Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
The Journal of Pathology
Volume 215, Issue 2, pages 164–174, June 2008
How to Cite
Tao, G.-Z., Li, D., Zhou, Q., Toivola, D., Strnad, P., Sandesara, N., Cheung, R., Hong, A. and Omary, M. (2008), Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation. J. Pathol., 215: 164–174. doi: 10.1002/path.2344
- Issue published online: 29 APR 2008
- Article first published online: 27 FEB 2008
- Accepted manuscript online: 27 FEB 2008 12:00AM EST
- Manuscript Accepted: 20 FEB 2008
- Manuscript Revised: 13 FEB 2008
- Manuscript Received: 11 JUL 2007
- National Institutes of Health, USA. Grant Number: DK47918
- National Institutes of Health Digestive Disease Center, USA. Grant Number: DK56339
- intermediate filaments;
- keratin 18;
- keratin 19;
- keratin 20;
- site-specific antibodies;
- protein degradation
Keratins 18 and 19 (K18/K19) are epithelial-specific intermediate filament proteins. Apoptosis induces caspase cleavage at the highly conserved K18 or K19 Asp237, which in K18 is preceded by cleavage at Asp396. We characterized the keratin N-terminal fragments that are generated upon caspase digestion of K18/K19 at Asp237 in order to study keratin dynamics during apoptosis. This was carried out by generating and characterizing antibodies selective to K18/K19 Asp237. K18 or K19 peptides that expose Asp237 in 234VEVD were used for rabbit immunization. The generated antibodies recognized cleaved but not intact K18/K19, exclusively, as determined by blotting or immunofluorescence staining of apoptotic human HT29 cells or livers isolated from Fas-Ab-injected mice. Antibodies to K18/K19 Asp237 recognized the common VEVD-motif as determined by immunoblotting of cells transfected with K18, K19 or K20. The K18/K19 VEVD-directed antibodies demonstrated sequential Asp396 then Asp237 K18 cleavage during apoptosis. Specific-keratin selectivity of the anti-Asp237 antibodies was confirmed by their inability to recognize K14 after UV-induced apoptosis in transfected cells. The Asp237-containing apoptotic keratin fragments are secreted into the medium of cultured HT29 cells and are stable up to 96 h after inducing apoptosis. Furthermore, the generated antibodies recognize keratin apoptotic fragments in sera of mice undergoing hepatocyte apoptosis and sera of patients with cirrhosis, and also recognize apoptotic cells in various epithelial human tumours. Therefore, the N-terminal caspase-generated K18 fragment is stable in tissues and biological fluids. The Asp237-directed antibodies provide a powerful tool to study apoptosis in human and mouse tissues, cells and serum, using a broad range of detection modalities. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.