Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis

A neo-epitope in cytokeratin 18 (CK18) that becomes available at an early c aspase cleavage event during apoptosis and is not detectable in vital epith elial cells is characterized. The monoclonal antibody M30, specific for thi s site, can be utilized specifically to recognize apoptotic cells, which sh ow cytoplasmic cytokeratin filaments and aggregates after immunohistochemis try with M30, while viable and necrotic cells are negative. The number of c ells recognized by the antibody increases after induction of apoptosis in e xponentially growing epithelial cell lines and immunoreactivity is independ ent of the phosphorylation state of the cytokeratins, The generation of the M30 neo-epitope occurs early in the apoptotic cascade, before annexin V re activity or positive DNA nick end labelling. In a flow cytometric assay, th e majority of the M30-positive cells appear in the 'apoptotic' subG1 peak. Tests with synthetic peptides define positions 387-396 of CK18, with a libe rated C-terminus at the caspase cleavage site DALD-S, as the ten-residue ep itope of M30, This epitope starts at the end of coil 2 of the predicted CK1 8 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site i n CK18 as a neo-epitope can be used for quantification of apoptotic epithel ial cells with immunocytochemical techniques and is applicable to both fres h and formalin-fixed material, Copyright (C) 1999 John Wiley & Sons, Ltd.