A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera

Apoptosis. 2003 Jun;8(3):263-8. doi: 10.1023/a:1023672805949.


We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30. The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA. The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries. We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors. The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds. We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release. Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo. These products can be measured in sera from cancer patients. We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal
  • Antineoplastic Agents / classification
  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects*
  • Apoptosis / physiology
  • Aspartic Acid / metabolism
  • Caspases / metabolism
  • Cell Line, Tumor
  • Cytochromes c / metabolism
  • Drug Evaluation, Preclinical / instrumentation
  • Drug Evaluation, Preclinical / methods*
  • Enzyme Inhibitors / pharmacology
  • Enzyme-Linked Immunosorbent Assay / instrumentation
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Epitopes / immunology
  • Humans
  • Keratins / blood
  • Keratins / immunology
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Neoplasm Recurrence, Local / blood
  • Neoplasms / blood
  • Neoplasms / drug therapy*
  • Peptide Fragments / blood
  • Predictive Value of Tests
  • Reproducibility of Results
  • Treatment Outcome


  • Antibodies, Monoclonal
  • Antineoplastic Agents
  • Enzyme Inhibitors
  • Epitopes
  • Peptide Fragments
  • Aspartic Acid
  • Keratins
  • Cytochromes c
  • Caspases