The M30 assay does not detect apoptosis in epithelial-derived cancer cells expressing low levels of cytokeratin 18

Abstract

The primary aim of this study was to compare measurement of apoptosis by M30 immunoreactivity (a biomarker for apoptosis) to other apoptosis assays (morphological assessment of nuclei, Annexin-V-FITC staining, DNA fragmentation and PARP cleavage) in vitro. Caspase-cleaved cytokeratin 18 (M30, ccK18) is only produced in epithelial cells and is regarded as a pharmacodynamic biomarker of apoptotic cell death because it is released from cells during apoptosis induced by chemotherapeutic agents. However, we have observed false negative results using this assay in clinical samples. Therefore, we tested its ability to accurately detect apoptosis in a panel of lung cancer cell lines with a range of clinically approved chemotherapeutic drugs. Three different non-small cell lung cancer (NSCLC) cell lines (A549, H1299, PC3) were used to correlate M30 levels with alternate apoptosis assays. Following successful induction of apoptosis, the A549 cell line showed an increase in M30 levels along with other well-known features of apoptosis, whilst H1299 and PC3 cell lines did not show an increase in M30 levels, even when apoptosis was detected by other means. Further analysis showed that H1299 and PC3 cell lines expressed much lower levels of cytokeratin 18 protein compared to the A549 cell line. Our results suggest that reliable detection of apoptosis via the M30 assay only works when sufficient levels of cytokeratin 18 are present in the cells. This means that the M30 assay may result in false negative results for apoptosis, and as such, the ELISA should be used in conjunction with other assays.