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

  • Research Article
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Tumor Biology

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.

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Abbreviations

ccK18:

Caspase-cleaved cytokeratin 18

K18:

Cytokeratin 18

M65:

Full-length cytokeratin 18

M30:

Caspase-cleaved cytokeratin 18

ATP:

Adenosine-5′-triphosphate

PARP:

Poly (ADP-ribose) polymerase

PI:

Propidium iodide

PBS:

Phosphate-buffered saline

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

ELISA:

Enzyme-linked immunosorbent assay

HRP:

Horseradish peroxidase

RPMI:

Roswell Park Memorial Institute medium, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ABTS:

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

FITC:

Fluorescein isothiocyanate

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Acknowledgments

This work was supported by BAP under grant no. KUAP(T)-2014/1. We appreciate the kind gift of the PC3 cell line from Assoc. Prof. Dr. Hakan Akca, Medical School of Pamukkale University, Turkey. F.M. Frame was funded by Yorkshire Cancer Research. Authors would like to thank Adam M. Hirst for critical evaluation of the manuscript.

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    Authors and Affiliations

    1. Department of Biology, Faculty of Arts and Sciences, Uludag University, Bursa, Turkey

      Buse Cevatemre & Mehmet Sarimahmut

    2. Department of Medical Biochemistry, Medical School, Uludag University, Bursa, Turkey

      Engin Ulukaya & Arzu Yilmaztepe Oral

    3. Department of Biology, YCR Cancer Research Unit, University of York, Heslington, North Yorkshire, YO10 5DD, UK

      Fiona M. Frame

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    1. Buse Cevatemre
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    Correspondence to Engin Ulukaya.

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    Buse Cevatemre and Engin Ulukaya contributed equally to this work.

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    Cevatemre, B., Ulukaya, E., Sarimahmut, M. et al. The M30 assay does not detect apoptosis in epithelial-derived cancer cells expressing low levels of cytokeratin 18. Tumor Biol. 36, 6857–6865 (2015). https://doi.org/10.1007/s13277-015-3367-5

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