title: 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.
authors.affiliation: Department of Biology, Faculty of Arts and Sciences, Uludag University, Bursa, Turkey.
authors.forename: Buse
authors.initials: B
authors.lastname: Cevatemre
authors.affiliation: Department of Medical Biochemistry, Medical School, Uludag University, Bursa, Turkey. eulukaya@uludag.edu.tr.
authors.forename: Engin
authors.initials: E
authors.lastname: Ulukaya
authors.affiliation: Department of Biology, Faculty of Arts and Sciences, Uludag University, Bursa, Turkey.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Sarimahmut
authors.affiliation: Department of Medical Biochemistry, Medical School, Uludag University, Bursa, Turkey.
authors.forename: Arzu Yilmaztepe
authors.initials: AY
authors.lastname: Oral
authors.affiliation: Department of Biology, YCR Cancer Research Unit, University of York, Heslington, North Yorkshire, YO10 5DD, UK.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Frame
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053538:Keratin-18 / Q000096:biosynthesis* / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D053538:Keratin-18; C561463:M30 cytokeratin-18 peptide, human; D010446:Peptide Fragments; D020169:Caspases
keywords: Anticancer drugs; Apoptosis; Lung cancer; Serum biomarker
references.citation: J Cell Sci. 1993 Jun;105 ( Pt 2):433-44
references.pmid: 7691841
references.citation: Cancer Res. 2004 Mar 1;64(5):1751-6
references.pmid: 14996736
references.citation: Invest New Drugs. 2002 Aug;20(3):253-9
references.pmid: 12201488
references.citation: Br J Cancer. 2010 Feb 2;102(3):577-82
references.pmid: 20051949
references.citation: Lung Cancer. 2007 Jun;56(3):399-404
references.pmid: 17316892
references.citation: Exp Cell Res. 2004 Jul 1;297(1):11-26
references.pmid: 15194421
references.citation: Cancer Res. 1995 Nov 15;55(22):5276-82
references.pmid: 7585588
references.citation: Tumour Biol. 2012 Dec;33(6):2201-8
references.pmid: 22890829
references.citation: Br J Pharmacol. 2008 Feb;153(4):646-56
references.pmid: 17876307
references.citation: Eur J Surg Oncol. 2009 Nov;35(11):1164-8
references.pmid: 19254831
references.citation: Biomed Pharmacother. 2014 May;68(4):477-82
references.pmid: 24755119
references.citation: Int Immunopharmacol. 2009 May;9(5):645-8
references.pmid: 19249390
references.citation: Clin Cancer Res. 2008 Feb 1;14(3):804-10
references.pmid: 18245542
references.citation: Nature. 1998 Jan 1;391(6662):96-9
references.pmid: 9422513
references.citation: Expert Rev Mol Diagn. 2010 Apr;10(3):353-9
references.pmid: 20370591
references.citation: J Natl Cancer Inst. 1992 Feb 5;84(3):165-74
references.pmid: 1371813
references.citation: J Cell Biol. 2000 Mar 20;148(6):1239-54
references.pmid: 10725337
references.citation: Int Immunopharmacol. 2010 Jul;10(7):719-22
references.pmid: 20362077
references.citation: Cell. 1997 Apr 18;89(2):175-84
references.pmid: 9108473
references.citation: Am J Pathol. 1993 Dec;143(6):1667-78
references.pmid: 7504888
references.citation: Clin Exp Med. 2013 Nov;13(4):289-95
references.pmid: 22825587
references.citation: Eur Arch Otorhinolaryngol. 1997;254(8):376-83
references.pmid: 9332893
references.citation: J Pathol. 1999 Apr;187(5):567-72
references.pmid: 10398123
references.citation: PLoS One. 2013 May 10;8(5):e64278
references.pmid: 23675532
references.citation: Biomed Pharmacother. 2005 Oct;59 Suppl 2:S359-62
references.pmid: 16507409
references.citation: Clin Transl Oncol. 2012 May;14(5):356-61
references.pmid: 22551541
references.citation: Nature. 1998 Jan 1;391(6662):43-50
references.pmid: 9422506
references.citation: PLoS One. 2013;8(1):e53532
references.pmid: 23341946
references.citation: Br J Cancer. 2006 Jun 5;94(11):1592-8
references.pmid: 16685278
references.citation: Neoplasia. 2008 Oct;10(10):1041-8
references.pmid: 18813353
references.citation: Anticancer Drugs. 2013 Jul;24(6):577-86
references.pmid: 23542749
references.citation: Annu Rev Biochem. 1994;63:345-82
references.pmid: 7979242
references.citation: Am J Pathol. 2009 Aug;175(2):808-16
references.pmid: 19628770
references.citation: Clin Cancer Res. 2007 Jun 1;13(11):3198-206
references.pmid: 17545523
references.citation: Cancer Invest. 2006 Nov;24(7):669-76
references.pmid: 17118776
references.citation: BMC Cancer. 2009 Mar 23;9:88
references.pmid: 19302716
references.citation: Clin Cancer Res. 1996 Nov;2(11):1879-85
references.pmid: 9816144
references.citation: Br J Cancer. 2009 Aug 4;101(3):410-7
references.pmid: 19603019
references.citation: Mol Cancer Res. 2005 Jul;3(7):365-71
references.pmid: 16046547
references.citation: J Cell Biol. 1997 Sep 22;138(6):1379-94
references.pmid: 9298992