title: Absence of keratins 8 and 18 in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: Cell line selective effects on cell invasion.
abstract: Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 mRNA levels being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription and mRNA or protein stability.
authors.affiliation: Department of Molecular & Integrative Physiology, University of Michigan Medical School, 7744 Medical Science Building II, 1301 E. Catherine, Ann Arbor, MI 48109.
authors.forename: Raymond
authors.initials: R
authors.lastname: Kwan
authors.affiliation: Department of Molecular & Integrative Physiology, University of Michigan Medical School, 7744 Medical Science Building II, 1301 E. Catherine, Ann Arbor, MI 48109.
authors.forename: Kok Sun
authors.initials: KS
authors.lastname: Looi
authors.affiliation: Department of Molecular & Integrative Physiology, University of Michigan Medical School, 7744 Medical Science Building II, 1301 E. Catherine, Ann Arbor, MI 48109.|Ann Arbor Health System VA Medical Center.
authors.forename: M Bishr
authors.initials: MB
authors.lastname: Omary
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D004847:Epithelial Cells / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D053538:Keratin-18 / Q000096:biosynthesis / Q000235:genetics*; D053533:Keratin-8 / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D014746:Vimentin / Q000096:biosynthesis*
chemical_list: D053538:Keratin-18; D053533:Keratin-8; C486595:Krt8 protein, mouse; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D014746:Vimentin; D046988:Proteasome Endopeptidase Complex; D001374:Azacitidine
keywords: CT26 cells; IEC-6 cells; Keratin 18; Keratin 8; Vimentin