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Contribution of Helicobacter hepaticus Cytolethal Distending Toxin Subunits to Human Epithelial Cell Cycle Arrest and Apoptotic Death in vitro
Author(s) -
Liyanage Namal P.M.,
Dassanayake Rohana P.,
Kuszynski Charles A.,
Duhamel Gerald E.
Publication year - 2013
Publication title -
helicobacter
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.206
H-Index - 79
eISSN - 1523-5378
pISSN - 1083-4389
DOI - 10.1111/hel.12084
Subject(s) - apoptosis , hela , cytolethal distending toxin , flow cytometry , cell cycle , cell cycle checkpoint , programmed cell death , transfection , microbiology and biotechnology , biology , recombinant dna , cell culture , protein subunit , cell growth , cell , toxin , gene , biochemistry , microbial toxins , genetics
Background Cytolethal distending toxin ( CDT ) is the only known virulence factor found in H . hepaticus , the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide C dt A , C dt B , and C dt C subunits produced by H . hepaticus CDT (Hhep CDT ) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated. Materials and Methods The temporal relationship between cell cycle and apoptotic death of human epithelial HeLa and INT 407 cells intoxicated with Hhep CDT holotoxin or reconstituted recombinant Hhep CDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant Hhep CDT protein subunits or increasing concentrations of individual recombinant Hhep CDT protein subunits transfected into HeLa cells was assessed at 72 hours post‐treatment by flow cytometry. Results Similar time course of Hhep CDT ‐induced G 2 / M cell cycle arrest and apoptotic death was found with both cell lines which reached a maximum at 72 hours. The presence of all three H hep CDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of H e L a cells with H hep C dt B , but not with H hep C dt A or H hep C dt C , resulted in a dose‐dependent G 2 / M arrest and apoptotic death. Conclusion All three subunits of H hep CDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and H hep C dt B subunit alone is necessary and sufficient for epithelial cell genotoxicity.