Get access

Contribution of Helicobacter hepaticus Cytolethal Distending Toxin Subunits to Human Epithelial Cell Cycle Arrest and Apoptotic Death in vitro

Authors

  • Namal P.M. Liyanage,

    1. School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA
    2. Vaccine Branch, National Cancer Institute, Bethesda, MD, USA
    Search for more papers by this author
  • Rohana P. Dassanayake,

    1. School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA
    2. Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA
    Search for more papers by this author
  • Charles A. Kuszynski,

    1. Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA
    Search for more papers by this author
  • Gerald E. Duhamel

    Corresponding author
    1. School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA
    • Reprint requests to: Dr. Gerald E. Duhamel, T4 012a Veterinary Research Tower, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850, USA. E-mail: ged36@cornell.edu

    Search for more papers by this author

Abstract

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 CdtA, CdtB, and CdtC subunits produced by H. hepaticus CDT (HhepCDT) 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 INT407 cells intoxicated with HhepCDT holotoxin or reconstituted recombinant HhepCDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant HhepCDT protein subunits or increasing concentrations of individual recombinant HhepCDT protein subunits transfected into HeLa cells was assessed at 72 hours post-treatment by flow cytometry.

Results

Similar time course of HhepCDT-induced G2/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 HhepCDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of HeLa cells with HhepCdtB, but not with HhepCdtA or HhepCdtC, resulted in a dose-dependent G2/M arrest and apoptotic death.

Conclusion

All three subunits of HhepCDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and HhepCdtB subunit alone is necessary and sufficient for epithelial cell genotoxicity.

Ancillary