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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
13:54

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Published on: October 21, 2012

Caspase activation by adenovirus e4orf4 protein is cell line specific and Is mediated by the death receptor pathway

A Livne1, R Shtrichman, T Kleinberger

  • 1The Gonda Center of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.

Journal of Virology
|January 3, 2001
PubMed

Insights

Adenovirus E4orf4 protein triggers apoptosis via the death receptor pathway in human cells. This process involves caspase-8 activation and reactive oxygen species generation, but not caspase-9.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Virology

Background:

  • Adenovirus E4orf4 protein induces apoptosis in transformed cells.
  • The E4orf4 apoptotic pathway was previously considered caspase-independent in certain cell lines.
  • The precise molecular mechanisms of E4orf4-induced apoptosis require further elucidation.

Purpose of the Study:

  • To investigate the role of caspases and the death receptor pathway in E4orf4-induced apoptosis in human cell lines.
  • To determine the involvement of reactive oxygen species (ROS) in the E4orf4 apoptotic cascade.

Main Methods:

  • Expression of Adenovirus E4orf4 protein in H1299 and 293T human cell lines.
  • Analysis of caspase activation, cytochrome c release, and apoptosis induction.
  • Utilized dominant-negative mutants of caspase-8 and FADD/MORT1.
  • Assessed ROS generation and its inhibition by Tiron.

Main Results:

  • E4orf4 induced caspase activation in H1299 and 293T cells.
  • Caspase activation was essential for apoptosis in 293T cells but not H1299 cells.
  • Inhibition of caspase-8 and FADD/MORT1 blocked E4orf4-induced apoptosis in 293T cells.
  • Cytochrome c was released, but caspase-9 was not required for apoptosis.
  • E4orf4-induced ROS accumulation was dependent on caspase-8 and FADD/MORT1.
  • Inhibition of ROS generation suppressed E4orf4-induced apoptosis.

Conclusions:

  • Adenovirus E4orf4 protein engages the death receptor pathway to induce apoptosis in human cells.
  • The E4orf4 apoptotic pathway involves caspase-8 activation and ROS generation.
  • Caspase-9 is not essential for E4orf4-mediated apoptosis, suggesting a distinct mechanism from the intrinsic pathway.

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