Related Experiment Video

Updated: Jan 26, 2026

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel
08:29

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Published on: May 14, 2018

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An ATG5 knockout promotes paclitaxel resistance in v-Ha-ras-transformed NIH 3T3 cells

Seong Yun Eom1, Sung-Hee Hwang1, Hojin Yeom1

  • 1Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, 22012, Republic of Korea.

Biochemical and Biophysical Research Communications
|April 8, 2019
PubMed

Insights

Autophagy gene ATG5 knockout (KO) confers paclitaxel resistance in cancer cells. This resistance is linked to the inability to produce a key autophagy protein, impacting cell cycle arrest and apoptosis.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Cancer Research

Background:

  • Autophagy, a cellular degradation process, has complex roles in cell survival and death.
  • ATG5 (autophagy related 5) is crucial for autophagosome formation, a key step in autophagy.
  • Paclitaxel is a widely used chemotherapy drug that induces cell cycle arrest and apoptosis.

Purpose of the Study:

  • To investigate the role of ATG5 in paclitaxel sensitivity.
  • To determine if ATG5 knockout affects paclitaxel-induced cell cycle arrest and apoptosis.
  • To explore the mechanism underlying ATG5-mediated paclitaxel resistance.

Main Methods:

  • CRISPR/Cas9 gene editing to create ATG5 knockout (KO) Ras-NIH 3T3 cells.
  • LC3 immunoblot and qRT-PCR to confirm ATG5 gene knockout.
  • Flow cytometry to analyze cell cycle distribution and apoptosis.
  • Overexpression of ATG5 cleavage product to assess functional restoration.

Main Results:

  • ATG5 knockout rendered Ras-NIH 3T3 cells resistant to paclitaxel via an ATP-binding cassette transporter-independent mechanism.
  • Paclitaxel induced less G2/M cell cycle arrest in ATG5 KO cells compared to parental cells.
  • The proportion of early apoptotic cells was significantly reduced in ATG5 KO cells treated with paclitaxel.
  • Restoration of ATG5 N-terminal cleavage product re-sensitized ATG5 KO cells to paclitaxel.

Conclusions:

  • ATG5 is essential for paclitaxel sensitivity in Ras-NIH 3T3 cells.
  • ATG5 knockout-mediated paclitaxel resistance is associated with impaired G2/M arrest and reduced apoptosis.
  • The inability to produce the truncated ATG5 (tATG5) protein is responsible for paclitaxel resistance in ATG5 KO cells.
Keywords:
ATG5 knockoutAutophagyMulti-drug resistancePaclitaxel

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