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Cytochrome c release and endoplasmic reticulum stress are involved in caspase-dependent apoptosis induced by G418

Q H Jin1, B Zhao, X J Zhang

  • 1Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, 320 YueYang Road, 200031.

Cellular and Molecular Life Sciences : CMLS
|July 9, 2004
PubMed

Insights

G418 antibiotic induces apoptosis in kidney cells via caspase activation. Two independent pathways, mitochondrial and endoplasmic reticulum stress, initiate this caspase-dependent cell death, crucial for understanding transfection selection mechanisms.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • G418 is widely used in eukaryotic cell transfection to select for neomycin resistance.
  • The precise mechanism by which G418 induces cell death remains largely unknown.
  • Understanding G418's effects is critical for optimizing gene transfer and selection protocols.

Purpose of the Study:

  • To elucidate the mechanism of G418-induced apoptosis in normal rat kidney cells.
  • To identify the specific signaling pathways and molecular players involved in G418-induced cell death.

Main Methods:

  • Treatment of normal rat kidney cells with G418.
  • Assessment of apoptotic features (cell shrinkage, nuclear fragmentation, caspase-3 activation).
  • Analysis of DNA fragmentation (low-molecular DNA ladder).
  • Pharmacological inhibition of caspases (z-VAD-fmk, z-LEHD-fmk) and calpain (E-64d).
  • Confocal microscopy to detect cytochrome c release.
  • Measurement of intracellular Ca2+ concentration.
  • Western blot analysis for m-calpain and procaspase-12 cleavage.

Main Results:

  • G418 induced typical apoptotic features and caspase-3 activation without DNA laddering.
  • The pan-caspase inhibitor z-VAD-fmk completely blocked G418-induced apoptosis.
  • Two independent pathways initiated G418-induced apoptosis: mitochondrial cytochrome c release and endoplasmic reticulum stress (Ca2+ increase, m-calpain and caspase-12 cleavage).
  • Both pathways converged on caspase-9 activation; its inhibition partially blocked apoptosis.
  • Calpain inhibition (E-64d) enhanced the block induced by caspase-9 inhibition.
  • Cleavage of caspase-9 and caspase-12 required simultaneous inhibition of both caspases and calpain, indicating pathway independence.

Conclusions:

  • G418 triggers a caspase-dependent apoptotic process in kidney cells.
  • Apoptosis is initiated through two distinct, independent pathways: mitochondrial and ER stress.
  • These findings provide crucial insights into the molecular mechanisms underlying G418 selection in cell culture.

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