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Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells
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Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic

Neda R Kasim1, Kateřina Kuželová, Aleš Holoubek

  • 1Department of Biological Sciences, Kent State University, Kent, OH, USA.

Apoptosis : an International Journal on Programmed Cell Death
|January 18, 2013
PubMed
Summary
This summary is machine-generated.

Actinomycin D induces apoptosis in HeLa cells with distinct blebbing and compact stages. This programmed cell death involves early blebbing, later caspase activation, and significant apoptotic volume decrease (AVD) driven by Na(+) accumulation.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Apoptosis is a critical biological process involving programmed cell death.
  • Apoptotic Volume Decrease (AVD) is a hallmark of apoptosis, but its early mechanisms are not fully understood.
  • Actinomycin D is a known inducer of apoptosis in various cell types.

Purpose of the Study:

  • To investigate the morphological and temporal events during actinomycin D-induced apoptosis in HeLa cells.
  • To characterize the role of blebbing and ion fluxes in the apoptotic process.
  • To examine the activation of caspases and mitochondrial membrane potential changes.

Main Methods:

  • Live fluorescence and transmission-through-dye microscopy for real-time cell volume measurements.
  • Induction of apoptosis using actinomycin D in HeLa cells.
  • Monitoring of mitochondrial membrane potential, chromatin condensation, and caspase activation.

Main Results:

  • Actinomycin D-induced apoptosis in HeLa cells occurs in two stages: early blebbing and late compact shape.
  • Blebbing precedes caspase-3 and 7 activation, suggesting an independent early mechanism.
  • Apoptotic Volume Decrease (AVD) of approximately 40% is observed, accompanied by intracellular Na(+) accumulation.
  • Low-sodium medium inhibits apoptosis, and most ion channel blockers are ineffective in preventing AVD.

Conclusions:

  • Blebbing is an early, caspase-independent event in actinomycin D-induced apoptosis.
  • Na(+) influx plays a crucial role in driving both apoptosis and AVD.
  • The developed microscopy technique allows for precise quantification of AVD in live cells.