Related Experiment Video

Updated: Oct 9, 2026

Evaluation of Caspase Activation to Assess Innate Immune Cell Death
10:23

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Published on: January 20, 2023

Caspase-3-dependent and caspase-3-independent pathways leading to chromatin DNA fragmentation in HL-60 cells

X W Meng1, M J Fraser, J M Feller

  • 1Department of Immunology and Rheumatology, Sydney Children's Hospital and School of Paediatrics, The University of New South Wales, Randwick, Australia.

Apoptosis : an International Journal on Programmed Cell Death
|March 3, 2001
PubMed

Insights

Etoposide-induced apoptosis in HL-60 cells involves caspases and CAD, while hydroxychloroquine triggers a caspase-3-independent pathway. A second nuclear nuclease is implicated in HCQ-induced DNA degradation.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Apoptosis is a crucial cellular process involving DNA fragmentation.
  • The nuclease CAD (caspase-activated DNase) is known to degrade DNA during apoptosis.
  • Different apoptotic stimuli may activate distinct molecular pathways.

Purpose of the Study:

  • To investigate the specific molecular mechanisms of apoptosis induced by etoposide (VP-16) and hydroxychloroquine (HCQ) in HL-60 cells.
  • To elucidate the roles of caspases and nucleases in VP-16 and HCQ-induced DNA fragmentation.
  • To identify potential differences in the apoptotic pathways activated by these two agents.

Main Methods:

  • HL-60 cells were treated with VP-16 or HCQ.
  • Apoptosis was assessed in the presence of broad-spectrum (Z-VAD-fmk) and caspase-3-specific (Z-DEVDfmk) inhibitors.
  • The effect of aurintricarboxylic acid (ATA) on apoptosis and DNA degradation was evaluated.
  • DNA degradation (laddering) was analyzed in isolated nuclei.

Main Results:

  • VP-16-induced apoptosis was caspase-dependent, requiring both caspase-3 and a second caspase for full CAD activation.
  • ATA did not inhibit VP-16-induced apoptosis, suggesting CAD's involvement.
  • HCQ-induced apoptosis was caspase-3 independent and inhibited by ATA, indicating CAD was not involved.
  • HCQ, but not VP-16, stimulated DNA degradation in isolated nuclei, pointing to a second, latent nuclear nuclease.

Conclusions:

  • VP-16 and HCQ induce apoptosis through distinct pathways in HL-60 cells.
  • VP-16 utilizes a caspase-dependent pathway involving CAD.
  • HCQ activates a caspase-3-independent pathway, likely involving a different nuclear nuclease for DNA degradation.

More Related Videos

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins10:24

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Published on: September 28, 2012

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation08:47

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

Published on: March 5, 2018

Related Concept Videos

Caspases01:24

Caspases

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside cells.
The Intrinsic Apoptotic Pathway01:31

The Intrinsic Apoptotic Pathway

Internal cellular stress, such as cellular injury or hypoxia, triggers intrinsic apoptosis. The B-cell lymphoma 2 (Bcl-2) family of proteins are the primary regulators of the intrinsic apoptotic pathway. For example, during DNA damage, checkpoint proteins, such as Ataxia Telangiectasia Mutated (ATM protein) and Checkpoints Factor-2 (Chk2) proteins, are activated. These proteins phosphorylate p53 which further activates pro-apoptotic proteins, such as Bax, Bak, PUMA, and Noxa, and inhibits...
DNA Damage Can Stall the Cell Cycle02:36

DNA Damage Can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
DNA Damage can Stall the Cell Cycle02:36

DNA Damage can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
The Extrinsic Apoptotic Pathway01:17

The Extrinsic Apoptotic Pathway

The extrinsic apoptotic pathway is initiated when extracellular death-inducing signals, such as specific cytokines, activate the death receptors expressed on the cell surface. The immune cells involved in this pathway are natural killer cells (NK cells) and cytotoxic T-lymphocytes. NK cells are critical in innate immune response, while cytotoxic T-lymphocytes are associated with adaptive immune response. These cells recognize specific receptors expressed on the altered cells and activate...