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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.
Abstract:
Apoptosis induced by etoposide (VP-16) in HL-60 cells was confirmed to be caspase-dependent. It was fully inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk. However, the caspase-3-specific inhibitor Z-DEVDfmk only partially inhibited apoptosis. This indicated that a second caspase is required in vivo for full activation of the apoptotic nucease CAD. Aurin tricarboxylic acid (ATA) did not inhibit VP-16-induced apoptosis. In contrast, apoptosis induced by hydroxychloroquine (HCQ) in HL-60 cells was caspase-3 independent and was fully inhibited by ATA. Thus, CAD does not appear to be involved in chromatin DNA degradation in this case. A second apoptotic nuclease is postulated to degrade the DNA, likely endo- exonuclease, an abundant nuclear enzyme that acts on both DNA and RNA and is present in latent form. HCQ, but not VP-16, stimulated DNA degradation ("laddering") in isolated nuclei. This indicates that the drug can act directly in the nuclei to trigger activation of the second latent apoptotic nuclease.
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.