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Selective activation of caspases during apoptotic induction in HL-60 cells. Effects Of a tetrapeptide inhibitor
1Amgen Inc., Thousand Oaks, California 91320-1789, USA.
Abstract:
Apoptosis is a highly regulated biochemical process that results in the selective death of cells. Members of the caspase family of cysteine proteases play a pivotal role in the effector phase of apoptosis. We show that, in HL-60 cells, the addition of either anisomycin, a protein synthesis inhibitor, or geranylgeraniol, an intermediate in the cholesterol biosynthetic pathway, results in a rapid and en masse induction of apoptosis. The levels of actin, p42 and p44 MAPK, JNK1, JNK2, p38, and PCNA were not substantially altered during this process. Although these treatments appear to function by diverse pathways, they both result in the processing and activation of caspase-3 (CPP32beta/Yama/Apopain). In contrast, no activation of caspase-1 (interleukin-1beta converting enzyme (ICE)) was observed. Furthermore, we obtained ambiguous results regarding the activation of caspase-2 (Ich-1) depending on the antibody used. Pretreatment of the cells with benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD.fmk), a tetrapeptide inhibitor of caspases, prevented the induction of apoptosis for 24 h. Even after 72 h of treatment, some cells were still alive and progressing through the cell cycle, suggesting that blockage of caspase activity is able to protect cells. These results suggest that selective activation of some caspases is necessary to induce apoptosis in HL-60 cells.
Insights
Anisomycin or geranylgeraniol induce apoptosis in HL-60 cells by activating caspase-3. Blocking caspase activity with zVAD.fmk protects cells, indicating selective caspase activation is crucial for apoptosis.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Apoptosis is a regulated process of programmed cell death.
- Caspases, a family of cysteine proteases, are key mediators of apoptosis.
- HL-60 cells are a human leukemia cell line often used in apoptosis research.
Purpose of the Study:
- To investigate the role of caspases in apoptosis induction by anisomycin and geranylgeraniol in HL-60 cells.
- To determine which specific caspases are activated by these treatments.
- To assess the effect of caspase inhibition on apoptosis.
Main Methods:
- Induction of apoptosis in HL-60 cells using anisomycin or geranylgeraniol.
- Analysis of caspase activation (caspase-1, -2, and -3) using specific antibodies and Western blotting.
- Assessment of protein levels for actin, MAPKs, and PCNA.
- Inhibition of apoptosis using the caspase inhibitor zVAD.fmk.
Main Results:
- Both anisomycin and geranylgeraniol rapidly induced apoptosis in HL-60 cells.
- These treatments specifically activated caspase-3 (CPP32beta/Yama/Apopain) but not caspase-1 (ICE).
- Caspase-2 (Ich-1) activation results were ambiguous. Caspase inhibition by zVAD.fmk protected cells from apoptosis.
Conclusions:
- Selective activation of specific caspases, particularly caspase-3, is necessary for anisomycin- and geranylgeraniol-induced apoptosis in HL-60 cells.
- Caspase activity is essential for the execution of apoptosis in this model.
- These findings contribute to understanding the molecular mechanisms of apoptosis regulation.