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Rapid induction of apoptosis during Kinesin-5 inhibitor-induced mitotic arrest in HL60 cells
Yangzhong Tang1, James D Orth, Tiao Xie
1Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. yangzhong_tan@hms.harvard.edu
Abstract:
Small molecule inhibitors of Kinesin-5 (K5Is) that arrest cells in mitosis with monopolar spindles are promising anti-cancer drug candidates. Clinical trials of K5Is revealed dose-limiting neutropenia, or loss of neutrophils, for which the molecular mechanism is unclear. We investigated the effects of a K5I on HL60 cells, a human promyelocytic leukemia cell line that is often used to model dividing neutrophil progenitors in cell culture. We found K5I treatment caused unusually rapid death of HL60 cells exclusively during mitotic arrest. This mitotic death occurred via the intrinsic apoptosis pathway with molecular events that include cytochrome c leakage into the cytoplasm, caspase activation, and Parp1 cleavage. Bcl-2 overexpression protected from death. We probed mitochondrial physiology to find candidate triggers of cytochrome c release, and observed a decrease of membrane potential (ΔΨm) before mitochondrial outer membrane permeabilization (MOMP). Interestingly, this loss of ΔΨm was not blocked by overexpressing Bcl-2, suggesting it might be a cause of Bax/Bak activation, not a consequence. Taken together, these results show that K5I induces intrinsic apoptosis during mitotic arrest in HL60 with loss of ΔΨm as an upstream event of MOMP.
Insights
Small molecule Kinesin-5 inhibitors (K5Is) cause rapid HL60 cell death during mitotic arrest via intrinsic apoptosis. Mitochondrial membrane potential loss precedes outer membrane permeabilization, revealing a novel mechanism for anti-cancer drug toxicity.
Area of Science:
- Cell Biology
- Cancer Biology
- Pharmacology
Background:
- Small molecule Kinesin-5 inhibitors (K5Is) are promising anti-cancer agents, arresting cells in mitosis.
- Clinical K5I trials show dose-limiting neutropenia, but the mechanism remains unclear.
- HL60 cells model neutrophil progenitor division and were used to investigate K5I effects.
Purpose of the Study:
- To elucidate the molecular mechanism of K5I-induced cell death in HL60 cells.
- To investigate the role of apoptosis and mitochondrial function in K5I toxicity.
- To determine if K5I-induced neutropenia is linked to mitotic arrest cell death.
Main Methods:
- Treatment of HL60 cells with a K5I.
- Analysis of cell death pathways, including apoptosis markers (caspase activation, Parp1 cleavage).
- Assessment of mitochondrial physiology, including membrane potential (ΔΨm) and outer membrane permeabilization (MOMP).
Main Results:
- K5I treatment induced rapid HL60 cell death specifically during mitotic arrest.
- Cell death occurred via the intrinsic apoptosis pathway, involving cytochrome c release and caspase activation.
- A decrease in mitochondrial membrane potential (ΔΨm) was observed before MOMP and was not prevented by Bcl-2, suggesting it triggers Bax/Bak activation.
Conclusions:
- K5I induces intrinsic apoptosis in HL60 cells during mitotic arrest.
- Loss of mitochondrial membrane potential is an upstream event of MOMP in K5I-induced apoptosis.
- This study provides a potential mechanism for K5I-related neutropenia observed in clinical trials.
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