Rapid induction of apoptosis during Kinesin-5 inhibitor-induced mitotic arrest in HL60 cells

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.

Figure 1

Cumulative survival curves of 12 cell lines treated with EMD534085. Phase-contrast and DIC time-lapse microscopy are taken for adherent and suspension cells respectively. In general, cells from hematological lineage (red curves) are more sensitive than cells from epithelial lineage (black curves).

Figure 2

EMD534085 induces rapid cell death in HL60 during mitotic arrest. A. Histograms of DNA contents in HL60 cells after EMD534085 treatment for 0, 8, 16, 24, 32, and 48 hrs. Cells are fixed, stained with propidium iodide (PI), and analyzed on BD FACSCalibur. B. Sub-2N population increases as 4N population accumulates during drug treatment. C. DIC time-lapse movie frames of DMSO (a – e) and EMD534085 treated (f – j) HL60 cells. Arrows point at nucleoi in interphase cells, which disappear when cells enter mitosis. Drug treated HL60 cells die during mitotic arrest. D. HL60 cells finish normal mitosis in less than 1 hr, whereas EMD534085 treated cells get arrested in mitosis for an average of 5 hrs.

Figure 3

EMD534085-induced cell death in HL60 during mitotic arrest occurs by the intrinsic pathway. A. Caspase-8, −9, −3, −7 are activated; Parp1 is cleaved; Mcl1 and XIAP are degraded in EMD534085-treated HL60 cells. Cells were synchronized using single thymidine block, and then were released to either vehicle alone or 500nM EMD534085 supplemented growth medium. Cells were harvested 4, 6, 8, 10, 12, 14, and 16 hrs after thymidine release for immunoblotting. B. Cytochrome c translocates from mitochondrial inter-membrane space to the cytoplasm during mitotic arrest. cytochrome c – red; microtubule – green; DNA – blue. C. Bax is activated in HL60 during EMD534085 treatment. D. Bcl-2 overexpression protects HL60 cells from EMD534085 treatment. (*: p-value < 0.05)

Figure 4

Neither Bim nor Bid knock-down affects HL60’s apoptotic response to EMD534085 treatment. Lentiviruses produced from 4 different shRNA against Bim and 4 different shRNA against Bid were used to infect HL60 cells. Flow cytometry over time shows the extent and timing of death (sub-2N) after K5I in either Bim- or Bid-knockdown cells is unchanged compared to parental lines.

Figure 5

Mitochondrial membrane potential decreases before MOMP in EMD534085-treated HL60 cells. A. Representative immunofluorescence images of HL60/Neo and HL60/Bcl-2 cells, co-stained for microtubules, MitoTracker-Red, and cytochrome c, in the absence or presence of EMD534085. Mitochondria are normal in the DMSO vehicle-treated cells, but in some EMD534085-treated cells a significant decrease in mitochondrial membrane potential is evident. B. Box-and-whisker plot of all average MitoTracker-Red fluorescent intensities in randomly scored normal mitotic and pre-MOMP mitotic arrest cells in HL60/Neo, HL60/Bcl-2, HeLa, and MCF7 lines, in the absence or presence of EMD534085. Statistical analysis shows significantly lower ΔΨ_m in pre-MOMP mitotic arrest cells than normal mitotic cells in HL60/Neo, HL60/Bcl-2, and HeLa, but not in MCF7. (*: p-value < 0.0005) C. Proposed temporal events in the cell death response of HL60 cells to EMD534085.