Mcl-1 dynamics influence mitotic slippage and death in mitosis

Abstract

Microtubule-binding drugs such as taxol are frontline treatments for a variety of cancers but exactly how they yield patient benefit is unclear. In cell culture, inhibiting microtubule dynamics prevents spindle assembly, leading to mitotic arrest followed by either apoptosis in mitosis or slippage, whereby a cell returns to interphase without dividing. Myeloid cell leukaemia-1 (Mcl-1), a pro-survival member of the Bcl-2 family central to the intrinsic apoptosis pathway, is degraded during a prolonged mitotic arrest and may therefore act as a mitotic death timer. Consistently, we show that blocking proteasome-mediated degradation inhibits taxol-induced mitotic apoptosis in a Mcl-1-dependent manner. However, this degradation does not require the activity of either APC/C-Cdc20, FBW7 or MULE, three separate E3 ubiquitin ligases implicated in targeting Mcl-1 for degradation. This therefore challenges the notion that Mcl-1 undergoes regulated degradation during mitosis. We also show that Mcl-1 is continuously synthesized during mitosis and that blocking protein synthesis accelerates taxol induced death-in-mitosis. Modulating Mcl-1 levels also influences slippage; overexpressing Mcl-1 extends the time from mitotic entry to mitotic exit in the presence of taxol, while inhibiting Mcl-1 accelerates it. We suggest that Mcl-1 competes with Cyclin B1 for binding to components of the proteolysis machinery, thereby slowing down the slow degradation of Cyclin B1 responsible for slippage. Thus, modulating Mcl-1 dynamics influences both death-in-mitosis and slippage. However, because mitotic degradation of Mcl-1 appears not to be under the control of an E3 ligase, we suggest that the notion of network crosstalk is used with caution.

Keywords: APC/C-Cdc20; Bcl-xL; Chromosome Section; FBW7; Taxol; spindle assembly checkpoint.

Figure 1. Mcl-1 is synthesized and degraded in mitosis

A. Cell fate profiles of RKO Cyclin B1 R42A cells following treatment with 0.1 μM taxol and 1 μg/ml tetracycline for 72 hours. B. Cell fate profiles of RKO Cyclin B1 R42A cells following a 24-hour transfection with Mcl-1 and Bcl-xL siRNAs before treatment with taxol, tetracycline (1 μg/ml) and MG132 (20 μM) or cycloheximide (CHX) (30 μg/ml) at 10 hours. The dotted line shows when MG132 and cycloheximide were added. Only cells that had entered mitosis 2.5 hours before drug addition were analyzed.

Figure 2. Analysis of E3 ligases implicated in mitotic degradation of Mcl-1

A. Cell fate profiles of RKO Cyclin B1 R42A cells treated with APC/C-Cdc20 inhibitors proTAME and/or Apcin for 72 hours using concentrations indicated. Zero hours represents mitotic entry. B. Cell fate profiles of RKO Cyclin B1 R42A cells co-treated with proTAME, Apcin and the Mps1 inhibitor AZ3146 for 72 hours. Zero hours represents mitotic entry. C. Cumulative death frequency of RKO Cyclin B1 R42A cells co-treated with taxol, tetracycline and proTAME/Apcin. Mann-Whitney U test, p > 0.05 D. Immunoblot of Mcl-1 levels in RKO Cyclin B1 R42A cells after 16 hours co-treatment with 0.1μM taxol, 1 μg/ml tetracycline and proTAME/Apcin at concentrations indicated. MG132 was added after 10 hours treatment. E. Cumulative death frequency graph of RKO Cyclin B1 R42A FBW7 RNAi cells exposed to proTAME/Apcin, 0.1μM taxol and 1 μg/ml tetracycline. Mann Whitney U test, ns p > 0.05. F. Immunoblot showing Mcl-1 and FBW7 levels in RKO Cyclin B1 R42A cells following transfection with FBW7 siRNA followed by exposure to proTAME/Apcin, 0.1μM taxol and 1 μg/ml tetracycline for 16 hours. Interphase sample was taken at zero hours.

Figure 3. Analysis of Mcl-1's putative D-box

A. Immunoblot of endogenous Mcl-1 (lower panel) and GFP-tagged Mcl1 (upper panel) following 24-hour 1 μg/ml tetracycline induction. B. Quantitation of time to mitotic death of RKO cells expressing Mcl-1^WT and Mcl-1^RALA following incubation with 0.1μM taxol and 1 μg/ml tetracycline. Zero hours represents mitotic entry. Mann Whitney U test, ns p > 0.05. C. Cumulative death frequency of RKO Cyclin B1 R42A cells transiently transfected with pLNCX2 plasmids expressing myc-tagged mCherry fused to Mcl-1 competitor fragments (a.a. 157-246) with or without the RALA mutation. Transfected cells were identified by fluorescent microscopy and tracked by phase contrast microscopy. Mann Whitney U test between mCherry WT and mCherry RALA, **** p < 0.0001. D. Quantitation of two independent immunoblots showing relative Mcl-1 levels in RKO Cyclin B1 R42A cells transiently transfected with the mCherry-Mcl-1 WT and RALA fragments exposed to 0.1 μM taxol for the times indicated.

Figure 4. Analysis of a lysine-less Mcl1

A. Immunoblots showing tetracycline-mediated induction of GFP-tagged wild type mouse Mcl-1 (WT) and a lysine-less mutant (ΔLys) whereby all 14 lysine residues are mutated to arginine. B. Quantitation showing time to death-in-mitosis for RKO cells expressing wild type Mcl-1 and the lysine-less mutant following exposure to 0.1 μM taxol. Transgenes were induced with 0.5 μg/ml tetracycline.

Figure 5. Mcl-1 levels influences slippage

A. Immunoblot showing Mcl-1 levels in DLD-1 cells arrested in mitosis. Cells were treated with AZ138 for 4 hours before mitotic shake-off into AZ138, then harvested at the times indicated. B. Immunoblot showing Mcl-1 and Bcl-xL levels following RNAi. C. Cell fate profiles of DLD-1 cells treated with AZ138 following Mcl-1 or Bcl-xL RNAi. Zero hours represents mitotic entry. D. Cumulative slippage frequency of DLD-1 cells treated with AZ138 following Mcl-1 RNAi. Mann Whitney U test, ** p < 0.01. E. Immunoblot of endogenous Mcl-1 and GFP-tagged Mcl-1 in DLD-1 cells following tetracycline induction. F and G. Cumulative slippage frequency of DLD-1 Mcl-1^WT and Mcl-1^RALA cells following induction with 0.5 μg/ml tetracycline and AZ138 exposure. Mann Whitney U test, **** p < 0.0001, ns p > 0.05.

Figure 6. Suppressing Mcl-1 rescues delayed slippage induced by Bax/Bak depletion

A. Immunoblot of Bak, Bax and Mcl-1 levels in DLD1 Mcl-1^WT cells following immunoprecipitation of GFP-AID-Mcl-1 induced with 0.5 μg/ml tetracycline. B. Immunoblot of Mcl-1, Bak and Bax levels 24 hours after transfection of the indicated siRNAs. C. Cumulative slippage frequency of DLD-1 cells treated with AZ138 following RNAi-mediated inhibition of Bak, Bax and Mcl-1. Mann Whitney U test, ** p < 0.01.

Figure 7. Overexpressing Mcl-1 levels delays both death in mitosis and slippage

A. Cell fate profiles and B, C, D quantification of time in mitosis for cells committed to death in mitosis (DiM), post-mitotic death or slippage in DLD-1 Mcl-1^WT and DLD-1 Mcl-1^RALA cell lines treated with 0.5 μg/ml tetracycline and 6.6 μM nocodazole. Zero hours represents mitotic entry. Mann Whitney U test, *** p < 0.001, ns p > 0.05.

Figure 8. Delaying slippage enhances post-mitotic apoptosis

A, B. Cell fate profiles of DLD-1 GFP-AID-Cyclin B1 R42A cells treated with 1 μM AZ138, 1 μg/ml tetracycline and 500 μM IAA. In panel (B) dotted lines show addition of IAA after 20 and 30 hours. C. Quantification of time to slippage. Mann Whitney U test, **** p < 0.0001.