The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

Abstract

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.

Figure 1

Fcp1 affects the duration of SAC-dependent mitotic delay. (a and b) HeLa cells transfected with a control pool of nontargeting siRNAs (Control siRNAs), with a siRNA pool targeting the Fcp1 3'-UTR (Fcp1 siRNAs) or with Fcp1 siRNAs and a siRNA-resistant, Flag-tagged, wild-type Fcp1 (+ Fcp1wt) expression vector, were synchronized at pro-metaphase. Detached cells were collected and released into taxol-containing fresh medium (time 0). Cell samples were either (a) taken at indicated time points and extracts separated on SDS-PAGE, blotted and probed for the indicated antigens (MPM-2, mitotic phosphoepitopes; P-Y15-Cdk1; Cdk1; Cyc B1, cyclin B1) or (b) phase contrast-photographed at the indicated time points. Scale bars, 10 μm. The bar graph shows a quantitation of adapted cells expressed as the percent of input cells that reattached to the dish bottom at the indicated time points of incubation in the taxol-containing medium; error bars indicate variability within three independent experiments. (c) Extracts of control, Fcp1 siRNA-treated and Fcp1 siRNA-treated+Fcp1wt-transfected HeLa cells were probed for the indicated antigens. (d) Untransfected, pro-metaphase-synchronized HeLa cells were released into the taxol-containing fresh medium (time 0) and sampled at indicated time points during further incubation. Indicated antigens were directly probed on cell extract blots except for P-T239-Wee1 and Wee1 that were probed on blots of Wee1 immunoprecipitates (IPs) from the relative cell extracts. Cells were also phase contrast-photographed at the indicated time points. Scale bars, 10 μm. Bar graph, quantitation of adapted cells expressed as the percent of input cells that reattached to the dish bottom at the indicated time points of incubation in taxol-containing medium; error bars indicate variability within three independent experiments. Line graph, densitometric quantitation of cyclin B1 and P-T239-Wee1 signals expressed as percent of signal remaining from time 0 of incubation. (e) Fcp1 siRNA-treated and Fcp1 siRNA-treated+Fcp1wt-transfected HeLa cells, synchronized at pro-metaphase, were released into the taxol-containing fresh medium (time 0) and sampled at indicated time points during further incubation. Indicated antigens were probed on blots of Wee1 IPs from the relative cell extracts. The data shown are representative of at least three independent experiments

Figure 1

Fcp1 affects the duration of SAC-dependent mitotic delay. (a and b) HeLa cells transfected with a control pool of nontargeting siRNAs (Control siRNAs), with a siRNA pool targeting the Fcp1 3'-UTR (Fcp1 siRNAs) or with Fcp1 siRNAs and a siRNA-resistant, Flag-tagged, wild-type Fcp1 (+ Fcp1wt) expression vector, were synchronized at pro-metaphase. Detached cells were collected and released into taxol-containing fresh medium (time 0). Cell samples were either (a) taken at indicated time points and extracts separated on SDS-PAGE, blotted and probed for the indicated antigens (MPM-2, mitotic phosphoepitopes; P-Y15-Cdk1; Cdk1; Cyc B1, cyclin B1) or (b) phase contrast-photographed at the indicated time points. Scale bars, 10 μm. The bar graph shows a quantitation of adapted cells expressed as the percent of input cells that reattached to the dish bottom at the indicated time points of incubation in the taxol-containing medium; error bars indicate variability within three independent experiments. (c) Extracts of control, Fcp1 siRNA-treated and Fcp1 siRNA-treated+Fcp1wt-transfected HeLa cells were probed for the indicated antigens. (d) Untransfected, pro-metaphase-synchronized HeLa cells were released into the taxol-containing fresh medium (time 0) and sampled at indicated time points during further incubation. Indicated antigens were directly probed on cell extract blots except for P-T239-Wee1 and Wee1 that were probed on blots of Wee1 immunoprecipitates (IPs) from the relative cell extracts. Cells were also phase contrast-photographed at the indicated time points. Scale bars, 10 μm. Bar graph, quantitation of adapted cells expressed as the percent of input cells that reattached to the dish bottom at the indicated time points of incubation in taxol-containing medium; error bars indicate variability within three independent experiments. Line graph, densitometric quantitation of cyclin B1 and P-T239-Wee1 signals expressed as percent of signal remaining from time 0 of incubation. (e) Fcp1 siRNA-treated and Fcp1 siRNA-treated+Fcp1wt-transfected HeLa cells, synchronized at pro-metaphase, were released into the taxol-containing fresh medium (time 0) and sampled at indicated time points during further incubation. Indicated antigens were probed on blots of Wee1 IPs from the relative cell extracts. The data shown are representative of at least three independent experiments

Figure 2

Effects of the Fcp1-Wee1-Cdk1 axis on SAC-dependent mitotic delay. (a) Prior to be pro-metaphase–synchronized, HeLa cells were treated with either nontargeting siRNA (Cont siRNAs) or with siRNAs targeting Wee1 3′-UTR (Wee1 siRNAs). Wee1 siRNA-treated cells were also complemented with 3XFlag-tagged Wee1 wild-type or T239D mutant (F-Wee1wt and F-Wee-T239D) expression vectors. Pro-metaphase-synchronized cells were released into taxol-containing fresh medium (time 0), sample taken at indicated time points and extracts probed for indicated antigens. (b) Wee1 expression levels in cells treated as described in a. (c) Pro-metaphase-synchronized HeLa cells were released into the taxol-containing fresh medium and taken at indicated time points. Mock or Wee1 IPs (Mk; Wee1) were performed from cell extracts and probed for indicated antigens. (d) Wee1 siRNA-treated HeLa cells transfected with F-Wee1wt or F-Wee1-T239D expression vectors or an empty vector (Mk) were pro-metaphase-synchronized, released into the taxol-containing fresh medium (time 0) and sampled at indicated time points. Anti-Flag IPs were performed from cell extracts and probed for indicated antigens. (e) Pro-metaphase-synchronized HeLa cells were released into the taxol-containing fresh medium (time 0) in the absence (Control) or presence of the Wee1 inhibitor MK-1775. Cell samples were either taken at indicated time points of incubation and extracts probed for indicated antigens or photographed in phase contrast at indicated time points. Scale bars, 10 μm. Bar graph: quantitation of adapted cells expressed as the percent of input cells that reattached to the dish bottom at the indicated time points of incubation in the taxol-containing medium; error bars indicate variability within three independent experiments. (f) Pro-metaphase-synchronized HeLa cells were released into taxol- and MK-1775-containing fresh medium and split into two portions. After 1-h incubation, one portion received the cdk1 inhibitor RO-3306, and incubation was prolonged for another hour for all portions. Cell extracts were directly probed for indicated antigens or processed for Cdc27 IPs that were subsequently probed for Cdc27, Cdc20 and Mad2 (Mk IP; mock IPs). Cells were also photographed in phase contrast at indicated time points. Bar graph: quantitation of adapted cells expressed as the percent of input cells that reattached to the dish bottom at the indicated time points of incubation in the taxol-containing medium; error bars indicate variability within three independent experiments. Scale bars, 10 μm. The data shown are representative of at least three independent experiments

Figure 3

Wee1 downregulation enhances degradation of the antiapoptotic Mcl-1 protein, caspase activity and cell death under taxol regimen. (a) The same cell extracts of the experiment described in Figure 2a were probed for the Mcl-1 protein (to follow SAC adaptation kinetics, Cyc B1 and Cdk1 were also reported from the experiment described in Figure 2a). (b and c) HeLa cells were treated as described in Figure 2. Then, 36 h post pro-metaphase release, either (b) cell death was measured by trypan blue exclusion and expressed in the bar graph as percent of total cells (error bars indicate variability within three independent experiments) or (c) Annexin-V positivity was scored by flow cytometry. In c, panels of flow cytometry analysis for Annexin-V staining of cell samples from a representative experiment. Samples of Wee1 siRNA-treated and reconstituted with mutant F-Wee1-T239D cells were also incubated with the caspase inhibitor z-VAD-fmk or the necroapoptosis inhibitor necrostatin-1 from the time of taxol addition (Wee1 siRNA-F-Wee1-T239D +Tax+z-VAD and +Tax+necro, respectively). The bar graph shows a quantitation of Annexin-V positivity expressed as percent of flow cytometry-analysed cells (error bars indicate variability within three independent experiments). The cutoff to count for Annexin-V positivity was established by imposing P3 marker between the end of the bell-shaped part and the brighter tail part of Annexin-V signal distribution histograms from control siRNA-treated cell populations incubated in the absence of taxol. (d) HeLa cells were treated as described in Figure 2e. Mcl-1, cyclin B1 (Cyc B1) and cdk1 were probed from extracts of cell samples taken at the indicated time points. (e) Pro-metaphase-synchronized HeLa cells were released just into fresh medium (Control), into fresh medium supplemented with MK-1775 (MK), into taxol-containing fresh medium (Tax) or into taxol-containing fresh medium supplemented with MK-1775 (Tax+MK). Left top graph, caspase activity was determined from cell samples 24 h post pro-metaphase release. Left bottom graph, cell death was measured 36 h post pro-metaphase release by trypan blue exclusion and expressed as percent of total cells. Right panels, flow cytometry analysis panels of Annexin-V staining of cell samples measured 36 h post pro-metaphase release from a representative experiment. Cell samples treated with taxol and MK-1775 (Tax+MK) were also incubated with z-VAD-fmk (+z-VAD) or necrostatin-1 (+necro) from the time of Tax+MK addition. The cutoff to count for Annexin-V positivity was established by imposing P3 marker between the end of the bell-shaped part and the brighter tail part of Annexin-V signal distribution histograms of control cell populations incubated in the absence of other treatments post-prometaphase release. Right bottom graph, quantitation of Annexin-V positivity expressed as percent of flow cytometry-analysed cells. Error bars indicate variability within three independent experiments

Figure 3

Wee1 downregulation enhances degradation of the antiapoptotic Mcl-1 protein, caspase activity and cell death under taxol regimen. (a) The same cell extracts of the experiment described in Figure 2a were probed for the Mcl-1 protein (to follow SAC adaptation kinetics, Cyc B1 and Cdk1 were also reported from the experiment described in Figure 2a). (b and c) HeLa cells were treated as described in Figure 2. Then, 36 h post pro-metaphase release, either (b) cell death was measured by trypan blue exclusion and expressed in the bar graph as percent of total cells (error bars indicate variability within three independent experiments) or (c) Annexin-V positivity was scored by flow cytometry. In c, panels of flow cytometry analysis for Annexin-V staining of cell samples from a representative experiment. Samples of Wee1 siRNA-treated and reconstituted with mutant F-Wee1-T239D cells were also incubated with the caspase inhibitor z-VAD-fmk or the necroapoptosis inhibitor necrostatin-1 from the time of taxol addition (Wee1 siRNA-F-Wee1-T239D +Tax+z-VAD and +Tax+necro, respectively). The bar graph shows a quantitation of Annexin-V positivity expressed as percent of flow cytometry-analysed cells (error bars indicate variability within three independent experiments). The cutoff to count for Annexin-V positivity was established by imposing P3 marker between the end of the bell-shaped part and the brighter tail part of Annexin-V signal distribution histograms from control siRNA-treated cell populations incubated in the absence of taxol. (d) HeLa cells were treated as described in Figure 2e. Mcl-1, cyclin B1 (Cyc B1) and cdk1 were probed from extracts of cell samples taken at the indicated time points. (e) Pro-metaphase-synchronized HeLa cells were released just into fresh medium (Control), into fresh medium supplemented with MK-1775 (MK), into taxol-containing fresh medium (Tax) or into taxol-containing fresh medium supplemented with MK-1775 (Tax+MK). Left top graph, caspase activity was determined from cell samples 24 h post pro-metaphase release. Left bottom graph, cell death was measured 36 h post pro-metaphase release by trypan blue exclusion and expressed as percent of total cells. Right panels, flow cytometry analysis panels of Annexin-V staining of cell samples measured 36 h post pro-metaphase release from a representative experiment. Cell samples treated with taxol and MK-1775 (Tax+MK) were also incubated with z-VAD-fmk (+z-VAD) or necrostatin-1 (+necro) from the time of Tax+MK addition. The cutoff to count for Annexin-V positivity was established by imposing P3 marker between the end of the bell-shaped part and the brighter tail part of Annexin-V signal distribution histograms of control cell populations incubated in the absence of other treatments post-prometaphase release. Right bottom graph, quantitation of Annexin-V positivity expressed as percent of flow cytometry-analysed cells. Error bars indicate variability within three independent experiments

Figure 4

Wee1 inhibition enhances AMCD-dependent cell death in stabilized cell cultures and in primary human adult lymphoblastic leukemia specimens. (a) Asynchronous HeLa cells were either mock-treated (Cont) or treated with MK-1775 (MK) or with taxol (Tax) or with a combination of MK-1775 and taxol (Tax+MK). Left graph, cell death was measured at 36 h from treatments by trypan blue exclusion and expressed as percent of total cells. Middle graph, quantitation of Annexin-V positivity expressed as percent of flow cytometry-analysed cells measured 36 h post-pro-metaphase release; cells were also incubated with z-VAD-fmk (+z-VAD) or necrostatin-1 (+necro) from the time of Tax+MK addition. The cutoff to count for Annexin-V positivity was established by imposing P3 marker between the end of the bell-shaped part and the brighter tail part of Annexin-V signal distribution histograms of control cell populations incubated in the absence of any treatment. Error bars indicate variability within three independent experiments. Right panels, indicated antigens were probed from cell extracts taken at the indicated time points from treatments. (b) Asynchronous TOM-1 and (c) MOLT-4 cells were either untreated (Cont) or treated with 20 nM MK-1775 (MK) or 10 nM vincristine (Vin) or with combinations of vincristine and MK-1775 (Vin+MK). Left graph, cell death was measured at 48 h from treatments by trypan blue exclusion and expressed as percent of total cells. Middle graph, quantitation of Annexin-V positivity expressed as percent of flow cytometry-analysed cells measured 48 h post-pro-metaphase release; cells were also incubated with z-VAD-fmk (+z-VAD) or necrostatin-1 (+necro) from the time of Vin+MK addition. The cutoff to count for Annexin-V positivity was established by imposing P3 marker between the end of the bell-shaped part and the brighter tail part of Annexin-V signal distribution histograms of control cell populations incubated in the absence of any treatment. Error bars indicate variability within three independent experiments. Right panels, indicated antigens were probed from extracts of cell samples taken at the indicated time points from the previously described treatments. (d) Percent of viability of leukemic blast specimens, deriving from bone marrow blood of four adult lymphoblastic leukemia patients (Patients 1–4), measured by trypan blue exclusion and expressed as percent of total cells at the indicated time points from the following additions: mock addition (Cont), 10 or 20 nM MK and (left graphs) 5 nM vincristine alone or in combination with 10 or 20 nM MK or (right graphs) 10 nM vincristine alone or in combination with 10 or 20 nM MK. Diagnosis and percent of leukemic blasts in the bone marrow mononuclear cell sample is indicated per patient; error bars indicate variability within triplicate assays