Substrate degradation by the anaphase promoting complex occurs during mitotic slippage

Abstract

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.

Figure 1

Activation of APC substrate degradation during mitotic slippage of synchronized HCT116 cells. (A) Cell cycle profile of HCT116 cells synchronized at G₁/S with thymidine (Thy), released and then treated with nocodazole (Noc, 500 ng/ml, 1.66 μM) at 1 hour after release from thymidine. Cells collected at the indicated times were stained with PI and with MPM2 antibody and examined by flow cytometry. Numbers in the MPM2 dot plots indicate the percent of MPM2 positive 4N cells. Cells progressing from G₂ to mitosis, and then exiting mitosis are indicated by the gain and loss of MPM2 staining in cells with 4N DNA content. 2N indicates G₁ DNA content. (B) Quantitation of the percent of MPM2 staining cells from three different experiments including that shown in (A). Error bars represent standard deviation. (C) Chromatin condensation as cells progress from G₂ to mitosis and decondensation as cells exit mitosis. Synchronized cells were treated with nocodazole as in (A), fixed and stained for DNA with Hoechst. Cells (n = 250/time point) with condensed chromatin were scored. The graph shows an average of two experiments. Representative fields of cells at different time during nocodazole exposure, stained with Hoechst are shown in the bottom panels. Cells with chromosomes condensation are easily distinguished. (D) Lamin border re-formation in cells upon mitotic slippage. Synchronized cells were treated with nocodazole as in (A), fixed and stained for nuclear borders with lamin B antibody and for DNA with Hoechst. Cells (n = 250/time point) with intact lamin rings around nuclei were scored. The graph shows an average of two experiments. Representative fields of cells at different time during nocodazole exposure are shown in the right panels. Red is Lamin B, and blue is DNA. (E) Extracts were made from HCT116 released from thymidine block and treated with nocodazole. Extracts were prepared from the experiment shown in (A). The level of APC substrates and of other proteins was examined by immunoblotting cell extracts with specific antibodies. The numbers on top indicate the hours following release from thymidine. Nocodazole was added at 1 hour after release. TPX2 and securin levels are low in G₁/S and increase as cells progress to G₂.^, An immunoblot for vinculin is shown as a loading control. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S2. Similar results were obtained using cell extracts from more than three independent experiments.

Figure 1

Activation of APC substrate degradation during mitotic slippage of synchronized HCT116 cells. (A) Cell cycle profile of HCT116 cells synchronized at G₁/S with thymidine (Thy), released and then treated with nocodazole (Noc, 500 ng/ml, 1.66 μM) at 1 hour after release from thymidine. Cells collected at the indicated times were stained with PI and with MPM2 antibody and examined by flow cytometry. Numbers in the MPM2 dot plots indicate the percent of MPM2 positive 4N cells. Cells progressing from G₂ to mitosis, and then exiting mitosis are indicated by the gain and loss of MPM2 staining in cells with 4N DNA content. 2N indicates G₁ DNA content. (B) Quantitation of the percent of MPM2 staining cells from three different experiments including that shown in (A). Error bars represent standard deviation. (C) Chromatin condensation as cells progress from G₂ to mitosis and decondensation as cells exit mitosis. Synchronized cells were treated with nocodazole as in (A), fixed and stained for DNA with Hoechst. Cells (n = 250/time point) with condensed chromatin were scored. The graph shows an average of two experiments. Representative fields of cells at different time during nocodazole exposure, stained with Hoechst are shown in the bottom panels. Cells with chromosomes condensation are easily distinguished. (D) Lamin border re-formation in cells upon mitotic slippage. Synchronized cells were treated with nocodazole as in (A), fixed and stained for nuclear borders with lamin B antibody and for DNA with Hoechst. Cells (n = 250/time point) with intact lamin rings around nuclei were scored. The graph shows an average of two experiments. Representative fields of cells at different time during nocodazole exposure are shown in the right panels. Red is Lamin B, and blue is DNA. (E) Extracts were made from HCT116 released from thymidine block and treated with nocodazole. Extracts were prepared from the experiment shown in (A). The level of APC substrates and of other proteins was examined by immunoblotting cell extracts with specific antibodies. The numbers on top indicate the hours following release from thymidine. Nocodazole was added at 1 hour after release. TPX2 and securin levels are low in G₁/S and increase as cells progress to G₂.^, An immunoblot for vinculin is shown as a loading control. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S2. Similar results were obtained using cell extracts from more than three independent experiments.

Figure 2

Activation of APC substrate degradation during mitotic slippage of asynchronized HCT116 cells. (A) Cell cycle profile of asynchronous (Asyn) HCT116 cells treated with nocodazole (500 ng/ml). Cells were stained with PI and with MPM2 antibody at the indicated times and examined by flow cytometry. Only PI is shown. (B) Quantitation of percent of MPM2 staining cells. The graph shows an average of three experiments including that shown in (A). (C) Quantitation of percent of cells with condensed chromatin. Cells treated as in (A) were fixed and stained for DNA with Hoechst. Cells (n = 250/time point) with condensed chromatin were scored. Chromosomes condensed as cells progress from G₂ to mitosis and decondensed as cells exit mitosis. The graph shows an average of two experiments. (D) Lamin border re-formation during mitotic slippage. Cells treated with nocodazole as in (A), were fixed and stained for nuclear borders with lamin B antibody and for DNA with Hoechst. Representative fields of cells stained for lamin B and DNA are shown. (E) Degradation of APC substrates during mitotic slippage of asynchronous cells treated with nocodazole. Asynchronous HCT116 cells were treated with nocodazole as in (A). Cell extracts prepared at the indicated time points were examined for the stability of APC substrates and the levels of other proteins by immunoblotting with specific antibodies. The timing of substrate degradation and mitotic slippage in asynchronous cells treated with nocodazole occurs some what later than that observed in nocodazole treated G₁/S synchronized cells. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S4. Similar results were obtained using cell extracts from three independent experiments. (F) Immunofluorescence anlaysis of Cyclin B degradation and lamin ring formation upon mitotic slippage. Asynchronous HCT116 cells were treated with nocodazole (500 ng/ml) for the indicated time. Cells were fixed and stained for lamin B, cyclin B and DNA (Hoechst) at the indicated time points. Representative fields of cells show that when cyclin B is present, cells are negative for lamin (left). Quantitation of cyclin B staining and lamin B staining in cells treated with nocodazole (right). The percent of cyclin B positive cells with and without lamin rings from two different experiments were scored. 250 cells were scored/time point. (G) Degradation of cyclin B is accompanied by suppression of Cdk associated kinase activity in nocodazole treated cells. Asynchronous cells were treated with 500 ng/ml nocodazole. Cdk1 and Cdk2 associated kinase activity was measured in Cdk1 and Cdk2 immunoprecipitates of cell extracts prepared at the indicated times. Histone H1 (H1) was used as a substrate. (H) Chromosome spreads from asynchronous HCT116 cells treated with nocodazole (500 ng/ml) for 24 hours. Representative image of chromosomes (left) showing chromatid arm separation (X or Y shape). Right panels show enlarged images of chromosomes from the left panel.

Figure 2

Activation of APC substrate degradation during mitotic slippage of asynchronized HCT116 cells. (A) Cell cycle profile of asynchronous (Asyn) HCT116 cells treated with nocodazole (500 ng/ml). Cells were stained with PI and with MPM2 antibody at the indicated times and examined by flow cytometry. Only PI is shown. (B) Quantitation of percent of MPM2 staining cells. The graph shows an average of three experiments including that shown in (A). (C) Quantitation of percent of cells with condensed chromatin. Cells treated as in (A) were fixed and stained for DNA with Hoechst. Cells (n = 250/time point) with condensed chromatin were scored. Chromosomes condensed as cells progress from G₂ to mitosis and decondensed as cells exit mitosis. The graph shows an average of two experiments. (D) Lamin border re-formation during mitotic slippage. Cells treated with nocodazole as in (A), were fixed and stained for nuclear borders with lamin B antibody and for DNA with Hoechst. Representative fields of cells stained for lamin B and DNA are shown. (E) Degradation of APC substrates during mitotic slippage of asynchronous cells treated with nocodazole. Asynchronous HCT116 cells were treated with nocodazole as in (A). Cell extracts prepared at the indicated time points were examined for the stability of APC substrates and the levels of other proteins by immunoblotting with specific antibodies. The timing of substrate degradation and mitotic slippage in asynchronous cells treated with nocodazole occurs some what later than that observed in nocodazole treated G₁/S synchronized cells. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S4. Similar results were obtained using cell extracts from three independent experiments. (F) Immunofluorescence anlaysis of Cyclin B degradation and lamin ring formation upon mitotic slippage. Asynchronous HCT116 cells were treated with nocodazole (500 ng/ml) for the indicated time. Cells were fixed and stained for lamin B, cyclin B and DNA (Hoechst) at the indicated time points. Representative fields of cells show that when cyclin B is present, cells are negative for lamin (left). Quantitation of cyclin B staining and lamin B staining in cells treated with nocodazole (right). The percent of cyclin B positive cells with and without lamin rings from two different experiments were scored. 250 cells were scored/time point. (G) Degradation of cyclin B is accompanied by suppression of Cdk associated kinase activity in nocodazole treated cells. Asynchronous cells were treated with 500 ng/ml nocodazole. Cdk1 and Cdk2 associated kinase activity was measured in Cdk1 and Cdk2 immunoprecipitates of cell extracts prepared at the indicated times. Histone H1 (H1) was used as a substrate. (H) Chromosome spreads from asynchronous HCT116 cells treated with nocodazole (500 ng/ml) for 24 hours. Representative image of chromosomes (left) showing chromatid arm separation (X or Y shape). Right panels show enlarged images of chromosomes from the left panel.

Figure 3

Proteasome inhibition prevents degradation of APC substrates in the continuous presence of nocodazole. Asynchronous (Asyn) HCT116 cells were treated with nocodazole (500 ng/ml), and the proteasome inhibitor LLnL (50 μM) was added at 18 hours. Cells were collected at the time points indicated, and the level of APC substrates was examined by immunoblotting cell extracts with specific antibodies. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S5. Similar results were obtained using cell extracts from two independent experiments.

Figure 4

APC substrate degradation upon mitotic slippage with different concentrations of nocodazole. (A) Asynchronous HCT116 cells were treated with 60, 200 and 500 ng/ml nocodazole for 15 hours. Cell were fixed and stained to visualize tubulin. Mitotic cells are shown. (B) Asynchronous HCT116 cells were treated with 60 ng/ml (0.2 μM) nocodazole. The level of APC substrates and of other proteins was examined by immunoblotting cell extracts with the indicated antibodies. The accelerated substrate degradation in low drug concentrations appears to correlate with greater retention of some spindle microtubules. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S6 (left). Similar results were obtained using cell extracts from two independent experiments. (C) Asynchronous HCT116 cells were treated with 200 ng/ml (0.66 μM) nocodazole. The level of APC substrates and of other proteins was examined by immunoblotting cell extracts with the indicated antibodies. Cell cycle profiles of HCT116 cells treated with 200 ng/ml nocodazole are shown. Numbers in the MPM2 dot plots indicate the percent of MPM2 positive 4N cells. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S6 (right). Similar results were obtained using cell extracts from two independent experiments.

Figure 5

APC substrate degradation during mitotic slippage in other cell lines. Asynchronous A549 (human lung carcinoma) cells (A) and U2OS (human osteocarcinoma) cells (B) were treated with 100 ng/ml nocodazole for the indicated time. This concentration was found to be optimal in preliminary experiments with these cells. APC substrates at different times after nocodazole treatment were examined by immunoblotting cell extracts with the indicated specific antibodies (left). Cells were stained with PI and MPM2 and examined by flow cytometry (right). Numbers in MPM2 dot plots are the percent of population in mitosis.

Figure 6

Downregulation of Cdh1 and Cdc20 prevents APC substrate degradation in the continuous presence of nocodazole. (A) Asynchronous (Asyn) HCT116 cells were transfected with control or with both Cdc20 and Cdh1 siRNA to downregulate Cdh1 and Cdc20. Immunoblots show effective suppression of both targets at 24 hours after transfection. (B) siRNA transfected cells obtained in (A) were treated with nocodazole (200 ng/ml). At 15 hours, cells were collected, stained with PI and with MPM2 antibody, and examined by flow cytometry to ensure that cells had progressed to mitosis. Numbers in the MPM2 dot plots indicate the percent of MPM2 positive 4N cells. (C) Extracts from siRNA transfected cells obtained from the experiment in (B) were examined for degradation of APC substrates by immunoblotting with the indicated specific antibodies. The quantitation of the levels of APC substrates at different time after nocodazole treatment is shown in Supplemental Figure S7. Similar results were obtained using cell extracts from two independent experiments.