Resistance of cancer cells to chemotherapy is a widespread and serious problem (Lee and Schmitt 2003). The experiments described in the preceding chapters demonstrated that impairment of the spindle checkpoint can confer resistance to spindle and DNA damaging agents, many of them commonly used for chemotherapy. A possible strategy to overcome this resistance could be to abrogate the spindle checkpoint, since it is essential for cell viability (Kalitsis et al. 2000, Michel et al. 2001, Kops et al. 2004, Iwanaga et al. 2007).
Therefore, our lab aimed to identify pharmacological inhibitors of the spindle checkpoint.
In fact, Gö6976, an indolocarbolozole compound structurally related to UCN-01, was identified as a potent spindle checkpoint abrogator in a screen for kinase inhibitors abrogating mitotic arrest (manuscript in preparation).
Severe repression of Bub1, BubR1 or Mad2 induces apoptosis
To confirm that severe spindle checkpoint impairment per se leads to apoptosis HeLa cells were transiently transfected with shRNA constructs targeting BUB1, BUB1B and MAD2.
The degree of repression of the targeted spindle checkpoint proteins was monitored by an assay determining the functionality of the spindle checkpoint.
Figure 43: Severe repression of spindle checkpoint components induces spontaneous apoptosis. HeLa cells were transiently transfected with shRNA constructs targeting BUB1, BUB1B and MAD2. a) 24 h post transfection the cells were treated with 150 nM nocodazole or 100 nM taxol for 16 h and the mitotic index was determined to control the inhibition of spindle checkpoint activity. Mean values and standard deviations from at least three independent experiments were determined. b) Caspase 3 activity of 1 x 105 cells was measured 48 h post transfection. The mean values and standard deviations from at least three independent experiments were calculated.
Results
The mitotic arrest of the transfected cells was measured by MPM2 FACS analysis after exposure to 150 nM nocodazole or 100 nM taxol for 16 h (Figure 43a). Depending on the amount of shRNA transfected (data not shown) the mitotic indices were significantly lower than in previous experiments (about 10% instead of 15-40%) and therefore indicated sufficiently low spindle checkpoint protein levels to ensure lethality (Figure 43a). Caspase 3 activation was measured 48 h after transfection and found to be significantly increased compared to the empty vector control (Figure 43b) verifying that severe repression of spindle checkpoint function leads to the induction of apoptosis.
Gö6976 overrides the spindle checkpoint independent of Cdk1
HCT116 wt cells had been used to identify Gö6976 as a novel spindle checkpoint abrogator. It should be tested whether Gö6976 had the same effect on mitotic arrest of spindle checkpoint impaired or TP53-/- cells. The mitotic indices of HCT116 wt, HCT116 TP53-/-, HCT116 MAD2+/- cells and the HCT116 MAD1kd cell clone 2-31 treated simultaneously with 150 nM nocodazole and 2 µM Gö6976 or DMSO for 16 h were determined by MPM2 FACS analysis (Figure 44a). Gö6976 reduced spindle checkpoint activation significantly in all cell lines. HCT116 wt and HCT116 TP53-/- cells arrested at levels normally seen upon nocodazole treatment of the spindle checkpoint compromised HCT116 cell lines. These cells, however, had an even further reduced spindle checkpoint response upon co-treatment with Gö6976 than after nocodazole treatment alone. Since Gö6976 impairs mitotic arrest and inhibition of Cdk1 during mitotic arrest results in exit from the arrest (Blagden and de Bono 2005), Cdk1 was tested as possible mitotic target of Gö6976. For the in vitro samples Cdk1/Cyclin B complexes were co-immunoprecipitated from nocodazole treated cells and incubated with the inhibitors for 30 min (see experimental scheme, left panel, Figure 44b). Incubation with 150 nM nocodazole and 200 µM ALLN to prevent mitotic exit in the presence of Gö6976, UCN-01 or roscovitine was carried out to determine inhibition of Cdk1 in living cells (see experimental scheme, right panel, Figure 44b). Gö6976 did not reduce Cdk1 kinase activity towards histone H1 in the in vitro assay and by only 9% in the in vivo assay. UCN-01, the negative control for Gö6976, did not diminish kinase activity in the in vitro assay and by just 8% in the in vivo assay, whereas the positive control roscovitine strongly inhibited Cdk1 (Figure 44b). These results suggest that Gö6976 overrides the spindle checkpoint independent of Cdk1 inhibition.
Results
Figure 44: Gö6976 overrides the mitotic spindle checkpoint. a) HCT116 wt, HCT116 TP53-/-, HCT116 MAD2+/- cells and the HCT116 MAD1kd cell clone 2-31 were treated sequentially with 150 nM nocodazole for 14 h and 2 µM Gö6976 or DMSO for 2 h and the mitotic index was determined by MPM2 FACS analysis. Mean values from at least three independent experiments were calculated. b) HeLa cells were treated as indicated in the experimental scheme. For the in vitro samples Cdk1/Cyclin B complexes were coimmunoprecipitated from nocodazole treated shake-off cells with the Cyclin B antibody and incubated with the inhibitors at concentrations of 50 µM roscovitine (RV), 1 µM Gö6976 (Gö) or 100 nM UCN-01 (UCN) for 30 min (left panel). Incubation with 150 nM nocodazole and 200 µM ALLN to prevent mitotic exit in the presence of 30 µM RV, 1 µM Gö6976 or 1 µM UCN-01 was done with living cells for the in vivo samples prior to the Cdk1/Cyclin B coimmunoprecipitation with the Cyclin B antibody (GNS-1, Santa Cruz, right panel). Cdk1 activity was determined in histone H1 kinase assays. One representative experiment is shown.
Gö6976 induces apoptosis in HCT116 cells
To test whether Gö6976 induces apoptosis and whether this effect depended on traversal through mitosis, Gö6976 was added to HCT116 wt cells 7 h after release from a double thymidine block just before cells entered mitosis and apoptosis was measured 7 h or 14 h later. Gö6976 induced caspase 3 activity in a concentration-dependent manner in fluorometric caspase 3 assays. 2, 10 and 20 µM Gö6976 led to a 1.3-fold, 3.0-fold and 4.2-fold increase in caspase 3 activity. Moreover, treatment with roscovitine prevented
Cdk1-Results
45a). Other hallmarks of apoptosis including PARP cleavage (Figure 45b) and DNA laddering (Figure 45c) were detectable after 14 h of Gö6976 treatment. Thus, the induction of apoptosis in response to pharmacological spindle checkpoint abrogation requires progression through mitosis.
Figure 45: Spindle checkpoint inhibition by Gö6976 results in the induction of apoptosis. 1 x 105 HCT116 wt cells synchronized at G1/S were released for 7 h and then treated with Gö6976 for 7 h or 14 h. a) Caspase 3 activation was measured in a quantitative fluorometric assay after 7 h treatment with 2, 10 or 20 µM Gö6976 or 30 µM roscovitine (RV) combined with DMSO or 20 µM Gö6976. Mean values and standard deviations from three independent experiments are shown.
b) PARP cleavage was detected on a Western blot. The result from one representative experiment is presented.
c) Internucleosomal DNA laddering was detected in a DNA laddering assay. The result from one repre-sentative experiment is shown.
A broad panel of human cancer cell lines is sensitive to Gö6976-mediated apoptosis
To exclude a cell line specific effect, a panel of human cancer cell lines was investigated for sensitivity towards Gö6976 treatment and compared with non-cancerous cells. A panel of cancer cell lines originating from different tissues and two non-cancerous cell lines were treated with 15 µM Gö6976 for 24 h and the activation of caspase 3 was determined in several experiments. The following cell lines including two non-transformed cell lines were tested: BJ-tert and HUVEC (human immortalized fibroblast and untransformed endothel cell lines, respectively), Ovca 429 and A2780 (ovary cancer), U2OS and SaOS-2 (osteosarcoma), A549 and H1299 (lung cancer), T47D (breast cancer), HeLa and C33A (cervix cancer) and four colon carcinoma cell lines: CaCo-2, SW-620, HT29 and HCT116 cells. Caspase 3 activity was induced in all cancer cell lines, indicating their susceptibility to Gö6976-induced apoptosis, whereas the non-cancerous cell lines did induce caspase 3
Results
greatly between the cancer cell lines from different tissues and between cell lines from the same tissue. HeLa, A549, Ovca429 and SW-620 cells were the most sensitive cell lines with caspase activation values of 44000, 35000, 29000 and 28000, whereas all other cancer cell lines exhibited inductions between 6000 and 20000 a.u. (Figure 46). In contrast, the non-cancerous cell lines showed caspase 3 activities below 4000 a.u. (Figure 46). Half of the cancer cell lines evaluated were above and half were below the mean value for all cancer cell lines tested for their reaction to Gö6976. Besides for their origin from different tissues, the cancer cell lines had been chosen for their differences in functionality of p53 and spindle checkpoint to get an impression, if Gö6976-mediated apoptosis would display selectivity for these features. However, the lack of p53 did not correlate with sensitivity to Gö6976. A correlation between normal spindle checkpoint status and Gö6976 sensitivity was apparent for the three most sensitive cell lines (HeLa, A549 and Ovca429 cells).
Figure 46: Various human cancer cell lines are sensitive to Gö6976-induced apoptosis. 6 x 104 cells of the indicated cell lines were seeded 24 h prior to treatment and were subsequently treated with 15 µM Gö6976 for 24 h. Caspase 3 activation was determined, the background values were subtracted and mean values and standard deviations from at least three independent experiments were calculated. The top cell lines (BJ-tert, HUVEC) are non-cancerous fibroblast cell lines, all other cell lines are cancer cell lines. The p53 and spindle checkpoint status of all cell lines is given as reported in the literature.
Results
However, as the cancer cell lines tested probably contain a lot more alterations to genes and cell cycle checkpoints than the known impairments of p53 or spindle checkpoint status, it is possible that many other unknown factors govern sensitivity to Gö6976-induced apoptosis.
To shed light on the role of spindle checkpoint function in Gö6976-mediated apoptosis, the isogenic HCT116 MAD2+/- cells and the HCT116 MAD1kd clone 2-31 cells were compared to HCT116 wt cells. The HCT116 cell lines were synchronized at G1/S by a double thymidine block, released for 7 h and treated with Gö6976 concentrations of 2.5, 5, 10, 15 and 20 µM and caspase 3 activity was determined. Interestingly, both spindle checkpoint impaired cell lines, HCT116 MAD2+/- cells and HCT116 MAD1kd cells, were slightly more sensitive to Gö6976 treatment than the HCT116 wt cells (Figure 47), suggesting that Gö6976-induced apoptosis in HCT116 cells can be even more effective in spindle checkpoint impaired cells than in spindle checkpoint proficient cells.
Figure 47: Gö6976-induced apoptosis in HCT116 cell lines is enhanced by spindle checkpoint impairment. 1 x 105 doubly thymidine blocked HCT116 wt, HCT116 MAD2+/- and HCT116 MAD1kd cell clones 2-31 were released for 7 h and then treated with Gö6976 for 7 h. The x-fold induction of caspase 3 activity and the standard deviation were calculated from values of least three independent experiments.
Taken together, the feasibility of spindle checkpoint abrogation as a strategy to overcome chemotherapy resistance was successfully demonstrated by the use of the newly identified pharmacological spindle checkpoint abrogator Gö6976. Treatment with Gö6976 mimics the lethal effect of severe repression of spindle checkpoint proteins. Gö6976 reduces the mitotic arrest in spindle checkpoint normal cells, but also in spindle checkpoint compromised cells and induces apoptosis upon passage through mitosis. It displays some selectivity for apoptosis induction in cancer cells over non-cancerous cells, suggesting to use it as a lead compound for anti-cancer drug development.
Discussion