Spindle checkpoint impairment is frequently observed in human cancer (Bharadwaj and Yu 2004), but intragenic mutations in the known spindle checkpoint genes appear to be rather rare (Bharadwaj and Yu 2004). However, diminished spindle checkpoint protein levels can lead to compromised checkpoint function and were found to be associated with cancer (Bharadwaj and Yu 2004) and to correlate with resistance to certain chemothera-peutic drugs (Masuda et al. 2003, Nitta et al. 2004, Sudo et al. 2004, Tao 2005, Vogel et al. 2005). Specifically, reduced protein levels of Mad1 or Mad2 in human cancers can occur alone or in combination (Wang et al. 2002), which suggests non-redundant functions of these proteins. Investigation of these non-redundant functions requires cell lines containing reduced Mad1 or Mad2 levels. Severe repression of spindle checkpoint proteins or homozygous deletion of spindle checkpoint genes leads to cell death indicating that their products are essential for viability (Kalitsis et al. 2000, Michel et al. 2001, Kops et al.
2004, Iwanaga et al. 2007). Therefore, previous studies could not investigate the consequences of Mad1 inactivation. Our approach was to mimic conditions in human cancers by reduction of Mad1 to a level compatible with long-term cell survival.
HCT116 colon carcinoma cells were particularly attractive for the generation of stable MAD1 knock down cell lines, because isogenic derivatives with a heterozygous gene deletion of MAD2 (Michel et al. 2001) or a homozygous deletion of TP53 (Bunz et al.
1998) were already available. These cell lines allowed functional comparisons in an isogenic background.
Stable HCT116 MAD1 knock down cells are spindle checkpoint impaired
For stable downregulation of MAD1 by expression of shRNAs which downregulate mRNA levels I cloned two constructs targeting different regions of the MAD1 mRNA into the pSUPER vector (Brummelkamp et al. 2002). HCT116 wt cells were stably co-transfected with the pSUPER-MAD1 and the pBabepuro vector coding for resistance to puromycin to allow selection with 5 µg/µl puromycin. As controls HCT116 wt cells were co-transfected with the empty pSUPER vector and the pBabepuro vector. The pSUPER vector containing
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which might indicate that the level of MAD1 downregulation reached was lethal to the cells. However, 31 independent cell clones of HCT116 wt cells transfected with the vector containing sequence si2 (targeting nucleotides 992 to 1010) and three independent cell clones for the controls containing the empty pSUPER vector were isolated. To identify cell lines with sufficient downregulation of MAD1 to impair spindle checkpoint function a functional screen was employed. Since Mad1 is an essential spindle checkpoint component (Vigneron et al. 2004), the spindle checkpoint activity in cells with reduced Mad1 levels should be markedly decreased compared to control cells expressing the empty vector.
Figure 10: Determination of spindle checkpoint impairment of stable HCT116 MAD1 knock down cell clones. HCT116 MAD1 knock down clones (MAD1kd) were generated by stable co-transfection of HCT116 wt cells with pSUPER-MAD1 and pBabepuro vectors. As controls HCT116 wt cells were co-transfected with pSUPER and pBabepuro vectors. The spindle checkpoint function of 31 independent HCT116 MAD1kd cell clones (numbered 1-31), HCT116 wt + pSUPER cell clone 1 (wt + pS) and HCT116 MAD2+/- cells was assessed by treatment with 150 nM nocodazole for 14 h and determination of the mitotic index by MPM2 FACS analysis. The controls and the MAD1kd cell clones 2-2 and 2-31 are highlighted in black. The horizontal line marks a mitotic index of 25% indicating an ideal degree of spindle checkpoint impairment for further experiments. One representative experiment is shown.
A total of 31 independent MAD1 knock down cell lines as well as HCT116 wt + pSUPER (clone 1) and HCT116 MAD2+/- cells were treated with 150 nM nocodazole for 14 h and
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the mitotic index was determined by MPM2 FACS analysis. In agreement with previous results the HCT116 wt cell line 1 carrying the control vector exhibited a mitotic index of 72.8% and the HCT116 MAD2+/- cells had a mitotic index of 23.7% (Figure 10). The mitotic indices of the 31 MAD1 knock down (MAD1kd) cell clones varied from 23.3% to 68.8% (Figure 10). Five HCT116 MAD1kd cell clones showed spindle checkpoint impairment comparable to the HCT116 MAD2+/- cells: HCT116 MAD1kd 2-2, 2-16, 2-26, 2-28 and 2-31 (mitotic index of 23.3%, 24.7%, 25.7%, 23.0%, 24.2%, respectively, (Figure 10). Two of these cell lines were selected for all following experiments: MAD1kd 2-2 and 2-31. The HCT116 wt + pSUPER (clone 1) was chosen as a control.
The level of Mad1 protein downregulation was determined by Western blotting.
Expression levels of HCT116 MAD1kd 2-2 and 2-31 cell clones were quantified compared to control cells (HCT116 + pSUPER clone 1), and parental cells (HCT116 wt). Mad1 signals were normalized for actin expression and the Mad1 protein level was calculated.
HCT116 MAD1kd cell lines 2-2 and 2-31 showed a 35.3% and 49.6% reduction of protein levels respectively (Figure 11), while HCT116 MAD2+/- cells displayed a loss of 20% on protein level (Michel et al. 2001) and also on mRNA level (my own Northern blot data, not shown). Surprisingly, the degree of Mad1 protein downregulation had to exceed that of Mad2 to yield an identical degree of spindle checkpoint impairment in the FACS-based functional assay.
Figure 11: Quantitation of reduced Mad1 protein levels in HCT116 MAD1 knock down cells on Western blot. Western blots of lysates from asynchronously growing cells of two independent HCT116 MAD1kd cell clones (2-2, 2-31), empty vector control cells (HCT116 + pSUPER clone 1) and parental cells (HCT116 wt) were used to calculate the mean of four independent experiments. Mad1 signals were normalized for actin expression and the Mad1 protein level was calculated in percent of HCT116 wt + pSUPER expression (indicated below the blot). The Western blot was probed with antibodies directed against Mad1 (Campbell et al. 2001) and actin. One representative Western blot is shown.
Asynchronously growing HCT116 MAD1kd cells clones 2-2 and 2-31 and control cell lines were analyzed for their DNA content and their mitotic index to detect possible cell cycle defects.
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Initial characterization of untreated HCT116 MAD1kd cell lines revealed no gross growth defects. The overall cell cycle distribution was undisturbed including the amount of mitotic cells compared to controls (Figure 12). Subcultivation ratios for HCT116 MAD1kd cells 2-2 and 2-31 were identical to the ratios for the other HCT116 cell lines, indicating unaltered growth rates (data not shown).
Figure 12: Mad1 downregulation does not alter normal cell cycle distribution of HCT116 MAD1 knock down cell lines. Asynchronously growing HCT116 MAD1 knock down cells (MAD1kd clones 2-2, 2-31) were tested for cell cycle distribution. HCT116 wt + pSUPER cells (wt + pSUPER clone 1) and HCT116 MAD2 +/-cells served as controls. DNA content distribution of the cell populations is presented as PI FACS profiles and the mitotic index determined by MPM2 FACS is given above the corresponding profiles.
HCT116 MAD1 knock down cells and HCT116 MAD2
+/-cells have an impaired spindle checkpoint response to spindle damaging agents
The spindle checkpoint is activated upon damage induced by spindle damaging agents.
Nocodazole and the chemotherapeutically used Vinca alkaloids (e.g. vincristine, vinblastine) interfere with spindle dynamics and prevent generation of attachment and tension by depolymerizing the mitotic spindle at high doses. Taxanes (e.g. taxol, docetaxel) interfere with spindle dynamics and stabilize the microtubules at high doses. Since microtubules capture kinetochores by a stochastic process of growth and shrinkage (dynamic instability) and pull chromosomes to the spindle poles by shrinkage, microtubule stabilization interferes with the generation of tension between kinetochores (Jordan and Wilson 2004). Monastrol inhibits the motor protein Eg5 responsible for centrosome migration. The result of monastrol treatment is a monopolar spindle leading to a lack of tension between kinetochores (Mayer et al. 1999). Chromosome alignment is disturbed by spindle damaging agents, cells arrest in metaphase and die later by apoptosis (Jordan and Wilson 2004, Zhou and Giannakakou 2005). Cells treated with spindle damaging agents leave the spindle checkpoint-mediated mitotic arrest after prolonged spindle checkpoint
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activation even if they possess a functional spindle checkpoint, a process termed mitotic slippage, which is accelerated in spindle checkpoint impaired cells (Chen et al. 2003).
The contribution of Mad1 to mounting and maintenance of mitotic arrest upon various kinds of spindle damage was assessed. HCT116 MAD1kd cells, HCT116 MAD2+/-, HCT116 wt and HCT116 TP53-/- cell lines were treated with 150 nM nocodazole, 100 nM taxol or 70 µM monastrol for 16 h and the mitotic arrest was monitored by MPM2 FACS analysis (Figure 13). Since the HCT116 wt + pSUPER clone 1 behaved similarly to HCT116 wt cells and the HCT116 MAD1kd 2-2 cells behaved similarly to HCT116 MAD1kd 2-31 cells the corresponding data was not shown. Treatment with nocodazole, taxol and monastrol yielded comparable results in each cell line (Figure 13). HCT116 wt and HCT116 TP53-/- cell lines exhibited an intact spindle checkpoint indicated by a maximal mitotic arrest of about 80% in response to the drugs after 16 h, which slowly decreased during time course measurements for 48 h in 8 h intervals (data not shown). HCT116 TP53-/- cells maintained the mitotic arrest approximately 8 h longer than HCT116 wt cells indicating that TP53 is not required for mitotic arrest, but might be involved in mitotic slippage. The mitotic indices of HCT116 MAD1kd and HCT116 MAD2+/- cell lines after 16 h were less than 50%
of spindle checkpoint proficient cells and mitotic slippage was completed after 24 h of treatment – significantly faster than in the spindle checkpoint proficient cell lines.
Figure 13: Spindle checkpoint impair-ment of HCT116 MAD1 knock down cells or HCT116 MAD2+/- cells upon spindle damage. The ability of HCT116 MAD1kd cells, MAD2+/- cells and the controls HCT116 wt and TP53-/- cells to arrest in mitosis upon treatment with 150 nM nocodazole, 100 nM taxol or 70 µM monastrol was determined by MPM2 FACS analysis after 16 h from at least three independent experiments.
Taken together, for the first time stable MAD1 knock down cell lines were generated and characterized. Mad1 is required for spindle checkpoint function, since a reduction to 50-65% of the Mad1 protein levels in HCT116 wt cells impairs the mounting and maintenance of mitotic arrest upon spindle damage. The same reduction in Mad1 levels, however, does
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damage, namely spindle depolymerization, spindle stabilization and a monopolar spindle, depends on normal levels of Mad1. Lowered Mad1 levels cause an impaired spindle checkpoint response leading to mitotic arrest defects, and premature mitotic slippage upon spindle damage. The experiments clearly show that MAD1 and MAD2 fulfill identical functions in the spindle checkpoint-mediated arrest upon challenge with various spindle damaging agents. The downregulation of either one is sufficient to induce mitotic arrest defects and premature mitotic slippage.
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