In the present study, morphological analysis indicated that ten breast cancer cell lines could be divided into two groups: those that display a prominent mitotic arrest after treatment with 1 μM nocodazole, 100 nM vincristine, or 100 nM colchicine (type A cells), and those that do not (type B cells). Nontransformed HMECs exhibited a similar dichotomy. Further experiments demonstrated that type B cells arrest in mitosis at low drug concentrations but undergo p21-associated G1 and G2 arrests at higher drug concentrations. These results have potentially important implications for current understanding of the actions of spindle poisons.
Because type A cells exhibited the expected response to microtubule-depolymerizing agents, much of the present analysis focused on type B cells. Several observations suggested that these cells have intact mitotic checkpoints. First, type B cells arrested in mitosis after treatment with paclitaxel (Figure d, Figure c, and data not shown), another agent that activates the mitotic checkpoint (35
). Second, type B cells arrested in mitosis at lower concentrations of nocodazole, vincristine, or colchicine (Figure , a and b, and data not shown), demonstrating that the machinery responsible for mitotic arrest was functionally intact. Third, BrdU labeling (Table ) and cyclin analysis (Figure e and Figure e) indicated that these cells failed to reenter G1
, a cell-cycle phase they would be expected to enter if the mitotic checkpoint malfunctioned.
Defects in the checkpoint protein Chfr also fail to account for the behavior of type B cells. Upon treatment with 1.5 μM nocodazole, 1.5 μM colcemid, or 5 μM paclitaxel during G2
, tumor cell lines lacking Chfr rapidly accumulate in mitosis, whereas cells expressing Chfr exhibit a 6-hour delay before entering mitosis (45
). The failure of type B cells to accumulate in mitosis when followed for as long as 72 hours after treatment with 1 μM nocodazole or 100 nM vincristine (Figure , b and c, and data not shown) clearly distinguished type B cells from Chfr-deficient cells as well.
Further investigation revealed that MDA-MB-468 cells, a prototypic type B cell line, arrested in both G1
after treatment with 1 μM nocodazole or 100 nM vincristine. The presence of a G1
arrest was indicated by the persistence of cells with 2N DNA content and high cyclin E levels (Figure , c and d, and Figure , d and e) despite the failure of 4N cells to undergo cytokinesis (Table ). The presence of a G2
arrest was indicated by the accumulation of 4N cells (Figure , c and d) with interphase morphology (Figure a and Figure ), high levels of cytoplasmic cyclin B (Figure , e and f, and Figure e), and focal staining for CENP-F (Figure , b, c, and g). Although microtubule-disrupting agents have been observed to induce accumulation in a tetraploid G1
-like state following mitotic delay and abnormal mitotic exit (11
), to our knowledge this is the first report of premitotic G1
arrest induced by these agents.
These premitotic G1
arrests were associated with increased expression of p21 (Figure ), a Cdk inhibitor that plays critical roles in G1
arrests after DNA damage (26
). Several observations raised the possibility that p21 might contribute to the behavior of the type B cells. First, elevated p21 levels were observed in both G1
cell populations (Figure e). Second, p21 levels increased after treatment with nocodazole, vincristine, or colchicine, but not paclitaxel, which failed to induce G1
arrests (Figure d and Figure a). Third, p21 levels were significantly elevated (four- to eightfold) only after treatment with nocodazole concentrations that caused the G1
arrests in type B cells (Figure , a and f). Finally, p21 levels increased in additional type B cells (Figure g) but not type A cells (Figure h). Although these observations establish a correlation between p21 elevation and the observed G1
arrests, more definitive evidence that p21 participates in type B behavior will require the examination of p21–/–
cells. Unfortunately, parental cells corresponding to both currently available p21–/–
cell lines (mouse fibroblasts and HCT116 colon cancer cells) exhibit type A behavior in response to nocodazole (refs. 27
, and data not shown), making these models unsuitable for assessing the role of p21 in the type B response.
The events leading from microtubule disturbance to p21 upregulation in type B cells require further study. The failure of other DNA damage–responsive polypeptides such as GADD45 to accumulate (Figure a) distinguishes nocodazole-induced p21 upregulation from a DNA damage response. Moreover, the accumulation of p21 in MDA-MB-468 cells, which contain mutant p53 (41
), suggests that nocodazole-induced p21 upregulation does not depend on p53 function.
Consistent with this latter conclusion, no relationship between p53 status and nocodazole-induced cell-cycle effects was observed. In particular, cells with p53 mutations exhibited both type A (MDA-MB-231, SKBr3, HS0578T) and type B (MDA-MB-468, T47D) behavior. Likewise, as described above, differences in microtubule stability did not track with type A versus type B behavior. Instead, the observation that different HMEC isolates also exhibit this dichotomous behavior (Figure e and Figure c) raised the possibility that allelic polymorphism in a currently unidentified gene determines the cell-cycle response to microtubule disruption.
The results presented above have potentially important implications for current efforts to study mitotic checkpoint function in cancer cell lines. Recent studies have demonstrated that approximately 50% of colon cancer cell lines fail to arrest upon exposure to 0.7 μM nocodazole. Although BUB1
mutations were demonstrated in two of these lines, extensive analysis failed to identify additional mutations in mitotic checkpoint genes in a variety of cancer cell lines (48
). Our results indicate that the use of high-dose nocodazole to screen for mitotic checkpoint defects in breast cancer lines results in false positives because a number of lines arrest in G1
before reaching mitosis. Whether similar limitations apply to other cell types remains to be determined.
In summary, the present observations lead to a number of unexpected conclusions. First, microtubule-depolymerizing agents can cause simultaneous G1 and G2 arrests before some cells ever reach mitosis. Second, this type B phenotype correlates with p53-independent induction of p21. Third, type B behavior occurs in some HMEC isolates as well as breast cancer cell lines, raising the possibility that it reflects normal phenotypic variation rather than cancer-associated checkpoint loss. Collectively, these observations suggest that microtubule-depolymerizing agents have effects that are more complicated and more diverse than previously appreciated.