• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. May 15, 2007; 104(20): 8334–8339.
Published online May 8, 2007. doi:  10.1073/pnas.0703164104
PMCID: PMC1895950
Cell Biology

Surveillance mechanism linking Bub1 loss to the p53 pathway


Bub1 is a kinase believed to function primarily in the mitotic spindle checkpoint. Mutation or aberrant Bub1 expression is associated with chromosomal instability, aneuploidy, and human cancer. We now find that targeting Bub1 by RNAi or simian virus 40 (SV40) large T antigen in normal human diploid fibroblasts results in premature senescence. Interestingly, cells undergoing replicative senescence were also low in Bub1 expression, although ectopic Bub1 expression in presenescent cells was insufficient to extend lifespan. Premature senescence caused by lower Bub1 levels depends on p53. Senescence induction was blocked by dominant negative p53 expression or depletion of p21CIP1, a p53 target. Importantly, cells with lower Bub1 levels and inactivated p53 became highly aneuploid. Taken together, our data highlight a role for p53 in monitoring Bub1 function, which may be part of a more general spindle checkpoint surveillance mechanism. Our data support the hypothesis that Bub1 compromise triggers p53-dependent senescence, which limits the production of aneuploid and potentially cancerous cells.

Keywords: aneuploidy, senescence, simian virus 40

Bub1 belongs to a small group of kinases that are implicated in regulating the spindle checkpoint (1, 2). This checkpoint is critically important for monitoring bivalent attachment of sister chromatids to spindle microtubules, as well as loss of tension across sister chromatids at mitotic metaphase. Proper functioning of the checkpoint is paramount to ensure the fidelity of chromosome segregation (3). The ultimate target of the checkpoint is inhibition of the anaphase promoting complex/cyclosome (APC/C), which is an E3 ubiquitin ligase that normally degrades key mitotic substrates like cyclin B and securin to trigger anaphase onset. Bub1 is evolutionarily conserved and was first identified in fission yeast genetic screens (4). Together with other checkpoint proteins like Bub3, Mad1, Mad2, BubR1 (Mad3 in yeast), and Mps1, Bub1 localizes to the kinetochore, which is a complex protein structure assembled at the centromere during mitosis. The kinetochore is believed to play a central role in the checkpoint by generating a “wait anaphase” signal (3, 5). In yeast, Bub1 clearly plays an essential role for spindle checkpoint function, but in mammalian cells there are conflicting reports as to whether Bub1 is critically required for the spindle checkpoint (2, 69). This controversy might be reconciled by hypothesizing that a partial, RNAi-mediated depletion of Bub1 triggers the spindle checkpoint, whereas a more complete silencing abrogates the checkpoint (2).

Importantly, mutations in Bub1 occur in a small number of colorectal cancer cell lines, where it is believed to contribute to chromosomal instability and aneuploidy (10). Apparently independent from its role in the spindle checkpoint, Bub1 was also demonstrated to be required for metaphase chromosome congression, because its depletion led to chromatid misalignments (2, 8). Finally, recent demonstrations indicate that Bub1 protects centromeric cohesion via its targeting of the Shugoshin (SgoI) protein (11, 12).

Despite significant progress toward understanding the function of Bub1, its relationship to the p53 tumor suppressor pathway has remained uncharacterized, in part because most studies were conducted in HeLa cells, which lack a functional p53. Because tumor cell studies have suggested that inhibition of the spindle checkpoint might be a feasible therapeutic approach for cancer treatment (3, 13), the result of spindle checkpoint perturbation in normal cells has become an important point but remains an infrequently studied subject. Here we have studied Bub1 function in normal human fibroblasts and found that specific suppression of Bub1 by lentiviral expression of short hairpin RNAs (shRNAs) or via its interaction with simian virus 40 (SV40) large T antigen (LT) (14) activates a p53-dependent premature senescence response.


LT Fragment Unable to Bind pRB and p53 Drives Cells into Premature Senescence Dependent on Bub1 Binding.

Previously we demonstrated that the LT viral oncoprotein binds Bub1 through LT residues 89–97 (14). Genetic analysis indicated that this binding can contribute to cellular transformation and spindle checkpoint dysfunction. To assess the function of Bub1 binding in the absence of SV40 effects on p53 and pRB, we used the naturally occurring SV40 17k gene product that also contains the well characterized mutation K1 (E107K) (15), known to prevent pRB family binding. The 17k viral protein comprises LT residues 1–131 but ends with four different amino acids (16). Because the p53 binding site is contained within the LT C terminus (17), 17k does not associate with p53. We then compared the effect of this product to one also lacking the Bub1 binding site (K1/dl89–97). After retroviral infection of normal BJ/tert human fibroblasts, expression levels of 17k K1 and 17k K1/dl89–97 proteins were equivalent, as shown in Fig. 1a. We found that a significant proportion of the cells expressing 17k K1 were large and flat in morphology, reminiscent of senescent cells. To investigate this phenotype further, we performed in situ β-gal assays at pH 6, a common test for senescent cells (18). Strikingly, the majority of cells expressing 17k K1 were senescence-associated (SA) β-gal-positive, consistent with a premature senescence response (Fig. 1b). However, expression of the corresponding Bub1 binding mutant (17k K1/dl89–97) failed to induce SA β-gal accumulation. This indicated that interaction with Bub1 was required to actively drive human fibroblasts into premature senescence. The appearance of SA β-gal-positive cells correlated with accumulation of p21CIP1, a key downstream effector of p53 (19, 20) (Fig. 1a). Because p53 is a major regulator of cellular senescence (20), we examined whether p53 inactivation could prevent the response. We derived BJ/tert cell lines stably expressing a dominant negative form of p53 (p53DD) via a retroviral vector (21). As shown in Fig. 1b, expression of the p53DD dominant negative mutant together with 17k K1 in BJ/tert cells significantly decreased the number of SA β-gal-positive cells but did not completely lower it to background level. Taken together, our results indicate that LT binding to Bub1 induces a premature senescence response that is, to a large part, p53-dependent.

Fig. 1.
Expression of a LT fragment incapable of binding pRB and p53 induces a premature senescence response via Bub1 binding. (a) BJ/tert cells expressing an empty vector (pBabe), 17k K1, or 17k K1/dl89–97 were examined by Western blot analysis for 17k, ...

Suppression of Bub1 in Normal Human Fibroblasts Results in p53/p21CIP1-Dependent Premature Senescence.

To examine the generality of our result, we wanted to determine the functional consequences of Bub1 inactivation in the absence of LT. Although most previous studies addressed Bub1 function in tumor cell lines, we felt it was important to continue to use the BJ/tert cells, because these normal human fibroblasts lack the mutations present in tumor cell lines. To obtain efficient silencing of Bub1 in normal human fibroblasts, we used a lentiviral delivery system to express shRNAs targeting Bub1 (22). We obtained from the RNAi Consortium (Cambridge, MA) pLKO.1 vectors containing 10 different shRNA sequences targeting Bub1. The two shRNAs exhibiting the greatest knockdown, referred to as Bub1 D3 and Bub1 D9, were selected for further study. As shown in Fig. 2a, immunoblotting with a Bub1 antibody demonstrated stable knockdown of Bub1 by ≈90% in BJ/tert cells at 1 week after lentiviral infection and puromycin selection. Cells in which Bub1 was knocked down were large and flat in morphology, again reminiscent of a senescence phenotype.

Fig. 2.
Suppression of Bub1 attenuates cell growth. (a) BJ/tert cells infected with lentivirus encoding either Bub1 D3 or D9 shRNA were compared with a control vector, pBabe. Whole-cell lysates were analyzed by immunoblotting for Bub1 and p21CIP1 by using vinculin ...

Senescent cells are characterized as undergoing a permanent growth arrest (23), in contrast to quiescent cells, which respond to mitogens. Therefore, we examined the growth properties of BJ/tert cells featuring Bub1 knockdown and compared them with those of the same cell lines additionally expressing p53DD. As shown in Fig. 2b, a growth curve demonstrates that the BJ/tert cells expressing the Bub1 D9 shRNA grew significantly more slowly than the ones expressing an empty vector. However, this growth defect can be partially rescued by expression of p53DD, consistent with the p53 dependence of the growth-inhibitory phenotype.

To further investigate the response, we conducted SA β-gal assays. Strikingly, a large proportion of the cells in which Bub1 expression was suppressed with either the Bub1 D3 or Bub1 D9 shRNAs were β-gal-positive, suggestive of a premature senescence response (Fig. 3a). Identical results were observed with one additional lentiviral shRNA that decreased Bub1 expression and with a fourth distinct shRNA, which was expressed from the retroviral pSuper-retro vector (data not shown). In contrast, a Bub1 shRNA that failed to suppress Bub1 (D5 or D6) or an empty pLKO.1 vector both failed to increase the frequency of SA β-gal-positive cells above the background [supporting information (SI) Fig. 6 and data not shown].

Fig. 3.
Suppression of Bub1 induces a p53/p21CIP1-dependent premature senescence response. (a) Representative photographs are shown of SA β-gal in BJ/tert or BJ/tert p53DD cells expressing either D3 or D9 shRNA. (b) Expression of p21CIP1 was knocked down ...

As previously emphasized, the p53/p21 pathway plays a major role in regulating cell senescence. As in the SV40 case, suppression of Bub1 expression by either D3 or D9 shRNA also induced the expression of p21CIP1, consistent with the induction of a cellular senescence program through p53 (Fig. 2a). Accordingly, expression of the dominant negative p53DD mutant completely prevented the senescence response (as monitored by SA β-gal staining; Fig. 3a) seen after silencing of Bub1 expression by either the D3 or D9 shRNA. Furthermore, retroviral expression of a p21CIP1 shRNA blocked the SA β-gal induction after Bub1 silencing (Fig. 3b). This suggests that p21CIP is a key effector of p53 in inducing senescence upon Bub1 depletion. To corroborate our findings in BJ/tert cells, we also introduced the Bub1 D3 or D9 shRNAs into IMR-90 or WI-38 cells, which are different strains of human fibroblasts. We found that, in IMR-90 and WI-38 cells, specific suppression of Bub1 also led to widespread accumulation of SA β-gal (SI Fig. 7). Taken together, we find that Bub1 suppression induces a p53/p21-dependent premature senescence response in several strains of human fibroblasts.

Bub1 Levels Decline in Replicatively Senescent Human Fibroblasts but Restoring Bub1 Does Not Extend Lifespan.

Because silencing of Bub1 provoked premature cellular senescence, we were interested in determining whether Bub1 levels decrease as cells reach replicative senescence due to telomere attrition upon passaging. We could not examine our BJ/tert cells, because these already express telomerase and are immortal. When examining IMR-90 cells, we found that aged cells that were approaching replicative senescence exhibited significantly reduced levels of Bub1 (Fig. 4a). This was also true in WI-38 cells (Fig. 4b). Because Bub1 levels are high in BJ/tert cells, the immortalization process may have selected for preservation of Bub1 expression, or, alternatively, it is attributable to differences among various human fibroblast strains (BJ versus WI-38/IMR-90) (24).

Fig. 4.
Aged human fibroblasts express significantly less Bub1 and BubR1. (a) IMR-90 cells at early passage or approaching replicative senescence were analyzed for Bub1 and BubR1 expression by using tubulin as a loading control. Passage number refers to number ...

Additionally, we examined the levels of the Bub1-related kinase BubR1, which also participates in spindle checkpoint regulation (25) but is not directly targeted by LT (SI Fig. 8). As shown in Fig. 4 a and b, BubR1 levels were also significantly diminished in IMR-90 and WI-38 cells. The level of Bub3 protein, which is associated with Bub1, was not altered (SI Fig. 9). Another marker of senescent cells, p16INK4a (23) was up-regulated in replicatively senescent cells but not noticeably altered in Bub1-depleted cells (SI Fig. 9). Because we have shown that reduction in Bub1 levels results in senescence, it is plausible that this down-regulation in aging cells may contribute to replicative senescence, hence providing a physiological context for our knockdown experiments. As shown in Fig. 4c, Bub1 could be ectopically expressed. However, unlike LT (26, 27), ectopic expression of HA-tagged Bub1 in presenescent IMR-90 cells was not sufficient to allow a significant extension of their lifespan (Fig. 4d). Sometimes ectopic expression of a protein might not suffice for lifespan extension; yet, it may restart DNA synthesis in senescent cells. However, in contrast to LT (28), adenovirus-mediated Bub1 expression did not induce DNA synthesis in senescent IMR-90 cells as measured by BrdU incorporation (SI Fig. 10). In fact, overexpression of Bub1 induced a premature senescence response similar to that resulting from Bub1 depletion (SI Fig. 11). Interestingly, either under- or overexpression of the Mad2 spindle checkpoint protein is known to induce genomic instability and oncogenic transformation (29, 30). Hence, any perturbation of expression level for a spindle checkpoint protein might elicit a similar response.

Loss of Bub1 Is Associated with a Compromised Spindle Checkpoint and Aneuploidy.

Given that Bub1 depletion induces premature senescence and that Bub1 is down-regulated in aging cells, we wanted to assess spindle checkpoint function and genomic instability.

Because dependence of the spindle checkpoint on Bub1 in mammalian cells has been questioned (8), we first wanted to address whether the BJ/tert cells with severely reduced Bub1 levels possess a fully functional spindle checkpoint. For that purpose, we applied 100 ng/ml nocodazole for various lengths of time to BJ/tert cells expressing p53DD together with either the control Bub1 D6 shRNA, which fails to suppress Bub1 expression, or the Bub1 D3/D9 shRNAs. At each time point, the mitotic index was determined by identification and quantitation of condensed chromosomes by using Hoechst 33342 staining. As shown in Fig. 5a, both the Bub1 D3 and D9 shRNAs significantly attenuated the mitotic arrest at all time points after nocodazole addition, although the D3 shRNA had an apparently greater effect when compared with the D6 control. Similar results were obtained in an independent checkpoint experiment. Thus, suppression of Bub1 expression in BJ/tert cells expressing p53DD is associated with a compromised spindle checkpoint.

Fig. 5.
Suppression of Bub1 expression causes a compromised spindle checkpoint and severe aneuploidy in human fibroblasts. (a) BJ/tert p53DD cells expressing either the Bub1 D6 (control) or D3/D9 shRNA were treated at different times with 100 ng/ml nocodazole, ...

A compromise of the spindle checkpoint would be expected to trigger chromosomal instability and, in turn, aneuploidy. To examine chromosome numbers, we performed karyotype analysis. Because Bub1 silencing causes a compromise of the spindle checkpoint, the traditional treatment with the microtubule inhibitor colchicine was avoided. Instead, to arrest cells in metaphase, we treated with the proteasome inhibitor MG-132 after synchronization. As shown in Fig. 5b, BJ/tert cells expressing p53DD exhibited some aneuploidy as would be expected. However, this was dramatically increased by expression of Bub1 D3 or D9 shRNA. Fig. 5c shows representative chromosome spreads from the p53DD, p53DD Bub1 D3, and p53DD Bub1 D9 cell lines. The latter two cell lines displayed a high incidence of spreads with very few chromosomes and also increased frequency of premature sister chromatid separation, a hallmark of spindle checkpoint-deficient cells and Bub1-deficient cells in particular (11). Hence, the loss of Bub1 strongly cooperates with p53 inactivation to elicit rampant aneuploidy.


Two independent lines of investigation described here connect Bub1 status to premature senescence. SV40 has been repeatedly used to provide insights into cellular control mechanisms. LT has, for example, been extensively used to immortalize rodent cells and to further understanding of cellular immortalization mechanisms (31). Here we find that, in the absence of pRB and p53 inactivation, LT can also drive cells into premature senescence if it binds Bub1. This observation might reconcile the previously described paradox that Bub1-binding mutants of LT can immortalize rodent cells more efficiently even than wild-type LT (14). Our observations suggest that LT must bind pRB and p53 not only to drive infected cells into the cell cycle but also to counter a premature senescence response that LT induces by inactivating Bub1. These results also suggest that the role of Bub1 binding needs to be considered in assessing the ability of LT N-terminal transgenic constructs such as dl1137 (32) to induce tissue- or cell type-specific tumors. Finally, these results raise the question of what purpose Bub1 binding serves in the normal life of the virus. Interestingly, preliminary data suggest that Bub1 binding is actually required for viral replication (data not shown).

The generality of the SV40 result connecting Bub1 loss to induction of senescence was demonstrated by knocking down Bub1 expression in normal human diploid fibroblasts with shRNA. Bub1 suppression by lentiviral expression of two different shRNAs triggers a p53-dependent pathway leading to cellular senescence in normal human fibroblasts. This response is apparently not mediated by telomere shortening, because it occurs equally in BJ fibroblasts expressing telomerase (BJ/tert) and in IMR-90/WI-38 cells that lack exogenous telomerase. Several criteria support the notion that we are observing a genuine premature senescence response. The cellular morphology of Bub1-depleted cells is characteristic of senescent cells, and the senescence marker SA β-gal is prominent in a significant proportion of the cells. Growth curves indicate that cells in which Bub1 is silenced are retarded in their growth, consistent with a proliferative arrest. In addition, the p21CIP1 protein that acts as a downstream effector of p53 in mediating cell cycle arrest is up-regulated and required for the observed senescence response as previously noted in other systems (19, 20). Expression of a dominant negative p53 blocked the appearance of SA β-gal-positive cells and restored normal morphology and proliferation, consistent with a p53-dependent response. Analysis of p53 Ser-15 phosphorylation revealed only a minor increase and no change in level of total p53 (data not shown), which is consistent with some p53-dependent senescence responses (33).

Because siRNA depletion of other spindle checkpoint genes, such as Mad2 or BubR1, was found to be lethal in human cancer cells within few cell divisions (25, 34), it might seem surprising that cells with ≥90% reduction in Bub1 are viable and show no overt signs of apoptosis. However, several points could explain this observation. First, even though the knockdown of Bub1 is almost complete, we do observe a subset of the population where Bub1 can still be detected, albeit weakly, at the kinetochore by immunofluorescence analysis (data not shown). It is possible that this low level of Bub1 could support certain functions and thus behave very differently from a true null phenotype. Hypomorphic mice with as little as 10% of the normal level of BubR1 are born alive and develop into adults, indicating that an extremely low level of spindle checkpoint proteins can be tolerated (35). Second, most previous studies have been conducted in tumor cell lines (HeLa). Conceivably, normal human fibroblasts may be more tolerant of suppression of spindle checkpoint proteins. Previous studies of Plk1 silencing that used lentiviral shRNAs have demonstrated that lethality occurs predominantly in cancer cells, not in normal cells (36). If normal cells are indeed more tolerant of Bub1 depletion, there might exist a meaningful therapeutic index for Bub1 inhibition in cancer treatment.

Our observations that Bub1 suppression in a primary cell system can lead to cellular senescence are consistent with previous reports demonstrating that hypomorphic expression of BubR1 can induce premature aging in the mouse and premature senescence in cultured fibroblasts (35). Additionally, we have found that reduction in BubR1 levels via lentiviral expression of shRNA in BJ/tert cells induces SA β-gal, which is largely blocked by p53DD (SI Fig. 12). Furthermore, it has been reported that mice doubly haploinsufficient for the spindle checkpoint genes Bub3/Rae1 also age prematurely, and fibroblasts derived from these mice undergo premature senescence (37). Taken together, it is reasonable to speculate that the senescence response seen here might be a general feature of interference with spindle checkpoint proteins. Indeed, a targeted complete knockout of Mad2 was only permissible at the cellular level when p53 was simultaneously lost (38). Such a response could limit the transforming potential of the aneuploid cells resulting from spindle checkpoint dysfunction.

What might be the nature of the lesion that triggers the p53-dependent response leading to premature senescence and aging? We observe that Bub1 loss in BJ/tert p53DD cells led to a compromise of the spindle checkpoint and concomitant aneuploidy as determined by karyotype analysis. One possibility is that a p53-dependent G1 checkpoint might be triggered after an aberrant mitotic exit. A “tetraploidy checkpoint” has been hypothesized to exist to explain responses seen after cytochalasin B-induced tetraploidization (39). However, more recent observations have seriously questioned the authenticity of this checkpoint (40, 41). Nevertheless, much evidence still exists that tetraploid cells have different fates depending on their p53 status (42, 43). A great deal of aneuploidy is seen in Bub1-deficient cells, which might trigger p53 activation. Arguing against a causal correlation between senescence and aneuploidy, there was significant aneuploidy in Mad2+/− mouse embryo fibroblasts but no obvious premature senescence (37). Alternately, it is possible that defects in Bub1's functions in either metaphase congression or in centromeric cohesion might be involved in generating the critical signal (2, 8, 11). Loss of Bub1 might lead to kinetochore or spindle damage, either of which might trigger a p53-dependent response to eliminate damaged and potentially oncogenic cells. Because Bub1 might play a role in chromosome segregation independent of the spindle checkpoint, this could either directly or indirectly, via generation of aneuploidy, elicit a p53-dependent response. Finally, we cannot completely exclude the possibility that the senescence phenotype linked to Bub1 suppression is due to a Bub1 requirement for localization of another spindle checkpoint component (8, 44, 45).

Bypass of senescence is likely to be an important, possibly even critical, step in the genesis of cancer (20). Although premature senescence triggered by oncogenes like activated Ras was initially viewed by some as an in vitro cell culture phenomenon, there is now ample evidence that it constitutes a physiological mechanism for tumor suppression. Several mouse models such as knockin of an oncogenic Ras allele, expression of an oncogenic B-Raf, or acute inactivation of the PTEN tumor suppressor have demonstrated that senescence occurs in vivo in the premalignant condition but not in malignant tumors (4648).

Most cancer cells are aneuploid, often as a result of chromosomal instability that in turn may derive from loss, mutation, or deregulation of spindle checkpoint proteins (3). We found that cells in which Bub1 was suppressed and that expressed the dominant negative p53 did not grow in soft agar, suggesting that additional changes are needed for transformation (data not shown). Aneuploidy might be a driving force in tumorigenesis because of copy number changes in oncogenes and tumor suppressor genes. Loss of Bub1, and perhaps other spindle checkpoint proteins, triggers a p53-dependent senescence pathway, which may serve to limit proliferation of aneuploid and potentially oncogenic cells, perhaps in a conceptually similar manner to the BRCA1 tumor suppressor gene, which also elicits p53-dependent senescence (49). An even more exciting prospect is the possibility that loss of Bub1 or other spindle checkpoint proteins modulates the sensitivity of cells to DNA damaging agents or anti-tubulin drugs in a manner that could be exploited for cancer therapy.

Materials and Methods

Detailed protocols for all of the methods used are given in SI Materials and Methods.

Cell Culture.

293FT, Phoenix amphotropic, WI-38, or IMR-90 cells were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FCS. BJ/tert cells were cultivated in 80% DMEM/20% medium 199 supplemented with 15% FCS. Aged IMR-90 cells (passage 29) were kindly provided by S. Lowe and M. Narita, whereas aged WI-38 cells [population doubling (PD) 52] were a kind gift from P. D. Adams.

Growth Curve.

A total of 104 cells was plated in duplicate in 24-well dishes. At 5 h after plating (time point 0) and at 1, 2, and 3 days after plating, the cells were fixed in 4% paraformaldehyde and subsequently stained with 0.1% crystal violet dissolved in 10% ethanol/90% water. After staining and thorough washing, the dye was extracted with 10% acetic acid, and absorbance at 590 nm was measured. Similar results were obtained in two independent experiments.

Viral Infections.

Lentiviral pLKO.1 vectors were packaged by transfection into 293FT cells by using FuGENE 6 (Roche Diagnostics, Basel, Switzerland) according to the published protocol (22). Infections with lentivirus were performed for 4 h in the presence of 8 μg/ml polybrene. Phoenix amphotropic cells were used for the packaging of pBabe-puro retroviral vectors, and these were transfected according to the N, N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (BES) calcium phosphate protocol. At 48 h after infection, cells were selected and then maintained at all times in 3 μg/ml puromycin.

SA β-Gal Assay.

SA β-gal activity was determined according to the published protocol (18). Briefly, cells were fixed for 15 min in 0.2% glutaraldehyde, washed with PBS, and then stained overnight at 37°C with freshly made staining solution (40 mM citric acid/sodium phosphate, pH 6.0/5 mM potassium ferricyanide/5 mM potassium ferrocyanide/150 mM NaCl/2 mM MgCl2/1 mg/ml X-Gal).

Supplementary Material

Supporting Information:


We thank S. Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), M. Narita (Cambridge Research Institute, Cambridge, United Kingdom), O. Pereira-Smith, R. Agami (The Netherlands Cancer Institute, Amsterdam, The Netherlands), P. D. Adams (Fox Chase Cancer Center, Philadelphia, PA), R. Freire, and C. Lee for kind assistance with reagents. We thank W. Hahn (Dana–Farber Cancer Institute) for critically reading the manuscript and for supplying the lentivirus shRNA vectors used here. This work was supported by National Institutes of Health Grants PO1-CA50661 and CA30002 (to T.M.R.) and PO1-CA50661 and CA34722 (to B.S.) as well as by Wellcome Trust Project Grant 072672 (to P.S.J.).


short hairpin RNA
simian virus 40
SV40 large T antigen


Conflict of interest statement: In compliance with Harvard Medical School guidelines on possible conflict of interest, we disclose that T.M.R. has consulting relationships with Novartis Pharmaceuticals.

This article contains supporting information online at www.pnas.org/cgi/content/full/0703164104/DC1.


1. Yu H, Tang Z. Cell Cycle. 2005;4:262–265. [PubMed]
2. Meraldi P, Sorger PK. EMBO J. 2005;24:1621–1633. [PMC free article] [PubMed]
3. Kops GJ, Weaver BA, Cleveland DW. Nat Rev Cancer. 2005;5:773–785. [PubMed]
4. Hoyt MA, Totis L, Roberts BT. Cell. 1991;66:507–517. [PubMed]
5. Rieder CL, Cole RW, Khodjakov A, Sluder G. J Cell Biol. 1995;130:941–948. [PMC free article] [PubMed]
6. Roberts BT, Farr KA, Hoyt MA. Mol Cell Biol. 1994;14:8282–8291. [PMC free article] [PubMed]
7. Taylor SS, McKeon F. Cell. 1997;89:727–735. [PubMed]
8. Johnson VL, Scott MI, Holt SV, Hussein D, Taylor SS. J Cell Sci. 2004;117:1577–1589. [PubMed]
9. Tang Z, Shu H, Oncel D, Chen S, Yu H. Mol Cell. 2004;16:387–397. [PubMed]
10. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B. Nature. 1998;392:300–303. [PubMed]
11. Tang Z, Sun Y, Harley SE, Zou H, Yu H. Proc Natl Acad Sci USA. 2004;101:18012–18017. [PMC free article] [PubMed]
12. Kitajima TS, Kawashima SA, Watanabe Y. Nature. 2004;427:510–517. [PubMed]
13. Schmidt M, Medema RH. Cell Cycle. 2006;5:159–163. [PubMed]
14. Cotsiki M, Lock RL, Cheng Y, Williams GL, Zhao J, Perera D, Freire R, Entwistle A, Golemis EA, Roberts TM, et al. Proc Natl Acad Sci USA. 2004;101:947–952. [PMC free article] [PubMed]
15. Kalderon D, Smith AE. Virology. 1984;139:109–137. [PubMed]
16. Zerrahn J, Knippschild U, Winkler T, Deppert W. EMBO J. 1993;12:4739–4746. [PMC free article] [PubMed]
17. Kierstead TD, Tevethia MJ. J Virol. 1993;67:1817–1829. [PMC free article] [PubMed]
18. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et al. Proc Natl Acad Sci USA. 1995;92:9363–9367. [PMC free article] [PubMed]
19. Brown JP, Wei W, Sedivy JM. Science. 1997;277:831–834. [PubMed]
20. Dimri GP. Cancer Cell. 2005;7:505–512. [PMC free article] [PubMed]
21. Shaulian E, Zauberman A, Ginsberg D, Oren M. Mol Cell Biol. 1992;12:5581–5592. [PMC free article] [PubMed]
22. Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, Piqani B, Eisenhaure TM, Luo B, Grenier JK, et al. Cell. 2006;124:1283–1298. [PubMed]
23. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Cell. 1997;88:593–602. [PubMed]
24. Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, Campisi J. EMBO J. 2003;22:4212–4222. [PMC free article] [PubMed]
25. Kops GJ, Foltz DR, Cleveland DW. Proc Natl Acad Sci USA. 2004;101:8699–8704. [PMC free article] [PubMed]
26. Neufeld DS, Ripley S, Henderson A, Ozer HL. Mol Cell Biol. 1987;7:2794–2802. [PMC free article] [PubMed]
27. Shay JW, Wright WE. Exp Cell Res. 1989;184:109–118. [PubMed]
28. Gorman SD, Cristofalo VJ. J Cell Physiol. 1985;125:122–126. [PubMed]
29. Hernando E, Nahle Z, Juan G, Diaz-Rodriguez E, Alaminos M, Hemann M, Michel L, Mittal V, Gerald W, Benezra R, et al. Nature. 2004;430:797–802. [PubMed]
30. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W, Dobles M, Sorger PK, Murty VV, Benezra R. Nature. 2001;409:355–359. [PubMed]
31. Manfredi JJ, Prives C. Biochim Biophys Acta. 1994;1198:65–83. [PubMed]
32. Saenz Robles MT, Symonds H, Chen J, Van Dyke T. Mol Cell Biol. 1994;14:2686–2698. [PMC free article] [PubMed]
33. Chan HM, Narita M, Lowe SW, Livingston DM. Genes Dev. 2005;19:196–201. [PMC free article] [PubMed]
34. Michel L, Diaz-Rodriguez E, Narayan G, Hernando E, Murty VV, Benezra R. Proc Natl Acad Sci USA. 2004;101:4459–4464. [PMC free article] [PubMed]
35. Baker DJ, Jeganathan KB, Cameron JD, Thompson M, Juneja S, Kopecka A, Kumar R, Jenkins RB, de Groen PC, Roche P, van Deursen JM. Nat Genet. 2004;36:744–749. [PubMed]
36. Liu X, Lei M, Erikson RL. Mol Cell Biol. 2006;26:2093–2108. [PMC free article] [PubMed]
37. Baker DJ, Jeganathan KB, Malureanu L, Perez-Terzic C, Terzic A, van Deursen JM. J Cell Biol. 2006;172:529–540. [PMC free article] [PubMed]
38. Burds AA, Lutum AS, Sorger PK. Proc Natl Acad Sci USA. 2005;102:11296–11301. [PMC free article] [PubMed]
39. Andreassen PR, Lohez OD, Lacroix FB, Margolis RL. Mol Biol Cell. 2001;12:1315–1328. [PMC free article] [PubMed]
40. Uetake Y, Sluder G. J Cell Biol. 2004;165:609–615. [PMC free article] [PubMed]
41. Wong C, Stearns T. BMC Cell Biol. 2005 Feb 15; doi: 10.1186/1471-2121-6-6. [PMC free article] [PubMed] [Cross Ref]
42. Fujiwara T, Bandi M, Nitta M, Ivanova EV, Bronson RT, Pellman D. Nature. 2005;437:1043–1047. [PubMed]
43. Castedo M, Coquelle A, Vivet S, Vitale I, Kauffmann A, Dessen P, Pequignot MO, Casares N, Valent A, Mouhamad S, et al. EMBO J. 2006;7:2584–2595. [PMC free article] [PubMed]
44. Sharp-Baker H, Chen RH. J Cell Biol. 2001;153:1239–1250. [PMC free article] [PubMed]
45. Meraldi P, Draviam VM, Sorger PK. Dev Cell. 2004;7:45–60. [PubMed]
46. Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Benguria A, Zaballos A, Flores JM, Barbacid M, et al. Nature. 2005;436:642. [PubMed]
47. Michaloglou C, Vredeveld LC, Soengas MS, Denoyelle C, Kuilman T, van der Horst CM, Majoor DM, Shay JW, Mooi WJ, Peeper DS. Nature. 2005;436:720–724. [PubMed]
48. Chen Z, Trotman LC, Shaffer D, Lin HK, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, et al. Nature. 2005;436:725–730. [PMC free article] [PubMed]
49. Cao L, Li W, Kim S, Brodie SG, Deng CX. Genes Dev. 2003;17:201–213. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...