Surveillance mechanism linking Bub1 loss to the pathway

Ole V. Gjoerup*†, Jiaping Wu‡, Devin Chandler-Militello‡, Grace L. Williams‡, Jean Zhao‡, Brian Schaffhausen§, Parmjit S. Jat¶, and Thomas M. Roberts†‡

*Molecular Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; ‡Department of Cancer Biology, Dana–Farber Cancer Institute and Harvard Medical School, 1 Jimmy Fund Way, Boston, MA 02115; §Department of Biochemistry, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111; ¶Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, United Kingdom

Communicated by Elliott D. Kieff, Harvard University, Boston, MA, April 9, 2007 (received for review September 26, 2006) Bub1 is a kinase believed to function primarily in the mitotic spindle centromeric cohesion via its targeting of the Shugoshin (SgoI) checkpoint. Mutation or aberrant Bub1 expression is associated protein (11, 12). with chromosomal instability, , and human cancer. We Despite significant progress toward understanding the func- now find that targeting Bub1 by RNAi or simian virus 40 (SV40) tion of Bub1, its relationship to the p53 tumor suppressor large T antigen in normal human diploid fibroblasts results in pathway has remained uncharacterized, in part because most premature senescence. Interestingly, cells undergoing replicative studies were conducted in HeLa cells, which lack a functional senescence were also low in Bub1 expression, although ectopic p53. Because tumor cell studies have suggested that inhibition of Bub1 expression in presenescent cells was insufficient to extend the spindle checkpoint might be a feasible therapeutic approach lifespan. Premature senescence caused by lower Bub1 levels de- for cancer treatment (3, 13), the result of spindle checkpoint pends on p53. Senescence induction was blocked by dominant perturbation in normal cells has become an important point but negative p53 expression or depletion of p21CIP1, a p53 target. remains an infrequently studied subject. Here we have studied Importantly, cells with lower Bub1 levels and inactivated p53 Bub1 function in normal human fibroblasts and found that became highly aneuploid. Taken together, our data highlight a role specific suppression of Bub1 by lentiviral expression of short for p53 in monitoring Bub1 function, which may be part of a more hairpin RNAs (shRNAs) or via its interaction with simian virus general spindle checkpoint surveillance mechanism. Our data sup- 40 (SV40) large T antigen (LT) (14) activates a p53-dependent port the hypothesis that Bub1 compromise triggers p53-dependent premature senescence response. senescence, which limits the production of aneuploid and poten- Results tially cancerous cells. LT Fragment Unable to Bind pRB and p53 Drives Cells into Premature Senescence Dependent on Bub1 Binding. ͉ ͉ Previously we demon- aneuploidy senescence simian virus 40 strated that the LT viral oncoprotein binds Bub1 through LT residues 89–97 (14). Genetic analysis indicated that this binding ub1 belongs to a small group of kinases that are implicated can contribute to cellular transformation and spindle checkpoint Bin regulating the spindle checkpoint (1, 2). This checkpoint dysfunction. To assess the function of Bub1 binding in the is critically important for monitoring bivalent attachment of absence of SV40 effects on p53 and pRB, we used the naturally sister chromatids to spindle microtubules, as well as loss of occurring SV40 17k product that also contains the well tension across sister chromatids at mitotic metaphase. Proper characterized mutation K1 (E107K) (15), known to prevent pRB functioning of the checkpoint is paramount to ensure the fidelity family binding. The 17k viral protein comprises LT residues of segregation (3). The ultimate target of the 1–131 but ends with four different amino acids (16). Because the checkpoint is inhibition of the promoting complex/ p53 binding site is contained within the LT C terminus (17), 17k cyclosome (APC/C), which is an E3 ubiquitin ligase that nor- does not associate with p53. We then compared the effect of this mally degrades key mitotic substrates like cyclin B and securin to product to one also lacking the Bub1 binding site (K1/dl89–97). trigger anaphase onset. Bub1 is evolutionarily conserved and was After retroviral infection of normal BJ/tert human fibroblasts, first identified in fission yeast genetic screens (4). Together with expression levels of 17k K1 and 17k K1/dl89–97 proteins were other checkpoint proteins like Bub3, , , BubR1 equivalent, as shown in Fig. 1a. We found that a significant (Mad3 in yeast), and Mps1, Bub1 localizes to the , proportion of the cells expressing 17k K1 were large and flat in which is a complex protein structure assembled at the centro- morphology, reminiscent of senescent cells. To investigate this ␤ mere during . The kinetochore is believed to play a central phenotype further, we performed in situ -gal assays at pH 6, a common test for senescent cells (18). Strikingly, the majority of role in the checkpoint by generating a ‘‘wait anaphase’’ signal (3, ␤ 5). In yeast, Bub1 clearly plays an essential role for spindle cells expressing 17k K1 were senescence-associated (SA) -gal- checkpoint function, but in mammalian cells there are conflict-

ing reports as to whether Bub1 is critically required for the Author contributions: O.V.G. designed research; O.V.G., J.W., D.C.-M., and G.L.W. per- spindle checkpoint (2, 6–9). This controversy might be recon- formed research; P.S.J. contributed new reagents/analytic tools; O.V.G., G.L.W., J.Z., B.S., ciled by hypothesizing that a partial, RNAi-mediated depletion P.S.J., and T.M.R. analyzed data; and O.V.G., B.S., P.S.J., and T.M.R. wrote the paper. of Bub1 triggers the spindle checkpoint, whereas a more com- Conflict of interest statement: In compliance with Harvard Medical School guidelines on plete silencing abrogates the checkpoint (2). possible conflict of interest, we disclose that T.M.R. has consulting relationships with Novartis Pharmaceuticals. Importantly, mutations in Bub1 occur in a small number of Abbreviations: shRNA, short hairpin RNA; SV40, simian virus 40; LT, SV40 large T colorectal cancer cell lines, where it is believed to contribute to antigen; SA, senescence-associated. chromosomal instability and aneuploidy (10). Apparently inde- †To whom correspondence may be addressed. E-mail: [email protected] or thomas࿝roberts@ pendent from its role in the spindle checkpoint, Bub1 was also dfci.harvard.edu. demonstrated to be required for metaphase chromosome con- This article contains supporting information online at www.pnas.org/cgi/content/full/ gression, because its depletion led to chromatid misalignments 0703164104/DC1. (2, 8). Finally, recent demonstrations indicate that Bub1 protects © 2007 by The National Academy of Sciences of the USA

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anti-vinculin

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b BJ/tert 17k K1 BJ/tert 17k K1/dl89-97

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 BJ/tert p53DD 17k K1 for Bub1 and p21CIP1 by using vinculin as a loading control. (b) Growth curves were performed by using BJ/tert or BJ/tert p53DD cells expressing either a control vector or the Bub1 D9 shRNA. Absorbance at 590 nm reflects cell number.

ing 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 Fig. 1. Expression of a LT fragment incapable of binding pRB and p53 induces stable knockdown of Bub1 by Ϸ90% in BJ/tert cells at 1 week 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 after lentiviral infection and puromycin selection. Cells in which Western blot analysis for 17k, p21CIP1, and vinculin expression. (b) Represen- Bub1 was knocked down were large and flat in morphology, tative photographs are shown of SA ␤-gal activity in BJ/tert cells expressing 17k again reminiscent of a senescence phenotype. CELL BIOLOGY K1 or 17k K1/dl89–97 or in BJ/tert p53DD cells expressing 17k K1. 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 positive, consistent with a premature senescence response (Fig. BJ/tert cells featuring Bub1 knockdown and compared them with 1b). However, expression of the corresponding Bub1 binding those of the same cell lines additionally expressing p53DD. As mutant (17k K1/dl89–97) failed to induce SA ␤-gal accumula- shown in Fig. 2b, a growth curve demonstrates that the BJ/tert tion. This indicated that interaction with Bub1 was required to cells expressing the Bub1 D9 shRNA grew significantly more actively drive human fibroblasts into premature senescence. The slowly than the ones expressing an empty vector. However, this appearance of SA ␤-gal-positive cells correlated with accumu- growth defect can be partially rescued by expression of p53DD, lation of p21CIP1, a key downstream effector of p53 (19, 20) (Fig. consistent with the p53 dependence of the growth-inhibitory 1a). Because p53 is a major regulator of cellular senescence (20), phenotype. we examined whether p53 inactivation could prevent the re- To further investigate the response, we conducted SA ␤-gal sponse. We derived BJ/tert cell lines stably expressing a domi- assays. Strikingly, a large proportion of the cells in which Bub1 nant negative form of p53 (p53DD) via a retroviral vector (21). expression was suppressed with either the Bub1 D3 or Bub1 D9 As shown in Fig. 1b, expression of the p53DD dominant negative shRNAs were ␤-gal-positive, suggestive of a premature senes- mutant together with 17k K1 in BJ/tert cells significantly de- cence response (Fig. 3a). Identical results were observed with creased the number of SA ␤-gal-positive cells but did not one additional lentiviral shRNA that decreased Bub1 expression completely lower it to background level. Taken together, our and with a fourth distinct shRNA, which was expressed from the results indicate that LT binding to Bub1 induces a premature retroviral pSuper-retro vector (data not shown). In contrast, a senescence response that is, to a large part, p53-dependent. Bub1 shRNA that failed to suppress Bub1 (D5 or D6) or an empty pLKO.1 vector both failed to increase the frequency of SA Suppression of Bub1 in Normal Human Fibroblasts Results in p53/ ␤-gal-positive cells above the background [supporting informa- p21CIP1-Dependent Premature Senescence. To examine the gener- tion (SI) Fig. 6 and data not shown]. ality of our result, we wanted to determine the functional As previously emphasized, the p53/p21 pathway plays a major consequences of Bub1 inactivation in the absence of LT. Al- role in regulating cell senescence. As in the SV40 case, suppres- though most previous studies addressed Bub1 function in tumor sion of Bub1 expression by either D3 or D9 shRNA also induced cell lines, we felt it was important to continue to use the BJ/tert the expression of p21CIP1, consistent with the induction of a cells, because these normal human fibroblasts lack the mutations cellular senescence program through p53 (Fig. 2a). Accordingly, present in tumor cell lines. To obtain efficient silencing of Bub1 expression of the dominant negative p53DD mutant completely in normal human fibroblasts, we used a lentiviral delivery system prevented the senescence response (as monitored by SA ␤-gal to express shRNAs targeting Bub1 (22). We obtained from the staining; Fig. 3a) seen after silencing of Bub1 expression by RNAi Consortium (Cambridge, MA) pLKO.1 vectors contain- either the D3 or D9 shRNA. Furthermore, retroviral expression

Gjoerup et al. PNAS ͉ May 15, 2007 ͉ vol. 104 ͉ no. 20 ͉ 8335 Downloaded by guest on September 30, 2021 a Bub1 D9 Bub1 D3

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Bub1 D9 Bub1 D3

BJ/tert p53DD

b Bub1 D3 Bub1 D3 + p21-F

BJ/tert

t

21-F BJ/ter p Anti-p21 Anti-tubulin

Fig. 3. Suppression of Bub1 induces a p53/p21CIP1-dependent premature senescence response. (a) Representative photographs are shown of SA ␤-gal in BJ/tert or BJ/tert p53DD cells expressing either D3 or D9 shRNA. (b) Expression of p21CIP1 was knocked down with a pSuper-retro (blasticidin) vector (p21-F), followed sequentially by lentiviral knockdown of Bub1 using the D3 shRNA. SA ␤-gal assays were conducted 10 days after lentiviral infection.

of a p21CIP1 shRNA blocked the SA ␤-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 Fig. 4. Aged human fibroblasts express significantly less Bub1 and BubR1. (a) D9 shRNAs into IMR-90 or WI-38 cells, which are different IMR-90 cells at early passage or approaching replicative senescence were strains of human fibroblasts. We found that, in IMR-90 and analyzed for Bub1 and BubR1 expression by using tubulin as a loading control. WI-38 cells, specific suppression of Bub1 also led to widespread Passage number refers to number of passages after receiving cells from accumulation of SA ␤-gal (SI Fig. 7). Taken together, we find American Type Culture Collection (ATCC). (b) WI-38 cells at early passage or that Bub1 suppression induces a p53/p21-dependent premature approaching replicative senescence were analyzed for Bub1 and BubR1 ex- senescence response in several strains of human fibroblasts. pression by using tubulin as a loading control. PD refers to the number of population doublings. (c) Bub1 levels were assayed by immunoblotting in young and old IMR-90 cells (passages 16 and 34, respectively) and in cells Bub1 Levels Decline in Replicatively Senescent Human Fibroblasts but ectopically expressing HA-Bub1. (d) IMR-90 cells expressing an empty vector, Restoring Bub1 Does Not Extend Lifespan. Because silencing of HA-Bub1, or LT were serially passaged until replicative senescence. Population Bub1 provoked premature cellular senescence, we were inter- doublings (PDs) were calculated based on cell counts at each time point. ested 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 9). Another marker of senescent cells, p16INK4a (23) was up- express telomerase and are immortal. When examining IMR-90 regulated in replicatively senescent cells but not noticeably cells, we found that aged cells that were approaching replicative altered in Bub1-depleted cells (SI Fig. 9). Because we have senescence exhibited significantly reduced levels of Bub1 (Fig. shown that reduction in Bub1 levels results in senescence, it is 4a). This was also true in WI-38 cells (Fig. 4b). Because Bub1 plausible that this down-regulation in aging cells may contribute levels are high in BJ/tert cells, the immortalization process may to replicative senescence, hence providing a physiological con- have selected for preservation of Bub1 expression, or, alterna- text for our knockdown experiments. As shown in Fig. 4c, Bub1 tively, it is attributable to differences among various human could be ectopically expressed. However, unlike LT (26, 27), fibroblast strains (BJ versus WI-38/IMR-90) (24). ectopic expression of HA-tagged Bub1 in presenescent IMR-90 Additionally, we examined the levels of the Bub1-related cells was not sufficient to allow a significant extension of their kinase BubR1, which also participates in spindle checkpoint lifespan (Fig. 4d). Sometimes ectopic expression of a protein regulation (25) but is not directly targeted by LT (SI Fig. 8). As might not suffice for lifespan extension; yet, it may restart DNA shown in Fig. 4 a and b, BubR1 levels were also significantly synthesis in senescent cells. However, in contrast to LT (28), diminished in IMR-90 and WI-38 cells. The level of Bub3 adenovirus-mediated Bub1 expression did not induce DNA protein, which is associated with Bub1, was not altered (SI Fig. synthesis in senescent IMR-90 cells as measured by BrdU

8336 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703164104 Gjoerup et al. Downloaded by guest on September 30, 2021 incorporation (SI Fig. 10). In fact, overexpression of Bub1 a induced a premature senescence response similar to that result- ing 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 transforma- tion (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 b 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 expres- sion, or the Bub1 D3/D9 shRNAs. At each time point, the mitotic index was determined by identification and quantitation of condensed 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 percentage of all chr spreads obtained in an independent checkpoint experiment. Thus, sup- pression of Bub1 expression in BJ/tert cells expressing p53DD is associated with a compromised spindle checkpoint. c p53DD D9 D9 D3 A compromise of the spindle checkpoint would be expected to trigger chromosomal instability and, in turn, aneuploidy. To examine chromosome numbers, we performed karyotype anal- ysis. Because Bub1 silencing causes a compromise of the spindle checkpoint, the traditional treatment with the microtubule in- hibitor colchicine was avoided. Instead, to arrest cells in meta- CELL BIOLOGY phase, we treated with the proteasome inhibitor MG-132 after Fig. 5. Suppression of Bub1 expression causes a compromised spindle check- synchronization. As shown in Fig. 5b, BJ/tert cells expressing point and severe aneuploidy in human fibroblasts. (a) BJ/tert p53DD cells expressing either the Bub1 D6 (control) or D3/D9 shRNA were treated at p53DD exhibited some aneuploidy as would be expected. How- different times with 100 ng/ml nocodazole, and mitotic arrest was monitored ever, this was dramatically increased by expression of Bub1 D3 by assaying the mitotic index. (b) BJ/tert p53DD cells alone or expressing D3/D9 or D9 shRNA. Fig. 5c shows representative chromosome spreads Bub1 shRNA were arrested in mitosis by treatment with MG-132 after syn- from the p53DD, p53DD Bub1 D3, and p53DD Bub1 D9 cell chronization. Metaphase chromosome spreads were prepared, and chromo- lines. The latter two cell lines displayed a high incidence of some numbers were counted from 101 p53DD cells, 209 p53DD Bub1 D3 cells, spreads with very few chromosomes and also increased fre- and 242 p53DD Bub1 D9 cells. An independent experiment with freshly quency of premature sister chromatid separation, a hallmark of derived cell lines yielded similar results. (c) Representative chromosome spindle checkpoint-deficient cells and Bub1-deficient cells in spreads are shown from cells expressing p53DD or additionally expressing the particular (11). Hence, the loss of Bub1 strongly cooperates with Bub1 D3 or D9 shRNA. p53 inactivation to elicit rampant aneuploidy. Discussion normal life of the virus. Interestingly, preliminary data suggest that Bub1 binding is actually required for viral replication (data Two independent lines of investigation described here connect not shown). Bub1 status to premature senescence. SV40 has been repeatedly The generality of the SV40 result connecting Bub1 loss to used to provide insights into cellular control mechanisms. LT induction of senescence was demonstrated by knocking down has, for example, been extensively used to immortalize rodent cells and to further understanding of cellular immortalization Bub1 expression in normal human diploid fibroblasts with mechanisms (31). Here we find that, in the absence of pRB and shRNA. Bub1 suppression by lentiviral expression of two dif- p53 inactivation, LT can also drive cells into premature senes- ferent shRNAs triggers a p53-dependent pathway leading to cence if it binds Bub1. This observation might reconcile the cellular senescence in normal human fibroblasts. This response previously described paradox that Bub1-binding mutants of LT is apparently not mediated by telomere shortening, because it can immortalize rodent cells more efficiently even than wild-type occurs equally in BJ fibroblasts expressing telomerase (BJ/tert) LT (14). Our observations suggest that LT must bind pRB and and in IMR-90/WI-38 cells that lack exogenous telomerase. p53 not only to drive infected cells into the but also to Several criteria support the notion that we are observing a counter a premature senescence response that LT induces by genuine premature senescence response. The cellular morphol- inactivating Bub1. These results also suggest that the role of ogy of Bub1-depleted cells is characteristic of senescent cells, and Bub1 binding needs to be considered in assessing the ability of the senescence marker SA ␤-gal is prominent in a significant LT N-terminal transgenic constructs such as dl1137 (32) to proportion of the cells. Growth curves indicate that cells in which induce tissue- or cell type-specific tumors. Finally, these results Bub1 is silenced are retarded in their growth, consistent with a raise the question of what purpose Bub1 binding serves in the proliferative arrest. In addition, the p21CIP1 protein that acts as

Gjoerup et al. PNAS ͉ May 15, 2007 ͉ vol. 104 ͉ no. 20 ͉ 8337 Downloaded by guest on September 30, 2021 a downstream effector of p53 in mediating cell cycle arrest is or in centromeric cohesion might be involved in generating the up-regulated and required for the observed senescence response critical signal (2, 8, 11). Loss of Bub1 might lead to kinetochore as previously noted in other systems (19, 20). Expression of a or spindle damage, either of which might trigger a p53- dominant negative p53 blocked the appearance of SA ␤-gal- dependent response to eliminate damaged and potentially on- positive cells and restored normal morphology and proliferation, cogenic cells. Because Bub1 might play a role in chromosome consistent with a p53-dependent response. Analysis of p53 segregation independent of the spindle checkpoint, this could Ser-15 phosphorylation revealed only a minor increase and no either directly or indirectly, via generation of aneuploidy, elicit change in level of total p53 (data not shown), which is consistent a p53-dependent response. Finally, we cannot completely ex- with some p53-dependent senescence responses (33). clude the possibility that the senescence phenotype linked to Because siRNA depletion of other spindle checkpoint , Bub1 suppression is due to a Bub1 requirement for localization such as Mad2 or BubR1, was found to be lethal in human cancer of another spindle checkpoint component (8, 44, 45). cells within few cell divisions (25, 34), it might seem surprising Bypass of senescence is likely to be an important, possibly even that cells with Ն90% reduction in Bub1 are viable and show no critical, step in the genesis of cancer (20). Although premature overt signs of . However, several points could explain senescence triggered by oncogenes like activated Ras was ini- this observation. First, even though the knockdown of Bub1 is tially viewed by some as an in vitro cell culture phenomenon, almost complete, we do observe a subset of the population where there is now ample evidence that it constitutes a physiological Bub1 can still be detected, albeit weakly, at the kinetochore by mechanism for tumor suppression. Several mouse models such immunofluorescence analysis (data not shown). It is possible as knockin of an oncogenic Ras allele, expression of an onco- that this low level of Bub1 could support certain functions and genic B-Raf, or acute inactivation of the PTEN tumor suppressor thus behave very differently from a true null phenotype. Hypo- have demonstrated that senescence occurs in vivo in the prema- morphic mice with as little as 10% of the normal level of BubR1 lignant condition but not in malignant tumors (46–48). are born alive and develop into adults, indicating that an Most cancer cells are aneuploid, often as a result of chromo- extremely low level of spindle checkpoint proteins can be somal instability that in turn may derive from loss, mutation, or tolerated (35). Second, most previous studies have been con- deregulation of spindle checkpoint proteins (3). We found that ducted in tumor cell lines (HeLa). Conceivably, normal human cells in which Bub1 was suppressed and that expressed the fibroblasts may be more tolerant of suppression of spindle dominant negative p53 did not grow in soft agar, suggesting that checkpoint proteins. Previous studies of Plk1 silencing that used additional changes are needed for transformation (data not lentiviral shRNAs have demonstrated that lethality occurs pre- shown). Aneuploidy might be a driving force in tumorigenesis dominantly in cancer cells, not in normal cells (36). If normal because of copy number changes in oncogenes and tumor cells are indeed more tolerant of Bub1 depletion, there might suppressor genes. Loss of Bub1, and perhaps other spindle exist a meaningful therapeutic index for Bub1 inhibition in checkpoint proteins, triggers a p53-dependent senescence path- cancer treatment. way, which may serve to limit proliferation of aneuploid and Our observations that Bub1 suppression in a primary cell potentially oncogenic cells, perhaps in a conceptually similar system can lead to cellular senescence are consistent with manner to the BRCA1 tumor suppressor gene, which also elicits previous reports demonstrating that hypomorphic expression of p53-dependent senescence (49). An even more exciting prospect BubR1 can induce premature aging in the mouse and premature is the possibility that loss of Bub1 or other spindle checkpoint senescence in cultured fibroblasts (35). Additionally, we have proteins modulates the sensitivity of cells to DNA damaging found that reduction in BubR1 levels via lentiviral expression of agents or anti-tubulin drugs in a manner that could be exploited shRNA in BJ/tert cells induces SA ␤-gal, which is largely blocked for cancer therapy. by p53DD (SI Fig. 12). Furthermore, it has been reported that mice doubly haploinsufficient for the spindle checkpoint genes Materials and Methods Bub3/Rae1 also age prematurely, and fibroblasts derived from Detailed protocols for all of the methods used are given in SI these mice undergo premature senescence (37). Taken together, Materials and Methods. it is reasonable to speculate that the senescence response seen here might be a general feature of interference with spindle Cell Culture. 293FT, Phoenix amphotropic, WI-38, or IMR-90 checkpoint proteins. Indeed, a targeted complete knockout of cells were grown in DMEM (Invitrogen, Carlsbad, CA) supple- Mad2 was only permissible at the cellular level when p53 was mented with 10% FCS. BJ/tert cells were cultivated in 80% simultaneously lost (38). Such a response could limit the trans- DMEM/20% medium 199 supplemented with 15% FCS. Aged forming potential of the aneuploid cells resulting from spindle IMR-90 cells (passage 29) were kindly provided by S. Lowe and checkpoint dysfunction. M. Narita, whereas aged WI-38 cells [population doubling (PD) What might be the nature of the lesion that triggers the 52] were a kind gift from P. D. Adams. p53-dependent response leading to premature senescence and aging? We observe that Bub1 loss in BJ/tert p53DD cells led to Growth Curve. A total of 104 cells was plated in duplicate in a compromise of the spindle checkpoint and concomitant an- 24-well dishes. At5hafterplating (time point 0) and at 1, 2, and euploidy as determined by karyotype analysis. One possibility is 3 days after plating, the cells were fixed in 4% paraformaldehyde that a p53-dependent G1 checkpoint might be triggered after an and subsequently stained with 0.1% crystal violet dissolved in aberrant mitotic exit. A ‘‘tetraploidy checkpoint’’ has been 10% ethanol/90% water. After staining and thorough washing, hypothesized to exist to explain responses seen after cytochalasin the dye was extracted with 10% acetic acid, and absorbance at B-induced tetraploidization (39). However, more recent obser- 590 nm was measured. Similar results were obtained in two vations have seriously questioned the authenticity of this check- independent experiments. point (40, 41). Nevertheless, much evidence still exists that tetraploid cells have different fates depending on their p53 status Viral Infections. Lentiviral pLKO.1 vectors were packaged by (42, 43). A great deal of aneuploidy is seen in Bub1-deficient transfection into 293FT cells by using FuGENE 6 (Roche cells, which might trigger p53 activation. Arguing against a causal Diagnostics, Basel, Switzerland) according to the published correlation between senescence and aneuploidy, there was sig- protocol (22). Infections with lentivirus were performed for 4 h nificant aneuploidy in Mad2ϩ/Ϫ mouse embryo fibroblasts but no in the presence of 8 ␮g/ml polybrene. Phoenix amphotropic cells obvious premature senescence (37). Alternately, it is possible were used for the packaging of pBabe-puro retroviral vectors, that defects in Bub1’s functions in either metaphase congression and these were transfected according to the N, N-bis(2-

8338 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703164104 Gjoerup et al. Downloaded by guest on September 30, 2021 hydroxyethyl)-2-aminoethane sulfonic acid (BES) calcium phos- We thank S. Lowe (Cold Spring Harbor Laboratory, Cold Spring phate protocol. At 48 h after infection, cells were selected and Harbor, NY), M. Narita (Cambridge Research Institute, Cambridge, then maintained at all times in 3 ␮g/ml puromycin. United Kingdom), O. Pereira-Smith, R. Agami (The Netherlands Can- cer Institute, Amsterdam, The Netherlands), P. D. Adams (Fox Chase Cancer Center, Philadelphia, PA), R. Freire, and C. Lee for kind SA ␤-Gal Assay. SA ␤-gal activity was determined according to the assistance with reagents. We thank W. Hahn (Dana–Farber Cancer published protocol (18). Briefly, cells were fixed for 15 min in 0.2% Institute) for critically reading the manuscript and for supplying the glutaraldehyde, washed with PBS, and then stained overnight at lentivirus shRNA vectors used here. This work was supported by 37°C with freshly made staining solution (40 mM citric acid/sodium National Institutes of Health Grants PO1-CA50661 and CA30002 (to phosphate, pH 6.0/5 mM potassium ferricyanide/5 mM potassium T.M.R.) and PO1-CA50661 and CA34722 (to B.S.) as well as by ferrocyanide/150 mM NaCl/2 mM MgCl2/1 mg/ml X-Gal). Wellcome Trust Project Grant 072672 (to P.S.J.).

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