Regulation of Cyclin-Dependent Kinase Activity During Mitotic Exit and Maintenance of Genome Stability by P21, P27, and P107

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Regulation of cyclin-dependent kinase activity during mitotic exit and maintenance of genome stability by p21, p27, and p107

Taku Chibazakura*†, Seth G. McGrew‡§, Jonathan A. Cooper§, Hirofumi Yoshikawa*, and James M. Roberts‡§

*Deparment of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan; and ‡Howard Hughes Medical Institute and §Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98019

Communicated by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, February 4, 2004 (received for review October 28, 2003) To study the regulation of cyclin-dependent kinase (CDK) activity bind to and inactivate mitotic cyclin–CDK complexes (15, 16). during mitotic exit in mammalian cells, we constructed murine cell These CKIs accumulate and persist during mid-M-to-G1 phase ͞ lines that constitutively express a stabilized mutant of cyclin A until they are phosphorylated by Sic1 Rum1-resistant G1 cyclin- (cyclin A47). Even though cyclin A47 was expressed throughout CDKs, which initiates their ubiquitin-dependent degradation at mitosis and in G1 cells, its associated CDK activity was inactivated the G1-to-S phase transition (17–19). Thus, Sic1 and Rum1 after the transition from metaphase to anaphase. Cyclin A47 constitute a switch that controls the transition from a state of low associated with both p21 and p27 during mitotic exit, implicating CDK activity to that of high CDK activity, thereby regulating these proteins in CDK inactivation. However, cyclin A47 was fully mitotic exit and S phase entry. This parallels the activity of ؊/؊ ؊/؊ inhibited during the M-to-G1 transition in p21 p27 fibro- APC-Cdh1, and indeed these two pathways constitute redundant blasts. Also, the CDKs associated with cyclin A47 were not inacti- oscillators that dictate the timing of CDK activation and inac- vated by phosphorylation at tyrosines. The protein responsible for tivation during the cell cycle (20). For example, a nondegradable CDK inactivation during mitotic exit in p21͞p27 null cells was the mutant of Clb5 does not disrupt cell-cycle progression, although Rb family member, p107. p107 bound to cyclin A47 when p21 and it is lethal when expressed in sic1-null yeast (21, 22). In Dro- p27 were absent, and cyclin A47–CDK activity was not inactivated sophila, the inhibitory protein Roughex (Rux) regulates the CELL BIOLOGY ؊/؊ ؊/؊ ؊/؊ during the M-to-G1 transition in p21 p27 p107 null fibro- activity of mitotic cyclin–CDK complexes. Rux binds to and blasts. Enforced expression of cyclin A in cells lacking all three CDK inactivates cyclin A- and B-CDK1 (23). It contributes to mitotic inhibitors induced rapid tetraploidization, indicative of mitotic exit in Drosophila embryo (24) and to G1 arrest of the cells in the failure͞endoreduplication. We concluded that cyclin proteolysis morphogenetic furrow of developing eye disk (25). Like Sic1, and CDK inhibitors constitute redundant pathways that control Rux prevents a nondegradable cyclin A mutant from inducing S cyclin A–CDK activity during mitotic exit in mammalian cells and phase (26). that loss of these pathways can cause genetic instability. It is not known whether there are pathways in addition to cyclin degradation that control CDK activity during mitotic exit in mammalian cells. To examine this, we have constructed scillation in cyclin-dependent kinase (CDK) activity is a murine cell lines that express a stabilized mutant of cyclin A at basic mechanism controlling progression through the eu- O nearly physiological levels. This has enabled us to identify new karyotic cell cycle (1, 2). Beginning in late G , rising amounts of 1 pathways that regulate cyclin A–CDK activity during mitotic exit CDK activity first trigger DNA replication and, later, mitosis. and to explore how cell-cycle regulation is disrupted when these Then, at the conclusion of anaphase, CDK activity is extin- pathways are inactivated. guished to allow for mitotic exit, reassembly of preinitiation complexes at replication origins, and establishment of a G1 Methods state (3). Mice. p21Ϫ/Ϫ and p107Ϫ/Ϫ mice were provided by T. Jacks Three mechanisms for controlling CDK activity during mitotic (Massachusetts Institute of Technology, Cambridge). exit have been identified in organisms ranging from yeast to humans: cyclin degradation, tyrosine phosphorylation, and CDK Cells. Mouse embryonic fibroblasts (MEFs) were isolated and inhibitor (CKI) binding. The anaphase-promoting complex propagated as described (27). NIH 3T3 cells (clone 7) were (APC) is a multisubunit E3 ubiquitin–protein ligase that initiates provided by C. Sherr (St. Jude Children’s Hospital, Memphis, the proteasomal degradation of cyclins (4–6). Both the A and B TN). Human cyclin A2 cDNA was provided by B. Clurman (Fred type cyclins contain a motif, the D box (destruction box) (7), that Hutchinson Cancer Research Center, Seattle) and mutagenized is required for its ubiquitination by the APC (8–10). Cyclin (28). Cyclin A2 cDNAs were N-terminally tagged with 6ϫ myc destruction is initiated at metaphase by one form of the APC, epitope and transduced into passage 3–5 MEFs by using retro- APC-Cdc20, and completed at anaphase by another form, viral vectors (29) at a multiplicity of infection of Ͼ2, and stably APC-Cdh1 (11). APC-Cdh1 remains active after mitotic exit has infected cells were pooled (Ͼ105 clones). For synchronization at been completed, creating a persistent state of low CDK activity, M phase, cells were treated with 2 mM thymidine for 16–20 h and called G1. At the end of G1, APC-Cdh1 is inactivated, thereby transferred into medium containing no thymidine and 0.1 ␮g͞ml allowing the reaccumulation of cyclin A and the onset of DNA nocodazol for 8–10 h; mitotic cells were then harvested by replication. shake-off and released into nocodazol-free medium. CDKs can also be inhibited by Wee1-related tyrosine kinases. Phosphorylation of Cdk2 on tyrosine 15 contributes to the timing Microscopy. For immunofluorescence microscopy (30), cells were of cyclin B degradation and the rate of mitotic exit in the early fixed with 4% paraformaldehyde in PBS for 10 min and perme- cleavage cycles of Xenopus embryos (12, 13). This pathway does not seem to be important in budding yeast (14), and its role in other organisms is not known. Abbreviations: CDK, cyclin-dependent kinase; D box, destruction box; MEF, mouse embry- CKIs have been shown to regulate CDK activity during mitosis onic fibroblast; APC, anaphase-promoting complex; CKI, cyclin-dependent kinase inhibitor. in yeast and Drosophila. In yeasts Saccharomyces cerevisiae and †To whom correspondence should be addressed. E-mail: [email protected]. Schizosaccharomyces pombe, CKIs Sic1 and Rum1, respectively, © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400655101 PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4465–4470 Downloaded by guest on September 28, 2021 Fig. 1. A stabilized D box mutant of human cyclin A2. (a) D box sequences in various cyclin A and cyclin B1 proteins. (b and c) NIH 3T3-derived cell lines stably expressing myc-tagged (MT) wild-type (cycA WT) or the mutant (cycA47) cyclin A protein were released from nocodazole block. At the indicated times, endogenous and exogenous cyclin A proteins were detected by anti-cyclin A immunoblotting (b), and the cell-cycle distribution was analyzed by ﬂow cytometry (c).

abilized with 0.2% Triton X-100 in PBS for 5 min at room CD20 antibody (BD Biosciences, San Jose, CA), fixed with 80% temperature. Cells were stained with anti-myc tag monoclonal ethanol, and stained with propidium iodide (36). Flow cytometry antibody PL14 (Medical and Biological Laboratories, Nagoya, was performed by using FACSCalibur cytometer and Japan) and FITC-conjugated anti-mouse IgG antibody (Roche CELLQUEST software (BD Biosciences), and data were gated for Diagnostics). FITC (CD20)-positive cells.

Preparation of Cell Extracts and Immunoprecipitation. Cells were Results rinsed once with TBS (pH 7.4) and lysed on ice in RIPA buffer Constitutive Expression of Cyclin A During Mitotic Exit. We have (10 mM Tris⅐HCl, pH 7.4͞0.15 M NaCl͞0.5% Nonidet P-40͞50 focused on regulation of the activity of human cyclin A2, which mM NaF͞1 mM Na-vanadate͞10 mg/ml each of aprotinin, is essential for normal mitotic cell cycles. To prevent cyclin leupeptin, and pepstatin). Immunoprecipitation and histone H1 destruction during mitotic exit, and thereby determine whether kinase assays were performed as described (31, 32) by using the there are additional pathways for regulating cyclin A-associated following antibodies: anti-myc tag 9E10 (31), anti-cyclin A (33), kinase activity, we constructed human cyclin A2 mutants that anti-Cdk2 (32), and anti-p21 (C-19), anti-p27 (N-20), anti-Cdc2 contained alanine substitution or deletion mutations in the D (clone 17), and anti-p107 (C-18) from Santa Cruz Biotechnol- box region. One of these, cyclin A47 (cycA47), contained three ogy. alanine substitutions at amino acid residues which are most conserved in the D boxes of A- and B-type cyclins (Fig. 1a). In Vivo Radiolabeling and Phosphoamino Acid Analysis. Cells were CycA47 and wild-type cyclin A were expressed in mouse NIH synchronized with nocodazol, and mitotic cells (4 ϫ 106) were 3T3 fibroblasts as myc epitope-tagged proteins by retroviral rinsed twice with phosphate-free medium plus 10% FBS dia- transduction. The levels of expression of these exogenous cyclins lyzed against 0.15 M NaCl and split (2 ϫ 106 cells each) into 4 closely approximated the amount of endogenous cyclin A2 in 32 ͞ ml of the same medium containing 37 MBq͞ml [ P]orthophos- S G2 phase cells (Fig. 1b, 16-h time point). D box-dependent phate (ICN), with and without 0.1 ␮g͞ml nocodazol. When degradation of cyclin A commences at metaphase and continues Ͼ95% of the cells completed cytokinesis (3–4 h after the release through G1. When the established cell lines were synchronized from nocodazol), they were rinsed twice with TBS (pH 7.4), and at prometaphase with the microtubule destabilizing agent no- extracts were prepared. CDK proteins in the anti-Cdk2 and codazole, cycA47 accumulated to a substantially higher level anti-Cdc2 immunoprecipitates, and corresponding proteins in than endogenous cyclin A (Fig. 1b). Moreover, the cycA47 the anti-myc tag immunoprecipitates, were extracted from the protein was inefficiently degraded after the cells were released gel and subjected to phosphoamino acid analysis (34). from nocodazole-induced prometaphase arrest, and it remained present throughout the G1 phase of the cell cycle. In contrast, Transfection and Flow Cytometry. Cyclin cDNAs were subcloned both the endogenous cyclin A2 and wild-type exogenous cyclin into pCS2MT (35) and myc-tagged and cotransfected with A2 proteins were completely degraded and absent from G1 pCMVCD20 plasmid expressing cell-surface antigen CD20 (36) phase cells. by using the modified Ca-phosphate method (31), with 10 ␮g Cells expressing the cycA47 protein exited mitosis normally. each of the pCS2MT-cyclin plasmids and 2 ␮g of pCMVCD20 By 80 min after release from the metaphase block, most cells ϫ 5 per 4 10 cells in 100-mm dishes. Sixteen hours after the expressing either cycA47 or wild-type cyclin A2 had a G1 DNA transfection, cells were refed and cultured for an additional 24 h. content (Fig. 1c), and there was no evidence that binucleated Harvested cells were incubated with FITC-conjugated anti- cells accumulated (data not shown). Thus, mitotic exit seemed

4466 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400655101 Chibazakura et al. Downloaded by guest on September 28, 2021 genotypes, whereas the wild-type cyclin A2 protein was degraded. CycA47-associated CDK activity was inactivated in early G1 in MEF strains of all three genotypes (Fig. 3a) and in MEFs from p21ϩ/ϩ p27ϩ/ϩ littermates (data not shown). Therefore, neither p21 nor p27 was essential for the repression of the cycA47-associated CDK activity during the transition from M to G1. We also investigated the contribution of tyrosine phosphorylation to CDK regulation during mitotic exit by determining the in vivo phosphorylation state of the cycA47-associated CDKs. NIH 3T3 cells stably expressing the myc-tagged cycA47 protein were synchronized in prometaphase and released into G1 in the continuous presence of [32P]orthophosphate. The phosphoamino acid composition of the CDK proteins coimmuno- precipitated with the cycA47 protein indicated that they were phosphorylated at serine and threonine but not at tyrosine residues (Fig. 3b). Parallel phosphoamino acid analysis on total Cdk2 and Cdc2 proteins immunoprecipitated with their specific antibodies revealed some phosphotyrosine in Cdc2 in early G1 cells (Fig. 3b), presumably representing either free Cdc2 or Cdc2 Fig. 2. Cyclin A-associated proteins and CDK activity. The NIH 3T3 derivatives associated with residual cyclin B. This was consistent with were grown asynchronously (As) or synchronously as in Fig. 1, and the extracts previous observations (42, 43). Our results suggested that inhib- from cells under nocodazole block (M), 120 min after the release (G1), and 16 h itory phosphorylation on tyrosine does not make a major after the release (S) were immunoprecipitated (IP) with anti-myc antibodies contribution to CDK inactivation in cyclin A complexes during and then immunoblotted (IB) for the indicated proteins or assayed for the mitotic exit. histone H1 kinase activity. Regulation of Cyclin A Activity by the Rb-Related Protein p107. CELL BIOLOGY Previous results have shown that the Rb-related proteins p107 unaffected by the constitutive presence of cyclin A2 during M and p130 can bind to and inhibit CDKs (44). Because p130 has and G1, although overexpression of superphysiological amounts a physiological role in regulating Cdk2 activity in G0 cells that of stabilized cyclin A2 can cause mitotic abnormalities and even express low amounts of p27 (45), we asked whether p107 might a block to cell-cycle progression (37–40). have a parallel role in CDK regulation in G1 cells. CycA47 was immunoprecipitated from M, G1, and S phase cells that were Regulation of Cyclin A-Associated Kinase Activity During Mitotic Exit. p21Ϫ/Ϫ, p27Ϫ/Ϫ,orp21Ϫ/Ϫ p27Ϫ/Ϫ (Fig. 4a). There was little p107 The observation that mitotic exit proceeded normally despite stably associated with cycA47 in control NIH 3T3 cells and in constitutive cyclin A expression suggested that cyclin A-associ- MEFs that lacked either p21 or p27. In contrast, a complex of ated kinase activity might be regulated independently of cyclin p107 and cycA47 was readily detected in G1 cells that lacked both destruction. To address this, we immunoprecipitated the exog- p21 and p27 and was also present at lower stoichiometry in S enous cyclin A proteins and measured their associated CDK phase p21Ϫ/Ϫ p27Ϫ/Ϫ cells (Fig. 4a). activity. Wild-type cyclin A-associated CDK activity was high in The mutually exclusive interaction between cyclin A and metaphase and decreased to an undetectable level in early G1 either p21͞p27 or p107 was confirmed in transfected cells. Cyclin coincidentally with cyclin degradation (Fig. 2). The cycA47- A was transiently overexpressed in HEK293 cells, and its asso- associated CDK activity was also extinguished in early G1 cells, ciation with endogenous p107 was measured by coimmunopre- even though a substantial amount of cyclin A protein remained cipitation. A complex between p107 and cyclin A was detected, and was bound to Cdk2 (Fig. 2). Immunofluorescence showed but formation of this complex was prevented by simultaneous that the cycA47 protein was localized in nuclei of newly divided overexpression of either p21 or p27 (Fig. 4b). Similar observa- G1 cells (data not shown). These results showed that mechanisms tions have been reported previously (46). other than cyclin destruction were able to down-regulate CDK These observations suggested that p107 might regulate CDK activity during mitotic exit in mammalian cells. activity during mitotic exit and perhaps be the primary regulator In addition to cyclin destruction, mechanisms of CDK inhi- in cells lacking p21 and p27. To test this, we studied CDK bition include association with CKIs and tyrosine phosphoryla- regulation in MEFs lacking all three inhibitory proteins. p21Ϫ/Ϫ, tion (41). We observed that both the p21 and p27 CKIs were p27Ϫ/Ϫ, and p107Ϫ/Ϫ mice were interbred, and MEFs were associated with cyclin A in metaphase cells and that the portion prepared from triple knockout progeny. However, using retro- of cyclin A that escaped proteolysis was associated with these viral vectors, we were unable to establish any triple knockout cell CKIs in G1 cells (Fig. 2). Indeed, the amount of p27 associated lines that stably expressed either wild-type cyclin A or cycA47, with stabilized cycA47 further increased in G1 cells when cyclin suggesting that even relatively moderate overexpression of cyclin A-associated kinase activity became inactive (Fig. 2). We could A protein might be lethal in this genetic background. not detect any p57 associated with cyclin A in these cells (data To circumvent this problem, we exposed these triple knockout not shown). These results showed that cyclin A associated with MEFs to high titer retroviral stocks at an multiplicity of infection p21 and p27 during mitotic exit and suggested that these CKIs of Ͼ5, such that nearly 100% of the recipient cells were might be responsible for the repression of the cycA47-associated successfully transduced and expressed exogenous cyclin A pro- CDK activity in G1 cells. This was tested by using early passage tein without the need for subsequent selection (data not shown). Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ MEFs derived from p21 / , p27 / , and p21 / p27 / knockout Under these conditions, the abundance and activity of wild-type mice. Wild-type cyclin A2 and cycA47 were introduced as cyclin A was down-regulated during mitotic exit as we had seen myc-tagged proteins by retroviral transduction. As we had in MEFs of other genotypes. However, the CDK activity asso- observed in NIH 3T3 cells, the cycA47 protein was relatively ciated with cycA47 was not effectively down-regulated in G1 cells Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ stable during the M-to-G1 transition in MEFs of all three of p21 p27 p107 MEFs (Fig. 5a). Therefore, p107 is

Chibazakura et al. PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4467 Downloaded by guest on September 28, 2021 Fig. 3. Regulation of cyclin A-associated CDK activity in G1 phase. (a) CycA47 abundance and its associated H1 kinase activity were analyzed during the M-to-G1 Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ transition in p21 , p27 , and p21 p27 MEFs. The extracts from nocodazole-blocked (M) and early G1-phase (G1) cells were immunoprecipitated and analyzed as in Fig. 2. (b Upper) Scheme of the in vivo 32P-labeling procedure (see Methods for details). (Lower) Phosphoamino acid analysis of the CDK proteins recovered from the immunoprecipitates after the in vivo 32P labeling. Horizontal and vertical arrows represent the ﬁrst and second electrophoresis, respectively.

required for repression of the cycA47-associated CDK activity alone is sufficient, as no mitotic abnormalities were observed when both p21 and p27 are absent. when only one or the other was disabled. However, simultaneous Despite the constitutive activation of cycA47, Ͼ90% of the inactivation of both pathways causes rapid and severe genetic cells completed cytokinesis after release from a nocodazole- instability. induced prometaphase arrest (Fig. 5a). However, stable activa- We probed the mechanisms that regulate CDK activity during tion of cyclin A in p21Ϫ/Ϫ p27Ϫ/Ϫ p107Ϫ/Ϫ MEFs was associated mitosis in murine cells by constructing cell lines that expressed with genetic instability. Whereas transient overexpression of a mutant version of cyclin A, cycA47, that was relatively stable wild-type cyclin A or cycA47 in NIH 3T3 cells (Fig. 5b)orin compared to endogenous cyclin A. CycA47 contained three wild-type MEFs (data not shown) caused only a modest increase alanine substitution mutations within the D box, which did not ͞ completely prevent cyclin A proteolysis but sufficiently disabled in the percentage of S and G2 M phase cells, transient overexpression of either wild-type or cycA47 in p21Ϫ/Ϫ p27Ϫ/Ϫ p107Ϫ/Ϫ it so that some cyclin A protein was present and bound to CDK MEFs resulted in a remarkable decrease in diploid G cells and throughout mitosis and in G1 cells. The amount of exogenous 1 cycA47 expressed by these cells was not greater than endogenous an increase in tetraploid cells (Fig. 5b). This was not accompa- cyclin A, which likely accounted for our success in producing nied by an increase in binucleated cells (data not shown), these viable cell lines, whereas previous studies employing cyclin suggesting that the rapid appearance of tetraploid cells was due ͞ A overexpression observed cyclin A-dependent mitotic arrest to mitotic failure and or endoreduplication. (37–40). Discussion In wild-type fibroblasts, the kinase activity associated with cycA47 was inactivated during mitotic exit. This correlated with Destruction of mitotic cyclins by ubiquitin-dependent proteolysis its assembly into complexes containing the CKIs p21 and p27 and is a universal mechanism for regulating CDK activity and mitotic not with CDK phosphorylation on tyrosine. These results suggest exit (11). In addition, CKIs Rux and Sic1 collaborate with cyclin that the p21 and p27 participate in CDK regulation during destruction to ensure timely CDK inactivation during mitotic mitosis. However, genetic experiments designed to confirm this exit in Drosophila and yeast, respectively (21, 22, 24–26). We now showed that p21 and p27 were not essential mitotic CKIs. Thus, show that CKIs also perform this function in mammalian cells. the kinase activity associated with cycA47 was just as effectively Our major conclusion is that cyclin destruction and CDK inhibited in p21Ϫ/Ϫ p27Ϫ/Ϫ cells as it was in control cells. inhibition are redundant pathways for controlling cyclin A Previous work showed that the Rb-related proteins p107 and activity during mitotic exit in mammalian cells. Either pathway p130 not only regulate E2F-related transcription factors but can

4468 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400655101 Chibazakura et al. Downloaded by guest on September 28, 2021 Fig. 4. p107 binds to cyclin A in the absence of p21 and p27. (a) NIH 3T3- and the indicated MEF-derived cell lines, stably expressing the myc-tagged cycA47 CELL BIOLOGY protein, were synchronized with nocodazole, and the extracts from nocodazole-blocked (M), early G1-phase (G1), and S-phase (S) cells were immunoprecipitated with anti-myc antibodies and immunoblotted for myc-tagged cycA47 and p107. (b) HEK293 cells were transfected with the combinations of myc-tagged wild-type cyclin A-, p21-, and p27-expressing vectors as indicated, and the cell extracts were immunoprecipitated with anti-myc tag antibodies and immunoblotted for the indicated proteins.

also bind to and inhibit CDKs (44). It has also been shown that activation of cycA47-CDK in M and G1 phase cells, deletion of p130 can substitute for p27 and inhibit Cdk2 in mitogen-starved, p107 in combination with p21 and p27 was sufficient to dereg- p27Ϫ/Ϫ fibroblasts (45). We now show that p107 has a parallel ulate cycA47–CDK activity. In MEFs, p107 seemed to perform function in regulating CDK activity during the M-to-G1 transi- this function only when p21 and p27 were absent because no p107 tion. In cells lacking both p21 and p27, but not in wild-type cells, was bound to cycA47 in M-phase p21Ϫ/Ϫ or p27Ϫ/Ϫ MEFs. It p107 was associated with cyclin A during mitotic exit. Moreover, remains to be determined whether p107 might normally perform whereas deletion of p21 and p27 alone did not cause constitutive this function in somatic cells that express lesser amounts of p21

Fig. 5. p107 regulates cycA47-associated CDK activity. (a Upper) The p21Ϫ/Ϫ p27Ϫ/Ϫ p107Ϫ/Ϫ MEF cells, infected with the myc-tagged cyclin A-carrying retroviruses, were synchronized with nocodazole, and the extracts from nocodazol blocked cells (M) and 2.5 h after release from arrest (G1) were analyzed as in Fig. 3. (Lower) Anti-myc tag immunostaining (green) of MEFs expressing myc-tagged cycA47. MEFs were ﬁxed 1.5 and 2.5 h after the release from nocodazole block. DNA was stained with Hoechst 33342 (blue). (b) NIH 3T3 cells and the p21Ϫ/Ϫ p27Ϫ/Ϫ p107Ϫ/Ϫ MEFs were transiently cotransfected with the CD20-expressing vector and the expression vectors carrying no cyclin (vec), myc-tagged wild-type cyclin A (MTcycA WT), or the cycA47 mutant (MTcycA47). Cell-cycle distribution of the transfected (CD20-positive) cells was analyzed by ﬂow cytometry.

Chibazakura et al. PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4469 Downloaded by guest on September 28, 2021 and p27. Another possibility is that the role of p107 in controlling the cells to exit mitosis without separation of sister chromatids CDK activity may become critically important in pathophysio- (24). We observed a similar phenotype in MEFs lacking p21, logical settings, such as tumor cells, in which cyclins are over- p27, and p107. Overexpression of even modest amounts of cyclin Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ expressed and p21 and p27 are present in aberrantly low A was lethal in p21 / p27 / p107 / MEFs. Moreover, tran- amounts. sient and ectopic cyclin A expression in the triple knockout In yeast and Drosophila, the CKIs Sic1 and Rux, respectively, MEFs caused the cells to rapidly become tetraploid, suggestive play crucial roles in regulating mitotic exit, and loss of those of a failure to separate sister chromatids during mitotic exit or pathways results in genetic instability (24, 47, 48). Neither Rux endoreduplication of DNA. This does not seem to be due to a nor Sic1 is essential because their roles in CDK regulation during deregulation of the E2F transcription program (49), as we observed no difference between p21Ϫ/Ϫ p27Ϫ/Ϫ and p21Ϫ/Ϫ mitotic exit are at least partially redundant with APC-mediated Ϫ Ϫ Ϫ Ϫ cyclin destruction, but both sic1 and rux mutant cells display p27 / p107 / MEFs in the expression of E2F-regulated genes, ␣ mitotic abnormalities. rux mutant cells are delayed at metaphase such as DNA polymerase and cyclin E (data not shown). (24), and sic1 mutant cells are delayed in mid-anaphase and In conclusion, we show that two redundant pathways control display a high frequency of chromosome nondisjunction cyclin A–CDK activity during mitotic exit in mammalian cells: and breakage (47, 48). p21, p27, and p107 are also nonessential, cyclin destruction and CKIs. The CKIs responsible for mitotic and we are continuing to explore whether their loss, alone or in CDK regulation have all been implicated in tumor suppression. combination, causes mitotic defects. Initial observations did not Their roles in mitotic exit may contribute to their tumor sup- reveal a substantial delay in the timing of mitotic exit in triple pressor function, as corruption of this pathway can cause genetic knockout cells compared to wild-type MEFs (see Fig. 5a). instability. The mitotic abnormalities associated with sic1 and rux mutations are greatly exacerbated if the functionally overlapping We thank M. Ohtsubo, B. Clurman, E. Firpo, E. Randel, M. Fero, D. Miller, J. Kato, and members of the Roberts laboratory for helpful cyclin destruction pathway is also disabled, for example by discussions, technical assistance, and materials. This work was supported expression of a nondegradable cyclin mutant or by ectopic by grants from the National Institutes of Health and from the Japan overexpression of a wild-type mitotic cyclin. For instance, ex- Society for the Promotion of Science (11660328). J.M.R. is an Investi- pression of a D box mutant of cyclin A in rux mutant cells causes gator of the Howard Hughes Medical Institute.

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