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provided by Elsevier - Publisher Connector Current Biology, Vol. 12, 1817–1827, October 29, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)01225-3 and Form an Inducible Barrier that Protects Cells against E-cdk2 Deregulation

Alex C. Minella,1,2 Jherek Swanger,1 Eileen Bryant,1 E-cdk2 is rate limiting and is required for G1 progression, Markus Welcker,1 Harry Hwang,1 and cyclin E abundance oscillates during the and Bruce E. Clurman1,2,3 [2–5]. A pulse of cyclin E transcription in late G1 is regu- 1Divisions of Clinical Research and Human Biology lated by activity, and this in part determines cyclin Fred Hutchinson Cancer Research Center E periodicity [6]. Cyclin E is an unstable protein, and 2 Department of Medicine ubiquitin-mediated proteolysis also determines its University of Washington School of Medicine abundance. Cyclin E turnover is regulated by site-spe- Seattle, Washington 98109 cific cyclin E phosphorylations, by binding to cdk2, and by the Fbw7/SCF ubiquitin ligase [7–10]. The (Rb) is phosphorylated Summary by - and cyclin E-associated cdks during G1 progression. Progressive phosphorylation inactivates Background: Cyclin E, in conjunction with its catalytic Rb, and this promotes entry. Most cancer cells partner cdk2, is rate limiting for entry into the S phase of contain mutations in the Rb-cyclin D pathway, although the cell cycle. Cancer cells frequently contain mutations the precise mechanisms through which these mutations within the cyclin D-Retinoblastoma protein pathway that promote tumorigenesis are largely unknown [11]. These lead to inappropriate cyclin E-cdk2 activation. Although mutations include gain-of-function mutations of cyclin deregulated cyclin E-cdk2 activity is believed to directly D and cyclin E and the loss of function of two classes contribute to the neoplastic progression of these can- of cdk inhibitor proteins that inhibit Rb phosphorylation cers, the mechanism of cyclin E-induced neoplasia is (the Cip/Kip and INK4 families), as well as Rb itself. unknown. Virtually all of these mutations may lead to inappropriate Results: We studied the consequences of deregulated cyclin E-cdk2 activity, which may then contribute di- cyclin E expression in primary cells and found that cyclin rectly to cell transformation. E initiated a p53-dependent response that prevented In established cell lines, ectopic cyclin E expression excess cdk2 activity by inducing expression of the causes faster progression through , prolongs p21Cip1 cdk inhibitor. The increased p53 activity was the duration of S phase, and leads to decreased mitogen not associated with increased expression of the requirements, altered cell size, and increased cell satu- tumor suppressor. Instead, cyclin E led to increased p53 ration density [4, 5, 12, 13]. Ectopic cyclin E expression serine15 phosphorylation that was sensitive to inhibitors in established cell lines has also been associated with of the ATM/ATR family. When either p53 or p21cip1 genetic instability and abnormal centrosome duplication was rendered nonfunctional, then the excess cyclin E [13, 14]. These activities have been suggested to under- became catalytically active and caused defects in S lie the association between cyclin E deregulation and phase progression, increased ploidy, and genetic insta- the development of cancer. bility. Deregulated cyclin E expression has also been linked Conclusions: We conclude that p53 and p21 form an to tumorigenesis in vivo. Cyclin E abnormalities have inducible barrier that protects cells against the deleteri- been described in many human cancers, and cyclin E ous consequences of cyclin E-cdk2 deregulation. A re- status has been correlated with poor clinical and/or his- sponse that restrains cyclin E deregulation is likely to tologic indices [15–18]. Mice with transgenes directing be a general protective mechanism against neoplastic cyclin E expression in the mammary epithelium develop transformation. Loss of this response may thus be re- breast carcinomas [19]. However, these tumors devel- quired before deregulated cyclin E can become fully oped in only 12% of animals with cyclin E transgenes oncogenic in cancer cells. Furthermore, the combination and after a long latent period. Similarly, transgenic mice of excess cyclin E and p53 loss may be particularly with cyclin E expression directed to the thymus did not genotoxic, because cells cannot appropriately respond spontaneously develop lymphomas, but they exhibited to the cell cycle anomalies caused by excess cyclin increased lymphomagenesis induced by carcinogens E-cdk2 activity. [20]. Thus, cyclin E deregulation was not sufficient for the development of these cancers, but it may have directly contributed to the multistep process of tumorigenesis. Introduction In order to further characterize the consequences of cyclin E deregulation in normal cells, we enforced cyclin Cell cycle transitions are catalyzed by the cyclin-depen- E expression in primary human and murine fibroblasts. dent kinases (cdks). Cdks are composed of a catalytic Unexpectedly, we found that prolonged expression of subunit (the cdk) and a regulatory subunit (a cyclin), and cyclin E in primary fibroblasts led to decreased cdk2 cyclin binding activates the cdk [1]. Progression through activity and inhibited, rather than promoted, cell cycle the G1 phase of the mammalian cell cycle is driven by progression. The decreased cdk2 activity was associ- the D-type , which activate the cdk4 and cdk6 ated with increased p21cip1 expression, and this re- kinases, and cyclin E, which activates cdk2. Cyclin quired intact p53 function. Furthermore, both p53 and p21 were required to suppress ectopic cyclin E in mouse 3 Correspondence: [email protected] embryo fibroblasts (MEFs). The activation of p53 by Current Biology 1818

(Figure 1). The increased p21 abundance was due to increased p21 mRNA, and not altered p21 turnover (Fig- ures S1B and S1C). In contrast, there was no increased abundance of the related p27Kip1 cdk inhibitor (not shown). Thus, deregulated cyclin E expression in pri- mary human fibroblasts led to increased p21cip1 abun- dance and cdk2 inactivation. Because p21 is a key transcriptional target of p53, we next examined if the increased p21 abundance resulted from increased p53 activity. Figure 2A shows that cyclin E overexpression in primary human foreskin fibroblasts (HFFs) caused increased p53 protein abundance that paralleled p21 abundance. To determine if increased p53 activity was required for the p21 induction, we first transduced cells with a vector encoding the human pap- illoma virus E6 protein (HPV-E6), which abrogates p53 function by catalyzing p53 degradation [21]. Thus, HPV- E6-expressing cells are effectively p53 null, although E6 may have activities in addition to p53 turnover. We found that expression of HPV-E6 completely inhibited the in- duction of p21 by cyclin E in WS1 human diploid fibro- blasts, HFFs, and WI-38 cells (Figures 2B and 2C and not shown). Furthermore, HPV-E6 expression reversed the inhibition of total cdk2 and -cdk2 activity Figure 1. Deregulated Cyclin E Expression in Primary Human Fibro- blasts Leads to cdk2 Inhibition and Increased p21cip Expression caused by cyclin E and allowed the cyclin E to form WI-38 cells were transduced with retroviral vectors expressing active kinase complexes in all three primary fibroblast cyclin E, a hyperactive cyclin E mutant, cyclin E (T380A), or control isolates (Figure 2C and not shown). The loss of p21 vector. Cells were harvested 2 weeks after transduction and selec- expression did not significantly alter the amount of cdk2 tion. Western analysis of cyclin E and p21 abundance, and total that coprecipitated with cyclin E, but it instead resulted cdk2 activity measured in an anti-cdk2 immunoprecipitate kinase in a larger proportion of active cyclin E-cdk2 complexes assay, are shown. (Figure 2D). These data are consistent with the idea that the induction of p21 was due to increased p53 activity. The suppression of cdk2 activity resulting from cyclin cyclin E was associated with increased p53 serine 15 E expression was also p53 dependent, because, in the phosphorylation that was inhibited by caffeine or wort- absence of p53, this response no longer occurred. mannin, suggesting that it resulted from the ATM/ATR family of kinases. Moreover, when p53 function was Both p21 and p53 Are Required disabled, excess cyclin E-cdk2 activity caused aberrant for the Suppression of Ectopic Cyclin cell cycle regulation and genetic instability. These data E in Primary Mouse Fibroblasts indicate that excess cyclin E in primary cells initiates a Because HPV-E6 may have pleiotropic effects in addi- homeostatic p53-dependent program that limits cdk2 tion to p53 degradation, we also used MEFs with tar- activity via induction of p21. This response protects geted deletions in either p21 or p53 to directly test the normal cells from inappropriate cyclin E-cdk2 activity. role of these proteins in suppressing deregulated cyclin E activity [22, 23]. We found that, whereas ectopic cyclin Results E had minimal kinase activity in normal MEFs, the exoge- nous cyclin E was fully active in cells lacking either p21 Deregulated Cyclin E Expression in Primary or p53 (Figures 3A and 3B). Similarly, cyclin E overex- Human Fibroblasts Causes the Induction pression caused profound cell cycle anomalies in p21 of p53 and p21 null and p53 null MEFs compared with normal cells (Fig- We constructed retroviral vectors encoding either cyclin ures 3A and 3B). Enforced cyclin E expression only E or cyclin E (T380A), a mutant that is hyperactive be- minimally altered the cell cycle profile of asynchronous cause it is resistant to ubiquitin-mediated proteolysis normal MEFs (Figure 3A). Like other workers, we consis- [7, 10]. Transduction of WI-38 primary human diploid tently detected a small fraction of 8N cells in normal fibroblasts with either cyclin E or cyclin E (T380A) vectors MEFs [24]. In contrast, cyclin E expression in either p21 led to significant cyclin E overexpression that exceeded or p53 null MEFs had two major effects on the cell the amount of endogenous cdk2 (Figures 1 and S1A, see cycle: accumulation of cells in S phase and an increased the Supplementary Material available with this article proportion of cells with higher ploidy, with cells reaching online). Although cyclin E normally activates cdk2, we 16N DNA content (Figures 3A and 3B). Thus, in primary paradoxically found that WI-38 cells with prolonged murine fibroblasts, the suppression of ectopic cyclin E cyclin E expression contained substantially reduced activity required p21 and p53 function. In contrast, MEFs cdk2 kinase activity (Figure 1). Concomitant with the with deletions of either p19ARF or the p27Kip1 cdk inhib- inactivation of cdk2, we observed a large increase in itor resembled wild-type cells and were quite resistant the abundance of the endogenous p21cip1 cdk inhibitor to excess cyclin E (Figure 3C and not shown). p53-Dependent Cyclin E Suppression 1819

Figure 2. The Induction of p21 Expression in Primary Human Fibroblasts Is Dependent upon p53 Function (A) Human foreskin fibroblasts (HFF) were lysed 2 weeks after transduction and selec- tion with the indicated vectors. Western anal- ysis of p53 and p21 expression is shown. (B) WS1 cells were transduced with either control virus (LXSH) or a vector expressing HPV-E6 prior to transduction with the indi- cated vectors. Expression of cyclin E and p21 is shown. (C) HFFs transduced with either control or E6 vectors were then transduced with the indi- cated vectors. Western analysis of cyclin E and kinase assays for cyclin E-cdk2, cyclin A-cdk2, or total cdk 2 activity is shown. (D) HFFs were transduced with the indicated vectors. Lysates were immunoprecipitated with anti-cyclin E antibody after normalizing total protein for total cyclin E protein. The amount of cdk2 (p-cdk2 indicates active thre- onine 160 phosphorylated cdk2) and p21 bound to cyclin E is shown.

The Induction of p53 by Cyclin E Is Associated creased p14ARF expression at any point after transduc- with Increased p53 Serine 15 Phosphorylation, tion (not shown). Thus, the increased p53 abundance but Not Increased p14ARF Expression resulting from cyclin E expression was not associated Enforced expression of dominant such as with increased p14ARF expression. and ras in MEFs also initiates a response involving Another common mechanism of signaling cellular p53 and p21 induction, followed by premature senes- stress to p53 is phosphorylation, and many distinct cence [11]. This response is mediated by the 19ARF phosphorylation sites affect p53 function in different protein (p14ARF in human cells), which increases in physiologic contexts [27, 28]. We thus examined site- abundance and prevents p53 degradation, in addition specific p53 phosphorylation in cyclin E-expressing to other activities. p14ARF is an E2F target gene (in at cells and found that phosphorylation of p53 on serine least some cell types), and the increased ARF expres- 15 was significantly elevated compared to control cells sion under these conditions may be due to increased (Figure 4B). In contrast, no increased p53 phosphoryla- E2F activity [25]. Because cyclin E overexpression may tion was noted on several other sites, including serine also lead to increased E2F activity through phosphory- 20 (not shown). Because the total abundance of p53 lation of the Retinoblastoma protein, we examined increased, it was difficult to determine if serine 15 phos- p14ARF expression in HFFs and WI-38 cells transduced phorylation specifically increased in response to cyclin by cyclin E vectors. In agreement with a recent report, E, or if this simply reflected increased p53 abundance. we could not detect p14ARF protein in primary human We thus treated control cells with the proteasome inhibi- fibroblasts, either in control or cyclin E-transduced cells tor MG-132 so that the amount of p53 in the control (not shown) [26]. We then used a sensitive real-time PCR cells approximated the amount of p53 in the cyclin assay to examine p14ARF mRNA abundance. WI-38 and E-expressing cells. The cyclin E-expressing cells con- HFF cells contained approximately 20% of the amount tained significantly more p53 serine 15 phosphorylation of p14ARF mRNA detected in HeLa cells, which are compared with the MG-132-treated controls, and this known to overexpress p14ARF (Figure 4A). No increased indicated that cyclin E expression led to a specific in- p14ARF mRNA was observed in cells expressing cyclin crease in p53 serine 15 phosphorylation (compare lanes E, although the assay was sensitive enough to detect 2 and 3, Figure 4C). 2-fold changes in p14ARF expression at this level of Several members of the PI3 kinase family are physio- expression (Figure 3A and not shown). We also assayed logically important p53 serine 15 kinases. The ataxia- p14ARF mRNA expression very early after transduction telangiectasia protein, ATM, and the related ATR protein with cyclin E retroviruses, in case there was a transient both phosphorylate p53 on serine 15 after DNA damage, pulse of p14ARF induction, but we failed to detect in- as well as in response to other cellular stresses such as Current Biology 1820

Figure 3. Both p21 and p53 Are Required to Suppress Deregulated Cyclin E Expression in Primary Murine Embryo Fibroblasts (A and B) MEFs derived from p21 ϩ/ϩ, p21 Ϫ/Ϫ, and p53 Ϫ/Ϫ littermates were transduced with the indicated viral vectors. Asynchronous cell cycle profiles, cyclin E abundance, and cyclin E-associated kinase activity are shown. (C) MEFs derived from p19ARF null embryos were transduced with the indicated viruses and were harvested for . The ploidy of the various cell populations is indicated (2N, 4N, 8N, 16N). replication blocks [29, 30]. DNA-PK also phosphorylates ther drug (Figure 4C). Furthermore, when we performed serine 15 in vitro, but its physiologic importance is less a time course analysis after wortmannin treatment of clear [31]. We treated cells with two distinct pharmaco- HFFs expressing cyclin E, we found that the abundance logic inhibitors of these enzymes, caffeine and wort- of p21 decreased roughly in parallel with serine 15 phos- mannin. We found that the amount of p53 serine 15 phorylation (Figure 4D). These data are consistent with phosphorylation in cyclin E-overexpressing cells was the idea that p53 serine 15 phosphorylation, at least in substantially decreased after a short treatment with ei- part, drives p21 expression. To further define the rele- p53-Dependent Cyclin E Suppression 1821

Figure 4. The Induction of p53 by Cyclin Is Associated with Increased p53 Serine 15 Phosphorylation, but Not Increased p14ARF Expression (A) HFF or WI-38 cells were transduced with either control (Babe) or cyclin E vectors, and RNA was harvested after 2 weeks. A quantitative real-time PCR assay was used to determine the amount of p14ARF mRNA compared with a HeLa cell standard. (B) HFFs were transduced as indicated, and Western analyses of p53 serine 15 phosphorylation (as detected by a phospho serine 15-specific antibody), total p53, and p21 are shown. (C) HFFs transduced with cyclin E (T380A) were treated with either caffeine or wortmannin. Control cells were treated with the proteasome inhibitor Mg-132 to increase total p53 abundance to levels comparable to cyclin E (T380A)-expressing cells. Western analysis of serine 15 phosphorylation and total p53 is shown. (D) HFFs were transduced with cyclin E vector and were treated for the indicated times with wortmannin prior to harvesting. Western analysis of serine 15 phosphorylation, total p53, p21, and cdk2 expression is shown. (E) ATM null primary fibroblasts (GM1627) were transduced with the indicated vectors, and total p53 abundance and p53 serine 15 phosphoryla- tion were examined. (F) Human foreskin fibroblasts and ATM null fibroblasts transduced and passaged in parallel. p21 expression in passage 4 cells is shown. Current Biology 1822

Table 1. Cytogenetic Analysis of WI-38 Cells 2 Weeks after Transduction with the Indicated Vectors Vector Karyotype (20 Total) Babe ϩ E6 14/20 normal 1/20 add (4p) 1/20 dic (4;8) 1/20 del(11q),dic(21;22) 1/20 dic(2;11) 1/20 del(Xq) 1/20 add(Xp) Cyclin E ϩ E6 14/20 normal 1/20 add(20p) 1/20 del(5q) 1/20 del(7q),t(X;9) 1/20 del(5q),del(6),ϩr 1/20 add(20q) 1/20 add(10p) Cyclin E(T380A) ϩ E6 13/20 normal 1/20 dic(3;11),Ϫ21,ϩmar 1/20 ϩmar 1/20 del(1),t(6;16) *1/20 46,XX,del(1)q11,del(3)(q21),del(18)(p11) *1/20 49,XX,Ϫ7,add(9)(q34),Ϫ16,ϩ21,ϩr,ϩmarx3 *1/20 80 Ͻ3nϾ,XXX,der(1)t(1:11)(p36;q13), ϩdel(1),Ϫ2,Ϫ3,Ϫ4,Ϫ5, del(5)(q23), ϩdel(6)(p21),ϩ7,del(8)(q13q22),ϩ9x2,Ϫ10, Ϫ12,ϩ13,der(17)t(17;18),ϩ18,der(18)t(17;18),ϩ21x2, ϩ22,ϩmarx5 *1/20 45,XX,add(X)(q28),del(1)(p22), del(7)(q22),add(13)(p11),Ϫ13,ϩmar

In each sample, 20 metaphases were analyzed. Asterisks denote metaphases containing complex cytogenetic abnormalities.

vant p53 serine 15 kinase, we transduced primary AT nentially growing HFFs expressing cyclin E, HPV-E6, fibroblasts (which are derived from a patient with ataxia both, or neither were irradiated or mock irradiated and telangiectasia and lack functional ATM protein) with the were harvested 24 hr later (Figure 5A). Wild-type cells cyclin E retroviral vectors. We found that the induction arrested in G1 and G2 following irradiation. Cyclin E of p21, p53, and p53 serine 15 phosphorylation occurred expression increased the proportion of S and G2 cells in the AT cells indistinguishably from normal human in the cycling cells but had no effect on either the G1 fibroblasts (Figures 4E and 4F). Thus, ATM is not essen- or G2 arrest after irradiation. In contrast, HPV-E6 expres- tial for this response. sion almost completely prevented the G1 arrest, and the irradiated cells arrested instead in G2. Furthermore, Deregulated Cyclin E-cdk2 Activity Causes when cells were transduced with both E6 and cyclin Genetic Instability and Delayed S Phase E vectors, they arrested in G2 following ␥-irradiation, Progression, but Does Not Impair indicating that ectopic cyclin E, even in combination the G1 and G2 Checkpoints with E6, does not override the G2 checkpoint. Reed and colleagues found that prolonged expression To further investigate the effects of cyclin E on S of cyclin E in established cells caused genetic instability phase entry, HFFs expressing cyclin E, E6, or neither that was manifested primarily by chromosome loss [13]. were density arrested, then released and either irradi- In order to determine if deregulated cyclin E activity in ated or mock irradiated. As expected, a large proportion primary cells also caused genetic instability, we ana- of the cells expressing HPV-E6 expression entered S lyzed WI-38 cells 2 weeks after transduction with cyclin phase after ␥-irradiation (Figure 5B). In contrast, cyclin E retroviruses. In these experiments, cells were first E expression in HFF did not lead to abnormal G1 check- transduced with the HPV-E6 virus to insure that the point control, and there was no entry into S phase. Simi- cyclin E was fully active. We found that the elevated larly, we found that, when cells were continuously la- cyclin E-cdk2 kinase activity associated with the expres- beled with BrDU after irradiation, nearly 50% of the E6- sion of cyclin E (T380A) rapidly caused massive chromo- expressing cells synthesized DNA, compared with only somal abnormalities in a substantial fraction of the cells 5% of the cells expressing cyclin E (Figures 5C and S2). analyzed (Table 1). In contrast, when parallel experi- Thus, deregulated cyclin E activity does not grossly alter ments were performed in control WI-38 cells that did the G1 and G2 checkpoints that sense DNA damage not express E6, sufficient metaphases could not be ob- after ␥-irradiation in HFFs, and this is unlikely to be the tained for cytogenetic analysis due to the severe inhibi- mechanism through which cyclin E promotes genetic tion of WI-38 proliferation caused by prolonged cyclin instability. Although there was a slight increase in BrDU E and cyclin E (T380A) expression. incorporation in irradiated cyclin E expressors versus We next examined the integrity of the G1 and G2 controls, this finding may simply reflect the slightly checkpoints in primary cells expressing cyclin E. Expo- higher proportion of cells in S phase in cells expressing p53-Dependent Cyclin E Suppression 1823

Figure 5. Enforced Cyclin E Expression Does Not Impair G1 and G2 Checkpoint Controls (A) HFFs transduced with the indicated vec- tors were irradiated (10 Gy) or mock irradiated and were analyzed by flow cytometry 24 hr after irradiation. (B) HFFs transduced with the indicated vec- tors were density arrested, then trypsinized and irradiated (10 Gy), or mock irradiated, and replated. Flow analysis was performed 24 hr after replating. (C) HFF transduced with the indicated vectors were density arrested, then trypsinized and irradiated (20 Gy) or mock irradiated, and re- plated. BrDU was added 1 hr after replating, and incorporation was measured by flow cy- tometry 24 hr later.

cyclin E compared with controls after the density arrest these experiments in HPV E6-expressing cells to segre- and is of unclear significance (data not shown and Figure gate the consequences of excess cyclin E-cdk2 activity 5B). We also examined cyclin E overexpressors for DNA from cell cycle affects resulting from the p53-dependent breaks and did not observe any increased TUNEL positi- response to excess cyclin E. When these cultures were vity in cells expressing cyclin E compared with controls synchronized in early S phase with aphidicolin and then (data not shown). released, we found that cyclin E did not affect the timing Finally, we found that deregulated cyclin E activity of S phase entry after release (Figure 6). Instead, we caused defective S phase progression. We preformed found that the rate at which cyclin E-expressing cells Current Biology 1824

Figure 6. Enforced Cyclin E Expression De- lays S Phase Progression in Synchronized Cells HFFs were first transduced with LXSH-E6, then with the indicated vectors. Cells were treated with aphidicolin overnight, then washed and placed in normal growth media for the indicated time periods.

progressed through S phase to G2 was decreased com- cyclin E-dependent p21 expression is an induced re- pared with control cells. Whereas almost half of the sponse. In MEFs, however, although both p21 and p53 cyclin E-expressing cells remained in S phase 12 hr are required to suppress excess cyclin E, the amount after aphidicolin release, almost all of the control cells of endogenous p21 synthesis is sufficient to inhibit the progressed to G2 by this time. Thus, deregulated cyclin activity of the ectopic cyclin E without additional p21 E-cdk2 activity, and not the p53-dependent response induction. This likely reflects the high rate of p53-depen- to cyclin E, impaired S phase progression in primary dent p21 synthesis that occurs in MEFs shortly after human fibroblasts. being placed in culture, which limits their continued pro- liferation in vitro [33]. We have observed increased p53 serine 15 phosphor- Discussion ylation in cells with excess cyclin E that is sensitive to both wortmannin and caffeine. We directly tested the Although cyclin E normally activates cdk2, we found role of the ATM protein and found that ATM null fibro- that prolonged and deregulated cyclin E expression in blasts accumulated serine 15 phosphorylated p53 and primary fibroblasts paradoxically inhibited cdk2. This induced p21 in response to cyclin E. However, although was because deregulated cyclin E expression initiated it is not essential, it is important to realize that ATM may a homeostatic cellular response that “buffered” the ex- function redundantly with other kinases and contribute cess cyclin E by inducing p21 expression and thereby to this response in normal cells. One such kinase may be limiting inappropriate cdk2 activation. Disruption of ei- the ATR protein. ATR is involved in signaling replication ther p53 or p21 rendered this response nonfunctional, blocks to p53, and the ATR knockout phenotype in mice and the excess cyclin E became fully active. has revealed that ATR probably plays an essential role in Initially we thought it likely that the p14ARF protein normal cell cycle progression [30, 34, 35]. Mechanisms mediated this response. However, we could not detect other than p53 serine 15 phosphorylation may also con- increased p14ARF mRNA or protein in cells with cyclin tribute to the activation of p53 by cyclin E. For example, E-induced p53 activation. Furthermore, cells with cyclin Schreiber and colleagues have found that ATR inhibition E overexpression hyperaccumulated in S and G2 phases, decreased p53 serine 15 phosphorylation after DNA but not in G1, as is usually observed in cells with p14ARF damage but did not prevent p53 activation [36]. Instead, induction. Finally, p19ARF null mouse embryo fibro- p53 and ATR acted independently and synergistically blasts were not hypersensitive to cyclin E overexpres- in the establishment of an S phase checkpoint response. sion. Thus, the accumulated data do not support a role Analogously, the caffeine/wortmannin-sensitive ser- for p14ARF in the signaling pathway that links cyclin E ine15 phosphorylation might comprise only a portion of deregulation and p53 induction in human fibroblasts. In the response that couples cyclin E deregulation to p53 fact, the signals that induce p14ARF in human fibro- activation. blasts are quite different than those in MEFs, and two How might deregulated cyclin E cause S phase abnor- recent studies found that oncogenic ras does not lead malities that activate an S phase checkpoint? In yeast, to human p14ARF induction [26, 32]. S phase-promoting cyclins inhibit the transition of repli- Although the essential features of the p53-p21 system cation origins to the prereplicative state [37]. Further- in restraining excess cyclin E activity are conserved in more, when early-firing origins are inhibited by hydroxy- human and murine fibroblasts, there is at least one dif- urea, then the stalled early origins inhibit late origins ference that deserves comment. In human fibroblasts, through a checkpoint that requires the Mec1 protein p53-Dependent Cyclin E Suppression 1825

(the budding yeast ATM/ATR homolog) [38]. Similarly, primary cells from excess cyclin E activity. When this inhibition of ATR function in a human cell line by a ki- barrier is breached through the loss of either p53 or p21, nase-inactive ATR mutant renders these cells hypersen- then deregulated cyclin E expression results in elevated sitive to treatments that prolong DNA synthesis, and cyclin E-cdk2 activity and causes severe cell cycle cyclin E overexpression was synthetically lethal with anomalies and genetic instability. ATR inhibition [39]. Thus, perhaps cyclin E deregulation leads to aberrant licensing of replication origins, and Experimental Procedures the resultant S phase progression defect may be sensed Cell Lines, Tissue Culture, Drug Treatments, Cell Cycle by a protein such as ATR, which then enforces an S Analyses, TUNEL Assay, and Cytogenetics phase checkpoint. Furthermore, the stalled replication Cells and cell lines were obtained as follows: WI-38 and WS-1, origins associated with this prolonged S phase may be ATCC; HFFs, W. Carter (FHCRC); ATM mutant fibroblasts (GM1627), fragile and constitute the precursors to genetic instabil- J. Simon (FHCRC); p21Ϫ/Ϫ and p21 ϩ/ϩ MEFs, J. Roberts (FHCRC); ity. Another mechanism through which enforced cyclin p53Ϫ/Ϫ and p53 ϩ/ϩ MEFs, C. Kemp (FHCRC); p19ARF Ϫ/Ϫ and ϩ ϩ Ϫ Ϫ E expression might impair normal cell cycle progression p19ARF / MEFs, M Roussell (St. Judes); P27 / MEFs, M. Fero (FHCRC). All cells were grown in DMEM with 10% FCS. Drug is by cyclin A-cdk2 inhibition, since we have found that treatments: caffeine (5 mM) and wortmannin (50 ␮M) were placed cyclin A-cdk2 activity (and cyclin A expression) drops in growth media for 3 hr prior to cell harvest. For the induction of substantially in cells with ectopic cyclin E expression p53, cells were treated with MG-132 (Calbiochem, 5 ␮g/ml) for 1 hr (Figures 2C and S3). However, cyclin E-induced cell cy- prior to harvest. To measure S phase transit, cells were synchronized cle anomalies persist in E6-expressing cells with high with aphidicolin (Calbiochem, 5 ␮g/ml), then released into media levels of cyclin A-cdk2 kinase activity, so cyclin A-cdk2 containing nocodazole (Sigma, 40 ng/ml). Cell cycle analyses: cells were trypsinized and fixed in cold 70% ethanol, then washed and activity cannot be the principle cause of the cyclin resuspended in PBS containing propidium iodide and RNase A. E-associated S phase phenotype. Analyses were performed on a Becton Dickinson FACSCAN and One unanswered question regarding the cyclin E-p53 utilized Multicycle and Cellquest software. G1 and G2 checkpoint homeostatic pathway concerns the signal that maintains assays: cells were trypsinized after being density arrested for 4 p21 expression once cyclin E-cdk2 activity has been days, or after asynchronous growth, and were irradiated from a initially suppressed. That is, if the signal from cyclin E Cesium source. Cells were harvested 24 hr after irradiation. BrDU labeling: cells were incubated continuously in 20 ␮M BrDU (Sigma) to p53 is simply excess cyclin E-cdk2 activity, once p21 after replating following mock treatment or irradiation at the indi- is induced, this signal should be quenched. However, cated dosage. After 24 hr, cell nuclei were isolated, and BrDU was our data indicate that p21 expression is constitutively detected with a fluorescein-conjugated monoclonal anti-BrDU anti- maintained once the response is activated. Perhaps body (Amersham). TUNEL staining was performed on HFF by using mechanisms other then elevated cyclin E-cdk2 kinase the In Situ Cell Death Detection Kit (Roche Diagnostics). The cytoge- activity per se might maintain p53 activation by enforced netic studies were performed by the University of Washington Clini- cal Cytogenetics Laboratory. cyclin E expression. For example, increased abundance of either monomeric cyclin E or cyclin E-cdk complexes, Retroviral Vector Cloning and Transduction or uncoupling of cyclin E expression from its normal cell DNAs encoding cyclin E or cyclin E (T380A) were cloned into pBabe- cycle regulation, might provide this signal. Puro [40]. PLXSH-E6 was provided by D. Galloway (FHCRC). Viral Our data indicate that inactivation of the p21-p53 stocks were prepared by transfecting appropriate vectors into the pathway in tumors may be necessary for deregulated Phoenix Ampho or Eco producer cells for transducing human or cyclin E expression to become fully oncogenic. Interest- mouse cell lines, respectively, and stocks were filtered and frozen (producer cells provided by G. Nolan, Stanford). Cells were trans- ingly, mice expressing a T cell-specific cyclin E trans- duced by viral supernatants in DME and 5 ␮g/ml polybrene and gene do not contain increased amounts of cdk2 activity were selected with puromycin (3 ␮g/ml for human cells, 5 ␮g/ml for in thymic extracts, whereas lymphomas induced in these MEFs) or hygromycin (400 ␮g/ml) 48 hr after transduction. mice do exhibit elevated cdk2 activity [20]. Perhaps loss of p53 function is one of the required events in the Antibodies neoplastic progression of these lymphomas and allows The following antibodies were used for Western blotting: mono- the exogenous cyclin E to form active complexes with clonal anti-p21 (Transduction Labs), monoclonal anti-human cyclin E HE12 (Pharmingen), anti-p53 (D01) (Santa Cruz Biotechnology), cdk2. The combination of cyclin E-cdk2 deregulation phospho-p53 Ser15 antibody (9284) (Cell Signaling), and anti-phos- and p53 loss may be particularly potent at inducing pho p53 Sampler Kit (Ser6, Ser9, Ser15, Ser20, Ser37, Ser46, Ser392) genetic instability. This is because unchecked cyclin (Cell Signaling). The following antibodies were used for kinase E-cdk2 activity may promote genetic instability through assays: rabbit polyclonal anti-cyclin E, monoclonal anti-human the mechanisms discussed above, and the loss of p53 cyclin E (HE111), rabbit polyclonal anti-cyclin A (gift of J Roberts function greatly impairs the cell’s ability to respond to [FHCRC]), and rabbit polyclonal anti-cdk 2 (M2) (Santa Cruz Biotech- nology). this damage appropriately. This model also suggests that tumors with deregulated cyclin E-cdk2 activity may Immunoblotting and Kinase Assays be very susceptible to therapies that modulate or restore Cells were lysed in NP-40 buffer (0.5% Np-40, 10 mM Tris [pH 7.4], p53 or ATR function. 0.15 M NaCl, 10 mg/ml each of aprotonin, leupeptin, and pepstatin, 50 mM NaF, 1 mM Na vanadate). Lysates were quantitated, electro- phoresed, and transferred to PVDF membranes as described [7]. Conclusions After incubation with primary antibodies, proteins were visualized Deregulated cyclin E expression in primary fibroblasts by incubation with HRP-conjugated anti-rabbit or anti-mouse sec- ondary antibodies as appropriate (1:10,000, Amersham), followed initiates a cellular response leading to p53 and p21 acti- by ECL per the manufacturer’s instructions (Pierce). IP kinase vation and thereby preventing excessive cdk2 activity. assays: cell lysates normalized for protein concentration were incu- p53 and p21 thus form an inducible barrier that protects bated at 4ЊC for 1 with appropriate dilutions of primary antibodies Current Biology 1826

and 30 ␮l of a 1:1 slurry of PBS-Gammabind (Pharmacia). Precipi- cyclin E for proteolysis and is mutated in a breast cancer cell tates were washed, and phosphorylation of histone H1 was per- line. Nature 413, 316–322. formed as previously described [7]. 10. Won, K.A., and Reed, S.I. (1996). Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin- Quantitative Real-Time PCR dependent degradation of cyclin E. EMBO J. 15, 4182–4193. RNA was isolated from cultured cells (10-cm dishes) with Trizol 11. Sherr, C.J., and McCormick, F. (2002). The RB and p53 pathways reagent (Invitrogen), and cDNA was synthesized by oligo dT priming in cancer. Cancer Cell 2, 103–112. with TaqMan Reverse Transcription Reagents (Applied Biosystems) 12. Ohtsubo, M., and Roberts, J.M. (1993). Cyclin-dependent regu- as per manufacturer’s instructions. Quantitative real-time PCR was lation of G1 in mammalian fibroblasts. Science 259, 1908–1912. performed with a Prism 7700 sequence detector (Applied Biosys- 13. Spruck, C.H., Won, K.A., and Reed, S.I. (1999). Deregulated tems) in triplicate 50 ␮l reactions containing 5 ␮l cDNA, 25 ␮l2X cyclin E induces chromosome instability. Nature 401, 297–300. Taqman Universal Master mix (Applied Biosystems), and probe/ 14. Mussman, J.G., Horn, H.F., Carroll, P.E., Okuda, M., Tarapore, primer mix for p14arf (Applied Biosystems) or PGK (Applied Biosys- P., Donehower, L.A., and Fukasawa, K. (2000). Synergistic in- tems). Quantitation for each probe/primer combination was deter- duction of centrosome hyperamplification by loss of p53 and mined relative to serial dilutions of HeLa cell cDNA with Sequence cyclin E overexpression. 19, 1635–1646. Detection System Software (Applied Biosystems). 15. Dou, Q.P., Pardee, A.B., and Keyomarsi, K. (1996). Cyclin E–a better prognostic marker for breast cancer than cyclin D? Nat. Supplementary Material Med. 2, 254. Supplementary Material including data referenced in the text but 16. Erlanson, M., Portin, C., Linderholm, B., Lindh, J., Roos, G., and not included in the published manuscript is available at http:// Landberg, G. (1998). Expression of cyclin E and the cyclin- images.cellpress.com/supmat/supmatin.htm. dependent kinase inhibitor p27 in malignant lymphomas-prog- nostic implications. Blood 92, 770–777. Acknowledgments 17. Porter, P., Malone, K., Heagerty, P., Alexander, G., Gatti, L., Firpo, E., Daling, J. R., and Roberts, J.M. (1997). Expression of We thank our colleagues for their many kind gifts of reagents. We cell-cycle regulators p27Kip1 and cyclin E, alone and in combi- also thank John Diffley, Mike Kastan, Scott Lowe, Jim Roberts, nation, correlate with survival in young breast cancer patients. and Charles Sherr for helpful discussions. B.E.C. is a W.M. Keck Nat. Med. 3, 222–225. Distinguished Young Scholar in Medical Research. This work was 18. Steeg, P.S., and Zhou, Q. (1998). Cyclins and breast cancer. supported by National Institutes of Health (NIH) R01 CA84069 Breast Cancer Res. Treat. 52, 17–28. (B.E.C.) as well as NIH T32 CA80416-05 (H.C.H.) and T32 CA09515- 19. Bortner, D.M., and Rosenberg, M.P. (1997). Induction of mam- 17 (A.C.M). H.H is an E.D. Thomas Scholar of the Jose Carreras mary gland hyperplasia and carcinomas in transgenic mice ex- Foundation. M.W. is a fellow of the Lymphoma and Leukemia So- pressing human cyclin E. Mol. Cell. Biol. 17, 453–459. ciety. 20. Karsunky, H., Geisen, C., Schmidt, T., Haas, K., Zevnik, B., Gau, E., and Moroy, T. (1999). Oncogenic potential of cyclin E in T-cell Received: June 26, 2002 lymphomagenesis in transgenic mice: evidence for cooperation Revised: August 15, 2002 between cyclin E and Ras but not Myc. Oncogene 18, 7816– Accepted: September 6, 2002 7824. Published: October 29, 2002 21. Scheffner, M., Huibregtse, J.M., Vierstra, R.D., and Howley, P.M. (1993). 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