Induction of 7 by DNA damage attenuates function

Peter Jung*, Berlinda Verdoodt*, Aaron Bailey†, John R. Yates III†, Antje Menssen*, and Heiko Hermeking*‡

*Molecular Oncology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried, Germany; and †Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Edited by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, and approved May 18, 2007 (received for review October 26, 2006) The p53 tumor suppressor encodes a transcription factor, specificity (17). Association with the SCF7 complex is required which is translationally and posttranslationally activated after for cellular transformation by SV40 large T antigen (18). Cul7 is DNA damage. In a proteomic screen for p53 interactors, we found highly homologous to PARC (PARkin-like, cytoplasmic, p53- that the cullin Cul7 efficiently associates with p53. After binding protein), which negatively regulates p53 by cytoplasmic DNA damage, the level of Cul7 protein increased in a caffeine- sequestration (19). PARC has been shown to heterodimerize sensitive, but p53-independent, manner. Down-regulation of Cul7 with Cul7 (20). The two have nonoverlapping functions by conditional microRNA expression augmented p53-mediated because deletion of PARC in mice has no effect on viability (20), inhibition of progression. Ectopic expression of Cul7 whereas Cul7-deficient mice exhibit neonatal lethality with inhibited activation of p53 by DNA damaging agents and sensitized reduced size and vascular defects (21). cells to adriamycin. Although Cul7 recruited the F-box protein Using a proteomic approach, we identified Cul7 as a p53- FBX29 to p53, the combined expression of Cul7/FBX29 did not interacting protein. Down-regulation of Cul7 increased p53 promote ubiquitination and degradation of p53 in vivo. Therefore, activity. Furthermore, Cul7 expression was induced after DNA the inhibition of p53 activity by Cul7 is presumably mediated by damage in a p53-independent manner and resulted in suppres- alternative mechanisms. The interplay between p53 and Cul7 sion of p53 activity. Therefore, Cul7 presumably plays a role in resembles the negative feedback loop described for p53 and the DNA damage response by limiting p53 activity. Mdm2. Pharmacological modulation of Cul7 function may allow the sensitization of cancer cells expressing wild-type p53 to geno- Results toxic agents used in cancer therapy. Cul7 Interacts with p53 in Vivo. In a proteomic screen for p53- associated proteins, we identified Cul7 as one of the most cul7 ͉ Fbx29 ͉ cell cycle ͉ ͉ tumor suppression efficiently copurified proteins according to the mass-spectral sequence coverage obtained in multiple tandem affinity purifi- n response to genotoxic stress, numerous cell cycle checkpoints cations of p53 (unpublished data). Cul7 is highly homologous to Iare triggered that prevent the propagation of cells with dam- PARC, a previously identified p53 interacting protein [support- aged genomes (1). These checkpoints mediate cell cycle arrest to ing information (SI) Fig. 6A]. To confirm the association provide time for DNA repair, or, in the case of too severe between p53 and Cul7, we ectopically expressed both proteins in damage, facilitate . A failure to correctly respond to p53-deficient H1299 non-small cell lung cancer cells. By coim- DNA damage may lead to genomic instability, which may give munoprecipitation, we could show that Flag-tagged Cul7 spe- rise to cancerous cells. In line with this scenario, the mutational cifically associates with HA-tagged p53 protein (Fig. 1A). The inactivation of critical components of these checkpoints fre- same result was obtained with HEK 293 cells (SI Fig. 6B). quently occurs in cancer cells. The p53 transcription factor is a Furthermore, immunoprecipitation with a p53-specific antibody central component of the DNA damage checkpoint and medi- identified an association with endogenous Cul7 in the breast ates cell cycle arrest or apoptosis by activation of specific target carcinoma cell line MCF-7 but, as expected, not in p53-deficient , such as p21, 14-3-3␴, and Puma (2–4). p53 is encoded by H1299 cells (Fig. 1B). The association between endogenous p53 a tumor suppressor gene that is inactivated in Ϸ50% of all human and Cul7 was enhanced after induction of DNA damage by tumors (5). Genotoxic stress triggers rapid phosphorylation of addition of the topoisomerase II inhibitor etoposide (Fig. 1B). p53 by ATM (ataxia telangiectasia mutated) and other kinases The association of endogenous p53 with Cul7 was also observed such as CHK2 (6, 7), resulting in the accumulation and activation in the osteosarcoma cell line U-2OS (SI Fig. 6C). of the p53 protein. The activity of p53 is also regulated by localization and acetylation (reviewed in ref. 7). In nonstressed Cul7 Protein and mRNA Accumulate After DNA Damage. Because we cells, p53 is kept inactive by MDM2, which shields the N- found that the association between Cul7 and p53 was enhanced terminal transactivation domain of p53, but also acts as an E3 ligase that targets p53 for proteasomal degradation (8). Because MDM2 is a direct transcriptional target of p53, both genes Author contributions: P.J., A.M., and H.H. designed research; P.J., B.V., and A.B. performed constitute a negative feedback loop (9). Furthermore, the deg- research; J.R.Y. contributed new reagents/analytic tools; P.J., B.V., A.B., A.M., and H.H. radation of the p53 protein is also tightly regulated by other E3 analyzed data; and P.J. and H.H. wrote the paper. ligases, such as COP1, Pirh2, and p300 (10–12). ATM was The authors declare no conflict of interest. recently shown to phosphorylate COP1, which promotes self- This article is a PNAS Direct Submission. degradation of COP1 and p53 stabilization (13). Abbreviations: ATM, ataxia telangiectasia mutated; Cul7, Cullin 7; PARC, PARkin-like, Cullin 7 (Cul7) was originally discovered as a 185-kDa protein cytoplasmic, p53-binding protein; PIKK, phosphatidylinositol 3-kinase-related kinase; ATR, ATM- and Rad3-related; IP, immunoprecipitation; DOX, doxycycline. (p185) associated with the large T antigen of simian virus 40 ‡To whom correspondence should be addressed at: Max-Planck-Institute of Biochemistry, (SV40) (14). The C terminus of Cul7 harbors a BH3 domain, Molecular Oncology, Am Klopferspitz 18A, D-82152 Martinsried/Munich, Germany. E- which presumably promotes apoptosis (15). Together with Skp1, mail: [email protected]. Fbx29, and ROC1, Cul7 forms the SCF-ROC1 E3 ligase complex This article contains supporting information online at www.pnas.org/cgi/content/full/ (SCF7) (16). Furthermore, Cul7 was shown to form an E3 ligase 0609467104/DC1. with Cul1 and the F-box protein FBX29, which confers substrate © 2007 by The National Academy of Sciences of the USA

11388–11393 ͉ PNAS ͉ July 3, 2007 ͉ vol. 104 ͉ no. 27 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609467104 Downloaded by guest on September 27, 2021 Fig. 2. Increase of Cul7 protein in response to DNA damage. (A) Detection of Cul7 protein levels in MCF-7, U-2OS, and H1299 cancer cell lines by Western blot analysis. The respective cell lines were treated with etoposide (20 ␮M) for the indicated periods. (B) Detection of Cul7 protein levels in p53-deficient and wild- type HCT116 colon cancer cell lines (28) by Western blot analysis. Cells were MEDICAL SCIENCES treated with 20 ␮M etoposide for the indicated periods. (C) Detection of Cul7 protein in MCF-7 cells expressing wild-type p53 by Western blot analysis. Cells Fig. 1. Interaction between ectopic and endogenous Cul7 and p53 proteins. (A) were treated for the indicated periods with 20 ␮M etoposide alone or in combi- H1299 cells were transfected with constructs encoding HA-tagged p53 and Flag- nation with 5 mM caffeine. Caffeine was added 45 min before etoposide. tagged Cul7. Western blot analysis of whole cell extracts (WCL) and immunopre- cipitates [IP; obtained by using HA-specific, Flag-specific, or mouse preimmune serum (IgG)]. (B) Coimmunoprecipitation of endogenous p53 and Cul7 in MCF-7 H1299 cells after DNA damage (Fig. 2C and SI Fig. 8A). In the and H1299 cells. Whole cell extracts (WCL) and immunoprecipitates [IP; obtained absence of DNA damage, caffeine treatment had no significant with p53-specific antibodies (DO-1 and 1801) or mouse preimmune serum (IgG)] effect on Cul7 expression level (SI Fig. 8A Lower). Furthermore, were subjected to Western blot analysis with Cul7-specific monoclonal and caffeine also prevented the increase in Cul7 mRNA after DNA p53-specific polyclonal antibodies. *, specific signal for Cul7. damage (SI Fig. 8B). However, down-regulation of ATM and/or ATR by RNA interference had no effect on the induction of Cul7 after treatment of cells with etoposide (Fig. 1B), we analyzed by DNA damage (data not shown). Therefore, other PIKK family whether Cul7 mRNA and/or protein levels increase in response members may be involved in the induction of Cul7 protein and to DNA damage. Indeed, endogenous Cul7 protein significantly mRNA during the DNA damage response. increased in MCF-7 cells as early as 10 h after treatment with etoposide and reached maximum levels after 24 h (Fig. 2A). A Increased p53 Activity and G1 Arrest After Knockdown of CUL7. To similar increase in Cul7 protein was observed in the osteosar- study the effect of Cul7 down-regulation on p53 activity we used coma cell line U-2OS and also in the p53-negative cell line H1299 the pEMI (plasmid for episomal microRNA expression) vector (Fig. 2A). The increase in Cul7 protein level was accompanied system (23). MCF-7 cell lines stably harboring pEMI constructs, by an induction of Cul7 mRNA in MCF-7 (SI Fig. 7A) and which allow conditional expression of Cul7-specific microRNAs U-2OS cells (SI Fig. 7A). In contrast, the p53-negative cell line and mRFP from a bidirectional promoter, were generated. A cell H1299 showed no increase in Cul7 mRNA 14 h after DNA line displaying homogenous induction of mRFP 48 h after damage (SI Fig. 7A). This p53 dependence suggested that CUL7 addition of doxycycline (DOX) to medium is shown in Fig. 3A. may be a p53 target gene. However, activation of a tet-regulated The maximum knockdown of Cul7 was achieved 72 h after p53 allele in DLD-1 and H1299 cells did not affect Cul7 mRNA induction of microRNA expression whereas a nonsilencing expression, whereas p21 mRNA was induced as expected (SI Fig. microRNA had no effect on Cul7 protein levels (Fig. 3B). 7B and data not shown). Furthermore, Cul7 protein increased Interestingly, p21 protein levels increased after pEMI-mediated after DNA damage in HCT116 colon cancer cells deficient for Cul7 knockdown in untreated and in DNA damaged cells. The p53 with similar kinetics as in cells expressing wt p53 (Fig. 2B), amount of p53 protein was not significantly affected by the whereas Cul7 mRNA was not significantly affected by DNA down-regulation of Cul7. Expression of a nonsilencing mi- damage in these cell lines (SI Fig. 7C). Furthermore, the Cul7 croRNA in a control cell line did not affect p53 and p21 protein promoter region was not responsive to p53 in a transient reporter expression. The effect on p21 expression after Cul7 ablation was assay (data not shown), indicating that the increase of Cul7 also observed with pools of pEMI-vector containing U-2OS cells mRNA observed in a subset of cell lines after DNA damage is (SI Fig. 9A), ruling out clonal or cell type-specific effects. We mediated by an unknown factor. noticed that MCF-7 cells with down-regulation of Cul7 displayed Interestingly, treatment with 5 mM caffeine, an inhibitor of an increase in size and showed a change in morphology indicative phosphatidylinositol 3-kinase-related kinases (PIKK) such as ATM of a cell cycle arrest, which is consistent with the induction of the and ATR (ATM- and Rad3-related) (22), effectively blocked the CDK-inhibitor p21 (Fig. 3 A and B). Indeed, when we analyzed accumulation of Cul7 protein in MCF-7 cells and p53-negative the DNA content of these cell populations by flow cytometry,

Jung et al. PNAS ͉ July 3, 2007 ͉ vol. 104 ͉ no. 27 ͉ 11389 Downloaded by guest on September 27, 2021 Fig. 3. Effects of acute inactivation of Cul7. (A) MCF-7 cell lines with inducible expression of mRFP and Cul7-microRNA (MCF-7-EMI-Cul7-miRNA) were compared with cells expressing a nonsilencing microRNA (MCF-7-EMI- nonsilencing). Seventy-two hours after addition of DOX (500 ng/ml), mRFP expression and cell morphology were analyzed by life cell microscopy. (Mag- nification: ϫ200.) (B) Down-regulation of Cul7 enhances transactivation of p21 by p53. Shown is Western blot analysis of Cul7, p53, and p21 protein levels in MCF-7-EMI-Cul7-microRNA cells and the control cell line MCF-7-EMI- nonsilencing. DOX (500 ng/ml) was added for 72 h, and etoposide (20 ␮M) was added for the indicated periods.

MCF-7 cells with a knockdown of Cul7 showed an increased fraction of cells in G -phase and a decrease of cells in S-phase 1 Fig. 4. Cul7 inactivation augments p53 activity. (A) DNA content analysis by (Fig. 4A). When cells exhibiting a knockdown of Cul7 were flow cytometry after Cul7 knockdown in MCF-7 cells. Cells were treated with treated with etoposide an increase of cells arrested in G1 resulted 500 ng/ml DOX for 72 h. DNA damage was induced by addition of etoposide when compared with cells expressing a nonsilencing microRNA. (20 ␮M). MCF-7 cells with and without induction of Cul7-specific or a nonsi- Cells without microRNA induction or cells expressing a nonsi- lencing microRNA were treated with etoposide for the indicated periods. The lencing microRNA showed a significant increase in the 4N DNA experiment was repeated twice, and representative results are provided. The % cell cycle distributions are average results of two independent experiments. content after DNA damage indicating a predominant G2/M- 2N, ; 4N, G2/M phase. SI Fig. 9B shows a similar analysis 24 h after arrest (Fig. 4A and SI Fig. 9B). To determine whether the effects etoposide treatment. (B) Concomitant knockdown of Cul7 and p53 in MCF-7 of Cul7 down-regulation were p53-dependent, we transfected cells. MCF-7 EMI-Cul7microRNA cell line was transfected with p53-specific MCF-7 pEMI-Cul7microRNA cells with p53-specific or nonsi- (p53si) or nonsilencing (NonS) siRNAs. Twenty hours later, Cul7 knockdown lencing short interfering RNAs (siRNAs) before induction of the was induced by addition of DOX for 72 h. Cul7, p53, and ␤-actin protein levels Cul7-specific microRNA (Fig. 4B). Indeed, down-regulation of were analyzed by Western blotting. p53 partially rescued the effect of Cul7 knockdown resulting in an increased proportion of cells that were arrested in G /M and 2 Cul7/Fbx29 Does Not Exhibit E3-Ligase Activity Toward p53. The a decrease of cells in G in response to DNA damage compared 1 SCF7-E3 ligase complex contains the F-box protein FBX29 (16), with controls (Table 1). Taken together, these results show that which recruits the substrates for ubiquitination. Next, we ana- Cul7 limits the activity of p53. lyzed whether FBX29 forms a complex with p53 and Cul7. In a coprecipitation assay with ectopically expressed proteins, p53 Ectopic Expression of Cul7 Inhibits p53 Function. Because the Cul7 associated with FBX29 in a Cul7-dependent manner in H1299 protein level increased in response to DNA damage we analyzed cells (Fig. 5B). Similar results were obtained for endogenous p53 the effect of ectopic Cul7 expression on endogenous p53 and p21 in MCF-7 cells ectopically expressing Cul7 and FBX29 (SI Fig. protein levels. Induction of ectopic Cul7 expression by addition 11A). Furthermore, ectopic FBX29 was coprecipitated with p53 of DOX inhibited the increase in p53 and p21 levels after in a Cul7-dependent manner (Fig. 5B and SI Fig. 11B). In line etoposide treatment (Fig. 5A; for vector controls see SI Fig. with the inability of p53 to directly associate with FBX29, p53 10A). Furthermore, coexpression of Cul7 led to a reduced does not serve as a substrate for Cul7-mediated ubiquitination/ transactivation activity of p53 in a transient reporter assay (SI degradation: ectopic expression of Cul7 in combination with Fig. 10B). When cells ectopically expressing Cul7 were treated FBX29 did not promote ubiquitination of p53 in vivo (Fig. 5C). with the DNA damaging agent adriamycin, a topoisomerase II The same result was obtained when proteasomal activity was inhibitor, an increase in apoptosis was observed (SI Fig. 10 C and inhibited by MG132 to increase the sensitivity of the ubiquiti- D). This effect presumably resulted from an inability of cells to nation assay (SI Fig. 11C). Coexpression of the E3 ligase MDM2 stably arrest. Taken together, these results demonstrate that served as a positive control and led to an efficient mono- and Cul7 modulates the stabilization and activity of p53 during the polyubiquitination of p53 and decreased p53 levels (Fig. 5C and DNA damage response. SI Fig. 11C). Therefore, other mechanisms than increased

11390 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609467104 Jung et al. Downloaded by guest on September 27, 2021 Table 1. DNA content analysis by flow cytometry after Cul7 and MCF-7 RTS-1-Cul7: p53 knockdown in MCF-7 cells A 0 0 4 4 8 8 + etoposide (h) Cul7-specific - + - + - + DOX (48 h) miRNA Hours ϩ Cell cycle Nonsilencing p53-specific induction etoposide phase siRNA siRNA -Cul7 -p53 Ϫ 0G1 54.48 50.20 S 18.99 20.40 -p21 G2/M 23.09 27.00 - β-actin ϩ 24 G1 59.60 47.27 S 8.04 7.85 IP: anti-HA WCL G2/M 27.00 38.88 B ϩ 48 G1 53.47 41.10 + + + FBX29-VSV + + + FBX29-VSV S 8.85 8.85 + - + Flag-Cul7 + - + Flag-Cul7 G2/M 31.50 46.27 ++ - p53-HA The MCF-7-EMI-Cul7microRNA cell line was transfected with p53-specific or + + - p53-HA - FBX29-VSV nonsilencing siRNAs. Twenty hours later, Cul7 knockdown was induced by - FBX29-VSV addition of 500 ng/ml DOX 48 h before etoposide treatment for the indicated periods. The %-cell cycle distributions are average results of three indepen- - Flag-Cul7 dent experiments. * - Flag-Cul7 - p53-HA - p53-HA ubiquitination and subsequent proteasomal degradation are - α-tubulin presumably responsible for the inhibition of p53 by Cul7.

Discussion 92XB 2MDM 2MDM 2 +

C MDM Our analysis revealed that Cul7 is induced by DNA damage in 7l a caffeine-sensitive manner and negatively regulates p53 func- u - F C MEDICAL SCIENCES tion presumably by direct interaction. While this manuscript was -++++ in preparation, a direct interaction between ectopic p53 and Cul7 His-Ub was reported by others (24–26), which confirms our findings. +-+++p53-VSV Unlike the interaction between PARC and p53 (19), the Cul7/ p53 interaction does not seem to influence p53 localization (24). n

Cul7 is a central component of an SCF-ROC1 E3 ligase complex ) b

(SCF7), consisting of Skp1, Cul7, FBX29, and ROC1 (16). We U(

found that the substrate recognizing component of this complex, - 35 Fbx29, does not directly interact with p53, but associates indi- p rectly with p53 in a Cul7-dependent manner. In agreement with our findings, the Cul7 domains responsible for p53 and FBX29 binding are not overlapping (25, 27). Furthermore, we provide evidence that endogenous Cul7 acts as - WCL: p53 a negative regulator of p53 because acute down-regulation of Cul7 by a conditional RNA interference approach led to increased p21 Fig. 5. p53 accumulation is inhibited by Cul7. (A) Western blot analysis of protein levels, which augmented a DNA damage-induced G1 arrest MCF-7 cells stably transfected with pRTS-1 episomal expression vector (39) in a p53-dependent manner. In addition, p53 accumulation and encoding FlagCul7. See SI Fig. 10B for pRTS-1-Luciferase (pRTS-1-Ctrl) vector activity after DNA damage was compromised by ectopic Cul7 control experiment. Cul7 expression was induced by addition of 500 ng/ml expression. Disruption of p53 function was previously described to DOX for 48 h. Cells were treated with etoposide (20 ␮M) for the indicated sensitize human cancer cells to apoptosis induced by genotoxic periods. (B) H1299 cells were transfected with plasmids encoding p53, Cul7, drugs (28) and the p53/p21 axis was shown to be critical for and FBX29. Shown is Western blot analysis of whole cell extracts (WCL) and sustained cell cycle arrest after DNA damage (29). In agreement immunoprecipitates (IP; obtained with a HA-specific antibody). (C) In vivo ubiquitination assay for p53 in H1299 cells. Cells were transfected with with Cul7 acting as a negative regulator of p53 function, ectopic pMT107 [His-tagged (38)], pcDNA3-p53-VSV, and plasmids express- expression of Cul7 increased the apoptotic fraction of MCF-7 cells ing MDM2 or FBX29 plus Cul7, as indicated. For details, see Materials and after exposure to genotoxic drugs, presumably by preventing the Methods. Circle with an X, background signal originating from anti-VSV establishment of a stable p53-mediated cell cycle arrest. antibody. Bottom lane, Western blot analysis of whole cell lysate. Previously, a Cul7-mediated mono- and di-ubiquitination of p53 has been observed in vitro by using immunoprecipitated complexes of Cul7 and ectopic p53 from H1299 cells. However, nuclear p53 levels are increased. Therefore, Cul7 might associate the biochemical consequences of this p53 modification remained with p53 in the cytoplasm and may be involved in controlling its elusive (24). We could not confirm a ubiquitination of p53 by oligomerization status. Nevertheless, the exact mechanism by Cul7/FBX29 in vivo in H1299 cells. Because several E3 ligases which Cul7 inhibits the activity of p53 remains to be determined. have been shown to mono- and di-ubiquitinate p53 (11, 13–15), Cul7 knockout mice exhibit neonatal lethality with reduced size it is possible that immunoprecipitates of Cul7/p53 contain other and vascular defects (21), suggesting that Cul7 plays a nonessential E3-ligases responsible for the in vitro ubiquitination of p53 role in mammalian development, which would be consistent with a observed by Andrews et al. (24). The association of Cul7 with p53 function as a checkpoint component. Our data imply that deregu- was shown to occur with oligomeric forms of p53 (26) and lation of p53 activity might at least in part account for the reduced tetrameric p53 is less efficiently imported into the nucleus than proliferation of Cul7-deficient mouse embryo fibroblasts (21). its monomeric form (31). Ectopic Cul7 interfered with p53 Furthermore, p53 may contribute to the neonatal lethality observed activation in response to DNA damage when cytoplasmic and after deletion of Cul7 (21). Similar phenotypes have also been

Jung et al. PNAS ͉ July 3, 2007 ͉ vol. 104 ͉ no. 27 ͉ 11391 Downloaded by guest on September 27, 2021 observed in mice deficient for other negative regulators of p53, as DNA content was determined by flow cytometry (FACSCalibur; MDM2 and MDMX (30, 32, 33). In line with a positive role of Cul7 Becton Dickinson). in unperturbed cell proliferation mutational inactivation of Cul7 causes the 3-M syndrome, an autorecessive form of dwarfism Western Blot Analysis. Cell lysis and immunoblotting were done as characterized by pre- and postnatal growth retardation (27). Ac- described in ref. 36. For immunodetection, membranes were incu- cording to our results, it is possible that the increase in p53 activity, bated with antibodies specific for Cul7 (mouse hybridoma clone which may result from mutated, nonfunctional Cul7, contributes to SA12 (37), p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, the 3-M syndrome. CA), p21 (Ab-11; NeoMarkers, Fremont, CA), HA (clone HA.11; Negative feed-back loops represent a common mode of p53 Covance, Princeton, NJ), Flag (M2, F-3165; Sigma), P-Chk2 (Thr- regulation (8, 34). Several negative p53 regulators, e.g., 68, sc-16297-R; Santa Cruz Biotechnology), ␣-tubulin (clone MDM2, COP1, and Pirh2, are encoded by target genes of p53 DM1A; Sigma), or ␤-actin (A-2066; Sigma). Signals from HRP (9–11). Interestingly, Cullin proteins have been recently in- (horseradish-peroxidase)-coupled secondary antibodies were gen- volved in the negative regulation of the DNA damage response erated by enhanced chemiluminescence (PerkinElmer Life Sci- as ATR-mediated phosphorylation marks CHK1 for ubiquiti- ences, Boston, MA) and recorded with a CCD camera (440CF nation by SCF ligase complexes containing Cul1 or Cul4a imaging system, Eastman Kodak). thereby limiting the duration of ATR-CHK1 signaling after genotoxic or replicative stress (35). Here, inhibition of PIKK Coimmunoprecipitation. For immunoprecipitation (IP) of exog- family members by caffeine prevented the increase in Cul7 enous proteins, H1299, MCF-7, and HEK293 cells were trans- mRNA and protein after DNA damage. Therefore, we suggest fected by using a calcium phosphate method. Cells were lysed a model in which PIKK kinases not only activate p53 in at 4°C in lysis buffer [25 mM Tris⅐HCl, pH 8.0/150 mM NaCl/10 response to genotoxic stress but also increase the levels of a mM MgCl2/0.5% Nonidet P-40/2 mM sodium orthovanadate/1 negative regulator of p53. In summary, we show that the mM DTT/1 mM NaF/50 units/ml DNase I (Roche)/complete induction of Cul7 after DNA damage attenuates the activation mini protease inhibitor mixture (EDTA free, Roche)/0.25% of p53. Pharmacological modulation of Cul7 function may Phosphatase Inhibitor Mixture I (Sigma)]. Cleared lysates allow to sensitize cancer cells expressing wild-type p53 to were rotated at 4°C with the respective antibodies for 3 h, and genotoxic agents used in cancer therapy. 25 ␮l of Protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) was added for an additional 2 h. After washing Materials and Methods four times with 25 mM Tris⅐HCl (pH 8.0), 150 mM NaCl, 10 Cell Lines/Culture and Reagents. U-2OS osteosarcoma cells, MCF-7 mM MgCl2, and 0.5% Nonidet P-40, proteins were separated breast cancer, and H1299 small cell lung cancer cells were main- by SDS/PAGE and subjected to Western blot analysis. tained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitro- gen, San Diego, CA) containing 10% FBS and penicillin (100 In Vivo Ubiquitination. MCF-7 and H1299 cells were transfected by units/ml)/streptomycin (100 ␮g/ml). DLD1tTA and HCT116 colon using a calcium phosphate method with VSV-tagged p53, His- cancer cells and their derivatives were maintained in McCoy’s tagged ubiquitin [pMT107 (38)] and the respective E3 ligase or medium (Invitrogen) containing 10% FBS, penicillin (100 IE)/ empty vector control. MG132 (Axxora, San Diego, CA), if used, streptomycin (100 ␮g/ml), and, in case of DLD1tTA cells, 100 ng/ml was added at a final concentration of 20 ␮M 4 h before harvest. doxycycline. Doxycycline (Sigma, St. Louis, MO) was dissolved in Cells were harvested 36 h after transfection by scraping into lysis water (100 ␮g/ml). Etoposide (Sigma) was dissolved in DMSO (40 buffer (6 M guanidine-HCl/0.1 M NaHPO4/0.1 M Tris/5 mM mM) and used at a final concentration of 20 ␮M. Caffeine (Sigma) N-ethylmaleimide, pH 8.0) and sonicated to shear the DNA. was used at a final concentration of 5 mM. Lysates were incubated for8hat4°Cwith40␮l of Ni-NTA- agarose beads (Qiagen, Hilden, Germany), after which they were Plasmids and siRNAs. Information and sequences are available in washed twice with wash buffer A (8 M urea/10 mM Tris/0.1 M SI Materials and Methods. NaHPO4/0.05% Tween 20/5 mM N-ethylmaleimide, pH 8.0), followed by two washes in wash buffer B (8 M urea/10 mM Generation of Cell Lines. MCF-7 breast cancer cells or U-2OS cells Tris/0.1 M NaHPO4/0.05% Tween 20/5 mM N-ethylmaleimide, were transfected with pRTS-1 or pEMI plasmids by using pH 6.4), and one final wash with PBS. Ni-NTA-agarose bound FuGENE reagents (Roche, Basel, Switzerland). After 48 h, cells proteins were eluted by boiling 10 min in 2ϫ Laemmli buffer plus were selected in media containing 150 ␮g/ml hygromycin for 2 250 mM imidazole, separated by SDS/PAGE and subjected to weeks. For MCF-7 cells, single cell clones were obtained by Western blot analysis. limiting dilution, and homogenicity of the derived cell lines was tested by addition of 500 ng/ml doxycycline for 24 h and detection Microscopy. mRFP and phase contrast images of living cells were of mRFP by fluorescence microscopy. obtained on an inverted Axiovert 200M microscope (Zeiss, Oberkochem, Germany) by using Metamorph software (Uni- DNA Content Analysis by FACS. MCF-7 cells (5 ϫ 104) were plated versal Imaging). into T25 cell culture flasks. For analyses after siRNA trans- fections, cells were seeded and transfected in 12-well plates. We thank Stefan Mu¨ller (MPI of Biochemistry, Martinsried, Germany), Floating cells and trypsinized cells were collected by centrif- Gunter Meister (MPI of Biochemistry, Martinsried, Germany), Zhen- ϫ Qiang Pan (Columbia University, New York, NY), James DeCaprio ugation at 290 g for 7 min, fixed with ice cold 70% ethanol, (Dana–Farber Cancer Institute, Boston, MA), Bert Vogelstein (The and stored overnight on ice. After washing with PBS, 1 ml of Johns Hopkins University, Baltimore, MD), and Georg Bornkamm FACS solution [PBS/0.1% Triton X-100/60 ␮g/ml propidium (Institute for Clinical Molecular Biology and Tumor Genetics, GSF, iodide (PI)/0.5 mg/ml DNase free RNase (Roche)] was added Munich, Germany) for generously providing reagents. The H.H. labo- per sample and incubated at room temperature for 30 min. ratory is supported by the Max-Planck-Society.

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