Journal of Cell Science frmnindmttdp3poen.Teedt ih siti uuetio-aeteaytreigtemtn 5–a xsi cancer. in axis p53–Ras mutant the targeting therapy tailor-made future in assist might data These proteins. p53 mutated aforementioned uatp3udrae e noei ciiis Several of gain activities. p53 mutant oncogenic for account which new to in suggested mechanism, undertakes were the gain-of-function mechanisms of p53 a activity by normal mutant the or inhibits dominant- p53, a it either wild-type which in in activity now manner, its is exerts negative active it p53 Indeed, an mutant transformation. plays that malignant p53 accepted of mutant process the that in possibility role the accumulated raised interface is p53 tumors, DNA-binding mutant the that in the observation alter The of 1994). al., structure which p53 et (Cho the H179), includes orients R175, that group scaffold (e.g. second mutations The which conformational conformation. retaining while R273), p53 group DNA with R248, first interact wild-type sub-groups directly The (e.g. that rudimentary acids folding. mutations amino DBD affect DNA-contact two the of on into missense impact consists The divided their DNA. to the be according to p53 can of missense mutations binding are (DBD), impaired p53 domain an binding in DNA causing p53 mutations the the within reside of that mutations Most 2002). (Guimaraes usually sequence Hainaut, defective, p53 and is the within pathway mutations p53 genetic the of and because cells (Levine, cancer death malignancy most cell from In cell programmed 1997). the arrest, guarding induces thereby, cycle repair, p53 cell DNA activation, as its such Following processes signals. stress to response 1991). ( al., et p53 Hollstein are 1989; tumor-suppressor cancer (Bos, the human Ras in proto-oncogene encoding genes mutated gene frequently the proto-oncogenes. most in the and of genes Two accumulation tumor-suppressor the in by mutations characterized of is tumors of development The Introduction words: Key DNA- The (CGS). signature p53 gene mutant cancer-related different (p53 a that mutants of suggest pattern p53 we expression conformational study unique this region a From induce obscure. (p53 mechanisms to largely mutants the ways p53 remain However, different contact Ras documented. in with well H-Ras cooperate is with categories transformation, cooperate p53 cellular categories mutant to leading various Ras, the oncogenic which and by p53 mutant of expression Concomitant Summary work this to equally ` contributed authors *These 2 signature 1 gene cancer-related Solomon a Hilla induce the to regulate circuit differently Ras proteins mutant p53 Various 3144 o:10.1242/jcs.099663 doi: 3144–3152 125, Science Cell of Journal 2012 February 13 Accepted tme rmaprubdfnto ftep3tasrpintre gene, target transcription p53 the of function perturbed (p53 a from stemmed d Goldstein Ido uhrfrcrepnec ( correspondence for Author eateto aeil n nefcs ezanIsiueo cec,Rhvt710 Israel 76100, Rehovot Science, Israel of 76100, Institute Rehovot Weizmann Science, Interfaces, of and Institute Materials Weizmann of Biology, Department Cell Molecular of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. 5 satasrpinfco htacmltsadi ciae in activated is and accumulates that factor transcription a is p53 G245S i o fetCSepeso.Orfnig eefrhrcrooae nhmntmrdrvdcl ie xrsigRsadthe and Ras expressing lines cell tumor-derived human in corroborated further were findings Our expression. CGS affect not did ) a,Gi ffnto,Mtn p53 Mutant function, of Gain Ras, 1 1, a Brosh Ran , ,YsfBuganim Yosef *, [email protected] R248Q n p53 and 1 ylKalo Eyal , R273H R175H 1, xiie h ihs ee fCSepeso ycoeaigwt NF with cooperating by expression CGS of level highest the exhibited ) ,IaKogan-Sakin Ira *, ) n p53 and 1 sv Beatus Tsevi , TP53 H179R n the and ) nue h G yeeaigHRsatvt.Ti lvto nHRsactivity H-Ras in elevation This activity. H-Ras elevating by CGS the induced ) 1 elePomeraniec Leslie , 2 am Goldfinger Naomi , a ee ntehmngenome: human the in a pivotal three genes of are Ras There because development. tumor hyperactive during mutation becomes point 2009; which Rotter, proto-oncogene, and Ras Brosh 2007; Prives, 2007a; and al., 2009). Li Rotter, et and 2000; Buganim Weisz Rotter, and 2007; (Sigal Lozano, (e.g. NFY) proteins p73, (MAR), other motives p63, with interactions DNA and specific modification epigenetic with interaction MDR1, unique MYC, MST1), (e.g. activation transcriptional NF as such function, Eiaue l,18;Prd ta. 94.Ltrsuisreported studies Later 1984). transformation al., cellular et Parada induce 1984; to al., et cooperate (Eliyahu oncogene Ras and 2003). (Downward, and as RALGDS such and survival pathways PI3K signaling proliferation, MAPK, of induction uncontrolled by Over- aggressiveness causes tumor active. constitutively Ras remains therefore activated regulation and to facilitates sensitive RasGAPs less which by is Ras family, GTPase mutated, by When the enzyme hydrolysis. by GTP GDP (RasGAP) regulated is of protein state replacement non-active activating the the to facilitates Shifting (RasGEF) GTP. conformation which factor exchange family, active nucleotide is enzyme state its guanine active the the enables by non- to controlled Switching which (the 2003). GDP Barbacid, GTP, to and (Malumbres or binding its state) in on active active depends al., more activation et Ras are (Maher respectively 1995). N-Ras cells, in hematopoetic and and transformation cells K-Ras cellular epithelial of whereas mediator cells, potent was more fibroblast H-Ras a function, be and sequence to in found similarity high their of spite nte rti rqetymnpltdb acrclsi the is cells cancer by manipulated frequently protein Another al tde ncne eerhrvae htmtn p53 mutant that revealed research cancer in studies Early BTG2 k 2,tasrpinlrpeso eg T3 D9 and CD-95 ATF3, (e.g. repression transcriptional B2), ycnrs,teL oprgo ofrainlmutant conformational region loop L3 the contrast, By . 1 1 alAssia Yael , n ad Rotter Varda and HRAS 1 hlmMadar Shalom , k .Frhroe h Zn the Furthermore, B. , KRAS 1, eerhArticle Research ` and NRAS 1 , .In +2 Journal of Cell Science hr ari N sRA gis h idtp 5 ( p53 wild-type the against expressing (shRNA) [vector RNA vector shp53 hairpin an short with vectors: infected a following stably were the (Con), of vector one control empty its or (Ras) -a ciiy oal,teL opcnomtoa mutant conformational loop L3 the p53 Notably, activity. H-Ras acngnss n uae om fp3frhrfacilitate further during p53 mutated of not commonly forms has is Ras, mutated activated p53 between and and However, Because cooperation p53 carcinogenesis, 2009). mutant addressed. the al., of been et underlying forms Meylan common mechanism the 2007; cell a Land, as hitherto, such and al., et Ras, Song Xia 2006; and al., 2007; et p53 (Boiko survival both and proliferation by motility, regulated processes several irbat W-8cls vrxrsigteH-Ras the to overexpressing cells) (WI-38 of decided fibroblasts regulation toward the we on mechanisms mutants 2009), of p53 various CGS. Rotter, range the of effect wide and the examine various a (Buganim the that exhibit malignancy fact mutants the and H-Ras p53 mutations, point oncogenic synergistically by 2010). al., predominantly and et is Buganim 2005; inactivation al., that et p53 (Milyavsky expression molecules, by upregulated secreted mainly encoding CGS, the cluster, pro-cancerous gene a reported we Previously, exhibit patterns categories expression p53 CGS mutant diverse various the harboring Cells upregulated synergistically was of was it H-Ras that because when (CGS) transformation interest signature of we gene analysis particular steps this cancer-related In different 2010). the al., the of et identified Buganim clusters to 2005; al., identify related et to (Milyavsky are profiling that genomic wide genes a performed and categories. p53 mutant Ras between various crosstalk the the and underlie different that the details molecular of Ras. specific role with crosstalk the the on in focus categories to p53 and decided mutant (Buganim we mechanisms 2009), of range Rotter, wide a using transformation edcddt ou nrpeettv ee fti signature, this of genes representative on focus investigation, mainly further to For decided TREM-1]. we and CLECSF2 metallopeptidase (MMP-3), [matrix 2006; 3 CXCL2 proteins chemokines al., (ECM)-related (CXCL1, matrix 3 of et (IL-1 cellular and interleukins consists Wang 2 CXCL3)], 1, CGS 2005; 2002; and ligand The al., Richmond, motif) 2008). (C-X-C et and [chemokine Voronov, Minn Dhawan and 2005; H- 1999; activated Apte al., by al., induced et be et Mendes to secreted shown (Sternlicht of were them and consists Ras of functions, mainly pro-cancerous parts 2005; have least CGS to at al., shown The et were 2010). that (Milyavsky molecules al., cells et the Buganim in expressed concomitantly xsi acradmgtb sflfrrtoaebsddrug rationale-based for Results useful be might and design. cancer in axis uat tlz ifrn ehnsst nueteCGS. the the with cooperate induce mutations to DNA-contact p53 different NF mechanisms p53 the that the different show Whereas we study utilize this In mutants CGS. the of groups eety eetbihda nvtotasomto model transformation vitro in an established the we identify to Recently, was work this of motivation main the Thus, ota n,imraie rmr ua mroi lung embryonic human primary immortalized end, that To occurs tumors human in p53 of inactivation Because hs idnsetn u nweg ftemtn p53–Ras mutant the of knowledge our extend findings These k G245S aha,teZn the pathway, B CXCL1 i o emt fetteeaie pathways. examined the affect to seem not did V12 , IL1B n T5 nciaigppie(S5)were (GSE56) peptide inactivating WTp53 and and +2 eincnomtoa uat augment mutants conformational region MMP3 htrpeettedfeetsub- different the represent that , b L6adCF)adextra- and CSF2) and IL-6 , V12 oncogene TP53 V12 ) xrsigoeo h iems rqetp3mtn forms mutant p53 frequent most five the (p53 of one expressing mouse shRNA an the expressing (vector against vector control shCon an sequence], hs 5 uain ersn he rnia categories: principal Zn three the represent within mutations mutations conformational p53 These p53 Ras/p53 h ciiyo h H-Ras the al., inhibits et of p53 wild-type (Vojtek activity that assay] demonstrated the (RBD) recently pull- have domain activity we binding Ras 1993) the [Ras Using assay mutants. directly p53 is down various activity the H-Ras by whether mutants examined affected p53 we various the CGS, which the by induce mechanisms the reveal to order hc 5 a nce on h N-otc mutant DNA-contact the in down, cells in knocked observed as was level (p53 similar p53 a to which CGS the upregulated uatp3tps codnl,floigmtn 5 knock p53 ( genes mutant different CGS following representative express human- Accordingly, the endogenously down, types. six that p53 in lines mutant CGS cell the tumor-derived of expression mutant-p53-dependent 1B,C). (Fig. expression (p53 salse ellnsuigtrerpeettv ee ( genes representative three using IL1B lines cell established Fg B.Spotn hsosrain efudta the that found we H-Ras observation, activated this of levels Supporting 2B). (Fig. n rti Fg AB.Ntby el xrsighg CGS high expressing cells Ras/p53 mRNA Notably, (Ras/shp53, H-Ras 2A,B). levels of (Fig. levels protein comparable and exhibited cells expressing eincnomtoa uat(p53 mutant conformational region eas G xrsinwsdpneto H-Ras on dependent was expression CGS Because various the categories by p53 regulated mutant differently is activity H-Ras sa netbihdW-8cls motnl,alH-Ras all Importantly, RBD cells. the WI-38 performed established we on assay cells, mutant-p53-expressing various ihretn.Itrsigy h 3lo einconformational region loop L3 (p53 the mutant Interestingly, extent. higher Zn the p53 Whereas Ras/ different patterns. levels expression (Con/shCon, the distinct protein had Interestingly, counterparts and forms 1B,C). mutant mRNA Fig. control the Con/shp53; their at shCon, both with CGS compared the upregulated cells) of Ras/shp53 as expression to referred (hereafter down knock ta. 00.T esr h ciiylvl fH-Ras of levels activity the measure To (Buganim 2010). expression al., CGS of et inhibition in resulting state, loading T ees hra el xrsiglwCSlvl (Ras/ levels CGS low expressing Ras/p53 cells shCon, whereas levels, GTP oe fato o ahmtn 5 yei euaigteCGS. the regulating in type p53 mutant each for action of DNA- modes to p53 our CGS. rise the the whereas give induce supports down, further knock mutants mutations p53 result contact conformational as levels This p53 CGS similar the S1). SKBR-3; that Hs-578-T, Fig. hypothesis (NCI-H23, material the mutants expressing supplementary cells in conformational knock expression DNA-contact CGS p53 p53 the p53 whereas affected hardly NCI-H322), the down SW-480, expressing (SW-620, cells mutations in only downregulated htclsepesn h H-Ras the expressing cells that ofrhrsrnte hsosrain eeaie the examined we observation, this strengthen further To et ecmae h xrsinlvl fteCSi the in CGS the of levels expression the compared we Next, loehr hs aadmntaeuiu hrceitc and characteristics unique demonstrate data these Altogether, H179R R248Q G245S R175H , aiu oe fp3mtnsi acr3145 cancer in mutants p53 of roles Various MMP3 R175H ,cnomtoa uainwti h 3lo region loop L3 the within mutation conformational ), n N-otc uain (p53 mutations DNA-contact and ) p53 , n p53 and G245S .I gemn ihorpeiu aa efound we data, previous our with agreement In ). n a/h5 el splmnaymtra Fig. material (supplementary cells Ras/shp53 and H179R el xiie nyamnrefc nteCGS the on effect minor a only exhibited cells ) G245S R273H p53 , Noxa xiie o -a–T levels H-Ras–GTP low exhibited ) el peuae h G oamuch a to CGS the upregulated cells ) G245S V12 oigsqec)o ihavector a with or sequence) coding V12 R175H p53 , noeeb euigisGTP- its reducing by oncogene eesmlri Ras/p53 in similar were V12 loehbtdhg H-Ras– high exhibited also ) R248Q noeeaogwt p53 with along oncogene CXCL1 R175H n p53 and 2+ R248Q p53 , ein(p53 region , IL1B R273H p53 , H179R , V12 i.1A). 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V12 2+ - , , , Journal of Cell Science ihH-Ras with hti epnil o h ue-lvto fteCSi p53 in the CGS in addressed the be descriptions. of will results issues super-elevation following two the These mutants. for DNA-contact responsible is that ehns ywihtedfeetp3mtn om euaeH- the regulate forms being mutant one Together, p53 first different Ras levels. the the issues; which CGS by interesting augment mechanism two mutants to raise p53 results H-Ras–GTP mechanism DNA-contact these moderate different the a only that use suggesting exhibited 2B), CGS, (Fig. levels of levels highest T2aditreewt t aaiiyt idt H-Ras to bind to capability its with interfere and BTG2 eety esgetdta h 5 agtgene, target p53 the that suggested we Recently, Zn The Ras/p53 Interestingly, S2). 3146 h el Fg A.Tesm hnmnnwsosre when observed was phenomenon in same accumulated The they 1A). although (Fig. 3A) cells (Fig. the expression its the affected reduce reduced of could cells levels Ras/shp53 mutants expression although p53 Surprisingly, these expression. whether examined we 92.I re oeuiaetemcaimb hc h p53 the which al., by et mechanism p53 the (Shaulian that elucidate manner shown to Zn dominant-negative order was a In it 1992). in Additionally, act 2010). mutants al., et (Buganim 2+ V12 eincnomtoa uat nueH-Ras induce mutants conformational region ciiy n h eodbigteadtoa mechanism additional the being second the and activity, 2+ ora fCl cec 2 (13) 125 Science Cell of Journal eincnomtoa uat neatwith interact mutants conformational region V12 n eitstep3dpnetCSsuppression CGS p53-dependent the mediates and BTG2 h iemtn 5 om,hardly forms, p53 mutant five the , R248Q el,wihepesdthe expressed which cells, BTG2 V12 interacts , activity, BTG2 V12 ersnaiemtn 5 osrcsit 5-ulcl line downstream cell three p53 the p53-null the of levels a targets introduced mRNA the into measured we constructs and (H1299), activity, p53 constructs p53 p53 mutant mutant representative wild-type the that some possibility retain the exclude To S3A). emntrdtemN eeso elkonp3target, p53 well-known a of levels mRNA p21 the monitored we 5 eeldta h mn cd e2 n r2 r essential are Trp23 and of Leu22 acids analysis amino the mutational that Because revealed wild-type p53 capability. suppressing or transactivation rather BTG2 of mechanism p53 expression different the by inhibiting the expression than CGS proteins, the upregulated p53 under intact. p53, mutant mostly system, wild-type remains endogenous our condition, ectopic the basal in of express that, activity DNA transcriptional suggest cells following results mechanism although these p53 attenuate dominant-negative Together, can a damage. forms p53 by mutant activity various the that indicating nuto ftep3treswsatnae ytevrosp53 various the the by agent, attenuated p53 was of p53-stabilizing cisplatinum, targets excepts p53 with mutants, and the treated of DNA-damage were Fig. induction cells material well-known mRNA supplementary when the 3B; a contrast, affect (Fig. not By targets did p53 S3B). forms the p53 of mutant levels all p53, wild-type hs bevtossgetta h aiu uatp3forms p53 mutant various the that suggest observations These WAF BTG2 as nw as known (also n p21 and WAF G245S CDKN1A sepce,adi otatt the to contrast in and expected, As . yorpyasy(right). assay by zymography measured were MMP3 levels and protein (left) assay ELISA by measured p53 ie eeaaye ywsenblotting. western by cell analyzed WI-38 were established lines the in levels protein ie n ersnaiersl sshown. is result ( representative a three and least times at performed were The experiments duplicates. the of represent deviation bars standard Error lines. established cell the WI-38 in QRT-PCR were by CGS, measured the from genes representative CXCL1 ( as control. used loading was protein housekeeping GAPDH 5 uat:p53 following mutants: the p53 a and or (shCon), (shp53) vector p53 control against infected shRNA stably the were with cells the Then, cells. WI-38 into stably infection retroviral were by (Con) introduced vector empty control a ifrn 5 uat oprtswt H- with Ras of cooperates expression mutants or p53 down different knock p53 1. Fig. C XL n IL-1 and CXCL1 ) splmnaymtra i.S4), Fig. material (supplementary R248Q V12 , oidc CGS. a induce to upeetr aeilFig. material supplementary ; IL1B n p53 and and B R175H R273H MMP3 RAlvl of levels mRNA ) b p53 , .( rti eeswere levels protein A three , H-Ras 5 n H-Ras and p53 ) H179R V12 p53 , Rs or (Ras) G245S , Journal of Cell Science o examined. not to normalized bands, Ras–GTP GTRD.Wsenbo nlsssostepoenlvl fatv Ras, active of levels and protein p53, the Ras, shows total analysis blot Western (GST–RBD). ue oteRsbnigdmi RD yglutathione by (RBD) were domain that beads binding using Ras lines the cell WI-38 to indicated fused the in out ( carried least shown. was is at activity result performed representative a were and represent experiments times bars The three Error duplicates. 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Fig. reduction substantial material a in supplementary resulted p65 5A; of (Fig. downregulation measuring Notably, by levels estimated was protein levels of p65 its capacity down the knock The to expression. of siRNA CGS the member p53-mediated p53 a mutant DNA-contact p65, on effect down the knocked of we activity NF end, the this To mediates mutants. that factor the 3148 k aiy ntevrosW-8cl ie n esrdits measured and lines cell WI-38 various the in family, B ora fCl cec 2 (13) 125 Science Cell of Journal on httespridcino h G xre oeyb the by solely status exerted (p53 we CGS p53 mutants Interestingly, the p53 of of S5B). 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(Fig. and TNF- ng/ml 20 ( with QRT-PCR. treated by measured were levels utemr,w eosrt htdfeettpso mutant of types different that demonstrate we Furthermore, CXCL2 ora fCl cec 2 (13) 125 Science Cell of Journal eemaue yQRT-PCR. by measured were A W60clswr rnfce ihete iN gis p65 against siRNA either with transfected were cells SW-620 ) k oidc h CGS. the induce to B k a -eedn aha ofrhrinduce further to pathway B-dependent o 4hus n RAlvl of levels mRNA and hours, 24 for B , C W40adSB- el were cells SKBR-3 and SW-480 ) CXCL1 V12 nidcn h CGS the inducing in , W60 W40and SW-480 SW-620, R248Q IL1B and n p53 and MMP3 k activity B IL8 , CXCL1 R273H mRNA ) p53 yep3caatrsissc ssaiiainadDAbinding DNA and wild- stabilization the as of such some characteristics retains partially p53 CGS, type the of levels expression eas u nvtotasomto oe sbsdo human on Therefore, based 1995). is al., model H-Ras transformation et cells, fibroblast vitro (Maher primary in than oncoproteins our transformation cellular Ras because of H- other mediator cells, effective the more fibroblast a similar in is share Moreover, Ras and 2003). identity), (Downward, sequence functions (85% homologous highly 2001). Fersht, and (Bullock affinity eeae nvv eehtrzgu o 5,sgetn that tumors suggesting and p53, vitro for in heterozygous grown were cells vivo WI-38 in the generated (data Both mice nude shown). into injected not when formation tumor by measured eedn -a ciiyihbto.Nvrhls,w suggest targets, p53- we the Nevertheless, gene Zn mediate the inhibition. p53 to that activity found wild-type H-Ras recently of we dependent which levels BTG2, expression including the reduce eepandb hi pcfccaatrsis o example, For characteristics. specific their p53 could by categories mutant p53 the mutant explained in various the be differences between the action of Therefore, to mode according conformation. categories structural several into their divided be can they however, pathway, p53 the with with functions. interact interfere tumor-suppressive its to could inhibit ability mutants and the p53 By protein, function. to BTG2 of leads gain that (e.g. 2009) p53 proteins Rotter, and mutant other (Buganim and NF-Y) Mre11, p53 by p73, protein– p63, mutant of demonstrated expression between that p53, studies the that interaction other affect mutant protein with not observation agree did and of CGS, codons, the the W23 capability L22, by the transactivation mutating supported This the CGS. is and damaging H-Ras mechanism active of proposed levels higher enabling therefore hntpsb ifrn en;ete ycoeaigwt the with cooperating by either means; NF these different augment can by mutations of p53 phenotypes different function The tumor-suppressive overridden. cancer-related the is a and p53 of induced, expression is the signature mutated, gene is p53 when process, are oncogene, H-Ras of also the in predictive exploiting fibroblast (supplementary a model, in transformation obtained mutants results vitro the Therefore, conformational S1). then Fig. p53 material expression CGS expressing p53 higher with cells showed along also oncogene K-Ras mutants the DNA-contact harbor that lines cell derived o 5 Vre ta. 97.Itrsigy tsol be should it Interestingly, p53 p53 various 1997). of of the expressing (except al., loss cells mutants established et of undergo our heterozygous that were (Varley 40% mentioned that not that tumors p53 is to did demonstrate for rise hypothesis gave al., patients and This sufficient et heterozygosity, syndrome tumorigenesis. is Varley state toward with Li-Fraumeni heterozygous cells accordance p53 the in Li-Fraumeni a drive in thus, wild-type mutant the to as and of of protein, levels effect cells, tumor-suppressive high p53 the the that overcome speculate in 1990). can to p53 al., present tempting is et are it (Malkin syndrome, p53 wild-type cancer both mutant whereby of situation and a incidence represents the model high predisposes our Because This a alleles. p53 to the patients of one in mutation germline h he a aiymmes -a,HRsadNRs are N-Ras, and H-Ras K-Ras, members, family Ras three The oto 5 uain eiewti h N idn domain; binding DNA the within reside mutations p53 of Most ncnlso,w ugs htdrn h transformation the during that suggest we conclusion, In iFamn ydoei aiildsre soitdwt a with associated disorder familial a is syndrome Li-Fraumeni k H179R rti rb qecigteatvt fBTG2. of activity the squelching by or protein B neatwt T2addsutisbnigt H-Ras, to binding its disrupt and BTG2 with interact ) +2 G245S eincnomtoa 5 uat (p53 mutants p53 conformational region hc norsse adyeeae the elevates hardly system our in which , K Rsocgn-eie cancer. -Ras-oncogene-derived R245S a uoiei phenotype, tumorigenic a had ) V12 a sd e,human-cancer- Yet, used. was R175H and Journal of Cell Science N a ees rncie sn i-T(i a,Jrslm sal and Israel) SuperMix Jerusalem, qPCR instrument Green SYBR Du 7300 Lab, Platinum ABI using (Bio an (Macherey-Nagel, CA) with Carlsbad, Bio-RT performed Biosystems, kit was (Applied using QRT-PCR primers. 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SW-480 al., fibroblasts lines, et supplemented cell (Milyavsky lung previously carcinoma embryonic described colon as human maintained and primary established immortalized infections retroviral The and transfections culture, Cell the and pRetroSuper-shmNOXA-Blast the pBabe-H-Ras shRNA-Blast, pRetroSuper-p53 The Plasmids were anti- anti- MA); 1801) CA); Billerica, Cruz, Dundee, and Santa (Chemicon, School, Biotechnology, Medical (DO-1 GAPDH Cruz and (Santa anti-p53 Hospital anti-p65 (Ninewells anti-H-Ras, used: Lane Scotland); David were by antibodies provided kindly primary following The plasmids and compounds Antibodies, Methods and Materials its and status p53 on pathways. based regulatory is that distinct tools therapy important cancer distinguish provide tailor-made might to for categories capability mutant the p53 the and between junction cellular important this 40% patients. in as syndrome tumorigenesis, Li-Fraumeni for of mechanisms other utilize cells these Tem cetfc A S) oca (Sigma). Ponceau USA), MA, Scientific, Tikva, (Thermo Petach (Pharmachemie, TNF- cisplatinum Israel), (Sigma), (DMSO) sulfoxide Dimethyl Compounds shows S2 specific Table material using Supplementary instructions sequences. mutations. primer specific manufacturer’s all the pWZL- the create the to to on primers conducted according was construct Biotechnology), using blast-p53 Cruz construct (Santa PLXSN- the PWZL-Blast mutagenesis from p53 the directed ORF various into p53 The wild-type construct Israel). the neo-p53 sub-cloning Institute, by by Weizmann was constructed (The constructed that were vector Peles vectors pGEX-GP1/3 were Elior the by into plasmids (ORF) provided frame kindly expression reading open GST-BTG2 BTG2 the cloning Israel). University, Ilan noeigtentr ftemlil ahasivle in involved pathways multiple the of nature the Uncovering a V12 RDsses inaoi,M) eCd lesann reagent staining Blue GelCode MN), Minneapolis, systems, (R&D Hgocntut eeknl rvddb oo iseg(Bar- Ginsberg Doron by provided kindly were constructs -Hygro b tbln(im,S.Lus MO). Louis, St. (Sigma, -tubulin Bam m lqo ftetotal the of aliquot g Irsrcin Site- restriction. HI ¨ren, rs,R n otr V. Rotter, and R. Brosh, L. J. Bos, Ivtoe) pcfcpieswr eindfrtefloiggenes: following the for designed were IL1B primers Specific (Invitrogen). uai,Y,Slmn . as . ite,D,Ncmn,I,Bat . aa,S., Madar, M., Brait, I., Nachmany, D., Kistner, Y., Rais, H., V. Solomon, Y., Buganim, Rotter, and Y. Buganim, R. B. Williams, I., V. Krivokrysenko, V., O. Razorenova, S., Porteous, D., A. Boiko, elcniindmdawr olce n etiue t20,800 at centrifuged and collected were media Cell-conditioned el (2 Cells assay zymography IL-1 and CXCL1 pe .N n ooo,E. Voronov, and N. R. Apte, References information the at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.099663/-/DC1 online of available material Helen made Supplementary and be Norman Institute. Weizmann the may the of at that incumbent Research Cancer the Chair use is Professorial Asher R. any V. for herein. EC contained authors’ liable The Community. the European not the reflects of is those publication necessarily not This and 223151, views 7104370402]. [number FP7-INFLACARE number and EC grant 7034640901]; Community number Center from [grant European Abraham Therapy grant the and Yad Excellence Diagnosis Institute, of Cancer Research Center for Medical a Attendant by supported Flight was research This Funding blotting western and assay binding glutathione BTG2 The assay, pull-down activity Ras S1). to normalized vrih.Tepoen eeeue ihsml ufr eaae nSDS- on separated 2003). al., described buffer, et as (Milyavsky conducted sample al. was cell et with blotting of Milyavsky Western eluted amounts by beads, analyzed. Equal GST–Empty and were gels, or buffer. proteins polyacrylamide GST–BTG2 lysis GST–RBD, RBD The the cold overnight. with in incubated lysed and were (Frangioni were extracts previously Cells described as 1993). prepared Neel, were beads fused GST–BTG2 the n IL-1 and otiigplarlmd es hn eswr nuae vrih in overnight incubated were gelatin- 37 at on gels (Sigma), separated Brij of Then, were 0.01% media determination containing gels. cell buffer For developing of polyacrylamide instructions. volumes equal manufacturer’s containing levels, the protein to MMP3 GRO- according Human systems), the using (ELISA) omsi rlin leR5 n5 ctcai n 0 ehnl odetect (0.1–0.5% to methanol) solution 10% Blue and acid Coomassie acetic activity. with 5% catalytic in of stained R250 areas Blue then Brilliant were Coomassie Gels enzymes. odti,I,Kl,E,Aa,N tal. et N. Adam, E., Kalo, I., Goldstein, clinic. the for implications Ras- of field. suppression p53 mutant in BTG2 of role major a reveals transformation. p53 induced of function suppressor V. A. Gudkov, and muoilg n immunotherapy? and immunobiology , MMP3 aiu oe fp3mtnsi acr3151 cancer in mutants p53 of roles Various 6 10 b 18) a noee nhmncne:areview. a cancer: human in oncogenes ras (1989). 5 rtiswr eetdb nyelne muoobn assay immunosorbent enzyme-linked by detected were proteins eegono i-elpae ihsrmfe E o 2hours. 72 for MEM serum-free with plates six-well on grown were ) , CXCL2 GAPDH S b taseae(S)Rsbnigdmi RD S–B)and GST–RBD) (RBD; domain binding (GST)–Ras -transferase NF uat (p53 mutants ersin odlnsidct togrregulation. stronger indicate indicate lines bars Bold whereas repression. activation, indicate Arrows pathways. n p53 and Zn CGS. p53 the the induce CGS, in to circuit mutants Ras p53 of various regulation different the the by describing utilized model mechanisms schematic A 7. Fig. lvt H-Ras elevate nyelne muoobn sa n MMP3 and assay immunosorbent enzyme-linked a.Rv Cancer Rev. Nat. k 20) ytmtcsac o ontemmdaoso tumor of mediators downstream for search systematic A (2006). , .Mtn p53 Mutant B. IL8 frpie eune e upeetr aeilTable material supplementary see sequences primer (for ee Dev. Genes H179R 20) hnmtnsgi e oes esfo the from news powers: new gain mutants When (2009). , u.J Cancer J. Eur. BTG2 20) sitrekn1ago rbd‘u’i tumor in ‘guy’ bad or good a interleukin-1 Is (2008). R248Q anyihbtBG ucinadtherefore and function BTG2 inhibit mainly ) +2 20) 5:blnigtmu upeso and suppression tumour balancing p53: (2009). V12 p21 , a eincnomtoa uat (p53 mutants conformational region CC1o IL-1 or /CXCL1 9 ciiy hra 5 DNA-contact p53 whereas activity, 20 701-713. , G245S n p53 and muo.Rev. Immunol. 236-252. , WAF 5Spl1 Suppl 45 21) 5 euae h a ici to circuit Ras the regulates p53 (2010). osntafc n fteexamined the of any affect not does -a n 6.cN eeswere levels cDNA p65. and H-Ras , R273H anycoeaewith cooperate mainly ) 222 b 217-234. , acrRes. Cancer muosa is(R&D kits immunoassay 222-241. , ˚ ,t ciaeteMMP the activate to C, g ,at4 oidc the induce To 49 4682-4689. , ˚ .CXCL1 C. R175H CXCL1 , Journal of Cell Science akn . i .P,Srn,L . ruei .F,J,Nlo,C . i,D H., D. Kim, E., C. Nelson, Jr, F., J. Fraumeni, C., L. Strong, P., F. Li, D., Malkin, ae,J,Bkr .A,Mnig . ib .J n oet,I A. I. Roberts, and J. N. Dibb, M., Manning, A., D. Baker, J., Maher, G. Lozano, i,J,Trsy .K n eie .J. A. Levine, and K. A. Teresky, J., Lin, i .adPie,C. Prives, and Y. Li, J. C. A. C. Levine, Harris, and B. Vogelstein, D., Sidransky, P. M., Hollstein, Hainaut, and P. D. Guimaraes, rnin,J .adNe,B G. B. Neel, and V. J. 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