Leul€mia (2009), 1-10

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ORICINAT ARTICLE

Heat stress triggers apoptosis by impairing NF-xB survivar signaling in marignant B ceilsC Belardol,2, R Piva3 and M6 Santorol,2

lDepaftmenr of Bíolo|y, LJnive'itv of Rome ,Tot,Ygrgita, via della Ricerca scientjlica, !gm? ltaly; 2 tnstitute- of Neurobiologyand Mo.lecular Medicine, consiglio Naz,ionale^dellekicérche, via rossò dit-dvalierc' 100, ioa,e:'thi;;;e3 ólepa'itient otBiomedicat sciences and Humin oncorogy, (en,u fù crp"íi.intu1 n"nZ,r-i una u"aìiít iiJin'tétlua,"ùíi"iri,y ,tTuin, Turin, ltaly

Nuclear factor.KB (NF.XB) ls involvéd in multiplè aspects ofoncogeneais anal conlrols cancer coll survlval by promollnoantl.apoptotlc gene oxpres€ion. lho constitu ve àcitvation oiNF-KB in sévoÌal typea. of cancer6, includlng hematologlcalmalgnanctés, haa been lmpllcated In the réalstance to chamo.and. radlatlon _therapy, Wo have prevlously rèported thatcytol(lne- or vlrua.inducod NF"KB ac vation ls Inhlbttod bvch€mlcal and physical Inducèrs of thè hsat shock responsó(HSF). In lhls atudy we show th6i heat stress l;hlbltsconalllutiv.e NF.'{B DNA.bindlng ac vtty in dlfforont typos otB-cèll.mallgnancles, Includlng mul ple myeloma, aóilvatedB.c€llllko (ABC) typo of dtffuse targe B-ce tymphoma (DLBCL)and Burkltt'6 lymphoma proaenflng abèrana NÈ-xB reiulaflonlHeat.induced NF-XB Inhibl on toad6 to rapld downroguia on oftho antl.apoptotlc protoln collular Inhibitoi.of.apoptoóls proteln2 (clAP-2), foflowed by ac va on of caapase.3-rnd ctoaiago oflhe caapaae.3 substrate poly(adenostno dlphosphoto rlbóse)-polymorade (PARP), caualng masslve apoptosls under condÍ.uona that do not aff€ct vlablllty In cells not prosen ng NF{Baberatlon6. NF-KB Inhlbl on by the proteasome Inhlbltorbortozomlb and by ahort.hairpin RNA (shRNA) Interferoncerosults in Increasod senstÙvlty of Hs-Sultan B-cetilymphoma tohyperthormlc atre6s. Altogethof, thé rosults i;dléat€ thataggrgaslvo B-cell mallgnanclo€ presenllng cons tu ve NF.rBactivtty are aon_altlvo lo heat-lnduced apoptosls, and suggestlhat abo ant NF-XB regula on may bo a marker of heat sìieessonAlllvlty in cancer cells.Leuke[,rb advance online publication, j9 November 2OO9:doir10.1 038/1eu.2009.227Keywords: apoplosis; clAP,2; heat shock tactor; heat stfssslNF-xB

Introduction

Nuclear _ factoFKB (NF-kB) transcription factors comprise a

family of critical regulators of the innate and adaptive immuneresponse Ihal have an imporlanl role in promoling inffammation,lin the regulation of virus replication2 and in thè connol of cellprol i ferat ion and survival.3 NF-xB normally exists as an inactivecytoplasmic complex, whose predominant form is a hetero_dimer composed of p50 and p65 (RelA) subunits, bound toinhibitory proteins of the IKB family 0rcBs), and is induced inresponse to a variety of pathogenic stimuli, including ultravioletradiat ion, e\posure to proinf ldmmatory cytokines o; mitogens,and to bacterial or viral infect ion.l-3 lnduction usuallv req-uireslhe activation of Ihe lrB l inase , lKK/ comple^, which is

composed of two catalytic subunits (lKKa and IKKB) and theIKK/NEMO re8ulalory subunit. In the classicdl paihway, rhcacl ivat ion ol lKK0 causes the phosphorylat ion of IxBs ai si terlhat tr igger their polvubiqui l ination and degradalion by the,l6Íìproteasome complex. An altefnative pathway responds to theengagement of receptors for cltokines, such as lymphotoxin-por CD40, through the involvement of lKKd homodimers,a Botirpathways ult imately el ici t lhe degrddation of the NF_l(L;inhibitory peptides, resLrlt ing in nuclear translocation of NF_KEdlmers and theiÌ binding to DNA al specif ic KB sites, rapidlyInoucrng a vanety ot genes encoding. dmong others, celadhesion molecules/ inf lJmmatorv and chemotdctic cylol ines.cvtokine rpceptors and enzymes thal produce inf lammatory

Nuclear factor-xB activation has been connected with multi-ple aspects of oncogenesis, including the control of cellmigration,-cel l-cycle progression and dif ferentiat ion, ds well a:apoptosis. ' NF,|{B general ly acts as an anl i-apoptotic fa( tor, andsuppresses cel l dealh pdthways bv sw;166;n, on geneî thalhrnder pro-apoplot ic signals, including members of the B_cellleukemid/lymphoma 2 rBcl-2r family, lumor necrosis fa(tor(TNF) feceptor-associated factors 1 and 2, the FADD-like tl-l B_convert ing enzyme-inhibitory protein cFLIB cel lular inhibjtorot-apoptosis proteins 1 and 2 (clAp-t and clAp_2) andX-chromosome-l inked inhibitor-of-apoplosis protein,\ lApj..Nt-KB-driven anti-apoptotic action confers a trenìendousadvantage in the clonal selection of neoplastic cells. ln fact.sustained constitutive NF-KB activity has been found in severaltypes ot neollastic cells, including various hematologicalmalignancies.o In addit ion, act ivat ion of NF-rcB in cancer iel lslly chemotherapy or by radiation has been shown to induce themult i-drug resistdnce response, which impinges on lhe abi l i tu ofrhe therapy i tself to induce cel l dearh.- In rhi, perspecrive,inhibition of NF-xB is expected to be therapeutic in tirosetumorsin which Nt-r<B seems to have a unique survival role, such as

i:!lll" ryî].'"i: no.dskin s ,anJ non_Hodskin;s B_cetllymphomas'-" and Burkit t 's lvmphoma.

Our previous studies on the biological activity of cyclopente-none prostanoids led to the discovery of a cross-talk betweenlrlF-xB and heat shock factor typel (HSF1), a transcriptiontac,tor with a major fole in the control ofthe heat shock response(HSR). ' ' In humans, the HSR is regulated by a family of threelunctional ly dif ferent heal shock transcript ion factors.I-rbl lSFl, which mediates . ignal ing of btrese,induced st imuli , surhas elevated tempefatures, is generally found in the cytoplasm asan inert monomer lacking transcriptìonal activity. After exposureto heat shock and other types of stress, monomers oligomerizelo a tr imeric state, form stress-induced intranuclear-eranulesnnd bind to heat shock element (HSt) sequences lhat arelocated upstream of heat shock-responsive genes, switching on

Conespondence: Dr MC Sdntoro. Depd(ment of Bioloqy, Uni,"ersitvof .Rome Tor Ver8dra. Via del la Ricerca Scienl i f icd. R;me, 00t . ìJ ,I ta ly.E'mai l : santoro@bio,uniroma2. i tReceived 13 November 2008; revised 30 September 2009j accepted2 October 2009

Héal shock lmpails NF-ÍB slNival sìgialingc Eelardo ei a/

stress-.induced transcription of heat shock proteins (HSp),including members of the HSPZ0 and HSp90 Ìamil ies, HSp27and other proteins of the network.r7,r3 HSps act as molecularchaperones in assisting the correct folding, assembly, andintracel lular transport of nascent proteinsl as well

'as in

circumventing slress-induced degrddation and assreeation ormisfolded proteins that could be roxic to the cel l : lq ,d As l- tspsynthesis increases/ HSP70 and other chaperones relocalize inthe nucleus and bind to HSFL ledding lo dissor- iat ion of tr jmersand attenuation of the response.13,le

-

We have previously shown that different chemical andphysical inducers of the HS& including cyclopentencrneprostanoids, sodium arsenite, serine protease inhibitors as wellas heat shock i tselt . prevenl NF-KB activation tr ippered byr.vtokinec. mirogens or virLrses.rr?1 22 Start ing fó-m thr.seobservations, in this study we analyzed wheth;r heat stressmay also inhibit consti tut ive NF-KB activi ty, and examined i tsefpct on the survi\ ,al of B-cel l neopldsms.

Materials and methods

Cell culture, trcatments and in vitro transductionEleven human cell lines were used for this study: Burkitt,slymphoma HS-Sultan and BL-41, acute leukemia K562 cel ls andmult iple myeloma U266 (obtained from the American TissueCulture Collect ion, Manassas, VA, USA)j KMMt and RpMl 8226multiple myeloma (kindly provided by professor A Neri,Pol icl inico di Milano, Milan, t taly) j act jvated B-cel l- l ike (ABC)OCI-1y10 and U2932t and germinal cenrer B-cel l- l ike (CCB)OCI-l y7 and OCI-Ly1 9 dif fuse large B-cel l lymphoma rDt BCL;kindly provided by Dr F Bertoni, Oncology Inst i tute of SoulhcrnSwitzefland, Bel l inzona, Switzerland), In addit ion, a clone ofdrug-resistant ,J N3 multiple myeloma cells UJN3r), not present-ing NF-KB aberrations,23 was used. Cells were srown in RpÀ,11-1640 medium lCibco, Crand lsland, Ny. USAj, supplementedwith I0% fetal bovine serum, 2 mM L-glutamine and antibiot ics(lnvit fogen, Carlsbad, CA, USA) ar 3Z"C in a humidif iedatmosphere of 5% carbon dioxide. For heat treatments, cells(5 x 10'cells/ml) were subjected to heat shock at the indicatedtemperatures in a precision water bath-W14 (Crant lnstruments,Cambfidge, UK) at 24 h after plating. TNF-o, cycloheximide anddexamethasone were purchased from Sigma (St Louis, MO,USA) and bortezomib (Velcade) was from Millennium pharma-ceuticals (Cambridge, MA, USA). Construction of lentiviralvectors Lv-shp65, containing the human p65-specifjc short-hairpin RNA (shRNAr ol igonucleotide. and Lv-shi l , containinqthe mutated p65 target sequence, was described previously,2aH;gh-titer lentiviral vector stock was produced in 293T cells bycalcium phosphate-rnediated transfection of the modifiedtransfer vector and the packaging vectors, pMDLg/pRRE,pRSV-Rev and pMD2.VSVC. Virus harvested at 60h wasconcentfated using ultracentrifugation (.50000 x g), Virus titeiswere determined by medsuring the amount of human immuno-deficiency virus type 1 p24 antigen using enzymeJinkedimmunosorbent assay (PerkinElmer Life Science, Boston, MA,USA), HS-Suhan and K562 cel ls (1 x 105) were transduced with300 ng of lentiviral p24 in the presence of polybrene (8 pglml),

Western blot anàlysisEqqal amounts of protein (20pglsample) from whole-cellextracts pfepared after lysis jn extraction buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0,1olo Tri ton X-100, 5mM EDTA,1mM Na3VO4 and 1mM phenylmethyl-sulfonyl-f luoride and

prolease inhibitorcl .wFre. sepdraled using sodiunt dodccyl

sulfate-polyacrylamide gel eleclrophoresis, transferr0d ontonitrocel lulose membranes, dnd f i l ters were incubated with theprìmary antibodies, followed by decoration with horseradishperoxìdase-conjugated goat anti-mouse or anti_rabbit antibodjes(1:10000j Amersham, Arl ington Heights, lL, USA). Immuno_complex detection was performed using enhanced chemi_luminescence system (Supersignal-West, pierce, Rockfor, lL, USA),i19 lTig"r-*91î caplured Lrsing Luminescenrlmagc"/\naly/erLAS-3000 iFuj i f i lm Corporal ion, Tokyo, Japan). i ,ronoclonaldntibodies to a{ubulin were purchased from Sigma, to HSPZ0trom Stressgen (Victoria, BC, Canada) and to Bcl_2 from Upstate(Mil l ipore Corporation, MA, USA). Rabbit polyclonal antibodiesto p65. polyladenosine diphosphate r iboserpolymerase rpARpr,and to caspase-3- (procaspase and activated fragments p19/j Z)were purchased from Cell Signal ing Technolog/ rBeveriy, MA,USA), and to clAP-2 and XlAp from R&D Sysrems rtvt inneapolis.MN, USA),

Electrophoretic mobiliry shih assdy (EMSA)Aliquots of total erlracts I I2 Fg protejn/sdmple) in 0. l9o lr i tonX-100 lysis bufer were incubated with Jzp-labeled ,<82. orHSE " DNA probes in binding buffer for 30 min as describecl.26DNA-protein.complexes were analyzed using nondenaturing4% polyacrylamide gel electrophoresis, To determine théspecificity of NF-KB-DNA complexes, whole_cell extracts fromHS-Sultan cel ls were preincubated with 200ng of polyclonalantibodies specific for c-Rel, p65, p5O and pSU (Santa CrLrzBiotechnoloS, Santa Cruz, CA, USA), or for c-Jun (Santa CruzBìotechnology, as an unrelated antibody for I h beforeelectromobility supershift assay, euantitative evaluation ofNF-KB-KB and HSF-HSE complex formation was determinedusing Typhoon-8600 imager (Moleculaf Dynamics phoshor_lmager, MDP) (Amersham Biosciences, piscataway, NJ, USA)and lmageQuant software (Amersham Biosciences) (MDpanalysis), For control of equal loading, NF-KB and HSFÌ valueswere nofmalized to the level of the nonspecific protein_DNAcomplex in the same lane,

Analysis of apoptosísFor annexin-V staining, cel ls were suspended in staining buffer(10mM 4{2-hydroxyethyl)-1-piperazineethanesulfoniJ acid(HEPES) pH 7.4, 14OmM NaCl and 2.5mu Caclr) with Splannexin VJluorescein isothiocyanate or annexin V-allophyco_cyanin roniu8ate for the analvsis in p65-interfered cel ls rBer.ton-Dickinson, San ,ose, CA. USA,. Alternal ively, cel ls were slainedwith the mitochondrion permeable, vol lage-sensit ive dye retra-melhylrodamine methyl ester {Molecular probes, Eugerre, OR,USA) for rhe analysis or dpoploric DLBCL cel ls.)r Afiei I i min ofincub-ation in-the darlt cells were analyzed by flow cytometry(FACS) using FACScan (Becton-Dickjnson) and evaluated usinsCellQuest Program {Becton-Dickinsonr.

Reverse transc ption-PcRTotal RNA was extracted using the lRlzol reagent (lnvitrogen),according-to the manufacturer,s instructions, and processJcl asdescribed.2T Prìmers for HSPZ0 were 5/-CAC6iCATCAA-CCACCCACACA-3/ (sense) and 5/-CTCCAT-CCTCACCìATCCAcAcc-3, (antisense), and for Bcl-2 were si_cAccTcTccTccACCTCAC-3' (sense) and 5/- TTCACCCTCTCCACACACAT-3/lantisense). Primers for clAP-2 Ànd glyceraldehyde )-phosphatedehydrogenase were described previously.T

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Results and Dìscussion

lf pgltlelm ic stresl í nhibits constituti ve NF_KB activityin HS-Sultan B-cell lymphomaBurkitt's lymphoma is an aggressive B-cell tumor, which ofrenpresents aberrations in NF_KB regulation, whose hallmark is lheacrlvatfon. ot, the c-myc oncogene throLtgh a reciprocallranslocation thal juxtaposes lhe c_myc gene on cnromosome_Bro_on_e of lhe immunoglobulins loci. ,ó l t has been shown thatNf-Ku, activity is required for c_myc expression Lrnder thecontrot ot the immunoglobulin heavy chain enhancer, suggestinAînat Interterence with NF-KB function may represenìa successful approach to Burkitt,s lymphoma treatment.2sTo analyze the effect of heat stress on constitutive NF_KBDNA-binding activi ly, we used as a model the Hs_sultanSLrrkitt's lymphoma cell line, which was previously shown topresenl aberrant NF-rB regulat ion.s2a FiS_Sultan'cel ls weieerther kept at 37.C or subiecled to hyperthermic trealment (HT)dt 41, 43 ot 45"C for 45 min. Afler different periods of recoverval 37'C, whole-cel l extracts were analyzed ior HSF and NF_rÉDNA-binding activi ly using EMSA tFigLrre I ) . Exposure to 43 and45"L strongty actÌvated HsF (10_ and 12_ to ts_fold, respec_tively), whereas treatment at 4l "C caused only a modestaclivation of HSf (50./" above control;, as demonstrated by MDpanalysis (Figure 1a, bottom). As expected accordine'to thetransient,nature of HSFI activation,2e both the intensity-and thelength.of HSF activation were dependent on the temperatureused. Atter exposure to 41 anó 43"C, HSF DNA_binding activitvreturned to control level after 2h recovery at :2.C,íherea'safter treatment at 45.C HSF activation persisted ai elevatedlevels for at least 3 h.

As expected, HS-Sultan cells were found to show constitu_tively_high NF-KB DNA-binding activi ty, The major Nf-xB bandin Hs-S^ultan cells was shown to consist oi the p65/RelAsubunit.2a HT was found to cause an inhibition of constitutiveNF-KB activity/ whích was inversely correlated with HSFactìvation in Hs-Sultan cells. NF-KB activity was not signifi_cantly altefed in Hs-Sultan cells heat-shocked at 41 "C; ÈT at43 'C resulted in a transient inhibition of NF-KB, whereas HT at45'C resulted ìn a marked and persistent inhibition ofconsti luLive NF-KB DNA-binding activiry (Figure 1bt. The levelsof lhe RelA./p65 NF-KB subunit were not àltered under theseconditions (data not shown),

Heat-induced NF-rB inhibition k rapidly foltowed byapoptosis in HS-Sultan cellsThe relationship between HSR activation and inhibition ofconstitutive Nf-l<B was further analyzed in HS-sulfan cells andcompared with the effect on human acute leukemia K562 cells,which show low basal level of NF-KB activity,2a HS-sultan anúK562 cells were subjected to HT at 41, 43 o;r 45 "C. Íot 45 min.After a 3-h recovery period at 3Z "C, whole-cell exhacts wereanalyzed for HSF and NF-KB activity using EMSA. As expectedat this time of recovery, HSF1 activation was detected onlv inHs-Sultan cells subjected to 45.C HT (Figure 2a, top). U;derlhese condil ions, Nf-KB activily was markedly inhibited(fiBure 2a, middle), confirming the resulrs described above.K562 cells displayed a HSR similar to Hs-Sulran cells wirh astrong activation of HSF al 45 "C lFigure 2a, top). NF-KB DNA_bindinS activity was barely derectable in K562 c€lls at J7"C,and no appreciable difference was detected at any of thetemperatures tested (Figure 2a, middle). Next, we analyzed thelevels of apoptosis in heat,shocked Hs-sultan and K562 cellsusing FACS analysis of annexin V+ cells at 24h after heat

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Fígurc 1 Temperature-dependeni inhibition of contitutive NF-KBaclrvrry rs associated with heat shock factor type_l (HSFI) dctivation.nr-òunan cels were either*ept at 37"C or subjected to hear shockror 45 mrn at 4ì, 43, and 45 .C. After the indicateil periods of rerovervat.17"C,. whole-cell exlracts were analyzed for'HSF f"f ., fli_*fi(ot-uNA-olnding activity using EMSA. positions of HSF-HSf comDlex(|.l5fL conslatutave HSE_binding activity (CHBAI and nonspeiificpmletn-uNA Interaction INS) are indicated (a, topr. positiòns ofNF-KB-DNA INF-KB) and nonspecific protein_DNA (ni) complexes areIndicated fb, top). The levels of HSF and NF_KB DNA_bindiiìe adivirvwere quantitated using MDp analysis (a, b, bottomr and expies"ecl alrord InOuctton raj or percenLage lb) of control al 32"C. The results arerepresentanve oi two independent experiments with similar resuhs,

exposure, Concomitant with NF-r(B inhibition, a tenfoldincrease in apoptotic cells was detected in HS_Sultan cellssubjected to 45"C HT, as compared with untreated cells(FiSure 2a, bottom). The increase in apoptotic cel ls was alsodetî:ted.u:jlg TUNEL (terminal deoxynucleoridyl lransferase_mediated.dUTP nick-end labellingr analysis ldata not shown;.Conversely, K562 cells were resistanl to apoptosis induction atall temperatures tested.

The effects of hyperthermic treatment are strictly dependenton the increase in temperature above phvsiologicJl conditionsand lhe duration oi exposure. Consequently, we analyzedthe eflect ol lemperature versus length of treatment on HSF andNF-KB activi fy, and inducl ion of apoptosis in Hs_sultan andK562 cells. Cells were subiecred to 45"C HT for 20, .ì0 or45 min and, aftef 3-h recovery at 37 "C, whole-cell extracts wereanalyzed for HSF and NF-KB activity. As shown in Figure 2b,HSF wal strongly induced in both cell lines independentìy ofthetime of exposure; a 20-min HT at 45"C was sufficient tomarkedly inhibit constitutive NF-(B activity in HS_sultan cells,whereas no effect was found in K562 cells (Figure 2b, top).Analysis of annexin V cells at 6 and 24 h after

-HT coniirmed

that K562 cells are resistant to 45.C HT, whereas ìn Hs-sultancells a 20-min Hl at 45 "C was sufficient to induce a ninefoldincrease in apoptotíc cells at 24h after treatment (Figure 2b,bottom), An increase ìn heat exposure time (30 and 45min)

L€ukemia

H6at shock lmpalls NF.|{B suMvalslgnalingG Belardo ei a/

Fi8ure 2 Temperature-dependent NF"KB inhibition is èssociated withIn_ouc||on ot apoptosis in HS_Sullan B,.el l lymphoma. HS_sultan andK5bz cetts,were.€ither kept at JToC or subjected to heat shock forlrr mrn at,îhe Indlcaled temperatures, (aJ After 3_h recovery at 37 oC,wnore-ce[ exLracts were analyzed for heat shock factor (HbF; too) orNt-Ka (middte) activity using FMSA. At 24h after hypenhermicrrearment rHlJ, apoplosis was determined in parallel sanioles usinofAI-5 anatysis of annexin V cel ls rboflomr. (b) Hs_suhan and K5bicers were subjected to HT at 45.C for the indicated periods. After J_hrecovery dt J7'C, whole-cell extracls were analyzedior Hsf or Nrf_ìgd(tivity. In. parallel. samples, lhe number of a;nexin V c;lls w;;determinect al 6 and 24 h afler HT, and expressed as fold induction ofunteated confots. (c) BL-4 l, HS_Sullan ,ìnd K562 cells that were keplat-37"C or subjected to hear shock {45 min at 4s'Ct were analvzàJlàiNf-ku (top) or HSF tmiddlel activity after j_h recovery at 3ZiC. Thenumoer ot annexin V . cells was determined at 24 h after HT, andexpressed as a perc_enrage of untreated controls tUottorf. it" i!-tiìare repre9entative of two independent experimenb with similar results.

resulted in a marked enhancement of annexin V+ cells alreadyat6haftertreatment.

Temperdture and dLtration of heaL exposure are known to beregulated in a way lhat a decline of 1.C can be compensated bvdoubling Ihe erposure time for temperatur€s above 43 "C.30 Inlhrs context, we studied the effect of exposure of Burkifl,slymphoma cells at temperatures below 45 t for different tirneintervals-. Hs-Sultan cells were subjected to HT at 43 "C for 90,120 or 150 min, at 44 "C for 30, 60 or 90 min, or at 45.C foil0 min as a posit ive control. Al iquots of cel ls were anèlvzed forHSF and NF-KB activi tv at 3 h after the end oftreatmenu paral lelsamples were collected at 24 h after treatment to determine thelevel of the caspase-3 substrate pARp as a marker of apoptosis.As shown in Supplementary Figure 1a, in cel ls exposed to43.Cup to 150min, HSF activi ly returned lo basal levels after a J_hrecovery period. On the other hand, HSF activation persisted upto 3 h after the end of treatment in cells exposed to 44.C for 6t)or 90 min, and to 45 'C for 30 min. As expected, a short perìod at45 "C was suflicienr to cause NF-KB inhibition. The deàrease of

l'C^in .temperature required prolonging the exposure time

ro bu.mtn to obtain complete inhibit ion of NF_KB activi ly. Nosr8ni l icant NF-KB inhibit ion was detected at 4l.C up tol50min. Under al l condit ions, prolonged Ntf_ra inhibit ioiwas assooated_ with

.apoptosis inducl ion in HS_sultan cel ls, asÌndicated by pARp cleavage (Supplementary Figure tb).

Heat slress inhibits constitulive NF_KB activitv andrndu.ces apoptosis in different types ol B-cell'ma SnanctesWe tÈ'en analyzed whether heat stress could induce at)optosis inother types of malignant B cel ls andrvt"t f ,u, rf , i ,

" f t ! . ì-u"".associated with NF-KB inhibit ion. First, we clelermined Ihe slaLusotconfi lul Ìve NF-KB activìty in a dif ferent Burkin,s lymphomacel [ne,..ts1-41, wltich y?s previously reported to presentaberrènt NF-KB regulat ion.," BL_41, Hs_sultan and K562 cel lswere subjected to 45 "C HT for 45 min, HSF and NF_KB activi tvwas analyzed after a 3-h recovery at 32.C, whereas apopiosi'swas determined in^paral lel samples at 24h after rreatment. Asshown previously,.. BL-41 cells expressed constitutivelv hiahlevels_ot NF-rcB activi ty simjlar lo Hs_sullan cel ls rf i Í lure 2"c,top). Exposure to 45.C HT jnduced HSF activation in all celilines (Figure 2c, middle), and markedly inhibited constitutiveNF-KB activity in BL-41 and Hs-sultan cells, whereas no effect

I3s tounq in

.K56.2 cells (Figure 2c, top). Concomitanlly,f l r cat seo a stgnit icanl ìncrease in the number of annerinV-- cel ls in BL-41 and Hs_sulran cel ls, but nor in Ks62 ,-el ls{igure zc, oottom).

, There is€enomic evidence that aberrant NF_KB activi ty is a

nalmarK otaggressive tymphoma subtypes classif ied as DLBCL,and tn part icLrtar in the ABC lymphomas,Ir.r l .33

To establ ishwhether heal stress could induce apoptosis in DLBCI cclls alsoand whether this effect was associated with NF_KB inhibit ion,we used four DLBCL cell lines with different pafterns of NF_KBactivation: rwo ABC cel l l ines (OCt_t yt 0 dnd U2932) with hiphrevets ot constìtutive NF-KB aclivity, and two CCB cell linÉs(Ocl-ly7 and^9ct-Ly,19j rhal did not present this rype oiaberral ion." OCI-1y10, U29J2. OCI-Ly7 and OCIJylg cel lswere subjected to 45.C HT for 30min. FiSF andNF-KB activity was analyzed after a 3_h recovery at 37"C,whereas apoptosis was determined in paral lel samptes at Zhait€r lreatment by determining the number of tetramethylroda_mrne methyt esler-negative cells, and the level ol pARpdeavage. As expected, OCI-1y10 and U2932 cells, but not9cl;1y1.: j ìd^O_Ct: l I1.9 .cettE expressed constirul ivety high

1"""]::l\l lq ?NA-bindjnB activiry {Fi8ure 3a. ropr. Exposuieto 45'C HT induced HSF activation in al l cel i l ines, andmarkedly inhibited constitutive NF-KB activity in OCI_Ly1b andU2932 cells (FÍgure 3a). ABC cells resulted to be muóh moresensitive to heat stress than CCB cells, undergojng massiveapoptosis at 7 h after HT (Figures 3b and c).

Finally, we also analyzed the effecl of heat slress in severalcel l l ines of mult iple myeloma {MM). a dif ferent lype ofaggressive B-cel l malignancy characterized by constl iut ivelyhigh levels of NF-KB. U266, Rpwt_a226. KMMI and l lN3r vvcel ls were subjected to 45.C HT ior 30min. HSF and NF_KBactivity was analyzed after a 3,h recovery at 37"C, whereasapoptosjs was determined at 24 h after treatment. As expectecl,high levels of NÌ-KB aclivity were detected in U 266, Rpt Al_822éand KMM1 cel ls, whereas JJN3r did not present Lhis aberrat ionlsupplementary Figure 2a. top), Also in this case HT inducedHSF activation in al l cel l l ines and markedly inhibired Nf KBDNA-bindin8 activi ty in U266, RPMI-8226 and KMMI cel ls(5Lrpplementary Figure 2a). Concomitantly, HT sLrongly induced

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FiSure.3 Activated B-cell-like ÉBC.diffuse larSe-B-cell lymphomasI rJ-LnLLl aÌe hlghty sensitive to heat_induced apoplosis. ABC DLBCIOCI-Lyl0 and U2932 cel l l ines, and Serminal cènrir B-pel l- l ike {GCB;DLBCI OCI-Ly7 and OCI-Lylg cel ls that were kept at l7;C orsubiected to hear shock tjo mjn ar 45 "C) were analyzed for NF_KB(op) or HSF (bonom) DNA-bindinS acrivity using EMSA afler 3_hrecove-ry at 37 "C (a). The number of apoptoric cell-s was determinedusrng ttoì.ù cvtomelry afier staining with telramethykodamine methvlester (TMRM) at 7h after hyperthermic trealment ú). ln oarall;ìlsamplps, poly (ADP,ribose) polymerase (pARp) levelswere determinedurjng western blot analysis lcr. The arroì^ indicaLes the 85lDa pARpapoptotic fragment. The results are representative of two independentexperiments with similar Ìesults.

apoptosìs in U266l RPMI-8226 and KMM1 cells, whereas it hadno effect in JJN3r cells {Supplementary Figure 2b), Alrogether,these resuhs indicale that aggressive B-cel l maliqnlnciespresenting NF-KB-dependent disruption of survival s-iqnalinqare pÀrt icularly sensit ive to heat-ìnduced apoptosis,

Heat-induced apoptosis is independent of proteinsyntnestsIhe results described above show that inhibition of constitutiveNF-KB activity is strictly associated with HSF activation, aspreviously shown_ for- mitogen-, c)4oline- or virus-inducedNF-KB activation, '" ' ,r" Despite the large amount of l i teraluredescribing a cross-lalk between the HSi and NF-KB oathwavs,the molecular mechanism at the basis of this interaction remaínselusive. Several HSPs have been indicated as possible mediatorsof NF-KB inhibir ion. In panicular, HSPZ0, one of the mainmembers of the HSP famil, was recently shown to promoteTNF-mediated apoptosís by bindjng to the lKK,y-subunit of theIKK signalosome and inhibit ing NF-KB,dependent survival

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ABC.OLBCL GCB-DLBCL signaling,l5 To analyze whether expression of newly synthe_sized protein, and in panicular HSp70, was necessary to triggerth_e -pro-apopiotic signal in heat_shocked Hs_suilan c;-lls,K562 and Hs-Sultan cells were treated with cycloheximidÉ(100 pglml) or vehicle for 30 min at 37.C, and then subiected to45 'C HT for 45 min. After 3 h at lZ.C, whole_cell extrícts wereanalyzed for HSF and NF-KB acLivity. HSp70 levels and rhenumber ot annexin V- cells were determined at 24 h afier HTin-_parallel samples. As expected, heat shock inducedr-rsf aclivation dnd HSPZo expression in both Hs_sultan andK562 cells, whereas. it complelely inhibited NF_KB activiry inHS-Sultan cells, resulting in massive apoptosis selectively in theBurkifi 's lymphoma cell l ine (supplementary Figures 3a and bì.Cycloheximide rreatment did not inhibir HsF activation, burprevented HSP70 synthesis and resulted in a modest apoptosisincrease in both cell types. Interestingly, cycloheximicle wis notable lo prevent the HT-induced block of NF_KB activily andthe consequent apoptosis in Hs-sultan cells (SuoplemóntarvFigures 3a and b, bottom), These results indicatà that heai_induced inhibition of constitutive NF-(B activity does notregu,ire.de,nouo protein synthesis, and is independenl of thesynthesis of HSP70 or othei molecular chaperones.

Heat-induced NF rB in.hibi.tion leads to downregulationot anti-apoptotic protein clAp-2The presence of apoptotic Hs-sultan cells early (6 h) after heatfress (Figure 2b) prompled us to analyze the possible mechan_ismr triggering cell death during the first few hours after heatexposure, Hs-Sultan cells were sLrbiected to 45.C HT for45min, and analyzed for HSF and NF-rB activitv at differenrtimes after recovery at 32"C. Levels of the RelA,/o65 NF_KBsubunit, PARP, caspase-3 autocatalytic products p1g/17 anddifferent anti-apoptotic .proteins were determined in parallelsamples using westem blot, As shown in Figure 4a, under theseconditions HSFI DNA-binding activity was sustained for dt leastlh and remained detectable up to 6h afier HT; ìn lhe samesamples, NF-KB DNA-binding aclivity was inhìbìted immedì-ately after heat treatment and for at leaet Bh, whereas levels ofthe RelA,/p65 NF-KB subunit were not altered. Interestingly, acaspase-3-dependent apoptosis was induced as soon as 2_3hafter heaL stress, as indicated by the cleavage of pARp andprocessing ot procaspase-3 (figures 4a and cr. Rapid caspaseactivation may be in pan responsìble for prolonged Ni_rOinhibit ion, as lhe regularory subunir of tKK (tfkl or úeUOt hasbe_en_shown to be proteolysed by cellular caspases, leading toNF-KB inact ivat ion. 'b

We then evaluated the effect of heat shock on the level ofclAP-2 and XIAP anti-apoptotic proteins, which are direclinhibitors of caspase-3 activity and whose expression istranscriptionally regulated by NF-KB,]7 in the same samples.As. shown in Figures 4a and q ctAp-2 levels were rafidlyrcduced in Hs-Sultan cells with kinetics similar to caspàse_iactivation and PARP cleavage. XlAp levels were also decreased,but at later times (6h) after treatmen! whereas Bcl-2 levelsremained unchanged (Figure 4a). To analyze whether thedecrease in clAP-2 protein was a consequence of reducedmRNA levels after heat shoclt clAp-2, Bcl-2 and HSPZ0 mRNAwas evaluated in HS-Sultan cells at different times after HT usinsreverse lranscriptase-PCR. As shown in figure 4b, the amount o-lthe HsF-driven HSP70 mRNA increased in a time-deDendentmanner after trealment, as expected, No significant change inBcl-2 mRNA level was observed; conversèly, clAp-2 m"RNAlevel was dramatically decreased as soon as 30min after heatshock and remained barely detectable for the following 6 h.

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To determine whether similar effects could be detected inK562 cells, HS-Sultan and K562 cells were subiected to 45"CHT lor 45min, and after 2, 4 or 6h recovery at 32"C, wereanalyzed for caspase-3 and pARp processing, and anti_apoptoticprotein expression. Western blol analysis of pAhp andprocaspaqa-3, and FACS analysis of annexin-Vr cells at 24h,confirmed thèt programmed cell death was induced selectivelyin Hs-Sultan cells (Figures 4c and d). Analysis of the ant!apoptotic proteins showed that clAp-2 basal levels wereelevated in Hs-Sultan cells and barely detectable in K562 cells,whereas XIAP levels seemed Io be similar in both cell tvpes(Figure 4c). In HS-Sultan cells, the levels of clAp-2 Drotein werealready reduced at 2 h dfter HT, whereas XlAp levels decreasedat 6h, confirming the results described above; instead, clAp_2and XIAP levels were unchanged in K562 cells. These resultssupport the hypothesis that clAp-2 may have an important rolein resistance to caspase-3-induced apoptosis in HS-Éultan cells.lnterestingly, high levels of clAp-2 were previouslv shown inEpstein-Barr virus-bearing lymphoid malignancies, and clAp_2overexpression was correlated wilh resistance to caspdse_l-mediated apoptosis in some types ol B-lymphoid neoplasms,lsFurthermore, compounds that selectiveiy iarget clAi'_t/z fordegradation have been recently shown to induce lNFd_dependent apoptosis in cancer cells.3e XIAP was prevjouslvidentified as a key fac{or in,apoptosis resistance of ilodgkin,slymphoma-derived B cells.ao Although we also observ'ed adownregulation of XIAP in our model, this occurred at latertimes (6h) after the beginning of the heat-induced apoptoticprocess, suggesting a secondary role Ìn triggering apopiosìs.

lnhibition of NF-KB by bortezomib or downresulation ofendogenous p65 expression enhances heat_iíclucedapoptosis in Hs-Sultan cellsThe role of NF-|(B in heat-induced apoptosis ofmalignant B cellswas analyzed using lentiviral,mediated RNA in6derence toinduce a knockdown of the NF-r<B p65 subunit, whichrepresents.the,major constituent of the NF-KB complex in HS_Sultan cells,'za Lentiviral vecLors used to knockdown D65expression/ containing shRNA for the p65 gene (LV-shp65) ora sequence in which four-point mutations were introduced togenerate a control shRNA (tV*hC) and a green fluorescentprotein reponer, were described previously.za Hs_sultan anclK562 cells were mock-transduced or trinsduced with theLv-shp65 vector or with the mutated LV-shC vector as a control,After 72 h, approximately 90% of the cells were found to bepositive for green fluorescent protein/ as determined usinglluorescence microscopV. At 72h after transduction, HS_sultaiand K562 cells were either stìmuldted with TNF-a (50ngiml, orsubjected to HT for 45min at 43, 44 ot 45.C. Aft;r 24hrecovery at 37'C, the cells were analyzed using FACScan forapoptosis detection, and p65 levels were determined in whole_cell exlracts using western blol analysis, euantitalive determi_nation. ot p65 protein indicaled that p65 levels were onlypartially reduced (30-350/.) in LV-shp6s-transduced cells ascompared wìth LV-shCtransduced cells. In spite of the modestreduction, the downregulation oi p65 resulted in a cooperativeeffect in heat-treated Hs-Sultan cells, As shown in Fieure 5.apoptosis levels were higher in p6s-interiered HS-Suhi cellsthan in Lv-shc{ransduced or mock-transduced cells at all

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lti- tti.d["',"],flfi'l:":i{'itrl:í"j::',-.'îr"l,Fiftrlf:{'î4fl['ruirffi:uilf;l:iil{""i"::tll,j:::ii:,1rtr;:"i:l[:i#iiji'nni: î'*.n[:f,ii,s"iiln;*;*ml*;:[:n*;Ì.#','ni;n:,#1"]:tl"ì[y;iiliù*kltemperatures. tested A particulaly relevant. synergistic efect p6s-interfered and mock-transduced cells was instead detectedwas detected in cells subjected to a 43"c HT, which did not in K562 cells under all conditions iriguà lu,'-Àononlt. ,ninduce apoptosis in Lv-shc-transduced or,.mock-transduced addition, pes-rntenered Hs-sulran cells were found to be morecells (FiSure 5a, top, and 5b) No significant difference between sensitive io iNF-q,induced apoptosis, '."r,ùt *". in"i"*"o ou

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t ime bonezomib was found to inhibit NF_KB acLivi ly inHS-Sultan_ cells dose dependenrly, whereas dexamethi'sonenad no ettect, confjrming previous observations.2a lrr a parallelexperiment, Hs-Sullan and K562 cel ls treated for l6h wirhdifferent concentrations of bortezomib or with dexamethasonewere subjected to 43.C HT for 45 min. Analysis of annexin V+cells at 24h after HT indicated tha! in the absence of heattreahenl, bortezomib induced apoptosis in Hs_sultan cel lsstanng at lhe concenLration of 7.S and 10nM concomitanLlywith NF-KB inhibit ion (Figure 6b). As expected, a 43"C HTcauseo a modest increase in the number of apoptotic cel ls(Figure 6b,. The number of apoptotic ce s was increased inn5-5u an ce s pre_treated with 7.5nM bortezomib, whereastreatment with dexamethasone did not enhance the effect ofhyperthermic stress (Flgure 6b). lt should be noted thatpretreatment with bortezomib was unable to further enhanceapoptosis under conditions of maximal response to heat (45 minal 45 "C; data not shownl. Finally, neiiher borteromib nordexamethasone had any effect on NF_KB DNA-bindins activilvor apoptosis in K562 cel ls (Figures 6c and d),.

rhese observaLions sLrggest the interesting possibility that,In some types oî cancers, cotreatment of sub_optimal doses ofNF-KB inhibitors with mild heat sness may induce cell deathunder conditions in which the single treatment is not effective.

Conclusion

The HSR represents Lhe major defense mechanism that cells useagainst the deleterious effects of environmental and phvsiolo_Bical .stress,.which provoke cell damage because oi froteinmrsfotding, degradation and ìnsoluble aggregation.l6 However,the role of HSR in cell fate is bimodal, Whànever the damagécan be overridden with HSp chaperoning activity, the cell willswvtvej conversety/ the. stress response will trigger apoptosis.n5rs nave an extremely complex role in the regulation ofapoptosis. In particular, HSPZ0, HSp2Z and HSp90 were shownto inhibit aqoBtosis by.direcl physical interacrion with apoptoticmolecules'". , and their expression was l inked to cancerapoptosis,resistance induced by chemotherapy and radiationtherapy. '" In fact, i t was recenlly shown that inhibi l ion orsì lencing of HSFl leads to enhanced sensit ivi ty of some tvpes ofsolid lumors to hyperthermia or chemorher'apy.or-r ón Iheother hand, hyperthermia is one of the modalìtles used in theclinical sefling to treat various forms of malignancies, and hasbeen proven to be especially effective in combination withradiotherapy and chemotherapy in different types of cancer.oB.aeIn addjtion, several potent HSFI inducers provoke apoptosis insome types of cancer cel ls.2a,27 Recently, the cl inical appl ica_tion of heat as a coadjuvant in cancer treatment is gaining newinterest b^erause of the substantial technìcal improvementsachieved;3o however, the molecular mechanism. it if," ú"ri,of the

. differentia I sensitivity oJ different types of neoplasias to

hyperthermic lreatment are st i l l mainly unknown, consti tut ine am4or obtacle in the use of this type of therapy,

Starting from our early discovery of a cross-talk betweenHSF1 and NF-rB,rJ there is a large amount of evidence lhata variety of chemical HSR induceB, which include cyclopentenoneprostanoids, sodium arsenite, the serine proteàse inhibitorsN-d{osyl-rJysine chloromethyl ketone OLC() and 3,4-dichloro_isocoumarin, as well as heat shock itselt prevent NF-KBactivation triggered by cylokines, mitogens or viiuses.r6.2r,2r,14. In.rhis study, using as a model ÈS-Sulran cel ls, a B_celllymphoma. characterized by high levels of constitutive NF_KBactivity,",".u we show for the first time that heat stress is Àlso

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Fi$rre 6 The proteasome inhibitor bortezomib enhances heat-rnduced .apoptosis in HS-Suhan cells. HS_Sultan (a, bl and K562(c/ d) cetls were treated with the indicared doseg of bortezomib.crexamethasone (DexaMl or control diluent. After i6h, whole"cellextracts were analyzed for NF-I(B activity using EVS,q ta,

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levels ol NF-|fB DNA-binding aclivity were quantirated usinà MDpanatysrs {a/ c, bottom). ln a parallel experiment, HS-sultan ibt andK5b2 (Ol cetts treated with bortezomib or dexamethasone for 16hw'ere subjected to heat shock (HS) at 43 "C for 45 min (+) or kept at37'C (-), Aft€r 24h, the number of annexin Vi' cells wàs àetermììeJin,heat-shocked (empty barg or not heat-shocked tfitted Uaisj ààffsusinB,FACS anal/sis. The results are reprerentative oftwo indepóndàniexperiments with similar results,

twofold as compared with mock{ransduced or Lv_shc_trans_duced cel ls (Figure 5c). This resuk is in l ine with the well_knownrole of NF-KB in counteracting the apoptotic stimulus triggeredby TNF-o 41.

To confirm that NF-KB inhibition could enhance the effect ofmild heat treatment in Burkitt,s lymphoma cells, HS_sultan andK562 cells were treated with different concentrations of theproteasome inhibitor bortezomib (Velcade, Millenium pharma_ceuticals), which is known to block NF_KB activitv bvpreventing lKBd degradation,a2 or with the conventiona l'drugdexamethasone, After 16h, whole-cel l extracts were analyzeàfor NF-rB DNA.binding activi ty. As shown In f igure ea, aithis

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dble lo inhibit consti tut i \ ,e Nl,-KB DNA_bjnding activi ty in B_cellmalignancies. Heat- induced Nl,-KB inhibi l io; tura. ro ,upiJdownregulation of the anti-apoptotic protein clAp_2, followedbv a(t ival ion of caspase-3 dnd cleavdge of the ca<pase_3substraLe PARP, (ausing mdssive apoptosis under condit ionsthat do not affecl viabj l i ty in cel l . not presenting Nf_KBaberrat ions. NF-KB inhibit ion by the proteasome inhibitorbortezomib and by shRNA intelerence results in increasedsensit ivi l ) of lymphoma cel ls to hyperlhermic stress. We alsoshow that, in addit ion to HS-Sultèn cel l) , heat stress is able toinhibit confi tut ive NF-KB activi ty and induce massive apoptosisin other tvpes of aggressive B-rel l mdliqnancie. presentinoaberrat ions in the regulat ion of lhF nucledr,;rtor. These includEmult iple myeloma and the ABC subtype of DLBCL, the mostcommon type of non-Hodgkin,s lvmphoma. which represents anrmpondnt cl inical rhal lenge ,r. parienls wirh AtiC DLBCLrespond poorty to conventional chemotherapy and present adist inct ly inferior prognosis. l 1,3r,32

Altogelher. these f indings indi(dte thal aggressive B_cellmdIgnancles presenting consti lut ive Nf-KB activi ty ,rre sensit iveto heal- induced apoptosis, dnd .uggest the interestìng possibi l i tvlhal aberrani NF-KB regulat ion may bc a marker oiÉeat stresssensitÌvi ty in cancer cel ls.

Acknowledgements

Thjs work was supported by the ltaljan Ministry of University andScjenti f ic Research (MtUR), the l tal ian Ministry of publ ic Fiealth(lSS projects Lotta ai Tumori), the EU ETCOSANOX project andRe8ione Piemonte I 43b I projectr. Wp thanl Dr Crist ina f-erreri fortechnical help.

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I \ i H. ErSin M, Huang e, e:n JZ Amin HM, Maf inez RL et r/ .Analvsjs ol expression ol nlrcJear faLtor kappa B (NF_Lappa Br inmu rpte myetoma: downregutùrion of Nf"lappa B inducesapoptosis. 8rl Hdema.ol2OOl; l l ' t )79_296,q Bargou RC, Emmerich T. krapprìann D, Bonrn ef K, Maoara My.Arnold W era/. Consritut ive nuclear fa( toÉkdppaB_RelA i( t ivatronis required for . prol i ferat ion dnd quryival or 'Hodgkin,s diseasetumor cel ls. / C/in /nrestt997;1OO )e6t_2969.

l0 Pham lV, Tamdyo A. \ oshimura LC, Lo p. Tpay N. Reid pS et a/. ALu4u srgnatosome anchored in l ipid raf is leads to consti tut i \€a.t ivat ion of NF-kappaB and aurononor,s cel l growrh in B cel llymphomas. lmmunity 2OO2i 1 6t 37-50.

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supplementary Information accompanies the paper oh the Leukemia website (httpv/www.nature,com/reu)

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