PTEN status switches cell fate between premature senescence and apoptosis in glioma exposed to ionizing radiation

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1 ell Deth n Differentition () 8, & Mcmilln Publishers Limite All rights reserve 5-97/ PTEN sttus switches cell fte between premture senescence n poptosis in gliom expose to ionizing rition J-J Lee, B Kim, M-J Prk, Y-S Lee, Y-N Kim, BL Lee n J-S Lee, Loss of the tumor suppressor phosphtse n tensin homolog (PTEN) hs frequently been observe in humn glioms, conferring AKT ctivtion n resistnce to ionizing rition () n rug tretments. Recent reports hve shown tht PTEN loss or AKT ctivtion inuces premture senescence, but mny etils regring this effect remin obscure. In this stuy, we teste whether the sttus of PTEN etermine fte of the cell by exmining PTEN-eficient, U5, n U7, n PTEN-proficient LN8 n LN8 gliom cells fter exposure to. These cells exhibite ifferent cellulr responses, senescence or poptosis, epening on the PTEN sttus. We further observe tht PTEN-eficient cells with high levels of both AKT ctivtion n intrcellulr rective oxygen species (ROS) unerwent senescence, wheres PTEN-proficient LN8 cells entere poptosis. ROS were inispensble for inucing senescence in PTEN-eficient cells, but not for poptosis in PTENproficient cells. Furthermore, trnsfection with wil-type (wt) PTEN or AKT smll interfering RNA inuce chnge from premture senescence to poptosis n epletion of p5 or p prevente -inuce premture senescence in cells. Our t inicte tht PTEN cts s pivotl eterminnt of cell fte, regring senescence n poptosis in -expose gliom cells. We conclue tht premture senescence coul hve compenstory role for poptosis in the bsence of the tumor suppressor PTEN through the AKT/ROS/p5/p signling pthwy. ell Deth n Differentition () 8, ; oi:.8/c..9; publishe online November Glioms re the most common type of brin tumor, n gre IV glioblstoms re lmost universlly ftl. Tretment of glioblstoms typiclly involves surgicl resection in combintion with rition n lkylting gent-bse juvnt chemotherpy. However, even in instnces in which complete surgicl resection of theses tumors is possible, the tumor generlly recurs within yer, regrless of its initil response to tretment. Oncogenes (EGF, PDGF, n their receptors) n tumor suppressor genes (p6 INK, p ARF, PTEN, RB, n TP5) re involve in the evolution of glioblstoms, n ctivtion of the phosphtiylinositol -kinse (PIK) signling pthwy hs crucil role in their evelopment. The tumor suppressor gene PTEN encoes lipi phosphtse tht countercts the effect of PIK signling, thereby negtively controlling the ctivtion of this pthwy. Tumor suppressor PTEN is muttionlly n trnscriptionlly inctivte in mny ifferent tumor types, incluing glioblstom. A centrl noe in signling events ownstrem of PIK is controlle by the serine-threonine kinse AKT. Therefore, AKT is ctivte by PIK, which genertes phosphtiylinositol,, 5-trisphosphte, n is negtively regulte by phospholipi phosphtses PTEN. Hyperctivte AKT provies protection from poptosis n promotes uncontrolle cell cycle progression. 5 However, it hs recently been shown tht AKT ctivity increses with cellulr senescence, n tht inhibition of AKT extens the lifespn of primry culture humn enothelil cells. 6 ellulr senescence is n extremely stble form of cell cycle rrest, which is ctivte in response to stress, incluing oncogenic signling n telomere shortening. 7 The initil escription of cellulr senescence by Hyflick n Moorehe ws bse on the enurble nlysis of norml humn cells grown in vitro. 8 In contrst to cncer cells, they observe tht norml cells h finite prolifertive cpcity tht ene in stble n long-term cell cycle rrest. onsiering tht neoplstic trnsformtion involves events tht inhibit the progrm of senescence, tumor cells were believe to hve lost the bility to senescence. However, recent t hs shown tht tumor cells cn be reily inuce to unergo senescence by genetic mnipultion, or by tretment with chemotherpeutic rugs, rition, or ifferentition gents. 9 The importnce of cellulr senescence is incresingly being recognize s tumor suppression mechnism., Here, we show tht fter ionizing rition () exposure, PTEN cts s criticl eterminnt of cell fte between senescence n poptosis in the gliom cell lines, U5, U7, LN8, n LN8. The, U5, n U7 cells unerwent senescence by p inuction becuse of muttion Division of Rition ncer Reserch, Kore Institute of Riologicl n Meicl Sciences, Seoul, Kore; ollege of Phrmcy & Division of Life Science n Phrmceuticls, Ewh Womns University, Seoul, Kore; Peitric Oncology Division, Ntionl ncer enter, Gyeonggi-o, Kore n Deprtment of Antomy, Seoul Ntionl University ollege of Meicine, Seoul, Kore orresponing uthor: J-S Lee, Division of Rition ncer Reserch, Kore Institute of Riologicl n Meicl Sciences, Seoul 9-76, Kore. Tel: þ ; Fx: þ ; E-mil: jeslee@kcch.re.kr Keywors: premture senescence; poptosis; PTEN; gliom Abbrevitions: BrU, bromoeoxyuriine; H O, hyrogen peroxie; NA, N-cetyl-l-cysteine; ROS, rective oxygen species; RT, riotherpy; SA-b-Gl, senescence-ssocite b-glctosise; sirna, smll interfering RNA; wt, wil-type Receive.5.; revise.9.; ccepte.9.; Eite by JA ilowski; publishe online..

2 PTEN sttus shifts response 667 or eficiency of PTEN. However, LN8 n LN8 cells, which express wil-type (wt) PTEN, isplye poptotic, s oppose to senescent, phenotypes fter exposure. Further exmintions suggest tht premture senescence coul be n lterntive mechnism to prevent berrnt cell prolifertion, inste of poptosis, in the bsence of the tumor suppressor PTEN. Results PTEN sttus icttes premture senescent n poptotic phenotypes in gliom cells following exposure. To test whether the cellulr response of gliom to is ssocite with PTEN sttus, we expose severl gliom cell lines, incluing PTEN-eficient, U5, n U7 n the PTEN-proficient LN8 n LN8 cells to vrious oses of. We first exmine reltive cell numbers n cellulr morphologies, n observe tht ll cell types h ecrese cell numbers in ose-epenent mnner following (Figure ). Furthermore, microscopic nlyses inicte tht, U5, n U7 cells were positive for senescence-ssocite b-glctosise (SA-b-Gl), hllmrk of senescence, n for senescent morphology (lrge flttene shpe) (Figure b). In contrst, LN8 n LN8 cells becme susceptible to trypn blue stining, n isplye positivity, in ose-epenent mnner (Figure c). yclin-epenent kinse inhibitor p, one of senescence mrkers, ws rmticlly increse in PTEN-eficient, U5, n U7 cells, but not in PTEN-proficient LN8 n LN8 cells, n cleve poly (ADP-ribose) polymerse (PARP), which is n inictor of the biochemicl chnges ue to cspse ctivtion uring poptosis ws etecte only in LN8 n LN8 cells Reltive cell numbers 8 6 PTEN p (Gy) U5 U7 LN8 LN8 mt mt null wt wt wt mt mt mt mt b U5 U7 LN8 LN8 Gy 6 Gy 8 Gy Gy SA-β-Gl (Gy) U5 U7 LN8 LN8 c Trypn blue (Gy) U5 U7 LN8 8 6 D D D (Gy) LN8 LN8 LN8 U5 U7 LN8 LN8 (Gy) 6 8 P P P leve PARP p Figure Phosphtse n tensin homolog sttus etermines ifferent cellulr responses to ionizing rition (). The PTEN-eficient or -proficient gliom cell lines were trete with t 6, 8, or Gy. Reltive cell numbers (), SA-b-Gl ctivities (b), n trypn blue n -positive cells (c) were quntifie fter ys or inicte ys (for positivity). ells were photogrphe uner phse contrst microscopy n cell numbers were counte in hemocytometer uner microscope. () Western blot nlyses of p n cleve PARP. ws etecte s loing control. Quntittive results re presente s men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus control () or t inicte ys. mt, mutnt; wt, wil type ell Deth n Differentition

3 668 PTEN sttus shifts response U5 U7 LN8 LN8 Gy Gy Doxo μg/ml μg/ml b SA-β-Gl 8 6 leve PARP p Reltive cell numbers U5 U7 LN8 LN8 8 6 Doxo Doxo Doxo Doxo Doxo U5 U7 LN8 LN8 c 8 6 Doxo Doxo Doxo Doxo Doxo h h h h h h h h h h U5 U7 LN8 LN8 LN8 LN8 U5 U7 Doxo Doxo Doxo Doxo Doxo Figure Phosphtse n tensin homolog (PTEN)-eficient gliom cells ctivte the poptotic pthwy fter tretment with oxorubicin, but enter premture senescence fter ionizing rition () exposure. The PTEN-eficient or -proficient gliom cell lines were trete with or Gy of, n or mg/ml of oxorubicin. ellulr morphologies (upper pnel) n reltive cell numbers (lower pnel) (), SA-b-Gl ctivities (b), n -positive cells (c) were quntifie fter ys or n h (for positivity). ells were photogrphe uner phse contrst microscopy n cell numbers were counte in hemocytometer uner microscope. () Western blot nlyses of p n cleve PARP. ws etecte s loing control. Ech br in the grphs inictes men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus (Figure ). spse-/7 ctivity n pro-cspse clevge were lso increse in ose- n time-epenent mnners to exposure to in LN8 n LN8 cells (Supplementry Figure S). These t suggest tht ecrese cell numbers fter exposure to re ue to premture senescence in PTEN-eficient cells, n ue to poptosis in PTEN-proficient cells, regrless of p5 sttus. In PTEN-eficient P- n PTEN-proficient DU5 prostte cncer cells, we observe cellulr responses to tht were very similr to those in gliom (Supplementry Figure S). PTEN-eficient glioms opt ifferent finl cell ftes epening on stimulus type. As inuce senescence in PTEN-eficient cells n poptosis in PTEN-proficient cells, we next teste the effect of higher oses of n tretment with the genotoxic rug oxorubicin on the sme cell types. When trete with or Gy of or with or mg/ml oxorubicin, ll gliom cell numbers ecrese, s compre with proliferting control cells (Figure ). As escribe before, PTEN-eficient, U5, n U7 cells were positive for SA-b-Gl stining fter or Gy of ell Deth n Differentition

4 PTEN sttus shifts response 669 exposure, inicting tht they unerwent senescence (Figure b). However, oxorubicin tretment inuce poptosis in PTEN-eficient cells, s evience by Annexin V positivity n PARP clevge (Figure c n ), wheres PTEN-proficient LN8 n LN8 cells unerwent poptosis, regrless of stimulus type (Figure ). Levels of p increse fter ministrtion of high oses of, but not in oxorubicin-trete, U5, n U7 cells, n neither tretment inuce p expression in LN8 nor LN8 cells (Figure ). We coul not etect expression of other cell cycle regultory proteins such s p6 n p (t not shown), consistent with prior t showing they re elete in ll gliom cell lines use in this stuy except U7. leve PARP ws observe in oxorubicin-trete, U5, n U7 cells n in LN8 n LN8 cells trete with high oses of or oxorubicin. spse-/7 ctivity n pro-cspse clevge were etecte in oxorubicintrete, U5, U7, LN8, n LN8 cells (Supplementry Figure S). In contrst with the response to, these t emonstrte tht PTEN-eficient cells unerwent poptosis, not senescence, s result of oxorubicin tretment. This inictes tht PTEN-eficient cells coul ctivte either poptosis or senescence pthwys, epening on the type of cellulr stimulus. When we pplie hyrogen peroxie (H O ) to n LN8 cells to exmine cellulr responses to other stimuli in the presence or the bsence of PTEN, LN8 cells showe rmtic increses in the number of trypn blue positive cells n PARP clevge, n no inuction of p, s compre with cells (Supplementry Figure S). Rective oxygen species re essentil for the inuction of senescence in cells, but not for poptosis in LN8 cells. We next exmine moleculr chnges of senescence in PTEN-eficient cells, n those of poptosis in PTEN-proficient LN8 cells, over time following tretment. Both cell types h n immeite reuction in cell number n in morphologicl chnges, n s before, only cells h increse SA-b-Gl stining (Figure ). Furthermore, the increse in poptotic cells, which ws etecte using ssy, ws eviently observe in LN8 cells (Figure b), s we previously observe. As shown in Figure c, LN8 cells exhibite rmtic increses in PTEN, mutnt p5, n cleve PARP. However, phospho- AKT (S7) n p inuction were not etecte. In contrst, cells were chrcterize by grul increses in phospho-akt, wt p5, n p (Figure c). There were no etectble PARP clevge n PTEN inuction in cells. We teste for rective oxygen species (ROS) prouction in n LN8 cells to know whether there ws ifference in the levels of ROS between premture senescence n poptosis. Rective oxygen species increse in both cell lines, n cells exhibite significntly higher intrcellulr ROS levels thn LN8 cells (Figure, left pnel). As it hs been known tht ctive AKT coul reuce MnSOD n ctlse expression by inhibition of Forkhe box O / (FOXO/), 5 n AKT ctivtion ws etecte in cells fter exposure in this stuy, we next teste for levels of phospho-foxo/, MnSOD, n ctlse. We observe no effect of on FOXO/ phosphoryltion or levels of MnSOD, u/znsod, or ctlse in n LN8 gliom (Figure, mile pnel), inicting tht increse ROS levels were not ttribute to FOXO/ phosphoryltion or the ecrese of ntioxint enzymes in either of the cell lines. As mitochonril ROS re the mjor source of intrcellulr ROS, we next mesure fluorescence of MitoSOX Re s mitochonril superoxie inictor (Figure, right pnel). Fluorescence intensity of MitoSOX Re ws increse in both the cell lines n more significntly in cells, consistent with increse intrcellulr ROS levels. To verify the role of ROS in senescence or poptosis inuce by, we trete n LN8 cells with the ROS scvenger N-cetyl-l-cysteine (NA) before exposure (Figure ). Wheres NA blocke inuction of senescence in cells, it i not inhibit poptotic cell eth in LN8 cells (Figure b ). Reltive cell numbers were ecrese both in -trete n in NA n co-trete LN8 cells (Figure b), n the percentge of poptotic cells n PARP clevge were not recovere by tretment of LN8 cells with NA (Figure c n ). Likewise, we observe no increse in SA-b-Gl ctivity n p inuction in LN8 cells (Figure c n ). In contrst, pretretment of cells with NA before exposure to, reuce SA-b-Gl stining, wt p5 ctivtion, n p inuction, which h been inuce by exposure (Figure c n ). However, AKT ctivtion n PARP clevge were not ffecte here (Figure ), n totl cell number n percentge of poptotic cells were not increse (Figure b n c). Together, these t suggest tht ROS genertion, likely through the upstrem AKT n the ownstrem p5/p signling pthwys, is inispensble for the inuction of senescence phenotypes, but not poptosis, in gliom. Wil-type PTEN expression or AKT epletion shifts premture senescence to poptosis in -expose gliom. We clrifie the role of PTEN/AKT in cellulr senescence by trnsfecting either wt PTEN or AKT smll interfering RNA (sirna) into cells. Overll, we observe tht trnsfection of either wt PTEN or AKT sirna inuce poptosis inste of senescence s responses to exposure. Increses in SA-b-Gl ctivity n intrcellulr ROS levels, which were becuse of exposure, were reuce by wt PTEN expression (Figure 5, right pnel, n c). -positive cells n cspse-/7 ctivity were increse, n cleve PARP ws etecte inste of p inuction (Figure 5b n ). Furthermore, cells trnsfecte with wt PTEN lone showe poptotic morphology n PARP clevge (Figure 5, mile pnel, n ), n lso resulte in ecrese cell number n increse percentge of poptotic cells (Figure 5 n b). When we further exmine response in AKT sirna-trnsfecte cells, AKT epletion lso shifte cells from premture senescence to poptosis s response to (Figure 6), s observe in wt PTEN-expressing cells (Figure 5). AKT epletion resulte in ecreses in both SA-b-Gl ctivity n intrcellulr ROS levels (Figure 6 n c). positivity, PARP clevge, n cspse-/7 ctivtion in Figure 6b n were eviently increse becuse of AKT epletion, s oppose to p inuction n SA-b-Gl stining. These t confirm tht PTEN is criticl not only for the regultion of cell ell Deth n Differentition

5 67 PTEN sttus shifts response Reltive cell numbers LN8 () LN8 SA-β-Gl 8 6 LN8 () b 8 6 () LN8 c PTEN pakt (S7) p5 leve PARP p LN8 () DF-DA (fol increse) () LN8 leve PARP p-foxo/ MnSOD u/znsod tlse LN8 () MitoSOX fluorescence (fol of Increse) 6 5 () LN8 Figure ellulr responses to ionizing rition () iffer between PTEN-eficient n PTEN-proficient LN8 cells. n LN8 cells were trete with 8 Gy of n were nlyze s inicte t,, n ys post tretment. Reltive cell numbers (left pnel) n SA-b-Gl ctivities (mile n right pnels) () n - positive cells (b) were ssesse t inicte time intervls. ells were photogrphe uner phse contrst microscopy ys fter tretment n cell numbers were counte in hemocytometer uner microscope. (c) Western blot nlyses using the ntiboies inicte in the figure. ws etecte s loing control. () Intrcellulr ROS were mesure by flow cytometry fter DF-DA stining (left pnel), n mitochonril superoxie ws mesure by fluorescence with MitoSOX Re (right pnel). Western blot nlyses were performe using the ntiboies inicte in the figure (mile pnel). Quntittive results re presente s men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus t inicte ys for Figure n b; Po. versus for Figure vibility uner norml conitions, but lso for the inuction of senescence uner conitions of stress, through AKT. The p5/p signling pthwy is essentil for the inuction of -inuce senescent phenotypes in cells. We next teste if -inuce senescence requires signling of the p5/p pthwy in cells. We trnsfecte sirna specific for p5 into cells expressing wt p5, n looke for senescent phenotypes (Figure 7). In p5 sirna trnsfecte cells before exposure, p expression ws not inuce (Figure 7c), n cells showe ecrese SA-b-Gl ctivity (Figure 7, right pnel). When we exmine whether the bsence of p5 coul trigger poptosis inste of senescence, increse poptotic cells, cspse- /7 ctivity, n PARP clevge were not etecte (Figure 7b n c). Furthermore, increses in intrcellulr ROS n mitochonril superoxie, which were observe in cells fter exposure, were not ffecte in p5 sirnatrnsfecte cells (Figure 7, left n mile pnels). Decrese in S-phse entry, which ws etecte using bromoeoxyuriine (BrU) incorportion ssy, ws not reverte in p5 sirna-trnsfecte cells (Figure 7, right pnel). Although smll increse in SA-b-Gl positive cells ws observe in p5-eplete cells, both SA-b-Gl ctivity n morphologicl chnges were observe only fter exposure in control sirna-trnsfecte cells, inicting tht p5 is involve in -inuce senescence in cells. ells trnsfecte with p sirna before exposure showe phenotypes similr to those trnsfecte with p5 sirna (Figure 8). -inuce senescence phenotypes were not ell Deth n Differentition

6 PTEN sttus shifts response 67 DF-DA (fol increse) ^ NA NA + NA NA + b Reltive cell numbers NA NA + NA NA + LN8 LN8 c LN8 NA SA-β-Gl 8 6 NA NA + NA NA NA NA + NA NA + NA + LN8 LN8 LN8 pakt (S7) leve PARP p5 p NA NA + NA NA + Figure Ionizing rition ()-inuce premture senescence, but not poptosis, requires ROS ccumultion. The n LN8 cells were trete with 8 Gy of n were nlyze s inicte fter ys in the presence or bsence of mm NA. () Intrcellulr ROS were mesure by flow cytometry fter DF-DA stining. (b) Reltive cell numbers were counte in hemocytometer uner microscope. (c) ellulr morphologies (left pnel), SA-b-Gl ctivities (mile pnel), n percentge of Annexin V-positive cells (right pnel). ellulr morphologies were observe uner phse contrst microscopy n cell numbers were counte in hemocytometer uner microscope. () Western blot nlysis using the ntiboies inicte in the figure. ws etecte s loing control. Ech br in the grphs inictes men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus, Po. versus, insignificntly ifferent: P. versus, P. versus etecte, such s SA-b-Gl ctivity (Figure 8, mile n right pnels). Likewise, evience of poptosis ws not etecte fter p-sirna trnsfection, such s increses in poptotic cell, cspse-/7 ctivity, n PARP clevge (Figure 8b n c). Finlly, increse in intrcellulr ROS level originte from mitochonri n cell cycle rrest were not returne to control levels (Figure 8). These t suggest tht epletion of either p5 or p irects PTEN-eficient cells towrs cytosttic (tht is, cell cycle rrest) chrcteristics, rther thn to poptosis. When we nlyze cell cycle istribution, p5- or p- eplete cells exhibite increses in neuploi cells (N) (t not shown), which hs been reporte s cuse of cytostsis., Altogether, the p5/p signling pthwy is criticl for the inuction of senescent phenotypes by exposure in PTEN-eficient gliom. As we etecte increses in nother cell cycle inhibitor p7 in both p5 sirna- n p sirna-trnsfecte cells (Figure 7c n 8c), we next co-trnsfecte cells with p7 sirna n either p sirna or p5 sirna to exmine the role of p7 in such cytosttic stte. Trnsfection with p7 sirna i not ffect the cytosttic sttus of p5 sirna- or p sirnatrnsfecte cells (Figure 8e). This coul be becuse of itionl roles of PTEN, inepenent of AKT, such s ell Deth n Differentition

7 67 PTEN sttus shifts response Reltive cell numbers EV wt PTEN EV + wt PTEN + EV wt PTEN EV + wt PTEN + SA-β-Gl 8 6 EV wt PTEN EV + wt PTEN + b c EV wt PTEN EV + wt PTEN + spse /7 ctivity (fol of increse) EV wt PTEN EV + wt PTEN + DF-DA (fol increse) EV wt PTEN EV + wt PTEN + PTEN p-akt (Ser7) leve PARP p-prb p5 p EV wt PTEN Figure 5 Wil-type (wt) PTEN expression shifts the response from senescence to poptosis in cells. The cells were trnsfecte with wt PTEN or empty vector (EV) followe by tretment with 8 Gy. () Reltive cell numbers (left pnel), cellulr morphologies (mile pnel), n SA-b-Gl ctivities (mile n right pnels) were nlyze t ys fter exposure. (b) Apoptotic cells were ssesse by positivity n cspse-/7 ctivity t ys fter exposure. (c) Intrcellulr ROS levels were mesure by flow cytometry fter DF-DA stining t ys fter exposure. () Western blot nlysis using the ntiboies inicte in the figure. ws etecte s loing control. Ech br in the grphs inictes men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus EV, Po. versus EV þ, Po. versus wt PTEN EV + wt PTEN + protein phosphtse ctivity. 5 The mechnism regring the cytosttic sttus in p5- n p-eplete cells nees to be clrifie by further experiments. Discussion Senescent cells re chrcterize by irreversible cell cycle rrest, lthough they remin metboliclly ctive n cquire specific properties, such s lrge n flttene morphologicl chnges, SA-b-Gl stining bse on lysosoml b-glctosise ctivity t ph 6., resistnce to poptosis, n ltere gene expression. 6 More thn 5 yers fter Hyflick n Moorehe reporte replictive senescence in norml humn fibroblsts, recent evience hs suggeste tht cellulr senescence is efense mechnism ginst tumorigenesis in vivo. 7 In ition, s cellulr senescence is bunnt in premlignnt neoplstic lesions n tumor regression is ttribute to cellulr senescence, this process is consiere to hve crucil role in tumor tretment. 8,9 Inee, if cellulr senescence pthwys remin intct, senescence inuction coul compenste for the inctivtion of the poptotic pthwy n coul be triggere with low oses of chemotherpeutic rugs or. Therefore, it is possible tht improve tumor tretments coul be evelope by unerstning the mechnism of premture senescence. 9 Glioms re the most common mlignnt brin tumor in ults n re mong the most lethl of ll cncers., Glioblstoms contin number of clerly efine genetic lesions, which result in isruption of criticl cellulr signling pthwys tht regulte cell prolifertion. Activtion of the AKT oncogene by muttionl inctivtion of the PTEN tumor suppressor, or muttionl ctivtion of PIK, is common in glioblstoms. As these genetic lesions regulte biologicl n clinicl behviors of tumor cells, the resultnt isrupte signling pthwys represent ttrctive trgets for therpy. The im of this stuy ws to elucite whether PTEN sttus coul lter cellulr responses to using five ifferent gliom cell lines. We foun tht PTEN-eficient, U7, n U5 gliom cells, which hve n ctive AKT signling pthwy becuse of the bsence of PTEN, entere senescence following exposure. However, the PTEN-proficient cell lines LN8 n LN8 unerwent poptosis uner the sme tretment conitions. AKT hs crucil roles not only in the prolifertion n survivl of mmmlin cells, thereby promoting tumorigenesis, but lso for the inuction of premture senescence. 6,5 In this stuy, cells eplete of either p5 or p i not evelop senescent phenotype, but inste remine cytosttic s oppose to triggering poptosis in response to ctivte AKT. 6,7 In ition, cute PTEN inctivtion inuces growth rrest through the p5-epenent cellulr senescence pthwy both in vitro n in vivo. 8 PTEN/AKT might exert nother regultory function(s) of cell prolifertion through p5/p-inepenent pthwy. For exmple, s PTEN hs recently been reporte ell Deth n Differentition

8 PTEN sttus shifts response 67 Reltive cell numbers AKT Si + AKT Si + AKT Si + AKT Si + SA-β-Gl 8 6 AKT Si + AKT Si + b c AKT Si + AKT Si + spse /7 ctivity (fol of increse) 5 AKT Si + AKT Si + DF-DA (fol increse).5.5 AKT Si + AKT Si + PTEN pakt (Ser7) leve PARP p-prb p5 p AKT Si + AKT Si + Figure 6 Depletion of AKT shifts the response from senescence to poptosis in cells. The cells were trnsfecte with AKT sirna or control sirna followe by tretment with 8 Gy. () Reltive cell numbers (left pnel), cellulr morphologies (mile pnel), n SA-b-Gl ctivities (mile n right pnels) nlyze t ys fter exposure. (b) Percentges of -positive cells n cspse-/7 ctivity. positivity n cspse-/7 ctivity were ssesse n ys or ys fter tretment, respectively. (c) Intrcellulr ROS levels were mesure by flow cytometry fter DF-DA stining ys fter exposure. () Western blot nlysis using the ntiboies inicte in the figure. ws etecte s loing control. on, control; si, sirna. Ech br in the grphs inictes men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus on si, Po. versus on si þ, Po. versus AKT si to hve protein phosphtse ctivity in ition to lipi phosphtse ctivity, 5 PTEN my hve itionl roles in tumor biology, inepenent of AKT ctivity n the senescence pthwy. As we foun tht p7, nother cell cycle inhibitor, i not hve role in the cytosttic sttus of p5- n p-eplete cells, itionl experiments will nee to be performe before the etils regring this mechnism re unerstoo. Although the PIK/AKT pthwy is involve in both premture senescence n cell prolifertion, the specific mechnism relte to this process is not fully unerstoo. Here, we observe tht ifferent responses by gliom cell lines re epenent on PTEN sttus, n tht PTEN hs criticl role in switching cell fte between senescence n poptosis fter exposure. We lso foun tht epletion of AKT or scvenging of ROS prevente -inuce senescence in PTEN-eficient gliom. These t suggest tht the effect of PTEN on cellulr senescence is likely meite by increse intrcellulr ROS by AKT ctivtion. Since the iscovery tht H O cn inuce poptosis, mny reports hve emonstrte the importnce of ROS in the poptotic pthwy in vrious cell systems. 9, However, it hs lso been reporte tht significnt levels of ROS re prouce in senescing cells, but not in poptotic cells. Similr stuies hve shown tht ROS re lso prouce in cells unergoing poptotic-like cell eth, but tht the prouce ROS o not cuse poptosis. In this stuy, we observe tht ROS were not require to inuce poptosis in PTEN-proficient gliom. Recent reports suggeste tht constitutive ctivtion of AKT promotes senescence-like rrest of cell growth through inhibition of the trnscription fctor FOXO/. 6, Inhibition of FOXO/ increses intrcellulr ROS through the regultion of ntioxint enzymes levels, such s MnSOD, sestrin, n ctlse, n mny experiments hve shown tht ROS hve criticl role in etermining life spn n cellulr senescence in mmmlin cells. 5 In this stuy, ROS levels were elevte uring -inuce senescence, n the ROS scvenger NA reverte -inuce senescence phenotypes, incluing p5 ctivtion, p inuction, n SA-b-Gl ctivity. However, increses in intrcellulr ROS levels were not ttribute to the phosphoryltion of FOXO/ through AKT ctivtion. Riotherpy (RT) is one of mjor regimes for cncer therpy, n reserch in rition biology is currently focuse on ientifying more effective mens to increse RT efficcy. It is ccepte tht poptosis hs primry role in tumor regression fter RT, n mny stuies hve ttempte to elucite pplicble mechnisms to increse cells unergoing poptosis in RT-trgete tumors. 5,6 It hs been known tht poptosis is reily inuce in tumors erive from hemtopoietic, lymphoi, n germ cells, but some soli tumors erive from epithelil cells show resistnce to poptosis fter exposure. 7,8 Recent evience hs shown tht premturely inuces senescent phenotypes, inste of poptosis, before the Hyflick limit, which is known s stressinuce premture senescence. We lso previously reporte ell Deth n Differentition

9 67 PTEN sttus shifts response Reltive cell numbers p5 Si + P5 Si + + p5 Si p5 Si + SA-β-Gl 8 6 p5 Si + P5 Si + b 8 6 p5 Si + p5 Si + P spse /7 ctivity (fol of increse) p5 Si + p5 Si + P c p5 p leve PARP pakt (S7) p7 p5 Si + p5 Si + P DF-DA (fol increse) p5 Si + p5 Si + MitoSOX fluorescence (fol increse) p5 Si + p5 Si + Reltive BrU incorportion p5 Si + p5 Si + Figure 7 Knockown of p5 inhibits -inuce premture senescence. The cells were trnsfecte with p5 sirna or control sirna before 8 Gy of exposure. () Reltive cell numbers (left pnel) n SA-b-Gl ctivities (mile n right pnels) were quntifie ys fter exposure. (b) Percentge of -positive cells n cspse-/7 ctivity nlyze ys fter tretment. (c) Western blot nlysis using the ntiboies inicte in the figure. ws etecte s loing control. () Intrcellulr ROS levels (left pnel), mitochonril superoxie levels (mile pnel), n reltive BrU incorportion (right pnel) were mesure ys fter exposure. Intrcellulr ROS were mesure by flow cytometry fter DF-DA stining, n mitochonril superoxie ws mesure by fluorescence with MitoSOX Re. on, control; si, sirna; P, positive control ( cells trete with oxorubicin mg/ml for h). Quntittive results re presente s men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus on si, Po. versus on si þ, insignificntly ifferent (?): P. versus on si þ tht inuces premture senescence in brest, colon, n lung crcinom, n in tumor tissue of xenogrfte mice.,9 Therefore, unerstning premture senescence mechnisms coul provie helpful informtion for improving the efficcy of RT. In this stuy, we escribe for the first time etile moleculr mechnism of -inuce senescence in PTENeficient gliom. Premture senescence hs compenstory role in poptosis in the bsence of the tumor suppressor PTEN through the AKT/ROS/p5/p pthwy, wheres PTEN proficiency sensitizes gliom cells to poptosis, inepenently of p5 n ROS (Figure 8f). onclusively, our results ffirm tht RT-inuce premture senescence coul be n lterntive fil-sfe tretment option in poptosisresistnt gliom cells, n we expect our finings my i in the unerstning of the clinicl significnce of premture senescence in PTEN-eficient tumor cells. Mterils n Methos Mterils. LN8 n gliom cell lines were purchse from Americn Type ulture ollections (Mnsss, VA, USA) n U5, U7, n LN8 cell lines were obtine from Prof. S-J Lee in Hnyng University (Seoul, Kore). These cell lines were culture in Dulbecco s moifie Egle s meium (Hylone, Logn, UT, USA) supplemente with % fetl bovine serum (WelGENE, Degu, Kore) n 5 U/ml penicillin/streptomycin. The p5 ntiboy ws purchse from Novocstr Inc. (Newcstle, UK). Phospho-pRB, phspho-akt, n PARP ntiboies were obtine from ell Signling Inc. (Dnvers, MA, USA). Antiboies for p7, p, b-ctin, n HRP-conjugte got nti-mouse were purchse from Snt ruz Biotechnology (Snt ruz, A, USA). Hoserish peroxise-conjugte ntirbbit n nti-mouse ntiboies were obtine from Amershm Inc. (Uppsl, Sween). Wil-type PTEN construct ws generous gift by Dr. Y-E Whng in ell Deth n Differentition

10 PTEN sttus shifts response 675. Reltive cell numbers p Si + p Si + + p Si p Si + SA-β-Gl 8 6 p Si + p Si + b 8 6 p Si + p Si + P spse /7 ctivity (fol of increse) p Si + P p Si + c p5 p leve PARP pakt (S7) p7 p Si + p Si + P DF-DA (fol of increse) p Si + psi + MitoSOX fluorescence (fol of increse) p Si + psi + Reltive BrU incorportion p Si + psi + e p Si p5 Si Reltive cell numbers p Si + p7 Si + p Si + p5 Si + p Si p5 Si Apoptosis Senescence ytostsis p7 Si + Figure 8 Knockown of p inhibits -inuce premture senescence. The cells were trnsfecte with p sirna or control sirna before 8 Gy of exposure. () Reltive cell numbers (left pnel) n SA-b-Gl ctivities (mile n right pnels) were quntifie ys fter exposure. (b) Percentges of -positive cells n cspse-/7 ctivity nlyze t ys fter exposure. (c) Western blot nlysis using the ntiboies inicte in the figure. ws etecte s loing control. () Intrcellulr ROS levels (left pnel), mitochonril superoxie levels (mile pnel), n reltive BrU incorportion (right pnel) were mesure ys fter exposure. Intrcellulr ROS were mesure by flow cytometry fter DF-DA stining, n mitochonril superoxie ws mesure by fluorescence with MitoSOX Re. (e) ellulr morphologies n SA-b-Gl ctivities (left pnel) n reltive cell numbers (right pnel) in cells, which were trnsfecte with p5 sirna or p sirna in the presence or bsence of p7 sirna followe by tretment with 8 Gy. ellulr morphologies were observe uner phse contrst microscopy n cell numbers were counte in hemocytometer uner microscope t ys fter exposure. on, control; si, sirna; P, positive control ( cells trete with oxorubicin mg/ml for h). (f) A moel illustrting the role of PTEN in switching the cell fte between premture senescence n poptosis by. Quntittive results re presente s men±s.d. of three inepenent experiments. Significntly ifferent: Po. versus on si, insignificntly ifferent: P. versus on si þ for Figure 8, P. versus p si þ for Figure 8e, P. versus p5 si þ for Figure 8e p7 Si + 5 Si + f spse PTEN AKT ROS ROS ROS ROS ROS ROS p5 p Senescence phenotype University of North rolin School of Meicine (hpel Hill, N, USA). Smll interfering RNA uplexes ginst AKT, p, n p7 were purchse from Bioneer Inc. (Dejeon, Kore), n p5 sirna ws purchse from Snt ruz Biotechnology. N-cetyl-l-cysteine n oxorubicin were obtine from lbiochem (Sn Diego, A, USA), n,7 -ichlorofluorescin icette ws purchse from Invitrogen (rlsb, A, USA). ell Deth n Differentition

11 676 PTEN sttus shifts response Irrition. For irrition, cells were expose to g-ry with 7 s g-ry source (Atomic Energy of n Lt.) t ose rte of. Gy/min. Trypn blue stining. Trypn blue solution (.%) ws e to the cell suspension t :5 rtio, n incubte for 5 min t room temperture. ells excluing the stin were counte in hemocytometer uner microscope s vible cells. Mesurement of cspse-/7 ctivity. spse-/7 ctivity ws mesure by using the Apo-one Homogenous spse-/7 Assy kit (Promeg, Mison, WI, USA) following the protocol provie by the mnufcturer. In brief, cells were trypsinize n mixe with the sme volume of the Apo-one Homogenous spse-/7 regent. After incubtion t room temperture for h, cspse-/7 ctivities were mesure from the fluorescence t the excittion wvelength of 85 nm n the emission wvelength of 55 nm using the Fluoroskn Ascent Synergy (Biotex Instrument, Highln Prk, IL, USA). stining. Apoptosis-meite cell eth ws exmine using FIT- poptosis etection kit (BD Biosciences, Sn Diego, A, USA) ccoring to the mnufcturer s instructions. Briefly, the hrveste 5 cells were wshe with phosphte-buffere sline (PBS) n resuspene in ml bining buffer. Then, 5 ml of FIT n 5 ml of propoium ioie (PI) were e. Flow cytometric nlysis ws performe fter incubtion for 5 min in the rk. Dt cquisition n nlysis were performe in flow cytometer (FASliber: Becton-Dickinson, Frnklin Lkes, NJ, USA) using ellquest softwre (BD Biosciences, Sn Jose, A, USA). Bromoeoxyuriine incorportion ssy. Bromoeoxyuriine incorportion ws ssesse using colorimetric enzyme-linke immunosorbent ssy kit (Roche Applie Science, Ininpolis, IN, USA) ccoring to the mnufcturer s instructions. Propiium ioie stining. ells were fixe in 7% ethnol n wshe in PBS n then stine with.5 ml of PBS contining mg/ml RNse n mg/ml PI for min in the rk t room temperture. The t were nlyze using Multicycle softwre (Phoenix Flow Systems, Sn Diego, A, USA). Mesurements of ROS n mitochonril superoxie. ROS n mitochonril superoxie were mesure using the protocol escribe in Lee et l. 9 Western blot nlysis. For western blot nlysis, we followe the protocol escribe in Byun et l. Trnsfection. Trnsfection ws crrie out using Lipofectmine regent (Invitrogen) ccoring to the mnufcturer s instructions. RNA interference. ells were trnsfecte with nm sirna uplexes by using Lipofectmine RNAiMAX ccoring to mnufcturer s protocol (Invitrogen). Nonspecific sirna (AGGUAGUGUAAUGUUGUU; Bioneer, Inc., Dejeon, Kore) ws use s control for comprison. Senescence ssocite b-glctosise stining. We followe the protocol escribe in Dimri et l. Sttisticl nlysis. Differences between vrious experimentl groups were clculte using either Stuent s t-test or nlysis of vrince test for multiple comprisons. P-vlues of o. were consiere significnt. onflict of interest The uthors eclre no conflict of interest. Acknowlegements. This work ws supporte by the Nucler Reserch & Development Progrm n Riologicl Trnsltionl Reserch Progrm (RTR- ) of the Kore Science n Engineering Fountion grnt fune by the Koren government (MEST).. Hirose Y, Berger MS, Pieper RO. Abrogtion of the hk-meite G() checkpoint pthwy potentites temozolomie-inuce toxicity in p5-inepenent mnner in humn glioblstom cells. ncer Res ; 6: Ohgki H, Dessen P, Joure B, Horstmnn S, Nishikw T, Di Ptre PL et l. Genetic pthwys to glioblstom: popultion-bse stuy. ncer Res ; 6: Bleeker FE, Lmb S, Znon, vn Tilborg AA, Leenstr S, Troost D et l. Absence of AKT muttions in glioblstom. PLoS One 9; : e568.. Keniry M, Prsons R. The role of PTEN signling perturbtions in cncer n in trgete therpy. Oncogene 8; 7: Nogueir V, Prk Y, hen, Xu PZ, hen ML, Tonic I et l. Akt etermines replictive senescence n oxitive or oncogenic premture senescence n sensitizes cells to oxitive poptosis. ncer ell 8; : Miyuchi H, Minmino T, Tteno K, Kunie T, Toko H, Komuro I. Akt negtively regultes the in vitro lifespn of humn enothelil cells vi p5/p-epenent pthwy. EMBO J ; :. 7. Brig M, Schmitt A. Oncogene-inuce senescence: putting the brkes on tumor evelopment. ncer Res 6; 66: ollo M, Serrno M. Senescence in tumours: evience from mice n humns. Nt Rev ncer ; : Roninson IB. Tumor cell senescence in cncer tretment. ncer Res ; 6: Hornsby PJ. Senescence s n nticncer mechnism. J lin Oncol. 7; 5: Wu H, vn Riggelen J, Yetil A, Fn A, Bchirey P, Felsher DW. ellulr senescence is n importnt mechnism of tumor regression upon c-myc inctivtion. Proc Ntl Ac Sci USA 7; : 8.. Ishii N, Mier D, Merlo A, T M, Swmur Y, Diserens A et l. Frequent co-ltertions of TP5, p6/dkna, parf, PTEN tumor suppressor genes in humn gliom cell lines. Brin Pthol 999; 9: Dlton WB, Yu B, Yng VW. p5 suppresses structurl chromosome instbility fter mitotic rrest in humn cells. Oncogene ; 9: To Y, Leteur, Yng, Zhng P, steo M, Pierré A et l. Riosensitiztion by hir-, selective HK inhibitor. ell ycle 9; 8: i XM, To BB, Wng LY, Ling YL, Jin JW, Yng Y et l. Protein phosphtse ctivity of PTEN inhibite the invsion of gliom cells with epierml growth fctor receptor muttion type III expression. Int J ncer 5; 7: Schmitt A. ellulr senescence n cncer tretment. Biochim Biophys Act 7; 775: Xue W, Zener L, Miething, Dickins RA, Hernno E, Krizhnovsky V et l. Senescence n tumour clernce is triggere by p5 restortion in murine liver crcinoms. Nture 7; 5: Wu H, vn Riggelen J, Yetil A, Fn A, Bchirey P, Felsher DW. ellulr senescence is n importnt mechnism of tumor regression upon c-myc inctivtion. Proc Ntl Ac Sci USA 7; : Ventur A, Kirsch DG, McLughlin ME, Tuveson DA, Grimm J, Lintult L et l. Restortion of p5 function les to tumour regression in vivo. Nture 7; 5: Byun HO, Hn NK, Lee HJ, Kim KB, Ko YG, Yoon G et l. thepsin D n eukryotic trnsltion elongtion fctor s promising mrkers of cellulr senescence. ncer Res 9; 69: Mischel PS, loughesy TF. Trgete moleculr therpy of GBM. Brin Pthol ; : Wng Y, Zhu S, loughesy TF, Liu LM, Mischel PS. p5 isruption profounly lters the response of humn glioblstom cells to DNA topoisomerse I inhibition. Oncogene ; : Solomon DA, Kim JS, Jenkins S, Ressom H, Hung M, opp N et l. Ientifiction of p8 INKc s tumor suppressor gene in glioblstom multiforme. ncer Res 8; 68: Mischel PS, Shi R, Shi T, Horvth S, Lu KV, hoe G et l. Ientifiction of moleculr subtypes of glioblstom by gene expression profiling. Oncogene ; : Minmino T, Miyuchi H, Tteno K, Kunie T, Komuro I. Akt-inuce cellulr senescence: impliction for humn isese. ell ycle ; : Shiohr M, Gombrt AF, Bermn JD, Koike K, Komiym A, Koeffler HP. ytosttic effect of TNF on cncer cells is inepenent of pwaf. Oncogene 997; 5: Sgulenko V, Muth D, Sgulenko E, Pffhusen T, Schwb M, Westermnn F. thepsin D protects humn neuroblstom cells from oxorubicin-inuce cell eth. rcinogenesis 8; 9: hen Z, Trotmn L, Shffer D, Lin HK, Dotn ZA, Niki M et l. rucil role of p5-epenent cellulr senescence in suppression of Pten-eficient tumorigenesis. Nture 5; 6: Ds M, Mukherjee SB, Shh. Hyrogen peroxie inuces poptosis-like eth in Leishmni onovni promstigotes. J ell Sci ; : Quillet-Mry A, Jffrézou JP, Mnst V, Borier, Nvl J, Lurent G. Impliction of mitochonril hyrogen peroxie genertion in cermie-inuce poptosis. J Biol hem 997; 7: Song YS, Lee BY, Hwng ES. Distinct ROS n biochemicl profiles in cells unergoing DNA mge-inuce senescence n poptosis. Mech Ageing Dev 5; 6: Derouet-Hümbert E, Drãgn A, Hkki T, Bureik M. ROS prouction by renooxin oes not cuse poptosis in fission yest. Apoptosis 7; : 5. ell Deth n Differentition

12 PTEN sttus shifts response 677. Sun Y, St lir DK, Xu Y, rooks PA, St lir WH. A NADPH oxise-epenent reox signling pthwy meites the selective riosensitiztion effect of prthenolie in prostte cncer cells. ncer Res ; 7: Suzuki M, Boothmn DA. Stress-inuce premture senescence (SIPS): influence of SIPS on riotherpy. J Rit Res 8; 9: Dewey W, Ling, Meyn RE. Rition-inuce poptosis: relevnce to riotherpy. Int J Rit Oncol Biol Phys 995; : Ross GM. Inuction of cell eth by riotherpy. Enocr Relt ncer 999; 6:. 7. Henry JH, Potten S, Merritt A. Apoptosis inuce by high- n low-let ritions. Rit Environ Biophys 995; : Nomur T, Kinut M, Hongyo T, Nkjim H, Htnk T. Progrmme cell eth in whole boy n orgn systems by low ose rition. J Rit Res 99; : Lee JJ, Lee JH, Ko YG, Hong SI, Lee JS. Prevention of premture senescence requires JNK regultion of Bcl- n rective oxygen species. Oncogene ; 9: Dimri GP, Lee X, Bsile G, Acost M, Scott G, Roskelley et l. A biomrker tht ientifies senescent humn cells in culture n in ging skin in vivo. Proc Ntl Ac Sci USA 995; 9: Supplementry Informtion ccompnies the pper on ell Deth n Differentition website ( ell Deth n Differentition