Prolyl-4-hydroxylase 2 enhances hypoxia-induced glioblastoma cell death by regulating the gene expression of hypoxia-inducible factor-a

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1 OPEN Cittion: (214) 5, e1322; doi:1.138/cddis & 214 Mcmilln Pulishers Limited All rights reserved /14 Prolyl-4-hydroxylse 2 enhnces hypoxi-induced gliolstom cell deth y regulting the gene expression of hypoxi-inducile fctor- W Sun,1, W Jelkmnn 1 nd R Depping 1 Oxygen deprivtion (hypoxi) is common feture of solid tumors in dvnced stges. The primry cellulr trnscriptionl responses to hypoxi re minly medited y the trnscription fctor hypoxi-inducile fctor (HIF). HIF consists of n oxygenlile -suunit (HIF-1,-2) nd stle -suunit (ARNT). Prolyl-4-hydroxylse 2 () is known s n importnt meditor of the oxygen-dependent degrdtion of HIF- suunits. As HIF- suunits re not confirmed to e the only sustrtes of, it is unknown whether regultes HIF-1 nd HIF-2 y intercting with other intrcellulr molecules. In this study, we found tht in the gliolstom cells, mintins the gene expression of HIF-1 in dependence of nucler fctor jb nd suppresses the gene expression of HIF-2 through HIF-1. The -medited degrdtion of HIF-1 nd HIF-2 seems less importnt. Furthermore, enhnces hypoxi-induced gliolstom cell deth y modulting the expression of the HIF trget genes glucose trnsporter 1, vsculr endothelil growth fctor-a nd Bcl-2 inding protein 3. Our findings show tht inhiits the dpttion of gliolstom cells to hypoxi y regulting the HIF- suunits in non-cnonicl wy. Modultion of ctivity might e considered s new wy to inhiit gliolstom progression. (214) 5, e1322; doi:1.138/cddis ; pulished online 1 July 214 Introduction Gliolstom (Gliolstom multiforme) is the most common nd most ggressvie primry rin tumor in dults. 1 Gliolstoms in dvnced stges generlly contin res with oxygen deprivtion (hypoxi) due to n imlnce etween the tumor growth nd the vsculriztion. 2,3 The primry trnscriptionl responses of the gliolsotm cells to hypoxi re minly medited y the trnscription fctor hypoxiinducile fctor (HIF). HIF is heterodimer consisting of n oxygen-lile -suunit (HIF-1, -2) nd stle -suunit (ARNT). Both suunits elong to the sic Helix-Loop-Helix PER-ARNT-SIM (HLH-PAS) fmily of trnscription fctors. HIF-1 nd HIF-2 re rpidly degrded y the protesome in the presence of oxygen. In hypoxi, the degrdtion of HIF-1 nd HIF-2 is suppressed through vrious mechnisms. HIF-1 nd HIF-2 re stilized, form heterodimers with ARNT (HIF-1/ARNT, HIF-2/ARNT) nd regulte the expression of vriety of genes y inding to the hypoxi-response elements. 4,5 HIF-1 nd HIF-2 ffect mny key spects of gliolstom progression including ngiogenesis, glucose metolism nd poptosis. 6 Incresed expression of HIF-2 in gliolstom tissues hs een reported to e ssocited with poor prognosis. 7 Therefore, HIF-1 nd HIF-2 represent ttrctive trgets for gliolstom therpy. The oxygen-dependent degrdtion of HIF-1 nd HIF-2 is minly medited y the HIF-Prolyl-4-Hydroxylses (PHDs). PHDs re dioxygenses tht require oxygen s co-sustrte. HIF-1 nd HIF-2 re hydroxylted y the PHDs t certin prolyl residues in the oxygen-dependent degrdtion domins nd susequently recognized y the von-hippel Lindu tumor suppressor protein (pvhl). pvhl is prt of multicomponent E3-ligse (pvhl-elonginb-elonginc-cul2-rx) nd trgets HIF-1 nd HIF-2 for protesoml degrdtion. The rte of HIF prolyl hydroxyltion is reduced in hypoxi, which enles HIF-1 nd HIF-2 to ccumulte to high levels. 8 Four PHDs (PHD1 4) hve een identified so fr. All PHDs re le to hydroxylte HIF-1 nd HIF-2 in vitro. 8 1 However, the expression levels nd the sustrte preferences of the PHDs vry etween tissues Besides, the PHDs were discussed to regulte severl HIF-independent pthwys It is uncler whether the PHDs regulte the stedy-stte levels of HIF-1 nd HIF-2 y intercting with other intrcellulr molecules in gliolstom cells. In this study, we focused on the indirect regultion of HIF-1 nd HIF-2 y Prolyl-4-hydroxylse 2 () in gliolstom cells. RNA interference studies showed tht in three humn gliolstom cell lines, oppositely regultes the gene expression of HIF-1 nd HIF-2 y mintining the synthesis of the NFkB suunit p5. knockdown cused significnt downregultion of HIF-1 expressionndmrked reduction of HIF-1 protein gliolstom cells. The - medited protesoml degrdtion of HIF-1 seemed less importnt. The mrna nd the protein contents of HIF-2 were elevted in the knockdown cells due to the downregultion of HIF-1 expression. Furthermore, promotes 1 Institute of Physiology, Center for Structurl nd Cell Biology in Medicine, University of Lueeck, Lueeck, Germny Corresponding uthor: W Sun, Institute of Physiology, Center for Structurl nd Cell Biology in Medicine, University of Lueeck, Rtzeurger Allee 16, Lueeck, Germny. Tel: ; Fx: ; E-mil: wenwen.sun@medizin.uni-lueeck.de Arevitions: BNIP3, cl-2 inding protein 3; GLUT, glucose trnsporter; HIF, hypoxi-inducile fctor; NFkB, nucler fctor kb; PHD, HIF-prolyl-4-hydroxylse; VEGF, vsculr endothelil growth fctor. Received ; revised ; ccepted ; Edited y C Munoz-Pinedo

2 2 suppresses the survivl of hypoxic GBM cells hypoxi-induced gliolstom cell deth y modulting the expression of the HIF trget genes glucose trnsporter 1 (GLUT1), vsculr endothelil growth fctor-a (VEGF-A) nd Bcl-inding protein 3 (BNIP3). Our findings show tht inhiits the dpttion of the gliolstom cells to hypoxi y regulting the HIF- suunits in non-cnonicl wy. Trgeted modultion of ctivity might e considered s new wy to inhiit the progression of gliolstoms. Results mintins the gene expression of HIF-1 in gliolstom cells. The gliolstom cells were trnsfected with sirna ginst. A sufficient knockdown ws chieved 24 h fter trnsfection (Figure 1). To study the role of in regulting the stedy-stte level of HIF-1, the cells were incuted for 2 h in normoxi or hypoxi (3% O 2 ) 48 h fter trnsfection. In ll three gliolstom cell lines, knockdown cused mrked reduction of HIF-1 in hypoxi. HIF-1 ws nerly undetectle in the normoxic cells. The influence of on HIF-1 protein in normoxi could therefore not e estimted (Figure 1). Quntittive RT-PCR showed tht the mrna of HIF-1 ws significntly decresed in the knockdown cells (Figure 1c). mintins the gene expression of the NFjB suunit p5 in gliolstom cells. To understnd how regultes the gene expression of HIF-1, we studied whether influences the ctivity of nucler fctor kb (NFkB), well-chrcterized regultor of HIF-1 trnscription. NFkB is dimeric trnscription fctor. The suunits p65 nd p5 were reported to hve the strongest inding ffinity to the HIF-1 promoter We confirmed the inding of p65 nd p5 to the HIF-1 promoter in the gliolstom cells with chromtin immunoprecipittion (dt not shown). As shown in Figure 2, the protein content of p5 in the knockdown cells ws mrkedly lower thn in the control cells. The mount of p5 in nucler extrct ws lso decresed. The sucellulr distriution of p5 seemed not to e ffected, s the protein contents of p5 were lmost evenly reduced in nucleus (y c. 7%) nd cytoplsm (y c. 6%; Supplementry Figures S1B nd C). Quntittive RT-PCR showed tht the gene expression of p5 ws reduced in the knockdown cells (Figure 2). The protein mount nd the sucellulr distriution of p65 were not chnged in the knockdown cells (Supplementry Figure S1A). The trnsctivtion ctivity of NFkB ws consequently decresed in the knockdown cells (Figure 2c). constitutively suppresses the gene expression of HIF-2 through HIF-1 in gliolstom cells. It ws reported tht HIF-1 suppresses the expression of HIF-2 expression in different cell lines including gliolstom cells. 25,26 Thus, we ssumed tht the expression of HIF-2 might e incresed in the knockdown cells due to the downregultion of HIF-1 expression. We first confirmed the regultion of HIF-2 expression y HIF-1 in the gliolstom cells. HIF-1 ws knocked down with sirna (Figure 3). Forty-eight hours fter trnsfection, the mrna content of HIF-2 ws significntly elevted in ll three gliolstom cell lines (Figure 3). We then studied the influence of knockdown on HIF-2 gene expression. As shown in Figure 4, the gene expression of HIF-2 ws significntly enhnced in the gliolstom 2h Nox 2h Hox 55kD 13kD HIF-1α [%]: c HIF-1α mrna rel.levels.5. neg.control 13kD 13kD 2h Nox 2h Hox HIF-1α 2h Nox 2h Hox HIF-1α Figure 1 mintins the gene expression of HIF-1., nd cells were trnsfected with non-specific sirna () or sirna ginst (). () Twenty-four hours fter trnsfection, ws detected y immunolotting. -Actin ws used s loding control. The results re representtive for three independent experiments. () Forty-eight hours fter trnsfection, cells were incuted for 2 h in normoxi (2% O 2 ) or hypoxi (3% O 2 ). HIF-1 ws detected y immunolotting. -Actin ws used s loding control. The results re representtive for three independent experiments. (c), nd cells were trnsfected with wter (), non-specific sirna () or sirna ginst (). Forty-eight hours fter trnsfection, totl mrna ws nlyzed for HIF-1 nd riosoml protein L28 expression y qrt-pcr. Normlized HIF-1/L28 rtios re shown. The dt re shown s the men±s.e.m. (n ¼ 3). Po.1, Po.1

3 suppresses the survivl of hypoxic GBM cells 3 p5 p5 mrna rel.levels.4.5. Neg. c FL/RL Figure 2 mintins the sl ctivity of NFkB. (), nd cells were trnsfected with non-specific sirna () or sirna ginst (). Forty-eight hours fter trnsfection, p5 ws detected y immunolotting. -Actin ws used s loding control. The results re representtive for three independent experiments. () nd cells were trnsfected with wter (), non-specific sirna () or sirna ginst (). Forty-eight hours fter trnsfection, totl mrna ws nlyzed for p5 nd riosoml protein L28 expression y qrt-pcr. Normlized p5/l28 rtios re shown. The dt re shown s the men±s.e.m. (n ¼ 3). Po.1. (c) cells were co-trnsfected with NFkB responsive firefly luciferse plsmids nd renill luciferse plsmids. Six hours lter, cells were trnsfected with sirna ginst nd incuted further for 48 h. Firefly luciferse ctivities (FL) were normlized to Renill luciferse ctivities (RL). The dt re shown s the men±s.e.m. (n ¼ 3). Po cells h fter sirna trnsfection (: 48 h, : 72 h). A mrked increse in HIF-2 protein level ws lso oserved in the knockdown cells (Figure 4). modultes the expression of HIF trget genes in gliolstom cells. We further studied whether ffects the expression of the HIF trget genes in hypoxic gliolstom cells. Forty-eight hours fter sirna trnsfection, the cells were incuted for 24 h in hypoxi (3% O 2 ). The expression levels of three HIF trget genes GLUT1, VEGF-A nd BNIP3 were studied. As shown in Figure 5, the expression of GLUT1 (Figure 5) nd VEGF-A (Figure 5) ws significntly upregulted in the knockdown cells. The gene expression of BNIP3 (Figure 5c) ws decresed. enhnces hypoxi-induced gliolstom cell deth. VEGF-A, GLUT1 nd BNIP3 re centrl regultors of the cellulr dpttion to hypoxi VEGF-A ws discussed to e cytoprotective independent of its ngiogenic potentil Thus, we further studied the influence of on hypoxi-induced gliolstom cell deth. Fortyeight hours fter sirna trnsfection, the cells were incuted for 48 h in normoxi or hypoxi (3% O 2 ). The experiments were performed in sence of FCS to minimize cell prolifertion. MTT (3-(4, 5-dimethylthizol-2-yl)-2, 5-diphenyl tetrzolium romide) ssy showed tht hypoxic incution cused viility reduction of the control cells y pproximtely 56% ()/43% (), wheres the viility of the knockdown cells ws only reduced y 25% () /18% (; Figure 6). We studied whether promotes poptotic gliolstom cell deth using DNA frgmenttion ELISA. As shown in Figure 6, the poptosis of the hypoxic gliolstom cells ws not significntly ffected y. Exogenous hs no impct on the gene expression of HIF-1 nd HIF-2 in gliolstom cells. We ttempted to study whether exogenous hs the opposite effect on HIF-1 nd HIF-2 expression. The gliolstom cells were trnsiently trnsfected with. The optiml trnsfection efficiency ws chieved 24 h fter trnsfection (Figure 7). Forty-eight hours fter trnsfection, the mrna contents of HIF-1 nd HIF-2 were nlyzed using qrt-pcr. As shown in Figures 7 nd c, the exogenous hd no significnt impct on the gene expression of HIF-1 nd HIF-2. The influence of knockdown on the gene expression of HIF-1, HIF-2 nd p5 is not cused y off-trget effect. To confirm tht the influence of knockdown on the gene expression of HIF-1, HIF-2 nd p5 is not cused y the off-trget effect of sirna, we performed knockdown studies with n independent sirna (#2). As shown in Supplementry Figure S2, the independent sirna hd the sme effect on the mrna levels of HIF-1, HIF-2 nd p5 in gliolstom cells. PHD1 mintins the low level of HIF-1 in normoxic gliolstom cells. To determine whether the low level of HIF-1 in normoxic gliolstom cells is mintined y other PHDs, we studied the influence of PHD1 nd PHD3 knockdown on the HIF-1 protein level in normoxi.

4 4 suppresses the survivl of hypoxic GBM cells 13kD 7kD HIF-1α [%]: HIF-2α mrna rel.levels HIF-1α kd 1 18 Contrtol HIF-1α kd Contrtol HIF-1α kd Contrtol HIF-1α kd HIF-1α Lmin A/C According to Henze et l., 11 the expression level of PHD4 in gliolstom cells is much lower thn other PHDs. It is therefore rther unlikely tht PHD4 hs mjor role in mintining the low level of HIF-1. As shown in Supplementry Figure S3, knockdown of PHD1 led to n ccumultion of HIF-1 in normoxic gliolstom cells. Hypoxic gliolstom cell extrcts were used s positive control. HIF-1 did not ccumulte in the PHD3 knockdown gliolstom cells (Supplementry Figures S4A nd B). PHD3 expression is not upregulted in knockdown gliolstom cells. To investigte whether PHD3 is involved in the downregultion of HIF-1 expression y, we studied the influence of knockdown on the gene expression of PHD3 using quntittive PCR. As shown in Supplementry Figure S4C, the gene expression of PHD3 ws not significntly ffected y knockdown. Discussion HIF is n importnt regultor of the cellulr dpttion to hypoxi. In this study, we show tht the oxygen sensor enzyme regultes the HIF-dependent hypoxic dpttion of gliolstom cells in non-cnonicl wy. knockdown led to significnt downregultion of HIF-1 expression nd mrked reduction of HIF-1 protein in 1 HIF-1α kd 8 HIF-1α kd Figure 3 HIF-1 suppresses the gene expression of HIF-2. (), nd cells were trnsfected with non-specific sirna () or sirna ginst HIF-1 (HIF-1 kd). Forty-eight hours fter trnsfection, cells were incuted for 2 h in hypoxi (3% O 2 ). HIF-1 ws detected y immunolotting. -Actin ws used s loding control. The results re representtive for three independent experiments. (), nd U343 cells were trnsfected with wter (), non-specific sirna () or sirna ginst HIF-1 (HIF-1 kd). Forty-eight hours fter trnsfection, totl mrna ws nlyzed for HIF-2 nd riosoml protein L28 expression y qrt-pcr. Normlized HIF-2/L28 rtios re shown. The dt re shown s the men±s.e.m. (n ¼ 3). Po.5, Po.1 gliolstom cells. A stiliztion of HIF-1 protein in the knockdown cells could not e oserved. As HIF-1 protein is inducile y cute hypoxi in the gliolstom cells, we ssume tht either does not medite the oxygendependent degrdtion of HIF-1 or the -medited HIF-1-degrdtion is only of importnce during cute hypoxic exposures. The downregultion of HIF-1 expression in the knockdown cells ws presumly cused y the decrese of NFkB sl ctivity. The interction etween nd the components of NFkB pthwy hs lredy een oserved in severl other tumor cell lines. 15,16,19 In the osteosrcom cell line LM8, knockdown lso cused reduction of the sl NFkB ctivity due to the suppressed expression of IkB kinse (IKK). 19 In the colon crcinom cell line HCT116 nd the cervix crcinom cell line HeL, ws reported to decrese the sl ctivity of NFkB. 15,16 These findings suggest tht the regultion of NFkB ctivity y is highly cell type specific. In the gliolstom cells, we could show tht mintins the sl NFkB ctivity y positively regulting the gene expression of the NFkB suunit p5. As the regultion of p5 gene expression in gliolstom cells is still uncler, the mechnisms y which mintins the p5 expression remins to e further studied. In severl other cell lines, the gene expression of p5 ws shown to e influenced y fctors such s humn immunodeficiency virus, tumor necrosis fctor-, pltelet-ctivting fctor nd N-Myc p5 ws lso discussed to prticipte in its own regultion. 35,38 It hs not een confirmed tht the utoregultory feedck loop of p5 gene expression exists in gliolstom cells. As the sucellulr distriution of p5 ws lmost not ffected y, it is rther unlikely tht regultes the gene expression of p5 y intercting with the upstrem components of NFkB pthwy like IkB nd IKK. Additionl studies need to e performed to understnd the interction etween nd NFkB pthwy in gliolstom cells. Although ws lso shown to mintin the sl ctivity of NFkB in the LM8 cells, the stedy-stte levels of HIF-1 ws not reduced in the knockdown LM8 cells. 19 This suggests tht in the LM8 cells, the protein level of HIF-1 is predominntly regulted y the /VHL xis, wheres the stedy-stte level of HIF-1 in the gliolstom cells is decisively dependent on the trnscription rte of HIF-1 gene. Besides, we cnnot exclude the possiility tht might mintin the gene expression of HIF-1 through other mechnisms in gliolstom cells. HIF-2 is nother importnt memer of the HIF fmily of trnscription fctors. Although HIF-1 nd HIF-2 shre some overlpping functions, they lso exhiit unique ctivities y regulting different trget genes. 39 In the knockdown gliolstom cells, the mrna nd protein levels of HIF-2 were incresed. Knockdown studies showed tht the expression of HIF-2 in gliolstom cells is constitutively suppressed y HIF-1 even under normoxic conditions. Therefore, we ssume tht the increse in HIF-2 expression ws cused y the downregultion of HIF-1 expression. Although NFkB ws lso reported to promote HIF-2 expression, the expression level of HIF-2 seems to e solely influenced y the NFkB suunit p65. 4 However, s little is

5 suppresses the survivl of hypoxic GBM cells 5 13kD HIF-2α mrna rel.levels h Nox 2h Hox Specific HIF-2α Figure 4 enhnces the gene expression of HIF-2. () nd cells were trnsfected with wter (), non-specific sirna () or sirna ginst (). Forty-eight to seventy-two hours fter trnsfection, totl mrna ws nlyzed for HIF-2 nd riosoml protein L28 expression y qrt-pcr. Normlized HIF-2/L28 rtios re shown. The dt re shown s the men±s.e.m. (n ¼ 3 6). Po.5. () cells were trnsfected with non-specific sirna () or sirna ginst (). Forty-eight hours fter trnsfection, cells were incuted for 2 h in normoxi or hypoxi (3% O 2 ). HIF-2 ws detected y immunolotting. -Actin ws used s loding control. The results re representtive for three independent experiments known out the regultion of HIF-2 expression, we cnnot exclude the possiility tht suppresses the HIF-2 gene expression through other trnscription fctors or signling molecules. The expression of GLUT1 nd VEGF-A ws reported to e regulted y oth HIF-1 nd HIF-2. The mrna levels of GLUT1 nd VEGF-A were significntly elevted in the knockdown gliolstom cells incuted in hypoxi. Although HIF-1 nd HIF-2 re oth indispensle for mintining the expression of GLUT1 nd VEGF-A, it seems tht when is inhiited with sirna, the loss of HIF-1 is fully compensted y the upregultion of the HIF-2 expression. The expression of hexokinse 2 (HK2) nd phosphofructokinse (PFK), the pcemker enzymes of glycolysis, ws not ffected in the knockdown cells (Supplementry Figure S5), suggesting tht suppresses the glucose uptke of hypoxic gliolstom cells without chnging the rte of glycolysis. Although HIF-1 ws shown to promote the gene expression of glycolytic enzymes in severl cell lines, 41 the expression levels of HK2 nd PFK in gliolstom cells correlte neither with the oxygen concentrtion nor with the protein levels of HIF-1 (dt not shown), which indictes tht the genes of these enzymes re not trgeted y HIF-1 in gliolstom cells. The expression of BNIP3 ws reported to e promoted specificlly y HIF-1. HIF-2 ws shown to suppress BNIP3 expression in renl crcinom nd heptocellulr crcinom cells. 26,42 knockdown cused downregultion of BNIP3 expression in hypoxic gliolstom cells. Although BNIP3 is known s n importnt meditor of hypoxi-induced GLUT1 mrna rel.levels VEGF-A mrna rel.levels.5. c BNP3 mrna rel.levels.5. Figure 5 modultes the expression of HIF trget genes. ( c): cells were trnsfected with wter (), non-specific sirna () or sirna ginst (). Forty-eight hours fter trnsfection, cells were incuted for 24 h in hypoxi (3% O 2 ). Totl mrna ws nlyzed for GLUT1, VEGF-A, BNIP3 nd riosoml protein L28 expression y qrt-pcr. Normlized GLUT1/L28 () VEGF-A/L28 () BNIP3/L28 (c) rtios re shown. The rtios were normlized to. The dt re shown s the men±s.e.m. (n ¼ 3). Po.5, Po.1, Po.1

6 6 suppresses the survivl of hypoxic GBM cells Viility rel.levels [%] % 56.5% Viility rel.levels [%] % 42.8% Nox Hox Nox Hox Nox Hox Nox Hox DNA Frgmenttion (O.D.) ns si Figure 6 modultes the survivl of gliolstom cells in hypoxi. () nd cells were trnsfected with non-specific sirna () or sirna ginst (). Forty-eight hours fter trnsfection, cells were incuted for 48 h in normoxi or hypoxi (3% O 2 ). Cell viility ws determined y MTT ssy. The dt re shown s the men±s.e.m (n ¼ 3). () cells were trnsfected with non-specific sirna () or sirna ginst (si). Forty-eight hours fter trnsfection, cells were incuted for 24 h in hypoxi (3% O 2 ). The poptosis ws determined using DNA frgmenttion ELISA. The dt re shown s the men±s.e.m. (n ¼ 6). NS, not significnt c HIF-2α mrna re.levels.5. pcdna pcdna endogenous exogenous ns HIF-1 mrna re.levels.5. pcdna ns Figure 7 Exogenous hs no impct on the gene expression of HIF-1 nd HIF-2. cells were trnsfected with empty vector (pcdna) or plsmid. () Twenty-four hours fter trnsfection, ws detected y immunolotting. -Actin ws used s loding control. The results re representtive for three independent experiments., c: Forty-eight hours fter trnsfection, totl mrna ws nlyzed for HIF-1, HIF-2 nd riosoml protein L28 expression y qrt-pcr. Normlized HIF-1/L28 () HIF-2/L28 (c) rtios re shown. The dt re shown s the men±s.e.m. (n ¼ 3). NS, not significnt cell deth, 29 it ws lso discussed to inhiit temozolomidend cermide-induced gliom cell deth. 3,31 Therefore, the promoting effect of on BNIP3 expression in gliolstom cells is proly not eneficil in ll cses. The ppliction of other therpeutic gents should e tken into considertion. knockdown further resulted in reduction of hypoxiinduced gliolstom cell deth, presumly due to the modulted expression of GLUT1, VEGF-A nd BNIP3. As the cells were incuted in sence of FCS to reduce prolifertion, we cnnot exclude the possiility tht might promote the prolifertion of gliolstom cells despite

7 suppresses the survivl of hypoxic GBM cells 7 its potentil to enhnce hypoxi-induced gliolstom cell deth. Hypoxi induces oth necrosis nd progrmmed cell deth. 43 DNA frgmenttion ELISA showed tht the poptosis of hypoxic gliolstom cells is not influenced y. We ssume the promotes hypoxi-induced necrotic cell deth nd other forms of progrmmed cell deth (such s utophgy nd necroptosis), ut not poptotic cell deth. According to Evns et l., 3 the hypoxi degrees in gliolstoms re in mjority of cses moderte (1 to.5% O 2 ). To simulte the in vivo sitution, ll hypoxic experiments were performed in 3% O 2. Interestingly, overexpressed hs no influence on the expression of HIF-1 nd HIF-2 in gliolstom cells. We ssume tht exogenous might not e fully iologiclly ctive. As we hve not chrcterized the sustrte tht is responsile for the non-cnonicl regultion of HIF-, we were not le to compre the enzymtic ctivities of exogenous nd endogenous towrd this specific sustrte. Moreover, we hve to consider the possiility tht the sequences of endogenous nd exogenous differ from ech other. Additionl studies hve to e performed. In conclusion, the oxygen sensor enhnces hypoxiinduced gliolstom cell deth y regulting the gene expression of HIF-1 nd HIF-2. The -medited protesoml degrdtion of HIF-1 nd HIF-2 seems to hve minor role. represents potentil prognostic mrker nd therpeutic trget for gliolstoms with severe hypoxi. As overexpressed hs no influence on the gene expression of HIF-1 nd HIF-2, it is necessry to find other wys to enhnce the effect of endogenous on gliolstom cell deth. Chrcteriztion of the interction prtners of in NFkB pthwy, the possile genetic normlities of nd the mechnisms y which HIF-1 suppresses HIF-2 gene expression in gliolstom cells would e oligtory. Mterils nd Methods Cell culture. The humn gliolstom cell lines nd were otined from the Americn Type Culture Collection. The humn gliolstom cell line ws otined from Cell Line Service (Eppelheim, Germny). Cells were cultured in DMEM (Gico, Drmstdt, Germny) supplemented with 1% FCS (Gico), 1 IU/ml penicillin nd 1 mg/ml streptomycin (PAA Lortories, Coele, Germny) in humidified tmosphere with 5% CO 2. For hypoxic tretments, cells were incuted in the presence of 3% O 2,5%CO 2 nd 94% N 2 t 37 1C. MTT ssy. The cell viility ws determined using the MTT (Sigm-Aldrich, Seelze, Germny) ssy. Cells were incuted with MTT solution (5 g/l) for 2 h t 37 1C nd then lysed with MTT lysis uffer (1% SDS, 1% HCl,.46% isopropnol). The opticl density ws detected with microplte reder (Thermo Scientific, Bonn, Germny) t 57 nm. Protein extrction. For the preprtion of whole cell extrcts, cells were wshed with ice-cold PBS nd lysed with ure lysis uffer contining 1 mm Tris HCl (ph 6.8), 7.6 M ure, 1 M glycerin, 1% SDS, 5 mm DTT nd protese inhiitor cocktil (Cliochem, Drmstdt, Germny) or cell lysis uffer contining 3 mm NCl, 1 mm Tris (ph 7.9), 1 mm EDTA,.1% IGEPAL nd protese inhiitor cocktil (Cliochem). Protein concentrtions were determined y Bio-Rd protein ssy (Bio-Rd, Munich, Germny) using ovine serum lumin s the stndrd. Immunolot nlysis. For immunolot nlysis, 3 6 mg protein ws sujected to 7.5 or 12% SDS-PAGE nd trnsferred onto nitrocellulose memrnes (Amershm Hyond-ECL, GE Helthcre, Freiurg, Germny). Memrnes were locked t room temperture for 1 h in 3% nonft dry milk in PBS nd then incuted with primry ntiodies t room temperture for 2 h. The following ntiodies were used: mouse monoclonl ntiodies ginst HIF-1 (1 : 1, BD Biosciences, Heidelerg, Germny), -ctin (1 : 5, Applied Biologicl Mterils, Richmond, British Columi, Cnd), -Tuulin (1 : 1, Snt Cruz, Heidelerg, Germny), rit polyclonl ntiodies ginst HIF-2 (1 : 1, GeneTex, Irvine, CA, USA), p5 (1 : 1, Snt Cruz), (1 : 5, Novus NB1-137, Cmridge, UK) nd histone H1.4 (1 : 1, Sigm-Aldrich), got polyclonl ntiody ginst Lmin A/C (1 : 1, Snt Cruz). After eing wshed with PBS-T, memrnes were incuted with horserdish peroxidse-conjugted nti-mouse, nti-rit or nti-got immunogloulin G ntiodies (1 : 2, Dko, Hmurg, Germny). Immunorective proteins were detected using ECL detection regents (Amershm ECL Western Blotting Detection Regents, GE Helthcre) nd X-ry films (Amershm Hyperfilm MP, GE Helthcre). RNA isoltion nd quntittive RT-PCR. Totl RNA ws extrcted using the 61 Nucleic Acid Prepsttion (Applied Biosystems, Drmstdt, Germny) following the mnufcturer s instruction. Totl RNA (2 5 ng) ws reverse trnscried with SuperScript II Reverse Trnscriptse (Invitrogen, Drmstdt, Germny) or SuperScript III Reverse Trnscriptse (Invitrogen) ccording to the mnufcturer s instructions. Quntittive RT-PCR ws performed in ABI Prism 7 Sequence Detection System (Applied Biosystems) using SensiMix SYBR Kit (Bioline, Luckenwlde, Germny) or Tqmn Gene Expression Assy (Applied Biosystems). The mrna levels were normlized to humn L28 mrna. The following primers nd ssys were used: humn GLUT1 forwrd, 5 -GGC CTT TTC GTT AAC CGC TT-3 ; humn GLUT1 reverse, 5 -AGC ATC TCA AAG GAC TTG CCC-3 ; humn L28 forwrd, 5 -ATG GTC GTG CGG AAC TGC T-3 ; humn L28 reverse, 5 -TTG TAG CGG AAG GAA TTG CG-3 ; humn VEGF-A forwrd, 5 -GCA GAA TCA TCA CGA AGT GG-3 ; humn VEGF-A reverse, 5 -GCA TGG TGA TGT TGG ACT CC-3 ; Tqmn Gene Expression Assy for HIF-1 (Hs936368_m1), HIF-2 (Hs126149_m1), BNIP3 (Hs969293_mH), p5 (Hs76573_m1). RNA interference. Cells were cultured to 6 7% confluence nd trnsfected with 3 nm sirna or BLOCK-iT Fluorescent Oligo (Invitrogen) s negtive control using Lipofectmine RNAiMAX (Invitrogen) ccording to the mnufcturer s instruction. sirna ginst HIF-1 (Invitrogen) nd (Dhrmcon, Drmstdt, Germny) were used. Trnsfection of plsmid DNA. Cells were cultured to 7 9% confluence nd trnsfected with plsmid DNA using Lipofectmine 2 (Invitrogen) following the mnufcturer s instruction. Reporter gene ssy. Cells were cultured to 6% confluence nd cotrnsfected with firefly luciferse plsmid contining five copies of NFkB inding sites (Promeg, Mnnheim, Germny) nd Renill luciferse plsmid s trnsfection efficiency control. Cells were wshed with ice-cold PBS nd lysed with pssive lysis uffer (Promeg). Luminescence ws mesured with Mithrs LB94 (Berthold Technologies, Bd Wildd, Germny). Firefly luciferse ctivities were normlized to Renill luciferse ctivities. Detection of DNA frgmenttion. Intrcellulr DNA frgmenttion ws detected using the Cellulr DNA Frgmenttion ELISA Kit (Roche Applied Science, Mnnheim, Germny) following the mnufcturer s instructions. The opticl density ws mesured t 45 nm with microplte reder (Thermo Scientific). Sttisticl nlysis. Dt re shown s mens±s.e.m. of t lest three independent experiments. Sttisticl nlysis etween the two groups ws performed y Student s t-test. Differences were considered significnt when Po.5 (Po.5; Po.1, Po.1). All sttistics were clculted using GrphPd Prism 5 (GrphPd Softwre, Witzenhusen, Germny). Conflict of Interest The uthors declre no conflict of interest.

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