Resistance to Hypoxia-Induced, BNIP3-Mediated Cell Death Contributes to an Increase in a...

ORIGINAL ARTICLE

Resistance to Hypoxia-Induced, BNIP3-Mediated Cell DeathContributes to an Increase in a CD133-Positive Cell

Population in Human Glioblastomas In Vitro

Ulf Dietrich Kahlert, PhD, Donata Maciaczyk, Fangping Dai, PhD, Rainer Claus, MD, Elke Firat, PhD,Soroush Doostkam, MD, Tomasz Bogiel, PhD, Maria Stella Carro, PhD, Mate Dobrossy, PhD,

Christel Herold-Mende, PhD, Gabriele Niedermann, MD, PhD, Marco Prinz, MD, PhD,Guido Nikkhah, MD, PhD, and Jaroslaw Maciaczyk, MD

AbstractIn addition to intrinsic regulatory mechanisms, brain tumor stemlike

cells (BTSCs), a small subpopulation of malignant glial tumorYderivedcells, are influenced by environmental factors. Previous reports showedthat lowering oxygen tension induced an increase of BTSCs expressingCD133 and other stem cellYrelated genes and more pronounced clo-nogenic capacity in vitro. We investigated the mechanisms responsiblefor hypoxia-dependent induction of CD133-positive BTSCs in glio-blastomas. We confirmed that cultures exposed to lowered oxygenlevels showed a severalfold increase of CD133-positive BTSCs.Both the increase of CD133-positive cells and deceleration of thegrowth kinetics were reversible after transfer to normoxic con-ditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirusE1B 19-kDa proteinYinteracting protein 3)Ydependent apoptosispreferentially in CD133-negative cells. In contrast, CD133-positivecells proved to be more resistant to hypoxia-induced programmedcell death. Application of the demethylating agent 5¶-azacitidine re-sulted in an increase of BNIP3 expression levels in CD133-positivecells. Thus, epigenetic modifications led to their better survival inlowered oxygen tension. Moreover, the, hypoxia-induced increaseof CD133-positive cells was inhibited after 5¶-azacitidine treatment.

These results suggest the possible efficacy of a novel therapy forglioblastoma focused on eradication of BTSCs by modifications ofepigenetic regulation of gene expression.

Key Words: Apoptosis, BNIP3, Brain tumor stemlike cells, CD133,Glioblastoma, Hypoxia, Promoter methylation.

INTRODUCTIONGlioblastoma (GBM) is the most common primary

malignant brain tumor in adult humans. Despite intensivemultimodal treatment, including surgery followed by the com-bination of radiotherapy and chemotherapy, most patients diewithin 1 to 2 years of diagnosis because of tumor progression(1, 2). Like many other solid tumors, malignant gliomas con-tain a small population of cells with stem cellYlike propertiesthat are presumably responsible for tumor initiation and relapseafter therapy (3, 4). These cells, termed brain tumor stemYlikecells (BTSCs), are defined as cells capable of initiating tumorswith karyotypic or genetic alterations, aberrant differentiationproperties, and multilineage differentiation capacity that permittheir in vitro and in vivo self-renewal and the generation ofnontumorigenic end cells (5).

The BTSCs exhibit increased chemoresistance that isattributed to a higher expression of genes encoding for DNA-repairing enzymes, such as O6-methylguanine methyltransfer-ase (4) and multidrug resistance genes, such as the breakpointcluster region pseudogene 1 (BCRP1) (6). Because of theireffective DNA damage reparation system, BTSCs are also lesssusceptible to radiation (7). Therefore, conventional therapiesthat reduce the rapidly growing cells of the tumor, but not ofthe cancer stem cells, do not prevent recurrence of tumors,which often have a higher fraction of tumor-initiating cellsand a more aggressive clinical course. Hence, the identifica-tion and eradication of BTSCs are prerequisites for the eventualcure of GBM.

Although imperfect, prominin-1 (CD133), a member ofa family of pentaspan transmembrane glycoproteins, is cur-rently accepted as a bona fide marker of BTSCs (3, 7, 8).Recent reports suggest that regulation of the CD133-positivetumor-initiating cell population by intratumoral and peritu-moral microenvironmental cues, particularly oxygen levels,

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From the Division of Stereotactic Neurosurgery (UDK, DM, TB, MD, GNikkhah), and Department of General Neurosurgery (FD, MSC, JM),University Freiburg Medical Center, Freiburg; Division of NeurosurgicalResearch, Department of Neurosurgery, University Heidelberg MedicalCenter, Heidelberg (CH-M); and Faculty of Biology, Albert-LudwigsUniversity of Freiburg (UDK); Department of Neuropathology, UniversityFreiburg Medical Center (SD, MP); BIOSS Center for Biological SignalingStudies, University of Freiburg (MP); and Radiation Oncology, UniversityMedical Center Freiburg (EF, G Niedermann), Freiburg, Germany; ClinicalGenetics Laboratory, Department of Pathomorphology, 10th Military Clin-ical Hospital, Bydgoszcz, Poland (TB); and Division of Hematology/Oncology, Department of Medicine I, University Medical Center Freiburg,Freiburg, Germany (RC).

Send correspondence and reprint requests to: Jaroslaw Maciaczyk, MD, De-partment of General Neurosurgery, University Freiburg Medical Center,Breisacher Str. 64, 79106 Freiburg, Germany; E-mail: [email protected]

This work was supported by the Comprehensive Cancer Freiburg SeedingGrant (Deutsche Krebshilfe) awarded to Jaroslaw Maciaczyk and GuidoNikkhah.

Supplemental digital content is available for this article. Direct URL citationsappear in the printed text and are provided in the HTML and PDF versionsof this article on the journal’s Web site (www.jneuropath.com).

Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.

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is involved in the homeostasis of neural stem cells (9, 10).Lowered oxygen tension plays a role in the maintenanceof pluripotency and inhibition of differentiation in humanembryonic stem cells (11) and promotes the proliferationof human CNS precursor cells (12). Hypoxic necrosis in amalignant brain tumor is a pathognomonic feature of GBM.

Tumor-associated hypoxia has also been reported to bea poor prognostic factor (13, 14). Lowered oxygen tensioninitiates an increase of a CD133-positive BTSC populationin vitro (15, 16), driven by hypoxia-inducible factors (HIFs)and by activation of specific early developmental programs(17, 18). Here, we present novel aspects of the hypoxia-dependent increase of CD133-positive BTSC population.Prolonged growth under hypoxia (93 days) resulted in theprogressive diminution of the total cell number and activationof BCL2/adenovirus E1B 19-kDa proteinYinteracting pro-tein 3 (BNIP3)Yinduced apoptosis. This seemed to be lesspronounced in the CD133-positive subpopulation, render-ing these cells more resistant to hypoxia-induced apoptosisand promoting their positive selection under lowered oxy-gen tension. In addition, CD133-positive cells expressedhigher levels of Beclin1 under hypoxia, suggesting an im-portant role of hypoxia-induced autophagy in contributingto better survival of BTSCs. Our results suggest that tar-geting hypoxia-dependent BNIP3 expression in BTSCs bydemethylation-inducing therapy might be a new therapeuticapproach for GBM.

MATERIALS AND METHODS

Clinical Data, Tissue Collection, and Cell LinesAll procedures described were approved by the local

ethical committee of the University of Freiburg and were inaccordance with the German law. Brain tumor samples frompatients with GBM were collected after surgical resection andgraded by neuropathologists.

First, necrotic areas and blood vessels within the sampleswere carefully dissected. The tissue was then cut into smallpieces, enzymatically separated into single-cell suspensionsby application of Accutase (Life Technologies, Millipore,Billerica, MA) with 0.1% trypsin (Worthington BiochemicalCorp., Lakewood, NJ) and 0.05% DNAse (Sigma-Aldrich,Munich, Germany) for 20 to 30 minutes at 37-C. After di-gestion, cells were plated in fresh media through a 40-Kmpore mash. By following this protocol, we established tumorcell lines named GB, FF, and KK (19). Cell lines NCH421kand NCH644 were established by C. Herold-Mende, Depart-ment of Neurosurgery, Heidelberg, Germany (20). Cell linesHSR-GBM1 and 040622 were kindly provided by A. Vescovi,Stemgen S.p.A., Milan, Italy (21). Cell line GBM10 was es-tablished by E. Bar, Department of Pathology, Johns HopkinsMedical Institutions, Baltimore, MD (22). HEK293T cellswere provided by M. Bredel, Department of Neurosurgery,University Medical Center Freiburg, Freiburg, Germany.

Cell CultureTissue-derived and established cell lines were cultured

in DMEM (Dulbecco modified Eagle medium)/F12 (3:1) me-dium (both from Life Technologies) supplemented with B27

(Life Technologies), 20 ng/mL of bovine fibroblast growthfactor, 20 ng/mL of recombinant human epidermal growthfactor (both R&D Systems, Minneapolis, MN), penicillin/streptomycin (Life Technologies), and 5 Kg/mL of heparin(Sigma-Aldrich), as previously described (8).

Two oxygen concentrations were applied, that is, 21%(normoxia) and 3% (hypoxia). Continuous surveillance ofhypoxic oxygen tension level was achieved by an automaticmonitoring system integrated in a Hera Cell incubator (ThermoScientific, Bonn, Germany). We chose 3% oxygen to studythe stemlike phenotype in GBM-derived cultures based on ourexperiences with the effects of this condition on neural stemcellYpromoting characteristics of fetal neural progenitors.

Normoxic and hypoxic cells were systematically sup-plied with fresh media. The continuous culture of each cellline was not longer than 10 to 16 passages.

Treatment With 5¶-AzacitidineCell cultures were pretreated with 10 Kmol/L 5¶-

azacitidine (AZA, Sigma-Aldrich) for 5 days in normoxia; freshAZA solution was added to the culture every second day.Afterward, cells were transferred for 4.5 days into hypoxiasupplied with fresh AZA, as previously described.

Immunocytochemistry glass coverslips were precoatedfor 3 hours with poly-l-ornithine (Sigma-Aldrich). The cellswere plated on the coverslips in a 24-well plate. For stainingof the free-floating cell aggregates, the spheres were not dis-sociated before they were carefully placed entirely onto cov-erslips. Spontaneous differentiation of GBM sphere cultureswas performed by plating dissociated cells in 10% fetal calfserum (Life Technologies)Ycontaining media for 5 days (ad-herent growth). After fixation, nonspecific binding was blockedwith a solution containing 4% goat serum (GE Healthcare,Buckinghamshire, UK) and 0.1% Triton X-100 (Sigma-Aldrich) in 1� PBS (Life Technologies) applied for 45 minutesat room temperature (RT). Subsequently, cells were incubatedwith primary antibodies in 1� PBS � 0.1% Triton X-100 (forcell surface markers without Triton X-100) and 1% goat se-rum (GE Healthcare) overnight at 4-C. Primary antibodiesused are listed in Table, Supplemental Digital Content 1,http://links.lww.com/NEN/A403. Secondary antibodies (goatanti-mouse and anti-rabbit conjugated with Alexa 488 or Alexa568, respectively, both 1:150; Molecular Probes, Eugene, OR)were applied for 1 hour (RT, dark) in 0.1 mol/L PBS solu-tion containing 4% goat serum. Nuclear dye DAPI (0.2 Kg/mL,Sigma-Aldrich) was included in the secondary antibody solu-tion. Negative controls were performed with the omission ofprimary antibodies.

ImmunohistochemistrySurgical tumor resection specimens from patients

with GBM were assessed for the presence of BNIP3-positivecells using rabbit polyclonal primary antibody (no. 38621,diluted 1:50; Abcam, Cambridge, MA). In brief, formalin-fixed paraffin-embedded samples were sectioned at 3-Kmthickness. After deparaffinization, the sections were subjectedto water steam for 40 minutes at pH 6 for antigen retrievaland stained with anti-BNIP3 primary antibody at 4-C over-night. Subsequently, the slices were incubated with secondary

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antibody (biotinylated polyclonal rabbit anti-mouse, no. E0464,1:200; Dako, Hamburg, Germany) for 60 minutes at RT.The BNIP3 was detected after incubation with horseradishperoxidaseYconjugated Avidin (1:1000, 45 minutes, RT) using3,3¶-diaminobenzidine solution (10 minutes, RT; Merck,Darmstadt, Germany).

Flow CytometryFor CD133 cell surface immunostaining, 5 � 105 cells

were retrieved from a single-cell suspension and labeled withanti-CD133/1-phycoerythrin (PE) antibody (no. 130-080-801;Miltenyi Biotec, Cologne, Germany) according to the manu-facturer’s instructions. The antibody solution consisted of anti-CD133/1-PE (1:11) plus FcR blocking reagent (1:11, MiltenyiBiotec) in 1� PBS. All CD133-positive fractions were gatedusing respective isotype (anti-mouse IgG2b-PE, no. 130-092-215; Miltenyi Biotec) as a control. Fluorescence-activated cellsorting (FACS) was performed on a FACSCalibur (BD Bio-sciences, Franklin Lakes, NJ) equipped with Cell Quest Prosoftware (BD Biosciences). Postprocessing data analyses wereperformed with FlowJo software (Tree Star, Inc., Ashland, OR).

For Annexin-V staining, 5 � 105 cells in single-cellsuspension were stained on CD133 staining with Annexin-Vkit (no. 130-092-052; Miltenyi Biotec) according to manu-facturer’s instructions. To detect late apoptotic/necrotic cells,7-aminoactinomycin was used.

Flow CytometryYActivated Cell SortingCell separation based on PE-coupled antibody selection

(against cell surface protein CD133/1) was performed on aMoFlow System (Beckman Coulter). The sorted cell fractionswere reanalyzed directly after separation on a FACSCaliburas previously described to prove successful purification.

Magnetic Cell SortingSeparation of CD133-positive/CD133-negative cells was

performed using direct magnetic beadsYbased separation de-scribed in the manufacturer’s protocol (Miltenyi Biotec). Inbrief, cells were washed with PBS, followed by magnetic

labeling with anti-CD133 MicroBeads (no. 130-050-801;Miltenyi Biotec) and separated through an LS column (no.130-042-401; Miltenyi Biotec) in a MidiMACS Separator(no. 130-042-302; Miltenyi Biotec). Enrichment of the 2 pro-portions was confirmed by FACS analyses with 5 � 105 cellson staining with mouse anti-CD133/2 (293C3)YPE (no. 130-090-853; Miltenyi Biotec) analog.

Generation of Lentiviral ConstructsFor stable genetic alteration, we used the third-

generation lentiviral packaging system (VSV-G; RRE, REV,originally distributed by Didier Trono [23]). The lentiviralplasmid carrying the shRNAs against BNIP3 was publiclyavailable (no. sc-37451-SH; Santa Cruz Biotechnology, SantaCruz, CA), as was for the control pLKO.1.sh.scramble plasmid(24) (no. 1864; Addgene, Cambridge, MA).

HEK293T cells were cultured to 80% confluence in10-cm Petri dishes. The cells were transiently transfectedwith 16 Kg of plasmid DNA using Lipofectamine 2000 (LifeTechnologies). The virus was collected 48, 72, and 96 hoursafter transfection and concentrated via ultracentrifugationusing ultraspeed centrifuge (Beckman Coulter, Brea, CA)at 22,500 rpm. Each virus production was concentrated in100 KL PBS and stored at -80-C. The virus titer was deter-mined according the protocol described by Sastry et al (25).

Lentiviral Infection of Target Cells and PositiveSelection for Infected Cells

To infect the GBM cultures, 3 � 105 cells were platedin a volume of 1 mL culture media. The cells were infectedwith a multiplicity of infection of 10. The puromycin resist-ance gene in the plasmid enabled an antibiotic selection(puromycin; Life Technologies) of the transfected cell pop-ulation starting 72 hours after infection.

RNA Extraction and Reverse TranscriptionTotal RNA was isolated using the RNeasy Mini kit

(Qiagen, Hilden, Germany) according to the manufacturer’s in-structions. Total RNA extracts were photometrically quantified

FIGURE 1. Schematic overview of the study. Patient-derived glial tumors were propagated after enzymatic and mechanic dis-sociation as free-floating neurosphere cultures in DMEM/F12 (3:1)-based media. Established cultures were subjected to 2 oxygentension levels: 21% (normoxia) and 3% (hypoxia). All cultures were analyzed for their metabolic activity (MTS), BTSC proportion(CD133-positive analyses via FACS), quantitative gene expression, protein levels, BNIP3 transcription knockdown, and promotermethylation analysis.

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with Biophotometer (Eppendorf, Hamburg, Germany). Reversetranscription was performed according to the manufacturer’sprotocol using the SuperScript III Reverse Transcriptase kit(Life Technologies). Complementary DNA was diluted withdouble-distilled H2O to a 10-ng/KL working solution andstored at -20-C.

Real-time Quantitative PolymeraseChain Reaction

The abundance of transcripts was determined by real-time quantitative polymerase chain reaction (PCR) on a MyiQReal-Time PCR Detection System (Bio-Rad Laboratories,Munich, Germany) caused by SYBR Green incorporation(Applied Biosystems, Foster City, CA). Primers were usedat a concentration of 10 pmol/primer (Table, Supplemen-tal Digital Content 2, http://links.lww.com/NEN/A404).All relative quantifications were normalized to the endog-enous housekeeping control gene A-actin. Values were auto-matically determined by the supplied software of the MyiQReal-Time PCR Detection System (Bio-Rad); for each ap-proach, the results were given as mean values T SD.

Methylation Analysis in Human BNIP3-CorePromoter Using Bisulfite Methylation-Specific/Unmethylation-Specific PCR andPyrosequencing

Isolation of genomic DNA was performed with genomicDNA/RNA All Prep Extraction Micro Kit (Qiagen) accordingto the manufacturer’s protocol. The concentration was assessedusing the Biophotometer (Eppendorf). The bisulfite conversionwas performed according to the manufacturer’s protocol (EZDNA Methylation-Gold Kit, Zymo Research, Irvine, CA).

To distinguish the methylated from unmethylated tar-geting region of the DNA, methylation-specific (MSP, 96 bp)PCR for human BNIP3 (located on human chromosome 10)was performed with primers containing CpG sites (CpG41to CpG31) adjacent to HIF-1 binding motif (ACGTG), aspreviously described (26Y28) (Figure, Supplemental DigitalContent 3, http://links.lww.com/NEN/A405). For the time-dependent BNIP3 methylation measurements under hypoxia,108 MSP primers were applied. The PCR reaction was per-formed using HotStart-plus master mix kit (Qiagen) with 30 ngof bisulfite-treated DNA, 200 mmol/L of each primer, 12.5 KL2� PCR master mix, 2.5 KL 10� CoralLoad (Qiagen). Theamplifications were performed with an initial denaturation stepat 95-C for 15 minutes followed by touchdown PCR withannealing temperatures from 60-C to 52-C (each cycle: 95-Cfor 30 seconds, variant temperature from 60-C to 52-C for30 seconds, 72-C for 30 seconds). The touchdown cycleswere increased from 5 to 25 and 72-C for 7 minutes as afinal extension step. The PCR products were checked by

2% agarose gel electrophoresis to verify the presence ofmethylated (100 bp) and unmethylated (96 bp) PCR products.

The MSP/unmethylation-specific PCR was performedusing the same primers, except for the biotinylated reverseprimers (for sequences, see Table, Supplemental Digital Con-tent 4, http://links.lww.com/NEN/A406). This was followedby examination of the methylation status at the targetingregion of BNIP3 (Figure, Supplemental Digital Content 3,http://links.lww.com/NEN/A405) by pyrosequencing on aPyroMark Q96 MD System (Qiagen) with the PyroMarkGold Q96 Reagent kit (Qiagen) according to the manu-facturer’s protocol. Briefly, the biotin-labeled PCR productswere captured by Streptavidin Sepharose HP (GE Healthcare)and denatured to produce single strands with a PyrosequencingVacuum Prep Tool (Qiagen). The sequencing primers wereannealed to single-stranded PCR products in annealing buffer at80-C for 2 minutes. The pyrosequencing assay and data anal-ysis were performed with the Pyro Q-CpG software (Qiagen).

Genome-Wide Methylation AnalysisGenomic DNA was bisulfite converted using the EZ

DNA Methylation Kit (Zymo Research). DNA methylationwas assessed on the HumanMethylation 450K Bead Chip(Illumina; San Diego, CA) according to the manufacturer’srecommendations. Data analysis was performed using theminfi Bioconductor package (http://www.bioconductor.org/packages/release/bioc/html/minfi.html). In brief, IDAT fileswere used as data source. Quality was controlled using den-sity plots and interrogating control probes. Probe locationswith detection values of p 9 0.01 and ‘‘rs’’ probes wereexcluded from the analysis. Signal intensities across the ar-ray were normalized using subset-quantile within array nor-malization, as previously reported (29). Probes with DNAmethylation differences more than 10% were used to markdifferentially methylated regions.

Western BlotWestern blotting was performed by standard semidry

procedure under denaturizing conditions. In brief, frozen cellsamples were homogenized in RIPA buffer then centrifugedat 12,000 rpm, and whole-cell lysate supernatant was sub-jected to protein quantification with the Dc Protein Assayaccording to the manufacturer’s protocol (Bio-Rad). Equalamounts of proteins were resolved on 10% sodium dodecylsulfateYpolyacrylamide gels and then transferred onto nitro-cellulose membranes. After 1 hour blocking with 5% milkpowder solution (Bio-Rad), the nitrocellulose membranes(GE Healthcare) were stained with primary antibody solutioncontaining 2% milk powder at 4-C overnight. The followingprimary antibodies were applied: BNIP3 (no. 38621, 1:100;Abcam), CD133 (W6B3C1) (no. 130-092-395, 1:200; MiltenyiBiotec), and A-actin (no. CS-130657, 1:1000; Santa Cruz

FIGURE 2. Growth curves of in vitro propagated GBMs under normoxia (21% oxygen) and hypoxia (3% oxygen). (A) The MTSassay absorption values of hypoxic cultures (corresponding to a slower increase of viable cells in all tumor lines) were lower than innormoxia cultures. * p e 0.05; ** p e 0.001. (B) All in vitroYpropagated GBMs contained a distinct CD133-positive subpopulationranging from above control value to more than 75%, as revealed by FACS-based analysis. Furthermore, independent of theirintrinsic CD133 content under 21% oxygen, all analyzed cultures showed a marked increase of CD133-positive cells under hypoxia,as shown by percentages of CD133-positive cells (left) and mean fluorescence intensity analysis (right). * p e 0.05.








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Biotechnology). The blots were then incubated with secondaryantibodies (anti-mouse, no. 474-1802; anti-rabbit, no. 474-1506;both from KPL, Gaithersburg, MD) conjugated to horseradishperoxidase for 1 hour at RT and developed with Immune-StarWestern C Kit (Bio-Rad) or Super Signal West Pico Chem-iluminescent Substrate (Thermo Scientific).

Cell Proliferation AnalysisMitochondrial activity for determining biologic active

cell mass was measured using CellTiter 96 AQueous OneSolution Cell Proliferation Assay according to the instruc-tions of the manufacturer (Promega, Mannheim, Germany).The individual values were retrieved through ELISA-based(Tecan, Mannedorf, Switzerland) absorption measurementsat 490 nm. In each proliferation experiment, we plated 10,000cells per 24 wells (except for the HSR-GBM1 ‘‘switchassay,’’ 20,000 per 24 wells).

3-(4,5-diMethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-Tetrazolium Salt MTS Switch Assay

In addition to the previously mentioned ‘‘classical’’MTS assay, the metabolic activity was measured in GBM-derived cultures proliferating under either hypoxic or nor-moxic conditions (Condition 1) during a period of 4 to 6 days,which were subsequently transferred into atmospheric orlowered oxygen concentrations (Condition 2), respectively,for another 4 to 6 days. The MTS measurement after culturecondition change was preceded with a 2-day adaptation periodin the new oxygen concentration.

Statistical AnalysisStatistical analyses were undertaken to compare the

differences of the various cell lines under the different con-ditions using multifactorial analyses of variance (Statisticaversion 5.1; Statsoft, Hamburg, Germany) followed by posthoc comparisons when appropriate using the Newman-Keulstest to identify the source of significant main effects andinteractions (for differences in metabolic activity under the 2oxygen conditions over time). Significance of the increase ofCD133-positive cells under hypoxia compared with normoxiaassessed via FACS was tested by 1-way analysis of variance.For quantitative PCR (q-PCR) results, means based on the CPvalue-raw data for each gene and condition were comparedwith Student t-test results using Statview 5.0 (SAS Insti-tute Inc., San Francisco, CA). The probability value was setat p e 0.05 for all comparisons. For a schematic overview ofthe experimental design, see Figure 1.

RESULTS

Hypoxia Decreases the Proliferation ofTumor-Derived Cell Cultures

The MTS-based analysis of the mitochondrial activityof tumor-derived cells cultured in 3% oxygen as comparedwith their counterparts cultivated under atmospheric oxygentension revealed faster growth of the latter. However, indi-

vidual cultures, derived from different tumors, reacted withdifferent sensitivities to low oxygen exposure. The moststriking effect was in the case of a GB showing an almost4-fold difference in viable cell number between both cultureconditions at Day 8. Furthermore, cultures with very fastcell turnovers under normoxia (i.e. GB and FF) showed agreater decrease in proliferation under hypoxia as comparedwith moderately proliferating lines (NCH421k). Prolongedcultivation of GBM-derived cells in 3% oxygen tensionresulted in progressive diminution of the total cell number.Representative MTS data are shown in Figure 2A.

Hypoxia Increases a CD133-PositiveCell Population

The FACS-based analysis of a putative stem cell pop-ulation based on CD133 expression in tumor-derived cultures

FIGURE 3. (A) Time course of hypoxia-induced changes ofCD133-positive cells. They rapidly increased within 24 hours(48% vs 30% in normoxia), with a further slower increase tomore than 52% after 56 hours in vitro (normoxia, 30%).Thereafter, the percentage of CD133-positive cells slowlydecreased. (B) After hypoxic exposure for 108 hours, the aug-mentation of the CD133-positive cell population was accom-panied by increased mRNA levels for the stem cellYrelated genesCD133, NANAOG, and SOX2. * p e 0.05.




(n = 6) grown under normoxic and hypoxic conditionsshowed an increase of CD133-positive cell population in thelatter condition. In GBM cultures with a low initial expres-sion of CD133-positive cells, their increase was very pro-nounced, including averaged augmentations of 4-fold (forGB) to 8-fold (for AH). Cell lines with an initial high per-centage of CD133-positive cells also showed an increase underhypoxia, although to a lesser extent (e.g. the NCH421k cellline [mean, 62%Y74% of all cells]) (Fig. 2B). Independentof the initial content of CD133-positive cells or its respectivelevel of mean fluorescence intensity, cell lines showed 200%to 300% increase of mean fluorescence intensity under a low-ered oxygen tension (Fig. 2B). This increase was most ap-parent after 3 to 3.5 days of exposure to hypoxia. However,

in our experimental setup, exposure to 3% oxygen for up to12 hours did not result in an increase of CD133-positive cellpopulation. Augmentation of the CD133-positive cell pop-ulation first appeared after 24 hours and reached its maximumafter 48 to 56 hours (Fig. 3A). Interestingly, the percentageof CD133-positive cells seemed to be stable up to 72 hoursin hypoxia and decreased gradually thereafter.

Hypoxia Increased the Expressionof Stem CellYRelated Genes

We next analyzed the expression of the stem cellYrelated genes CD133, NANAOG, and SOX2 in normoxicand hypoxic cultures. Real-time q-PCR data revealed a higherexpression of the examined genes under hypoxia and increased

FIGURE 4. The MTS results of the switch assay. (A) Cells grown under normoxia turned into slowly proliferating cultures whenexposed to lowered oxygen concentrations. (B) They regained their initial growth dynamic after transfer to atmospheric oxygentension. (C) Histograms of FACS-based analysis of CD133 expression in GBM-derived cultures. There was a parallel increase in theCD133-positive fraction after exposure to hypoxia and subsequent reduction on retransfer to 21% oxygen conditions.




protein levels confirmed by Western blot (Fig. 3B). The cellcultures could be divided into 2 groups with respect to theirfold increase of expression of these genes. GBM10 and GBshowed moderate upregulation of the mRNA levels of theexamined genes (2- to 20-fold increase), whereas FF andNCH421k showed a massively increased expression (mean,20- to 100-fold) (Fig 3B).

Hypoxia-Dependent Decrease of Growth Kineticand Augmentation of CD133-Positive CellPopulation Are Reversible

To assess the possible reversibility of the changes con-cerning the growth kinetic and CD133 expression dependingon actual oxygen tension conditions, GBM-derived cultureswere tested in an MTS switch assay. In concordance with pre-viously described cell proliferation experiments, fast dividingnormoxic cultures slowed their proliferation after transfer tohypoxia (Fig. 4A).

Interestingly, initially slow-growing hypoxic culturesturned into high-proliferating cultures when they were trans-ferred into normoxic culture conditions (Fig. 4B). In parallel,the CD133-positive augmentation observed under hypoxiaseemed to disappear (Fig. 4C).

Increased Proapoptotic Signals Under HypoxiaTo explain the slower proliferation of tumor-derived

cultures under hypoxia leading to progressive diminution oftotal cell numbers, we hypothesized that there was an increasedrate of cell death after oxygen deprivation. To test this, real-timeq-PCRYbased expression analysis of the gene encoding forthe hypoxia-induced proapoptotic BH3-only protein BNIP3in normoxic and hypoxic bulk cultures was performed. Thisanalysis showed higher transcript levels of up to 20-foldof BNIP3 (an HIF-1>Yregulated gene) in slowly proliferatinghypoxic tumor-derived cultures (GBM10, KK, NCH421k,FF, and GB) versus their normoxic counterparts; this wasconfirmed with Western blot data for BNIP3 (Fig. 5A).

Interestingly, a higher percentage of CD133-positivecells in hypoxic cultures coincided with the overall activa-tion of hypoxia-induced proapoptotic genes. Therefore, wesuspected that there was improved survival of BTSCsunder hypoxia as a consequence of different susceptibilityto apoptosis-initiating signals in CD133-positive versus CD133-negative cells and/or an induction of autophagy as a survival-promoting alternative metabolic program in CD133-positivecells. To assess possible differences in proapoptotic and anti-apoptotic responses to hypoxia in these cell subpopulations,we investigated the expression of several proapoptotic andantiapoptotic genes in both cell fractions on FACS-basedseparation after hypoxic exposure. All investigated cultures

showed lower proapoptotic gene expressions in the CD133-positive pools (observed downregulation in: NCH421k-BNIP3 28�, BAX 4.8�, NCH644- BNIP3 10.7� and BAX 2�,HSR-GBM1-BNIP3 3.7� and BAX 2.5�), whereas the ex-pression levels of survival-promoting BCL2 and SURVIVINwere much higher in these cell populations versus their CD133-negative counterparts (Fig. 5B, NCH421k-BCL2 8�, NCH644-BCL2 2.3�, and SURVIVIN 6.2�; Fig. 5C, HSR-GBM1-BCL23.2� and SURVIVIN 1.7�). The hypoxia-regulated expressionof BNIP3 was supported by immunohistochemical analysis ofpatient-derived GBM samples. The BNIP3-immunopositivecells were abundant in the vicinity of necrotic foci withinthe tumors and were less frequently found in areas of viabletumor cells (Fig. 5D).

The FACS-based assessment of apoptosis by Annexin-V binding revealed an increased signal in CD133-negativeversus CD133-positive cells (Fig. 5C). Moreover, the com-parison of genome-wide methylation status of bulk cells grownunder hypoxic and normoxic conditions revealed a stable epi-genomic status in most of the observed sites. However, whenanalyzing the relatively rare CpGs differentially methylated,we detected an overall slight tendency toward hypomethyla-tion under hypoxia. Interestingly, a high percentage of thesesites with differential epigenomic status could be attributedto genes or their regulatory units involved in cell cycle con-trol, DNA damage repair mechanisms, or cell death, includ-ing PACS2, FAM172A, ZAK, BCL6, BCL7C, ADK, TERT,and CUL4A. Screen shots from Illumina-array evaluationare shown in Figure, Supplemental Digital Content 5,http://links.lww.com/NEN/A407.

AZA Demethylated the BNIP3 Promoter andIncreased the Expression of BNIP3 in Hypoxia

One of the most common mechanisms regulating theexpression of BNIP3 is the methylation of its promoter abro-gating binding of HIF proteins and induction of gene tran-scription. Thus, the demethylation agent AZA should increasethe BNIP3 mRNA levels. To examine this hypothesis, bulknormoxic tumor-derived cultures were treated with 10 Kmol/LAZA 5 days before exposure to hypoxia and during the expo-sure to 3% oxygen; this was followed by gene expressionanalysis and promoter methylation analysis by pyrosequenc-ing. Indeed, AZA treatment resulted in marked induction ofexpression of BNIP3 and its homolog BNIP3L under hyp-oxia versus both normoxic and nontreated hypoxic cultures(Fig. 6A). Methylation assessment using pyrosequencing ofHSR-GBM1 and GBM10 bulk cultures revealed inductionof hypermethylation in the BNIP3 promoter target area afterpropagation in hypoxia, which was again absent after AZAtreatment, reaching levels similar to those observed in nor-

FIGURE 5. (A) Increased expression of the proapoptotic BNIP3 gene in bulk GBM cultures under hypoxic conditions (n = 5; upperpanel). There was an increased signal for BNIP3 by Western blot in representative lines (lower panel). (B) Quantitative geneexpression analysis of FACS-separated hypoxia-derived CD133-positive and CD133-negative cells showed greater expression ofantiapoptotic survival-promoting genes (BCL2 and SURVIVIN) in CD133-positive cells. Apoptosis-inducing BNIP3 and BAX weredownregulated as compared with the CD133-negative population. (C) Annexin-V staining (indicating ongoing apoptosis) wasgreater in CD133-negative cells than that in CD133-positive cells. (D) Immunohistochemistry of a GBM patient sample materialshowed accumulation of cells immunopositive for BNIP3 predominantly next to necrotic tumor areas as compared with more intacttumor parenchyma. This spatial distribution of BNIP3-positive cells presumably corresponded to an intratumoral oxygen gradient.





moxia (Fig. 6B). By analyzing the methylation status ofBNIP3 promoter in sorted CD133-positive/-negative sub-fractions, we failed to identify its hypermethylation in lessapoptotic BTSCs. However, only 1 BNIP3 promoter region(not covering the entire gene regulatory unit) was tested.

Whole-Genome Demethylation and BNIP3Silencing Abrogate Hypoxia-Induced Increaseof CD133-Positive Cell Subpopulation

After broad genome-wide demethylation via AZA treat-ment, the previously observed increase of CD133-positive cellsunder hypoxia could not be documented any longer. Thepercentage of CD133-positive cells in AZA-treated hypoxiccultures was in fact in some cases even mildly lower than incorresponding normoxic cultures (Fig. 7A). This indicates apotential activation of hypoxia-driven apoptotic genes nor-mally silenced in untreated CD133-positive BTSCs.

Furthermore, after knockdown of BNIP3 expression,the hypoxia-induced increase of the CD133-positive fractionversus normoxia was no longer present (Fig. 7B). Togetherwith the epigenetic modification, this suggests that BNIP3-mediated hypoxic-induced apoptosis is more relevant inCD133-negative cells, where knocking down BNIP3 expres-sion led to better survival of this cell compartment underoxygenic stress conditions.

DISCUSSIONGlioblastoma is the most common malignant glial tumor

characterized by a progressive clinical course with inevitablyfatal prognosis because of its resistance to conventional ther-apeutic modalities, including surgical resection followed by acombination of radiotherapy with different chemotherapeuticregimes (30, 31). In particular, the recently discovered sub-population of BTSCs responsible for initiation and furtherprogression of GBM has been shown to be resistant to standardtherapies (3, 6, 32). These cells are similar to non-neoplasticneural stem cells (9, 11, 33), and, as in embryogenesis (34),BTSCs seem to be influenced by oxygen concentration in thedevelopment of brain tumors (35Y37). Therefore, our currentwork was focused on the response of in vitroYpropagatedGBM-derived cultures to lowered oxygen concentration, in-vestigating especially the response of CD133-positive BTSCsto hypoxia. Despite recent controversies suggesting the tumor-initiating capacity of CD133-negative cells (38Y43), we usedprominin-1 (CD133) for identification of BTSC populationsand investigation of in vitro responses to hypoxia in GBM-derived cultures.

Both established cultures and freshly dissected tumortissue were propagated in a suspension culture system thatenabled long-term propagation of cells exhibiting markers ty-pical for neural stem cells (Figure, Supplemental Digital Con-tent 6, http://links.lww.com/NEN/A408). Similar to previousreports, we consistently observed significant increases of theCD133-positive subpopulation (4, 15, 44Y46) in hypoxia. Geneexpression analysis of cells cultivated in 3% oxygen alsoshowed overexpression of several stem cellYrelated genesas compared with their normoxic counterparts, suggesting

that proliferation of tumor-derived tissue in lowered oxygentension leads to enrichment of cells with stem cellYlike geneexpression profile, as has been shown by others (47). Thisprocess might be directly regulated by HIF-dependent induc-tion of the early developmental program (48). Recent reportsalso showed increased sphere-forming abilities and in vivoengraftment of the hypoxia-derived cultures that were attrib-uted to a stemlike cell population (15). We observed an initialincrease of CD133-positive cells in hypoxia, followed by theirrapid diminution in cultures transferred from lowered toatmospheric oxygen tensions, suggesting dynamic regulationof the CD133-positive cell population in an environment-dependant context. Griguer et al (16) argued that prominin-1functions as a marker of bioenergetic stress. According to thishypothesis, immediate transient and completely reversibleexpression of CD133 is a part of a ‘‘glycolytic response’’leading to significant metabolic changes. However, in ourexperiments, we observed a significant increase of CD133-positive cells first after 24 hours, with the most pronouncedeffect after 48 to 56 hours of culture in lowered oxygen con-centrations. In fact, mild hypoxia in our system versus 1%oxygen in others (15, 16) could be a reason why we observeda later onset of CD133 increase. To explain the slower growthand ultimately almost complete growth arrest of prolongedhypoxic cultures, we hypothesize that oxygen deprivationmight promote hypoxia-induced cell death via the HIF-1>/BNIP3Ydependent pathway (16, 49, 50). Indeed, we foundhigher transcript and protein levels of the proapoptotic BNIP3in bulk tumor cultures propagated in hypoxia and hypothe-size that CD133-positive cells are less prone to activate pro-grams for hypoxia-induced apoptosis. Indeed, CD133-positivecells overexpressed antiapoptotic BCL2 and SURVIVIN,whereas CD133-negative population was characterized bya higher expression of BNIP3; this was also confirmed byprotein analysis (Figure, Supplemental Digital Content 7,http://links.lww.com/NEN/A409). Concordantly, Annexin-Vbinding on hypoxic treatment of the cell cultures couldpredominantly be found in CD133-negative cells, indicatingpreferential induction of apoptosis in this cell population. Inaddition, we detected differential methylation of multiplegenes involved in cell cycle control, DNA repair, and ap-optosis in hypoxia versus normoxia. This unique methyl-ation pattern might be associated with a more aggressivetumor phenotype (51).

Immunohistochemical evaluation of patient-derivedGBM specimens revealed cells with positive BNIP3 sig-nal predominantly at the boundaries of tumor necrosis, pre-sumably corresponding to the lowest intratumoral oxygenconcentrations. This observation supports in vivo hypoxia-regulated expression of BNIP3 and suggests its role in res-ponse to lowered oxygen tensions.

One of the most efficient regulatory mechanisms con-cerning the BNIP3 expression is the epigenetic chromatinmodification leading to gene silencing through the methylationof its promoter (49, 52). In fact, the MSP sequencing analysisof the dedicated BNIP3 promoter region in GBM culturesproved its methylation exclusively under hypoxia starting from80 hours of exposure to lower oxygen tension. However, afterFACS sorting of CD133-positive and -negative populations,






FIGURE 6. (A) The BNIP3 and BNIP3L expression increase in hypoxic cultures of GBM after exposure to the demethylating agentAZA. This suggests that there is methylation-regulated epigenetic control of expression. (B) The MSP-converted PCR product(96 bp) was detected under hypoxic conditions and was absent after AZA treatment. The USP PCR band (100 bp) was detectedin all conditions. Pyrosequencing of biotinylated PCR products proved the lack of methylation under normoxia and hypoxia +AZA, whereas the BNIP3 promoter was methylated exclusively in hypoxia. (C) Hypermethylation was first observed starting from80 hours in hypoxic cultures.




FIGURE 7. (A) The FACS analysis showed abrogation of the CD133-positive cell increase in hypoxia after AZA treatment, wheresimilar BNIP3 expression levels in CD133-positive and CD133-negative populations were detected (B), as well as in response toshRNA-mediated BNIP3 knockdown.




we could not confirm hypermethylation in putative BTSCcompartment in the tested promoter region. Nevertheless,the BNIP3 transcript levels in CD133-positive cells weredecreased several fold versus CD133-negative counterparts.Moreover, whole-genome demethylation led to an increaseof BNIP3 mRNA in CD133-positive cells and abrogationof the hypoxia-induced increase of CD133-positive cells. Fromthese data, we conclude that AZA treatment might inducethe BNIP3 transcription in CD133-positive cells via epige-netically regulated mechanisms that remain to be elucidated.A possible explanation of the AZA effect includes BNIP3promoter demethylation affecting different CpG islands thatwere not tested in the current study. In concordance with themodel of pivotal role of BNIP3 expression in the regulationof CD133-positive population in hypoxia, shRNA-mediatedBNIP3 knockdown led to abrogation of a hypoxia-inducedincrease of the BTSC fraction. The silencing approach pre-dominantly targets this cell population, subsequently leadingto diminished induction of the apoptotic program.

In addition, analysis of hypoxia-derived CD133-positivecells showed an increased expression of BECLIN1 as com-pared with CD133-negative counterparts (Figure, Supplemen-tal Digital Content 8, http://links.lww.com/NEN/A410). Thisindicates a possible role for hypoxia-induced autophagy asa prosurvival selection mechanism. Altogether, we observed apossible alternative mechanism contributing to the increase.of CD133-positive population after hypoxia exposure. Ourin vitro data support the hypothesis that, besides the induc-tion of CD133 expression shown previously, this processmight depend also on positive selection of CD133-positiveBTSCs being less susceptible to hypoxia-induced programmedcell death.

Low susceptibility to hypoxia-induced BNIP3-drivenapoptosis in CD133-positive cells, together with a previouslyreported resistance to standard radiotherapy and chemotherapy,underlines the exceptional characteristics of BTSCs as a cellpopulation responsible not only for tumor initiation but evenmore importantly the recurrence and inevitably fatal course ofGBM. These observations might contribute to the furtherdevelopment of a novel therapeutic approach in which epi-genetic modifications aim at the elimination of BTSCs in GBMs.

ACKNOWLEDGMENTSThe authors thank C.G. Eberhart, Johns Hopkins Med-

ical Institutions, Baltimore, MD, for careful revision of thisarticle. The authors acknowledge A. Vescovi for the generousdonation of several cell lines used in this study.

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