PLZF/ZBTB16, a glucocorticoid response gene in acutelymphoblastic leukemia, interferes with glucocorticoid-inducedapoptosis

Muhammad Wasima,b,1, Michela Carleta, Muhammad Manshaa,b, Richard Greilc, ChristianPlonera, Alexander Trockenbachera, Johannes Rainera,b, and Reinhard Koflera,b,⁎aDivision Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Fritz-Pregl-Straße3, A-6020 Innsbruck, Austria.bTyrolean Cancer Research Institute, 6020 Innsbruck, Austria.cIII. Medical University Hospital, Paracelsus Medical University, 5020 Salzburg, Austria.

AbstractGlucocorticoids (GCs) cause cell cycle arrest and apoptosis in lymphoid cells which is exploited totreat lymphoid malignancies. The mechanisms of these anti-leukemic GC effects are, however,poorly understood. We previously defined a list of GC-regulated genes by expression profiling inchildren with acute lymphoblastic leukemia (ALL) during systemic GC monotherapy and inexperimental systems of GC-induced apoptosis. PLZF/ZBTB16, a transcriptional repressor, was oneof the most promising candidates derived from this screen. To investigate its role in the anti-leukemicGC effects, we performed overexpression and knock-down experiments in CCRF-CEM childhoodALL cells. Transgenic PLZF/ZBTB16 alone had no detectable effect on cell proliferation or survival,but reduced sensitivity to GC-induced apoptosis but not apoptosis induced by antibodies against Fas/CD95 or 3 different chemotherapeutics. Knock-down of ZBTB16 entailed a small, but significant,increase in cell death induction by GC. Affymetrix Exon array-based whole genome expressionprofiling revealed that PLZF/ZBTB16 induction did not significantly alter the expression profile,however, it interfered with the regulation of numerous GC response genes, including BCL2L11/Bim,which has previously been shown to be responsible for cell death induction in CCRF-CEM cells.Thus, the protective effect of PLZF/ZBTB16 can be attributed to interference with transcriptionalregulation by GC.

KeywordsAcute lymphoblastic leukemia; CCRF-CEM cell line; Exon array-based expression profiling;Functional gene analysis; Glucocorticoid-induced apoptosis; PLZF/ZBTB16

© 2010 Elsevier Ltd.This document may be redistributed and reused, subject to certain conditions.⁎Corresponding author at: Division of Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Fritz-Pregl-Straße 3,A-6020 Innsbruck, Austria. Tel.: +43 512 9003 70360; fax: +43 512 9003 73960. Reinhard.Kofler@i-med.ac.at.1Present address: Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan.This document was posted here by permission of the publisher. At the time of deposit, it included all changes made during peer review,copyediting, and publishing. The U.S. National Library of Medicine is responsible for all links within the document and for incorporatingany publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to be such by Elsevier,is available for free, on ScienceDirect.

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1 IntroductionGlucocorticoids (GCs) induce apoptosis in certain cells of the lymphoid lineage and aretherefore useful therapies in the treatment of lymphoid malignancies, most notably childhoodacute lymphoblastic leukemia (ALL) [1]. GCs mediate their effects via the GC receptor (GR),a ligand-activated transcription factor of the nuclear receptor super-family that resides in thecytoplasm and, upon ligand binding, translocates into the nucleus, where it modulates geneexpression via binding to specific DNA response elements or by protein–protein interactionswith other transcription factors [2–5]. A large number of genes have been identified that areregulated by GCs in lymphoid lineage cells in experimental systems [6] and related clinicalsamples [7,8], but the genes responsible for cell death induction and other effects of GCs onthe immune system are not well understood (for recent reviews see [6,9–12]).

One of the most interesting GC-regulated candidate genes identified in the above screen wasZBTB16 (zinc finger and BTB domain containing gene 16), also referred to as promyelocyticleukemia zinc finger (PLZF, reviewed in [13,14]). It was induced by GC in the majority ofchildren with precursor B-cell and T-cell ALL, in one patient with adult ALL and in peripheralblood lymphocytes from 2 healthy donors treated with GC, but not in 2 ALL cell lines [7].More recently, we re-analyzed these data using a normalization procedure that better resolvesregulations of low abundance genes (GC-RMA) [15] and also performed additional expressionprofiling studies of 14 more ALL children (J. Rainer, in preparation) and found that PLZF/ZBTB16 was induced by GC more than 2-fold in 23 of 27 such cases.

PLZF/ZBTB16 was initially identified as a fusion partner of retinoic acid receptor α (RARα)in a variant chromosomal translocation t(11;17)(q23;q21) that occurred in a subset of acutepromyelocytic leukemia patients [16,17]. PLZF/ZBTB16 is a transcriptional repressor of thePOK (POZ and Krüppel) family of proteins. It contains one BTB (Broad complex, Tramtrack,and Bric à brac)/POZ (poxviruses and zinc finger and Krüppel) domain at the NH2-terminalmoiety and 9C2H2 Krüppel-type zinc fingers at the carboxy-terminal end of the protein. ThePOZ/BTB domain mediates interactions with proteins such as transcriptional co-repressorsentailing chromatin remodeling and transcriptional silencing. The Krüppel-type zinc fingersconfer specificity of the repressor activity to particular promoters by interacting withcorresponding response elements in regulatory regions of genes repressed by PLZF/ZBTB16[18–21]. The hinge region of the protein contains a PEST domain with two consensus sites forCDK2-mediated phosphorylation that triggers ubiquitination and subsequent degradation ofPLZF through the ubiquitin-proteasome pathway [22]. The human PLZF/ZBTB16 gene mapsto chromosome 11q22–q23 with seven exons [23] distributed over a region of approximately200 kb. Although additional alternative transcripts encoding distinct proteins have beenreported [24], most recent NCBI and Ensembl databases contain 2 and 3 transcripts,respectively, that differ only in their 5′ untranslated region and thus encode the same protein.

Regarding its function, a natural mutation (luxoid) in, and knock-out of, the mouse homologueZfp145/ZBTB16 unraveled a crucial role in limb and skeletal patterning and spermatogonialstem cell maintenance [25–27]. PLZF/ZBTB16 has further been implicated in tumorsuppression in melanoma [28] and prostate cancer [29], ascribed to its ability to cause cell cyclearrest and induce apoptosis in certain cell systems. The complex effects of PLZF/ZBTB16 havebeen associated with transcriptional repression of numerous genes such as members of the Hoxfamily of transcription factors [25], kit [18], CRABPI [30], c-myc [31], CCNA2/Cyclin A,CDKN1B/p27/Kip1 [32] and possibly others [26,28,29,33,34]. Thus, its suggested role in cellcycle arrest and apoptosis induction in some systems [28,31,34,35], further supported apossible role of PLZF/ZBTB16 induction in the anti-leukemic GC response.

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In this study, we extended our analysis of GC regulation of PLZF/ZBTB16 to several leukemiccell lines and performed functional analyses on the role of PLZF/ZBTB16 in the anti-leukemiceffects of GC. We found that transgenic PLZF/ZBTB16 alone had no effect on cell proliferationand survival, but reduced sensitivity to apoptosis induced by the GC analogue dexamethasonein the CCRF-CEM model for T-ALL. Similarly, knock-down of PLZF/ZBTB16 in CCRF-CEM cells entailed a small, but significant, increase in cell death induction by dexamethasone.We further exploited whole genome expression profiling to derive a molecular explanation forthe protective effect of PLZF/ZBTB16 on GC-induced apoptosis.

2 Materials and methods2.1 Cell lines and tissue culture

The T-ALL cell lines CCRF-CEM-C7H2 [36], CEM-C7H2-2C8 [37], a CEM-C7H2 derivativewith constitutive expression of the tetracycline-regulated reverse transactivator, rtTA [38],MOLT4 (CRL-1582, ATCC, Rockville, MD), and Jurkat (untransfected and a rat GR-transfected derivative [39]), the precursor B-cell lines 697/EU-3 (ACC 42, DSMZ,Braunschweig, Germany), NALM6 (ACC 128, DSMZ), RS4;11 (ACC 508, DSMZ) and AT-1[40] (kindly provided by R. Panzer Grümeier, Vienna), and the Burkitt's lymphoma Daudi(CCL-213, ATCC) were cultured in RPMI 1640 supplemented with 10% fetal calf serum and2 mM L-glutamine at 37 °C, 5% carbon-dioxide and saturated humidity. HEK293T packagingcells (ATCC, Manassas, VA) were cultured in DMEM supplemented as above. The cells werefree of mycoplasma infection and their authenticity was verified by DNA fingerprinting, asdetailed previously [41]. Taxol was dissolved in DMSO (10 mg/ml), doxycycline (100 μg/ml)in phosphate buffered saline, doxorubicin (10 mg/ml) and mitomycin C (500 μg/ml) in destilledwater, and β-dexamethasone (10−4 M) in 100% ethanol. The final ethanol-concentration in thedexamethasone-treated and control cultures was maintained at 0.1%. All above reagents werefrom Sigma (Vienna, Austria).

2.2 Apoptosis determinations and cell cycle analysesApoptosis was determined by FACS analysis of propidum iodide (PI)-treated permeabilizedcells [42], as previously detailed [43]. Even though this method may over-estimate the rate ofapoptotic cells as compared to other methods, it allows simultaneous analysis of cell deathinduction and cell cycle progression in the same samples. Moreover, since this study addresseswhether PLFZ/ZBTB16 reduces or increases cell death, the absolute extent of apoptosis doesnot affect its conclusions. Cells were analyzed with a FACScan cytometer (Becton DickinsonBiosciences, San Jose, CA) in combination with CellQuest Pro software (Becton DickinsonBiosciences) acquiring forward scatter/sideward scatter, FL-2 (log), and FL-3 (linear). In FL-2,the percentage of nuclei with reduced DNA-content (subG1 peak) was assessed. For cell cycledetermination, the FACS data from PI-treated nuclei were analyzed using ModFit LT 2.0(Verity, Topsham, ME) with the following quality parameters: minimal number of eventsanalyzed: 10,000 and maximal %CV: 8. For the knock-down experiments, the percentage ofviable cells in culture was determined by staining cell suspensions with 1 μg/ml 7-amino-actinomycin D (7-AAD; Sigma–Aldrich) plus APC-coupled Annexin-V (Becton Dickinson)and analyzing the samples in a FACScan (Becton Dickinson). Cells staining positive forAnnexin-V and/or 7-AAD were defined as undergoing cell death.

2.3 ImmunoblottingOur immunoblotting procedure has been described in detail recently [44]. Briefly, proteinswere extracted from 5 × 106 cells in 100 μl RIPA-buffer, quantified by Bradford analysis,mixed with 40 μl loading buffer (4× SSB, 5% β-mercaptoethanol), denatured, fractionated ona 12.5% SDS-PAGE and electroblotted onto nitrocellulose. The membranes were incubatedovernight with rabbit polyclonal antibodies against PLZF/ZBTB16 (HPA001499, Prestige

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antibodies, Sigma, Vienna, Austria) or BCL2L11/Bim (#559685, Becton DickinsonBiosciences, San Jose, CA), or mouse monoclonal antibodies against α-tubulin (DM1A,CalBiochem, Nottingham, UK) as a loading control. Specifically bound antibodies weredetected with anti-rabbit or anti-mouse horseradish-peroxidase-conjugated secondaryantibodies (Amersham Pharmacia Biotech, Uppsala, Sweden) and visualised bychemiluminescence (ECL, Amersham) and subsequent exposure to AGFA Curix X-ray filmsfor 1 s to 30 min.

2.4 Generation of CCRF-CEM derivatives with PLZF/ZBTB16 overexpression and RNAi-mediated knock-down

2.4.1 Constructs for gene overexpression and knock-down—The lentiviralconditional expression construct pHR-tetCMV-ZBTB16 (U317) was generated using theGATEWAY™ technology (Invitrogen, Carlsbad, CA). The details of this procedure and thegeneration of stable clonal cell lines with tetracycline-regulated expression of cDNAs clonedinto such constructs has been described previously [45]. In brief, human PLZF/ZBTB16 mRNAwas PCR-amplified from IRAT p970B0341D6 (RZPD, Germany) using forward primer 5′-CAAAAAAGCAGGCTCCGCCACCATGGATCTGACAAAA-3′ and reverse primer 5′-CAAGAAAGCTGGGTCTTACACATAGCACAGGTAGAGG-3′, and (after a second PCRreaction to complete the attB sites) recombined into pDONR207 (Invitrogen) resulting inpENTR207-ZBTB16 (U316). The construct was sequence-verified and subsequentlyrecombined into the “destination vector” pHR-tetCMV-Dest-IRES-GFP (U192) to generatepHR-tetCMV-ZBTB16-ires-GFP (U317). The lentiviral shRNA-expressing constructs pHR-THT-ZBTB16-shRNA1-SFFV-GFP (U547), pHR-THT-ZBTB16-shRNA2-SFFV-GFP(U548) and pHR-THT-cocntrol-shRNA-SFFV-GFP (U288) were generated as describedpreviously [45]. In brief, complementary shRNA oligonucleotides directed against PLZF/ZBTB16 and a non-target nucleotide sequence (control shRNA) containing BamHI and HindIIIsticky ends were synthesized (MWG Ebersberg, Germany) sequences:

shRNA1 (PLZF-Exon7/U547): 5′-GATCCCCGCCACAAGCCCGAGGAGATTTCAAGAGAATCTCCTCGGGCTTGTGGCTTTTTGGAAA-3′;

shRNA2 (PLZF-Exon6; U548): 5′-GATCCCCCATCCACACGGGTGAGAAATTCAAGAGATTTCTCACCCGTGTGGATGTTTTTGGAAA-3′;

control shRNA (U288): 5′-GATCCCCGTATCATCTCTTGAATGATTTCAAGAGAATCATTCAAGAGATGATACTTTTTGGAAA-3′).

The annealed oligonucleotides were cloned into the BglII–HindIII sites of pENTR-THT(U191) and the THT-shRNA cassette was recombined into the lentiviral RNAi “destination”vector pHR-Dest-SFFV-eGFP (U218) to generate pHR-THT-shRNA-1-SFFV-GFP (U547),pHR-THT-shRNA-2-SFFV-GFP (U548) and pHR-THT-shRNA-control-SFFV-GFP (U288).

2.4.2 Generation of transduced CCRF-CEM derivatives—The above describedlentiviral constructs for gene overexpression or knock-down were transfected into 293Tpackaging cells along with the packaging plasmids pSPAX and pVSV-G (kindly provided byDidier Trono) and the lentivirus-containing supernatants used to transduce CCRF-CEMderivatives. For conditional PLZF/ZBTB16 overexpression, CEM-C7H2-2C8 (whichconstitutively express rtTA [37]) were transduced and two clonal cell lines, termed CEM-C7H2-2C8-ZBTB16#19 (ZBTB16#19) and CEM-C7H2-2C8-ZBTB16#58 (ZBTB16#58),derived by limiting dilution cloning. For knock-down experiments, the lentivirus-containingsupernatants were concentrated using poly-ethylene glycol [46] and used to transduce CEM-

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C7H2 and ZBTB16#58 cells. Over 85% GFP-expressing bulk populations (if required, afterenrichment by FACS-sorting) were used for further experiments.

2.5 Real-time RT-PCRFor PLZF/ZBTB16 mRNA quantification by real-time RT-PCR, 50 μl of diluted cDNA (2 ng/μl) were added to 50 μl of TaqMan Universal MasterMix (Applied Biosystems, Foster City,CA) and introduced into microfluidic cards containing real-time RT-PCR mixes for humanPLZF/ZBTB16 (Hs00232313_m1, Applied Biosystems) and TBP (Hs00427620_m1) asnormalization control according to the manufacturer's guidelines: After equilibration at RT,the channels were filled with 100 μl of reaction mix and centrifuged two times for 1 min at1000 rpm. Thereafter, the cards were sealed, loaded into the HT7900 real-time machine(Applied Biosystems), and run with a 2-step PCR thermo-protocol that included an initial 94.5 °C step for 10 min followed by 40 cycles of 97 °C for 15 s alternating with 60 °C for 1 min.Fluorescence signal intensities were read during the 60 °C temperature step. Primary real-timePCR data analysis was performed with SDS software version 2.2.1, and further analysis wasperformed in R (version 2.8). Data from 3 technically replicated measurements were averagedand normalized to the internal TBP control (Hs.00427620, Applied Biosystems). Log 2-foldchange values (M values) were calculated for 3 biological replicates by comparing normalizedreal-time RT-PCR data from GC-treated samples against data from the corresponding controlsamples. M values were averaged for the 3 biological replicates and p-values were calculated(Student's t-test) to test against the null hypothesis of no differential expression (mean M = 0).In some instances (knock-down experiments, determination of Bim mRNA), individual real-time RT-PCRs were performed using the same kit as above for PLZF/ZBTB16 andHs00197982_m1 for BCL2L11/Bim (Applied Biosystems). For individual assays, 20 ngtranscribed cDNA was amplified in TaqMan Universal MasterMix (Applied Biosystems) in96-well plates (94.5 °C for 5 min, 40 cycles of 94 °C for 15 s alternating with 60 °C for 1 min)and fluorescence signal intensities were read during the 60 °C temperature step (iQ5 MulticolorReal-Time PCR Detection System; Bio-Rad Laboratories Ges.m.b.H., Vienna, Austria).

2.6 Affymetrix Exon microarray-based data analysisTo determine PLZF/ZBTB16 response genes, C7H2-2C8-ZBTB16#19 and #58 cells werecultured in duplicate in the absence (set A) or presence (set B) of 400 ng/ml doxycycline for2, 6 or 24 h. Total RNA was prepared and 1.5 μg RNA subjected to expression profiling onExon 1.0 microarrays (total of 24 arrays) as detailed previously [47]. To assess the effect ofPLZF/ZBTB16 on the GC response, the above cell lines were cultured for 24 h in the absence(set C) or presence (set D) of 200 ng/ml doxycycline and subsequently exposed to 10−8 Mdexamethasone for 6 and 24 h and expression-profiled as above, resulting in a total of 15 arrays(one of the four 6 h replicates in set C was removed because it failed quality control).

The bioinformatic analysis of the microarrays was based on a custom CDF (CEL definitionfile) that allowed generating expression intensities of transcripts by summarizing the intensitysignals of the individual oligonucleotide probes on the microarray targeting a particulartranscript. In brief, the sequences of the >5 million 25 nt-long oligonucleotide probes on theAffymetrix Exon array (HuEx-1.0-st v2) were aligned to the genomic DNA sequence fromEnsembl (version 52_36n). For all probes with a single complete alignment and no additionalalignments with up to 1 mismatch, we determined whether the alignment was within the exonboundaries of a gene. Probes with sequences with a G/C content >18 were excluded from theanalysis. Exon boundaries and transcript definitions were taken from the Ensembl coredatabase for all known protein- and non-coding transcripts and from the ASTD database(version 1.1) [48] for all potential isoforms of a gene.

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Based on the above annotation, a CDF file was compiled defining the probes targetingindividual transcripts. Probes with no alignment (allowing 1 mismatch) to the human genomewhere defined as “background probes”. The resulting custom CDF contained 1,292,740 probesorganized in 131,642 transcript probe sets corresponding to 28,532 Ensembl genes (49,701and 88,789 transcript probe sets were based on annotations from the Ensembl core databaseand the ASTD database, respectively). 72,150 probes were defined as “background probes”.Pre-processing and all further data analyses were performed in R (version 2.8.0) using packagesfrom Bioconductor (version 2.3) [49] and the custom CDF file described above. The raw signalintensities where preprocessed using the GC-RMA algorithm [15], where the “backgroundprobes” were used to predict the non-specific binding potential of the oligonucleotide probes.After pre-processing, the transcript with the highest variance across all samples was selectedfor further analysis as representative for a given gene. The moderated t-test [50] was then usedto assess the significance for differential expression between the compared conditions. Theresulting raw p-values were adjusted for multiple hypothesis testing using Benjamini andHochberg's method for a strong control of the false discovery rate [51].

3 Results3.1 PLZF/ZBTB16 expression and GC regulation in different leukemia model systems

We have previously shown that PLZF/ZBTB16 is regulated by GC in numerous patients withALL, but not in 2 ALL cell lines [7]. To determine whether PLZF/ZBTB16 regulation isrestricted to the in vivo situation or occurs in vitro as well and, if so, whether it relates to GCsensitivity, we determined PLZF/ZBTB16 regulation by GC in various in vitro leukemiamodels using real-time RT-PCR for PLZF/ZBTB16 and 9 leukemia cell lines treated with10−7 M dexamethasone for 2, 6, 12 and 24 h in biological triplicates. As shown in Fig. 1, theextent of GC sensitivity in these cell lines was markedly different, i.e., untransfected Jurkatand MOLT4 T-ALL cell lines as well as AT-1 precursor B-ALL and Daudi Burkitt lymphomacells were resistant to GC-induced apoptosis, while all others were GC-sensitive, although withvarying kinetics. GR-transfected Jurkat cells died after 24 h exposure to 10−7 Mdexamethasone, the response in CEM-C7H2 was delayed about 24 h, NALM6 and RS4;11showed a somewhat slower death response than CEM-C7H2, while 697/EU-3 cells required96 h to reach ∼40% apoptosis. Effects of GC on cell cycle progression were also determined(Fig. 1) and showed that GC-induced apoptosis was frequently preceded or accompanied byan increase of cells in the G1 phase of the cell division cycle, whereas the cell lines resistantto GC-induced apoptosis were also resistant to the GC effects on the cell cycle. The notableexception was AT-1, in which G1 cell cycle arrest was observed in the complete absence ofapoptosis.

Basal PLZF/ZBTB16 expression levels among these cell lines differed by close to 3 orders ofmagnitude (Fig. 2a), reflecting the situation in children with ALL [7]. Expression ranged fromundetectable (697, not shown in Fig. 2) to levels similar to those of the “housekeeping gene”TBP (MOLT4). PLZF/ZBTB16 expression showed no correlation with GC sensitivity or T-or precursor B-cell origin of the cell lines. Significant, as defined by an M value >1 (M valuescorrespond to log 2 of fold induction) and a p-value of <0.05, and consistent (i.e., at all timepoints) GC-induction of PLZF/ZBTB16 was seen in NALM6, RS4;11, AT-1 and GR-transfected Jurkats (Fig. 2b). The latter finding showed that PLZF/ZBTB16 induction iscritically dependent on sufficient amounts of functional GR in the investigated cell line (Jurkatcells lacking transgenic GR express low levels of wild type GR but fail to auto-induce the GR[52]. Taken together, PLZF/ZBTB16 regulation by GC is not restricted to the in vivo situation,requires sufficient levels of functional GR and neither its regulation nor its expression appearto be clearly correlated with GC sensitivity.

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3.2 PLZF/ZBTB16 overexpression reduces, and its knock-down increases, GC-inducedapoptosis

To assess a possible contribution of PLZF/ZBTB16 to the anti-leukemic GC effects in ALL,we generated 2 stably-transduced derivatives of the CCRF-CEM T-ALL cell line withconditional expression of PLZF/ZBTB16, termed CEM-C7H2-2C8-ZBTB16#19 and #58(ZBTB16#19 and #58), respectively. As revealed by quantitative RT-PCR andimmunoblotting, PLZF/ZBTB16 mRNA and protein expression in these cell lines could becontrolled by the addition of the tetracycline analogue doxycycline to the culture medium (Fig.3). As little as 3.1 ng/ml doxycycline led to a detectable induction of PLZF/ZBTB16 mRNAthat increased up to 50 ng/ml and reached a plateau thereafter. Interestingly, close to maximalinduction was seen after only 2 h (Fig. 3a, the decrease in expression levels after 6 and 24 h at3.1 and 12.5 ng/ml doxycycline is probably explained by the half-life of doxycycline in theculture). Similar observations were made at the protein level (Fig. 3b), although the detectionlimit was somewhat higher (starting from 12.5 ng/ml doxycycline).

The above clonal cell lines were then used to determine whether PLZF/ZBTB16 expressionaffects cell survival or cell cycle progression and/or modulates the anti-leukemic effects ofGC. As shown in Fig. 4, transgenic expression of PLZF/ZBTB16 alone, even at levels thatclearly exceeded those induced by GC, had no detectable effect on survival or cell cycleprogression. However, it significantly reduced the extent of apoptosis induced by 10−7 M and,even more so, 10−8 M dexamethasone in this T-ALL in vitro model (Fig. 4a). GC-induced cellcycle arrest was also reduced, but this effect was less pronounced (Fig. 4b).

Since PLZF/ZBTB16 overexpression provided a protective effect, we investigated whetherknock-down of PLZF/ZBTB16 might increase GC-induced cell death. To this end, wegenerated 2 lentiviral shRNA constructs targeting either exon 7 (shRNA-1) or 6 (shRNA-2) ofPLZF/ZBTB16 and tested them in comparison with a negative control shRNA in C7H2-2C8-ZBTB16 cells. A clear, although incomplete, PLZF/ZBTB16 knock-down was observed bothon the mRNA and protein levels (Fig. 5a). Next we infected GC-sensitive CEM-C7H2 cells(the parent of the C7H2-2C8-ZBTB16 cell lines) with recombinant lentiviruses containingthese constructs. Since the PLZF/ZBTB16 levels in CEM-C7H2 cells are below the detectionlimit of the immunoblotting technique even after GC treatment, we could not verify the knock-down by Western technology. On the mRNA level, however, we observed a partial knock-down in this system as well (5 experiments resulting in a mean reduction of 1.6- and 2.1-foldfor shRNA-1 and -2, respectively). Corresponding to the extent of knock-down observed withthe 2 constructs, a small, but statistically significant increase in cell death induction wasobserved (Fig. 5b) further supporting the protective role of PLZF/ZBTB16 seen in theconditional overexpression experiments (Fig. 4a).

3.3 PLZF/ZBTB16 protects from GC-induced but not from several other forms of apoptosisNext we explored whether the protective effect of PLZF/ZBTB16 was specific for GC-inducedapoptosis or reflected a more general protective effect against cell death induction. To this end,we cultured ZBTB16#19 and #58 cells in the presence or absence of doxycycline (to inducetransgenic PLZF/ZBTB16 expression) and determined the extent of apoptosis elicited byantibodies against CD95/FAS (representative of an apoptosis inducer via the extrinsic pathway)or the chemotherapeutics taxol, doxorubicin, or mitomycin C (representatives of apoptosisinducers via the intrinsic pathway). As depicted in Fig. 6, there was no significant protectiveeffect against apoptosis induction by these 4 substances. Thus, within the limits of theinvestigated substances, the protective effect of PLZF/ZBTB16 appeared to be specific for GC-induced apoptosis.

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3.4 PLZF/ZBTB16 alone does not significantly change gene expression in CCRF-CEM cellsbut interferes with the GC-mediated transcriptional response

To test whether the transcriptional repressor PLZF/ZBTB16 protects cells from GC-inducedapoptosis by directly regulating expression of genes involved in cell death/survival decisions,we assessed the transcriptional response to transgenic PLZF/ZBTB16 in the above ALL invitro model by whole genome expression profiling. We cultured 2 biological replicates ofZBTB16#19 and #58 (expressing PLZF/ZBTB16 in a tetracycline-dependent manner) in theabsence (“set A”) or presence (“set B”) of 400 ng/ml doxycycline for 2, 6 and 24 h and subjectedthe corresponding RNAs to expression profiling on human Exon 1.0 microarrays (Affymetrix,Inc., total of 24 arrays) as indicated in Section 2. Unexpectedly, however, apart from PLZF/ZBTB16 itself, which served as an internal control, no significant regulations, as defined by amean M value better than ±0.7 and a Benjamini–Hochberg [53] adjusted p-value (pBH) of<0.05, were observed after 2 and 6 h of doxycycline exposure. After 24 h, a single gene reachedthe above level of significance, i.e., HBEGF (heparin-binding EGF-like growth factor) wasrepressed with a mean M value of −2.3 and a pBH of 0.0299. These data strongly argued againstthe hypothesis that the inhibitory effect of PLZF/ZBTB16 on GC-induced apoptosis was causedby direct transcriptional regulation of death/survival genes by PLZF/ZBTB16 (the completedata can be viewed at the GEO web page, series GSE15820).

Since PLZF/ZBTB16 has previously been shown to interfere with GC-induction of a transientlytransfected, GC-responsive reporter construct [54], we investigated whether PLZF/ZBTB16interfered with the transcriptional response to GCs. We determined Exon array-basedexpression profiles from ZBTB#19 and #58 cells treated in duplicate with the GC analoguedexamethasone together with, or in the absence of, doxycycline. Together with the expressionprofiles generated further above, we generated 4 sets of experimental conditions that allowedus to study the effect of PLZF/ZBTB16 on the transcriptional response to GC. As explainedabove, sets “A” and “B” corresponded to data derived from ZBTB16#19 and #58 cells culturedin duplicates in the absence (set A) or presence (set B) of doxycycline for 6 or 24 h. Sets “C”and “D” derived from ZBTB16#19 and #58 cells that were first cultured for 24 h in the absence(set C) or presence (set D) of doxycycline to induce PLZF/ZBTB16 and subsequently exposedto 10−8 M Dex for 6 and 24 h. Comparison between sets A (no doxycycline, no dexamethasone)and C (no doxycycline, but plus dexamethasone) was used to identify GC-regulated genes(defined by mean M values better than ±0.7 and pBH < 0.05) in this system in the absence oftransgenic PLZF/ZBTB16. After 6 h of GC exposure, 308 down- and 378 up-regulatedtranscripts fulfilled this criterion. After 24 h, 929 genes were down- and 717 up-regulated. Theregulation of these genes was then studied after induction of PLZF/ZBTB16 by comparing setB (plus doxycycline, but no dexamethasone) with set D (plus doxycycline and plusdexamethasone). As shown in Fig. 7, the above GC response genes were significantly lessregulated in the presence of doxycycline than in its absence, and this was true both for inducedand repressed genes. The inhibitory effect included genes such as BCL2L11/Bim, the inductionof which was previously shown to play a crucial role in GC-induced apoptosis in this modelsystem [45]. For this functionally relevant gene, the inhibitory effect of ZBTB16overexpression on GC-induction was verified on the mRNA level by quantitative real-timeRT-PCR (Fig. 8a) and on the protein level by immunoblotting (Fig. 8b). In conclusion, the datasupported the hypothesis that PLZF/ZBTB16 reduces GC-induced apoptosis by interferingwith the hormone's potential to regulate gene expression.

4 DiscussionThe transcriptional repressor PLZF/ZBTB16 was one of the most promising GC-regulatedcandidate genes for the anti-leukemic effects of GC: It was induced by GC during the in vivoresponse to systemic GC monotherapy in childhood ALL and, as shown in this study, in severalleukemia in vitro cell lines. However, the functional analyses performed in this study revealed

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that, at least in the CCRF-CEM T-ALL model, PLZF/ZBTB16 exhibited a protective effectrather than copying, or contributing to, the anti-leukemic GC effects. Attempts to understandthe molecular mechanism of this protective effect revealed that this bona fide transcriptionalrepressor failed to significantly affect gene expression in the investigated T-ALL model. Thisfinding was unexpected because PLZF/ZBTB16 has been reported to cause changes in geneexpression in several systems [26,28,29,33,34]. Even a specific search for regulation of someof the most prominent PLZF/ZBTB16 response genes, such as KIT, HOXB7, MYC, CCNA1/cyclin A, PBX1, CRABPI, BID or RUNX2, failed to provide evidence for transcriptionalactivity. This included a comparative analysis between the parental CEM-C7H2 cell line(assayed in biological triplicates on Exon arrays) [47] and ZBTB16#19 and #58 to assesspossible effects of the small levels of PLZF/ZBTB16 expressed as a consequence of someleakiness of the tetracycline-regulated expression constructs. Several explanations can beoffered for this phenomenon: In most instances [26,28,29,33], non-lymphoid tissues wereanalyzed and PLZF/ZBTB16 might require additional co-factors or post-transcriptionalmodifications, such as acetylation [55], that may not be present in lymphoid cells. The soleexpression analysis in lymphoid cells (Jurkat T-ALL cells [34]) indicated that PLZF/ZBTB16altered gene expression only at low cell density and after 72 h of PLZF/ZBTB16 induction.After 24 h and at “normal” cell densities, the findings in Jurkat cells were compatible withours.

Although apparently inactive as a transcription factor when tested alone, PLZF/ZBTB16significantly interfered with the transcriptional response to GC (Fig. 7). Although thetransgenic PLZF/ZBTB16 levels were considerably higher than the endogenous levels inCCRF-CEM cells, similar levels were observed in lymphoblasts from some children withprecursor B-ALL during systemic GC monotherapy [7] (and our unpublished data). Since bothgene inductions and repressions were attenuated in the presence of PLZF/ZBTB16, and sinceregulations of many transcription units were affected, a general mechanism of action seemslikely, such as sequestration of the activated GR by PLZF/ZBTB16. A more specificmechanism, such as binding of PLZF/ZBTB16 to the GR on the promoter and recruitment oftranscriptional repressors, would be expected to interfere with transcriptional induction but notrepressions and, hence, is less likely given that both repression and inductions were attenuated.A previous report suggested that PLZF/ZBTB16 might bind to, and inhibit DNA binding of,the GR [54]. Mutational analyses revealed that this property maps to the C-terminal portion ofPLZF/ZBTB16, which showed particularly strong GR binding ability. Current analyses in ourlab suggest that in at least in one of our cell line systems, GC might specifically induce exons3–7 (presumably via alternative promoter usage) that encode the above-mentioned particularlyactive portion of PLZF/ZBTB16.

A question of considerable interest is whether PLZF/ZBTB16 is a direct target of the GCreceptor (GR). Previous studies using PLZF/ZBTB16 promoter-driven reporter assays inprimary human endometrial stromal and myometrial smooth muscle cells, where PLZF/ZBTB16 is regulated by progesterone and GC, suggested that it is not [56]. We have performedAffymetrix promoter tiling array-based Chip-on-CHIP analyses using CCRF-CEM andNALM6 cells and failed to find evidence of direct binding of the GR to the PLZF/ZBTB16promoter (Rainer et al., in preparation). However, both negative evidences are limited by thesequences analyzed, i.e., the reporter study covered an ∼5 kb region upstream and the promotertiling array 7.5 kb upstream and 2.5 kb downstream of the putative start site.

In conclusion, we report that the transcriptional repressor PLZF/ZBTB16, one of the mostpromising GC-regulated candidate genes for the anti-leukemic effects of GC, did not contributeto, but rather protected CCRF-CEM leukemia cells from, the anti-leukemic GC effects.Although counterintuitive on first sight, induction of pro-survival signaling by GC in the courseof an apoptotic response is not unheard of. Thus, we observed repression of the pro-apoptotic

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BH3-only molecule PMAIP1/Noxa in children and experimental systems of GC-inducedapoptosis [45] and could show that this regulatory event is functionally relevant, since itinterferes with the kinetics of, and sensitivity to, GC-induced apoptosis [57]. Since GC areknown to mediate pro-survival effects in certain systems, such as erythroblasts [58], neutrophils[59,60] or solid tumors [61], the GC response may inherently be complex, containing pro- andanti-apoptotic components. The cellular context may then determine which of the 2 opposingresponses prevails. The growing understanding of this complexity might eventually allowspecific interference with the pro-survival component of the response, thereby increasing theefficacy of GC in the therapy of lymphoid malignancies.

AcknowledgmentsThe authors would like to thank Dr. S. Geley for discussions, M. Brunner, K. Götsch, B. Gschirr, A. Kofler, S.Lobenwein, and C. Mantinger for technical assistance, and M. Kat Occhipinti-Bender for editing the manuscript.Supported by the Austrian Science Fund (SFB-F021, P18747), the Higher Education Commission of Pakistan, andONCOTYROL, a COMET Center funded by the Austrian Research Promotion Agency (FFG), the TirolerZukunftsstiftung and the Styrian Business Promotion Agency (SFG). The Tyrolean Cancer Research Institute issupported by the “Tiroler Landeskrankenanstalten Ges.m.b.H. (TILAK)”, the “Tyrolean Cancer Aid Society”, variousbusinesses, financial institutions and the People of Tyrol.

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Fig. 1.GC-induced apoptosis and cell cycle arrest in various leukemia cell lines. CCRF-CEM-C7H2(C7H2), CEM-C7H2-2C8 (2C8, a C7H2 derivative stably transfected with the reversetetracycline-responsive transactivator rtTA), Jurkat (untransfected and transfected with ratGR), MOLT4, NALM6, 697 preB-cell recently renamed EU-3 (697/EU-3), RS4;11, AT-1 andDaudi cells were cultured in the presence of 10−7M dexamethasone (Dex “ + ”) or 0.1% ethanolas vehicle control (Dex “−”) for the indicated time and subjected to apoptosis (•—•) or cellcycle (G1: dark gray bars; G2/M: light gray bars; S: open bars) determination using flowcytometric analysis of propidium iodide-stained nuclei. Shown are mean values ±SD of specificapoptosis (apoptosis in GC-treated samples minus apoptosis in corresponding vehicle controls)or mean values of cells in the 3 phases of the cell cycle derived from biological triplicates. Insome cases, cell cycle determinations did not fulfill the required quality requirements due to

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high degree of apoptosis and hence were not included in the figure. The data for all cell linesexcept 2C8 have been reported previously [62].

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Fig. 2.Basal and GC-regulated PLZF/ZBTB16 expression in various leukemia cell lines. Theindicated cell lines were cultured for the indicated times in the presence of 10−7 Mdexamethasone or 0.1% ethanol as vehicle control, and analyzed for PLZF/ZBTB16 (and TBPas internal control) mRNA expression using quantitative real-time RT-PCR for PLZF/ZBTB16. (a) TBP-normalized PLZF/ZBTB16 expression levels in the absence ofdexamethasone expressed as minus (−) mean ΔCT (thus, “0” means CT values equal to thoseof TBP, negative values indicate increasingly lower expression). For each biological replicateof the 2 h vehicle controls, the CT value for TBP was subtracted from the corresponding PLZF/ZBTB16 CT value; subsequently, mean (mean ΔCT) and standard deviations were calculated.(b) GC regulation of PLZF/ZBTB16 in the indicated cell lines at the indicated time pointsexpressed as mean M values ± standard deviation (M-value of +1 indicates 2-fold up-regulation). Asterisks indicate pBH-values of <0.05 for the corresponding mean M value. Inthe 697/EU-3 precursor B-cell ALL line, basal PLZF/ZBTB16 levels were below the detectionlimit, however, PLZF/ZBTB16 became detectable after GC treatment (not shown in thegraphs).

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Fig. 3.Characterization of T-ALL cell lines with conditional PLZF/ZBTB16 expression. C7H2-2C8-ZBTB16#19 and #58 cells (expressing PLZF/ZBTB16 in a doxycycline-dependent manner)were cultured in the presence of increasing concentration of doxycycline (Dox) for theindicated time and analysed for PLZF/ZBTB16 (and TBP as internal control) mRNAexpression using quantitative real-time RT-PCR (a) or immunoblotting with antibodies againstPLZF/ZBTB16 and α-tubulin as loading control (b). The real-time RT-PCR data in (a) areexpressed as −ΔCT (i.e., CT value of PLZF/ZBTB16 minus CT value of TBP). Shown are meanvalues ±SD from both cell lines each analyzed in 3 biological and 3 technical replicates. (b) A

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representative dose response at the protein level for the 2, 6 and 24 h time points for theZBTB16#58 cell line.

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Fig. 4.PLZF/ZBTB16 alone has no effect on cell cycle progression and survival, but interferes withthe anti-leukemic properties of GC. ZBTB16#19 and #58 cells (expressing PLZF/ZBTB16 ina doxycycline-dependent manner) were cultured in the absence or presence of 10−7 or 10−8 Mdexamethasone (Dex), with and without 200 ng/ml doxycycline (Dox) for the indicated timeand subjected to apoptosis (a) or cell division cycle (b) determination using flow cytometricanalysis of propidium iodide-stained nuclei. Shown are mean values ±SD of cells in the subG1window (referred to as “apoptosis” in the graph in (a) or mean values of cells in the G1/G0phase of the cell division cycle (b) derived from biological triplicates of both cell lines (bothcell lines gave very similar results and hence were combined in single graphs). Statisticallysignificant (p < 0.05) differences between cells treated in the absence (+ dox) or presence (−dox) of doxycycline are indicated by an asterisk.

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Fig. 5.Knock-down of PLZF/ZBTB16 increases GC-elicited cell death. (a) C7H2-2C8-ZBTB16#58cells (ZBTB16#58) transduced by recombinant lentiviruses containing shRNA-expressingconstructs targeting exon 7 (shRNA1) or 6 (shRNA2) of PLZF/ZBTB16 or a control shRNA(shRNA-control) and enriched by FACS-sorting were cultured with 50 ng/ml doxycycline for24 h and analysed for PLZF/ZBTB16 (and TBP as internal control) mRNA expression usingquantitative real-time RT-PCR ((a), left panel) or immunoblotting with antibodies againstPLZF/ZBTB16 and α-tubulin as loading control ((a), right panel). ZBTB16 mRNA is expressedas −ΔCT (i.e., CT value of PLZF/ZBTB16 minus CT value of TBP). Shown are mean values±SD analyzed in 2 biological and 3 technical replicates. Statistically significant (p < 0.01)differences of PLZF/ZBTB16 mRNA expression levels between cells expressing ZBTB16-shRNAs and controls are indicated by an asterisk. b: CEM-C7H2 cells transduced by theindicated shRNAs as described above were cultured in the absence or presence of 10−7 or10−8 M dexamethasone (Dex) for 48 h and subjected to cell death determination using flowcytometric analysis of 7-AAD and APC-coupled Annexin-V stained cells. Shown are meanvalues ±SD of cells staining positive for Annexin-V and/or 7-AAD (referred to as “% celldeath” in (b) derived from biological triplicates. Statistically significant (p < 0.05) differencesbetween cells expressing the indicated shRNAs are indicated by an asterisk.

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Fig. 6.PLZF/ZBTB16 does not interfere with anti-CD95/FAS-induced apoptosis or apoptosis andcell cycle arrest induced by mitomycin C, doxorubicin, or taxol. ZBTB16#19 and #58 cells(expressing PLZF/ZBTB16 in a doxycycline-dependent manner) were cultured in the absenceor presence of 5, 10 or 20 ng/ml anti-CD95/FAS (AF), 0.6 or 3 μg/ml mitomycin C, 20 or100 ng/ml doxorubicin (Doxo), 1 or 10 μg/ml taxol, with and without 200 ng/ml doxycycline(Dox) for the indicated time and subjected to apoptosis (a) or, in the case of treatment withchemotherapeutics, cell cycle (b) determination using flow cytometric analysis of propidiumiodide-stained nuclei. Shown are mean values ±SD of cells in the subG1 window (referred toas “apoptosis” in the graphs) at the indicated time points derived from biological triplicates ofboth cell lines (both cell lines gave very similar results and hence were combined in singlegraphs).

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Fig. 7.PLZF/ZBTB16 interferes with GC regulation of gene expression. To determine PLZF/ZBTB16response genes, total RNA from ZBTB16#19 and #58 cells (expressing PLZF/ZBTB16 in adoxycycline-dependent manner) cultured in duplicate in the absence (set A) or presence (setB) of 400 ng/ml doxocycline for 6 or 24 h was subjected to expression profiling on 16 Exon1.0 microarrays. To assess the effect of PLZF/ZBTB16 on the GC response, the above celllines were cultured for 24 h in the absence (set C) or presence (set D) of doxycycline andsubsequently exposed to 10−8 M dexamethasone for 6 and 24 h and expression-profiled asabove. Comparison between sets A and C identified GC-regulated genes at 6 and 24 h,respectively. The full lines in the 4 panels show the distribution of the GC-regulated transcriptswith respect to the extent of regulation (mean M) in the absence of doxycycline (GC/no Dox).The dotted lines represent the regulation of the same transcripts comparing sets B and D, i.e.,when GC acts in the presence of induced PLZF/ZBTB16 levels (GC/Dox). The 4 panels depictup- or down-regulations after 6 or 24 h, respectively. Underneath the plots, mean M for allregulations in the corresponding plot ±SD are shown (|—•—|). In all 4 instances, the extent ofregulation was highly significantly (p ≪ 0.05) reduced in the presence of doxycycline-inducedPLZF/ZBTB16 as determined by paired Student's t-tests.

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Fig. 8.PLZF/ZBTB16 reduces GC-induction of the BH3-only molecule Bim. ZBTB16#58 cells(expressing PLZF/ZBTB16 in a doxycycline-dependent manner) were cultured in the absenceor presence of 200 ng/ml doxycycline (Dox, added 24 h prior to dexamethasone) and 10−7 Mdexamethasone (Dex) for 24 h and analysed for BCL2L11/Bim mRNA expression by real-time RT-PCR (a) or for PLZF/ZBTB16 and BCL2L11/Bim protein expression byimmunoblotting (b). (a) Regulation (ΔΔCT) of BCL2L11/Bim mRNA by GC in the presenceor absence of doxycycline-induced PLZF/ZBTB16 expression. For calculation of ΔΔCT, meanvalues of TBP-normalized BCL2L11/Bim expression (ΔCT) without GC were subtracted fromthe corresponding mean values in the presence of GC (thus, a ΔΔCT of +1 indicates 2-fold up-regulation). Shown are mean values ±SD of 2 biological replicates assayed 3 times in 3technical replicates per assay. (b) A representative immunoblot of the same cells analyzed forPLZF/ZBTB16 (top panel), BCL2L11/Bim (2nd panel: short exposure, i.e., 30 s; 3rd panel:long exposure, i.e., 4 min) and α-tubulin as loading control (bottom panel). The 3 major splicevariants of Bim (BimEL, BimL, Bim-S) are indicated in the long exposure.

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