1. Introduction & Objectives - hTERT is the catalytically active component of the telomerase complex [1] - Its expression correlates with telomerase activity and is restricted to >90% of cancers [2] - ectopic hTERT expression in epithelial cells induced mitogenic genes (EGFR & FGF2) [3] - an immediate proliferation stop was initiated by targeting the hTERT mRNA with ribozymes or antisense oligonucleotides (AS ‑ ODNs) [4-6] - our previous studies: AS-ODN-mediated hTERT knock-down in bladder cancer (BCa) cell lines leading to an immediate growth suppression [5,7] - aim of the study: changes in the mRNA expression profile of the cell line EJ28 after transfection with 2 hTERT AS-ODNs; 2 additional anti-hTERT siRNAs were also investigated - to differ between specific effects of the hTERT targeting constructs & effects caused by a general AS-ODN-mediated or siRNA-mediated mechanism, AS-ODNs & siRNAs targeted at the targets survivin (SVV) & vascular endothelial growth factor (VEGF) were used Materials & Methods Cell culture and transfection - the human BCa cell line EJ28 was transiently transfected with ODNs (Invitrogen) and siRNAs (Qiagen) (Table I) at 250 nM complexed using Lipofectin (LF) (Invitrogen) at a LF-nucleic acid-ratio of 3:1 (w/w) or DOTAP (Roche) at a DOTAP- nucleic acid-ratio of 4:1 (w/w). Combination of hTERT siRNAs and chemotherapeutic agents - Cisplatin (CDDP), Mitomycin C (MMC), diluted in culture medium, treatment scheme & analysis of apoptosis (annexin V assay) as described before [7]. Sample preparation for microarray hybridization - isolation of total RNA using RNeasy Mini Kit (Qiagen), agarose gel electrophoresis - single stranded cDNA (from 8 µg RNA) using a T7-OligodT24-primer (TibMolbiol) & SuperScript II (Invitrogen), secondary strand synthesis of cDNA using E.coli DNA Ligase, E.coli DNA Polymerase I, RNase H & dNTPs (all from Invitrogen) - purification of double stranded cDNA by GFX PCR DNA and Gel Purification Kit (Amersham Biosciences), copy into biotin-labeled cRNA (Megascript T7 kit, Ambion) - purification of cRNA using RNeasy columns (Qiagen) followed by quantification, fragmentation and hybridization on HG-U133 A arrays (Affymetrix) for 16 h at 45°C - scanning of the chips using a GeneArray scanner (Agilent) Data analysis and comparison strategies -All probe sets from each array were normalized to a target intensity of 500 - analysis of the output files by dCHIP 1.3 software (www.dchip.org) by PM-only model -comparisons for the identification of differentially expressed genes in cells treated with hTERT AS-ODNs or siRNAs in comparison to the controls (NS ‑ ODN, NS-si; Fig. 1) - 1st step: each AS-ODN array (ASt2206, ASt2331, AS ‑ SVV, AS ‑ VEGF) was compared to the NS-ODN array & each siRNA array (si ‑ hTERT1, si-hTERT2, si-SVV, si-VEGF) to the NS-si array. - fold changes used as cut offs (2.0 for AS-ODN, 1.7 for siRNA)  one specific gene list; lists for hTERT AS-ODN or siRNAs were compared to those for control constructs - 2nd step: generation of 2 lists of genes containing differentially expressed candidates for hTERT AS-ODNs and siRNAs, respectively - 3rd step: these hTERT gene lists were compared with those obtained by normalization of the SVV arrays & VEGF arrays to the NS controls - comparison of hTERT AS-ODN specific & hTERT siRNA specific gene lists (Fig. 1) Quantitative polymerase chain reaction (qPCR) analyses - hTERT mRNA quantification by LightCycler TeloTAGGG hTERT Quantification (Roche) - for RT (1 µg total RNA) Superscript II RT (Invitrogen) & random hexamer primers - cDNA dilution of 1:5 for qPCR assays (TaqMan Gene Expression; Applied Biosystems) at the LightCycler for selected candidate genes (ATF3, EGR1, RHOB, PDCD4, RAB31, ID2) - serially diluted PCR fragments (10E1-10E6) for the generation of calibration curves - primers & probes for the amplification of EGFR and PBDG in Table II, PCR assay for the reference gene TBP was adopted from Linja et al. [8], all qPCRs except for hTERT using the LC FastStart Master Hybridization Probe kit (Roche) & represent the mean of independent duplicates EGFR protein detection by Western blotting - lysis of BCa cells (5  10E4/sample) in loading buffer, incubation at 95°C for 5 min - WB analyses (7.5% PAA) using a monoclonal anti-EGFR ab (1:1,000) (Biosource),  -actin detection by a monoclonal anti-  -actin ab (1:8,000) (Sigma) as control; secondary anti-mouse-HRP ab (1:1,000) (Dako) & ECL system (Amersham Biosciences) Results Effects of hTERT AS-ODNs on target expression efficient knock-down of hTERT was shown by qPCR in EJ28 cells (Fig. 2) - both of the hTERT AS-ODNs ASt2206 & ASt2331 diminished expression of their target 12 h after transfection Expression profiling by microarrays after treatment with hTERT AS-ODNs - paired comparison analyses (Fig. 1), AS-ODNs targeted at SVV (AS-SVV) or VEGF (AS-VEGF) as controls - 2 hTERT AS-ODNs  total numbers of changed genes of 59 (ASt2206) & 101 (ASt2331); most of them upregulated (ASt2206: 66%, ASt2331: 90%; Fig. 3a) - 15 of 59 genes (25%) changed by ASt2206 were also affected by AS-SVV, whereas 25 genes (42%) affected simultaneously by AS-VEGF; concordance between ASt2331 & AS-VEGF higher (75/101, 75%) than that between ASt2331 & AS-SVV (36/101, 36%) - list of genes changed by both of the hTERT constructs (ASt2206, ASt2331)  (Table III) - comparison of this hTERT AS-ODN gene list with genes, whose expression was altered by AS-SVV & AS- VEGF  high degree of concordance: from the 28 genes, 22 genes (79%) were also up-regulated by AS- VEGF, 13 genes (46%) were co-affected by AS-SVV & 11 genes (39%) were altered together by all AS-ODNs (Table III) - 4 genes differentially expressed exclusively after hTERT AS-ODNs - a variety of genes known to be involved in stress response were induced by treatment with AS-ODNs against different targets (f.e. ATF3, CEBPB, Table III);  assumption: these effects may be independent of effects on AS-ODN targets Effects of various AS-ODNs on cell growth - both hTERT AS-ODNs reduced numbers of EJ28 within 24 h after transfection (Fig. 4) Influence of scrambled hTERT AS-ODNs on cellular viability & target expression - 4 additional control ODNs (SCR2, SCR3, SCR4, SCR5, derived from ASt2331) - scrambled SCR-ODNs compared to ASt2331 (Table I): scrambled SCR2 construct had neither an influence on cellular viability nor on hTERT expression (Fig. 5); SCR3 reduced the viability & the hTERT expression of EJ28 cells more efficiently than the primary ASt2331; SCR4 or SCR5 had no or little effect on viability, whereby SCR5 caused a moderate hTERT repression  beside the effects on target expression also target- independent effects of hTERT AS-ODNs contributed to the growth inhibition Effects of hTERT siRNAs on target expression of BCa cells -Reduction of the hTERT transcript numbers by si-hTERT1 & si-hTERT2 at 24 h (Fig. 2) Expression profiling by microarrays after treatment with hTERT siRNAs -total number of altered genes clearly differed between si-hTERT1 (41 genes) & si-hTERT2 (118 genes) (Fig. 3b), proportions of upregulated genes were 37% (si-hTERT1) & 44% (si-hTERT2) - 7 (17%) concordant genes were identified by comparing si-hTERT1 with si-SVV as well as by comparing si- hTERT1 with si-VEGF; of these, 3 genes (EREG, IL13RA2, RIG) were found in both of the comparisons, 10/118 (8%) & 7/118 (6%) genes were regulated in parallel by si-hTERT2/si-VEGF & si-hTERT2/si-SVV, F-box & FBXL11 & G3BP2 were altered by all siRNAs (Table IV) - 11 genes were regulated together by si-hTERT1 & si-hTERT2, whereof 7genes (64%, all  ) were exclusively altered by the hTERT si-RNAs & not by si-SVV/VEGF (Table IV) - hTERT siRNA gene list included 2 oncogenes: EGFR & FOSL1 - also the LAMC2 gene (involved in cell adhesion& migration)  Validation of the microarray data by quantitative PCR (qPCR) & Western blotting - for verification qPCRs for 7 genes with different changes on microarrays  FCs of qPCRs correlated well with that from microarrays (qPCRs more sensitive; Table V); transfection with both si-hTERT1/2 diminished EGFR protein after 24 h, si-VEGF (Fig. 6) & si-SVV (not shown) had no effect on EGFR protein content Effects of hTERT siRNAs in combination with CT on growth of EJ28 cells - no significant effects on viability, proliferation or apoptosis by si-hTERT1/2 (24 h) but decreased number of cells in S-phase 48 h after si-hTERT2 transfection (21% vs. 31% in the control) & increase of cells in G1 (70% vs. 59%) - an hTERT inhibition by si-hTERT2 followed by incubation with a relatively low concentration of MMC caused a decrease in the cell count of EJ28 cells (compared to si-hTERT2 or MMC treatment (Fig. 7a), reduction by si- hTERT2+MMC to 50% of the control (NS-si + MMC) after 72 h - rate of apoptosis was specifically  after si-hTERT2+MMC (33.7%) compared to NS ‑ si+MMC (17.3%) (Fig 7b) Conclusions - 2 AS ‑ ODNs (ASt2206, ASt2331) changed the expression of different genes mainly involved in stress response but without an association to telomerase function  immediate growth inhibition was caused, at least in part, by off-target effects; in comparison, siRNA-mediated blockade of hTERT was accompanied by the down- regulation of the oncogenes FOSL1 & EGFR - 2 microarray-based studies independently found an association of the expression of both EGFR & FOSL1 with metastatic phenotype [9,10]. - comparison of signatures of matched BCa tissue pairs  upregulation of FOSL1 in malignant tissues [11]; - upregulated expression of FOSL1 & EGFR correlates with CDDP resistance in ovarian cancer cells [12] - for the first time we show that repression of the hTERT transcript number downregulates EGFR expression both at the mRNA & protein levels  potential new function of hTERT in the regulation of EGFR-stimulated proliferation - siRNA-mediated hTERT  caused enhancement of the antiproliferative capacity of MMC & CDDP - hTERT promotes the growth of tumor cells not only by telomere lengthening but rather via multiple pathways References [1] Nakamura TM et al. Science 1997;277:955-9. [2] Poole JC et al. Gene 2001;269:1-12. [4] Smith LL et al. Nat Cell Biol 2003;5:474-9. [4] Saretzki G et al. Cancer Gene Ther 2001;8:827-34. [5] Kraemer K et al. Clin Cancer Res 2003;9:3794-800. [6] Folini M et al. Eur J Cancer 2005;41:624-34. [7] Kraemer K et al. J Urol 2004;172:2023-8. [8] Linja MJ et al. Cancer Res 2001;61:3550-5. [9] Modlich O et al. Clin Cancer Res 2004;10:3410-21. [10] Song B et al. World J Gastroenterol 2005;11 1463-72. [11] Dyrskjot L et al. Nat Genet 2003;33:90-6. [12] Macleod K et al. Cancer Res 2005;65:6789-800. [13] Fuessel S et al. J Urol 2004 171;2471-6. [14] Ning et al. Int J Oncol 2004 25; 1065-71. [15] Forster et al. Cancer Lett 2004 212;95-103. [16] Chen et al. Neoplasia 2000 2;235-41 a b Table II Primers and probes for qPCR Table I Sequences of nucleic acid constructs. All ODNs contained two phosphorothioates on the terminal nucleotides of the 5’-site and the 3’-site. The RNA target sequences (5’-3’) were shown for siRNAs. Scrambled nucleotides of the SCR-ODNs in comparison to ASt2331are depicted underlined. Fig.1 Schematic description of the comparative analysis of expression signatures by oligonucleotide microarrays. The cells were treated with AS-ODNs targeted at hTERT (ASt2206, ASt2331) and control AS-ODNs (AS-SVV, AS-VEGF) as well as with hTERT siRNAs (si-hTERT1, si-hTERT2) and control siRNAs (si-SVV, si-VEGF). Each AS-ODN and siRNA group was normalized to cells treated by NS-ODN or NS-si, respectively. Fig.2 Target-specific actions of AS-ODNs and siRNAs. The relative hTERT mRNA expression (hTERT/PBGD) was measured by qPCR at 12h (AS-ODNs) & 24h (siRNAs), respectively. The hTERT transcript numbers were normalized to those of NS-ODN and NS-si treated samples, respectively. Fig.3 Venn diagrams illustrating the numbers of differentially expressed genes and their overlaps. (a) 12 h after treatment with hTERT AS-ODNs. (b) 24 h after treatment with hTERT siRNAs. Each circle described the effects of a construct targeted at hTERT (solid line) or of a control construct (dashed line) directed at survivin (AS-SVV, si- SVV) or at the vascular endothelial growth factor (AS-VEGF, si-VEGF), each normalized to the NS-ODN or NS-si, respectively. Percentages of up-regulated genes are shown in brackets. Table III hTERT AS-ODN gene list. Shown are genes, regulated together by both of the hTERT AS- ODNs and the influence of AS-SVV and AS-VEGF on these genes. Negative fold changes indicate downregulation. Fig.4 Effects of transfection with hTERT AS-ODNs on EJ28 cell count. Fig.5 Correlation between viability and hTERT expression after treatment with the hTERT targeting ASt2331 and its modified counterparts. The viabilities and the hTERT/PBGD expression ratios were normalized to that of the NS-ODN samples. Table IV hTERT siRNA gene list. Shown are genes, regulated together by both of the hTERT siRNAs and the influence of si-SVV and si-VEGF on these genes. Negative fold changes indicate downregulation. Fig.6 EGFR protein detection. The protein lysates of EJ28 cells were generated 24h after transfection with si-hTERT1 (1), si-hTERT2 (2), si-VEGF (3) and NS-si (4).  -actin served as a control for equal loading. Table V Validation of differentially expressed genes identified by microarrays using qPCR. The expression values of ATF3 and EGR1 were normalized to the reference gene PBGD; all other expression values were normalized to TBP. Negative fold changes indicate downregulation. References: 1) Kraemer, 2003 [5]; 2) Fuessel, 2004 [13]; 3) Ning, 2004 [14]; 4) Forster, 2004 [15]; 5) Chen, 2000 [16] Fig.7 Chemosensitization of EJ28 cells by hTERT siRNA pretreatment. (a) reduction of cell count and (b) increased apoptosis after pretreatment with 200 nM si-hTERT2 followed by incubation with 0.5 µg/ml MMC or 2.0 µg/ml CDDP. Apoptosis was quantified as proportion of annexin V-posive cells, measured by FACS. 1234 EGFR  -Actin #220:Microarray Analyses in Bladder Cancer Cells: Inhibition of hTERT Expression Down-regulates EGFR K. Kraemer 1, U. Schmidt 1, S. Fuessel 1, A. Herr 2, O.W. Hakenberg 1, A. Meye 1, M.P. Wirth 1 1 Department of Urology, 2 Institute of Clinical Genetics, Medical Faculty, Technical University Dresden, Germany Supported by the Jürgen Manchot Foundation (Düsseldorf, Germany) http://urologie.uniklinikum-dresden.de Kai Kraemer: titabo@gmx.de or axel.meye@uniklinikum-dresden.de