A highly selective telomerase inhibitor limiting human cancer cell proliferation
2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês
10.1093/emboj/20.24.6958
ISSN1460-2075
Autores Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle17 December 2001free access A highly selective telomerase inhibitor limiting human cancer cell proliferation Klaus Damm Corresponding Author Klaus Damm Boehringer Ingelheim Pharma KG, Birkendorfer Strasse 65, D-88397 Biberach, Germany Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Ulrike Hemmann Ulrike Hemmann Genomics, Birkendorfer Strasse 65, D-88397 Biberach, Germany Present address: Aventis Pharma GmbH, Fraunhofer Strasse 13, D-82152 Martinsried, Germany Search for more papers by this author Pilar Garin-Chesa Pilar Garin-Chesa Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Norbert Hauel Norbert Hauel Chemistry, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Iris Kauffmann Iris Kauffmann Chemistry, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Henning Priepke Henning Priepke Chemistry, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Claudia Niestroj Claudia Niestroj Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Christine Daiber Christine Daiber Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Barbara Enenkel Barbara Enenkel Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Bernd Guilliard Bernd Guilliard Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Ines Lauritsch Ines Lauritsch Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Elfriede Müller Elfriede Müller Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Emanuelle Pascolo Emanuelle Pascolo Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Gabriele Sauter Gabriele Sauter Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Milena Pantic Milena Pantic University Medical Center, Department for Hematology/Oncology, Hugstetterstrasse 55, D-79106 Freiburg i. Br., Germany Albert-Ludwigs-University, Department of Biology, D-79106 Freiburg i. Br., Germany Search for more papers by this author Uwe M. Martens Uwe M. Martens University Medical Center, Department for Hematology/Oncology, Hugstetterstrasse 55, D-79106 Freiburg i. Br., Germany Search for more papers by this author Christian Wenz Christian Wenz Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland Search for more papers by this author Joachim Lingner Joachim Lingner Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland Search for more papers by this author Norbert Kraut Norbert Kraut Genomics, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Wolfgang J. Rettig Wolfgang J. Rettig Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5–11, A-1120 Vienna, Austria Search for more papers by this author Andreas Schnapp Andreas Schnapp Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Klaus Damm Corresponding Author Klaus Damm Boehringer Ingelheim Pharma KG, Birkendorfer Strasse 65, D-88397 Biberach, Germany Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Ulrike Hemmann Ulrike Hemmann Genomics, Birkendorfer Strasse 65, D-88397 Biberach, Germany Present address: Aventis Pharma GmbH, Fraunhofer Strasse 13, D-82152 Martinsried, Germany Search for more papers by this author Pilar Garin-Chesa Pilar Garin-Chesa Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Norbert Hauel Norbert Hauel Chemistry, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Iris Kauffmann Iris Kauffmann Chemistry, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Henning Priepke Henning Priepke Chemistry, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Claudia Niestroj Claudia Niestroj Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Christine Daiber Christine Daiber Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Barbara Enenkel Barbara Enenkel Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Bernd Guilliard Bernd Guilliard Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Ines Lauritsch Ines Lauritsch Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Elfriede Müller Elfriede Müller Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Emanuelle Pascolo Emanuelle Pascolo Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Gabriele Sauter Gabriele Sauter Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Milena Pantic Milena Pantic University Medical Center, Department for Hematology/Oncology, Hugstetterstrasse 55, D-79106 Freiburg i. Br., Germany Albert-Ludwigs-University, Department of Biology, D-79106 Freiburg i. Br., Germany Search for more papers by this author Uwe M. Martens Uwe M. Martens University Medical Center, Department for Hematology/Oncology, Hugstetterstrasse 55, D-79106 Freiburg i. Br., Germany Search for more papers by this author Christian Wenz Christian Wenz Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland Search for more papers by this author Joachim Lingner Joachim Lingner Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland Search for more papers by this author Norbert Kraut Norbert Kraut Genomics, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Wolfgang J. Rettig Wolfgang J. Rettig Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5–11, A-1120 Vienna, Austria Search for more papers by this author Andreas Schnapp Andreas Schnapp Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany Search for more papers by this author Author Information Klaus Damm 1,2, Ulrike Hemmann3,4, Pilar Garin-Chesa2, Norbert Hauel5, Iris Kauffmann5, Henning Priepke5, Claudia Niestroj2, Christine Daiber2, Barbara Enenkel2, Bernd Guilliard2, Ines Lauritsch2, Elfriede Müller2, Emanuelle Pascolo2, Gabriele Sauter2, Milena Pantic6,7, Uwe M. Martens6, Christian Wenz8, Joachim Lingner8, Norbert Kraut3, Wolfgang J. Rettig9 and Andreas Schnapp2 1Boehringer Ingelheim Pharma KG, Birkendorfer Strasse 65, D-88397 Biberach, Germany 2Oncology Research, Birkendorfer Strasse 65, D-88397 Biberach, Germany 3Genomics, Birkendorfer Strasse 65, D-88397 Biberach, Germany 4Present address: Aventis Pharma GmbH, Fraunhofer Strasse 13, D-82152 Martinsried, Germany 5Chemistry, Birkendorfer Strasse 65, D-88397 Biberach, Germany 6University Medical Center, Department for Hematology/Oncology, Hugstetterstrasse 55, D-79106 Freiburg i. Br., Germany 7Albert-Ludwigs-University, Department of Biology, D-79106 Freiburg i. Br., Germany 8Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland 9Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5–11, A-1120 Vienna, Austria *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6958-6968https://doi.org/10.1093/emboj/20.24.6958 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities. Introduction Telomerase is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends (Morin, 1989; Blackburn and Greider, 1995). In human cells, the enzyme comprises a high molecular weight complex with a template-containing RNA subunit (Feng et al., 1995) and protein components including the catalytic subunit human telomerase reverse transcriptase, hTERT (Harrington et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997). Telomerase activity has been demonstrated in immortalized cell lines and in 80–90% of human cancer specimens representing a range of cancer types (Counter et al., 1994; Kim et al., 1994; Shay and Bacchetti, 1997) and recently, human telomerase has been directly implicated in cellular immortalization and tumorigenesis (Bodnar et al., 1998; Hahn et al., 1999a). In most normal human cells, telomerase activity is low or not detectable, and telomeric DNA is progressively lost at a rate of 30–120 bp with each replication cycle (Harley et al., 1990; Hastie et al., 1990; Counter et al., 1992). Eventually, telomeres shorten to a critical length and lose their ability to protect the ends of chromosomal DNA (Counter et al., 1992; Blasco et al., 1997). Uncapped chromosomes are sensitive to degradation and fusion and can activate DNA damage checkpoints, thus potentially contributing to the replicative senescence and growth arrest observed in aged primary cultured cells (Hayflick and Moorhead, 1961). Indeed, it has been proposed that telomere length specifies the number of cell divisions a cell can undergo prior to senescence (Cooke and Smith, 1986; Harley, 1991). In cancer cells, the reactivation of telomerase is thought to stabilize telomere length, thereby compensating for the cell division-related telomere erosion and providing unlimited proliferative capacity to malignant cells (Counter et al., 1992; Kim et al., 1994). As a corollary to this hypothesis, the inhibition of telomerase in tumour cells should disrupt telomere maintenance and return malignant cells to proliferative crisis followed by senescence or cell death (Harley et al., 1990; Counter et al., 1992). Genetic experiments using a dominant-negative form of human telomerase demonstrated that telomerase inhibition can result in telomere shortening followed by proliferation arrest and cell death by apoptosis (Hahn et al., 1999b; Zhang et al., 1999). A challenge for the development of pharmaceutically useful telomerase inhibitors is the long lag period required to observe telomere attrition. Cellular growth arrest that depends on telomere shortening will require a series of cell division cycles to become apparent, and treatment may have to be given continuously for weeks to months, potentially in conjunction with other treatment modalities. Therefore, potency of action, selectivity, tolerability and suitable pharmaceutical formulations are formidable tasks to be met in telomerase drug design. Here we describe a novel structural class of non-peptidic, non-nucleosidic inhibitors of human telomerase that are highly potent and selective in vitro and pharmacologically active in the control of human cancer cell proliferation. Results Small molecules selectively inhibit telomerase activity in vitro To characterize small-molecule inhibitors of human telomerase, we used nuclear extracts prepared from HeLa cells to set up an in vitro telomerase activity assay (Morin, 1989; Schnapp et al., 1998). Our study focuses on compounds with the general structure shown in Figure 1A, for which systematic structure-activity correlations have been established (N.Hauel et al., manuscript in preparation). Two examples from this class of compounds, designated BIBR1532 {2-[(E)-3-naphtalen-2-yl-but-2- enoylamino]-benzoic acid} and BIBR1591 {5-morpholin- 4-yl-2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid}, inhibit the in vitro processivity of telomerase in a dose-dependent manner, with half-maximal inhibitory concentrations (IC50) of 93 and 470 nM, respectively (Figure 1B). The selectivity of BIBR1532 was assessed in a panel of DNA and RNA polymerases, including HIV reverse transcriptase, showing that none of these enzymes was inhibited at concentrations vastly exceeding the IC50 for telomerase (Figure 1C). As shown in the direct telomerase assay (Figure 1D), BIBR1532 can also inhibit recombinant, affinity purified telomerase, suggesting that it is indeed the catalytic activity of the telomerase enzyme which is the target for BIBR1532 inhibition. Figure 1.Specific and selective telomerase inhibitors. (A) Chemical structure of the BIBR compound class of inhibitors. BIBR1532, R = H; BIBR1591, R = morpholin-4-yl. (B) Dosis-dependent inhibition of telomerase activity by BIBR1532 (solid squares) and BIBR1591 (open circles). Assays were performed and quantified using a PCR-based protocol followed by a TCA precipitation step. The incorporated activity of samples with inhibitor was normalized to the control and plotted against the inhibitor concentration. (C) Selectivity profile of BIBR1532. Enzymatic activity was assayed in the presence of 0–50 μM BIBR1532 as described in Materials and methods. −, no effect at 50 μM. (D) Direct assay of telomerase activity. Telomerase was reconstituted with insect cell expressed hTERT and in vitro transcribed RNA, affinity purified and incubated in the presence of different concentrations of BIBR1532. Telomerase products were separated on a sequencing gel. Download figure Download PowerPoint Telomerase inhibitors induce telomere shortening in cancer cells The compounds also had no effect on short-term cell viability or proliferation, as determined in a 7 day cytotoxicity assay using concentrations 100-fold above the in vitro IC50 (i.e. 10 μM for BIBR1532, 50 μM for BIBR1591). To investigate the cellular consequences of long-term treatment with a telomerase inhibitor, we cultivated exponentially growing NCI-H460 lung carcinoma cells in the presence of BIBR1532 (10 μM) or BIBR1591 (50 μM), respectively. As a control, untreated cells or cells treated with the solvent alone were grown using the same culture conditions. Periodically, total DNA samples were prepared from treated and control cells, digested with frequently cutting restriction enzymes and the telomere length examined by Southern blotting. NCI-H460 cells exhibit a heterogeneous size distribution, with an average telomere length of 4 kb and a predominant range of 2 to 6 kb (Figure 2A). As cells are propagated in the presence of telomerase inhibitor, steady telomere shortening occurred. The average telomere restriction fragment (TRF) size shortened progressively from 4 to 1.5 kb at population doubling (PD) 140 (Figure 2A), corresponding to a telomere loss of 30 bp/PD. We observed a comparable erosion of the telomeres in HT1080 fibrosarcoma, MDA-MB231 breast carcinoma and DU145 prostate carcinoma cells similarly treated with telomerase inhibitor (Figure 2A). In contrast, untreated cells or cells exposed to solvent alone maintained a stable TRF size (Figure 2A). Figure 2.Telomerase inhibitors induce telomere shortening and limit cell proliferation. (A) Total genomic DNA prepared from untreated (lane 1), solvent- (lane 2) or inhibitor-treated (lanes 3 and 4) NCI-H460, HT1080, MDA-MB231 or DU145 cells was assessed for telomere restriction fragment size by Southern blot analysis with a telomeric probe. PD, population doubling; −, absence and +, presence of BIBR1532 or BIBR1591. (B) NCI-H460, HT1080, MDA-MB231 and DU145 cells were plated in 24-well plates in duplicate in the presence of 10 μM BIBR1532 or 50 μM BIBR1591 dissolved in 0.1% DMSO (closed symbols). Control cells were untreated (open triangles) or treated with corresponding solvent concentrations (open circles). Cultures were replated every 2–3 days to maintain log-phase growth and to calculate the growth rate. Download figure Download PowerPoint Telomerase inhibitors limit cancer cell proliferation The growth kinetics of inhibitor-treated cells initially did not differ from those of untreated or solvent treated control cells, regardless of the cell line used. NCI-H460 cell cultures in the absence or presence of telomerase inhibitor exhibited no or only minor differences in proliferation for more than 120 days of treatment (Figure 2B). However, after PD135 the inhibitor-treated cells slowed their growth and showed an almost complete inhibition of proliferation after additional 4–8 population doublings (Figure 2B). This telomerase inhibitor-induced growth arrest is apparently independent of functional p53 since similar growth curves, with an onset of cellular growth arrest after a significant lag-phase, were obtained for the p53-deficient HT1080, MDA-MB231 and DU145 cell lines (Figure 2B). The reduced proliferation capacity of telomerase-inhibited cells near growth arrest was further substantiated in colony formation assays, with about 50% reduction in colony formation for treated versus mock-treated NCI-H460 and HT1080 cells. As a control for compound specificity we also cultivated telomerase-negative, normal human lung fibroblasts as well as an osteosarcoma cell line (SAOS-2) that exhibits the alternative lengthening of telomere (ALT) phenotype (Bryan et al., 1995). Our results showed that inhibitor treatment had no effect on telomere length, growth kinetics and morphology for the entire time of treatment (8 weeks) in either cell line. Reversibility of inhibition In parallel cultures of the NCI-H460 cells shown in Figure 2B we observed very slowly proliferating cells that were overgrowing the resting, senescent cells in the culture plate, resulting in a flat but measurable growth rate (Figure 3A). Apparently, the treated cells enter senescence not in a parallel but rather in a sequential fashion, which may be due to the heterogeneity of their telomere lengths. We never observed spontaneous telomere lengthening or telomerase-independent growth attributable to the induction of an ALT phenotype. To determine the effect of inhibitor depletion, we transferred the treated cells to normal medium without drug starting at day 220. After a short delay of 3–4 days the cells exhibited a growth rate similar to the control culture (Figure 3A). Examination of the TRF sizes also revealed a rapid elongation of the telomeres in these cells (Figure 3B), demonstrating that the telomerase inhibition is fully reversible. Figure 3.Reversibility of inhibition. (A) NCI-H460 cells were cultivated in 24-well plates in the absence (open circles) or presence of 50 μM BIBR1591 (closed triangles). After 130 days, compound-treated cells were replated only when the culture dishes reached subconfluence. At day 220, these cells were washed, replated in medium without compound and the growth rate monitored for additional 50 days (open squares). (B) The median TRF size of inhibitor-treated NCI-H460 cells at day 220 corresponds to only 1.5 kb (lanes 1 and 2). Removal of the inhibitor and cultivation of these cells for 40 PD in absence of inhibitor leads to a significant telomere elongation (lane 3). Untreated control cells at day 260 are also shown for comparison (lane 4). Download figure Download PowerPoint Telomerase inhibitors induce a senescent phenotype The inhibitor-treated, late passage tumour cells showed distinctive morphological features associated with senescence of aged normal human cells. In contrast to untreated cell cultures (Figure 4A), the growth-arrested NCI-H460 cells (Figure 4B) became enlarged, often contained multiple nuclei, had a vacuolated cytoplasm and showed induction of senescence associated β-galactosidase activity (Figure 4C). Similar morphological alterations were observed with late-passage HT1080, MDA-MB231 and DU145 cells (data not shown). FACS analysis of inhibitor-treated NCI-H460 cells revealed an elevated forward and side scatter relative to untreated cells, which may reflect increased cell size and granularity (Figure 4D and E). The inhibitor-treated NCI-H460 cells also showed a heterogeneous cell cycle profile, with broad 2n and 4n peaks and a shift towards higher DNA content (Figure 4F and G). We performed extensive FACS analysis and TUNEL staining in p53 positive (NCI-H460) or p53 negative (HT1080, DU145, MDA-MB231) cell lines at different timepoints, but could not detect an increase in apoptosis comparing treated and control cells. Figure 4.Induction of a senescence phenotype. Phase contrast micrographs show the cellular morphology of (A) untreated and (B) inhibitor-treated NCI-H460 cells at PD162 and PD130, respectively. (C) Inhibitor-treated NCI-H460 cells from a separate inhibition experiment were stained for β-galactosidase activity at pH 6.0. Phenotypic changes in this experiment were already observed at PD72. (D–G) Flow cytometric analysis of NCI-H460 cells shown in (C). Dot density maps of PI-staining (x-axis) and 90-degree light scatter (y-axis) are shown for control (D), and inhibitor-treated cells (PD72) (E). DNA content analysis of control (F), and inhibitor-treated cells (PD72) (G). Download figure Download PowerPoint Inhibitor-treated cells exhibit telomere dysfunction We next analysed chromosomal metaphase spreads derived from late-passage NCI-H460 cells. Due to the reduced proliferative capacity and the correspondingly low mitotic index only eight metaphases were obtained from the inhibitor-treated cells, and these were compared qualitatively with 20 metaphases from control cultures. The loss of telomeric sequences was readily detected using the Q-FISH technique with a telomere-specific probe (Figure 5A and B) (Martens et al., 1998). Signal intensity, which has been shown previously to correlate directly with the number of TTAGGG repeats, was significantly (p <0.0001) reduced in the inhibitor-treated cells relative to control cells (Figure 5C). The histogram also shows an accumulation of short telomeres and an increased skewness in the distribution of telomere fluorescence (Figure 5C) which is very similar to the observations in pre-senescent fibroblasts (Martens et al., 2000). Furthermore, we observed an increase in chromosome end fusions (0.88/metaphase in treated versus 0.55/metaphase in control cells) as well as an increased number of chromosomes with no telomere signal at both sister chromatids in treated cells (mean: 3.75/metaphase) compared with control cells (mean: 1.1/metaphase). Figure 5.Telomere analysis of inhibitor-treated NCI-H460 cells. (A) Q-FISH analysis of metaphase chromosomes from inhibitor-treated NCI-H460 cells. DAPI-stained chromosomes and Cy-3-labeled telomeres are shown in blue and yellow, respectively. The NCI-H460 cell line is hypotriploid with seven marker chromosomes (modal chromosome number 57) and exhibits a characteristic chromosome with interchromosomal telomere signals. Arrowhead denotes missing telomeres; arrow denotes fused chromosomes; dashed arrow denotes interchromosomal telomere signals. (B) Details of end-to-end fusions from another metaphase with telomere signals present at the fusion site. (C) Histograms express the fluorescence intensity and frequency of all individual telomere spots from NCI-H460 derived metaphases. Twenty metaphases (n = 3740) were derived from control cultures (PD68) and eight metaphases (n = 1366) were derived from inhibitor treated cells (PD120). n is the number of individual telomere signals. The differences in mean fluorescence intensity (arbitrary fluorescence units ± SD) between the control cells (321 ± 160) and the treated cells (217 ± 101) were highly significant (p <0.0001). Download figure Download PowerPoint Microarray analysis of mRNA expression levels in senescent NCI-H460 cells To identify genes responsive to pharmacological telomerase inhibition and telomere shortening we determined changes in gene expression levels between inhibitor-treated and untreated NCI-H460 cells using DNA microarrays with the capacity to display transcript levels of 6817 known human genes. Total RNA was prepared from cells exposed to telomerase inhibitor for 7, 28 and 56 days, respectively, or until overt morphological changes characteristic for senescence were apparent. Analysis of the day 7, day 28 and day 56 timepoints revealed only minor variations in RNA transcript patterns without consistent changes. However, in the inhibitor-treated, senescent NCI-H460 cells we identified 302 (4.4%) genes showing at least a 2-fold difference in expression levels in a minimum of three out of four independent comparisons (for complete gene list see Supplementary data available at The EMBO Journal Online). The up- or down-regulated genes displaying the largest alterations in expression were assigned to functional categories (Tables I and II). Table 1. Transcriptional changes induced in NCI-H460 cells by long-term telomerase inhibitor treatment: down-regulation Accession No. Fold Δ Gene name Identified in context of senescence/ageing Cell cycle control U74612 −11.5 hepatocyte nuclear factor-3/fork head homolog 11A Ly et al. (2000) X51688 −11.4 cyclin A (*) Ly et al. (2000); Shelton et al. (1999) Z29066 −7.1 NEK2 X65550 −7.0 mki67a X13293 −6.3 B-MYB Chang et al. (2000); Ly et al. (2000) U05340 −4.9 p55CDC Ly et al. (2000) U33761 −3.9 cyclin A/CDK2-associated p45 (SKP2) U77949 −3.5 CDC6-related protein M25753 −3.4 cyclin B Chang et al. (2000); Ly et al. (2000); Shelton et al. (1999) X54941 −3.3 CKSHS1 Ly et al. (2000) D50914 −3.2 KIAA0124, similar to mouse BOP1 U65410 −3.1 MAD2 Chang et al. (2000) U01038 −2.9 PLK1 Chang et al. (2000); Ly et al. (2000) X05360 −2.5 CDC2 Chang et al. (2000); Shelton et al. (1999) U56816 −2.4 MYT1 Ly et al. (2000) X54942 −2.3 CKSHS2 DNA segregation, mitosis, chromatin assembly D38751 −31.0 kinesin-like DNA binding protein (KID) U20979 −7.4 chromatin assembly factor-I p150 subunit U61145 −7.1 enhancer of Zeste homolog 2 (EZH2) D38553 −5.4 XCAP-H condensin homolog Chang et al. (2000) D63880 −4.2 KIAA0159 X63692 −3.6 DNA (cytosin-5)-methyltransferase U09087 −3.4 thymopoietin β U18271 −3.4 thymopoietin U37426 −3.3 kinesin-like spindle protein (HKSP) Ly et al. (2000) X62534 −3.3 HMG2 Chang et al. (2000); Ly et al. (2000) D38076 −3.3 RANBP1 Ly et al. (2000) D00591 −3.2 RCC1 L07515 −3.0 heterochromatin protein homologue (HP1) M34458 −2.9 lamin B Chang et al. (2000) X85137 −2.9 kinesin-related protein U14518 −2.7 centromere protein-A (CENP-A) Chang et al. (2000); Ly et al. (2000) M97856 −2.5 histone-binding protein Ly et al. (2000) X67155 −2.5 mitotic kinesin-like protein-1 Ly et al. (2000) D80000 −2.5 SMC1L1 D43948 −2.1 TOG Ly et al. (2000) DNA synthesis, replication L16991 −11.1 thymidylate kinase (CDC8) Ly et al. (2000) M87338 −8.7 replication factor C HG2379-HT3996 −8.5 serine hydroxymethyltransferase D55716 −5.7 P1CDC47/MCM7 Chang et al. (2000) U40152 −3.5 origin recognition complex 1 Chang et al. (2000) X52142 −3.1 CTP synthetase X59618 −2.7 ribonucleotide reductase M2 polypeptide Chang et al. (2000) M63488 −2.7 replication protein A 70 kDa subunit M15205 −2.6 thymidine kinase Chang et al. (2000); Ly et al. (2000); Shelton et al. (1999) U00238 −2.5 glutamine PRPP amidotransferase J04031 −2.5 MTHFD1 U81375 −2.5 equilibrative nucleoside transporter 1 (hENT1) HG2846-HT2983 −2.3 dihydrofolate reductase Chang et al. (2000) X06745 −2.3 DNA polymerase α-subunit Chang et al. (2000) X54199 −2.2 GARS-AIRS-GART D84557 −2.2 MCM6 Transcription, RNA processing, translation initiation X89416 −14.8 protein phosphatase 5 D32002 −8.0 nuclear cap binding protein D12686 −7.1 eukaryotic initiation factor 4γ M85085 −6.6 cleavage stimulation factor U76421 −5.4 dsRNA adenosine deaminase DRADA2b X13482 −4.9 U2 snRNP-specific A′ protein X75918 −3.1 NOT U28042 −2.9 DEAD box RNA helicase-like protein M60784 −2.8 U1 snRNP-specific protein A U08815 −2.4 splicesomal protein (SAP61) L10838 −2.4 pre-mRNA splicing factor (SRP20) Ly et al. (2000) X67337 −2.2 HPBRII-4 M86737 −2.2 high mob
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