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Immortalized Human Urothelial Cells Genes Involved in Differentiation, Stem Cell Renewal, and Tumorigenesis Are Modulated in Telomerase- Immortalized Human Urothelial Cells Emma J. Chapman,1 Gavin Kelly,2 and Margaret A. Knowles1 1Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, United Kingdom and 2Bioinformatics and Biostatistics Service, Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, London, United Kingdom Abstract non–telomere effects of telomerase and provides further The expression of hTERT, the catalytic subunit of rationale for the use of telomerase inhibitors in UC. telomerase, immortalizes normal human urothelial cells (Mol Cancer Res 2008;6(7):1154–68) (NHUC). Expression of a modified hTERT, without the ability to act in telomere maintenance, did not Introduction immortalize NHUC, confirming that effects at telomeres The primary and well-documented role of telomerase is as are required for urothelial immortalization. Previous a reverse transcriptase that acts in the maintenance of studies indicate that inhibition of telomerase has an telomere length and structure. Up-regulation of telomerase immediate effect on urothelial carcinoma (UC) cell line expression occurs in the majority of urothelial carcinoma viability, before sufficient divisions to account for (UC) irrespective or stage or grade (1), suggesting that this telomere attrition, implicating non–telomere effects may be an early event in tumorigenesis. Normal human of telomerase in UC. We analyzed the effects of urothelial cells (NHUC) are immortalized by expression of telomerase on gene expression in isogenic mortal and hTERT, the catalytic subunit of telomerase. In contrast to hTERT-transduced NHUC. hTERT expression led to requirements for immortalization in other epithelial cell types consistent alterations in the expression of genes and despite the common loss of expression of p16 in UC, predicted to be of phenotypic significance in inactivation of the CDKN2A locus (encoding p16 and tumorigenesis. A subset of expression changes were p14ARF) was not observed (2). detected soon after transduction with hTERT and Non–telomere effects of hTERT expression have been persisted with continued culture. These genes (NME5, described in other cell types, some of which may be relevant to PSCA, TSPYL5, LY75, IGFBP2, IGF2, CEACAM6, XG, tumorigenesis in vivo (3, 4). Inhibition of telomerase as a NOX5, KAL1 HPGD , and ) include eight previously therapeutic strategy is generally based on the assumption that identified as polycomb group targets. TERT-NHUC lack of telomerase activity will result in continued cell division showed overexpression of the polycomb repressor and telomere attrition, which will eventually lead to replicative complex (PRC1 and PRC4) components, BMI1 and senescence or apoptosis (5). However, inhibition of telomerase SIRT1, and down-regulation of multiple PRC targets and has an immediate effect on UC cell line viability, before genes associated with differentiation. TERT-NHUC at 100 sufficient divisions to account for telomere attrition (6). This population doublings, but not soon after transduction, strongly implicates non–telomere effects of hTERT in bladder showed increased saturation density and an attenuated tumorigenesis and suggests that telomerase inhibition may be of differentiation response, indicating that these are not rapid therapeutic benefit. Thus, identification of the genes and acute effects of telomerase expression. Some of the pathways involved in the non–telomere effects of telomerase in changes in gene expression identified may contribute bladder and other cancers may highlight novel therapeutic or to tumorigenesis. Expression of NME5 and NDN was diagnostic targets. There is also data that links the expression of down-regulated in UC cell lines and tumors. Our data telomerase with the inhibition of cellular differentiation (7, 8). supports the concept of both telomere-based and This may be a non–telomere event and is an example of how telomerase expression could contribute to tumorigenesis by mechanisms discrete from its classic actions in telomere Received 11/19/07; revised 4/18/08; accepted 4/21/08. maintenance. Grant support: In part by Cancer Research UK (C6228/A5433). hTERT-immortalized NHUC (TERT-NHUC) are generally The costs of publication of this article were defrayed in part by the payment of diploid and have no chromosomal alterations (detectable by page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. array CGH or karyotyping; ref. 2). However, changes in gene Note: Supplementary data for this article are available at Molecular Cancer expression after telomerase expression have not been Research Online (http://mcr.aacrjournals.org/). investigated. Microarray analysis of gene expression in Requests for reprints: Margaret A. Knowles, Cancer Research UK Clinical Centre, St. James’s University Hospital, Beckett Street, Leeds LS97TF, United isogenic mortal NHUC and their hTERT-immortalized coun- Kingdom. Phone: 44-11320-64913; Fax: 44-11324-29886. E-mail: m.a.knowles@ terparts was done to examine the hypothesis that expression of leeds.ac.uk Copyright D 2008 American Association for Cancer Research. telomerase contributes to tumorigenesis in ways that are doi:10.1158/1541-7786.MCR-07-2168 additional to its effect on telomere length and structure. As 1154 Mol Cancer Res 2008;6(7). July 2008 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Gene Expression Analysis of TERT-NHUC 1155 TERT-NHUC had no detectable genetic alterations, we aimed it is possible that more subtle effects on telomere structure such to identify changes in gene expression that may have occurred as at the 3¶ overhang are required for immortalization (14). To via transcriptional mechanisms. Additionally, as TERT-NHUC determine whether immortalization of NHUC was due to effects provide the basis for an in vitro model of urothelial on telomere maintenance, cells were transduced to express transformation, it is important to determine whether alterations hTERT-HA. hTERT-HA has a carboxyl-terminal hemagglutinin in gene expression are present before other genes are (HA) tag and induces soluble telomerase activity but cannot act experimentally manipulated. in telomere maintenance, probably due to an inability to interact Several previous experiments have examined the effect of with the additional proteins required (15). Despite the induction hTERT on gene expression (9-12). There is little concordance of telomerase activity (7.2-fold compared with empty vector– between genes identified in these studies, which perhaps transduced cells), the expression of hTERT-HA did not lead to a reflects the cell type–specific pathways involved in immor- significant extension in the replicative life span of NHUC, talization. This is mirrored by the different combinations of confirming that telomere-dependent effects are required for the alterations seen in tumors arising within a particular tissue. immortalization of NHUC (Fig. 1). No previous study has examined changes in gene expression after telomerase expression in matched pairs of mortal and Telomerase Activity immortal epithelial cells from multiple donors. By repeating A low level of telomerase activity was detected in mortal the experiment in three biological replicates (derived from NHUC strains. Telomerase activity in each TERT-NHUC line three cell donors) and looking for changes in expression was quantified relative to that in the isogenic NHUC cell strain. consistent to multiple donors relative to their isogenic The ratios were 17, 9, and 6 for TERT-NHUC N, TERT-NHUC controls, inter-cell line differences should be minimal. This B, and TERT-NHUC A, respectively (Fig. 2A). experimental design should increase the power to detect genes whose expression is consistently altered after expres- Expression of hTERT Leads to Consistent and Stable sion of telomerase. We propose that the effects of telomerase Changes in Gene Expression in vivo are likely to be a combination and potentially Changes in gene expression of 2.0-fold or greater were synergistic effect of the classic actions in maintenance of identified following the comparison of hTERT-transduced and telomere length and structure coupled to its currently isogenic mortal strains of NHUC from three donors. This uncharacterized non–telomere effects. For this reason, we analysis was done soon after transduction with hTERT when chose to investigate the putative non–telomere effects of cells were still within their normal mortal life span [<18 telomerase in the biologically relevant context of fully population doublings (PD)] and also when cells were deemed functional telomerase. immortal and had undergone f100 and 250 PDs. The expression of hTERT led to a statistically significant down- Results regulation of 104 probe sets, early after transduction, in at Expression of a Modified hTERT without the Ability to least two of the three donors (Table 1). These comprised 87 Elongate Telomeres Does Not Immortalize NHUC genes and 17 unknown transcripts or open reading frames It has been unclear how expression of hTERT immortalizes (orf) or hypothetical genes. In cases in which a change in NHUC, as profound shortening of telomere length is not expression in cells from one of the three donors was not observed in NHUC at replicative senescence (2, 13). However, statistically
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