Oncogene (2004) 23, 3561–3571 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc Telomere erosion and chromosomal instability in cells expressing the HPV oncogene 16E6 Annemieke W Plug-DeMaggio*,1, Terri Sundsvold1, Michelle A Wurscher1, Jennifer I Koop1, Aloysius J Klingelhutz1,3 and James K McDougall1,2,4 1Cancer Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA; 2Departmentof Pathology, University of Washington, Seattle, WA 98195, USA Progression to advanced-stage cervical carcinomas is Introduction characterized by a recurrent pattern of chromosomal rearrangements. Structural chromosome rearrangements Human cancers are subject to ongoing chromosomal are generated through the fusion of broken chromosome changes as a result of defects in the checkpoints that ends. These chromosome breaks may be induced by normally ensure stability of the genome. Two types of mutagenic agents such as ionizing radiation, or chromo- chromosomal instability are recognized: (1) aneuploidy, some ends may be exposed through extensive telomere or change in chromosome copy number and (2) shortening. The human papilloma virus oncogene 16E6 structural aberrations of chromosomes. Chromosomal induces telomerase activity in human keratinocytes, a instability is an early event in the development of human model system for cervical tumor formation. The present (Heselmeyer et al., 1996, 1997; Kirchhoff et al., 1999; study explores the relationship between 16E6 expression, Matthews et al., 2000) papillomavirus (HPV) associated telomerase activity, and chromosomal instability. We anogenital carcinoma. Epidemiological studies have show that the frequency of anaphase bridges is dependent determined that high-risk type HPVs are the main on the level of telomerase activity in 16E6/E7-expressing etiological factors for cervical cancer (Zur Hausen, clones, and is the result of telomere shortening. High 2002). Immortalization of human keratinocytes, which frequencies of anaphase bridges, associated with low are the natural host cells of HPV infection, is dependent telomerase activity, correlate with increased chromosome on expression of the HPV oncogenes E6 and E7 instability. Anaphase bridge formation is also associated (Hawley-Nelson et al., 1989; Mu¨ nger et al., 1989). The with the presence of micronuclei, which are shown to E7 oncoprotein inactivates the retinoblastoma tumor contain unstable chromosomes frequently involved in suppressor (Rb) and the cyclin-dependent kinase (CDK) rearrangements. As anaphase bridges are observed in inhibitor p21 (Dyson et al., 1989; Helt et al., 2002). both high and low telomerase 16E6/E7 clones, but not Inactivation of Rb and p21 overrides cellular senescence in hTERT-expressing control clones, expression of 16E6 by allowing expression of genes required for entry and in these immortalized clones is not sufficient to stabilize transit through the S phase (Demers et al., 1996; Jones shortened telomeres completely. We suggest a model in et al., 1997). The E6 oncoprotein targets tumor which HPV-induced tumorigenesis may be dependent on suppressor p53 for degradation (Scheffner et al., 1990; persistent bridge–breakage–fusion cycles that allow for Werness et al., 1990). Lack of p53, a transcription factor continued genomic rearrangements. which mediates the expression of a variety of genes that Oncogene (2004) 23, 3561–3571. doi:10.1038/sj.onc.1207388 induce a growth arrest or apoptosis, abrogates the cellular Published online 12 April 2004 response to DNA damage (Lakin and Jackson, 1999). Structural chromosome rearrangements are generated Keywords: human papillomavirus; telomere erosion; through the fusion of broken chromosome ends. When HPV 16E6; chromosomal instability; tumorigenesis; both chromosome fragments include a centromere, the anaphase bridges resulting dicentric chromosome is broken again when the two centromeres are pulled in opposite directions by the mitotic spindle apparatus. Unless the resulting chromosome fragments are stabilized, this process will repeat itself in a breakage–fusion–bridge (BFB) cycle (McClintock, 1941). Chromosome breaks may be *Correspondence: AW Plug-DeMaggio, Fred Hutchinson Cancer induced by mutagenic agents such as ionizing radiation, Research Center, Cancer Biology Program, 1100 Fairview Avenue or chromosome ends may be exposed through extensive N, Seattle, WA 98109-1024, USA; E-mail: [email protected] telomere shortening (reviewed in Lundblad, 2000). 3Current address: Department of Microbiology, University of Iowa, Telomere dysfunction as a result of telomere erosion 3-403 BSB, Iowa City, IA 52242, USA 4In memory of Jim McDougall, our mentor and our friend has been shown to trigger extensive DNA fragmentation Received 28 July 2003; revised 9 October 2003; accepted 20 November and evolution of complex chromosome abnormalities in 2003; Published online 12 April 2004 human malignant tumors (Gisselsson et al., 2001a, b). In Chromosomal instability in the development of HPV AW Plug-DeMaggio et al 3562 addition, telomeric erosion induced BFB cycles, which genomic rearrangements, in addition to a failure in the were shown to play an important role in epithelial mitotic spindle checkpoint, which results in aneuploidy. carcinogenesis in mice (Chang et al., 2001). Stabilization of telomere length is a critical event in the immortalization of human cells and is associated Results with telomerase activity (McEachern et al., 2000). Regulation of telomerase activity is dependent on the Anaphase bridges and nuclear abnormalities in human reverse-transcriptase component of telomerase, hTERT keratinocytes expressing the HPV oncogenes 16E6 (Meyerson et al., 1997; Nakamura et al., 1997). and E7 Previously, it has been demonstrated that HPV 16E6 can upregulate hTERT transcription (Veldman et al., To study the role of HPV oncogenes in the induction of 2001). The present study explores the relationship genomic instability, human foreskin keratinocyte cul- between expression of HPV 16E6, telomerase activity, tures were infected with retroviral constructs that and genomic instability. We show that the frequency of expressed either HPV 16E6 alone, 16E7 alone, or 16E6 anaphase bridges is dependent on the level of telomerase and E7 together. Following selection, both pooled activity in 16E6/E7-expressing clones, and is a result of populations and clones were passaged in culture. HPV telomere shortening. High frequencies of anaphase 16E6-, 16E7- and 16E6/E7-expressing clones growing in bridges, associated with low telomerase activity, corre- plates were fixed, stained with DAPI to visualize DNA, late with increased chromosome instability. Anaphase and examined using an inverted fluorescent microscope. bridge formation is associated with the presence of Previously described mitotic abnormalities in 16E6/E7 micronuclei, which are shown to contain unstable cells include multipolar metaphases and metaphases chromosomes frequently involved in rearrangements. with lagging chromosomes (Duensing et al., 2000; Plug- As anaphase bridges were observed in both high and low deMaggio and McDougall, 2002). Cytological observa- telomerase 16E6/E7 clones, but not in hTERT-expres- tions also indicate the frequent presence of anaphase sing control clones, expression of 16E6 in these bridges in cells expressing HPV oncogenes. Anaphase immortalized clones is not sufficient to stabilize shor- bridges form between daughter nuclei when the cen- tened telomeres completely. We suggest a model in tromeres of a dicentric chromosome are pulled in which HPV-induced tumorigenesis may be dependent opposite directions by the mitotic spindle (Figure on persistent BFB cycles that allow for continued 1a, b). These chromatin bridges are usually resolved Figure 1 Abnormalities in HPV 16E6/E7-expressing keratinocytes. Fixed cells were stained with DAPI and examined using an inverted fluorescent microscope. (a, b) Anaphase bridge. (c) Micronuclei in interphase. (d) Chromatin string between interphase nuclei Oncogene Chromosomal instability in the development of HPV AW Plug-DeMaggio et al 3563 through chromosome breaks; however, they may also crisis; (2) high telomerase activity precrisis, reduced or remain as chromatin strings between two interphase low activity postcrisis; (3) low telomerase activity pre- cells (Figure 1d). Anaphase bridges are present at varying frequencies in cells expressing both 16E6 and E7 together, and become prominent around passage 15 a (Figure 2a). Cells expressing 16E7 alone have a 20 significantly higher frequency of anaphase bridges than 18 cells expressing 16E6 alone at passage 15 (Figure 2b). 16 Following selection, the frequency of anaphase bridges 14 varies between 0 and 3.5% at passage 4, similar to 12 control HFKs (Figure 2a). This suggests that the 10 * * expression of 16E6 may not have a direct effect on the 8 formation of anaphase bridges. If anaphase bridge 6 4 frequencies would have been equally high at this early % anaphase bridges 2 passage as compared to later passages, a direct effect of 0 HPV 16E6 on the formation of these bridges, for 24579262728381739 example by inducing chromosome breaks, could have HPV 16E6/E7-clones been suspected. However, our data clearly indicate that p4 (*p8) p15 this is not the case. b Cytological analysis of 16E6- and/or 16E7-expressing 30 clones also revealed the occurrence of micronuclei in interphase cells. Micronuclei are formed by nuclear 25 membrane formation around either a lagging chromo- 20 some or chromosomal