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Aneuploidy and Telomere Attrition Are Independent Determinants of Crisis in SV40-Transformed Epithelial Cells1

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Aneuploidy and Telomere Attrition Are Independent Determinants of Crisis in SV40-Transformed Epithelial Cells1 [CANCER RESEARCH 63, 5813–5820, September 15, 2003] Aneuploidy and Telomere Attrition Are Independent Determinants of Crisis in SV40-transformed Epithelial Cells1 Mihaela Velicescu, Jiamei Yu, Brittney-Shea Herbert, Jerry W. Shay, Eileen Granada, and Louis Dubeau2 University of Southern California/Norris Comprehensive Cancer Center, Department of Pathology, University of Southern California, Keck School of Medicine, Los Angeles, California 90089-9181 [M. V., J. Y., E. G., L. D.], and University of Texas Southwestern Medical Center, Department of Cell Biology, Dallas, Texas 75390-9039 [B-S. H., J. W. S.] ABSTRACT locations (14), and regional deletions and amplifications (15). Most of these abnormalities have been postulated to be a direct consequence of Replicative immortality is achieved in vitro by overcoming two mortal- the loss of telomeric sequences through the formation of dicentric ity checkpoints, M1 (senescence) and M2 (crisis). Cancer cells are thought chromosomes and breakage-fusion-bridge cycles (16). In contrast, to overcome M2 by activating telomerase, an enzyme believed to confer genomic stability in addition to maintaining telomeric sequences above a numerical chromosomal alterations leading to aneuploidy cannot be critical length. Here we show that a subset of cultured ovarian cystade- simply explained by a simple loss of telomeric sequences (17, 18). noma cells expressing SV40 large T-antigen, which allows bypassing of These alterations are linked to missegregation of the chromosomes, M1, develop a specific type of genomic instability, characterized by nu- possibly due to loss of p53 and RB proteins, which are essential merical (as opposed to structural) chromosomal alterations, that leads to regulators of the mitotic spindle assembly (19) and of normal centro- non-telomere-based premature growth arrest/crisis. Cells recover from some formation during mitosis (20, 21). The ectopic expression of this type of growth arrest and stabilize their ploidy status without telom- telomerase appears to protect against genomic instability (22). It has erase expression. In these cases, telomeres continue to shorten until a been suggested that telomere-based DNA damage may be prevented second, telomere-based growth arrest/crisis event is reached. Transfection by the stabilization of the telomere ends, whereas nontelomere DNA of the catalytic subunit of telomerase does not immortalize cells harboring damage may be more efficiently detected and repaired in cells ex- severe abnormalities in their DNA ploidy but results in immortalization of diploid cells. We conclude that changes in DNA ploidy constitute an pressing telomerase (23). This idea that telomerase expression may important determinant of growth arrest that is independent of telomere confer genetic stability is intriguing, given that this enzyme is ex- attrition in a subset of SV40 large T-antigen-expressing cystadenoma cells. pressed in most cancer cells, which are thought to be genetically more Reestablishment or emergence of ploidy stability, which is not always unstable than their normal counterparts that do not express telomerase. dependent on telomerase activation, is necessary for acquisition of the We sought to better understand the relationship between crisis, potential to achieve replicative immortality. telomere attrition, and development of aneuploidy because such un- derstanding might provide important insights into the mechanism of genetic instability in human cancers. Although our knowledge of in INTRODUCTION vitro mortality checkpoints comes largely from work done with fi- One of the most consistent differences between normal cells and broblasts, we used an epithelial cell model because most human cancer cells cultured in vitro is the fact that cancer cells can divide cancers arise in epithelial cells. We took advantage of established indefinitely, whereas normal cells have a limited life span (1). Most cultures of epithelial cells derived from benign ovarian epithelial normal cells lose telomeric DNA each time they undergo DNA tumors (cystadenomas), which express an adenovirus vector for SV40 synthesis due to their inability to replicate their chromosomal ends (2). large T-antigen that had become stably integrated into the host ge- Such telomere attrition is thought to trigger growth arrest signals that nome (24). This viral oncoprotein, which interferes with RB and p53 limit the life span of normal cells by activating two mortality check- proteins, allows bypassing of M1 but not of M2 (8). Two cell clones points known as senescence [M1] (3, 4) and crisis [M2] (5). M1 is derived from one SV40 large T-antigen-expressing cystadenoma cell characterized by absence of cell division due to inhibition of the cell strain called ML10 (24) recovered from M2 and became immortal cell cycle (6). Cells that overcome or bypass M1 due to loss of cell lines, providing a longitudinal model for studying the mechanisms cycle-inhibitory signals, such as the absence of functional RB or p53 associated with acquisition of ability to bypass crisis. Our observa- proteins, can extend their replicative life span but eventually reach a tions suggest that the form of genetic instability that is associated with second mortality checkpoint, M2, also known as crisis (7, 8). It is numerical chromosomal alterations, in contrast to that associated with thought that cells that overcome this checkpoint, usually through structural chromosomal changes, precedes significant telomere attri- activation of telomerase, an enzyme capable of maintaining telomeric tion in this cell culture model, implying that shortening of the te- sequences above a critical length, acquire the ability to grow indefi- lomeres is unlikely to play any significant role in the induction of this nitely (9–11). In support of these concepts, in vitro replicative im- form of instability. Our results further suggest that this form of mortality is achieved by ectopic expression of the human catalytic genomic instability, which is also prominent in most human cancers, subunit of telomerase (hTERT) in normal cells (12). is an independent mediator of crisis that is not overcome by telom- Crisis is often accompanied by widespread genomic instability (5) erase expression. These findings not only clarify the relationship characterized by both structural and numerical chromosomal alter- between crisis and genomic instability but also provide insights into ations. Structural alterations include fusions (13), nonreciprocal trans- potential mechanisms for the acquisition of aneuploidy during cancer development. Received 8/14/02; revised 6/25/03; accepted 7/8/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with MATERIALS AND METHODS 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grants CA 51167, CA 79750, CA70907, and Cell Lines and Strains and Culture Conditions. ML3, ML5, and ML10 CA14089. cell strains were established from primary cultures of benign ovarian epithelial 2 To whom requests for reprints should be addressed, at Department of Pathology, tumors (cystadenomas) that were infected with an adenovirus vector express- University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine of University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA ing SV40 large T-antigen (24). This vector had become stably integrated into 90089-9181. Phone: (323) 865-0720; Fax: (323) 865-0077; E-mail: [email protected]. the host genomes upon multiple reinfections (24). MCV39, MCV500, and 5813 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. DETERMINANTS OF IN VITRO CRISIS MCV50 cells were derived from ML10 as described in “Results.” HOC-7 cells ethanol and air dried. The slides were incubated with a hybridization mixture ␮ Ј Ј were obtained from Dr. Ronald N. Buick (University of Toronto; Ref. 25). All (20 l) containing 70% formamide, 3 -Cy3-conjugated (CCCTAA)3 2 - Ј Ј cells were grown in MEM (Invitrogen, Carlsbad, CA) supplemented with 10% deoxyoligonucleotide N3 -P5 phosphoramidate telomeric probe and FITC- FBS.3 The origin and characteristics of VA13 cells have been described conjugated centromeric probe (kindly provided by Geron Corp., Menlo Park, previously (26). CA), 0.25% (w/v) blocking reagent (Roche Molecular Biochemicals), and 5% Telomere Length Determination. Ten-␮g samples of genomic DNA di- MgCl2 in 10 mM Tris (pH 7.2) for 3 min at 78°C. The slides were then gested with RsaI/HinfIII restriction endonucleases were electrophoresed on incubated for2hatroom temperature and washed twice with 70% formamide, 0.8% agarose gels and transferred to Zetabind (Cuno, Inc., Meride, CT) 0.1% BSA, and 10 mM Tris (pH 7.2). After two washes with 0.15 M NaCl, membranes according to the manufacturer’s protocol. The membranes were 0.05% Tween 20, and 0.05 M Tris, the slides were dehydrated by a 2-min hybridized to a 32P-labeled probe consisting of the basic human telomeric incubation in ethanol, air dried in the dark, mounted with Vectashield con- taining 4Ј,6-diamidino-2-phenylindole (Vector Laboratories), and imaged us- sequence (TTAGGG)3 in Church buffer [500 mM NaPO4 (pH 6.8), 7% SDS, and1mM EDTA (pH 8.0)] at 42°C for 24 h. The membranes were washed ing a Zeiss Axioplan 2 microscope. twice for 10 min at room temperature in 2ϫ SSC, 0.5% SDS, followed by two ϫ 15-min washes in 0.2 SSC, 0.1% SDS. The hybridization signals were RESULTS visualized by phosphorimaging. Assay of Telomerase Activity. Telomerase activity was detected using the Numerical Chromosomal Alterations Precede Telomere Attri- TRAP assay (27) as described previously (28). tion in Ovarian Cystadenoma Cells Expressing SV40 Large T- Analysis of DNA Ploidy by Flow Cytometry. One million cells resus- antigen. Changes in DNA ploidy are a well-documented manifesta- pended in PBS were fixed in 70% ethanol. After centrifugation, the cell pellets tion of chromosomal instability in cells expressing SV40 large were resuspended in 1 ml of PBS, 10 ␮g/ml propidium iodide, and 100 ␮g/ml T-antigen (5, 29–31).
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