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[Cancer Research 63, 5813-5820, September 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Replicative immortality is achieved in vitro by overcoming two mortality checkpoints, M1 (senescence) and M2 (crisis). Cancer cells are thought to overcome M2 by activating telomerase, an enzyme believed to confer genomic stability in addition to maintaining telomeric sequences above a critical length. Here we show that a subset of cultured ovarian cystadenoma cells expressing SV40 large T-antigen, which allows bypassing of M1, develop a specific type of genomic instability, characterized by numerical (as opposed to structural) chromosomal alterations, that leads to non-telomere-based premature growth arrest/crisis. Cells recover from this type of growth arrest and stabilize their ploidy status without telomerase expression. In these cases, telomeres continue to shorten until a second, telomere-based growth arrest/crisis event is reached. Transfection of the catalytic subunit of telomerase does not immortalize cells harboring severe abnormalities in their DNA ploidy but results in immortalization of diploid cells. We conclude that changes in DNA ploidy constitute an important determinant of growth arrest that is independent of telomere attrition in a subset of SV40 large T-antigen-expressing cystadenoma cells. Reestablishment or emergence of ploidy stability, which is not always dependent on telomerase activation, is necessary for acquisition of the potential to achieve replicative immortality.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the most consistent differences between normal cells and cancer cells cultured in vitro is the fact that cancer cells can divide indefinitely, whereas normal cells have a limited life span (1) . Most normal cells lose telomeric DNA each time they undergo DNA synthesis due to their inability to replicate their chromosomal ends (2) . Such telomere attrition is thought to trigger growth arrest signals that limit the life span of normal cells by activating two mortality checkpoints known as senescence [M1] (3 , 4) and crisis [M2] (5) . M1 is characterized by absence of cell division due to inhibition of the cell cycle (6) . Cells that overcome or bypass M1 due to loss of cell cycle-inhibitory signals, such as the absence of functional RB or p53 proteins, can extend their replicative life span but eventually reach a second mortality checkpoint, M2, also known as crisis (7 , 8) . It is thought that cells that overcome this checkpoint, usually through activation of telomerase, an enzyme capable of maintaining telomeric sequences above a critical length, acquire the ability to grow indefinitely (9, 10, 11) . In support of these concepts, in vitro replicative immortality is achieved by ectopic expression of the human catalytic subunit of telomerase (hTERT) in normal cells (12) .

Crisis is often accompanied by widespread genomic instability (5) characterized by both structural and numerical chromosomal alterations. Structural alterations include fusions (13) , nonreciprocal translocations (14) , and regional deletions and amplifications (15) . Most of these abnormalities have been postulated to be a direct consequence of the loss of telomeric sequences through the formation of dicentric chromosomes and breakage-fusion-bridge cycles (16) . In contrast, numerical chromosomal alterations leading to aneuploidy cannot be simply explained by a simple loss of telomeric sequences (17 , 18) . These alterations are linked to missegregation of the chromosomes, possibly due to loss of p53 and RB proteins, which are essential regulators of the mitotic spindle assembly (19) and of normal centrosome formation during mitosis (20 , 21) . The ectopic expression of telomerase appears to protect against genomic instability (22) . It has been suggested that telomere-based DNA damage may be prevented by the stabilization of the telomere ends, whereas nontelomere DNA damage may be more efficiently detected and repaired in cells expressing telomerase (23) . This idea that telomerase expression may confer genetic stability is intriguing, given that this enzyme is expressed in most cancer cells, which are thought to be genetically more unstable than their normal counterparts that do not express telomerase.

We sought to better understand the relationship between crisis, telomere attrition, and development of aneuploidy because such understanding might provide important insights into the mechanism of genetic instability in human cancers. Although our knowledge of in vitro mortality checkpoints comes largely from work done with fibroblasts, we used an epithelial cell model because most human cancers arise in epithelial cells. We took advantage of established cultures of epithelial cells derived from benign ovarian epithelial tumors (cystadenomas), which express an adenovirus vector for SV40 large T-antigen that had become stably integrated into the host genome (24) . This viral oncoprotein, which interferes with RB and p53 proteins, allows bypassing of M1 but not of M2 (8) . Two cell clones derived from one SV40 large T-antigen-expressing cystadenoma cell strain called ML10 (24) recovered from M2 and became immortal cell lines, providing a longitudinal model for studying the mechanisms associated with acquisition of ability to bypass crisis. Our observations suggest that the form of genetic instability that is associated with numerical chromosomal alterations, in contrast to that associated with structural chromosomal changes, precedes significant telomere attrition in this cell culture model, implying that shortening of the telomeres is unlikely to play any significant role in the induction of this form of instability. Our results further suggest that this form of genomic instability, which is also prominent in most human cancers, is an independent mediator of crisis that is not overcome by telomerase expression. These findings not only clarify the relationship between crisis and genomic instability but also provide insights into potential mechanisms for the acquisition of aneuploidy during cancer development.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Strains and Culture Conditions.
ML3, ML5, and ML10 cell strains were established from primary cultures of benign ovarian epithelial tumors (cystadenomas) that were infected with an adenovirus vector expressing SV40 large T-antigen (24) . This vector had become stably integrated into the host genomes upon multiple reinfections (24) . MCV39, MCV500, and MCV50 cells were derived from ML10 as described in "Results." HOC-7 cells were obtained from Dr. Ronald N. Buick (University of Toronto; Ref. 25 ). All cells were grown in MEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS.3 The origin and characteristics of VA13 cells have been described previously (26) .

Telomere Length Determination.
Ten-µg samples of genomic DNA digested with RsaI/HinfIII restriction endonucleases were electrophoresed on 0.8% agarose gels and transferred to Zetabind (Cuno, Inc., Meride, CT) membranes according to the manufacturer’s protocol. The membranes were hybridized to a 32P-labeled probe consisting of the basic human telomeric sequence (TTAGGG)3 in Church buffer [500 mM NaPO4 (pH 6.8), 7% SDS, and 1 mM EDTA (pH 8.0)] at 42°C for 24 h. The membranes were washed twice for 10 min at room temperature in 2x SSC, 0.5% SDS, followed by two 15-min washes in 0.2x SSC, 0.1% SDS. The hybridization signals were visualized by phosphorimaging.

Assay of Telomerase Activity.
Telomerase activity was detected using the TRAP assay (27) as described previously (28) .

Analysis of DNA Ploidy by Flow Cytometry.
One million cells resuspended in PBS were fixed in 70% ethanol. After centrifugation, the cell pellets were resuspended in 1 ml of PBS, 10 µg/ml propidium iodide, and 100 µg/ml RNase. Fluorescence was measured on a Coulter Profile II flow cytometer (Beckman Coulter, Hialeah, FL) and analyzed using the MultiCycle software (Phoenix Flow Systems Inc., San Diego, CA).

FACS.
Cells cultured on plastic dishes were incubated in MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FBS in the presence of 10 µM Hoechst 33342 reagent (Molecular Probes, Inc., Eugene, OR) for 90 min at 37°C. After dissociation with 0.05% trypsin/0.02% EDTA, the cells were resuspended in MEM plus 20% FBS and 10 µM Hoechst 33342. The cells were kept at 4°C until sorted based on fluorescence intensity using a FACSar plus cytometer (Becton Dickinson, San Jose, CA).

TUNEL Assay.
Cells cultured on Lab-Tek chamber slides (Miles Scientific) were fixed in freshly prepared 4% methanol-free formaldehyde (Polysciences, Inc., Warrington, PA) in PBS for 25 min at 4°C. After incubation in equilibration buffer containing terminal deoxytransferase and fluorescein-labeled dUTP (Apoptosis Detection Kit; Promega, Madison, WI) at 37°C for 60 min inside a humidified chamber, the cells were protected from light and treated for 15 min at room temperature with 40 ml of 1 mg/ml propidium iodide solution freshly diluted in PBS. Cells with three or more green fluorescent nuclear signals seen under a fluorescence microscope were scored as positive for apoptosis.

Detection of APBs by Immunofluorescence.
Cells grown on coverslips overnight were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 before blocking with 3% BSA. Staining was performed with a PML and either a Rad51 or a TRF2 antibody (Santa Cruz Biotechnology) diluted in PBS with 0.5% sodium azide. Secondary antibodies included Alexa-Fluor 488-conjugated antimouse and 568-conjugated antirabbit immunoglobulins (Molecular Probes). The coverslips were mounted with Vectashield containing 4',6-diamidino-2-phenylindole (Vector Laboratories), and cells were examined using a Zeiss fluorescence microscope. The percentage of APBs were scored as the percentage of total cells with PML/Rad51 or PML/TRF2 colocalized in the nuclei.

Metaphase Spread Preparation and FISH.
Cultured cells were treated with 5 µg/ml Colcemid (Invitrogen) for 4 h and then harvested as usual. After a 30-min incubation in hypotonic 0.075 M KCl at 37°C, the cells were fixed and washed three times with methanol/acetic acid (3:1) and dropped onto slides. One- to seven-day-old slides were rehydrated in 1x PBS (pH 7.5) for 15 min at room temperature. The slides were fixed in 4% formaldehyde in PBS (pH 7.5) for 2 min and washed in 1x PBS three times (5 min each time). The slides were treated with1 mg/ml pepsin (pH 2) at 37°C for 10 min and washed twice for 2 min each time in 1x PBS. The slides were then fixed in the formaldehyde solution for 2 min and washed again in 1x PBS three times. Slides were dehydrated by 2-min serial incubations in 70%, 90%, and 100% ethanol and air dried. The slides were incubated with a hybridization mixture (20 µl) containing 70% formamide, 3'-Cy3-conjugated (CCCTAA)3 2'-deoxyoligonucleotide N3'-P5' phosphoramidate telomeric probe and FITC-conjugated centromeric probe (kindly provided by Geron Corp., Menlo Park, CA), 0.25% (w/v) blocking reagent (Roche Molecular Biochemicals), and 5% MgCl2 in 10 mM Tris (pH 7.2) for 3 min at 78°C. The slides were then incubated for 2 h at room temperature and washed twice with 70% formamide, 0.1% BSA, and 10 mM Tris (pH 7.2). After two washes with 0.15 M NaCl, 0.05% Tween 20, and 0.05 M Tris, the slides were dehydrated by a 2-min incubation in ethanol, air dried in the dark, mounted with Vectashield containing 4',6-diamidino-2-phenylindole (Vector Laboratories), and imaged using a Zeiss Axioplan 2 microscope.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Numerical Chromosomal Alterations Precede Telomere Attrition in Ovarian Cystadenoma Cells Expressing SV40 Large T-antigen.
Changes in DNA ploidy are a well-documented manifestation of chromosomal instability in cells expressing SV40 large T-antigen (5 , 29, 30, 31) . The presence and magnitude of such ploidy changes in ovarian cystadenoma cells expressing SV40 large T-antigen are illustrated in Fig. 1aCitation , which compares the DNA content of a strain of ovarian cystadenoma called ML10 after approximately 30 versus 42 PDs in vitro. Although the cells were predominantly diploid after 30 PDs, the majority had become either tetraploid or aneuploid after as little as 12 additional PDs. We estimated the percentage of aneuploid cells in cultured cystadenomas from the ratio of the number of nondiploid cells (excluding apoptotic cells) over the total number of nonapoptotic cells present based on flow cytometry data. Comparing the changes in ploidy status with changes in doubling time at various time points preceding crisis showed that the ploidy changes did not accumulate at a constant rate throughout the life span of the cells but took place primarily during the 5 PDs that preceded any noticeable increase in doubling time due to crisis (Fig. 1b)Citation . An abundance of aneuploid cells in this period was confirmed by examining the number of chromosomes in metaphase spreads prepared from these SV40-transfected cells (data not shown). These results are similar to what was observed by Romanov et al. (32) in primary cultures of normal breast epithelial cells.



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  		Fig. 1. Changes in DNA content and telomere length in ovarian cystadenoma cells expressing SV40 large T-antigen. a, ML10 cells, derived from an ovarian cystadenoma and transfected with an expression vector for SV40 large T-antigen, were stained with 10 µg/ml propidium iodide and examined for DNA content by flow cytometry after 30 and 42 PDs in vitro. b, ML10 cells that had been in culture for approximately 25 PDs were analyzed by flow cytometry to determine the percentage of cells with a nondiploid DNA content. These measurements were repeated every 3.3 PDs until the cells reached crisis. Doubling times were measured at each time point by growth curve analysis. c, telomere length was analyzed by Southern blotting in ML3, ML5, and ML10 ovarian cystadenoma cells after 20 PDs or after either 45, 40, or 50 PDs as indicated. ML3, ML5, and ML10 cells typically reach crisis after 40–45, 35–40, and 45–50 PDs, respectively.

 
We sought to determine whether the genomic instability resulting in these changes in DNA ploidy, which precede the onset of crisis, coincides with changes in telomere length. Although the current evidence suggests an important role for telomere attrition in the induction of crisis (17 , 18) , the role of the type of genetic instability that is associated with numerical chromosomal changes leading up to crisis is less clear. TRF length was examined by Southern blotting in three strains of ovarian cystadenomas, ML3, ML5, and ML10, after 20 PDs and after either 45 PDs (for ML3), 40 PDs (for ML5), or 50 PDs (for ML10) in vitro. These cell strains, none of which express telomerase, reach crisis after approximately 40–45, 35–40, and 45–50 PDs, respectively. One telomerase-positive ovarian carcinoma cell line (HOC-7) was also included in the analysis for comparison. Although a slight shortening in the average TRF length had indeed occurred in all three strains when they reached crisis, the magnitude of the mean telomere shortening (cell to cell variation) was minimal (Fig. 1c)Citation , suggesting that this type of crisis was either initiated by a few short telomeres or that crisis was not caused by telomere shortening at all. The average telomere length in ML10 cells at 20 PDs in this experiment was 8.60 ± 0.38 kb, whereas that of ML10 cells entering crisis (50 PDs) was 7.34 ± 0.10 kb. This size was much greater than that seen in HOC-7 cells (Fig. 1c)Citation , which had a calculated average telomere length of 2.71 ± 0.23 kb. Examination of the chromosomes in metaphase spreads of cystadenoma cells approaching crisis showed no evidence of end-to-end fusion in 200 cells examined.

Genetic Instability Resulting in Numerical Chromosomal Alterations Is a Determinant of Crisis Independent of Telomere Shortening.
The results of Fig. 1bCitation strongly suggest the existence of a close relationship between the development of alterations in DNA ploidy and the establishment of crisis. We investigated whether such alterations could by themselves play a causative role in the development of crisis. ML10 cells that had undergone approximately 25 PDs in vitro were stained with Hoechst 33342 dye, a fluorescent DNA binding compound able to diffuse intracellularly into living cells. The intensity of fluorescence emitted reflected cellular DNA content, allowing separation of different cell populations based on their ploidy status by FACS (Fig. 2a)Citation . Metaphase spreads prepared from cells that were put back in culture after having been subjected to such sorting procedures showed that 24% of the cells recovered in the nondiploid fraction had a normal number of chromosomes compared with 76% of the cells recovered in the diploid fraction (data not shown). Although the median number of chromosomes in the nondiploid cells was around 92 (i.e., near tetraploid), there was marked heterogeneity within the cell population, with the majority of the cells showing varying degrees of aneuploidy. There were no significant differences in the doubling time of the sorted diploid and nondiploid cells immediately after the sorting procedures (data not shown). However, growth curve analyses performed 10 PDs after the sorting procedures revealed a substantial increase in the doubling time of nondiploid cells compared with cells derived from age-matched diploid fractions (Fig. 2b)Citation . The sorted diploid cells maintained their logarithmic growth for an additional 10 PDs after the aneuploid cells reached crisis, at which point they also reached crisis. Thus, cells harboring severe ploidy changes reached crisis earlier than the age-matched diploid cells. This result was reproduced in seven independent cell sorting experiments. Growth arrest in the nondiploid cell fraction was not due to loss of SV40 large T-antigen because this antigen continued to be expressed in both cell fractions after the sorting procedures (Fig. 2c)Citation . The p53 and p21 proteins, which become elevated during in vitro crisis (data not shown), showed higher expression levels in the nondiploid cells (Fig. 2c)Citation . Although this may provide further support to the idea that crisis was more advanced in these cells, the contribution of potential differences in gene dosage due to ploidy changes involving the p53 and p21 loci has not been investigated. The telomeres were of similar sizes in both cellular fractions (Fig. 2d)Citation , suggesting that these differences in the timing of initiation of crisis were not necessarily due to differences in telomere attrition. Further evidence that the changes in in vitro kinetics represented crisis as opposed to senescence comes from lack of expression of senescence-associated ß-galactosidase, a well-established senescence-associated marker (33) , in cells undergoing growth arrest (data not shown).



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  		Fig. 2. Isolation and characterization of ML10 cell subpopulations fractionated based on differences in their DNA content. a, ML10 ovarian cystadenoma cells that had completed an average of 25 PDs in vitro were treated with 10 µM Hoechst 33342 reagent and separated into diploid and nondiploid fractions by FACS. R1 and R2 indicate the position of the sorting gates. b, the sorted diploid and nondiploid fractions were analyzed by growth curve analysis performed 10 PDs after the sorting procedures. The sorted diploid and nondiploid fractions were analyzed for expression of SV40 large T-antigen, p53, p21, and actin (used as protein loading control) by Western blotting (c) and for telomere length by Southern blotting (d).

 
Recovery from Crisis-based Chromosomal Instability Is Not Dependent on Telomerase Activation.
ML10 ovarian cystadenoma cells sorted based on their DNA content were kept in culture through their crisis period. Of 17 separate experiments with either sorted or unsorted cells, spontaneous recovery from crisis occurred twice, resulting in the establishment of two cell lines called MCV39 and MCV500. Both cell lines were derived from nondiploid fractions of sorted ML10 cells. Microscopic examination of metaphase spreads revealed that MCV39 had 57 chromosomes, whereas MCV500 had 67 chromosomes (data not shown). MCV39 cells were cultured for over 200 PDs and showed no change in their in vitro kinetics over this time period, attesting to their true immortality. In contrast, MCV500 cells showed logarithmic growth for only 35 PDs, at which point they underwent a second crisis. Recovery from this second crisis was observed only once in five separate attempts (using MCV500 cells that had been stored frozen in liquid nitrogen soon after their recovery from the first crisis), resulting in the establishment of a second immortal cell line, derived from MCV500, called MCV50.

The earliest time at which we were able to analyze TRF length in MCV39 and MCV500 cells was 20 PDs after they had recovered from crisis. Compared with the parental ML10 cells, MCV39 cells had considerably shorter telomeres at this time point (20 PDs) and expressed telomerase (Fig. 3)Citation . Telomere length and telomerase activity were essentially unchanged in this cell line 30 PDs later (Fig. 3)Citation . The severe imbalances in DNA ploidy that characterized the parental ML10 cells were no longer apparent in MCV39 cells, at least based on analysis of cellular DNA content by flow cytometry (Fig. 3)Citation . MCV500 cells differed from age-matched MCV39 cells in that they showed no measurable telomerase activity, and their average TRF length was substantially larger after 20 PDs (Fig. 3)Citation . Similarly to MCV39, however, they showed a relatively stable DNA profile compared with the parental ML10 cells (Fig. 3)Citation . Thus, telomerase reactivation was not required for overcoming the first crisis event in MCV500, nor was it required for conferring stability in DNA ploidy. The average TRF length decreased rapidly between the first and second crisis (Fig. 3)Citation . Recovery from the second crisis event occurred in cells with short TRF lengths and was accompanied by the appearance of telomerase activity (Fig. 3)Citation . This activity persisted at later passages, which showed no further changes in TRF length (Fig. 3)Citation .



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  		Fig. 3. Examination of telomerase activity and genetic stability after recovery from crisis. Sorted nondiploid ML10 cells were kept in culture approximately 4 weeks after reaching crisis, at which time two cell clones, MCV39 and MCV500, recovered from crisis. After 35 further PDs, MCV500 cells underwent a second crisis event. An immortal cell clone called MCV50 recovered from this second crisis. Top panel, Southern blot analysis of telomere length in the parental ML10 cells and in MCV39, MCV500, and MCV50 cells at various time points after their isolation (expressed as number of PDs). Middle panel, telomerase activity was examined by TRAP assay at selected time points in the various cell lines and strains. The characteristic ladders in the second, third, and fifth lanes indicate the presence of such activity. Bottom panel, examination of DNA content in the parental ML10 cells and in MCV39 and MCV500 cells by flow cytometry. G1 indicates the position of the peaks corresponding to cells in the G1 phase of the cell cycle.

 
Absence of ALT.
We were intrigued by the fact that whereas little telomere attrition took place before the initiation of ploidy-dependent crisis (pdM2) in all three cystadenoma cell strains examined, the rate of attrition appeared to be accelerated after recovery from pdM2 in MCV500 cells. We investigated the possibility that our cystadenoma cell strains developed ALT (34) in the absence of telomerase before the first crisis. Indeed, ALT mechanisms are common in SV40-transformed cell lines (35) . We therefore performed immunofluorescence staining of our cultured cells with antibodies against PML bodies and either Rad51 or a TRF2, which are components of APBs (36) . The percentage of cells containing such bodies, which was scored as the percentage of total cells with PML/Rad51 or PML/TRF2 colocalized in the nuclei (Fig. 4, a and b)Citation , was less than 1% for most of the cells examined. Another and perhaps more specific characteristic of ALT is the presence of marked heterogeneity in telomere length and extrachromosomal telomeric repeats (35) . These changes are readily apparent in Fig. 4dCitation , which shows the results of FISH experiments on VA13 cells using a probe for telomeric/centromeric DNA. These cells, which were derived from normal human lung fibroblasts transfected with SV40 large T-antigen, were previously shown to use ALT mechanisms (26) . In contrast, very few, if any, extrachromosomal telomeric repeats were observed and there was no evidence of long and heterogeneous telomeres in ML10 cells (Fig. 4c)Citation . We conclude that the lack of significant telomere attrition in cystadenoma cells approaching crisis was not due to the presence of ALT mechanisms.



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  		Fig. 4. Lack of ALT mechanisms in cultured ovarian cystadenomas. a and b, ML10 cells were reacted with primary antibodies against PML bodies and Rad51 (a) or TRF2 (b), followed by appropriate fluorescent secondary antibodies. Immunoreactivity for PML resulted in green fluorescence (long arrows), whereas reactivity for either Rad51 or TRF2 resulted in red fluorescence (short arrows). The figure shows superimposed photographs of the two immunostains, revealing very little nuclear colocalization of either PML/Rad51 or PML/TRF2 and, thus, no evidence of APBs. c and d, analysis of ML10 ovarian cystadenoma cells (c) and VA13 cells (d) by FISH using probes for telomeric/centromeric DNA. The results show a large number of extrachromosomal telomeric repeats (arrows; Ref. 26 ) as well as marked heterogeneity in telomeric lengths in VA13 cells, which use ALT mechanisms (26) , whereas such changes are absent in ML10 cells.

 
Consequences of Ectopic Expression of hTERT in Diploid versus Aneuploid Cells.
We further examined the role of telomerase on chromosomal stability in our cell culture model by introducing an expression vector for the catalytic subunit of the human telomerase cDNA (hTERT) into ML3, ML5, and ML10 cystadenoma cells. Several immortal clones of transfected cells were isolated that were essentially diploid and showed a normal and stable DNA profile when analyzed by flow cytometry (data not shown). Only a small proportion of aneuploid cells were detectable immediately after the DNA transfection procedures. These transfected aneuploid cells were separated from the more abundant diploid cells by FACS. Although we were able to obtain cell populations that were highly enriched for telomerase-positive aneuploid cells using such sorting procedures, the small number of diploid cells present in the sorted cell population outgrew the nondiploid cells in as little as 35 PDs (Fig. 5, a and b)Citation . The cells remained diploid thereafter, as attested by flow cytometry studies as well as by performing chromosome counts in metaphase spreads (data not shown). No appreciable differences in the doubling time could be detected in the two fractions based on growth curve analyses performed immediately after the cell sorting procedure (data not shown). TUNEL assay (data not shown) showed evidence of apoptosis in 34 ± 5% of the nondiploid cell fraction compared with 15 ± 4% of the diploid cell fraction (measured by determining the number of apoptotic cells/100 cells/field in four separate fields), suggesting that the selective disadvantage of nondiploid cells was due, at least in part, to increased apoptosis, which is one of the hallmarks of crisis.



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  		Fig. 5. Consequences of telomerase expression in diploid versus nondiploid cells. ML10 cells transfected with an expression vector for the catalytic subunit of telomerase were separated into diploid and nondiploid fractions by FACS. a and b, the DNA content of the nondiploid fraction was analyzed by flow cytometry 3 (a) and 35 (b) PDs after the cell sorting procedures. The results show that the aneuploid cells had been completely overgrown by the diploid cells by the time this mixed cell population had reached 35 PDs. c, one diploid and two aneuploid subclones, the latter containing 61 and 109 chromosomes, respectively, were isolated from the sorted aneuploid fraction by plating the cells onto 96-well microtiter plates at a density of 0.5 cell/well. All three clones expressed telomerase, as evidenced by positive TRAP assay results (data not shown). The figure shows growth curve analyses of each clone performed in parallel and initiated approximately 20–25 PDs after the cloning procedures, at which time the aneuploid clones were showing signs of crisis. The number of cells/dish was determined using a Coulter counter. Each point represents the average of triplicate dishes. The vertical bars represent SEs.

 
The fact that telomerase-transfected aneuploid cells were outgrown by the initially small proportion of diploid cells suggests that ectopic expression of telomerase could not immortalize cells harboring severe changes in DNA ploidy, although it was sufficient to immortalize cells with normal DNA content or with less severe ploidy changes. Subclonal populations of sorted telomerase-transfected cells were obtained and examined for their in vitro longevity to verify this hypothesis. Most of the subclones recovered from the sorted aneuploid fraction were diploid, further attesting to the selective advantage of such cells over the more abundant aneuploid cells. Two aneuploid subclones, containing 61 and 109 chromosomes, respectively, were cultured in parallel with an age-matched diploid subclone. Telomerase expression was verified in all three subclones by the TRAP assay (data not shown). The two aneuploid subclones started showing signs of crisis after an estimated 20–25 PDs following the cloning procedures (Fig. 5c)Citation . Such changes were noticed 3–5 PDs earlier in the subclone with 109 chromosomes compared with the one with 61 chromosomes (data not shown). The aneuploid subclones could not be propagated further after this experiment, attesting to the fact that they had not been immortalized by transfection with the catalytic subunit of telomerase.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results clearly show that a specific type of genetic instability associated with numerical chromosomal alterations is an independent determinant of crisis in ovarian cystadenoma cells expressing SV40 large T-antigen. Such cells underwent crisis after accumulation of severe changes in their DNA ploidy and before extensive telomere attrition had taken place. This lack of significant telomere attrition in the period preceding crisis was not due to ALT mechanisms. Separation of cells that had undergone severe ploidy changes from those that had remained diploid using FACS showed that crisis occurred earlier in the nondiploid fraction, despite similar telomere lengths in all fractions. Recovery from ploidy-dependent crisis as well as from this specific type of genetic instability did not depend on telomerase expression because MCV500 cells, which underwent two separate crisis events, did not express this enzyme after recovery from the first event, although their ploidy status had become stable. It is only after recovery from the second crisis event and after further telomere shortening, 35 PDs later, that induction of telomerase expression was observed in those cells. Expression of telomerase in ovarian cystadenoma cells expressing SV40 large T-antigen could confer in vitro immortality only to cells that had recovered or escaped from ploidy-dependent crisis. We conclude that there are at least two determinants of crisis in this epithelial cell culture system: one that is ploidy dependent (pdM2); and another that is telomere dependent (tdM2). These results are summarized in the diagram shown in Fig. 6Citation , which illustrates the independent contributions of alterations in DNA ploidy and telomere attrition to the crisis phenomenon.



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  		Fig. 6. Model: telomere attrition and abnormalities in DNA ploidy are independent determinants of crisis. We propose, based on our results obtained with SV40 large T-antigen-expressing ML10 cystadenoma cells, that after bypassing M1 due to overcoming of the action of cell cycle-regulatory proteins such as p53 and p16/RB, cells resume their logarithmic growth until they show manifestations of chromosomal instability leading to alterations in DNA ploidy. When severe, these alterations induce pdM2 characterized by increased apoptosis. Cells that adapt to these ploidy changes or those that never develop changes of high enough severity to induce crisis can resume or sustain logarithmic growth until further telomere attrition leads to a second crisis event, independent of pdM2, called tdM2. Recovery from tdM2 requires the establishment of a mechanism for telomere maintenance such as telomerase activation.

 
Our results emphasize the distinction between genetic instability associated with structural versus numerical chromosomal alterations, both of which are hallmarks of cancer. An association between structural abnormalities and telomere attrition and a protective role of telomerase against such changes have been demonstrated in previous studies using normal cells (12) , cells carrying p53 mutations (37) , and cells transformed with SV40 large T-antigen (22) . It has also been suggested that any form of non-telomere-based DNA damage may be more efficiently detected and repaired in cells expressing telomerase (23) . Our results suggest that the concept of a role for telomerase in controlling genetic instability should not be generalized to all forms of instability. The apparent increase in ploidy stability seen in cells expressing telomerase may simply reflect the fact that telomerase expression only confers immortality to cells that already acquired such stability by recovering from pdM2, providing them with a selective advantage over genetically more unstable subpopulations. This idea is in complete agreement with earlier results (17) , in which human mammary epithelial cells expressing SV40 large T-antigen were observed to contain heterogeneous populations of cells, some of which were chromosomally diploid, and others that were grossly aneuploid. In this previous study (17) , the epithelial cells that escaped crisis were almost universally chromosomally diploid, suggesting that escape from crisis only occurred in this subpopulation of cells.

The mechanisms responsible for the development of either tetraploidy or aneuploidy in cells harboring abnormalities in the p53 pathway may come from impairment in a postmitotic cell cycle checkpoint that normally prevents endoreduplication and arrests cells with abnormally replicated DNA or missegregated chromosomes (19 , 38, 39, 40) . Defects in both p53 (20 , 21) and RB (20) could also lead to aneuploidy due to the role of these proteins in monitoring normal centrosome duplication during mitosis. Although we have not systematically investigated the molecular determinants of ploidy-based crisis, it is possible that the increase in the levels of the p21 protein, which parallels changes in DNA ploidy, may have been important in mediating this phenomenon. Although the existence of p53-independent mechanisms for p21 induction is well established, the elevated levels of p53 in cells carrying severe ploidy changes may have been sufficient to overwhelm the SV40 large T-antigen, resulting in p21 protein induction. Ploidy changes may be perceived as a type of DNA damage by the cellular machinery, resulting in growth arrest through telomere-independent mechanisms. A role for the p53 and p21 proteins in crisis was suggested recently by Romanov et al. (32) . This conclusion is also supported by earlier results from Kiyono et al. (41) , who showed that expression of telomerase could not immortalize cells transfected with E6, which neutralizes p53 and often results in ploidy changes, but led to immortalization in cells transfected with E7, in which p53 function is intact, and ploidy status usually remains stable. Although coexpression of both E6 and E7 in the presence of telomerase resulted in immortalization, the ploidy status of the cells was not examined in this study (41) , raising the possibility that immortalization had taken place only in cells that had remained diploid or near diploid. Premature p16-mediated growth arrest independent of telomere attrition has been documented in presenescent epithelial cells and keratinocytes lacking essential nutrients (42 , 43) , as well as in cells treated with agents that cause DNA double-strand breaks (44) . It is unlikely that these mechanisms played an important role in mediating ploidy-dependent growth arrest in our cell culture system because the absence of a functional RB protein deprived p16 of its downstream substrate. In addition, the fact that our cultured SV40 large T-antigen-expressing cystadenomas can be propagated 40–60 PDs in vitro and that a subset of cells can be immortalized by expression of telomerase argues against a growth arrest due to depletion of essential nutrients.

Although the exact relevance of the various mortality checkpoints observed in vitro to the development of human cancers is not clear, evidence that human cancers arise from cells that have recovered from the equivalent of in vitro crisis includes the facts that (a) cancer cells characteristically show shorter telomeres than normal tissues and almost invariably express telomerase (27 , 45 , 46) ; (b) genetically engineered mice used as models to study crisis in vivo are the only transgenic mice known to develop a spectrum of tumors similar to that found in the adult human population (14) ; and (c) chromosomal abnormalities associated with early stages of breast cancer are similar to those present in late-passage human mammary epithelial cells that spontaneously overcome senescence in culture (32) . Our results, which emphasize the role of numerical chromosomal alterations in the crisis phenomenon, may therefore have implications for our understanding of the mechanism of acquisition of such numerical alterations in human cancers. According to the classical theories of tumor progression first advanced by Foulds (47) and Nowell (48) , the profound genomic alterations that characterize cancer cells occur randomly, followed by elimination of changes that are detrimental to cell survival and selection of those associated with a growth advantage. Our results illustrate the fact, supported by a number of studies (5 , 29, 30, 31 , 49) as well as from conclusions based on computer modeling (50) , that aneuploidy, although normally associated with cells that have undergone malignant transformation, can occur before such transformation and even before acquisition of replicative immortality, at least in the presence of a permissive genetic background such as in cells harboring a p53 mutation. The contribution and importance of chromosomal changes acquired through these mechanisms to the establishment of the cancer genome as well as to the cancer phenotype itself remain to be investigated.


   ACKNOWLEDGMENTS
 
We thank Dr. R. A. Weinberg from the Whitehead Institute of Biomedical Research for providing us with an expression vector for the telomerase catalytic subunit, Sergei Gryaznov of Geron Corporation for FISH probes, Dr. Axel Schönthal from University of Southern California for critical review of the manuscript, and Dr. Daniel Weisenberger for assistance in preparing the manuscript. We also thank the FACS Core of the USC/Norris Comprehensive Cancer Center for help with the cell sorting procedures.


   FOOTNOTES
 
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 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Cancer Institute Grants CA 51167, CA 79750, CA70907, and CA14089. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine of University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9181. Phone: (323) 865-0720; Fax: (323) 865-0077; E-mail: ldubeau{at}usc.edu Back

3 The abbreviations used are: FBS, fetal bovine serum; TRF, telomere restriction fragment; PD, population doubling; pdM2, ploidy-dependent M2; tdM2, telomere-dependent M2; ALT, alternative lengthening of telomeres; TRAP, telomerase repeat amplification protocol; FACS, fluorescence-activated cell sorting; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PML, promyelocytic leukemia; APB, ALT-associated PML body; FISH, fluorescence in situ hybridization. Back

Received 8/14/02. Revised 6/25/03. Accepted 7/ 8/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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