Effects of reconstitution of telomerase activity on telomere

© 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 18 1953–1961

Effects of reconstitution of telomerase activity on telomere maintenance by the alternative lengthening of telomeres (ALT) pathwayJennifer V. Grobelny, Michelle Kulp-McEliece and Dominique Broccoli*

Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA

Received May 8, 2001; Revised and Accepted June 24, 2001

Telomere length maintenance is essential for cellularimmortalization, and thus tumorigenesis. Most humantumors and immortal cell lines maintain their telomericDNA via the activity of a specialized reverse tran-scriptase, telomerase. Stabilization of telomeric repeattracts may also be achieved through a telomerase-inde-pendent mechanism, referred to as alternative length-ening of telomeres (ALT). ALT cells are telomerasenegative and are characterized by extremely long andheterogeneously sized telomeres and novel multi-protein structures called ALT-associated PML nuclearbodies which are unique to ALT cells. To determine ifreconstitution of telomerase activity suppressed ALTand restored wild-type telomere lengths, we introducedthe catalytic subunit of telomerase into two ALT celllines. Initially, two clonal lines exhibited enrichment ofshorter telomeres while maintaining a population ofultra-long telomeres similar to that observed in theparental line, suggesting that telomerase is stabilizingthe shorter telomeres in the population. Telomerelength in the third clonal line was not detectablydifferent from that in the parental cell line. One clonalline with a phenotype of shorter telomeres maintainedthis pattern over time in culture while the second grad-ually reverted to the parental ALT telomere lengthpattern, concurrent with reduction of telomeraseactivity. All clones continued to maintain ALT-associ-ated PML nuclear bodies regardless of whether telom-erase was present. The data suggest that introductionof telomerase activity alone is not sufficient tocompletely repress ALT, that telomerase acts preferen-tially on the shortest telomeres in the culture and thatthe ALT and telomerase pathways may be presentconcurrently in mammalian cells.

INTRODUCTION

Telomeres are specialized structures that confer stability to theends of linear chromosomes. In most eukaryotes, telomeres are

composed of tandem repeats of short G-rich sequencescomplexed with telomere specific binding proteins (reviewedin 1). In somatic cells with a finite proliferative potential, telom-eric sequences are lost at each cell division (2,3). In contrast,maintenance of telomeric DNA permits continued prolifer-ation of eukaryotic cells (4–7) linking cellular immortality tothe maintenance of telomeric DNA repeats.

In most tumors and immortalized cell lines, telomere loss isbypassed through the action of telomerase (reviewed in 8), areverse transcriptase that utilizes an RNA moiety as thetemplate for the addition of telomeric repeats onto the 3′ endsof linear DNA molecules (reviewed in 9). However, a subset oftumors and immortal cell lines utilize a telomerase-inde-pendent mechanism, termed alternative lengthening of telom-eres (ALT), to maintain telomeric DNA (10,11) and therebycircumvent the telomere length-dependent limitation onproliferation. Recent evidence suggests that telomeres in cellsthat utilize ALT may be maintained via homologous recombin-ation and copy switching between telomeric tracts (12). Celllines that utilize the ALT pathway have two characteristics inaddition to being telomerase negative: they contain ultra-long,heterogeneously sized telomeric arrays (10) and have novelmultiprotein structures, called ALT-associated PML nuclearbodies (APBs), in which telomeric proteins and DNA co-localize with the PML nuclear body (13). APBs appear in cellscoordinately with the appearance of the ultra-long telomericrestriction fragments, linking these structures to telomeremaintenance by the ALT pathway.

Mutation of telomerase in the yeast Saccharomyces cerevisiaeresults in telomere loss and eventual culture senescence (14).Rare survivors do arise for these populations and fall into twoclasses, termed Type I and Type II; this survival is dependentupon RAD52 (15). These survivors also fall into two classeswith respect to telomere structure (15,16). Type I survivorsdisplay short telomere tracts interspersed between subtelomericY′ elements, indicating that recombination between Y′ elementsis the underlying mechanism for telomere stabilization. Type IIsurvivors have long stretches of telomeric repeats which areprobably maintained via non-reciprocal recombinationbetween two telomeres. Interestingly, reintroduction of telom-erase into telomerase-deficient yeast strains results in restor-ation of wild-type telomeres (16). This occurs rapidly in the

*To whom correspondence should be addressed. Tel: +1 215 728 7133; Fax: +1 215 728 2741; Email: [email protected] authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First AuthorsPresent address:Jennifer V. Grobelny, Neuronyx, Inc., 1 Great Valley Parkway, Suite 20, Malvern, PA 19355, USA

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Type I survivors and with slower dynamics in Type II survivorswhere restoration of wild-type telomere length requires severalhundred population doublings. These data indicate that gener-ation of either class of survivors did not require additionalmutation of the proteins that usually regulate telomere lengthand indicate that telomerase is the preferred mode of telomeremaintenance in yeast.

Repression of the ALT pathway has also been observed inmammalian cells (17,18). In these experiments somatic cellhybrids were created between ALT cell lines and telomerase-positive immortal cell lines or primary mortal cell lines. Theresult was suppression of ALT in the hybrids, suggesting thatinter-telomere recombination is normally either suppressed orless efficient than telomerase at maintaining telomeres. It hasbeen demonstrated previously that telomerase activity can bereconstituted in ALT cell lines by exogenous expression ofcomponents of the telomerase holoenzyme (19). However, thefate of telomeres and the ALT pathway was not investigated inthese experiments. To determine whether telomerase alonewas sufficient to suppress ALT and restore wild-type telomerelengths, we introduced the catalytic subunit of telomerase,hTERT, into two ALT cell lines. Similar experiments havebeen carried out by others (18,20,21). In two cases, reconstitu-tion of telomerase activity did not alter characteristics of theALT pathway (18,21), whereas in the third report, ALT wassuppressed in two of the derived cell lines (20). In the experi-ments described here, two clonal lines initially exhibitedenrichment of shorter telomeres while maintaining a popula-tion of ultra-long telomeres similar to that observed in theparental line. Telomere length in the third clonal line was notdetectably different from that in the parental cell line. In theclonal lines exhibiting shorter telomeres, one maintained thispattern over time in culture while the second gradually lost theshorter telomeres and reverted to the parental pattern of ultra-long telomeres concurrent with reduction of telomeraseactivity. All clones maintained APBs over the course of theseexperiments. The data suggest that telomerase activity alone isnot sufficient to repress ALT in these cell lines and areconsistent with telomerase acting preferentially on the shortesttelomeres in the culture.

RESULTS

Identification of telomerase positive clones

The catalytic subunit of telomerase, hTERT, was introducedinto two cell lines derived from human ovarian surface epithe-lium (HOSE), HIO118 and HIO107, by transfection to inducetelomerase activity. These cell lines have previously beencharacterized and demonstrated to use the ALT pathway fortelomere maintenance (22). Transient transfection with thehTERT construct resulted in readily detectable telomeraseactivity, indicating that these lines express the RNA subunit oftelomerase, hTER (data not shown). Following selection andisolation of clonal cell lines, the TRAP assay was used to iden-tify those clones that contained in vitro detectable telomeraseactivity. Three cell lines, HIO118A5, HIO118D6 andHIO107C6, were identified which contained telomeraseactivity (Fig. 1A, lanes 1–12 and data not shown). As expected,inclusion of RNase in the reaction inhibited formation ofTRAP assay products. Two cell lines, HIO118A6 and

HIO107B5, which were hygromycin resistant but did notexhibit detectable telomerase activity were also isolated(Fig. 1, lanes 13–21 and data not shown). Extract from thetelomerase-positive HeLa cell line was included to demon-strate that the lack of detectable telomerase activity in theHIO118A6 and HIO107B5 cell lines was not due to a diffus-ible inhibitor (Fig. 1A, lanes 14, 15, 17, 18, 20 and 21). Thelack of telomerase activity in these cell lines is probably aresult of the integration of the construct such that hTERT is notexpressed. These lines provided internal controls for alter-ations observed in the telomerase positive clones isolated fromthe same transfection.

The level of telomerase activity present in the HIO118A5,HIO118D6 and HIO107C6 cell lines was quantitated relativeto the levels of telomerase activity in HeLa cells. It has beendemonstrated previously that the TRAP assay is inhibited athigher levels of protein extract (23). For our HeLa cell extracts,this occured at a level between 0.5 and 1 µg of protein extract(Fig. 1B). Likewise, titrations were carried out for the proteinextracts from each clonal line to establish that the assay wasconducted at levels that exhibited increasing product withincreasing amount of extract used (data not shown). Based onthese results, quantitation of telomerase activity was carriedout using 0.1 µg of each extract and was compared to thatpresent in 0.1 µg of HeLa cell extract (Fig. 1C). TheHIO118A5 and HIO107C6 cell lines had relatively stablelevels of telomerase activity over the time course of theseexperiments. For the HIO118A5 cell line telomerase activityranged from 1.2 to 1.5 times that present in HeLa cell extract,whereas for the HIO1097C6 cell line, telomerase activityranged from 0.5 to 0.8 times that present in HeLa cell extract.In contrast, the HIO118D6 cell line initially had almost1.8 times the level of telomerase activity as is present in HeLacells; this activity gradually decreased over time in cultureuntil it was completely absent by 80 population doublings(PDs).

Telomere length analysis

Telomere length in each clonal line was analyzed by Southernblot analysis at various times during culture (Fig. 2). Both theHIO118 and HIO107 parental cell lines exhibit the ultra-longtelomeres characteristic of cells that utilize the ALT pathwayfor telomere maintenance (Fig. 2) (10). At the earliest possiblepoint for analysis, 16 PDs, telomere length in both HIO118telomerase positive subclones, HIO118A5 and HIO118D6,had altered from that in the parental HIO118 cell line exhib-iting an increased hybridization intensity of lower molecularweight DNA fragments (Fig. 2A, bracket). These data indicatethat the presence of telomerase activity alters the telomere sizedistribution in ALT cells.

To determine if the telomere array size changes observed atearly PD were retained, telomeric DNA was analyzed in theHIO118 derived subclones over ∼70 PD. In the HIO118A5clone, shorter telomeres persisted in the population for thelength of these experiments (Fig. 2A). In contrast, in theHIO118D6 cell line telomeres gradually lengthened withincreased time in culture and eventually reverted to a patternsimilar to that in the parental HIO118 cell line (Fig. 2).Importantly, neither cell line exhibited complete loss of theparental ALT type terminal restriction fragment pattern, with

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Figure 1. Introduction of in vitro telomerase activity in ALT cells. (A) An example of a TRAP assay used to detect telomerase activity in whole-cell extractsprepared from the HIO118 clones A5, D6 and A6 at the indicated PDs. The assay was carried out using 0.5 µg of whole-cell extract in each reaction. Inclusion ofRNase in the reaction inhibits formation of telomerase products by destroying the RNA template molecule, hTER (lanes 2, 4, 6, 8, 10, 12, 15, 18, 21, 23). Mixing0.5 µg of telomerase-positive HeLa cell extract with 0.5 µg of extract from the telomerase-negative HIO118A6 did not affect the level of HeLa cell telomeraseactivity in the reaction (compare lanes 14, 17 and 20 with lane 22), demonstrating the absence of a diffusible inhibitor. (B) Quantitation of telomerase activity inHeLa cell extracts. The TRAP assay was carried using the indicated amounts of protein extract in the absence (–) or presence (+) of RNase. An example of a TRAPgel is shown in the left panel and quantitation of that gel is shown in the right panel. The total amount of reaction products generated under each condition isexpressed as a percentage of the maximum amount obtained. Note that at higher protein levels the formation of TRAP assay products is inhibited. (C) Quantitationof the level of telomerase activity present in the HIO118A5, HIO118D6 and HIO107C6 cell lines at the indicated PDs relative to the amount present in HeLa cellextracts. The assays were carried out using 0.1 µg of protein extract, established previously by titration experiments to be below the level of protein that inhibitsformation of TRAP assay products.


significant hybridization at the limit of mobility of conven-tional agarose gels over the course of these experiments. Thesize distribution of telomeric restriction fragments was alsocharacterized in the telomerase negative HIO118A6 clone. Asexpected based on absence of telomerase activity, the distributionof telomeric fragments appeared unchanged relative to theparental line (Fig. 2A).

To further characterize the size changes suggested byconventional gel eletrophoresis in the HIO118A5 andHIO118D6 cell lines, telomere length was analyzed by pulsedfield gel electrophoresis (PFGE) (Fig. 2B). This analysisindicated gradual shortening of the longest telomeric frag-ments in the HIO118A5 cell line and gradual lengthening ofthe telomeric fragments in the HIO118D6 cell line. Telomereattrition of the longest telomeres in the HIO118A5 occursdespite levels of telomerase activity greater than that present inHeLa cells.

To establish the degree of length diversity that accompaniesclonal variation, telomere length was analyzed in threeadditional clonal cell lines that were independently derivedfrom the parental HIO118 cell line following transfection withan unrelated vector that did not contain the hTERT openreading frame. Telomere length was similar to that in theparental HIO118 cell line in all three clonal lines as assessedby Southern blot analysis following PFGE (data not shown).These results suggest that the changes in telomere lengthobserved in HIO118A5 and HIO118D6 at early PDs are due tothe presence of biologically active hTERT rather than to clonalvariation.

Telomere length was also determined in the telomerase-positiveHIO107C6 and telomerase-negative HIO107B5 clonal celllines. In contrast to what was observed with the HIO118 celllines, no change in terminal restriction fragment lengths wasobserved for the HIO107C6 cell line, despite the presence ofin vitro telomerase activity (Fig. 2A). As expected and similarto the results obtained in the HIO118 background, the telom-erase-negative HIO118B5 and three independently derivedcell lines exhibited no detectable change in the distribution ofterminal restriction fragments (Fig. 2A and data not shown).

Telomerase activity does not affect the frequency of APBs

Cell lines that utilize the ALT pathway for telomere main-tenance contain large multiprotein complexes in which telom-eric proteins and DNA co-localize with the PML nuclear body,called APBs (13). To determine whether forced expression oftelomerase in ALT cell lines resulted in inhibition of thismarker of the ALT pathway, we carried out indirect immuno-fluorescence and determined the frequency of these structuresin logarithmically growing cells. All of the clonal cell linesgenerated here contained APBs, irrespective of whether or notthey had detectable telomerase activity (Fig. 3; Table 1). Thefrequency of APBs varied both among the clonal lines andwithin each cell line at different PDs after clonal isolation. Toconfirm that this variation was within a range consistent withthat due to clonal variation, we determined the frequency ofAPB-positive cells in five independently derived HIO118clonal cell lines and eight independently derived HIO107clonal cell lines following transfection with a vector that did

Figure 2. Southern blot of telomeric restriction fragments from DNA prepared at the indicated PDs. (A) Conventional agarose gel electrophoresis. The bulk of thetelomeric signal in the HIO118 and HIO107 parental cell lines is >20 kb, consistent with these cultures utilizing the ALT pathway for telomere maintenance. Notethat there is increased hybridization of lower molecular weight fragments at early population doublings in the telomerase-positive HIO118A5 and HIO118D6clones (bracket), whereas the telomerase-positive HIO107C6 clone showed no change in the pattern of telomeric hybridization. The telomeres became longer withtime in culture in the HIO118D6 clone. As expected, the telomerase-negative clones HIO118A6 and HIO107B5 did not exhibit any detectable changes in telomerichybridization pattern. No DNA was visible on the gel following staining with ethidium bromide, indicating complete digestion of genomic DNA. The position ofmolecular weight standards in kilobases is indicated along the left of the membrane. (B) PFGE. The HIO118A5 cell line exhibits gradual telomere attrition overtime in culture that is not detectable in (A), whereas the HIO118D6 cell line exhibits gradual telomere lengthening consistent with the pattern observed in (A).


not contain the hTERT open reading frame. In both back-grounds, the frequency of APB-positive cells varied from<10% of the cells in the population up to ∼30% of the cells inthe population (data not shown). Thus, the variation in thefrequency of APB-positive cells observed in the hTERT-derived cell lines is within that accompanying clonal variation.

We, and others, have shown previously that the frequency ofAPB-positive cells in the population is increased in culturesenriched for cells in the G2 phase of the cell cycle (22,24). Todetermine whether this aspect of APB regulation was alteredby the presence of telomerase, the telomerase-positiveHIO118A5 and HIO118D6 and the telomerase-negativeHIO118A6 cell lines were arrested in G2/M by exposure to themicrotubule poison, nocodazole. FACS analysis was carriedout to confirm the enrichment of cells with a G2 DNA contentin the nocodazole-treated cultures (Fig. 4A). The frequency ofAPB-positive cells was determined in the arrested populationand compared to that in parallel untreated cultures. Thefrequency of APB-positive cells was increased to a similarlevel in the G2/M arrested cellular populations regardless of

whether the cell line exhibited detectable telomerase activity(Fig. 4B). The extent of enrichment, ∼3-fold, is similar to thatreported previously for the parental cell lines (22). Theseresults indicate that telomerase activity does not affect thepresence or cell cycle regulation of APBs.

Analysis of end-protection function

Telomeres serve the essential function of providing stability toends of linear chromosomes. One phenotype exhibited by cellsthat have lost telomere end-protection function is the end-to-end fusion of chromosomes (25,26). During anaphase, thesefusions are manifested as bridges of unresolved DNA betweenthe separating daughter nuclei. An increase in the frequency ofanaphase bridges is seen in primary cultures as they approachcrisis and is correlated with extremely short telomeres (25).When the fate of a single marked telomere in ALT cells wascharacterized it exhibited gradual shortening over time inculture, punctuated by rapid increases in length and resumptionof telomere attrition (27). This cyclical behavior suggests thatALT acts preferentially on telomeres that reach a criticallyshort length and that a steady state level of cells exhibitingcompromised telomere function might be a feature of culturesthat utilize ALT for telomere maintenance.

To determine whether expression of telomerase resulted inincreased telomere stability, we analyzed the frequency ofanaphase cells that exhibited fusions in the parental HIO118and HIO107 cell lines, all hTERT derivative cell lines and anindependently derived cell line, HIO114, which activatedtelomerase spontaneously upon immortalization (Table 2). Theparental ALT cell lines have a higher frequency of anaphasebridges than does HIO114, consistent with there being a higherlevel of telomere malfunction in these cell lines. The frequencyof anaphase bridges in the clonal lines analyzed here wassimilar regardless of the presence of telomerase activity. Thelowest frequency of bridges (reduced by almost 2-fold fromthat in the parental cells) was present in the HIO118A5 cellline, which maintained the highest consistent level of telom-erase activity during the course of these experiments.However, the HIO118A5 cell line still had a higher frequencyof anaphase bridges than did the telomerase-positive HIO114cell line. In the HIO118D6 cell line, anaphase bridges occurredat a frequency similar to that in the parental cell line, althoughthis cell line also had relatively high levels of telomerase

Table 1. Frequency of cells in logarithmically growing cultures of each cell line which contained APBs at the indicated PD

Cell Line PD Telomerase APB-positive/Total (%)

HIO118 241 Negative 22/108 (20.0)

HIO118A5 6 Positive 2/64 (3.1)

48 5/57 (8.8)

72 9/79 (11.4)

HIO118D6 6 Positive 3/81 (3.7)

48 1/68 (1.5)

68 5/68 (7.4)

HIO118A6 6 Negative 3/58 (10.3)

46 4/60 (6.7)

68 14/72 (19.4)

HIO107 244 Negative 27/109 (24.8)

HIO107C6 4 Positive 9/69 (13.0)

24 18/64 (28.1)

HIO107B5 12 Negative 9/116 (7.8)

Figure 3. Indirect immunofluorescence of HIO118A5 cells demonstrating the presence of APBs, a characteristic of cell lines that utilize the ALT pathway fortelomere maintenance. In the left panel, the PML protein (green) is detected with a polyclonal goat antibody, while in the center panel the double-stranded telomereDNA binding protein hTRF1 (red) is detected in the same cells with a polyclonal rabbit antibody. The arrowheads in the third panel indicate two cells in this fieldthat contain APBs in which hTRF1 co-localizes with the PML nuclear body (yellow). DNA is stained with DAPI (blue).


activity (0.8 times that present in HeLa cells at the time ofanalysis). The HIO107C6 cell line did not exhibit an alterationin the frequency of anaphase bridges relative to the HIO107parental cell line or to the telomerase-negative HIO107B5 cellline.

DISCUSSION

Maintenance of telomere repeat arrays is essential for cellularimmortality. To date, two mechanisms which may be utilizedby human cells to circumvent telomere length-dependentlimitations on proliferation have been characterized, telom-erase activation and ALT. Recent evidence suggests telomerestabilization by way of the ALT pathway may occur throughnon-reciprocal recombination of telomeres (12). The genesregulating ALT remain elusive, but based on results fromsomatic cell hybrids (17) and from chromosome transferexperiments (28), the existence of one or more repressors ofALT has been suggested. In S.cerevisiae, it has been noted thatreintroduction of telomerase into telomerase-deficient strainsthat contain long telomeres results in reversion to wild-typetelomere lengths (16). These data suggest that in yeast the tran-sition to recombination-based telomere maintenance did notrequire mutations in constitutive components of the telomerecomplex. While these data do not directly address whether thesurvivor pathway is repressed in S.cerevisiae, they areconsistent with telomerase being a more efficient mode oftelomere replication. To determine whether telomerase is ableto restore wild-type telomere length regulation and/or suppressALT in human cells, we introduced the catalytic subunit oftelomerase, hTERT, into two ALT cell lines. The resultingtelomerase-positive clones exhibited alterations in telomerelength while maintaining characteristics of the ALT pathway.

The discovery of unique multiprotein complexes withinALT cells has provided a valuable tool to characterize the pres-ence of this pathway in cell lines. The presence of APBs withina culture is tightly linked to activation of ALT, as they aredetected with the simultaneous appearance of the ultra-longtelomeres characteristic of ALT (13). The frequency of cells incultures that contain APBs is variable between cultures.Furthermore, cells containing APBs are more common when

the population is enriched in the G2-phase of the cell cycle(22,24). All the clonal cell lines derived here retained APBsand exhibited a G2-dependent increase in the frequency ofAPB-positive cells regardless of whether or not telomerasewas present. Similar results have been observed in additionalALT cell lines that have been modified through exogenousaddition of telomerase components to force the expression oftelomerase (18,21), suggesting that ALT and telomerase maybe present concurrently in human cells. In contrast, it hasrecently been reported that forced expression of telomerasemay occasionally result in clonal cell lines that do not retaincharacteristics of the ALT pathway (20). This apparent contra-diction, together with the differences observed in the linesderived here with respect to telomere length dynamics,suggests that modifiers are present which dictate the suscepti-bility of a given cell line to telomerase or ALT.

Telomeres are essential for chromosome end protection andloss of this function is manifested as end-to-end fusion ofchromosomes (25,26). In S.cerevisiae, it has been suggestedthat short telomeres are the initiating substrate for telomereelongation through the recombination based survivor pathwayand that telomerase is able to suppress this pathway by elonga-ting these critically short telomeres (16). We analyzed thefrequency of anaphase bridges, a manifestation of end-to-endfusions, in the parental cell lines, the clonal lines derived hereand a cell line derived from the same tissue, human ovariansurface epithelium, that spontaneously activated telomeraseupon immortalization. All the cell lines analyzed had a greaterfrequency of anaphase bridges than was present in the telom-erase-positive HIO114 cell line. This outcome is in contrast towhat would be expected if telomerase is rescuing short telom-eres resulting in increased genome stability. However, it is alsopossible that the bridges observed here are occurring throughsome mechanism other than telomere malfunction, e.g. thesecell lines may have a high frequency of spontaneously occur-ring double-stranded DNA breaks leading to chromosomefusions and subsequent bridge formation in anaphase that isindependent of the ALT pathway.

Although the cell lines analyzed here retained characteristicsof the ALT pathway, the presence of telomerase activityresulted in changes in telomere length in the HIO118 derived

Figure 4. The frequency of cells that contain APBs is increased in cells arrested in the G2/M phase of the cell cycle. (A) Cell cycle profiles of untreated (Log) ornocodazole-treated (Nz) cells. Cell line is indicated at the top of the figure; x-axis, DNA content; y-axis, number of events. (B) Quantitation of the frequency ofcells in the population containing APBs. There is a 3-fold enrichment in the number of cells containing APBs relative to the frequency of APB-positive cells in alogarithmically growing culture (log, set to one) when cells are blocked in G2/M phase of the cell cycle by exposure to nocodazole (Nz) regardless of whethertelomerase is present.


subclones. However, no alterations in telomere length in thetelomerase-positive HIO107C6 cell line were observed. Thismay reflect differences in the level of telomerase activity or intelomere length regulation in the HIO107 parent as comparedto the HIO118 parent, e.g. by harboring different mutations intelomere-binding proteins. Alternatively, the lack of an effectof telomerase expression on telomere length in the HIO107C6clone may reflect differences in the regulation of the ALTpathway in the two parental cell lines, perhaps through loss ofdifferent repressors that are differentially sensitive to ectopictelomerase expression. Finally, it is a possibility that the telom-erase activity observed in vitro in the HIO107C6 cell line isunable to act on telomeres in vivo. Examples of alleles ofhTERT that exhibit dissociation of in vitro telomerase activityand in vivo telomere maintenance have been described previ-ously (29). However, this explanation is unlikely givenprevious demonstrations that the hTERT construct used inthese experiments is able to stabilize the telomeres of a numberof cell lines (29,30) and the changes in telomere lengthobserved in the HIO118 subclones.

Both the HIO118A5 and HIO118D6 cell lines had appar-ently experienced a rescuing of the shortest telomeres in theculture during the early points of culture expansion, asevidenced by increased hybridization of lower molecularweight telomeres at the earliest analyzable timepoint. Alterna-tively, the increased hybridization of lower molecular weighttelomeric fragments in the 5–23 kb size range in these cell linesmight indicate gradual telomere shortening in these cultures.However, FISH analysis of telomerase-modified ALT celllines carried out by two other laboratories indicates a preferen-tial increase of telomeric signal on chromosome ends that hadpreviously had very weak or undetectable hybridization signals(18,21). The simplest interpretation of these data would be thattelomerase acts preferentially on short telomeres in the culture.In the HIO118A5 cell line, continual telomere attrition of thelargest telomeric fragments was observed. Telomere short-ening in the presence of telomerase activity may be forcedthrough overexpression of telomere-binding proteins. Thus, itis conceivable that the long telomeres in ALT cell lines recruitsufficient protein to prevent telomerase from acting on themsuch that stabilization and equilibration of telomere arrays onlyoccurs when an equilibrium is reached between the amount oftelomere-binding proteins and the level of telomerase activity.

Despite telomere shortening, the HIO118A5 cell line retainedAPBs, suggesting that ALT was not completely suppressed byexogenous expression of telomerase.

In contrast to what was observed in the HIO118A5 cell line,the HIO118D6 cell line exhibited gradual telomere elongationwith continued time in culture and had a telomeric hybridiza-tion pattern consistent with ALT by 68 PDs. Although theHIO118D6 cell line eventually lost all detectable telomeraseactivity, the telomere lengthening observed in this cell lineoccurred in the presence of telomerase activity at levels equiva-lent to that present in HeLa cells. Because telomere length wasanalyzed at the population level it is not possible to determinewhether the rate of telomere growth is consistent withcontinued action of telomerase or with rapid growth of a fewtelomeres in the population at each division. These datasuggest that differences exist between the two HIO118 derivedclonal lines with respect to telomere length regulation. Thismay be due to the difference in the level of telomerase activitybetween these two cell lines or to factors that affect the relativeefficiency of telomerase versus ALT as a means of telomerelength regulation. The alterations in telomere length observedin the HIO118 clones are unlikely to be due to clonal variationbecause we did not detect changes in telomere length in theHIO118A6 clone, or in three additional clones derivedfollowing transfection with a vector that did not contain thehTERT open reading frame.

Finally, similar to a previous observation (11), we haverecently characterized an ALT cell line derived from a telomerase-positive tumor (A.Tosolini, A.De Rienzo, J.V.Grobelny,S.C.Jhanwar, D.Broccoli and J.R.Testa, manuscript inpreparation) raising the possibility that telomerase and ALTmay co-exist in some tumors. The importance of telomeremaintenance in ensuring continued proliferation and preva-lence of telomerase in human tumors has made this enzyme anattractive target for developing new drug strategies to combatcancer. If telomerase and ALT do, in fact, co-exist in sometumors, this would have important implications for theresponse of tumors to telomerase inhibitors.

MATERIALS AND METHODS

Cell lines and culture conditions

The HIO118 and HIO107 human ovarian surface epitheliumcell lines were maintained in a 1:1 mixture of Media 199 andMCDB-105 media, supplemented with 4% fetal bovine serumand 0.2 IU/ml pork insulin (Novagen). The pBabehygro-hTERT construct, a gift from Dr R.Weinberg (29) was trans-fected into parental lines using the FUGENE6 reagent (Roche)following the manufacturer’s specifications. HIO118 cloneswere selected with 50 µg/ml hygromycin and the HIO107clones were selected with 100 µg/ml hygromycin. Following2–3 weeks of selection, colonies were picked and transferred to24-well dishes. Upon reaching confluence, the clonal lineswere seeded into 6-well dishes and this was assigned aspopulation doubling 1. Cells were subcultured 1:4 for theremaining culture period. Cultures were arrested in G2/Mphase by addition of nocodazole (Sigma) to 1.5 µg/ml for 24 hand harvested for analysis.

Table 2. Frequency of cells in each cell line which contained anaphase bridges at the indicated PD

aThe HIO114 cell line activated telomerase spontaneously upon immortalization.

Cell Line PD Bridge-positive/Total (%)

HIO118 210 43/101 (42.6)

HIO118A5 72 7/27 (25.9)

HIO118D6 68 41/98 (41.8)

HIO118A6 68 37/106 (34.9)

HIO107 213 21/100 (21.0)

HIO107C6 28 20/100 (20.0)

HIO107B5 26 21/70 (30.0)

HIO114 229a 12/111 (11.0)


Analysis of telomerase activity

Telomerase activity was assessed using the TRAP assay.Whole-cell protein extracts were prepared as describedpreviously (31). Protein concentrations were determined usingthe Bradford assay (Bio-Rad). The TRAP assay was used todetect telomerase activity as described previously (23,32). Allreactions were carried out using 0.1–5.0 µg of protein extractin duplicate without or with inclusion of 20 ng RNase. RNasesensitivity was used to ensure that reaction products were dueto telomerase. In the case of the HIO118A6 extracts that lacktelomerase activity, the reactions were carried out with theaddition of 0.5 µg of telomerase-positive HeLa extract toconfirm the absence of a diffusible inhibitor of telomeraseactivity.

Southern analysis

Genomic DNA was extracted following standard procedures.Restriction enzyme digestion of genomic DNA was carried outwith HinfI and RsaI, the resulting fragments were resolved on0.7% agarose gels and transferred to Hybond N membranes asdescribed (31,33). Alternatively, genomic DNA was resolved byPFGE on 1% agarose/0.5× TBE gels using a CHEF DRIIapparatus (Bio-Rad) at 6 V/cm for 18 h with a 5 s constantswitch time. Telomeric restriction fragments were detectedfollowing hybridization with oligonucleotides complementaryto the telomeric repeats, TTAGGG and AATCCC, as describedpreviously (34). 100 ng of each oligonucleotide was labeled atits 5′ end using T4 polynucleotide kinase and [γ-32P]ATP.

Indirect immunofluorescence

Cells were seeded directly onto coverslips and immunofluores-cence carried out as described previously (35). Briefly, cellswere fixed in 3.7% formaldehyde/1× PBS and permeabilizedwith 0.5% NP-40/1× PBS. For detection of anaphase bridges,DNA was stained with 0.2 µg/ml 4,6-diamino-2-phenylindole(DAPI) at this time. Detection of APBs was carried out usingan affinity-purified rabbit polyclonal antibody raised against apeptide contained in the N-terminal domain of hTRF1 diluted1:200 and a goat polyclonal antibody against the N-terminus ofPML (N-19; Santa Cruz) diluted 1:5000. Antibodies weredetected using tetramethyl isothiocyanate-conjugated donkeyanti-rabbit IgG and fluorescein isothiocyanate-conjugateddonkey anti-goat IgG (Jackson ImmunoResearch). Thesecondary antibodies did not cross-react. Following incubationwith secondary antibody, the DNA was stained with 0.2 µg/mlDAPI. Coverslips were observed using an Eclipse E800epifluorescent microscope (Nikon) and images captured usinga three-color CCD camera (Optronics; DEI-750 CE) and ScionImage software.

FACS analysis

Cells were collected by trypsinization, washed twice withPBS/2 mM EDTA and fixed in cold 70% ethanol. Cells werestained with propidium iodide (50 mg/ml) and analyzed usinga Becton-Dickinson FacScan and CellQuest software.

ACKNOWLEDGEMENTS

We thank R.Reddel and S.Bacchetti for sharing results prior topublication, and R.Weinberg for the pBabehygrohTERTconstruct. D.B. is an Ellison Medical Foundation Scholar and aV Foundation Scholar. M.K-M. was supported by a fellowshipfrom the NIH (CA-09035-25). This work was supported bygrant RPG-00-259-01-GMC (A.C.S., D.B.) and an appropri-ation from the Commonwealth of Pennsylvania (F.C.C.C.).

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Effects of reconstitution of telomerase activity on telomere