A Novel Telomere Structure in a Human Alternative ... · 1Department of Medicine, ... by elevated rates of telomere recombi-nation, and by nuclear structures called ALT-associated

A Novel Telomere Structure in a Human Alternative Lengthening

of Telomeres Cell Line

Robert A. Marciniak,1,2David Cavazos,

1Richard Montellano,

1Qijun Chen,

4

Leonard Guarente,3and F. Brad Johnson

4

1Department of Medicine, University of Texas Health Science Center at San Antonio, 2South Texas Veterans Health Care System,San Antonio, Texas; 3Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts; and4Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Abstract

Cancer cells require mechanisms to maintain telomeres. Mostuse telomerase, but 5% to 20% of tumors use alternativelengthening of telomeres (ALT), a telomerase-independentmechanism that seems to depend on recombination. ALT ischaracterized by amplification of telomere TTAGGG repeats tolengths beyond 50 kb, by elevated rates of telomere recombi-nation, and by nuclear structures called ALT-associatedpromyelocytic leukemia bodies. In Saccharomyces cerevisiae ,survivors of telomerase inactivation also use recombination tomaintain telomeres. There are two types of survivors, whichdiffer in telomere structure. The first possesses telomererepeats and the YVsubtelomeric element amplified together asa tandem array at chromosome termini (type I), and the otherpossesses amplification of telomeric repeats alone (type II),similar to previously described human ALT cells. Here, wedescribe the first human ALT cell line having ‘‘tandem array’’telomeres with a structure similar to that of type I yeastsurvivors. The chromosome termini consist of a repeat unitcontaining ff2.5 kb of SV40 DNA and a variable amount ofTTAGGG sequence repeated in tandem an average of 10 to 20times. Similar to previously described ALT cells, they showevidence of telomere recombination, but unlike standard ALTcells, they lack ALT-associated promyelocytic leukemia bodiesand their telomeres are transcribed. These findings haveimplications for the pathogenesis and diagnosis of cancer.(Cancer Res 2005; 65(7): 2730-7)

Introduction

Most immortalized cancer cells use telomerase to maintaintelomeres through repeated rounds of cell division, but somecancer cells instead use alternative lengthening of telomeres (ALT;refs. 1–3). ALT is particularly common in certain sarcomas,including osteosarcomas, and in glioblastoma multiforme (4–6).The type of telomere maintenance mechanism is a strongprognostic indicator in glioblastoma multiforme (5) but not inosteosarcoma (4). Furthermore, it is anticipated that ALT cancerswill not be responsive to telomerase inhibitors and that the use ofsuch inhibitors may select for the emergence of ALT in tumors (7).The ALT mechanism is not fully understood, but seems to involve

telomere recombination (7–10) and is characterized by telomeres ofheterogeneous length that have size distributions that extendfrom <2 to >20 to 80 kb (3, 11). ALT cells are also characterizedby ALT-associated promyelocytic leukemia bodies (APBs), whichare nuclear structures containing telomere DNA, promyelocyticleukemia (PML) protein, TTAGGG repeat binding factors 1 (TRF1),and 2 (TRF2), and a variety of recombination proteins includingRAD51 and the Werner and Bloom syndrome proteins (3, 12–14).In Saccharomyces cerevisiae , inactivation of telomerase causes

most cells to die from telomere dysfunction, but rare cells surviveand maintain telomeres by recombination (15). There are twosurvivor classes, types I and II, that are distinguished by their geneticrequirements and telomere structures. Type I survivors depend onRAD51 and have telomeres comprising a tandem array of amplifiedunits consisting of YV subtelomeric elements and telomere repeats,whereas type II survivors depend on RAD50 and SGS1 , the yeasthomologue of the Werner and Bloom syndrome helicase genes, andhave amplification primarily of telomere repeats alone (14, 16–18).All human ALT cells thus far described have a telomere structuresimilar to yeast type II survivors. Here, we describe a novel humanALT cell line with a structure similar to yeast type I survivors.

Materials and Methods

Cell culture. Cell lines AG00780 (Werner mutant primary fibroblast),AG11395 (SV40-transformed Werner mutant fibroblast) and WI38 75.1

(SV40-transformed fetal lung fibroblast, AG07217) were obtained from the

Coriell Cell Repository (Camden, NJ). HeLa (human cervical carcinoma,

catalog number CCL-2) cells were obtained from the American TypeCulture Collection (Manassas, VA). WV (SV40-transformed Werner mutant

fibroblast; ref. 19) was kindly provided by Prof. Sydney Shall (Cell and

Molecular Biology Laboratory, University of Sussex, Sussex, UnitedKingdom). Cells were grown in 5% CO2 under conditions recommended

by the supplier.

Telomerase enzymatic assays. Telomerase enzymatic activity was

detected using the TRAPeze telomerase detection kit (Chemicon, Temecula,CA). Fluorescently labeled products were detected using a Typhoon imager

(Amersham, Piscataway, NJ).

Quantititative PCR for telomerase reverse transcriptase mRNA.

Total cellular RNA was prepared using the RNeasy mini kit (Qiagen,

Valencia, CA) and cDNA was prepared using the high-capacity cDNA

archive kit (Applied Biosystems, Foster City, CA). Human telomerase reverse

transcriptase (hTERT) mRNA was detected using a Taqman-MGB probe

(Applied Biosystems, Foster City, CA). Primers used were 5V-GACATGGA-GAACAAGCTGTTTGC-3V, 5V-AACAAGAAATCATCCACCAAACG-3V, and 5V-ATTCGGCGGGACGGG-3V (Taqman-MGB probe, FITC-conjugated), which

flank the exon 9/10 boundary of the hTERT cDNA. Amplification conditions

were 30 seconds at 95jC and 60 seconds at 60jC in a Mx3000P thermal

cycler (Stratagene, La Jolla, CA). Glyceraldehyde-3-phosphate dehydroge-

nase mRNA levels were measured under the same conditions as an internal

control (Assays on Demand, Applied Biosystems). The amplification

efficiency of the hTERT primers was 96%.

Requests for reprints: F. Brad Johnson, Department of Pathology and LaboratoryMedicine, University of Pennsylvania School of Medicine, 405A Stellar Chance Labs,422 Curie Boulevard, Philadelphia, PA 19104-6100. Phone: 215-573-5037; Fax: 215-573-6317; E-mail: [email protected] or Robert A. Marciniak, Department ofMedicine, University of Texas Health Science Center at San Antonio, 7703 Floyd CurlDrive, MSC 7884, San Antonio, TX 78229-3900. Phone: 210-567-4777; Fax: 617-507-3491;E-mail: [email protected].

I2005 American Association for Cancer Research.

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Telomere Southern blots. Total genomic DNA was prepared using theBlood & Cell Culture DNA Mini Kit (Qiagen). Total DNA (2 to 5 Ag) weredigested with the indicated endonucleases and resolved on a 0.5% agarose

gel. 32P-d(CCCTAA)3 probe was prepared using polynucleotide kinase.

For terminal restriction fragment (TRF)analyses (Fig. 1), gels were dried andhybridization done in the gel as described (20). For standard Southern blots

(Fig. 3), nucleic acids were transferred to a GeneScreen Plus membrane

(Perkin-Elmer, Boston, MA) using a PosiBlot Pressure Blotter apparatus

(Stratagene), and hybridized to probe. The weighted average of thehybridization signal was used to determine mean telomere length: A (ODi

� lengthi) / A (ODi). For two-dimensional Southern blots, all procedures

were done as described (21) using 10 Ag of total cellular DNA digested with

BamHI, BglII, EcoRI, and XbaI. For SV40 hybridization, SV40 PCR product(nucleotides 232-654) was 32P-labeled by random priming, hybridized, and

washed per standard protocols as described (22). Quantitation of Southern

blots was done with Imagequant software analysis of phosphoimager scansand using average local background subtraction.

Cloning of telomere repeat. To isolate terminal array DNA from most

genomic sequences, genomic DNA from cell line AG11395 was digested with

BamHI, BglII, EcoRI, Sac I, and XbaI (which do not cleave within theterminal array), the products were resolved on 0.5% agarose gel, and DNA at

limit mobility was isolated. This DNA was then cut with SphI (which waspredicted to cut once within the array), and the fragments were then ligated

to double-strand oligonucleotide P1-SphI (5V-TCGCCCGCACCCTTAATT-TACGCATG-3Vand 5V-CGTAAATTAAGGGTGCGGGCGT-3V). Unligated oligos

were removed by filtration through Sephadex G25 (Pharmacia). Ligationproducts were then linearly amplified with Platinum Taq (Invitrogen,

Carlsbad, CA; 30 cycles, 56jC for 45 seconds, 72jC for 5 minutes, and 94jCfor 45 seconds) using primer P2-TEL [5V-GCATTCGACTGCTGCCAAT-GAGG(CCCTAA)5-3V], which introduced a second unique primer site forsubsequent ‘‘nested’’ PCR. Finally, the target DNA was exponentially

amplified using primers P1 (5V-TCGCCCGCACCCTTAATTTAC-3V) and P2

(5V-ATTCGACTGCTGCCAATGAGG-3V; 30 cycles, 56jC for 45 seconds, 72jCfor 5 minutes, and 94jC for 45 seconds). The products of this reaction werecloned into T-vector (Promega, Madison, WI), and sequenced. Once the

identity of the sequence integrated at telomeres was determined, additional

PCR clones were obtained and sequenced by direct PCR from AG11395genomic DNA using SV40-specific primers and P2-TEL and primer P1-TEL

[5V-CGTCGCCCGCACCCTTAATTTAC(TTAGGG)5-3V].Fluorescence in situ hybridization. For telomere repeat fluorescence

in situ hybridization (FISH), a Cy3-conjugated peptide nucleic acid (PNA)probe [(CCCTAA)3; Applied Biosystems] was used as described (23). For

Figure 1. An unusual telomere structure in animmortalized Werner syndrome mutant cell line. Cellsincluded WI38 75.1 (telomerase-negative,SV40-immortalized human fibroblasts), HeLa(telomerase-positive cervical carcinoma cells), AG00780(Werner syndrome primary fibroblasts), and AG11395(SV40-immortalized derivative of AG00780). A, TRF lengthanalysis; 2 Ag of genomic DNA from each of the cell linesindicated were digested with Hin fI and Rsa I, resolved on a0.5% agarose gel and the products analyzed by in-gelhybridization using a d(CCCTAA)3 probe. The mean TRFlengths of AG0780 and AG11395 are 8 and 2.6 kb,respectively; B, TRAP performed on the indicated amountsof cell extracts. The lowermost band (IC ) is an internalPCR control. The absence of TRAP inhibitors wasassessed by mixing 100 ng each of HeLa and AG11395extracts; C, quantitative reverse transcriptase-PCRanalysis of hTERT mRNA levels relative to HeLa cells.Columns, mean; bars, F SD of three independentmeasurements; D, comparison of TRF lengths producedby restriction endonucleases with six-base recognitionsequences. Genomic DNA (2 Ag) from the indicated celllines was digested as indicated and the products analyzedby in-gel hybridization as in (A).

SV40 Sequences at Telomeres in ALT Cells

www.aacrjournals.org 2731 Cancer Res 2005; 65: (7). April 1, 2005



SV40 FISH, an equimolar mixture of probes was prepared from plasmidpBRSV40 (American Type Culture Collection): a 2,214 bp NgoM IV fragment,

and 1,935 and 1,446 bp PvuII fragments. These fragments overlap the

T antigen coding sequence and the SV40 regulatory region. After gel

purification, the fragments were labeled with biotin using the NEBlotPhototope Kit (New England Biolabs, Beverly, MA). Slide preparation,

hybridization, and detection were done following standard protocols (22).

RNase pretreatment of mitotic spreads was done after fixation onto

microscope slides by incubation for 30 minutes at 37jC with 20 ng RNase A(Invitrogen) diluted in 40 mL of PBS. Slides were then washed thrice for 2

minutes in PBS, and processed for PNA-FISH as described above.

Indirect immunofluorescence. Fixation and indirect immunofluores-

cence were done as described (24). Antibodies used and dilutionswere mouse monoclonal anti-PML (1:100; Santa Cruz Biotech, Santa

Cruz, CA), affinity purified rabbit anti-TRF1 (1:100; Alpha Diagnostic, San

Antonio, TX), Cy3-conjugated donkey anti-rabbit IgG (1:300; AmershamBiosciences, Piscataway, NJ) and FITC-conjugated donkey anti-mouse IgG

(1:100; Vector Labs, Burlingame, CA).

Dot blot of telomere RNAs. Total RNA from the indicated cell lines was

prepared as described above, and concentration was determinedby absorbance at 260 nm. RNA was blotted onto GeneScreen Plus

(Perkin-Elmer). Hybridization was done with biotinylated d(CCCTAA)3 or

d(TTAGGG)3 probes (IDT, Coralville, IA), and probe detected using the

Detector horseradish peroxidase chemiluminescent blotting kit (KPL,Gaithersburg, MD). Blots were treated with blocking buffer and then

hybridized to probe for 6 hours at 37jC. The presence of telomere RNAs was

also verified by qPCR on cDNA prepared cell lines, using conditions describedpreviously (25).

Results

Immortalized AG11395 cells have unusually short telomereTTAGGG repeat binding factors and lack telomerase activity.As part of an analysis of genetic requirements for telomeremaintenance in human cells, we found that an SV40-transformed,immortalized cell line (AG11395, ref. 26), which lacks the Wernersyndrome helicase (WRN, ref. 24), seemed to have unusually shorttelomeres. Standard TRF analysis, which involves a Southern blot ofRsaI and HinfI-digested genomic DNA hybridized with a telomererepeat probe, d(CCCTAA)3, indicated a mean TRF length of 2.4 kb(Fig. 1A). In comparison, the mean TRF length of the primaryfibroblast cell line (AG00780), from which cell line AG11395 wasderived, was 8.0 kb, and TRF lengths of the WI38 75.1 ALT cell lineextended above 25 kb (Fig. 1A). Although the TRF length of AG11395cells was stable through more than 40 population doublings andupon subcloning (data not shown), the cells lacked detectabletelomerase activity as measured by the telomere repeat amplifica-tion protocol (TRAP) assay (27). Mixing protein extracts fromAG11395 with extracts from telomerase-positive HeLa cells showedthat AG11395 does not contain an inhibitor of TRAP activity (Fig.1B). To confirm the absence of telomerase activity in AG11395, wedid quantitative reverse-transcriptase PCR for the hTERT mRNA(Fig. 1C). AG11395 had 0.14% of the level of hTERT mRNA found inHeLa cells, and was similar to the level measured in the WI38 75.1ALT cells. The maintenance of telomere length in AG11395 withouttelomerase therefore indicates the activity of an ALT mechanism.Telomere repeat sequences of AG11395 are interspersed

with non–telomere repeat sequences. AG11395 cells lack theRecQ helicase WRN, and the S. cerevisiae RecQ homologue, Sgs1p,is required for the generation of type II survivors of telomerasedeletion in yeast (14, 18, 28). The type I survivors that emergewithout Sgs1p have a telomere structure that consists of a tandemarray with units that each contain both a non–telomere repeatsequence (the YV-element) and telomere repeats; therefore, we

reasoned that the short TRF profile of AG11395 might reflect asimilar telomere structure. That is, the non–telomere repeatsequence might contain restriction sites for RsaI and HinfI, andthus the TRF profile would represent the length of the telomererepeats within one unit. In this case, it should be possible to findendonucleases that do not cut within the non–telomere repeatsequences in the array, and these enzymes should reveal the trueTRF length. Furthermore, endonucleases that cut once within thearray should yield unit-sized fragments (or the spectrum of unit-sized fragments if the individual units differ in size). This modelalso predicts that a similar pattern of fragments should beproduced by different endonucleases with unrelated recognitionsequences if each cuts once within the repeat unit.To test this model, we digested genomic DNA from AG11395

with a panel of endonucleases that have six base recognitionsequences (Fig. 1D). As predicted, some enzymes yielded long ALT-like TRF lengths (e.g., AflII in Fig. 1D). The TRF length of theparental AG00780 line digested with AflII is not longer than thoseproduced by other endonucleases (Fig. 1D), indicating that theincreased TRF length of AG11395 was not due to scarcesubtelomeric AflII sites. Furthermore, the pattern of TRF fragmentsare remarkably similar for several enzymes (e.g., SphI, NdeI, andEcoRV), consistent with these enzymes having a single recognitionsite within each unit of the array; the varying size of the fragmentscould be due to heterogeneity in the lengths of telomere repeat ornon–telomere repeat components of each unit, or both.SV40 sequences are associated with telomere repeat DNA in

AG11395. We cloned the sequences associated with the telomererepeats using the strategy outlined in Fig. 2A (also see Materials andMethods). Briefly, telomere DNA was separated from bulk genomicDNA, digested with SphI (predicted to cut once within each arrayunit), ligated to adaptors and amplified by PCR using adaptor-specific and telomere repeat-specific primers. This procedureproduced a smear of products, including two predominant productsat 1.4 and 1.7 kb (Fig. 2B), which were then cloned. These clonesyielded information on the nature of the non–telomere repeatsequences and the centromeric junction between these sequencesand telomere repeat sequences. This information was used to designprimers to amplify and clone fragments containing the telomericjunction. Sequencing of six clones containing the centromericjunction and five clones containing the telomeric junction revealedthat the telomere repeat sequences in each unit are interrupted by afragment of the SV40 viral genome that extended from nucleotide1696 through nucleotide 0/5243 and continued to nucleotide 4469 ofthe circular SV40 genome, abbreviated SV40 4469 to 1696 (Fig. 2C).The centromeric and telomeric junction sequences were the same inall clones analyzed. The SV40 fragment contains the regulatoryregion, which includes the origin of replication and the early and latepromoter sequences (Fig. 2D). Degenerate telomere repeats (e.g.,TTACGG) were found within the first 45 nucleotides flanking theSV40 DNA, but the remaining telomeric repeats matched thestandard TTAGGG repeat. The fact that all clones had the sameSV40-telomere repeat junctions indicates that a single SV40integration event had occurred, followed by propagation alonga chromosome and between chromosomes. Although the SV40sequence did not contain an intact large Tantigen coding sequence,large T antigen was present on immunoblot (data not shown),indicating that the original SV40 sequences used to transform theparental cell line were extant elsewhere in the genome.The majority of telomere repeats and SV40 sequences are

closely associated in AG11395. The association of the majority of

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SV40 sequences with telomere repeats was also shown by Southernblot (Fig. 3A). Endonucleases that do not cut within the array unitsyielded long, heterogeneous TRFs that had identical or nearlyidentical patterns of hybridization with SV40 and telomere probes;those enzymes that cutwithin the unit but do not cut both 5Vand 3Vofthe SV40 hybridization target also showed nearly identical patternsof hybridization to both probes (cf. SphI, EcoRV; Fig. 3A). Enzymesthat cut multiple times within each unit—and cut between thelocation of the SV40 hybridization probe and the telomere repeats—dissociated the patterns of hybridization with the two probes. Themultiple bands in the HinfI/RsaI, DraI or HindIII digested DNAhybridized to the telomere repeat probe showed substantialheterogeneity in the amount of telomere repeat sequence within aunit repeat. In contrast, the single bands observed for these digestshybridized to an SV40 probe indicated less heterogeneity in the SV40sequences contained within the unit repeat. Thus, the heterogeneityin array unit length seems to be caused by variation in the length oftelomere repeats within each unit; in samples cut with enzymes thatonly cut the array unit once (e.g., SphI; Fig. 3A), the long size of somefragments indicates infrequent SV40 sequences at some arrays,whereas the decline of hybridization of the telomere repeat probe to

short fragments that hybridize strongly to the SV40 probe indicatesthat SV40 sequences may be interrupted by very short (and possiblyeven no) telomere repeat DNA in other arrays. In addition, thefinding that there is little heterogeneity in the size of SV40 fragmentsin the arrays is consistent with the constancy of sequence at theSV40-TTAGGG junctions, and indicates that a single primordialintegration event of the SV40 fragment into telomere repeat DNAwas followed by propagation of the SV40 sequence along andbetween chromosomes.SV40 and telomere repeat sequences are found at chromo-

some termini in AG11395. Although the sequence and Southernanalyses showed an association between SV40 and telomere repeatsequences in AG11395, they do not show where in the genome thesesequences are located. PNA-FISH for telomere repeats (23)confirmed that the majority of TTAGGG repeats are near thechromosome ends and showed large inter- and intrachromosomalvariation in hybridization signal in the AG11395 cell line (Fig. 3B),similar to that observed at standard ALT telomeres. FISH analysiswith SV40 regulatory region probes (Fig. 3C) confirmed that themajority of SV40 hybridization signal was seen near the chromo-some ends. The strength of telomere repeat and SV40 signals at

Figure 2. SV40 sequences are associated with telomere repeats sequences in AG11395. A, cloning of repeat array by ligation-anchored PCR. To separate telomeresfrom bulk genomic DNA, AG11395 was digested with five different endonucleases that do not cut within the telomere tandem arrays, and then limit mobility fragmentswere isolated from agarose gels. The DNA was then digested with Sph I, which cuts once in the non–telomere repeat sequences, and ligated to an adaptor with aprimer binding site (P1 ). Linear amplification with a second primer containing telomere repeats and a different primer binding site (P2 ) was followed by PCR with P1and P2 primers. The products, containing the junction of non–telomere repeat and telomere repeat sequences on the centromeric side of the telomere repeatssequences, were then cloned and sequenced. Further amplifications to obtain the telomeric junction are described in Materials and Methods; B, ethidium bromidestained gel of PCR products produced by primers P1 and P2. The shorter (1.4 kb) band contained an internally deleted SV40 sequence, which likely reflects a PCRartifact (data not shown); C, map of the SV40 genomic fragment linked to telomere repeats. Sequences linked to the telomere repeats are in boldface. The SV40 openreading frames and origin of replication (ori) are shown; D, sequence integrated at telomeres. The SV40 sequences at the telomere junction sequences are boxed .Only single types of SV40/telomere junctions were found in sequencing six centromeric junction and five telomeric junction clones.





chromosome termini varied between chromosomes, and between pand q arms within the same chromosome. No SV40 hybridizationsignal near the chromosome ends was seen in metaphases fromWI38 75.1, another SV40-transformed ALTcell line (data not shown).

AG11395 cells lack alternative lengthening of telomeres–associated promyelocytic leukemia bodies. APB have been usedas a morphologic marker of ALT because they are found only inimmortalized cells that lack telomerase and that have telomeres ofextremely heterogeneous length (3, 6, 12). APBs are characterized bythe colocalization of the PML protein with telomere repeat bindingfactors, including TRF1 (3). APBs were readily detected in WI38 75.1ALT cells, as indicated by colocalization of the PML and TRF1proteins (Fig. 4A-C), but no such colocalization was seen in anyAG11395 cell (Fig. 4D-F). Thus, AG11395 cells lack ALT-associatedPML bodies.Telomere repeat DNA is transcribed in AG11395. While

analyzing metaphase spreads, we noticed that 88% of AG11395interphase nuclei had an unusually bright and diffuse pattern oftelomere PNA-probe hybridization visible on short exposures(Fig. 5A). Given a possible role for extrachromosomal circularDNAs in generating tandem array structures via a rolling-circleintermediate (3, 15, 21, 29), we first assessed whether extrachro-mosomal DNAs containing telomere and SV40 sequences werepresent in AG11395. Two-dimensional gel electrophoresis andSouthern analysis showed extrachromosomal circular speciescontaining telomere repeat and SV40 DNA in AG11395 (Fig. 5D ;and data not shown). Similar species containing telomere repeatbut not SV40 DNA were also present in the ALT lines WI38 75.1and WV. All three ALT lines had higher levels of circular speciesthan the non-ALT BJ-hTERT line. However, the level of circularspecies in AG11395, although the highest among the linesexamined, were still <0.2% of telomere repeat DNA and so couldnot account for the diffuse telomere PNA staining of AG11395nuclei. Given the presence of the SV40 promoters integratedadjacent to telomere sequences, we next assessed whether RNAtranscripts containing telomere repeat sequences were present.RNase A treatment prior to hybridization to the telomere PNAprobe eliminated the diffuse nuclear hybridization signal (cf. Fig.5A and B). Telomere DNA-hybridization signals at chromosometermini remained detectable, if a standard length (i.e., longer)exposure was used to acquire the FISH signal (Fig. 5C). Toconfirm that the diffuse nuclear hybridization signal was due tothe presence of RNA, we did dot blot hybridization of totalcellular RNA isolated from this cell line (Fig. 5E). A C-strandtelomere probe hybridized to immobilized total cellular RNAisolated from AG11395, but not to total cellular RNA from HeLa(telomerase-positive) or WI38 75.1 (ALT) cells. When a G-strandtelomere probe was used, no hybridization signal from total cellularRNA was observed (Fig. 5E). This indicates either that the telomererepeats are transcribed only in the direction yielding G-richtranscripts, or that the C-rich transcripts, if transcribed, are notstable and do not accumulate.

Figure 3. SV40 and (TTAGGG)n repeats exist in a tandem array structure nearchromosome ends in AG11395. A, evidence for the tandem array structure oftelomere repeat and SV40. High molecular weight genomic (2 Ag) DNA fromAG11395 was digested with the enzymes indicated, and analyzed by Southernblot of a 0.5% agarose gel. Hybridized to a telomere-specific hybridization probe(left ). The same blot rehybridized to an SV40 regulatory region probe (right ).Enzymes used are indicated above each lane, and size standards (in kilobases)are shown. ‘‘Bam/Bgl/Eco/Xba ’’ indicates simultaneous digestion with BamHI,Bgl II, EcoRI, and Xba I; B, telomere repeats are near chromosome ends in cellline AG11395. FISH to mitotic chromosomes with a Cy3-labeled PNA telomereprobe (red ), counterstained with 4V,6-diamidino-2-phenylindole (blue ). Anenlarged view is shown in the inset; C, SV40 sequences are near chromosomeends in cell line AG11395. FISH with a FITC-labeled SV40 regulatory regionprobe (green ). An enlarged view is shown in the inset.

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Discussion

The telomere structure of the AG11395 cell line, consisting of anaverage of 10 to 20 tandem repeats of a SV404469-1696-(TTAGGG)<500unit at chromosome ends, is the first example of ‘‘tandem array’’telomeres in human cells. A similar telomere structure was alsodescribed in a murine embryonic stem cell line in whichtelomerase had been inactivated through disruption of the Terctelomerase RNA template gene and the cells then passaged throughcrisis; in this line, the non–telomere repeat component of the arrayconsisted of an interspersed repetitive element from the mousegenome rather than SV40 sequence (30). Both of these cases havetelomeres that bear structural similarity to those of S. cerevisiaetype I survivors of telomerase deletion, and they show that tandemarray telomeres can stabilize chromosome termini in the absenceof telomerase in mammalian cells.In yeast lacking the Sgs1p RecQ family helicase, only type I

survivors of telomerase deletion emerge (14, 18, 28). Because thereis evidence that the mammalian WRN and BLM RecQ helicasesparticipate in telomere maintenance (31–35), including standardALT (36), it is possible that the absence of WRN in AG11395contributed to the development of its type I-like telomeres.However, if WRN does participate in the standard ALT mechanism,there is no absolute requirement for WRN because the only otherimmortalized cell line lacking WRN, WV (19), has a standard ALTphenotype (Fig. 5D).5 A possible role for WRN in regulating ALT isintriguing because sarcomas account for much of the increasedcancers in Werner syndrome (37) and because sarcomas are oftencharacterized by ALT (3, 6).Although there are differences between the tandem array

telomeres of AG11395 and previously described ALT cells, thereare also similarities. The presence of the same SV40 sequences inassociation with at least the majority of the telomere repeatsindicates that telomere recombination led to the propagation of thissequence between chromosome termini. The ability of a markedtelomere to propagate among different telomeres in AG11395 also

Figure 4. Absence of APBs in AG11395. APBs can be detected bycolocalization of PML antigen with TRF1. A-C, indirect immunofluorescentanalysis of the ALT cell line WI38 75.1 with mouse anti-PML (A ) and rabbitanti-TRF1 (B ) shows extensive colocalization typical of ALT cell lines (C ).Indirect immunofluorescence analysis of cell line AG11395 with mouse anti-PML(D ) and rabbit anti-TRF1 (E ) does not reveal significant colocalization (F ).

Figure 5. AG11395 contains telomere RNA transcripts. A, telomere PNA-FISHof cell line AG11395. When performed under standard conditions, most cellsshow diffuse, bright nuclear hybridization; B, telomere PNA-FISH after RNase Adigestion. AG11395 cells were treated as in (C ) except slides were incubatedwith RNase A prior to hybridization. The exposure of (A ) and (B) is the same andis shorter than exposures for other cell lines; C, a standard exposure of theRNase A treated AG11395 in (B) reveals probe hybridization at chromosomeends. The inset is an enlargement of the cell immediately above; note metaphasechromosomes at top of inset; D, two-dimensional gel electrophoresis andSouthern blotting for telomere repeat DNA in BJ-hTERT and the SV40 T antigenimmortalized lines WI38 75.1, AG11395, and WV. The first and seconddimensions and linear size standards (in kilobases) are indicated. Numbers onthe right are the ratios of telomere repeat-hybridizable circular versus linear DNA.Circular species were primarily nicked species based on precise comigration withnicked circular markers added to genomic samples prior to electrophoresis, andthey extended beyond 16 kb in length (data not shown). Reprobing of the sameblots with SV40 regulatory region sequences revealed the same pattern inAG11395, but only discrete spots on the linear arcs for WI38 75.1 and WV, andno hybridization to BJ-hTERT (data not shown); E, AG11395 contains RNAtranscripts containing TTAGGG repeats. Total RNA or genomic DNA from theindicated cell lines were blotted with probes for either the G-strand (top ) orC-strand (bottom ) telomere repeats. Only cell line AG11395 contains detectableRNA transcripts containing (TTAGGG) repeats; no transcripts of thecomplementary strand were detected. The masses of RNA or DNA spotted ontothe membranes are indicated. Total DNA was blotted and detected as a controlfor the integrity of the telomere sequence hybridization probes.5 R.A. Marciniak and F.B. Johnson, unpublished data.





supports this conclusion (38). Furthermore, the presence of elevatedlevels of extrachromosomal circular DNA containing telomererepeat and SV40 sequences suggests the possible use of such speciesto amplify telomere ends by rolling circle replication, as suggestedpreviously (3, 15, 21, 29). FISH analyses of metaphase chromosomesrevealed heterogeneity in the apparent length of the tandem arraysamong chromosome termini, similar to standard ALT and alsoconsistent with a recombination-based mechanism of telomeremaintenance. Recombination might thus underlie both forms ofALT, and it remains to be determined if there are differences in theparticular recombination pathways employed, as there are for type Iand II survivors in yeast. Furthermore, it is also possible that reversetranscription of transcripts containing more than one array unit,followed by integration into an existing array, might contribute tomaintenance of the arrays.What is the significance of the SV40 sequences in the tandem

telomeres? First, their association with most or all of the telomererepeat DNA implies that the SV40 sequences provide a selectiveadvantage in this context, although the nature of this function isunclear. It is possible that the 3V ends of replication intermediatesgenerated by the origin of replication provide substrates for telomererecombination. Alternatively, the origin might help maintainepisomal species containing telomere repeat and SV40 sequencesand thus facilitate a rolling circle mechanism of telomereamplification; our observation that AG11395 had the highest levelsof circular species among the three ALT lines that we examined isconsistent with this possibility. Second, the SV40 early promoter isoriented so that it would give rise to G-rich, rather than C-rich,telomere repeat transcripts, and it is likely that it is responsible forthe transcripts observed in AG11395. The transcription of telomericsequences is remarkable because telomeres are generally transcrip-tionally silenced (39). However, yeast type I survivors do transcribe

the subtelomeric YVelement (40), which is silenced in normal cells.Thus, loss of telomere silencing might be a general feature of tandemarray telomeres. Alternatively, the episomal telomere DNA speciesmight have a chromatin structure permissive to transcription andthus be the source of the telomere repeat-containing transcripts.Third, the telomeric SV40 sequences are very likely derived simplyfrom the SV40 sequences used to transform the primary cells thatgave rise to AG11395 (26). Nonetheless, certain human tumorscontain SV40 DNA (41) and it will be of interest to test if any of thesehave SV40 sequences at telomeres.Although AG11395 is immortal and lacks detectable telomerase

activity, and is thus ALT, it lacks the APBs and long telomeres instandard TRF assays characteristic of standard ALT cells. One orboth of these markers of ALT are used to screen tumors for the ALTphenotype, and so the finding that nonstandard ALT exists inhuman cells is of importance for the diagnosis of tumor type.Although standard ALT clearly accounts for some cases oftelomerase-negative tumors, there are tumors that are neithertelomerase-positive nor standard ALT, for example up to half ofglioblastomas (5). It will be of interest to screen such tumors for theexistence of tandem array telomeres, and to determine if such anonstandard ALT mechanism correlates with prognosis orresponses to different therapies.

Acknowledgments

Received 8/10/2004; revised 1/3/2005; accepted 1/14/2005.Grant support: Grants from the National Institute on Aging (K08AG000775), the

Concern Foundation, the Veterans Administration, and by ACS IRG 78-002-24.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Sheila Stewart and Nina Luning Prak for their insight, as well as membersof the Marciniak, Guarente, and Johnson labs for discussions and comments on themanuscript, and Roger Reddel for communicating unpublished observations.

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2005;65:2730-2737. Cancer Res Robert A. Marciniak, David Cavazos, Richard Montellano, et al. Lengthening of Telomeres Cell LineA Novel Telomere Structure in a Human Alternative

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A Novel Telomere Structure in a Human Alternative ... · 1Department of Medicine, ... by elevated rates of telomere recombi-nation, and by nuclear structures called ALT-associated