Oncogene (1998) 16, 1825 ± 1838  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human ®broblasts expressing human papillomavirus type 16 E6 oncoprotein

Leonid Filatov, Vita Golubovskaya, John C Hurt, Laura L Byrd, Jonathan M Phillips and William K Kaufmann

Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295, USA

Cell cycle checkpoints and tumor suppressor gene functions Introduction appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal Chromosomal instability is common in cancer cells destabilization was monitored in relation to telomere (Holliday, 1989; Hartwell and Kastan, 1994). Not only structure, lifespan control and G2 checkpoint function. do cancers display abnormalities of chromosomes, be Replicative senescence was inactivated in secondary they polyploidy, aneuploidy, interstitial deletions and cultures of human skin ®broblasts by expressing the human ampli®cations, or a single marker chromosome, but papillomavirus type 16 (HPV-16) E6 oncoprotein to malignant cells also appear to acquire such abnormal- inactivate p53. Chromosome aberrations were enumerated ities at increased rates in comparison to their normal during in vitro aging of isogenic control (F5neo) and HPV- progenitors. The mechanisms of genetic instability in 16E6-expressing (F5E6) ®broblasts. We found that cancer cells are, therefore, of considerable interest. structural and numerical aberrations in chromosomes were Chromosome instability in human cells may be caused signi®cantly increased in F5E6 cells during aging in vitro by defects in various elements of DNA metabolism and ¯uorescence in situ hybridization (FISH) analysis using including replication, chromosome segregration, repair chromosome-speci®c probes demonstrated the occurrence and recombination processes (Hartwell, 1992; Cohen of rearrangements involving chromosome 4 and 6 in and Levy 1989; Coquelle et al., 1997). Chromosomal genetically unstable F5E6 cells. Flow cytometry and aberrations and alterations in DNA ploidy can be karyotypic analyses revealed increased polyploidy and induced by treatments with various drugs that damage aneuploidy in F5E6 cells only at passages 416, although DNA, including chemotherapeutic agents and chemical these cells displayed defective mitotic spindle checkpoint carcinogens, or by exposure to ionizing and ultraviolet function associated with inactivation of p53 at passages 5 radiations. Remarkably, expression of viral oncopro- and 16. G2 checkpoint function was con®rmed to be teins that inactivate tumor suppressor gene functions gradually but progressively inactivated during in vitro aging also induces chromosomal aberrations (Stewart and of E6-expressing cells. Aging of F5neo ®broblasts was Bacchetti, 1991; Chang et al., 1997), implying that documented during in vitro passaging by induction of a chromosome stability is preserved by tumor suppressor senescence-associated marker, pH 6.0 lysosomal b-galac- gene expression. tosidase. F5E6 cells displayed extension of in vitro lifespan The tumor suppressor genes p53 and pRB serve and did not induce b-galactosidase at high passage. Erosion within checkpoint circuits that regulate cell division. of telomeres during in vitro aging of telomerase-negative Cell cycle checkpoints represent positions of control F5neo cells was demonstrated by Southern hybridization that ensure the completion of dependent events in the and by quantitative FISH analysis on an individual cell cell division cycle and provide more time for DNA level. Telomeric signals diminished continuously as F5neo repair before DNA replication and mitosis (Hartwell cells aged in vitro being reduced by 80% near the time of and Kastan, 1994). Two DNA damage-responsive replicative senescence. Telomeric signals detected by FISH checkpoints act to delay the G1?S and G2?M cycle also decreased continuously during aging of telomerase- phase transitions (Kaufmann and Paules, 1996). negative F5E6 cells, but telomeres appeared to be stabilized Inactivation of p53 ablates G1 checkpoint function at passage 34 when telomerase was expressed. Chromoso- and is associated with gene ampli®cation and mal instability in E6-expressing cells was correlated chromosomal instability in human ®broblasts (Yin et (P50.05) with both loss of telomeric signals and al., 1992; Livingstone et al., 1992; White et al., 1994). inactivation of G2 checkpoint function. The results suggest Ataxia telangiectasia cells that are defective in both G1 that chromosomal stability depends upon a complex and G2 checkpoint functions display enhanced interaction among the systems of telomere length main- frequencies of spontaneous and radiation-induced tenance and cell cycle checkpoints. chromosomal aberrations (Taylor et al., 1976; Zam- petti-Bosseler and Scott, 1981; Ejima and Sasaki, Keywords: chromosome; instability; telomere; cell cycle; 1986). Defects in G2 checkpoint function were checkpoint associated with enhanced frequencies of radiation- induced chromosome breaks in a panel of human cancer lines (Schwartz et al., 1996). Cell cycle Correspondence: WK Kaufmann checkpoints, consequently, appear to preserve genetic Received 14 August 1997; revised 4 November 1997; accepted 5 stability and suppress carcinogenesis (Hartwell, 1992; November 1997 Kaufmann and Kaufman, 1993). Chromosomal instability in HPV-16E6-expressing fibroblasts L Filatov et al 1826 The clastogenic e€ects of oncogenic viruses have telomeres may be sensed as irreparable DNA damage been related in part to their abilities to bind to and signals by the p53-dependent G1 checkpoint (Dulic et inactivate tumor-suppressor genes (Stewart and Bac- al., 1994; Kaufmann and Paules, 1996). Telomere chetti, 1991; Chang et al., 1997). Certain viruses, such length can be maintained in immortal cells, stem cells as SV40, adenovirus and oncogenic strains of human and germ-line cells by a ribonucleoprotein enzyme, papillomavirus (HPV), alter the functions of the telomerase, which adds new telomeric repeats to protein products of p53 and pRB. In the case of chromosome ends (for review see Greider and Black- HPV-16, the E6 gene product targets p53 for ubiquitin- burn, 1985; Kim et al., 1994). An alternative mediated proteolysis (Sche€ner et al., 1990; Galloway mechanism for telomere maintenance in immortal et al., 1994). A strain of HPV with low oncogenicity human cell lines may involve recombination (Bryan et (HPV-6) expresses an E6 protein that is unable to al., 1995; Rogan et al., 1995). Recently a human target p53 for proteolysis and fails to inactivate the G1 telomeric-repeat binding factor TRF1 was shown to be checkpoint (Galloway et al., 1994). Carcinogenesis by involved in telomere length regulation (van Steensel HPV-16 therefore appears to depend upon inactivation and de Lange, 1997). It was proposed that the binding of p53 function by its E6 gene. HPV-16E6-immorta- of TRF1 controls telomere length in cis by inhibiting lized human urothelial cells displayed high frequencies the action of telomerase at the ends of individual of unstable chromosomal aberrations and stable telomeres (van Steensel and de Lange, 1997). marker chromosomes as an example of the chromoso- To explore further the mechanisms of chromosomal mal instability that develops in HPV-16E6-expressing instability in HPV-16E6-expressing cells that lack the cells (Rezniko€ et al, 1994). By using an amphotropic replicative senescence checkpoint function, we have retrovirus to induce synchronous expression of HPV- monitored cellular aging during in vitro proliferation 16E6 in neonatal human skin ®broblasts, it was by assay of senescence-associated b-galactosidase possible to monitor the kinetics of cell cycle expression and by quantitative analysis of telomere checkpoint inactivation and chromosomal destabiliza- structure. In an attempt to identify the origin of tion under the in¯uence of a viral oncoprotein. di€erent chromosome aberrations we analysed chro- Expression of the HPV-16E6 oncoprotein in neonatal mosomal instability in relation to telomere structure human diploid ®broblasts inactivated the G1 check- and G2 checkpoint function. The development of point (Dulic et al., 1994), but the G2 checkpoint was chromosomal abnormalities was correlated (P50.05) una€ected initially (Paules et al., 1995; Levedakou et with both telomere erosion and attenuation of G2 al., 1995) and cells were normal cytogenetically (White checkpoint function in HPV-16E6-expressing cells. et al., 1994). However, as E6-expressing cells aged in Chromosomal stability appears to depend upon a vitro they displayed increased frequencies of chromo- complex interaction among the systems of telomere somal abnormalities including telomere associations, length maintenance and cell cycle checkpoints. chromosomal aberrations and aneuploidy (White et al., 1994; Kaufmann et al., 1997). This chromosomal instability was associated with concurrent attenuation Results of G2 checkpoint function (Paules et al., 1995; Kaufmann et al., 1997). The results suggested that Age-dependent appearance of chromosomal instability in oncogene-mediated inactivation of p53 can lead to F5E6 ®broblasts subsequent genetic alterations during cellular aging that deregulate the G2 checkpoint and induce We used two human ®broblast lines, F5neo and F5E6, chromosomal instability. to study chromosomal destabilization during in vitro Chromosomal instability also has been associated aging. F5E6 cells lacked G1 checkpoint function due to with telomere shortening during cellular aging in vitro. inactivation of p53 by the HPV-16E6 oncoprotein Progressive erosion of telomeres in precrisis SV40 (Dulic et al., 1994; Kaufmann et al., 1997). Degrada- virus-transformed cells and p53-de®cient Li ± Fraume- tion of p53 protein was con®rmed by Western blotting ni Syndrome (LFS) ®broblasts was associated with with p53-speci®c antibody (results not shown) and increased numbers of telomere associations, dicentric inactivation of G1 checkpoint function was con®rmed chromosomes, and ring chromosomes (Counter et al., by ¯ow cytometry after ionizing radiation treatment

1992; Rogan et al., 1995). Telomeres are T2AG3 repeats (Kaufmann et al., 1997). Normal F5neo and p53- complexed with specialized proteins that are located at negative F5E6 ®broblasts displayed in vitro population the ends of all eukaryotic chromosomes. Telomeres are expansion typical of cells of these types (Shay et al., essential for genetic stability. Telomeres di€erentiate 1993; White et al., 1994). F5neo ®broblasts underwent chromosome ends from double-stranded breaks and 65 population doublings between passages 3 and 27, protect them from aberrant recombination and after which senescence occurred. F5E6 cells had nuclease degradation (McClintock, 1941; for reviews undergone 66 population doublings by passage 22 see Healy, 1995; Ishikawa, 1997). In normal human and at passage 27 (population doubling level 88) F5E6 cells that lack expression of the enzyme telomerase, the cells were at the stage of crisis in which population ends of chromosomes gradually shorten with advanc- expansion was stable even though cells were dividing. ing age in vivo and with increasing number of passages Unexpectedly, F5E6 cells resumed population expan- in vitro due to the inability of DNA polymerases to sion by passage 34 and threafter population expansion replicate the 3' ends of linear DNA (Olovnikov, 1971; was continuous. Healy, 1995). Telomere shortening below a certain In a previous analysis of these cells (Kaufmann et length was proposed to induce irreversible cell cycle al., 1997) G2 checkpoint function and chromosomal arrest known as replicative cell senescence (Harley et aberrations were charted continuously as F5neo and al., 1990). Chromosome ends with critically eroded F5E6 cells aged through their in vitro proliferative life Chromosomal instability in HPV-16E6-expressing fibroblasts LFilatovet al 1827 spans. This experimental design required that cytoge- increased numbers of aneuploid metaphases with both netic and checkpoint analyses be performed over a 6 losses and gains of chromosomes (Figure 1b). The month interval as cells were passaged weekly. During F5E6 line also had increased percentages of cells with this analysis cells were cryopreserved at various 3-4n chromosome numbers (hypotetraploids and passage levels. Subsequent re-establishment of low tetraploids) at passages 22, 27 and 34 (Figure 2b). and intermediate passage preparations enabled syn- The passage 27 F5E6 cells in crisis displayed severely chronous assessment of chromosomal aberrations and aberrant ploidies with increased percentages of cells G2 checkpoint function in cells of various ages. with 5-13n chromosome numbers (Figure 2b and Additional evaluation of a senescence marker and Figure 3b). The reduced fraction of highly polyploid telomere structure permitted evaluation of the associa- metaphases in the postcrisis F5E6 cells at passage 34 tion between cellular aging and chromosomal instabil- probably re¯ects the emergence of one or a few clonal ity. We ®rst quanti®ed chromosomal aberrations in the sublines that maintain hypodiploid and hypotetraploid cell lines at various phases of their in vitro proliferative karyotypes. F5neo cells maintained 2n chromosome life span to determine the reproducibility of our content up to near senescence at passage 27. previous measurement (Kaufmann et al., 1997). Structural chromosomal abnormalities were infre- F5E6 cells, but not F5neo cells, displayed signi®cant quent in F5neo cells at all passages. Among 50 diploid age-dependent abnormalities of chromosome number metaphases sampled at each passage of F5neo cells, the and structure. The results of analyses of chromosome percentage with a chromosome aberration was 2 ± 6% numbers are summarized in Figure 1 and Figure 2. (results not shown). F5E6 ®broblasts exhibited similar F5neo cells had diploid numbers of chromosomes low frequencies of aberrations in diploid metaphases at (46+1) at all passages (Figure 1a), as did F5E6 cells at low passage levels (5 and 16) (Table 1). Starting with passages 5 and 16 (Figure 1b). At passages above 16 passage 22 the numbers of chromosomal aberrations in F5E6 cells with near 2n chromosome number displayed near-diploid F5E6 cells were progressively and sig- ni®cantly increased. Examples of the types of aberrations seen in F5E6 are shown in Figure 3. Structural aberrations included telomere associations, dicentrics, breaks, exchanges, ring chromosomes and a

a

b

b

Figure 1 Distribution of F5neo and F5E6 cells with near diploid chromosome numbers. (a) F5neo cells at passages 5, 16, 22 and 27 (cummulative population doubling levels 8, 42, 50 and 65, respectively). (b) F5E6 cells at passages 5, 16, 22, 27 and 34 (population doubling levels 8, 47, 66, 88 and 94 respectively). 50 Figure 2 Polyploidy analysis in F5neo and F5E6 cells. (a) F5neo metaphases were analysed for each passage number, except for cells at passages 5, 16, 22 and 27. (b) F5E6 cells at passages 5, 16, passage 34, where 33 metaphases were analysed. For chromosome 22, 27 and 34. 500 metaphases were examined for analysis of number 46: P50.001 in F5E6p22, 27 and p34 vs F5E5p5 cells. polyploidy for each passage level except for p34, where 50 For chromosome numbers, 37 ± 47: P50.03 in F5E6p22, 27 and metaphases were examined. For 3 ± 4 N, P50.0001 in F5E6p22, 34 vs F5E6p5 cells. For chromosome numbers, 47 ± 52: P50.002 27 and 34 vs F5E6p5. For 5 ± 13 N, P50.0001 in F5E6 p27 vs in F5E6p27 vs F5E6p 5 cells (Chi-square test) F5E6 p5 (Chi-square test) Chromosomal instability in HPV-16E6-expressing fibroblasts L Filatov et al 1828

a b

c d

Figure 3 Structural and numerical chromosome aberrations in F5E6 ®broblasts at passage 27. (a) Part of a Giemsa-stained metaphase with a chromatid break (single arrow), chromatid exchange (double arrow) and telomere association (triple arrow). (b) Polyploid DAPI-stained metaphase with dicentrics (arrows), ring chromosome (arrowhead) and chromosome breaks (double fragments). (c,d) FISH with directly labeled chromosome-speci®c probes (chromosome 4 ± spectrum green, chromosome 6 ± spectrum orange). (c) Metaphase showing translocation involving part of chromosome 4 (small arrowhead), normal chromosome 4 (arrow) and deleted isodicentric chromosome 4: idic(4)(q32?):4pter?cen?4q32?::4q32??cen?4pter (large arrowhead), and a translocation involving part of chromosome 6 (wide arrow). (d) Tetraploid cell with four normal chromosomes 6 (red), one normal chromosome 4 (green, arrow) and four translocations involving chromosome 4 (arrowheads) Chromosomal instability in HPV-16E6-expressing fibroblasts LFilatovet al 1829 Table 1 Chromosomal aberrations in F5E6 ®broblasts Aberrations Telomere Breaks Chd Ring Total % aberrant Passage associations Dicentrics Chm Chd Exch Chm (freq.) cells p5 0 0 0 1 0 0 1 (0.02) 2 p16 1 1 1 1 0 0 4 (0.08) 8 p22 2 3 1 1 0 1 8 (0.16) 14*a p27 4** 5** 4 7 2 2 24 (0.48) 28*b p34 0 0 3 3 0 0 6 (0.18) 15*c Abbreviations: Chm ± chromosome, Chd ± chromatid, P ± passage, Exch ± exchange, Freq. ± frequency. Fifty metaphases with diploid chromosome numbers (46+1) were examined for every passage level (except, for passage 34, where 33 metaphases were analysed). *For total % aberrant cells: P50.05 vs F5E6p5 (Chi-square test). aat p22, 1 cell (2%) had 2 aberrations, dicentric+Chd break. bAt p27, 4 cells (8%) had 2 aberrations: 2 cells with Ring Chm+Chd break, 1 cell with Chd exchange+Chm break, 1 cell with telomere association+Chd break; 3 cells (6%) had 3 aberrations: 1 cell with 2 dicentrics+Chm break, 1 cell with telomere association+dicentric+chd break, 1 cell with 2 chd breaks+dicentric. cAt p34, 1 cell (2%) had Chm break+Chd break. **For telomeric associations and dicentrics: P50.01 vs F5E6 p5 and p34 (Chi-square test)

translocations. The numbers of telomere associations M compartment. When treated with colcemid, F5E6 and dicentrics increased progressively with aging of cells at passage 9 showed a sevenfold accumulation of F5E6 ®broblasts (Table 1). No such aberrations were cells in the diploid G2/M compartment (from 7 ± 51%) found at passage 5 whereas at passage 27 ®ve dicentrics and increased polyploidization (22% of cells in and four telomere associations were observed. F5E6 tetraploid S, G2 and M compartment). F5neo controls cells also displayed a similar age-dependent increase in also displayed sevenfold accumulation in the diploid chromatid chromosome breaks, ring chromosomes, G2/M compartment during incubation with colcemid and exchanges between passages 5 and 27. The post- but only 2% of cells were in the tetraploid S, G2 and crisis F5E6 at passage 34 displayed increased M compartment (Figure 4). Passage 25 F5E6 cells frequencies of chromosome and chromatid breaks, displayed 16% tetraploid cells when incubated with but no telomeric associations or dicentrics were seen colcemid (data not shown). However, in the absence of in the 33 diploid metaphases that were scored. The colcemid treatment the high passage F5E6 cells also percentages of F5E6 cells with telomeric associations displayed increased fractions of cycling cells with or dicentrics were signi®cantly di€erent at passages 27, tetraploid DNA content (14% of cells in tetraploid S, in which 7 of 50 of metaphases were positive, and G2 and M) as compared to F5neo controls (Figure 4). passage 34, in which no metaphases were positive (Chi- The 14% of passage 25 F5E6 cells in the tetraploid S, square, P50.05). G2 and M compartment may be matched by an equal or larger percentage of tetraploid G1 phase cells that are superimposed on the diploid G2/M compartment. Rearrangements of chromosomes 4 and 6 in F5E6 Thus, the ¯ow cytometry analyses supported the ®broblasts at passage 27 cytogenetic data showing increasing numbers of G-banding of F5E6 cells was used for preliminary polyploid F5E6 cells during in vitro aging. detection of rearranged chromosomes (not shown). Observation of rearrangements involving chromosomes Inactivation of G2 checkpoint function in F5E6 4 and 6 led us to perform FISH with chromosome- ®broblasts during aging speci®c probes. Two near-tetraploid metaphases with examples of rearrangements of these chromosomes are As a further control G2 checkpoint function was shown in Figure 3c and d. The rearrangements with quanti®ed by enumerating the fractions of cells in chromosome 4 included one deleted isodicentric mitosis 2 h after treatment with g-irradiation (Table 2). (Figure 3c) and several di€erent translocations F5neo cells at passage level 5 had a strong G2 (Figure 3d). A rearrangement involving a portion of checkpoint response with no cells escaping radiation- chromosome 6 is shown in Figure 3c. induced G2 delay. G2 checkpoint function remained unchanged as F5neo ®broblasts aged from passage 5 to 27. In F5E6 ®broblasts at passage 5 G2 checkpoint Mitotic spindle checkpoint function function was normal, as only 4% of G2 phase cells The analyses of chromosome numbers (Figure 2) entered mitosis 2 h after 1.5 Gy of g-rays. As F5E6 cells revealed increased frequencies of polyploid meta- aged in vitro they displayed progressive attenuation of phases in aging F5E6 cells. Polyploidization can imply G2 checkpoint function in response to g-irradiation, as inactivation or deregulation of mitotic spindle check- was shown previously (Paules et al., 1995; Kaufmann et point function (Cross et al., 1995; Minn et al., 1996; al., 1997). When F5E6 cells were in crisis at passage DiLeonardo et al., 1997). The mitotic spindle level 27, 100% of irradiated cells excaped the G2 checkpoint was assayed by incubating cells in colcemid checkpoint and entered mitosis 2 h after 1.5 Gy. to inhibit microtubule polymerization and quantifying initiation of DNA synthesis after 24 h (Cross et al., Aging of F5neo ®broblasts con®rmed by b-galactosidase 1995; DiLeonardo et al., 1997) (Figure 4). In the assay absence of colcemid, F5neo and F5E6 cells at passage 9 displayed quite similar pro®les of incorporation of To monitor the kinetics of cellular aging during in vitro BrdU, with 1 ± 2% of cells in the tetraploid S, G2 and passaging of the ®broblast lines, we assayed the Chromosomal instability in HPV-16E6-expressing fibroblasts L Filatov et al 1830 senescence-associated marker, pH 6.0 b-galactosidase Terminal restriction fragment length analysis in F5neo (Dimri et al., 1995). When tested at passage 5, both lines and F5E6 ®broblasts displayed few b-galactosidase-positive cells (Figure 5). As cells aged they displayed di€erent levels of Southern hybridization analysis of the telomere- expression of b-galactosidase. Virtually all F5neo cells associated terminal restriction fragment (TRF) is expressed pH 6.0 b-galactosidase activity at passage 27. shown in Figure 7. TRF length and hybridization In contrast, few if any F5E6 cells expressed this marker intensity decreased with cell aging in vitro.At of senescence when in crisis at passage 27 (Figure 5). passage 9, TRF length in F5neo and F5E6 cells did not di€er substantially. As F5neo cells aged in culture, the TRF shortened modestly and the Expression of telomerase in F5E6 ®broblasts only at late intensity of hybridization with the telomere-speci®c passages probe was diminished. The TRF length decreased We assayed for telomerase activity in F5neo ®broblasts more extensively in F5E6 cells than in F5neo cells at passages 10 and 26 and F5E6 ®broblasts at passages (Figure 7). At passage 34, the greatest mass of 10, 26 and 34 using TRAP (Figure 6). Telomerase telomere-probe-hybridizing DNA in F5E6 cells was activity was not detected at passage 10 in F5neo and less than 2 kb in length (Figure 7). The TRF length F5E6 cells, while at passage 26 some telomerase activity remained constant at this level in F5E6 cells, as it was detected in F5E6 but not in F5neo cells. Telomerase did not decrease further at higher passages (data not activity was relatively high at passage 34 in F5E6 cells. shown).

Figure 4 Attenuation of mitotic spindle checkpoint function in F5E6 ®broblasts. F5neo and F5E6 cells at passages 9 and 25 in logarithmic growth phase were incubated for 24 h with and without 0.27 mM colcemid. Addition of BrdU for the last 2 h of colcemid incubation allowed the determination of the fraction of cells that were synthesizing DNA. Fixed cells were processed for two-parameter ¯ow-cytometric analysis of DNA content (propidium iodide) vs incorporation of BrdU (FITC). The small boxes enclose diploid cells in G2/M and tetraploid cells in G0/G1, the large boxes enclose tetraploid cells in S, G2 and M. The percentages of cells in these boxes are indicated

Table 2 Attenuation of G2 checkpoint function in F5E6 cells during in vitro aging Mitotic indexa F5neo % escaping G2 F5E6 % escaping G2 Passage sham % g-Irradiated checkpointb sham % g-Irradiated checkpointb 5 1.8 0 0 3.4 0.15 4 16 1.6 0.05 3 3.4 0.8 24* 22 1.1 0 0 10.4 4.8 46* 27 0.15 0 0 5.5 5.8 100* aThe percentage of cells in mitosis 2 h after sham treatment or irradiation with 1.5 Gy g-rays (number mitotic cells/ total number cells counted, n=2000). bThe percentage represents the fraction of irradiated G2 phase cells that failed to delay entry into mitosis 2 h after irradiation with 1.5 Gy g-rays. *P50.01 vs F5E6 p5 Chromosomal instability in HPV-16E6-expressing fibroblasts LFilatovet al 1831

Figure 5 Expression of b-galactosidase in aged F5neo but not F5E6 cells. Assay of pH 6.0 b-galactosidase was done as described in Materials and methods. (a) F5neo cells at passage 5, (b) F5E6 cells at passage 5. (c) F5neo cells at passage 27, (d) F5E6 cells at passage 27. Phase-contrast photomicrographs were image-captured and a composite generated using Macintosh Photoshop software

percent of diploid metaphases with structural chromo- In situ analysis of telomere erosion in F5neo and F5E6 somal aberrations (Table 1); percent of polyploid cells during in vitro aging (42n) metaphases (Figure 2b); and mean number of In order to establish the kinetics of telomere shortening telomere-speci®c spots in diploid metaphases (Figure 9, during in vitro aging on an individual cell basis, we Table 3). All but one comparisons indicated signi®cant used FISH with a human telomere probe, which is a correlations (P50.05). The only pairwise comparison direct method to determine telomere length (Henderson that was not correlated with a high probability was the et al, 1996; Therkelsen et al., 1995; Lansdorp et al., percent of polyploid metaphases versus telomere spot 1996). The number and size of telomeric signals as well number. The greatest correlation was between the as their intensity are functions of telomere length percentages of cells escaping the G2 checkpoint and (Lansdorp et al., 1996). The total number of visible diploid cells with chromosomal aberrations telomeric signals over diploid metaphases (FITC- (P=0.0005). positive dots at chromatid termini) decreased with in vitro aging at similar rates in normal and E6-expressing ®broblasts (Figure 8a ± g, Table 3). However, starting Discussion with passage 22 the di€erence in the number of telomeric signals between F5neo and F5E6 ®broblasts Chromosomal instability in HPV16E6-expressing hu- was signi®cant (P50.05) (Figure 9). At passage 27 near man skin ®broblasts lacking p53-dependent G1 the time of replicative senescence, the number of checkpoint function was correlated with both telo- telomeric signals detected in mitotic F5neo cells was mere erosion and inactivation of G2 checkpoint reduced by 80% relative to the signals observed at function. By quantitative FISH analysis on an passage 5. The average number of FITC-positive individual cell basis it appeared that telomeres eroded signals in F5E6 cells was reduced still further from at similar rates in diploid F5neo and F5E6 cells, with 159 (at passage 5) to 21 (at passage 27), representing a approximately 80% loss of telomeric sequences at the decrease of 87% when F5E6 cells were in crisis (Figure time of replicative senescence in F5neo cells. F5E6 9). A method for enumeration of telomere intensity cells bypassed the replicative senescence checkpoint, based on photodetection yielded an equivalent result con®rmed by expression of 20 additional population (R2=0.995, P50.01) (Table 3). doublings before crisis (Shay et al., 1993; Rogan et al., 1995), lack of expression of the senescence-associated marker, pH 6.0 lysosomal b-galactosidase (Dimri et Correlations between inactivation of G2 checkpoint al., 1995) and more severe loss of telomeric sequences. function, telomere erosion and chromosomal instability in G2 checkpoint function was normal soon after E6-expressing ®broblasts expression of HPV-16E6 but progressively degraded The following data were analysed for correlations: during aging of F5E6 cells. F5neo cells had no percent of cells evading the G2 checkpoint (Table 2); changes of karyotype during in vitro aging and no Chromosomal instability in HPV-16E6-expressing fibroblasts L Filatov et al 1832

p9 p21 p27 p34 p10 p26 p34 /Hind lll λ F5E6 F5E6 F5E6 F5E6 F5 F5 F5 F5 F5E6 F5 F5E6 F5E6 MDAH041 Lysis Buffer 23.1 —

9.4 —

6.5 —

4.4—

2.3 — 2.0 —

Figure 7 Terminal restriction fragment length analysis in F5neo and F5E6 cells. Genomic DNA was isolated from F5 and F5E6 cells at the passages indicated and digested with HinfI and RsaI enzymes. The digested DNA was separated by electrophoresis on Figure 6 Telomerase activity was examined in F5neo and F5E6 a 0.8% agarose gel and hybridized to digoxigenin-labeled cells. Telomerase activity was examined in F5neo cells at passage (TTAGGG)3 probe. Molecular weight markers (Lambda DNA/ 10 and 26 and in F5E6 cells at passages 10, 26 and 34 by PCR- HindIII digests) are indicated on the left. The image is a based TRAP assay as described in Materials and methods. composite of two di€erent exposures to optimize visualization MDAH 041 (an immortal LFS ®broblast line) was used as a of hybridization in the high passage cells positive control and lysis bu€er was used as a negative control

loss of G2 checkpoint function. In contrast, F5E6 cells al., 1993; Bond et al., 1994; White et al., 1994). A at low passage (5) had normal karyotype, but starting previous analysis by Shay et al. (1993) suggested that with passage 22, the number of cells with structural inactivation of both p53 and pRB was required for and numerical chromosome aberrations increased immortalization of human ®broblasts. However, wild progressively. The p53-dependent mitotic spindle type pRB was observed in one line of spontaneously checkpoint was defective in F5E6 cells starting in immortalized LFS ®broblasts (Rogan et al., 1995). early passages when chromosomes were normal, These immortal ®broblasts had no detectable p16INK4 indicating that this checkpoint alteration also was protein (possibly equivalent to inactivation of pRB) insucient for chromosome instability in F5E6 cells. and expressed chromosome destabilization of the types Thus, signi®cant correlations were observed among described here (Rogan et al., 1995). Other genetic telomere erosion, inactivation of G2 checkpoint events leading to elevated expression of cyclins, cyclin- function, and structural and numerical chromosome dependent kinases (CDK) and CDK inhibitors may abnormalities during the aging of cells lacking G1 override pRB and contribute to escape from senescence checkpoint function. (Grana and Reddy, 1995). The sequence of alterations The results that the HPV-16E6-expressing ®broblasts in chromosome structure and number observed in displayed extended lifespan in vitro at late passages are F5E6 cells closely resembled those previously docu- consistent with data on spontaneous immortalization mented during stages of immortalization of human of LFS ®broblasts (Rogan et al., 1995). It has been ®broblasts induced by SV40 virus (Counter et al., 1992) shown that loss of wt p53 by either mutation or and epithelial cells induced by HPV-16E6 (Klingelhutz deletion results in a ®nite increase in the in vitro et al., 1994). In both of these examples, severe proliferative potential of human ®broblasts (Shay et chromosomal instability developed during cellular Chromosomal instability in HPV-16E6-expressing fibroblasts LFilatovet al 1833 aging, peaking at the time of crisis, when most cells Telomeres eroded with age in vitro in both F5neo died. Post-crisis lines expressing telomerase and stable and F5E6 cells, reaching a stable stage at passage 34 in telomeres also appeared to have partially stabilized F5E6 cells, when telomerase was expressed. Klingel- chromosomes. hutz et al. (1994) measured telomere length in relation

Figure 8 FISH analysis of telomere erosion in F5neo and F5E6 cells. Digoxigenin-labeled `all-human-telomeric' probe was used for FISH analysis. FISH analysis was performed under identical conditions for cells at each passage level. After detection with antidigoxigenin-FITC, chromosomes were counterstained with propidium iodide. Fluorescence photomicrographs were image- captured and a composite generated using Macintosh Photoshop software. (a,b,c) F5neo metaphases from passages 5, 16 and 27, respectively. (d,e,f,g) F5E6 metaphases from passages 5, 16, 27 and 34, respectively Chromosomal instability in HPV-16E6-expressing fibroblasts L Filatov et al 1834 decrease in intensity and detectability of telomeric signals during in vitro aging (Table 3). Interchromoso- mal heterogeneity in telomere signals (Landsorp et al., 1996) in aged cells suggests that shortening of certain chromosome telomeres below a critical length could trigger cell senescence (Healy, 1995). E6-expressing cells did not undergo cell senescence, but as telomeres continued to erode in the highly proliferative aging population, the frequency of telomere associations and dicentric chromosomes increased until cells reached crisis at or about passage level 27. Chromosomal end- to-end associations and fusions were found to increase progressively over several generations of mTR (mouse telomerase RNA) knockout mice, as telomeres were eroded (Blasco et al., 1997). Our data also indicate that telomere shortening is associated with chromosome destabilization in human cells lacking p53-dependent Figure 9 Telomere erosion dynamics in F5neo and F5E6 cells. G1 checkpoint function. Metaphases were scanned after FISH with digoxigenin-labeled telomere probe. The number of telomeric FITC-signals was Several types of chromosome aberrations were expressed as a function of cell aging. The values shown are observed in E6-expressing ®broblasts, including means+standard deviations (n=10). Telomeres eroded signifi- telomeric associations, dicentrics, ring chromosomes, cantly in F5E6 cells as compared to F5neo cells at high passages breaks, chromatid exchanges and translocations. (p22 and 27, Student's t-test, P50.05). FITC-labeled metaphases were also evaluated by a photomicroscopic method, which These aberrations may be divided into two groups determined the number of seconds needed to achieve sucient depending on their putative origins: the ®rst group exposure (Table 3) may be linked to telomere erosion and includes telomere associations and dicentrics; the second group may be linked to inactivation of G2 checkpoint function and includes breaks, exchanges, Table 3 Quantitation of telomeric FITC signals during cellular translocations and ring chromosomes. Both groups of aging aberrations developed progressively as F5E6 cells aged Mean # of signals Exposure time to crisis, while the ®rst group appeared to be Cell line/passage (+s.d.) (s+s.d.)** diminished post-crisis (Table 1). F5neo p5 160+791+7One of the functions of telomeres is to prevent end- F5E6 p5 159+768+5to-end associations and fusions between chromosomes. F5neo p16 109+7 132+7 When two chromosomes lose telomeric DNA they F5E6 p16 108+4 136+7 F5neo p22 69+6 191+9 may associate at their termini as a ®rst step that F5E6 p22 61+6* 193+8 precedes formation of a true dicentric chromosome. F5neo p27 31+6 230+9 When the centromeres of the dicentric chromosome F5E6 p27 22+7* 235+6 segregate towards the two daughter poles during F5E6 p34 16+6 245+8 anaphase, a chromosome break may occur between *P50.05 vs F5neo at same passage. **Exposure time is inversely the two centromeres. The new chromosome ends are proportional to the intensity of FITC-telomeric signals in the non-telomeric and if carried through another replica- metaphase. Linear regression of mean # of signals against exposure time yielded a correlation coecient (R2) of 0.995, P50.001 tion cycle may again fuse to form another dicentric. A cycle of chromatid bridge-breakage and fusion was originally described by McClintock for maize chromo- somes (McClintock, 1941; see Coquelle et al., 1997 for to immortalization of cervical epithelial cells and found recent discussion) but without the ®rst apparent step that telomeres gradually shortened in the pre-senes- of forming chromosome end-to-end associations, and cence normal and precrisis E6-expressing cells. In this model has been invoked to explain the mechan- contrast to precrisis cells, E6/E7-immortalized cells isms of gene ampli®cation in cells incubated with generally showed elongated telomeres suggesting that DNA metabolic poisons (Coquelle et al., 1997). The arrest of telomere shortening may be important for number of telomere associations and dicentrics was HPV-associated immortalization. In our study the TRF signi®cantly increased with the aging of F5E6 length had decreased extensively in F5E6 cells near the ®broblasts, consistent with a view that progressive time of crisis. The TRF has been shown to be telomere erosion in cells that lack p53 and G1 composed of telomere and telomere-like sequences as checkpoint function leads to the formation of these well as non-telomeric sequences (Counter et al., 1992; structures. A connection between telomere associa- Henderson et al., 1996). Thus, TRF size distributions tions/dicentrics and telomere erosion is also suggested re¯ect chromosomal and cellular heterogeneity in both by the observation that in postcrisis F5E6 cells, which

T2AG3 and non-T2AG3 components. In situ hybridiza- expressed telomerase to stabilize telomeres, the tion with a telomere-speci®c probe quanti®ed changes frequency of cells with these lesions was signi®cantly in telomere size on an individual cell level in the reduced. Interestingly, ataxia telangiectasia (AT) cell diploid population of dividing cells. This method is lines that are defective in G1 and G2 checkpoint particularly useful for small populations of cells and functions (Kaufmann and Paules, 1996; Rotman and when applied to the F5neo and F5E6 ®broblasts, Shiloh, 1997), displayed increased frequencies of quantitative telomere FISH showed a continuous chromosome end-to-end-associations (Pandita et al., Chromosomal instability in HPV-16E6-expressing fibroblasts LFilatovet al 1835 1995, 1996). An inverse correlation between telomere ism of chromosomal endoreduplication in murine cells length and chromosome end associations was observed indicated that p53 acts to prevent initiation of DNA in AT cells (Pandita et al., 1995). synthesis in cells that pass from mitotic arrest directly Another origin of dicentric chromosomes as well as into G1, without having completed the anaphase and chromosome translocations and deletions are DNA telophase steps of mitosis (Minn et al., 1996). In normal double-strand breaks occuring during G1 phase in the human ®broblasts, the p53-dependent mitotic spindle non-telomeric regions of chromosomes. These may checkpoint exercises stringent control over initiation of occur at fragile sites (Yunis and Soreng, 1984; Coquelle DNA synthesis. As shown in Figure 4, less than 2% of et al., 1997), constitutive heterochromatin regions, and normal ®broblasts endoreduplicate DNA after 24 h in chromosomal regions (mostly R-bands) containing incubation with colcemid. The nature of the signal that clusters of Alu-family repeats (Filatov et al., 1987, induces p53 to arrest S phase entry in colcemid-treated 1991); Korenberg and Rykowski, 1988). AT cells also cells has not been determined. It is notable that expressed higher frequencies of chromosomal breaks at polyploidization of E6-expressing ®broblasts was also metaphase (Pandita et al., 1995). correlated with chromosomal aberrations and attenua- The second group of chromosomal aberrations tion of G2 checkpoint function. Although the E6 cells found in F5E6 cells includes chromosome breaks, displayed the ability to endoreduplicate when stressed translocations, chromatid breaks, chromatid ex- with the mitotic poison colcemid, they maintained a changes and ring chromosomes. The chromatid-type diploid karyotype at least through passage 16. This aberrations are especially interesting as these originate result raises the possibility that some element of genetic during S phase from lesions in only one of the parental destabilization associated with chromosomal damage or DNA strands or from double-strand breaks introduced attenuation of G2 checkpoint produces a stress on the into late S and G2 phase cells. The appearance of mitotic spindle checkpoint system, thereby selecting for chromatid-type aberrations in F5E6 metaphases im- outgrowth of polyploid cells. plies some defect in DNA damage response or DNA The absence of numerical and structural chromo- repair. As chromosomal aberrations were highly some alterations in p53-negative E6-expressing cells at correlated with inactivation of G2 checkpoint func- low passages shows that there is no simple relation- tion, which serves to prevent cells from entering mitosis ship between p53 loss or mutation and chromosomal with broken chromosomes, it is possible that loss of instability (Yin et al., 1992; Livingstone et al., 1992; this checkpoint permits cells to enter mitosis with White et al., 1994; Lengauer et al., 1997). In our damage that would normally arrest growth. G2 study, low passage p53-negative E6 cells with checkpoint function remained attenuated in post-crisis attenuated mitotic spindle checkpoint function ex- F5E6 cells that express telomerase and this may pressed a functional G2 checkpoint and displayed a account for the persistent chromosome and chromatid normal telomere length distribution. Only in aged aberrations. Spontaneously immortalized LFS fibro- cells with attenuated G2 checkpoint and eroded blasts and SV40-immortalized ®broblasts also display telomeres was chromosomal destabilization mani- attenuation of G2 checkpoint function indicating that fested. The results suggest that chromosomal destabi- the defect in this checkpoint post-crisis is independent lization in telomerase-negative cells that express of telomerase and stabilization of telomeres (Kauf- HPV16 E6 may involve selective outgrowth of G2 mann et al., 1995; Paules et al., 1995). checkpoint-defective cells during a phase of telomere The ring chromosomes seen in this series may have crisis. Expression of telomerase post-crisis was two di€erent origins. Usually ring chromosomes are associated with incomplete normalization of chromo- accompanied by double fragments. In such cases the somal numbers and structure suggestive of persistent origin of the ring chromosome is a result of double- chromosomal instability. strand breaks in both chromosome arms followed by In summary, the simultaneous analysis of checkpoint fusion of these newly formed chromosome ends. functions and telomere structure provided new insights Another way to form a ring chromosome is by to the mechanisms of chromosome destabilization in telomere shortening. The ring chromosome shown in human cells. The results indicate that there is a Figure 3b did not have an accompanying double complex interplay between telomere erosion and fragment and may be formed by this mechanism. attenuation of G2 checkpoint function that accom- We found an involvement of chromosomes 4 and 6 panies the instabilities for both chromosome numbers in translocations with other chromosomes in F5E6 cells and structure. The observation that chromosomal at passage 27, which is consistent with the literature on instability was associated with both telomere erosion chromosomes involved in senescence and aging (Smith and inactivation of G2 checkpoint function precludes a and Pereira-Smith, 1996; Ning et al., 1991; Solinas- conclusion as to which is the rate-limiting step. Toldo et al., 1997). One of the chromosome 4 Targeted inactivation of G2 checkpoint function in rearrangements was an isodicentric chromosome with low passage E6-expressing cells with long telomeres breakpoints located very close to the telomere on the may enable determination of the rate-limiting step in q-arm. This idodicentric could have been formed by chromosome destabilization. telomere shortening and chromatid fusion. E6-expressing cells also displayed highly signi®cant changes in chromosome numbers and ploidy suggestive Materials and methods of non-disjunction errors and mitotic spindle abnorm- alities (Fukasawa et al., 1996). However, signi®cant Cells ploidy changes did not occur until there had been The diploid human ®broblast line, NHF5, was derived substantial telomere erosion and attenuation of G2 from foreskin and grown in minimal essential medium checkpoint function. A recent analysis of the mechan- supplemented with 10% fetal bovine serum, L-glutamine Chromosomal instability in HPV-16E6-expressing fibroblasts L Filatov et al 1836 and antibiotics as previously described (Paules et al., 1995; Probe preparation, hybridization, postwashing and detec- Kaufmann et al., 1997). Primary cultures of NHF5 cells tion The slides from the cells at passage 27 were used for were infected with a replication-defective amphotropic FISH with Spectrum Orange chromosome 6 and Spectrum retrovirus containing a neomycin-resistance gene and Green chromosome 4 probes. Two microliters of Spectrum designated, F5neo, or with the retrovirus containing the Green WCP 4 and 2 ml of Spectrum Orange WCP 6 probes HPV-16E6 oncogene, and designated, F5E6 (Kaufmann et were mixed with 2 ml of water and 14 ml of hybridization al., 1997). Infected cells were selected by growth in G418. bu€er (50% formamide/10% dextran-sulfate/26SSC). F5E6 and isogenic control F5neo cells were provided by Dr WCP 4 and WCP 6 DNA was denatured 5 min at 738C. Denise Galloway (Fred Hutchinson Cancer Research The denatured probe was added to slides, sealed under Center). These cells were passaged every week using a glass coverslip with rubber cement and incubated overnight 1 : 8 split ratio. At various passage levels during in vitro at 378C. Postwashing was performed at 458Cinthree population expansion, aliquots of cells were cryopreserved. changes of 50% formamide/26SSC, pH 7.0 for 5 min After secondary cultures of F5neo cells underwent each, in 26SSC, pH 7.0 for 5 min and ®nally at 26SSC/ senescence arrest, cultures of F5neo and F5E6 cells were 0.1% NP-40 for 5 min (Filatov et al., 1996). Slides were reconstituted from aliquots frozen at passage levels 4, 15, counterstained with DAPI (4'-6-diamidino-2-phenildole) in 21 and 26. Re-constituted lines were used generally within Antifade solution (18 ml per slide; Vysis, Inc.). two passages. F5E6 cells that had been in culture continuously up to passage 34 were also included in some analyses. FISH with telomeric DNA probe The slides from F5-neo cells at passages 5, 16, 22, 27 and Mitotic spindle and G2 checkpoint assays F5-E6 cells at passages 5, 16, 22, 27 and 34 were used for FISH with an `all-human-telomeres' probe (Oncor, Inc.). The dependence of initiation of DNA synthesis on Digoxigenin-labeled telomeric probe was hybridized and completion of mitosis was assayed by incubating log- detected according to the supplier's protocol: 30 mlofprobe phase cells with 0.27 mM colcemid for 24 h. During the were denatured for 5 min at 738C, added to slides, and ®nal 2 h of incubation BrdU was added at 10 mM. Fixed incubated overnight at 378C. The hybridization signals were cells were analysed for DNA content and incorporation of detected by incubation with ¯uorescein-labeled anti-digox- BrdU by ¯ow cytometry as previously described (Kauf- igenin antibody for 5 min at 378C, followed by amplifica- mann et al., 1997). G2 delay was assayed by ¯uorescence tion with rabbit anti-sheep and ¯uorescein-labeled anti- microscopy, also as described in Kaufmann et al. (1995). rabbit antibodies for 15 min each. The chromosomes were Log-phase cells were irradiated with 1.5 Gy or sham- counterstained with propidium iodide (0.03 mg/ml; Oncor, treated (as controls) then incubated for 2 h at 378Cbefore Inc.). Cells were viewed and photographed with a Zeiss ®xation with methanol/acetic acid (3 : 1). After staining photomicroscope (Axiophot 20) equipped for epifluores- with propidium iodide, mitotic cells were enumerated by cence. Kodak Ektachrome color slide 400 ®lm was used for ¯uorescence microscopy and expressed as a percentage of photography. Telomere spot numbers were determined for the total (mitotic index). A minimum of 2000 cells were 10 diploid metaphases per sample. A photomicroscopic counted for each sample. To ensure an unbiased analysis, method for quanti®cation of telomere signal intensity over samples were assigned coded random numbers before diploid metaphases was also employed (Lansdorp et al., quantitation of mitotic index. 1996). Exposure time (in s), that is inversely proportional to intensity, was automatically measured by the Zeiss Cytogenetics photomicroscope exposure system. Fibroblasts were split 1 : 4 1 day before harvesting when colcemid (0.05 mg/ml) was added for 25 min to arrest cells Telomere terminal restriction fragment (TRF) analysis in metaphase. Cells were detached with trypsin/EDTA, Genomic DNA was extracted from cells by a standard incubated in 0.075 M KCl for 20 min at 378C, and ®xed in three changes of methanol:acetic acid (3 : 1). Fixed cell protocol. Cell pellets were resuspended in 1 vol of lysis pellets were used for slide preparation. Slides were air dried bu€er, containing 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, at least 2 days at 378C before using. For chromosome 25 mM EDTA, pH 8.0, 0.5% sodium dodecyl sulfate, aberrations and polyploidy study, slides were stained with 0.1 mg/ml proteinase K. The samples were incubated at 508C for 12 ± 16 h, extracted with phenol/chloroform/ Giemsa solution (Gibco, BRL) in 0.06 M phosphate bu€er (pH 6.8, 1 : 30) for 4 min at room temperature. At each isoamyl alcohol and precipitated with ethanol. The DNA passage level 50 metaphases were examined for quantita- was dissolved in Tris-EDTA bu€er. RNA was removed by tion of chromosome aberrations and chromosome num- adding DNase free-RNase (1 mg/ml) and incubating for 1 h bers, and 500 metaphases were analysed for ploidy, except at 378C. Genomic DNA was digested with the restriction for passage 34 F5E6 cells, in which 33 metaphases were enzymes HinfIandRsaIfor16hat378C. The digested examined for cytogenetics and 50 metaphases for determi- DNA was extracted with phenol-chloroform, ethanol nation of ploidy. precipitated and dissolved in Tris-EDTA bu€er. DNA was electrophoresed in an 0.8% agarose gel in Tris-borate- EDTA bu€er for 16 h. Southern hybridization was Fluorescence in situ hybridization (FISH) performed with a non-radioactive digoxigenin-labeled (TTAGGG) probe at 438C for 18 h using the Genius Probes The probes used for chromosome painting were 3 Spectrum Green WCP 4 (for chromosome 4) directly System protocol (Boehringer Mannheim). labeled with ¯uorescein-12-dUTP and Spectrum Orange WCP 6 (for chromosome 6) directly labeled with Telomerase assay rhodamine (Vysis, Inc.). For analysis of telomere erosion an all-human telomeric probe labeled with digoxigenin Cell extracts for assay of telomerase were prepared as (Oncor, Inc.) was used. described (Golubovskaya et al., 1997). The concentration of protein was measured for each extract with bicinchro- Slide-preparation Air-dried slides were aged two to three ninic acid protein assay kit (Pierce Chemical Co). weeks prior to FISH, denatured in 70% formamide/ Telomerase activity was detected by the PCR-mediated, 26SSC, pH 7.0 at 728C for 2.5 min and gradually telomeric-repeat ampli®cation protocol (TRAP) described dehydrated in cold ethanol series (70%, 85%, 100%). by Kim et al., 1994). The initial telomerase extension Chromosomal instability in HPV-16E6-expressing fibroblasts LFilatovet al 1837 reaction and the secondary PCR reaction were performed 5mM potassium ferrocyanide, 150 mM NaCl and 2 mM in di€erent tubes (Bryan et al., 1995; Nakayama et al., MgCl2. 1997), as described (Golubovskaya et al., 1997). b-galactosidase assay Acknowledgements Senescent ®broblasts were identi®ed by assay of pH 6.0 b- We would like to thank Dr JM Mason for his critical galactosidase as described by Dimri et al. (1995). Brie¯y, reading of this manuscript and helpful comments and cells were washed in PBS, ®xed for 20 min in 2% discussion. We are grateful to members from Medical formaldehyde, 0.2% glutaraldehyde, washed again in PBS Illustrations and Photography Department for expert and incubated for several hours in staining solution: 1 mg/ assistance in preparation of the ®gures. This work was ml X-Gal (5-bromo-4-chloro-3-indolyl b-D-galactoside), supported by Public Health Service grant CA42765 40 mM citric acid, 40 mM sodium phosphate, pH 6.0, (WKK).

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