Amplification, Increased Dosage and in Situ Expression of the Telomerase

Ampli®cation, increased dosage and in situ expression of the telomerase RNA gene in human cancer

Anja I Soder1, Stacey F Hoare1, Sharon Muir1, James J Going2, E Kenneth Parkinson3 and W Nicol Keith1

1CRC Department of Medical Oncology, University of Glasgow, CRC Beatson Laboratories, Alexander Stone Building, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD; 2Glasgow University Department of Pathology, Glasgow Royal In®rmary, Castle Street, Glasgow G4 0SF; 3Beatson Institute for Cancer Research, CRC Beatson Laboratories., Garscube Estate, Switchback Road, Glasgow G61 1BD, UK

Telomere length is maintained by the enzyme, telomer- Telomerase activity is essential for the immortal ase, which has been linked to cellular immortality and phenotype of certain human tumour cells with short tumour progression. However, the reasons for the high telomeres but not for the proliferation of normal cells, levels of telomerase found in human tumours are suggesting that telomerase antagonists may be eective unknown. We have mapped the human telomerase anticancer agents (Feng et al., 1995; Harley et al., RNA gene, (hTR), to chromosome 3q26.3 and show 1990; Norton et al., 1996; Parkinson, 1996; Rhyu, the hTR gene to be ampli®ed in four carcinomas, (2/33 1995). However, it is unclear whether high telomerase cervix, 1/31 head and neck, 1/9 lung). In addition, activity in human tumour cells is due to reactivation increased copy numbers of the hTR locus was also following genetic alteration when the cells become observed in 97% of tumours. By in situ hybridisation, the immortal following prolonged telomeric attrition, or histological distribution of high levels of hTR expression whether the targets for human carcinogenesis are rare could be demonstrated in a lung tumour and its telomerase-positive stem cells which selectively expand metastasis with hTR ampli®cation. These results are during tumour progression as a result of a the ®rst report of genetic alterations involving a known concomitant block in terminal maturation (Counter component of telomerase in human cancer. Indeed, it is et al., 1992; Greaves, 1996; Kim et al., 1994; Shay and also the ®rst report of the ampli®cation of a speci®c Wright, 1996a). Nevertheless, it is generally agreed locus within the chromosome 3q region frequently subject that genetic alterations to known components of to copy number gains in human tumours. In addition, we telomerase in human tumours in vivo would support also show for the ®rst time the histological distribution the reactivation hypothesis (Greaves, 1996; Shay and of the RNA component of telomerase in human tumours. Wright, 1996a). The reintroduction of an intact human chromosome 3 into a real carcinoma cell line Keywords: telomerase; gene ampli®cation; in situ inactivates telomerase, restoring telomeric attrition hybridisation; gene expression; hTR and senescence (Ohmura et al., 1995) and the E6 region of human papillomavirus 16 upregulates telomerase (Klingelhutz et al., 1996). However, it is unclear whether either of these mecahnisms operate in Introduction vivo, or whether they are the result of a direct eect on telomerase. The ends of mammalian chromosomes, the telomeres, Telomerase is composed of two proteins (of 80 and are important for stabilising the chromosome during 95 kd in Tetrahymena (Collins et al., 1995)) and an replication but because of the end replication problem RNA component (Greider and Blackburn, 1985), the these structures suer attrition in the absence of the human counterpart of which, (hTR), has recently been enzyme telomerase (Bacchetti and Counter, 1995; Shay cloned (Feng et al., 1995). hTR is often, but not and Wright, 1996b). As telomerase is inactivated before always, overexpressed in immortal human cells and or soon after birth (Wright et al., 1996), telomeric tumours. However, expression studies, like the attrition is experienced by all normal somatic cells biochemical detection of telomerase activity, do not studied and has been suggested as a mechanism by distinguish between the selection and reactivation which human cells count replication events and limit hypotheses (Avilion et al., 1996; Bacchetti and their clonal proliferation by replicative senescence Counter, 1995; Feng et al., 1995; Greaves, 1996; Shay (Harley et al., 1990). The presence of telomerase in and Wright, 1996a,b). A priori one might anticipate immortal human cell populations and malignant that hTR could act as an oncogene (Kipling, 1995) if tumours and its general absence prior to this stage, the reactivation hypothesis were true and the concen- has led to the suggestion that cellular immortalization tration of hTR RNA in a particular tumour became and telomerase reactivation are essential for the rate limiting for telomerase activity. Point mutation progression and clinical lethality of most human and gene ampli®cation are known mechanisms of tumours, but not for their formation (Edington et al., oncogene activation and genetic instability in solid 1995; Kim et al., 1994). tumours. Therefore, we decided to map the chromosomal position of the hTR gene to test whether it Correspondence: WN Keith corresponded to regions of duplication and/or ampli- Received 29 November 1996; revised 24 January 1997; accepted ®cation in human tumours in vivo. The results show 27 January 1997 that this is indeed the case. hTR amplification AI Soder et al 1014

Figure 1 (a) Localisation of the hTR locus to chromosome 3q. (i), Double hybridisation of the hTR P1 clone (green) and a probe for the telomeric region of chromosome 3p (red) to normal metaphase chromosomes (blue). (ii), Partial metaphase spread showing hybridisation of the hTR probe (green) to both chromosome 3. The counterstain for the chromsomes is DAPI (blue) and (iii) shows inversion of the DAPI staining to give a G-like band pattern homologues, allowing localisation of the probe signal to 3q26.3. Ampli®cation of hTR in carcinomas. (b) Interphase cytogenetic analysis of hTR ampli®cation in a cervical carcinoma. The clusters of ampli®ed hTR sequences are visualised in green, the nuclei in red. (c) Interphase cytogenetic analysis of hTR ampli®cation in a lung tumour. The ampli®ed hTR sequences are visualised in green, the nuclei in red. Note the dispersed appearance of the ampli®ed hTR sequences within the nuclei in comparison to the cervix sample shown in (b). (d) Co-localisation of the hTR locus with the region of ampli®cation on chromsome 3 in a lung carcinoma. Labelled genomic DNA extracted from the lung tumour with a known ampli®cation of hTR, (c above), was cohybridised with the hTR probe to normal metaphase chromosomes. The images of chromosome 3 are shown with the hybridisation signal for the tumour DNA in red, (i), hTR probe in green, (ii), the DAPI stained chromosome 3 in blue, (iii), and the 3-colour merge is shown in (iv). (dv), shows a typical intensity plot for ¯uorescence along the pro®le of chromsome 3 (blue trace) generating a visual representation of the hybridisation site for hTR (green trace) relative to genomic sequences ampli®ed in the lung tumour (red trace). Note the coincidence in peak ¯uorescence for both the hTR probe and the chromosome 3 amplicon present in the tumour genome. (e) As a control, labelled genomic DNA extracted from a lung tumour without ampli®cation of hTR, was co-hybridisation with the hTR probe to normal metaphase chromosomes. The images for hTR amplification AI Soder et al 1015 extrachromosomal origin for the ampli®ed hTR Results sequences in this tumour (Figure 1c). Ampli®ed regions may encompass large chromoso- Chromosomal localisation of the hTR gene mal segments. In order to examine the physical The human telomerase RNA gene has recently been relationship between the map position of the hTR cloned and tentatively mapped to the long arm of locus and the region ampli®ed in these tumours, chromosome 3 (Feng et al., 1995). Due to the genomic DNA isolated from two of the tumours (one importance of the hTR gene and the high frequency cervix and the lung sample), was used as complex in with which chromosome 3 is implicated in the situ probes, and reverse in situ hybridisation back onto progression of some solid tumours (Arnold et al., normal metaphase chromosomes carried out as a 1996; Brzoska et al., 1995; Heselmeyer et al., 1996; double hybridisation with dierentially labelled hTR Iwabuchi et al., 1995; Levin et al., 1994, 1995; Ried et probe (Hoare et al., 1977; Joos et al., 1993). Figure 1d al., 1994; Speicher et al., 1995), we decided to more (lung), and f (cervix) show typical intensity plots for precisely map the hTR locus. A P1 clone containing ¯uorescence along the pro®le of chromosome 3, hTR sequences was obtained as described in the generating a visual representation of the hybridisation Methods and the chromosomal localisation deter- site for hTR relative to the genomic sequences mined by ¯uorescence in situ hybridisation (Figure ampli®ed in the carcinomas. From the intensity 1a). The hTR probe was localised by fractional length pro®les shown in Figure 1d and f the regions measurements (McLeod and Keith, 1996), (Flpter), to ampli®ed on chromosome 3q in the tumour genomes the long arm of chromosome 3 with a mean Flpter can quite clearly be identi®ed and the peak ¯uorescence value of 0.84 (number of chromosomes examined=23, intensities correspond to that of the site for the standard error=0.02) which approximates to chromo- localisation of the hTR probe (Hoare et al., 1997). some 3q26. The map position of hTR to chromosome Thus the hTR locus is within the most highly ampli®ed 3q26.3 was con®rmed by DAPI banding of the region in the tumour genomes and is therefore a hybridised chromosomes. candidate for the target locus ampli®ed on chromosome 3q. As shown above, we have demonstrated amplifica- Ampli®cation of the hTR gene in solid tumours tion of the hTR locus in 4/73 tumours. Clearly it is Recent data from comparative genomic hybridisation important to determine how speci®c the ampli®cation (CGH), of a number of solid tumour types including is for hTR. The question is therefore, do any samples cervix, lung and squamous cell carcinoma of the head have an amplicon on chromosome 3q which excludes and neck, (SCC-HN), show frequent copy-number hTR, suggesting the presence of another locus near to gains and localised ampli®cations on the long arm of hTR as the driver for ampli®cation. This issue was chromosome 3 (Arnold et al., 1996; Brzoska et al., addressed in two ways, ®rstly, by an extension of the 1995; Heselmeyer et al., 1996; Iwabuchi et al., 1995; reverse in situ hybridisation approach, and secondly by Levin et al., 1994, 1995; Ried et al., 1994; Speicher et carrying out interphase cytogenetics with chromosome al., 1995), whereas others such including breast and 3q probes in the locality of hTR. Of the four tumours colon rarely show these chromosome 3 changes (Ried with hTR ampli®cation, (de®ned by FISH), sucient et al., 1995, 1996). However, as yet no speci®c genes genomic DNA was available from three for reverse in have been reported to be included within the situ hybridisation, and all three showed evidence of amplicons. Having mapped hTR to chromosome chromosome 3q ampli®cation by reverse in situ 3q26.3, hTR gene copy number was quanti®ed by hybridisation, (examples of two of these are shown in interphase cytogenetic analysis on frozen sections from Figure 1d and f). Of the 69 tumours without 73 biopsies from cervix, lung and head and neck ampli®cation of hTR (de®ned by FISH), sucient cancers using the cloned hTR sequences. Interestingly, genomic DNA was available from 41 for reverse in situ the hTR gene was ampli®ed in four carcinomas, (2/33 hybridisation, as an extension of the approach shown cervix, 1/31 SCC-HN, 1/9 lung). Figure 1b and c show in Figure 1d ± g. Using this approach none of the 41 examples of hTR ampli®cation in one of the cervical DNA samples from tumours without hTR amplifica- carcinoma samples and the lung carcinoma. The tion had evidence of ampli®cation of chromosome 3q pattern of hybridisation in the cervix and SCC-HN material by reverse in situ hybridisation. The second cancer indicated that the ampli®ed sequences were approach was to carry out interphase cytogenetics likely to be present as integrated blocks, (Figure 1b). using two P1 clones neighbouring the hTR locus. The However, in the lung carcinoma, the hybridisation P1 clone, RMC03P017, has a FLpter map position of signal was dispersed over the nucleus which suggests an 0.83 (3q25-q26, and contains the marker D3S1268) and

chromosome 3 are shown with the hybridisation signal for the tumour DNA in red, (i), hTR probe in green, (ii), the DAPI stained chromosome 3 in blue, (iii) and the 3-colour merge is shown in (iv). (ev) shows a typical intensity plot for the ¯uorescence along the pro®le of chromosome 3 (blue trace) generating a visual representation of the hybridisation site for hTR (green trace) relative to genomic sequences in the lung tumour (red trace). Note that no evidence of an ampli®cation on chromosome 3 is seen in the control hybridisations (ei) and (ev) in comparison to (di) and (dv) above. (f) shows co-localisation of the hTR locus with the region of ampli®cation on chromosome 3 in a cervical carcinoma. (f) shows a typical intensity plot for ¯uorescence along the pro®le of chromosome 3 (blue trace) generating a visual representation of the hybridisation site for hTR (green trace) relative to genomic sequences ampli®ed in the cervical carcinoma (red trace). Note the coincidence in peak ¯uorescence for both the hTR probe and the chromosome 3 amplicon present in the tumour genome. (g) shows the intensity plot for the control hybridisation with DNA from a cervical carcinoma without ampli®cation of the hTR locus. (h) shows interphase cytogenetic analysis of cervix sample, C47, with the P1 clones: (i) RMC03P017; (ii) hTR; (iii) RMC03P061 hTR amplification AI Soder et al 1016 the P1 clone RMC03P061, has a FLpter map position In situ detection of the essential RNA component of of 0.92 (3q28, and contains the gene Bc16) (LBL/ telomerase UCSF Resource for molecular Cytogenetics, http:// rmc-www.lbl.gov/). hTR has a FLpter map position of To examine expression of hTR we used in situ 0.84 and using double hybridisations on lymphocyte hybridisation with a riboprobe labelled with 35S. This metaphase spreads, the signal for marker RMC03P017 which lies closest to hTR cannot be resolved from that of hTR suggesting they lie close together on Table 1 Summary of copy number gains of the hTR locus in HN- chromosome 3 (data not shown). Sucient sections SCC and cervix cancer were available to carry out hybridisations on the two Mean number of % of nuclei Min. number Max. number cervix samples, C47 and C60 which have ampli®cation hTR signals/ with 42 sig- signals/ signals/ Tumour nucleus nals/nucleus nucleus nucleus of the hTR gene. Neither RMC03P017 or RMC03P061 were ampli®ed and examples of the hybridisations with HN1 4 90 2 6 the three P1 clones (hTR, RMC03P017 and HN2 3 70 1 5 RMC03P061) on cervix sample C47 are shown in HN3 3.4 80 1 6 HN4 2.7 54 1 6 Figure 1h. Thus by a combination of interphase HN5 4.3 96 2 8 cytogenetics and reverse in situ hybridisation, the only HN6 2.3 30 1 5 3q amplicons detected cover hTR, consistent with the HN7 3.7 80 2 10 suggestion that hTR is a good candidate for the target HN8 3.2 62 1 6 locus ampli®ed on chromosome 3q. HN9 3.3 66 1 8 HN10 4.8 88 1 10 HN11 2 24 1 4 Copy number gains of the hTR locus in solid tumours HN12 2 18 1 4 HN13 2 22 1 4 During the analysis of the high level ampli®cations HN14 2.5 48 1 4 HN15 2 28 1 4 described above, it became apparent that in addition to HN16 2.5 50 1 5 these tumours, many samples had subpopulations of HN17 1.8 6 1 3 nuclei with a greater than normal hTR copy number. HN18 2.5 44 1 5 Therefore, in order to quantitate hTR copy number, a C1 1.8 14 1 3 group of tumours showing high quality hybridisations C2 2.1 22 1 4 C3 2.7 54 1 6 and good morphology were selected for further study C4 2.9 48 1 10 using previously established criteria (Murphy et al., C5 2.9 56 1 6 1995a). From Table 1 it can be seen that of 18 SCC- C6 3.5 76 1 8 HN biopsies, 17 showed subpopulations of nuclei C7 3.8 84 2 8 C8 3.6 68 1 6 (ranging from 18 ± 90%), with more than two C9 2.4 38 1 5 hybridisation sites for hTR per nucleus. Of 12 cervix C10 2.7 58 1 5 biopsies, all contained subpopulations of nuclei C11 2.4 44 1 4 (ranging from 14 ± 84%) with greater than two C12 2.7 64 1 4 hybridisation sites for hTR per nucleus. The range in Eighteen SCC-HN biopsies, (HN1 ± 18) and 12 cervical carcinomas, number of hTR signals per nucleus in each tumour (C1 ± 12), were analysed for hTR copy number by FISH. For each also varied, and although some of this variation is sample, the mean hTR copy number per nucleus, the % of nuclei within each biopsy with more than two hybridization sites, (%42), likely to be due to nuclear truncation (Murphy et al., and the minimum and maximum number of hybridization sites per 1995a) nuclei with up to a maximum of 10 copies of nucleus, are shown. More than 10% of the nuclei had to show hTR were detected (Table 1). greater than two signals for the hTR probe, to be evaluated as having Possible mechanisms of increased hTR copy number a gain of hTR sequences, (Murphy et al., 1995a) in these samples include polysomy and isochromosome formation. To obtain information on the mechanism and indirectly to evaluate whether structural changes may occur on chromosome 3, the ratio of hTR copies to chromosome 3 centromeric sequences in each tumour was determined. A ratio of one suggests equality of hTR gene copies and chromosome 3 centromeric sequences, a ratio of 1.5 suggests a gain of one copy of hTR relative to chromosome 3 centromere. From Figure 2 it can be seen that seven out of 18 SCC-HN and one out of 12 cervical carcinomas hava a ratio of hTR signals to chromosome 3 centromere signals greater than 1.5 indicating a structural change such as isochromosome 3q or a duplication of part 3q encompassing hTR. Indeed, preliminary data from FISH analysis of SCC-HN cell Figure 2 Gains in hTR copy number in cervix cancers and lines with the hTR probe in our laboratory support squamous cell carcinomas of the head and neck (SCC-HN). The these interpretations (data not shown). The remaining ratio for hTR copy number to chromosome 3 centromeric- SCC-HN biopsies and cervical carcinomas have a ratio sequence copy number is shown for 18 SCC-HN biopsies and 12 between 0.5 ± 1.5 and so increased hTR copy number in cervix biopsies. A ratio of one suggests equal numbers of hTR gene copies and chromosome 3 centromeric sequences, a ratio of these tumours is likely to be due to polysomy of 1.5 suggests a gain of one copy of hTR relative to chromosome 3 chromosome 3. centromere hTR amplification AI Soder et al 1017 allowed us to examine the histological distribution of tumour and its metastasis with ampli®cation of the the RNA component of the telomerase enzyme. We hTR gene (Figure 3a, c and e) and not in a lung have concentrated on two individuals with lung tumour and its metastasis without hTR ampli®cation cancer, L2, is hTR ampli®ed and is shown in Figure (Figure 3f). In addition, within a tumour with hTR 1c, with the reverse in situ data shown in Figure 1d. ampli®cation, regions of high levels of expression are L3 does not have hTR ampli®cation and is shown in clearly distinguishable from those with low or Figure 1e. From the tumour archives we were able to undetectable (Figure 3a, c and e). Thus, the normal obtain sections from both the primary tumours and a squamous epithelium is negative as in an area of lymph node metastasis from both individuals. Thus in squamous metaplasia of the bronchial epithelium total four tumour blocks were examined. From Figure (Figure 4d). Interestingly, high levels of telomerase 3 it can be seen that the histological distribution of RNA expression are maintained in a lymph node essential RNA component of telomerase can be metastasis from a primary tumour with high levels of detected by in situ hybridisation and that high levels expression (Figure 4a, c and e). Expression is nuclear of telomerase RNA expression are found in a lung localised and detected in all tumour cells.

Figure 3 Histological distribution of the telomerase RNA component in lung tumours. Expression of the hTR gene was detected by in situ hybridisation with a riboprobe labelled with 35S. (a) Primary lung tumour, L2, with ampli®cation of the hTR locus, antisense probe. Note signal over tumour cells and not detectable in normal tissue. (b) Primary lung tumour, L2, with ampli®cation of the hTR locus, sense, (control), probe. Adjacent section to (a) above same area viewed. (c) Primary lung tumour, L2, with ampli®cation of the hTR locus, antisense probe. Note signal over tumour cells and not detectable in normal tissue. (d) Primary lung tumour, L2, with ampli®cation of the hTR locus, antisense probe. Area of tumour with squamous metaplasia of the bronchial epithelium shows no detectable hTR expression. (e) Lymph node metastasis form lung tumour, L2, with ampli®cation of the hTR locus, antisense probe. Note signal over tumour cells and not detectable in normal tissue. (f) Primary lung tumour, L3, which does not have ampli®cation of the hTR locus, antisense probe. No detectable hTR expression. No detectable hTR expression found in the lymph node metastasis from L3 either hTR amplification AI Soder et al 1018 Discussion component may be an early event in tumour progression (Avilion et al., 1996; Blasco et al., 1995, Telomere stabilisation through telomerase activity is 1996; Feng et al., 1995). Given that telomerase is a probably critical for survival and sustained replication multi-component enzyme, genetic events leading to re- of tumour cells and this with its almost ubiquitous activation of the RNA element may occur prior to expression in tumour cells make telomerase inhibition reactivation of the protein elements (Collins, 1996), an attractive anticancer strategy (Bacchetti and and we have presented a number of genetic events such Counter, 1995; Norton et al., 1996; Parkinson, 1996; as ampli®cation and isochromosome formation which Shay and Wright, 1996b; Wright and Shay, 1995; may contribute to this. Once the genes for the protein Rhyu, 1995). Indeed, the recent demonstration that elements of human telomerase have been cloned and telomerase inhibition through the expression of mapped, the temporal sequence and combination of antisense hTR transcripts leads to cell crisis supports events leading to telomerase up regulation can be telomerase antagonism as an important new strategy analysed in full. Interestingly, telomerase RNA is for the suppression of tumour growth (Feng et al., overexpressed in a number of tumours which would 1995). However, while many tumours that have been be consistent with hTR being an active component of assayed for telomerase activity (Bacchetti and Counter, the amplicon (Avilion et al., 1996). Therefore, it was 1995; Shay and Wright, 1996b), it is still unclear important to determine whether a tumour with hTR whether high levels of telomerase are a result of genetic ampli®cation expressed the telomerase RNA compo- reactivation or to the selection of pre-existing nent, as this would provide some evidence to support telomerase positive cells (Greaves, 1996; Shay and the idea that the ampli®ed hTR gene has some Wright, 1996a). At present progress is limited by a lack biological eect. To examine expression of hTR, we of molecular reagents but the human telomerase RNA used in situ hybridisation. This allowed us to examine component has been cloned (Feng et al., 1995). Since for the ®rst time the histological distribution of the the RNA component is a proven target for antagonism essential RNA component of the telomerase enzyme in and subject to novel oligonucleotide-based gene human tumours. This is an important issue in therapeutics (Feng et al., 1995; Norton et al., 1996), examining the role for telomerase in the development it is clearly important to investigate this gene in human of immortal or metastatic clones from a telomerase tumours. negative solid tumour, and central to clarifying Mapping the hTR locus to chromosome 3q26.3 whether the selection of telomerase positive cells or places it within a region of the human genome telomerase activation occurs in tumours (Shay and frequently subject to both localised ampli®cations and Wright, 1996a,b; Greaves, 1996). Thus, the in situ whole arm or whole chromosome gains in solid detection of the telomerase RNA component comple- tumours (Arnold et al., 1996; Brzoska et al., 1995; ments the interphase cytogenetic approach used to Heselmeyer et al., 1996; Iwabuchi et al., 1995; Levin et study the hTR gene. Interestingly, high levels of al., 1994. 1995; Ried et al., 1994; Speicher et al., 1995). telomerase RNA expression were found in a lung We used interphase cytogenetics to study the hTR tumour and its metastasis with ampli®cation of the locus as the most direct method to quantitate hTR hTR gene (Figure 3), suggesting that the expression is a copy number per tumour cell and the optimal stable phenotype. An advantage of the in situ detection approach to analyse the distribution of genetic change of the telomerase RNA component, is that its in histologically de®ned areas (Murphy et al., 1995a,b). histological distribution can be assessed. The data These are important considerations for tumour shown in Figure 3 clearly demonstrates the dierential heterogeneity and the selection of cells with an in expression between the tumour and adjacent normal immortal phenotype. We found the hTR gene to be tissue, with telomerase RNA expression detected in all ampli®ed in four out of 73 carcinomas analysed and the tumour cells and nuclear localised. In addition, the this is the ®rst report on the ampli®cation of a speci®c lung tumour studied which had high levels of locus within the chromosome 3q region and the ®rst expression in areas of tumour, also had regions of description of a genetic alteration of a known squamous metaplasia, a potential precursor lesion to component of human telomerase in vivo. Indeed, hTR lung cancer, and this did not have detectable levels of is a promising candidate for the target locus ampli®ed expression. Thus, in the future it may be possible to on chromosome 3q given the role of telomerase in trace the evolution of immortal sub-clones within cellular senescence (Kipling, 1995; Wright and Shay, tumours and detect rare clones in apparently benign 1995). Although a neighbouring locus cannot at this lesions or normal tissue and identify the cell type. stage be ruled out, no region of chromosome 3q In addition to the ampli®cation of the hTR locus, ampli®cation was detected in 41 tumours examined increased copies of hTR were found in 97% of cervical which did not have ampli®cation of hTR. Moreover, carcinomas and SCC-HN studied in detail and this was the co-localisation of peak ¯uorescence for the hTR likely to be due to polysomy of chromosome 3 and probe and the chromosome 3 ampli®cation unit in the isochromosome 3q formation. These data are consis- lung and cervical carcinomas as shown in Figure 1d tent with the chromosome 3 data from karyotypic and and f demonstrates how close hTR lies to the centre of CGH analysis of SCC-HN and cervical cancer the ampli®ed sequences and supports hTR as a (Brzoska et al., 1995; Hermsen et al., 1996; Hesel- candidate locus, as does the interphase cytogenetic meyer et al., 1996; Speicher et al., 1995; Sreekantaiah analysis of hTR ampli®ed tumours with neigbouring et al., 1994; Van Dyke et al., 1994) and suggest markers. dysregulation of hTR expression may occur by a It has been noted that telomerase RNA expression number of genetic routes including ampli®cation, can be detected in the absence of telomerase enzyme polysomy and isochromosome 3q formation. Thus, activity and indeed upregulation of the RNA possible mechanisms of telomerase activation and hTR amplification AI Soder et al 1019 escape from senescence, include imbalance between the published sequence). Clone 9913 was con®rmed as gain of hTR copies and the loss or titration out of containing hTR sequences by the sequencing of PCR telomerase repressors, for example isochromosome 3q products generated using clone 9913 as a template for would result in loss of the chromosome 3p repressor, primers hTRg and hTRh and primers hTRe, (a 20 bp oligo (Ohmura et al., 1995) with a gain of one copy of hTR. starting at base 404 of the published sequence) and hTRf, (a 20 bp oligo corresponding to bases 637 ± 656 of the Furthermore, ampli®cation of hTR may titrate out published sequence). other repressors. In the future it will be important to expand our studies to link the genetic alterations at and around the hTR locus with the expression of the FISH analysis telomerase RNA component as this will help clarify by Probe labelling, in situ hybridisation and probe detection which mechanisms telomerase activity is ultimately were as previously described (Murphy et al., 1995a; Soder et regulated in tumour cells. al., 1995) using the Hybaid Omnislide system (Hybaid Ltd, The studies presented here used in the main, in situ Teddington, UK). Chromosome 3-speci®c centromeric technologies to analyse the hTR locus and its probes were purchased from Appligene-Oncor (Durham, expression, and clearly show the importance of UK) and Cytocell Ltd (Banbury, UK). Chromosome 3p heterogeneity within the tumour sections, therefore telomere probe was purchased from Appligene-Oncor new quantitative in situ approaches to telomerase RNA (Durham, UK). Fluorescence was analysed and images expression and activity will have to be developed to captured using confocal microscopy or a CCD camera. Original unedited images were stored on optical disks and investigate the relationship between the hTR gene, its have been retained. Normal lymphocyte interphase nuclei expression, and telomerase activity. However, genetic and metaphases were used as hybridisation controls to events altering the expression of hTR or the expression ensure probe speci®city and that the hybridisation eciency of the RNA component of telomerase, may mark cells was greater than 95%. Chromosome 3 and hTR gene copy in the early stages of disease progression with the number in individual cancer cells was determined by FISH potential to become immortal through a telomerase analysis of interphase nuclei. Quantitative evaluation of activation pathway, prior to the detection of the hybridisations was carried out on 50 nuclei according to telomerase activity, and may be of more prognostic previously described criteria (Murphy et al., 1995a,b). For use than the enzyme activity itself, which is present in tumours described in Table 1, more than 10% of the nuclei most solid tumours (Shay and Wright, 1996b). Indeed, had to show greater than two signals for the hTR probe, to be evaluated as having a gain of hTR sequences (Murphy et the in situ approach we have taken to analyse the hTR al., 1995a). A tumour was de®ned as having ampli®cation of locus and its expression is ideally placed to be the hTR locus when the density of hybridisation signal developed within the diagnostic setting. precluded the counting of individual hybridisation spots A strength of our analysis of the hTR locus and its (this was greater than 10 hybridisation sites per nucleus), expression is that all the alterations described have and the pattern of hybridisation was consistent with been identi®ed in tumour biopsy samples rather than ampli®cation as discussed previously (Murphy et al., cell lines. Now that we have described a series of 1995a,b; Coutts et al., 1993; Robertson et al., 1996). In molecular changes involving a component of the addition normal areas of tissue within the tumour blocks telomerase ribonuclear enzyme we can start to build served as internal controls. Speci®city of the P1 probe 9913 representative cell line models to understand the role of for detection of hTR ampli®cation was con®rmed by Southern blot using a small PCR generated probe these genetic changes in the regulation of telomerase corresponding to TRC3 sequences in hTR (Feng et al., activity and test anti-telomerase therapies under 1995) (data not shown). The two chromosome 3q P1 clones, clinically relevant conditions. RMC03P017 and RMC03P061, were obtained from the LBL/UCSF Resource for molecular Cytogenetics, (http:// rmc-www.lbl.gov/). The P1 clone, RMC03P017, has a FLpter map position of 0.83, a band map position of Materials and methods 3q25-q26 and contains the marker D3S1268. The P1 clone RMC03P061, has a Flpter map position of 0.92, a band map Tissue samples position of 3q28 and contains the marker Bcl6 (LBL/UCSF Seventy-three carcinomas of the head and neck, lung and Resource for molecular Cytogenetics, http://rmc- cervix were analysed in the study and 5 m frozen sections www.lbl.gov/). hTR has a FLpter map position of 0.84 were available from previous studies. Sections were stored and a band map position of 3q26.3 as shown in this study. at 7208C until use. Of the 33 cervical carcinomas, 32 were Flpter is the distance from the probe location to the end of squamous and one an adenocarcinoma. Of the 31 head and the short arm of a chromosome divided by the total length neck carcinomas, 30 were squamous, one a papillary of the chromosome (McLeod and Keith, 1996; Hoare et al., adenocarcinoma. All nine lung carcinomas were squamous 1997). Published Flpter maps are available from the non-small cell. All four carcinomas which had amplifica- Resource for Molecular Cytogenetics at Lawrence Berkeley tion of hTR were squamous. National Laboratories and the University of California, San Francisco (Internet connection, http://rmc-www.lbl.gov/) and also in Bray-Ward and colleagues (Bray Ward et al., Cloning of hTR 1996). A genomic clone containing the human telomerase RNA gene was identi®ed by screening the Du Pont Merck Pharmaceutical Company human foreskin ®broblast P1 Reverse in situ hybridisation (REVISH) using genomic DNA as library DMPC-HFF#1-279-B8, clone name 9913. Screening a probe (Hoare et al., 1997) was carried out by Genome Systems Inc, St Louis, Co-localisation of tumour amplicons with the position of Missouri, using PCR primers directed to the hTR genomic the hTR locus on chromosome 3 was carried out by co- sequence (Feng et al., 1995), primer hTRg (a 20 bp oligo hybridising labelled tumour DNA with the cloned hTR starting at base 430 of the published sequence), and primer probe (Hoare et al., 1997; Joos et al., 1993). Analysis of hTRh (a 21 bp oligo corresponding to bases 691 ± 711 of digitised images was carried out using IPLab Spectrum hTR amplification AI Soder et al 1020 software with SmartCapture extensions from Digital TRC3 (Feng et al., 1995) were cloned into pCR-Script Scienti®c Ltd (Cambridge UK). GraphPolygon was used (Stratagene Ltd, Cambridge, UK), and the inserts to produce an intensity plot of ¯uorescence along the sequenced. Autoradiographic exposures were from 1 to 3 pro®le of the chromosome where the width of the weeks. Within each experiment, sections were hybridised chromosome was determined in pixels and the average with the same batch of probe, exposed for the same length intensity over the width plotted (Hoare et al., 1997; of time and processed together. McLeod and Keith, 1996). Thus, a visual representation for the localisation of hTR on chromosome 3 relative to genetic imbalances present in the test tumour is generated. At least four metaphase spreads were examined and representative ¯uorescence intensity plots shown in Figure Acknowledgements 1d ± g. The authors are grateful to Ms N Morrison for help with hTR localisation and Dr RJ Akhurst and Ms E Due for help with the RNA in situ hybridisation. This work was Detection of hTR gene expression by in situ hybridisation supported by the Cancer Research Campaign (UK) and Paran sections (5 m), were subjected to RNA in situ Glasgow University. Some of the equipment used in this hybridisation using 35S-labelled antisense or sense hTR- study was purchased as a result of grants from the speci®c riboprobes (Fitzpatrick et al., 1990). In order to University of Glasgow Medical Research Funds and generate the riboprobe vectors, sequences corresponding to donations from the White Lily Group.

References

Arnold N, Hagele L, Walz L, Schempp W, P®sterer J, Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Bauknecht T and Kiechle M. (1996). Genes Chrom. Ho PLC, Coviello GM, Wright WE, Weinrich SL and Cancer, 16, 46 ± 54. Shay JW. (1994). Science, 266, 2011 ± 2015. Avilion AA, Piatyszek MA, Gupta J, Shay JW, Bacchetti S Kipling D. (1995). Nature Gen., 9, 104 ± 106. and Greider CW. (1996). Cancer Res., 56, 645 ± 650. Klingelhutz AJ, Foster SA and McDougall JK. (1996). Bacchetti S and Counter CM. (1995). Int. J. Oncol., 7, 423 ± Nature, 380, 79 ± 82. 432. Levin NA, Brzoska P, Gupta N, Minna JD, Gray JW and Blasco MA, Funk W, Villeponteau B and Greider CW. Christman MF. (1994). Cancer Res., 54, 5086 ± 5091. (1995). Science, 269, 1267 ± 1270. Levin NA, Brzoska PM, Warnock ML, Gray JW and Blasco MA, Rizen M, Greider CW and Hanahan D. (1996). Christman MF. (1995). Genes Chrom. Cancer, 13, 175 ± Nature Gen., 12, 200 ± 204. 185. BrayWard P, Menninger J, Lieman J, Desai T, Mokady N, McLeod HL and Keith WN. (1996). Br. J. Cancer, 74, 508 ± Banks A and Ward DC. (1996). Genomics, 32, 1±14. 512. Brzoska PM, Levin NA, Fu KK, Kaplan MJ, Singer MI, Murphy DS, Hoare SF, Going JJ, Mallon EEA, George WD, Gray JW and Christman MF. (1995). Cancer Res., 55, Kaye SB, Brown R, Black DM and Keith WN. (1995a). J. 3055 ± 3059. Natl. Cancer Inst., 87, 1694 ± 1704. Collins K, Kobayashi R and Greider CW. (1995). Cell, 81, Murphy DS, McHardy P, Couts J, Mallon EA, George WS, 677 ± 686. Kaye SB, Brown R and Keith WN. (1995b). Int. J. Cancer, Collins K. (1996). Cur. Opin. Cell Biol., 8, 374 ± 380. 64, 18 ± 26. Counter CM, Avilion AA, Lefeuvre CE, Stewart NG, Norton JC, Piatyszek MA, Wright WE, Shay JW and Corey Greider CW, Harley CB and Bacchetti S. (1992). EMBO DR. (1996). Nature Biotech., 14, 615 ± 619. J., 11, 1921 ± 1929. Ohmura H, Tahara H, Suzuki M, Ide T, Shimizu M, Yoshida Coutts J, Plumb JA, Brown R and Keith WN. (1993). Br. J. MA,TaharaE,ShayJW,BarrettJCandOshimuraM. Cancer, 68, 793 ± 800. (1995). Jpn. J. Cancer Res., 86, 899 ± 904. Edington KG, Loughran OP, Berry IJ and Parkinson EK. Parkinson EK. (1996). Br. J. Cancer, 73, 1±4. (1995). Mol. Carcinogen., 13, 254 ± 265. Rhyu MS. (1995). J. Natl. Cancer Inst., 87, 884 ± 894. Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu Ried T, Petersen I, HoltgreveGrez H, Speicher MR, Schrock CP, Adams RR, Chang E, Allsopp RC, Yu J, Le S, West E, Du Manoir S and Cremer T. (1994). Cancer Res., 54, MD, Harley CB, Andrews WH, Greider CW and 1801 ± 1806. Villeponteau B. (1995). Science, 269, 1236 ± 1241. Ried T, Just KE, HoltgreveGrez H, Du Manoir S, Speicher Fitzpatrick DR, Denhez F, Kondaiah P and Akhurst RJ. MR, Schrock E, Latham C, Blegen H, Zetterberg A, (1990). Develop., 109, 585 ± 595. Cremer T and Auer G. (1995). Cancer Res., 55, 5415 ± Greaves M. (1996). Trends Gen., 12, 127 ± 128. 5423. Greider CW and Blackburn EH. (1985). Cell, 43, 405 ± 413. Ried T, Knutzen R, Steinbeck R, Blegen H, Schrock E, Harley CB, Futcher AB and Greider CW. (1990). Nature, Heselmeyer K, Du Manoir S and Auer G. (1996). Genes 345, 458 ± 460. Chrom. Cancer, 15, 234 ± 245. Hermsen MAJA, Joenje H, Arwert F, Welters MJP, Robertson KW, Reeves JR, Smith G, Keith WN, Ozanne Braakhuis BJM, Bagnay M, Westerveld A and Slater R. BW, Cooke TG and Stanton PD. (1996). Cancer Res., 56, (1996). Genes Chrom. Cancer, 15, 1±9. 3823 ± 3830. Heselmeyer K, Schrock E, Du Manoir S, Blegen H, Shah K, Shay JW and Wright WE. (1996a). Trends Gen., 12, 129 ± Steinbeck R, Auer G and Ried T. (1996). Proc. Natl. Acad. 131. Sci. USA, 93, 479 ± 484. Shay JW and Wright WE. (1996b). Cur. Opin. Oncol., 8, 66 ± Hoare SF, Freeman CA, Coutts JC, Varley JM, James L and 71. Keith WN. (1997). Br. J. Cancer, 75, 275 ± 282. Soder AI, Hopman AHN, Ramaekers FCS, Conradt C and Iwabuchi H, Sakamoto M, Sakunaga H, Ma YY, Carcangiu Bosch FX. (1995). Cancer Res., 55, 5030 ± 5037. ML, Pinkel D, YangFeng TL and Gray JW. (1995). Speicher MR, Howe C, Crotty P, Du Manoir S, Costa J and Cancer Res., 55, 6172 ± 6180. Ward DC. (1995). Cancer Res., 55, 1010 ± 1013. Joos S, Scherthan H, Speicher MR, Schlegel J, Cremer T and Lichter P. (1993). Human Gen., 90, 584 ± 589. hTR amplification AI Soder et al 1021 Sreekantaiah C, Rao PH, Xu L, Sacks PG, Schantz SP and Wright WE, Piatyszek MA, Rainey WE, Byrd W and Shay Chaganti RSK. (1994). Genes Chrom. Cancer, 11, 29 ± 39. JW. (1996). Dev. Gen., 18, 173 ± 179. Van Dyke DL, Worsham MJ, Benninger MS, Krause CJ, Wright WE and Shay JW. (1995). Trends Cell Biol., 5, 293 ± Baker SR, Wolf GT, Drumheller T, Tilley BC and Carey 297. TE. (1994). Genes Chrom. Cancer, 9, 192 ± 206.