GENES, CHROMOSOMES & CANCER 11:171-177 (1994)

Telomere Length Variation in Normal and Malignant Human Tissues Holger Schmitt, Nikolaus Blin, Heinrich Zankl, and Harry Scherthan

Division of Human Biology and Human Genetics University of Kaiserslautern. Kaiserslautern Germany (H Schm H Z H Sche ) Division of Molecular Genetics University of Tubingen Tubingen Germany (H Schm N 5 )

Tissue and tumor specific length variation of telomere (TAGGG) , repeats was studied in DNAs from various normal and malignant tissues. D N A was isolated from bone marrow and blood cells, malignant tissues, and established tumor cell lines. Nonisotopic Southern hybridization revealed a reduction of telomere repeat arrays in 14 of the 35 tumors analyzed. However, other cases (60%) showed no reduction, or even an increase, in telomeric length. Our finding of elongated telomere stretches in several tumors of different origin compared with normal tissue is in contrast to previous reports describing a general shortening of terminal repeat length in colorectal cancer and neuroblastoma. We tentatively conclude that there is no general tendency to telomere reduction in malignant tissues. Genes Chromosom Cancer I /:/7/-/77 (1994). 6 1994 Wiley-Liss, Inc.

I N T R O D U C T I O N

T h e end of the linear eukaryotic chromosome is capped by a specialized nucleoprotein structure, the telomere, which exhibits particular functional features that prevent chromosome ends from deg- radation, fusion, and recombination (for review see Zakian, 1989; Blackburn, 1991). At the very end of the chromosomes these properties are brought about by the presence of long stretches of G-rich repeats. T h e human telomeric DNA terminates in numerous tandem arrays of a simple repetitive se- quence, (TTAGGG),,; on average, 500-2,000 of these hexamers are present at each chromosome telomere (hloyzis et al., 1988; Allshire et a]., 1989).

The size of the terminal repeat arrays varies de- pending on several biological processes. A progres- sive reduction of the number of telomere repeats has been reported in vitro and, as a function of cellular aging, in vivo (Harley et al., 1990; Hastie et al., 1990; Counter et al., 1992; Guerrini et al., 1993). After a certain number of cell divisions, a critical telomere length rnay be reached, resulting in deletions of essential sequences from subtelo- meric regions: This rnay lead to cell death. Thus, a causal relationship between gradual telomere shortening during cellular senescence and the rep- licative capacity of cells is being discussed (Harley, 1991; Levy et al., 1992).

Losses of terminal repeats during cellular prolif- eration due to incomplete replication can be coun- teracted by telomere elongation by the ribonucle- oprotein enzyme telomerase, which is capable of adding telomere repeats to eukaryotic chromosome ends de novo (for review see Blackburn, 1992). In

human germline cells, telomeres are significantly longer and more homogeneous than those from so- matic tissues, and do not decrease in length during aging (Allshire et al., 1988; Cross et al., 1989; Hastie and Allshire, 1989). Presumably, long te- lomeres are actively maintained in the germline by telomerase activity, but shorten gradually in so- matic cells due to a developmental inactivation of this enzyme (Harley et al., 1992). A causal role for telomere shortening in cellular senescence would further imply that immortalized cells should ex- hibit telomerase activity to stabilize telomeres and to escape cell death (reviewed by Harley, 1991). Telomerase activity has indeed been detected in unicellular eukaryotes, where a constant telomere length is maintained (Greider and Blackburn, 1985). Expression of the same enzyme activity in human cells has been identified in extracts of the permanent tumor cell line HeLa (Morin, 1989) as well as in immortal, virally transformed embryonic kidney cells (Counter et al., 1992).

Since many tumors are characterized by an in- creased rate of cell division and uncontrolled pro- liferation, this enhanced activity may lead to a shortening of telomeres in cancer cells compared with those in the tissue of origin. Loss of telomeric sequences associated with tumor development has been reported in a breast carcinoma, two Wilnis’ tumors (de Lange et al., 1990), colorectal carci-

Received Januav 12. 1994; accepted June 6 , 1994. Address reprinr requests to H. Schmitt, Ilivision of Molecular

Genetics, L‘nivcrsit? of Tubingen. Wilhelmstr. 27, 1) 72071 Ti- bingen. Gcrnian)-.

0 1994 Wiley-Liss, Inc.

I 72 SCHMITT ET AL.

noma (Hastie et al., 1990), and neuroblastoma (Hiyama et al., 1992). Such a reduction of telomere length may result in a destabilization of the DNA molecule so that chromosomes lacking telomeres may be prone to terminal fusion (Hastie and All- shire, 1989). In a variety of benign and malignant human tumor tissues, nonspecific telomere associ- ations and fusions occur at high frequency, similar to those observed in senescent cells and some non- neoplastic disorders (Kovacs et al., 1987; Pathak et al., 1988; Saltman et al., 1989). T h e mechanisms of the formation of these random telomeric associ- ations are still unknown, but a correlation of telo- meric fusions with a reduction in telomere length was recently demonstrated in renal tumors (Holz- mann et al., 1993) and in two human tumor cell lines (Saltman et al., 1993).

T o investigate the telomere length variation in human tissues and tumors of different origin, we estimated the lengths of terminal restriction fragments (TRFs) that contain the telomeric (TTAGGG), repeats. T o this end, nonisotopic Southern hybridization was applied using digoxi- genin labeled telomere oligonucleotides as probes. Hybridization signals were detected by a chemilu- minescence system. Rehybridization of the blots with the minisatellite (CAC), probe (Zischler et al., 1989) was performed to monitor experimental variation.

MATERIALS AND METHODS

Tissue Samples and Tumor Cell Lines

Tissue samples of 35 different tumors (carcino- mas and adenomas) were obtained as frozen archi- val material (no blood samples were available) through the Departments of Pathology and Urol- ogy of the University of the Saar, Homburg/Saar. Tumor tissue was characterized by histological ex- amination (Drs. G. Seitz and B. Wullich, personal communication). Origins and abbreviations are listed in Figure 4. Some tissues allowed a separa- tion of the peripheral and the central parts, and in a few cases tissue adjacent to the tumor region was available. Peripheral blood (pb) and bone marrow (bm) were obtained from six unrelated, healthy do- nors (Pl : female [f.], 30 years [y]; P2: male [m.], 28 y; P3: f., 32 y; P4: m., 16 y; P5: f., 67 y; P6: m., 35 y). Embryonic tissue (e l , e2) was isolated from abortion material of the 10th to 12th weeks of gestation, obtained through the City Hospital of Kaiserslautern. T h e permanent human tumor cell lines R T 4 (transitional-cell papilloma, urinary bladder), Namalwa (lymphoblastoid cell line de-

rived from a biopsy of a Burkitt's lymphoma), K562 (chronic myelogenous leukemia), H T 1080 (fibro- sarcoma), and HeLa (epitheloid cervical carci- noma) were obtained from ATCC (Rockville, MD, USA).

DNA Preparation

Approximately 100 mg of each tissue sample was minced with scalpels and suspended in 15 ml saline buffer (75 mM NaCI, 25 mM EDTA, pH 8.0), 1% SDS. Peripheral blood was supplemented with 3 volumes of lysis buffer (155 mM NH,CI, 10 mM KHCO,, 0.1 mM EDTA, pH 7.4) and incubated for 15 min on ice. Lymphocytes were pelleted by centrifugation (900 X g, 10 min) and resuspended in 5 ml SE, 1% SDS. High molecular weight DNA was isolated by salt-chloroform extraction as de- scribed earlier (Mullenbach et al., 1989). Genomic DNA concentration was estimated following elec- trophoresis by comparing band intensities using video-densitometry analysis (Herolab, Wiesloch, Germany). DNA samples were digested with HinfI or A h 1 (New England Biolabs, Beverly, MA, USA) to completion. Then 5 pg of restricted DNA per lane was separated by horizontal electrophoresis on 0.7% agarose gels (1 V/cm, 24 hr) and transferred to positively charged nylon membranes (Boehringer Mannheim, Germany) using a vacuum transfer ap- paratus (Millipore, Bedford, MA, USA).

Nonisotopic Southern Hybridization

Nonisotopic labeling of oligomere-DNA probes was achieved by introducing hapten modified dUTP at the 3' ends of the oligonucleotides us- ing terminal transferase. T h e telomeric probe (TTAGGG), (Moyzis et al., 1988) and the mini- satellite probe (CAC), (Zischler et al., 1989) were labeled with digoxigenin-1 1-dUTP (Boehringer Mannheim, Gemany) or with biotin-11-dUTP (Sigma, Munchen, Germany). Then 15 pmoles of each oligonucleotide were tailed in the presence of 0.02 mM digoxigenin-11-dUTP and 0.12 mM dATP using 50 U of terminal transferase (Boeh- ringer Mannheim) as described earlier (Scherthan et al., 1992). After ethanol precipitation, oligonu- cleotides were dissolved a t 4 ng/pl in T E (10 mM Tris-HCI, pH 7.5, 1 mM EDTA). T h e biotin la- beling reaction was performed in an analogous way using 0.02 mM biotin-11-dUTP and 0.03 mM dATP.

Successful hapten incorporation was monitored by dot blot assay. Nylon membranes (200-250 cm') were prehybridized in 40 ml of 5 x SSC, 2% blocking reagent (Boehringer Mannheim), 0.1 %

CHROMOSOMAL TELOMERE VARIATION IN TUMORS I 73

N-lauroylsarcosine, 0.02% SDS, and 1 pg/ml E . roli tRNA for 1 hr a t 55°C. Prehybridization solu- tion was discarded, 5 ml of the same solution con- taining digoxigenated telomeric oligonucleotide probes at 15 ng/ml was added, and hybridization was performed at 55°C overnight. Posthybridiza- tion washes were 2 x 5 min in 2 x SSC, 0.1% S D S at room temperature, followed by 2 x 15 min in 0.1 x SSC, 0.1% SDS at 42°C. T h e conditions for fluorescence in situ hybridization with biotinylated telomeric oligonucleotides were published previ- ously (Scherthan, 1990).

Chemiluminescent Detection and Rehybridization

Chemiluminescent detection was performed as described by the manufacturer of the D I G lumi- nescent detection kit (Boehringer Mannheim). Prior to x-ray film exposure (Kodak X-OMAT AR), filters were sealed in plastic bags and preincubated for 5-15 min at 37°C. Thereafter, filters were stored wetted with T E at 4°C until reuse. For re- probing the filters were briefly washed in sterile redistilled water and subsequently incubated 2 x 15 min in 0.2 M N a O H , 0.1% SDS at 39°C for stripping. After a short equilibration in 2x SSC, rehybridization with the digoxigenated minisatel- lite probe (CAC) 5 was performed according to the manufacturer’s instructions (Boehringer Mann- heim) with the following modifications. Prehybrid- ization was carried out for 1.5 hr, and hybridization was performed at 42°C. Stringent washes were 2 x 5 min at room temperature and 2 x 15 min at 42°C in 4 x SSC. 0.1% SDS.

RESULTS

T h e telomeric losses in malignant cells were in- vestigated by nonisotopic blot analysis. Genomic D N A was isolated from normal peripheral blood lymphocytes, bone marrow, permanent tumor cell lines, and a number of tumors of different origin. DNA samples were digested to completion with restriction enzymes HinfI or AIuI, and length vari- ations of terminal restriction fragments ( T R F s ) were examined by probing with digoxigenated te- lomeric oligomere (TTAGGG), (Fig. 1). T h e ob- served length distributions of telomeric repeats in the examined tissues are summarized in Figures 3 and 4. All experiments were repeated with A h I - digested D N A samples and showed results compa- rable to the HinfI data. D N A loading and complete digestion were checked by rehybridizing the same filters to the fingerprint probe (CAC), (Fig. 2).

T h e distribution of T R F s in blood lymphocytes and bone marrow of all healthy donors (age 28-35

years) ranged from 4 to 15 kb. T w o cases, however, deviated from this general picture: T h e peripheral blood sample of a 67-year-old female exhibited no T R F s above 10 k b (Fig. 3) and showed a decrease in intensity of the hybridization signal. T h e DNA of a 16-year-old male displayed a maximum telo- mere length of 17 kb. Both fetal samples showed a maximum telomere length of more than 23 kb, with absence of short telomeres below 7 kb. T h e hybridization signal in this sample was stronger and more homogenous than in any other.

All permanent tumor cell lines investigated were found to have dramatically shortened TRFs of 1-7 kb, with the exception of H e L a cells, which ex- hibited an increased TRF range (1-15 kb). Fur- thermore, the cell lines with a reduced telomere length showed a considerably decreased intensity of the hybridization signal, indicating the loss of telomere repeats (Fig. 1). Rehybridization of the same filters with the minisatellite probe (CAC), proved that reductions of telomere length did not result from general DNA degradation or different amounts of D N A transferred to the filters, since in all samples bands of repetitive sequences above 10 k b were observed and signal intensity was compa- rable in all lanes. T h i s was further confirmed by fluorescence in situ hybridization (FISH) with bi- otinylated telomeric oligonucleotides to metaphase chromosomes of peripheral blood lymphocytes and tumor cells. Distinct signals were observed on the majority of chromosome termini from normal pe- ripheral blood lymphocytes, whereas the chromo- some termini of the permanent tumor cell lines Namalwa and K 562 exhibited only few and weak FISH signals (data not shown). T h e s e observations further support the results of the Southern blot analysis, which indicated substantial loss of telo- mere repeats in cell lines maintained in culture for a long time.

T h e length of telomeric repeats in D N A samples of the examined human tumors from various tis- sues fell within a wide range (from 1 k b to more than 23 kb). T h e maximum telomere length varied from 3 k b to more than 23 kb. In 35 tumors of different origin, we found 14 samples (40%) show- ing telomere reduction, whereas another 10 cases (29%) showed no length variation compared with the telomeres of peripheral blood lymphocytes. In- terestingly, 11 samples (31%) exhibited an in- creased telomere length above 15 k b (Fig. 4). T h e r e was no general tendency to maintain com- parable telomere length, even within the same tu- mor type. In the majority of the malignant tissue samples, extraordinarily short telomeres below 4

I 74 SCHMITT ET AL.

Figure I. Nonisotopic Southern blot analysis of terminal restriction fragments performed on Hinfl-digested genomic DNA samples with digoxigenated (TAGGG), oligonucleotides. Lane I, peripheral blood sample of a healthy donor (PI ); lanes 2-4, gastric carcinoma (T IS, T 19, TZI); lane 5 meningioma (HA47I); lanes 6-9, permanent tumor cell

lines (HeLa, HT1080. RT4 Namalwa). A DNA size marker (indicated in kilobase pairs, kb) is shown in the flanking lanes.

Figure 2. Rehybridization of the filter in Figure I with the minisat- ellite probe (CAC),. Lanes as in Figure I.

kb were detectable, something that was not ob- served in healthy human tissues.

DISCUSSION

Terminal restriction fragments of genomic DNAs that contain the very ends of the telomeres are heterogeneous in size and appear in Southern blot analysis as a heterodispersed smear that ex- tends over several kilobase pairs (de Lange et al., 1990). Terminal heterogeneity exists at individual chromosome ends and seems to be an intrinsic fea- ture of human chromosomes (Moyzis et al., 1988). T o investigate the variation of telomere repeat lengths in various human tumors, tissues, and per- manent cell lines, we performed nonisotopic blot hybridization with the digoxigenin labeled telo- meric probe (TTAGGG), to restriction enzyme di- gested genomic DNAs prepared from 44 different tumor and healthy tissue samples. HinfI and A h 1 were chosen for digestion, as they cleave fre-

quently in human genomic DNA but not within telomeric sequences; they therefore leave the ter- minal repeats intact (Hastie et al., 1990).

T h e T R F lengths in peripheral blood lympho- cytes from various healthy donors were analyzed first. In these DNA samples (including one bone marrow sample) of 28- to 35-year-old individuals of either sex, T R F length distribution was constant, showing no variability between individuals. Based on our data and results published earlier (Moyzis et al., 1988), we considered a size distribution from 4 to 15 kb to represent an average length for telo- mere restriction fragments in healthy tissues of middle-aged humans. T R F s from two embryonic tissue samples exhibited strong signals from about 7 kb to more than 23 kb, thus indicating the pres- ence of long homogeneous stretches of TTAGGG repeats. In the blood cells of a 16-year-old boy, T R F s from 4 to 17 kb were detected, in contrast to the telomere length distribution observed in the

kb 25

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CHROMOSOMAL TELOMERE VARIATION IN TUMORS

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Figure 3. Length distribution of terminal restriction fragments in normal human tissues (pb. peripheral blood; bm. bone marrow: e. embryonic tissue) and permanent tumor cell lines (cl).

lymphocvtes of a 67-year-old woman, which ranged from 4 to 10 kb. We used these data, which are in accordance with previous findings (Hastie et al., 1990), as a reference for our investigation of telo- meric length in tumor samples.

A drastic reduction in the size of TRFs and sig- nal intensitv was observed in the permanent tumor cell lines Namalwa, RT4, K 562, and H T 1080. These losses of terminal TTAGGG repeats in im- mortal cell cultures are probably a consequence of incomplete replication of chromosome ends and thus reflect the immense number of cell divisions these cell lines had undergone (Counter et al., 1992). An exception to the telomere shortening was found for the cervical carcinoma cell line HeLa, which exhibited heterogeneous TRFs rang- ing from 1 kb up to 15 kb with increased signal intensity, suggesting de novo synthesis of long stretches of TTAGGG repeats at various chromo- some ends. This finding is in agreement with the detection of telomerase activity in HeLa cells (Morin, 1989; de Lange et al., 1990).

During tumor development, the genome of can- cer cells undergoes great changes, which may lead to aneuploidies and other karyotype alterations (Mitelman, 1991). This may include unspecific te- lomere associations (Kovacs et al., 1987; Pathak et al., 1988; Saltman et al., 1989), similar to those found in senescent cells and some non-neoplastic disorders (Benn, 1976; Taylor et al., 1981). T h e enhanced proliferation rate of many tumors may lead to an increased replication-dependent short- ening of terminal repeat sequences in aberrant cells compared with healthy tissues. Loss of telomeric

sequences during tumor development (de Lange et al., 1990; Hastie et al., 1990; Hiyama et al., 1992) and a correlation between the reduction of TTAGGG repeats and telomeric fusions have been reported (Holzmann et al., 1993; Saltman et al., 1993). Thus, cancer cell chromosomes are likely to undergo a replication-dependent decrease of telo- mere length and fuse at their ends to form dicen- trics (Counter et al., 1992).

Since until now only few tumor types have been examined with regard to their telomere repeat number, we investigated the length distribution of TRFs in a variety of human tumors of different origin and found that it ranged from 1 kb to more than 23 kb. Forty percent of all tumor samples showed a reduced maximum telomere length (<15 kb). Another subset of the tumors (29%) showed TRFs up to 15 kb, indicating no length alteration when compared with healthy tissues. T o our sur- prise, nearly one third of all tumor samples exhib- ited TRFs exceeding 15 kb. Even different sam- ples from the same tumor type did not show a common tendency, neither to telomere reduction nor to elongation (Fig. 4). Interestingly, a fraction of extremely short TRFs (below 4 kb) was de- tected in nearly all tumor samples (Fig. 4), a find- ing that was not observed in healthy tissues. Our finding of long telomere stretches in several tumors of different origin is in contrast to previous reports that a general shortening of terminal repeat length occurs in particular cancers (de Lange et al., 1990; Hastie et al., 1990; Hiyama et al., 1992), but in accordance with recently published data about te- lomere lengthening in human intracranial tumors

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Figure 4. Length distribution of terminal restriction fragments in human malignant tissues: gl, glioma; mca, mamma carcinoma; mb, medulloblastoma; men, meningioma; oca, ovarial carcinoma; pch. pheochromocy- toma; gca, gastric carcinoma; pad, prostate adenoma; pca, prostate carcinoma; n, corresponding normal tissue. Peripheral (p) or central (c) localization of the sample in the tumor is indicated.

CHROMOSOMAL TELOMERE VARIATION IN TUMORS I77

(Nurnberg et al., 1993). Our data suggest that there is no general tendency to telomere reduction in malignant tissues.

ACKNOWLEDGMENTS

T h e authors are grateful to G. Seitz, Depart- ment of Pathology; B. Wullich, Department of Ilrology; and K. Holzrnann, Department of Hu- man Genetics, Homburg, for several tumor and DNA samples. The study was supported in part by an H. Dietz Krebsstiftung grant and an EC travel grant (Biomed 1 Program B14).

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