Oncogene (2001) 20, 3835 ± 3844 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas Christina Scheel1, Karl-Ludwig Schaefer1, Anna Jauch3, Monika Keller3, Daniel Wai1, Christian Brinkschmidt1, Frans van Valen2, Werner Boecker1, Barbara Dockhorn-Dworniczak1 and Christopher Poremba*,1 1Gerhard-Domagk-Institute of Pathology, WestfaÈlische Wilhelms University, MuÈnster, Germany; 2Department of Experimental Orthopedics, WestfaÈlische Wilhelms University, MuÈnster, Germany; 3Department of Human Genetics, Ruprecht Karls University, Heidelberg, Germany Telomere maintenance is regarded as a key mechanism in most cases this is achieved by the activation of in overcoming cellular senescence in tumor cells and in telomerase (Greider and Blackburn, 1987; Counter et most cases is achieved by the activation of telomerase. al., 1998). Earlier investigations revealed telomerase However there is at least one alternative mechanism of activity (TA) in 90% of human tumors, but not in telomere lengthening (ALT) which is characterized by normal tissues (Kim et al., 1994; Shay and Bacchetti, heterogeneous and elongated telomeres in the absence of 1997). telomerase activity (TA). We evaluated the prevalence of However, the speci®c association of telomerase with TA, gene expression of telomerase subunits and ALT in cancer progression is being challenged. The frequency relation to telomere morphology and function in matrix and biologic importance of TA seem to be subjected to producing bone tumors and in osteosarcoma cell lines tumor-type dependent variation (Wynford-Thomas, and present evidence of a direct association of ALT with 1999). Stem cells (Harle and Boukamp, 1996), telomere dysfunction and chromosomal instability. stimulated lymphocytes (Broccoli et al., 1995; Hiyama Telomere ¯uorescence in situ hybridization (T-FISH) in et al., 1995; Counter et al., 1995) and dierent highly ALT cells revealed elongated and shortened telomeres, proliferative tissues (Hiyama et al., 1996; Takubo et al., partly in unusual con®gurations and loci, dicentric 1997; Bryan et al., 1997) exhibit TA. Moreover, an marker chromosomes and signal-free chromosome ends. alternative mechanism of telomere lengthening (ALT) Free ends give rise to end-to-end associations and may exists in a subset of human tumors and immortalized induce breakage-fusion-bridge cycles resulting in an cell lines. ALT is characterized by the presence of increased number of complex chromosomal rearrange- elongated and heterogeneous telomeres in the absence ments, as detected by multiplex-FISH (M-FISH). We of TA and is supposed, though not yet elucidated, to propose that ALT cannot be seen as an equivalent to elongate telomeres through recombination-based inter- telomerase activity in telomere maintenance. Its associa- chromosomal exchanges of sequential material (Bryan tion with telomere dysfunction and chromosomal in- et al., 1995; Biessmann and Mason, 1997; Shay and stability may have major implications for tumor Bacchetti, 1997; Bryan et al., 1997). progression. Oncogene (2001) 20, 3835 ± 3844. Our purpose was to evaluate the prevalence of TA and ALT in matrix producing bone tumors in regard Keywords: telomere maintenance; osteosarcomas; telo- to telomere morphology and function. We ®rst laid merase activity; alternative lengthening of telomeres; emphasis on the analysis of the frequency of TA in chromosomal instability osteo- and chondrosarcomas, benign lesions with various histology, and osteosarcoma cell lines by the telomere repeat ampli®cation protocol (TRAP). Gene Introduction expression of the enzyme's catalytic subunit hTERT (Meyerson et al., 1997; Takakura et al., 1998; Telomeres shorten with each round of cell division Ramakrishnan et al., 1998) and its endogenous RNA (Olovnikov, 1971; Watson, 1972). Telomere mainte- template hTR (Avilion et al., 1996; Ito et al., 1998; nance is regarded as a key mechanism in overcoming Sumida et al., 1999) was performed by quantitative cellular senescence through immortalization in tumor real-time RT ± PCR. Secondly, we detected ALT in cells (Wright et al., 1989; Counter et al., 1992) and primary osteosarcomas and osteosarcoma cell lines by telomere length analysis using Southern blotting. Analysis of telomere morphology and function was performed by telomere PNA-¯uorescence in situ *Correspondence: C Poremba, Gerhard-Domagk-Institute of hybridization (T-FISH). Finally we investigated the Pathology, WestfaÈ lische Wilhelms-University, Domagkstrasse 17, occurrence of molecular cytogenetic aberrations char- 48149 MuÈ nster, Germany Received 4 January 2001; revised 27 March 2001; accepted 2 April acteristic for either TA or ALT by comparative 2001 genomic hybridization (CGH) in osteosarcomas and ALT is associated with chromosomal instability C Scheel et al 3836 osteosarcoma cell lines and by 24-color multiplex FISH expression (Table 3). The osteosarcoma cell lines U2- (M-FISH) in osteosarcoma cell lines. As a guardian of OS and ZK-58 had no detectable TA in the TRAP genome integrity, TP53 gene status in the osteosarco- assay and no hTERT expression. ZK-58 presented ma cell lines was analysed. marginal hTR expression and U2-OS had no detectable hTR expression. Neuroblastoma cell lines Lan-5 and SK-PN-DW, used as positive controls in T-FISH and telomere length analysis, showed high levels of TA. To Results exclude false negative results, protein extracts of telomerase positive tumors were mixed with protein Frequency of TA in matrix producing bone tumors extracts of telomerase negative tumors at dierent Sixty-eight bone tumors were assayed for TA by the dilutions. No inhibition of the PCR reactions of the TRAP assay (Table 1). 15/29 osteosarcomas (52%) TRAP assay was detected. compared to 16/23 chondrosarcomas (70%) showed TA. 6/16 histologically dierent benign lesions (38%), ALT, TA and telomere morphology all of chondrogenic origin, presented minimal TA. TA levels were signi®cantly lower than in any of the The 4/7 cell lines with high TA (HOS, MNNG, OST, examined malignant tumors. When examining the SJSA-1) had short telomeres, with a mean length of corresponding histological slides, bone marrow cells 3.5 kb (Figure 1). Cell lines U2-OS and ZK-58 and in®ltration of lymphocytes in some regions of these displayed no TA and no hTERT expression. Telomere lesions were found. Contamination with these cells in length analysis of these cell lines by Southern blotting the corresponding samples could explain our ®ndings revealed a hybridization signal smear stretching from of minimal TA (Counter et al., 1995; Harle and 52 ± 23 kb with the strongest hybridization signal Boukamp, 1996). present at 23 kb, thus representing the ALT phenotype 15/68 primary tumors (eight osteosarcomas, seven (Bryan et al., 1997). Saos-2 exhibited an intermediate chondrosarcomas) were assayed for telomerase core ALT type with low TA, marginal hTERT and hTR component (hTERT, hTR) gene expression by quanti- expression, but elongated and heterogeneous telomeres tative real-time RT ± PCR (Table 2). 8/8 tumors with (strongest hybridization signal at 23 kb). TA (100%) were positive for hTERT gene expression To exclude that ALT is a cell culture artifact, DNA and 7/7 tumors lacking TA (100%) had no detectable of two primary high grade osteoblastic osteosarcomas hTERT expression. All tumors revealed hTR expres- obtained from tumor biopsies without any pretreat- sion with a tendency to higher expression in telomerase ment was included for telomere length analysis (Figure positive tumors, consistent with the literature (Avilion 1). One tumor without TA and without hTERT et al., 1996; Ito et al., 1998). expression revealed elongated and heterogeneous Cell lines MNNG, HOS, OST and SJSA-1 displayed telomeres with a mean length of 23 kb, representing high TA with varying, but signi®cantly higher levels of the ALT phenotype. The other tumor, with inter- hTERT and hTR gene expression when compared to mediate TA and high hTERT expression, had Saos-2, which exhibited low TA and hTERT/hTR gene telomeres with a mean length of 4 kb. Fluorescence in situ hybridization with a telomere specific Table 1 Frequency of telomerase activity in matrix producing bone PNA-probe (T-FISH) tumours Twenty metaphases each of neuroblastoma cell line Entity Number Telomerase Activity % Lan-5 and osteosarcoma cell line OST (both with high Total number 68 37 54 TA), ALT cell lines U2-OS and ZK-58, intermediate Malignant tumors 52 31 60 type cell line Saos-2 and a normal human lymphocyte Osteosarcoma 29 15 52 high grade 25 13 52 culture were examined. Aberrations present in at least low grade 2 0 0 50% (10 metaphases) are described. Chromosomes of post chemo 4 1 25 Lan-5, OST or the human normal lymphocyte culture relapse 2 0 0 essentially showed four ¯uorescent hybridization not further class. 2 1 50 signals at telomeric positions of similar ¯uorescent Chondrosarcoma 23 16 70 GI 6 5 83 intensity (Figure 2a). In Saos-2, U2-OS and ZK-58, GII 13 7 54 ¯uorescence intensity diered greatly in chromosomes GIII 4 4 100 of the same metaphase and between chromosome ends. Benign lesions*166*38Hybridization spots with a higher and a lower intensity En/Chondroma 7 5 71 Eosinophilic granuloma 3 0 0 than in Lan-5, OST and the lymphocyte culture were Giant cell tumor 1 0 0 found (Figure 2b). Desmoplastic fibroma 1 0 0 Large, dicentric marker chromosomes indicating Aneurysmatic bone cyst 2 0 0 end-to-end