Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas Christina Scheel 1 , Karl-Ludwig Schaefer 1 , Anna Jauch 3 , Monika Keller 3 , Daniel Wai 1 , Christian Brinkschmidt 1 , Frans van Valen 2 , Werner Boecker 1 , Barbara Dockhorn-Dworniczak 1 and Christopher Poremba* ,1 1 Gerhard-Domagk-Institute of Pathology, Westfa Èlische Wilhelms University, Mu Ènster, Germany; 2 Department of Experimental Orthopedics, Westfa Èlische Wilhelms University, Mu Ènster, Germany; 3 Department of Human Genetics, Ruprecht Karls University, Heidelberg, Germany Telomere maintenance is regarded as a key mechanism in overcoming cellular senescence in tumor cells and in most cases is achieved by the activation of telomerase. However there is at least one alternative mechanism of telomere lengthening (ALT) which is characterized by heterogeneous and elongated telomeres in the absence of telomerase activity (TA). We evaluated the prevalence of TA, gene expression of telomerase subunits and ALT in relation to telomere morphology and function in matrix producing bone tumors and in osteosarcoma cell lines and present evidence of a direct association of ALT with telomere dysfunction and chromosomal instability. Telomere ¯uorescence in situ hybridization (T-FISH) in ALT cells revealed elongated and shortened telomeres, partly in unusual con®gurations and loci, dicentric marker chromosomes and signal-free chromosome ends. Free ends give rise to end-to-end associations and may induce breakage-fusion-bridge cycles resulting in an increased number of complex chromosomal rearrange- ments, as detected by multiplex-FISH (M-FISH). We propose that ALT cannot be seen as an equivalent to telomerase activity in telomere maintenance. Its associa- tion with telomere dysfunction and chromosomal in- stability may have major implications for tumor progression. Oncogene (2001) 20, 3835 ± 3844. Keywords: telomere maintenance; osteosarcomas; telo- merase activity; alternative lengthening of telomeres; chromosomal instability Introduction Telomeres shorten with each round of cell division (Olovnikov, 1971; Watson, 1972). Telomere mainte- nance is regarded as a key mechanism in overcoming cellular senescence through immortalization in tumor cells (Wright et al., 1989; Counter et al., 1992) and in most cases this is achieved by the activation of telomerase (Greider and Blackburn, 1987; Counter et al., 1998). Earlier investigations revealed telomerase activity (TA) in 90% of human tumors, but not in normal tissues (Kim et al., 1994; Shay and Bacchetti, 1997). However, the speci®c association of telomerase with cancer progression is being challenged. The frequency and biologic importance of TA seem to be subjected to tumor-type dependent variation (Wynford-Thomas, 1999). Stem cells (Harle and Boukamp, 1996), stimulated lymphocytes (Broccoli et al., 1995; Hiyama et al., 1995; Counter et al., 1995) and dierent highly proliferative tissues (Hiyama et al., 1996; Takubo et al., 1997; Bryan et al., 1997) exhibit TA. Moreover, an alternative mechanism of telomere lengthening (ALT) exists in a subset of human tumors and immortalized cell lines. ALT is characterized by the presence of elongated and heterogeneous telomeres in the absence of TA and is supposed, though not yet elucidated, to elongate telomeres through recombination-based inter- chromosomal exchanges of sequential material (Bryan et al., 1995; Biessmann and Mason, 1997; Shay and Bacchetti, 1997; Bryan et al., 1997). Our purpose was to evaluate the prevalence of TA and ALT in matrix producing bone tumors in regard to telomere morphology and function. We ®rst laid emphasis on the analysis of the frequency of TA in osteo- and chondrosarcomas, benign lesions with various histology, and osteosarcoma cell lines by the telomere repeat ampli®cation protocol (TRAP). Gene expression of the enzyme's catalytic subunit hTERT (Meyerson et al., 1997; Takakura et al., 1998; Ramakrishnan et al., 1998) and its endogenous RNA template hTR (Avilion et al., 1996; Ito et al., 1998; Sumida et al., 1999) was performed by quantitative real-time RT ± PCR. Secondly, we detected ALT in 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 hybridization (T-FISH). Finally we investigated the occurrence of molecular cytogenetic aberrations char- acteristic for either TA or ALT by comparative genomic hybridization (CGH) in osteosarcomas and Oncogene (2001) 20, 3835 ± 3844 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: C Poremba, Gerhard-Domagk-Institute of Pathology, WestfaÈ lische Wilhelms-University, Domagkstrasse 17, 48149 MuÈnster, Germany Received 4 January 2001; revised 27 March 2001; accepted 2 April 2001