Cytogenet Cell Genet 85:196–199 (1999)

Chinese hamster telomeres are comparable insize to mouse telomeresP. Slijepcevica and M.P. Handeb

a Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex (UK), andb The Terry Fox Laboratory, British Columbia Cancer Research Center, Vancouver, BC (Canada)

Supported in part by a grant from UKCCCR to P.S. Both authors contributed equallyto this work.

Received 1 February 1999; revision accepted 31 March 1999.

Request reprints from Dr. P. Slijepcevic, Department of Biology and Biochemistry,Brunel University, Uxbridge, Middlesex, UB8 3PH (UK);telephone: +44-1895-274-000 ext 2109; fax: +44-1895-274- 348;e-mail: predrag.slijepcevic@brunel.ac.uk.

ABC E-mail karger@karger.chFax + 41 61 306 12 34http://www.karger.com

© 1997 S. Karger AG, Basel0301–0171/99/0854–0196$17.50/0

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Abstract. We report here the results of a telomere lengthanalysis in four male Chinese hamsters by quantitative fluores-cence in situ hybridization (Q-FISH). We were able to measuretelomere length of 64 (73%) of 88 Chinese hamster telomeres.We could not measure telomere length in chromosome 10 or inthe short arms of chromosomes 5, 6, 7 and 8 because of theoverlaps between the interstitial and terminal telomeric signals.Our analysis in the 73% of Chinese hamster telomeres indicate

that their average length is F38 kb. Therefore, Chinese ham-ster telomeres are comparable in length to mouse telomeres, butare much longer than human telomeres. Similar to previous Q-FISH studies on human and mouse chromosomes, our resultsindicate that individual Chinese hamster chromosomes mayhave specific telomere lengths, suggesting that chromosome-specific factors may be involved in telomere length regulation.

Telomeres are specialized nucleoprotein complexes at chro-mosome termini implicated in oncogenesis and cellular ageing.Telomere length in human chromosomes is typically 10–20 kb(Harley et al., 1990). By contrast, mouse chromosomes containmuch longer telomeres, ranging from 20 to 150 kb when esti-mated by Southern blot analysis (Kipling and Cooke, 1990) or10 to 60 kb when estimated by quantitative fluorescence in situhybridization (Q-FISH) (Zijlmans et al., 1997). The paucity ofrestriction sites in the subtelomeric regions of mouse chromo-somes may limit Southern blot analysis as a means of determin-ing the lengths of terminal telomeric repeats (Zijlmans et al.,1997). Thus, Q-FISH is the technique of choice for estimating

telomere length in mouse chromosomes. An additional advan-tage of Q-FISH is that telomere length may be accurately esti-mated in species exhibiting interstitial telomeric sites (ITSs) intheir chromosomes. The Chinese hamster (Cricetulus griseus)genome (2n = 22) contains a total of 18 ITSs located in thepericentromeric regions of chromosomes 3–10 and X (Slijep-cevic et al., 1996). Almost all available data involving Chinesehamster telomeres are based on studies employing immortal-ized cell lines (Alvarez et al., 1993; Bertoni et al., 1994; Panditaand de Rubeis, 1995). These cell lines show a high degree ofkaryotypic alteration (Slijepcevic et al., 1996), as well as a largereduction in telomere length in comparison with primary celllines in early passages (Slijepcevic and Bryant, 1995). Informa-tion on telomere length in Chinese hamster cells in vivo is cur-rently unavailable. Therefore, extrapolations based on dataobtained from studies employing immortalized Chinese ham-ster cell lines may not necessarily reflect the in vivo situation.For the purposes of examining differences in telomere mainte-nance between the Chinese hamster, mouse, and human ge-nomes and of establishing the evolutionary implications ofthese comparisons that emerged from a recent study (Smilenovet al., 1998), it is important to determine telomere length inChinese hamster in vivo.

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Materials and methods

Chinese hamsters were purchased from B+K Universal Ltd., Hull, UK.Chromosome spreads were prepared directly from bone marrow cells. Ani-mals were killed at the age of 10 mo, following the intraperitoneal adminis-tration of Colcemid (3.5 mg/kg body weight) for 1 h. Chromosomes wereprepared directly from bone marrow cells flushed from both femora andspread onto slides. Q-FISH analysis was performed as described earlier (Sli-jepcevic et al., 1997; Zijlmans et al., 1997). Briefly, chromosomes werehybridized with the Cy3-labeled (CCCTAA)3 peptide nucleic acid (PNA)oligonucleotide and observed with a Zeiss Axioplan microscope equippedwith a CCD camera. Separate images were taken for DAPI (chromosomes)and Cy3 (telomeres) and subjected to telomere fluorescence measurementsby using the TFL TELO software developed by S. Poon and P. Lansdorp(Terry Fox Laboratory, Vancouver, Canada). About 10–15 metaphases persample were analyzed for individual telomere measurements. Telomere fluo-rescence intensity is expressed so that one telomere fluorescence unit (TFU)corresponds to 1 kb of (TTAGGG)n sequences according to results from sim-ilarly hybridized and analyzed plasmids with a defined (TTAGGG)n length(Martens et al., 1998).

Results and discussion

To estimate telomere length in vivo, we used four male Chi-nese hamsters. Owing to the presence of ITSs in the Chinesehamster genome, Southern analysis may not be suitable for tel-omere length measurements in this species. As an alternative,we used Q-FISH. A metaphase spread showing telomeric sig-nals at all chromosome ends is presented in Fig. 1. Telomerelength could not be measured by Q-FISH in chromosome 10 oron the short arms of chromosomes 5, 6, 7, and 8 because ofoverlaps between the ITSs and terminal telomeric repeats(Fig. 1). We were able, however, to estimate telomere length byQ-FISH in the rest of the chromosomes (Table 1). Therefore,our analysis covers 64 of 88 telomeres (73%) in the Chinesehamster genome (Table 1). In line with previous Q-FISH stud-ies involving mouse and human chromosomes (Zijlmans et al.,1997; Martens et al., 1998), our results indicate heterogeneityin telomere length among Chinese hamster chromosomes. Spe-cific chromosomes in different animals had similar lengths,suggesting the involvement of chromosome-specific factors in

telomere length regulation. For example, the Yp telomeres pro-duced the strongest fluorescence in all four animals, leading toan estimated telomere length of 74–94 kb (Table 1). By con-trast, the 2p and 5q telomeres were relatively short in all ani-mals, ranging from 16 to 26 kb in 2p and from 13 to 27 kb in 5q(Table 1). We also observed variations in telomere lengthbetween individual animals (Table 1), in agreement with simi-lar findings in humans and mice (Zijlmans et al., 1997; Martenset al., 1998). In addition, the long arms had longer telomeresthan the short arms in most chromosomes (Table 1). However,

Fig. 1. A metaphase spread from a bone marrow Chinese hamster cellshowing telomeric signals at all chromosome ends.

Table 1. Telomere length estimation in four male Chinese hamsters by Q-FISHa

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198 Cytogenet Cell Genet 85:196–199 (1999)

Table 2. Average telomere length in all fourChinese hamstersa

the Y chromosome was a notable exception to this rule (Ta-ble 1).

Based on our Q-FISH analysis, the average telomere lengthin vivo in Chinese hamster chromosomes is estimated to beF38 kb (Table 2), ranging from 13 to 95 kb (Table 1). Althoughour estimate of telomere length in the Chinese hamster is basedon the analysis of 64 of 88 telomeres, the fact that we have ana-lyzed the majority (73%) of the telomeres on the chromosomesand that, with the exception of chromosome 10, all chromo-somes were covered by the analysis, leads us to believe thattelomere lengths in the mouse and Chinese hamster are verysimilar and that Chinese hamster telomeres are, on average,much longer than human telomeres. Telomere length, as esti-mated by Q-FISH in mouse chromosomes, varies between 10and 60 kb (Zijlmans et al., 1997). The average telomere lengthin human chromosomes, as estimated by Q-FISH in 16 indi-viduals, is F5 kb (Martens et al., 1998).

All immortalized Chinese hamster cell lines that have beenanalyzed to date have extremely short telomeres, estimated tobe in the region of 1 kb (Slijepcevic and Bryant, 1995; Slijep-cevic et al., 1997). Assuming that the average telomere length invivo in the Chinese hamster is F38 kb (Table 2), and given thefact that telomere length in any individual, karyotypically nor-mal chromosome originating from any immortalized Chinesehamster cell line is estimated to be F1 kb (Slijepcevic andBryant 1995), we speculate that an extensive loss of telomericsequences occurs during proliferation and immortalization ofChinese hamster cells in vitro. For example, in the case of Chi-nese hamster chromosome 1 (Tables 1 and 2), the estimatedloss of telomeric sequences in immortalized cell lines, in com-parison with primary cells, is greater than 35 kb per telomere.This loss of telomeric sequences occurs in the presence of telo-merase activity. Both primary cells and cell lines establishedfrom Chinese hamster and other rodents express strong telo-merase activity throughout their lifespan (Newbold, 1997).This suggests that exonuclease activity, which normally partici-pates in telomere length maintenance in yeast (Wellinger et al.,1996) and probably also in mammalian cells (Makarov et al.,

1997), may be particularly strong in immortalized Chinesehamster cells, leading to the loss of telomeric sequences even inthe presence of telomerase activity. Alternatively, Chinesehamster cells may show down-regulated tankyrase activity.Tankyrase, a recently identified protein, is a negative regulatorof TRF1 (TTAGGG repeat factor 1), which is a negative regu-lator of telomerase (Smith et al., 1998). Another possibility isthat telomerase-independent mechanisms that contribute totelomere length maintenance in postsenescent cells (Bryan etal., 1995) may be suppressed in immortalized Chinese hamstercells.

Despite their very short telomeres, chromosomes from im-mortalized Chinese hamster cell lines do not show an increasedfrequency of end-to-end chromosome fusions (Slijepcevic et al.,1997), as would be expected from studies on human chromo-somes. It appears that a few telomeric repeats present at thechromosomes from immortalized Chinese hamster cell linesare sufficient for telomere function, i.e., to prevent end-to-endchromosome fusions. The recently cloned gene encoding theChinese hamster TRF1 protein shows a higher degree ofhomology to the human rather than the mouse gene (Smilenovet al., 1998). It has been speculated that this evolutionary dif-ference may be due to the relative shortness of the telomeres inhuman and Chinese hamster cells in comparison to those inmouse cells (Smilenov et al., 1998). This speculation was basedon the use of immortalized cell lines, such as the Chinese ham-ster ovary (CHO) cell line, in which the telomeres are estimatedto be F1 kb long (Slijepcevic and Bryant, 1995; Slijepcevic etal., 1997). However, our results indicate that Chinese hamstertelomeres are, in fact, much longer than human telomeres invivo and are comparable in size to mouse telomeres (Tables 1and 2). Therefore, the speculation made by Smilenov et al.(1998) may not be correct, and evolutionary differences in theTRF1 gene among the three species may not be due to differ-ences in their telomere lengths. Taken together, our observa-tions suggest that the Chinese hamster may be an interestingmodel for exploring the basis of mammalian telomere lengthregulation.

Acknowledgements

We would like to thank P.M. Lansdorp and S. Poon for allowing us to usetheir TFL TELO software for telomere length estimation by Q-FISH.

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