Mutagenesis, Vol. 17, No. 1, 67-72,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene
1 Departamento de Genética, Faculdade de Medicina, 2 Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras and 3 Grupo de Imunogenética Molecular, Departamento de Genética and Faculdade de Odontologia, Universidade de São Paulo, Campus de Ribeirão Preto, SP, Brasil and 4 Center for Radiological Research, Columbia University, New York, NY, USA
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A large proportion of human malignancies have been associated with activating mutations in the ras oncogene (Bos, 1989
). Recently, Chin et al. (1999) provided genetic evidence that Ha-ras is important for both the genesis and maintenance of solid tumors in the mouse melanoma model. The induction of activated Ha-ras gene expression in murine fibroblasts (NIH 3T3 cells) results in genomic instability, which can be detected as aberrant chromosomes at the next mitosis (Denko et al., 1994). Many of the characteristics of ras transformation appear to result from inappropriate Ras signaling disturbing normal cell cycle controls and a requirement for cooperating genes seems to be necessary to induce tumor formation in human cells (Weitzman and Yaniv, 1999); this is in accordance with the well-known multi-step nature of tumor development. However, the role of ras in cell growth control and/or genomic instability still remains unclear.
The ends of chromosomes in most eukaryotes are protected by telomeres, which comprise (TTAGGG)n repeats. The maintenance of telomere length and function requires telomerase holoenzyme, a specialized reverse transcriptase, as well as telomere-associated proteins (Blackburn, 1991; Greider, 1996; deLange and dePinho, 1999; deLange and Jacks, 1999). It has been demonstrated that telomerase holoenzyme activity is directly linked to the intracellular levels of hTERT, the telomerase catalytic subunit (Counter et al., 1998; Koyanagi et al., 2000). Most somatic tissues and primary cells possess low or undetectable telomerase activity (Shay and Bacchetti, 1997) and telomeres shorten with each cell division in vivo and in vitro, until they reach a critical length that induces cellular senescence (Harley et al., 1990). The activation of a senescence program through signals generated by short telomeres seems to operate through pathways involving the Rb and p53 tumor suppressor genes (Artandi and dePinho, 2000). Reactivation of telomerase and stabilization of the telomere appear to be concomitant with the attainment of immortality in tumor cells (Kim et al., 1994). Telomere repeats are also detected at intrachromosomal sites in a variety of vertebrate species and they may represent ancestral telomere fusion events or amplification of the repeat sequences present in ancestral karyotypes as latent telomeres (Meyne et al., 1990).
Stabilization of telomere length by telomerase may be required for immortalization (Counter et al., 1992). Since telomeres are required to maintain chromosome stability and integrity (Zakian, 1995), it seems obvious that chromosomes or chromosome fragments lacking telomeres at the end or uncapped chromosomes can fuse with each other. This has been observed in Rodentia (Nanda et al., 1995), in irradiated mammalian cells in vitro (Boei and Natarajan, 1996) and in irradiated tumor cells (Joo et al., 1998). Recently, this assumption has been tested and proved in telomerase knockout mice and mouse cells lacking telomerase upon successive rounds of cell divisions (Blasco et al., 1997; Hande et al., 1999a; Niida et al., 2000).
We have used immortalized ras-transfected fibroblasts (B61 cells) and the parental A31 cells (which are spontaneously transformed in culture) to study the incidence of chromosomal rearrangements involving telomeric DNA sequences by FISH (fluorescence in situ hybridization) and whether they can be correlated with ras mRNA overexpression in the transfected cells.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cell lines
The Balb/3T3 cell lines, clones A31 and B61, were provided by Prof. Dr Mari C.Sogayar (Instituto de Quimica, Universidade de São Paulo, São Paulo, Brazil). Clone A31 was obtained by clonal selection from spontaneously transformed cells that became independent of growth factors (Armelin et al., 1988
; Kovary et al., 1989). Clone B61 was generated from A31 cells which were co-transfected with the Ej-ras oncogene (plasmid pEJ 6.6) and with the bacterial neo gene (plasmid HSV-neo); the transfectants were selected by resistance to the neomycin analog G418, conferred by the neo gene (Armelin et al., 1988). The B61 transfectant cell line harbors 50–100 integrated EJ-ras copies per cell (Kovary et al., 1989).
Cell culture and metaphase preparations
Balb/3T3 cell clones A31 and B61 were kept frozen in nitrogen. For the experiments the cells were thawed and cultured at 37°C in HAM's F-10/DEM medium with 10% fetal bovine serum; the growing cells were sub-cultured every 3 or 4 days and used at earlier passages (maximum of 10 passages) so as to avoid the influence of late passages on genomic instability. The metaphase spreads were obtained from exponentially growing cells (two independent cultures) and prepared by the conventional method: treatment with colcemid (0.1 µg/ml) for 2 h, hypotonic shock (1% sodium citrate) for 10 min and fixation with methanol:acetic acid (3:1). The cell suspension was dropped onto cleaned slides and dried at room temperature. The metaphase preparations were aged for 2 days before processing for FISH.
Fluorescence in situ hybridization (FISH)
In situ hybridization with Cy3-conjugated PNA (peptide nucleic acid) oligonucleotide (CCCTAA)3 on the metaphases was performed according to Ziljmans et al. (1997) and Hande et al. (1999a). The slides were washed in phosphate-buffered saline (PBS), fixed in 4% formaldehyde for 2 min and washed again in PBS for 3x5 min. The slides were incubated in pepsin (1 mg/ml) for 10 min at 37°C. Fixation with formaldehyde and PBS washes were repeated and the slides were dehydrated in an ethanol series. The target DNA and the telomeric PNA probes were denatured simultaneously at 80°C for 3 min in a hybridization mixture containing 70% formamide and 0.3–0.5 µg/ml PNA probe in 10 mM Tris, pH 7.2, for 15 min each. Additional washes in 0.05 M Tris, 0.15 M NaCl, pH 7.5, and 0.05% Tween 20 solution were also performed. The preparations were dehydrated in an ethanol series and counterstained with 0.25 mg/ml 4',6-diamidino-2-phenylindole (DAPI) in anti-fade solution (Vectashield; Vector Laboratories).
Cytogenetic analysis
Chromosomal analysis of Giemsa-stained metaphases was performed for both cell lines, A31 and B61, to determine the mean chromosome number for each. The metaphase spreads were prepared for FISH and analyzed with a fluorescence Axiophot microscope (Carl Zeiss, Germany) equipped with appropriate fluorescence filters. The rearrangements involving telomeric DNA sequences were analyzed using an in situ imaging system (ISIS; Metasystems, Germany).
A lack of telomeric signals (LTS) at chromosome ends was observed in either one (LTS') or both chromatids (LTS''). Robertsonian fusion-like configurations (RLC), types I and II, were classified according to Boei and Natarajan (1996). Interstitial telomeric sequences (ITS), single (ITS') or double (ITS''), were scored at both non-telomeric sites and in the centromeres of RLC. LTS and ITS were scored in 50 metaphases, while an additional 30 metaphases were analyzed for RLC, so as to provide a higher chance of detecting RLC with ITS, which was a less frequent event, mainly in A31 cells.
Analysis of ras mRNA expression
Total RNA was isolated from exponentially growing A31 and B61 cells using the Trizol reagent (Gibco BRL Life Technology). The plasmid DNA with Ha-v-ras cDNA inserts transformed in Escherichia coli DH5
was amplified as described by Sambrook et al. (1989) and used as probe. Probe labeling with 32P was done by random priming. Dot blot analysis was performed according to Sambrook et al. (1989) using a Hybond N+ nylon membrane (Amersham Pharmacia). The filters were exposed to a Fuji imaging plate and incorporation of 32P was quantified by phosphorimaging (Cyclone; Packard).
Analysis of cell cycle kinetics
Cell cycle analysis was performed by staining the cells with propidium iodide and applying the detergent–trypsin method (Vindelov et al., 1983). 10 000 cells were analyzed per cell line using a FACScan flow cytometer (Becton Dickinson, USA) and the cells distributed in the different phases of the cell cycle according to the DNA content, which was performed on gated nuclei using the software program CellFit provided by Becton Dickinson.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cytogenetic analysis on metaphase spreads prepared from A31 and B61 cells showed a characteristic hyperdiploidy and the mean chromosome numbers were 70 ± 12.43 and 75 ± 21.01, respectively. The cell cycle kinetics were analyzed by flow cytometry. The two cell lines differed in the distribution of cells in different phases, with B61 cells presenting a higher percentage of cells undergoing S phase than A31 cells (Figure 1
). These results confirmed that the ras-transfected cells (clone B61) exhibit a higher level of chromosome instability than the parental A31 cells.
|
Chromosomal alterations involving telomeric regions were detected with high accuracy using FISH with a PNA telomeric probe. The main types of alterations were RLC types I and II, ITS, which were observed in one (ITS') or both chromatids (ITS''), and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS''), as shown schematically in Figure 2
. High frequencies of these kinds of chromosomal alterations were found (Tables I and II), mainly RLC type I or II, which occurred at higher frequencies in B61 cells (1.95/cell) compared with A31 cells (0.25/cell), and LTS (5.9 and 17.5/cell, for A31 and B61 cells, respectively). Interestingly, ITSs were detected at both non-telomeric sites and in the centromeres of RLC. In the case of those located at non-telomeric sites, A31 and B61 cells presented 4.1 and 5.7 ITS/cell, respectively. Although B61 cells showed a much higher frequency of RLCs, RLCs with ITSs located in the centromeres were more frequent in A31 cells (Table I). Different types of RLC and the presence of ITSs at different positions along the chromosome, as well as LTSs, are illustrated in Figure 3.
|
|
|
|
), lack of telomere signals in three chromosomes (
) and RLC type I with ITS in the centromere ( 
). (B) ITS located at non-telomeric sites in both chromatids ( ras mRNA expression in transfected and non-transfected mouse cells was analyzed by dot blot (Figure 4
). The quantification of 32P by phosphorimaging confirmed the higher level (46%) of ras expression in B61 cells, as compared with A31 cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cytogenetic analysis and the distribution of cells in different phases of the cell cycle indicated that the ras-transfected cells (clone B61) exhibited a higher level of chromosome instability compared with the parental A31 cells, with a higher percentage of cells undergoing S phase.
In the present study chromosomal rearrangements were efficiently detected by FISH analysis with a telomeric PNA probe. Previously, many authors demonstrated that mouse cells are especially suitable for this type of analysis, since their chromosomes have exceptionally long telomeres (Blasco et al., 1999; Hande et al., 1999a; Niida et al., 2000). LTSs and RLCs were the most frequent aberrations observed, with the ras-transfected B61 cells presenting much higher frequencies of rearrangements when compared with A31 cells, while ITSs located at non-telomeric sites were 28% higher in B61 cells. RLCs without telomeric signals were found at higher frequencies in B61 cells. Robertsonian translocations at variable sites in the pericentromeric regions of human acrocentrics have been reported by Cheung et al. (1990), indicating that both reciprocal translocations and fusions/fissions may possibly result in Robertsonian interchanges. Telomeric sequences in the centromeric region of house mouse Robertsonian metacentric chromosomes were not detected in earlier studies (Narayanswami et al., 1992; Schubert et al., 1992; Nanda et al., 1995), even when molecular and cytogenetic analyses were performed (Garagna et al., 1995). Schubert et al. (1992) suggested that the mechanism of Robertsonian rearrangement leading to abnormal chromosome numbers may not consist solely of a simple fission or fusion of chromosomes without a concomitant gain or loss of chromatin material.
The use of PNA telomeric probes clearly showed a certain percentage of RLCs presenting telomeric signals in the centromeric regions, although RLCs without telomeric sequences were the most frequent in B61 cells. These results support the assumption that complete or partial loss of telomeric repeats occurs leading to chromosome fusion and consequently the formation of RLC, with the possibility of retaining a variable number of telomeric repeats which cannot be resolved by in situ hybridization. In the present study the use of a PNA telomeric probe and the detection of a hybridization signal by an imaging system provided higher resolution and allowed the detection of RLCs showing faint telomeric signals located both at non-telomeric sites and in the centromeres of RLC. The presence of telomere signals at the fusion point in the case of RLCs has recently been observed in DNA repair-deficient mice and mouse cell lines (Bailey et al., 1999; Hande et al., 1999b, 2001; di Fagagna et al., 2001).
It has been suggested that telomere length is not the only factor determining chromosome fusigenic potential in mammalian cell lines (Saltman et al., 1993; Slijepcevic et al., 1997). The progression of normal human cells towards immortalization and tumorigenicity may lead to a reduction in the capacity of telomeres to prevent chromosome fusions (Hastie et al., 1990; Counter et al., 1992). Since the mouse cell lines used in this study present the characteristics of immortalized cells with abnormal karyotypes, or tumorigenic potential in the B61 clone (Kovary et al., 1989), the high frequency of RLCs without telomeric repeats is compatible with the role of telomeres in preventing chromosome fusion and maintaining chromosome stability. This indicates that most RLCs may originate from considerable loss of telomeric repeats, so that the remaining telomeric signals cannot be visualized by the FISH method.
The presence of telomeric DNA sequences located in centromeric regions in RLC types I and II may be explained by partial loss of telomeric repeats, so that fusion of the ends of both chromosomes reconstitute a short telomeric site. Alternatively, the addition of telomere repeats by telomerase could be another explanation for the origin of the interstitial telomeric repeats observed in the present study with mouse cell lines, taking into account that telomerase may be able to heal chromosomes in vivo (Harrington and Greider, 1991), and evidence reported for human cells (Murnane and Yu, 1993; Flint et al., 1994) as well as irradiated mouse splenocytes (Hande et al., 1998). Although the addition of telomere repeats (>200 bp) at a broken chromosome site was demonstrated in X-irradiated cells, it was not clear whether they were newly formed telomeric sequences added by telomerase or acquired from other chromosomes (Hande et al., 1998). Thus, a mechanism of telomere healing may also explain the presence of interstitial telomeric repeats observed in both cell lines.
The existence of telomeric sequences at non-telomeric sites of submetacentric and metacentric chromosomes has been detected in the Indian muntjac (Lee et al., 1993), in Sigmodon mascotensis and Mus dunnii (Meyne et al., 1990). In addition, interstitial telomere repeats were detected in Chinese hamster cell lines (Balajee et al., 1994) and were shown to constitute hot-spots for radiation-induced chromosome breaks (Balajee et al., 1996; Slijepcevic and Bryant, 1998). Interstitial telomeres are also present in the highly recombinogenic chicken cell line DT40 (Hande, unpublished data). RLCs type II can also be involved in the formation of telomeric sequences at non-telomeric sites, since they present two centromeres susceptible to the breakage–fusion–bridge mechanism.
Thus, in the present study a high level of chromosome instability was observed in B61 transfectant cells overexpressing ras mRNA. Besides the higher variation in chromosome number and the characteristic cell cycle progression (with a large proportion of cells in S phase), B61 cells showed a high instability involving telomeric sequences. As a whole, the results indicate that cells over-expressing the ras gene exhibit high instability involving telomeric sequences and, consequently, a tendency to form RLCs, as a consequence of the high proliferation capacity conferred by the increase in ras expression in B61 cells.
Extra copies of the ras gene were found to be integrated in tandem arrays when metaphase spreads were submitted to FISH with a biotinylated ras probe (unpublished data) and analysis of ras mRNA expression showed an increase of 46% in B61 compared with parental A31 cells. This is in agreement with an earlier report on the capacity of a mutant Ha-ras transgene to contribute to the process of genomic instability (Denko et al., 1994). It was demonstrated that oncogenic ras and telomerase were sufficient to create human tumor cells in vitro after disruption of the intracellular pathways regulated by the simian virus 40 large T antigen (Hahn et al., 1999), but whether the same pathways occur in rodent cells has not yet been demonstrated.
We also found that both mouse cell lines, A31 and B61, express p53 mRNA when RNA was extracted from exponentially growing cells under normal conditions (data not shown). Murine somatic cells express telomerase activity and have much longer telomeres than their normal human counterparts, which lack telomerase activity (Kipling and Cooke, 1990; Zijlmans et al., 1997). According to Gollahon et al. (1998), ras-transformed LFS 087 cells formed tumors in nude mice without reactivation of telomerase activity. This is in agreement with earlier results obtained by Blasco et al. (1997), who reported that telomerase-deficient cells can be immortalized in culture, transformed by viral oncogenes and generate tumors in nude mice (while untransformed cells did not form tumors).
In mouse cell lines deficient for telomerase Hande et al. (1999a) verified many chromosomes lacking detectable telomere signals at late passages, as well as a significant increase in end-to-end fusions, and these cells also showed heterogeneous fluorescence intensity values which increased with population doublings, thus suggesting an ALT mechanism. Similarly, in the present work heterogeneous telomere signals were observed when different chromosomes were compared within the same metaphase, thus reflecting heterogeneous telomere length. Probably, the lack of telomere signals observed for a number of chromosomes or chromatids in each metaphase may correspond to very short telomeres, which are unable to be visualized by FISH, but still have the minimum number of TTAGGG repeats required for chromosome stabilization, with a tendency to form RLCs when a critical length is reached in the next cell division.
Therefore, the higher frequency of chromosomes ends lacking telomeric sequences in the transfectant B61 clone and the presence of ITSs and RLCs observed in the present study might indicate a defective capping mechanism. Wei et al. (1999) demonstrated that even in the presence of active telomerase, overexpression of the Ha-ras oncogene can induce a cellular senescence program in normal human fibroblasts. Recently, Jones et al. (2000) demonstrated for the first time, in primary human cells, the existence of a telomere-independent mechanism of ras-induced senescence, additional or alternative to the telomere clock. Rodent cells express high levels of telomerase, which contributes to their increased susceptibility to immortalization and malignant transformation. In the present study overexpression of the Ha-ras gene in B61 cells may be associated with an increase in chromosomal instability after successive cell divisions; this is in agreement with the results obtained by Denko et al. (1994), but these authors verified a rapid increase in genomic instability within one cell cycle.
Since it was demonstrated that B61 cells exhibit a higher percentage of cells in S phase and a shorter cell cycle when compared with the parental cells, it is reasonable to speculate that a deficient telomere capping mechanism occurs due to the rapid cell cycle progression of B61 cells, leading to a high heterogeneity in telomere length, as verified by the different intensities of the signals at the chromosome ends. But to what extent this depends on telomerase status remains to be investigated. Though telomerase maintains functional telomeres, there is evidence that telomere length can also be maintained by an alternative pathway (ALT) which is telomerase independent (Bryan et al., 1997; Artandi and dePinho, 2000). This alternative mechanism has been clearly demonstrated in mouse embryonic fibroblasts and embryonic stem cells in which the telomerase RNA gene was deleted (Hande et al., 1999a; Niida et al., 2000). Furthermore, recent reports showed that DNA repair proteins are also involved in the maintenance of telomeric repeats, to prevent chromosome fusions, at least in mouse cells (Bailey et al., 1999; di Fagagna et al., 1999, 2001; Hande et al., 1999b, 2001).
Thus, the present study showed a high chromosome instability involving telomeric DNA sequences in ras- transfected cells overexpressing ras mRNA, which may be a consequence of a rapid cell cycle progression associated with a deficient telomere capping mechanism. Furthermore, the interstitial telomere sequences located randomly in the genome (at non-telomeric sites) might have been produced in proliferating cells as a consequence of a breakage–fusion–bridge mechanism occurring in unstable RLCs presenting more than one centromere and subsequent addition of telomeres by chromosome healing or telomere–telomere associations.
|
Acknowledgments |
|---|
We are grateful to Prof. Dr Mari C.Sogayar for providing the Balb/3T3 cell lines and the Ha-ras insert. This research was supported by FAPESP (grant no. 97/12939-5), FINEP, CNPq and Pró-Reitoria de Pesquisa–USP (fellowship to S.S.M.).
|
Notes |
|---|
* These authors contributed equally to this paper.
5 To whom correspondence should be addressed at: Departmento de Biologia, FFCLRP-usp, Avenida Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brasil. Tel: +55 16 602 3827; Fax: =55 16 633 0069: Email: etshojo{at}usp.br
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Armelin,H.A., Armelin,M.C.S. and Kovary,K. (1988) Neoplastic transformation of 3T3 mouse embryo cells with c-myc and c-Ha-ras-1 oncogenes. Brazilian J. Med. Biol. Res., 21, 1155–1161.[Web of Science][Medline]
Artandi,S.E. and dePinho,R.A. (2000) A critical role for telomeres in suppressing and facilitating carcinogenesis. Curr. Opin. Genet. Dev., 10, 39–46.[Web of Science][Medline]
Bailey,S.M., Meyne,J., Chen,D.J., Kurimasa,A., Li,G.C. and Lehnert,B.E. (1999) DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc. Natl Acad. Sci. USA, 96, 14899–14904.
Balajee,A.S., Joo,O.H. and Natarajan,A.T. (1994) Analysis of restriction enzyme-induced chromosome aberrations in the interstitial telomeric repeat sequences of CHO and CHE cells by FISH. Mutat. Res., 307, 307–313.[Web of Science][Medline]
Balajee,A.S., Dominguez,I., Bohr,V.A. and Natarajan,A.T. (1996) Imunofluorescent analysis of the organization of telomeric DNA sequences and their involvement in chromosomal aberrations in hamster cells. Mutat. Res., 372, 163–172.[Web of Science][Medline]
Blackburn,E.H. (1991) Structure and function of telomeres. Nature, 350, 569–573.[Medline]
Blasco,M.A., Lee,H.W., Hande,M.P., Samper,E., Lansdorp,P.M., DePinho,R.A. and Greider,W.C. (1997) Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell, 91, 25–34.[Web of Science][Medline]
Boei,J.J.W.A. and Natarajan,A.T. (1996) Classification of X-rays-induced Robertsonian fusion-like configurations in mouse splenocytes. Int. J. Radiat. Biol., 69, 421–427.[Web of Science][Medline]
Bos,J.L. (1989) Ras oncogene in human cancer: a review. Cancer Res., 49, 4682–4689.
Bryan,T.M., Marusic,L., Bacchetti,S., Namba,M. and Reddel,R.R. (1997) The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit. Hum. Mol. Genet., 6, 921–926.
Cheung,S.W., Sun,L. and Featherstone,T. (1990) Molecular cytogenetic evidence to characterise breakpoint regions in Robertsonian translocations. Cytogenet. Cell, Genet., 54, 97–102.[Web of Science][Medline]
Chin,L., Tam,A., Pomerantz,J., Wong,M., Bardeesy,N., Shen,Q., O'Hagan,R., Pantginis,J., Zhout,H., Horner,J.W., Cordon-Carlo,C., Yancopoulos,G.D. and DePinho,R.A. (1999) Essential role for oncogenic Ras in tumour maintenance. Nature, 400, 468–471.[Medline]
Counter,C.M., Avillion,A.A., LeFeuvre,C.E., Stewart,N.G., Greider,C.W., Harley,C.B. and Bascchetti,S. (1992) Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J., 11, 1921–1929.[Web of Science][Medline]
Counter,C.M., Meyerson,M., Eaton,E.N., Ellisen,L.W., Caddle,S.D., Haber,D.A. and Weinberg,R.A. (1998) Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene, 16, 1217–1222.[Web of Science][Medline]
DeLange,T. and dePinho,R.A. (1999) Unlimited mileage from telomerase? Science, 283, 947–949.
DeLange,T. and Jacks,T. (1999) For better or worse? Telomerase inhibition and cancer. Cell, 98, 273–275.[Web of Science][Medline]
Denko,N.C., Giaccia,A.J., Stringer,J.R. and Stambrook,P.J. (1994) The human Ha-ras oncogene induces genomic instability in murine fibroblasts within one cell cycle. Proc. Natl Acad. Sci. USA, 91, 5124–5128.
di Fagagna,F.D., Hande,M.P.,Tong,W.M., Lansdorp,P.M., Wang,Z.Q. and Jackson,S.P. (1999) Functions of poly(ADP-ribose) polymerase in controlling telomere length and chromosomal stability. Nature Genet., 1, 76–80.
di Fagagna,F.D., Hande,M.P., Tong,W.M., Roth,D., Lansdorp,P.M., Wang,Z.Q. and Jackson,S.P. (2001) Effects of DNA nonhomologous end-joining factors on telomere length and chromosome stability in mammalian cells. Curr. Biol., 11, 1192–1196.[Web of Science][Medline]
Flint,J., Craddock,C.F., Villegas,A., Bentley,D.P., Williams,H.J., Galanello,R., Cao,A., Wood,W.G., Ayyub,H. and Higgs,D.R. (1994) Healing of broken human chromosome by the addition of telomeric repeats. Am. J. Hum. Genet., 55, 505–512.[Web of Science][Medline]
Garagna,S., Broccoli,D., Redi,C.A., Searle,J.B., Cooke,H.J. and Capanna,E. (1995) Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area. Chromosoma, 103, 685–692.[Web of Science][Medline]
Gollahon,L.S., Krausk,E., Wu,T.-A., Yim,S.O., Strong,L.C., Shay,J.W. and Tainsky,M.A. (1998) Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts. Oncogene, 17, 709–717.[Web of Science][Medline]
Greider,C.W. (1996) Telomere length regulation. Annu. Rev. Biochem., 65, 337–365.[Web of Science][Medline]
Hahn,W.C., Counter,C.M., Lundberg,A.S., Beijersbergen,R.L., Brooks,M.W. and Weinberg,R.A. (1999) Creation of human tumour cells with defined genetic elements. Nature, 400, 464–468.[Medline]
Hande,M.P., Lansdorp,P.M. and Natarajan,A.T. (1998) Induction of telomerase activity by in vivo X-irradiation of mouse splenocytes and its possible role in chromosome healing. Mutat. Res., 404, 205–214.[Web of Science][Medline]
Hande,M.P., Samper,E., Lansdorp,P.M. and Blasco,M.A. (1999a) Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice. J. Cell Biol., 144, 589–601.
Hande,M.P., Slijepcevic,P., Silver,A., Bouffler,S., van Buul,P., Bryant,P.E. and Lansdorp,P.M. (1999b) Elongated telomeres in SCID mice. Genomics, 56, 221–223.[Web of Science][Medline]
Hande,M.P., Balajee,A.S., Tchirkov,A., Wynshaw-Boris,A. and Lansdorp,P.M. (2001) Extra-chromosomal telomeric DNA in cells from Atm–/– mice and patients with ataxia-telangiectasia. Hum. Mol. Genet., 10, 519–528.
Harley,C.B., Futcher,A.B. and Greider,C.W. (1990) Telomeres shorten during ageing of human fibroblasts. Nature, 345, 458–460.[Medline]
Harrington,L.A. and Greider,C.W. (1991) Telomerase primer specificity and chromosome healing. Nature, 353, 451–454.[Medline]
Hastie,N.D., Dempster,M., Dunlop,M.G., Thompson,A.M., Green,D.K. and Allshire,R.C. (1990) Telomere reduction in colo-rectal carcinoma and with ageing. Nature, 346, 866–868.[Medline]
Jones,C.J., Kipling,D., Morris,M., Hepburn,P., Skinner,J., Bounacer,A., Wyllie,F.S., Ivan,M., Bartek,J., Wynford-Thomas,D. and Bond,J.A. (2000) Evidence for a telomere-independent `clock' limiting ras oncogene-driven proliferation of human thyroid epithelial cells. Mol. Cell. Biol., 20, 5690–5699.
Joo,O.H., Hande,M.P., Lansdorp,P.M. and Natarajan,A.T. (1998) Induction of telomerase activity and chromosome aberrations in human tumour cell lines following X-irradiation. Mutat. Res., 401, 121–131.[Web of Science][Medline]
Kipling,D. and Cooke,H.J. (1990) Hypervariable ultra-long telomeres in mice. Nature, 347, 400–402.[Medline]
Kim,N.W., Piatyszek,M.A., Prowse,K.R., Harley,C.B., West,M.D., Ho,P.L.C., Coviello,G.M., Wright,W.E., Weinrich,S.L. and Shay,J.W. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science, 266, 2011–2015.
Koyanagi,Y., Kobayashi,D., Yajima,T., Asanuma,K., Kimura,T., Sato,T., Kida,T., Yagihashi,A., Kameshima,H. and Watanabe,N. (2000) Telomerase activity is down regulated via decreases in hTERT mRNA but not TEP1 mRNA or hTERC during differentiation of leukemic cells. Anticancer Res., 20, 773–778.[Web of Science][Medline]
Kovary,K., Armelin,M.C. and Armelin,H.A. (1989) Ha-Ras-1 oncogene dosage differentially affects Balb/3T3 cells growth factor requirement and tumorigenicity. Oncogene Res., 4, 55–64.[Web of Science][Medline]
Lee,C., Sasi,R. and Lin,C.C. (1993) Interstitial localisation of telomeric DNA sequences in the Indian muntjac chromosomes: further evidence for tandem chromosome fusions in the karyotypic evolution of the Asian muntjacs. Cytogenet. Cell Genet., 63, 156–159.[Web of Science][Medline]
Meyne,J., Baker,R.J., Hobart,H.H., Hsu,T.C., Ryder,O.A., Ward,O.G., Wiley,J.E., Wurster-Hill,D.H., Yates,T.L. and Moyzis,R.K. (1990) Distribution of non telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes. Chromosoma, 99, 3–10.[Web of Science][Medline]
Murnane,J.P. and Yu,L.C. (1993) Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break. Mol. Cell. Biol., 13, 977–983.
Nanda,I., Schneider-Rasp,S., Winking,H. and Schimid,M. (1995) Loss of telomeric sites in the chromosomes of Mus musculus domesticus (Rodentia: Muridae) during Robertsonian rearrangements. Chromosom. Res., 3, 399–409.
Narayanswami,S., Dogget,N.A., Clark,L.M., Hildebrand,C.E., Weier,H.U. and Hamkalo,B.A. (1992) Cytological and molecular characterisation of centromeres in Mus domesticus and Mus spretus. Mamm. Genome, 2, 186–194.[Web of Science][Medline]
Niida,H., Shinkai,Y., Hande,M.P., Matsumoto,T., Takehara,S., Tachibana,M., Oshimura,M., Lansdorp,P.M. and Furuichi,Y. (2000) Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA. Mol. Cell. Biol., 20, 4115–4127.
Saltman,D., Morgan,R., Cleary,M.L. and De Lange,T. (1993) Telomeric structure in cells with chromosome end association. Chromosoma, 102, 121–138.[Web of Science][Medline]
Sambrook,J., Fritch,E.F. and Maniats,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Vol. 1, Ch. 7, pp. 7.53–7.56.
Schubert,I., Schriever-Schwemmer,G., Werner,T. and Adler,I.-D. (1992) Telomeric signals in Robertsonian fusion and fission chromosomes: implications for the origin of pseudoaneuploidy. Cytogenet. Cell Genet., 59, 6–9.[Web of Science][Medline]
Shay,J.W. and Bacchetti,S. (1997) A survey of telomerase activity in human cancer. Eur. J. Cancer, 33, 787–791.
Slijepcevic,P. and Bryant,P.E. (1998) Chromosome healing, telomere capture and mechanisms of radiation-induced chromosome breakage. Int. J. Radiat. Biol., 73, 1–13.[Web of Science][Medline]
Slijepcevic,P., Hande,M.P., Bouffler,S.D., Lansdorp,P. and Bryant,P.E. (1997) Telomere length, chromatin structure and chromosome fusigenic potential. Chromosoma, 106, 413–421.[Web of Science][Medline]
Vindelov,L.L., Christensen,I.J. and Nissen,N.I. (1983) A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry, 3, 323–327.[Web of Science][Medline]
Wei,S., Wei,W. and Sedivy,J.M. (1999) Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic Ha-Ras in normal human fibroblasts. Cancer Res., 59, 1539–1543.
Weitzman,J.B. and Yaniv,M. (1999) Rebuilding the road to cancer. Nature, 400, 401–402.[Medline]
Zakian,V.A. (1995) Telomeres: beginning to understand the end. Science, 270, 1601–1607.
Zijlmans,J.M., Martens,U.M., Poon,S.S.S., Raap,A.K., Tanke,H.J., Ward,R.K. and Lansdorp,P.M. (1997) Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc. Natl Acad. Sci. USA, 94, 7423–7428.
Received on April 9, 2001; accepted on September 13, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. S. Agapova, J. L. Volodina, P. M. Chumakov, and B. P. Kopnin Activation of Ras-Ral Pathway Attenuates p53-independent DNA Damage G2 Checkpoint J. Biol. Chem., August 27, 2004; 279(35): 36382 - 36389. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||