Skip Navigation


Human Molecular Genetics Advance Access originally published online on May 11, 2005
Human Molecular Genetics 2005 14(13):1785-1794; doi:10.1093/hmg/ddi185
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
14/13/1785    most recent
ddi185v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Jeyapalan, J. N.
Right arrow Articles by Royle, N. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeyapalan, J. N.
Right arrow Articles by Royle, N. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Activation of the ALT pathway for telomere maintenance can affect other sequences in the human genome

Jennie N. Jeyapalan1, Helen Varley1, Jenny L. Foxon1, Raphael E. Pollock2, Alec J. Jeffreys1, Jeremy D. Henson3, Roger R. Reddel3 and Nicola J. Royle1,*

1Department of Genetics, University of Leicester, Leicester, LE1 7RH, UK, 2MD Anderson Cancer Centre, Houston, Texas, TX 77030 USA and 3Cancer Research Unit, Children's Medical Research Institute, Sydney, NSW 2145, Australia

* To whom correspondence should be addressed. Tel: +44 162522270; Fax: +44 162523378; Email: njr{at}leicester.ac.uk

Received February 22, 2005; Revised April 13, 2005; Accepted May 4, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Immortal human cells maintain telomere length by the expression of telomerase or through the alternative lengthening of telomeres (ALT). The ALT mechanism involves a recombination-like process that allows the rapid elongation of shortened telomeres. However, it is not known whether activation of the ALT pathway affects other sequences in the genome. To address this we have investigated, in ALT-expressing cell lines and tumours, the stability of tandem repeat sequences known to mutate via homologous recombination in the human germline. We have shown extraordinary somatic instability in the human minisatellite MS32 (D1S8) in ALT-expressing (ALT+) but not in normal or telomerase-expressing cell lines. The MS32 mutation frequency varied across 15 ALT+ cell lines and was on average 55-fold greater than in ALT– cell lines. The MS32 minisatellite was also highly unstable in three of eight ALT+ soft tissue sarcomas, indicating that somatic destabilization occurs in vivo. The MS32 mutation rates estimated for two ALT+ cell lines were similar to that seen in the germline. However, the internal structures of ALT and germline mutant alleles are very different, indicating differences in the underlying mutation mechanisms. Five other hypervariable minisatellites did not show elevated instability in ALT-expressing cell lines, indicating that minisatellite destabilization is not universal. The elevation of MS32 instability upon activation of the ALT pathway and telomere length maintenance suggests there is overlap between the underlying processes that may be tractable through analysis of the D1S8 locus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human cells that express the ALT pathway (ALT+) have very heterogeneous length telomere repeat arrays (1Go), and single telomeres shorten gradually in culture but undergo rapid elongation, gaining many kilobases of DNA, probably in a single extension event (2Go). A class of human telomere mutations, seen only in ALT+ human cells, are characterized by truncation of the progenitor allele and fusion to a telomere repeat array from elsewhere. The breakpoints in such mutant telomeres can occur very close to the start of the telomere repeat array and locate to consensus TTAGGG or sequence variant repeats. These ALT-associated telomere mutations most likely arise through a recombination-like mechanism (3Go). Similar mechanisms for telomere length maintenance have been discovered in a variety of other species, some of which lack telomerase activity (4Go,5Go). Yeast (Saccharomyces cerevisiae) normally maintains telomere length via telomerase but strains deficient in either the RNA or reverse transcriptase component of telomerase can survive crisis by activation of recombination-based pathways dependent on the RAD52 gene (6Go,7Go). Yeast survivors can be subdivided into two types (8Go). Type I survivors show gross amplification of subterminal repeat sequences called Y' elements but the chromosomes still terminate with a telomere repeat array. Y' element amplification is dependent on the RAD52 and RAD51 genes (9Go). Yeast Type II survivors are dependent on the RAD52, RAD50 and other genes and have heterogeneous length telomeres that show a similar pattern of shortening and sudden elongation to that observed in human ALT+ cells (6Go,10Go).

Knowledge of the proteins involved in the human ALT pathway is largely indirect and primarily from the study of specialized PML bodies found in a proportion of ALT+ cells (reviewed in 11). These bodies are nuclear structures found in many cell types and contain the PML protein (11Go) and other components, some of which may be cell type specific. ALT-associated PML bodies (APBs) contain extra-chromosomal telomeric DNA sequences and the telomere binding proteins TRF1 and TRF2, which are not found in PML bodies of other cell types. APBs also contain a range of proteins involved in DNA damage repair and replication, some of which are found in PML bodies in other cell types (12Go–14Go). It is not clear, however, what role APBs play in the recombination-like mechanism that underlies telomere elongation in ALT+ cells.

The recombination-like events in ALT+ cells affect telomere repeat arrays but not subterminal sequences (15Go) and it appears that more general homologous recombination is not elevated in ALT-expressing cells (16Go). However, the effect of activation of the ALT pathway on other tandem repeat loci has not been investigated extensively in human cells. To address this, we have studied the instability of minisatellite loci in ALT+ and ALT– cells. Initially we selected the well-characterized hypervariable human minisatellite MS32 (locus D1S8). MS32 is located interstitially on chromosome 1 (1q42.3) and is composed of a tandem array of 29 bp repeat units that are GC-rich (62%) (17Go). Alleles vary in size from 12 to more than 800 repeat units. Mutations that alter the number of repeats can be assayed at the single DNA molecule level by PCR amplification across the array, followed by size resolution in an agarose gel. The MS32 minisatellite has a high male germline mutation rate (0.81% per sperm) with the majority of mutants (>80%) arising from cross-over or conversion-like events between alleles most likely arising at meiosis (18Go). These germline exchange events cluster at one end of the repeat array and appear to be driven by a meiotic recombination hotspot centred ~200 bp from the start of the repeat array (19Go). The frequency of somatic mutations in blood DNA is much lower (~0.004% per haploid genome) and the mutations tend to be simple insertion or deletion events distributed throughout the array (20Go).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To investigate the frequency of MS32 length mutations in normal (telomerase– ALT–), ALT+ and telomerase+ cell lines, we PCR-amplified MS32 alleles from many clonal DNA samples derived from single cells from six different cell lines. All three ALT+ cell lines (WI38VA13/2RA, IIICF/a2, JFCF.6T-1J.11E) showed a very high level of MS32 instability, with 45% of alleles analysed in the WI38VA13/2RA ALT+ clones showing clear length changes compared to <2% in the telomerase+ (HT1080, NT2D1) or normal (WI38) cell lines (Fig. 1A; Table 1). In some of the ALT+ clonal DNA samples (111CF/a2, in particular), multiple faint bands were amplified and clear progenitor alleles (alleles present in the cell that seeded the clone) could not be identified. This suggested that the MS32 minisatellite continued to be extremely unstable during expansion of the clones. To investigate this instability further, we amplified multiple small aliquots of genomic DNA (small pool-PCR) (21Go) from a variety of cell lines to analyse the frequency of mutant MS32 molecules. Very few molecules containing MS32 length mutations were observed in the WI38 normal (ALT– telomerase–), IIICF/a2 precrisis (ALT– telomerase–), JFCF6-T.5K precrisis (ALT– telomerase–), HT1080 (telomerase+) and NT2D1 (telomerase+) cell lines analysed. In contrast, extraordinary instability of the MS32 minisatellite was seen in three ALT+ cell lines analysed [IIICF/a2 (ALT+), WI38VA13/2RA (ALT+), JFCF6-T.1J/11E (ALT+)] (Fig. 1B and data not shown).





View larger version (200K):
[in this window]
[in a new window]
  		Figure 1. The MS32 minisatellite is unstable in cell lines expressing the ALT pathway. (A) The MS32 minisatellite was analysed from clonal DNA samples from the cell lines indicated, with each track containing DNA from a different clone. MS32 alleles were amplified using the allele-specific primer H1G resulting in the detection of single alleles from the H1G/C heterozygous cell lines, WI38 and WI38VA13/2RA. Both alleles were amplified from the HT1080 cell line, which is homozygous for H1G, but only the 5.5 kb allele is shown as the large allele (8.2 kb) does not amplify well in the presence of the shorter allele. PCR products were detected by agarose gel electrophoresis and Southern blot hybridization. (B) Single molecule mutation analysis in ALT+ and ALT– cell lines. Multiple DNA aliquots containing the indicated number of MS32 molecules were amplified using the universal primers MS32B and MS32E and the PCR products analysed, as above, to detect mutant molecules. Progenitor alleles are arrowed. Note the absence of a clear progenitor in the IIICF/a2 cell line and the presence of three progenitors in the WI38VA13/2RA cell line. (C) Small-pool PCR analysis of MS32 in ALT+ cell lines established independently from two precursor cell lines (left, IIICF/a2; right, JFCF6-T).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Minisatellite mutation frequencies in clonal DNA samples from normal, ALT+ and telomerase+ cell lines
 
MS32 mutation frequencies vary in ALT+ cell lines
Accurate mutation frequencies at the MS32 locus were determined for 20 cell lines: 14 unrelated cell lines plus six cell lines that arose as independent immortalization events from a common progenitor line (JFCF6-T cell lines). The mutation frequencies were determined using small-pool PCR to count mutant MS32 molecules and Poisson analysis of single molecule dilutions to estimate the overall number of amplifiable molecules analysed (Table 2) (21Go). The limitation of size resolution in agarose gels means that the mutation frequencies are underestimates. The mutation frequencies of the four telomerase+ cell lines and the GM02096 (telomerase– ALT–) cell line were low (0–0.014; average 0.0062) whereas the mutation frequencies in the 15 ALT+ cell lines varied widely (0.034–0.93, average 0.345). Interestingly, the five ALT+ cell lines that arose from the same precursor also showed a wide range of MS32 mutation frequencies (JFCF6-T cell lines Table 2 and Fig. 1C). The reason for the considerable variation in MS32 mutation frequencies among the ALT+ cell lines is unknown but the length of the repeat array may be a factor, as some short alleles are more stable than longer alleles in ALT+ cell lines (e.g. IIICF lines, Fig. 1C, left). There are, however, some exceptions; for example, the JFCF6-T.1J/11E cell line has longer alleles that are more stable that those in the JFCF6-T.1J/1D cell line (Fig. 1C, right and data not shown).


View this table:
[in this window]
[in a new window]
  		Table 2. MS32 mutation frequencies established by small-pool PCR in a variety of cell lines
 
The MS32 minisatellite is highly unstable in a proportion of ALT+ soft tissue sarcomas
DNA samples from 18 adult sarcomas were assayed (blindly) for MS32 instability, as discussed earlier. The ALT status, based on telomere length, length heterogeneity, detection of APBs, and telomerase activity, had been determined previously for these 18 tumour samples (22Go). Table 3 shows that three of eight ALT+ tumours showed very high MS32 instability whereas none of 10 ALT– samples showed high MS32 instability, giving an overall concordance of 72% between MS32 instability and ALT expression. High MS32 instability could pass undetected if the ALT+ tumour sample contained a significant amount of DNA from normal cells, but the proportion of DNA from normal cells would have to be >50%. Instead it seems more likely that high MS32 instability is only seen in a subset of ALT+ tumours and it raises the possibility that these ALT+ tumours are a distinct subset that may show wider effects of genome instability. Interestingly, the three tumours with high MS32 instability also showed APBs in a large proportion of cells (Table 3).


View this table:
[in this window]
[in a new window]
  		Table 3. MS32 instability in DNA samples from sarcomas
 
ALT+ cell lines do not show instability at all minisatellites in the genome
To determine whether the instability of the MS32 minisatellite was indicative of genome-wide instability of hypervariable GC-rich minisatellites in ALT-expressing cells, we analysed four other minisatellites in the clone-derived DNA samples from two ALT+ and two telomerase+ cell lines (Table 1). The four minisatellites, MS31A (D7S21), CEB1(D2S90), MS205 (D16S309) and B6.7 are all located in terminal regions of human chromosomes and all show germline instability with reported mutation rates of 1.2, ~13, 0.5 and 7% per sperm, respectively (23Go–26Go). However, none of these minisatellites showed elevated instability in ALT+ clones, and indeed the frequency of mutant alleles at these loci was similar to the frequency of mutant MS32 alleles in telomerase+ cell lines. Clearly, not all minisatellites are highly unstable in ALT-expressing cells.

Comparison of the MS32 mutation rate and mechanism in ALT+ cell lines and the germline
The MS32 mutation rate per cell division was determined by clonal expansion of single ALT+ cells from the IIICF/a2 (ALT+) and WI38VA13/2RA (ALT+) cell lines, for an estimated number of population doublings. Following DNA extraction, mutation frequencies were determined for three or four clones from each cell line and used to estimate the mutation rate per cell division (Table 4). The average mutation rate for the IIICF/a2 (ALT+) cell line (0.94% per cell division) was higher than for the WI38VA13/2RA (ALT+) cell line (0.36% per cell division), consistent with the mutation frequencies for these cell lines (Table 2). Variation in the size of the progenitor alleles between clones might affect the mutation rate; for example, the larger and smaller alleles in IIICF/a2 (ALT+) clone 5G5 have different mutation rates (Table 4).


View this table:
[in this window]
[in a new window]
  		Table 4. Mutation rates per cell division for the WI38VA13/2RA and IIICF/a2 ALT+ cell lines
 
Minisatellite variant-repeat mapping by PCR (MVR–PCR) (27Go) maps the order of sequence-variant repeats in the array. PCR priming within the repeat array is initialized by tagged MVR primers that each anneal to one of the four sequence variant repeats (E,e,Y,y) commonly found in MS32 repeat arrays. The tagged MVR primers include a non-complementary sequence at the 5' end (TAG) that is used in conjunction with an MS32-adjacent primer to drive the amplification. Four PCRs, each containing the MS32-adjacent primer, a low concentration of one of the four MVR tagged primers and the TAG primer, generate amplicons that are subsequently size separated to reveal the distribution of sequence variant repeats along the MS32 array. MVR maps can be generated for single alleles when a heterozygous SNP adjacent to the minisatellite is exploited for allele-specific amplification. Comparison of mutant and progenitor MS32 MVR maps has revealed the molecular mechanisms that underlie germline and somatic instability at this locus (20Go,21Go). Therefore, we used MVR-mapping to compare MS32 alleles in the WI38 normal and WI38VA13/2RA ALT+ cell lines. Both cell lines are heterozygous for the H1C/G SNP, 311 bp from the start of the MS32 repeat array. The WI38VA13/2RA (ALT+) cell line has three copies of the MS32 minisatellite in most cells: two associated with the H1C allele and one with the H1G allele. Partial MS32 MVR-maps were generated from both alleles in the WI38 normal cell line (Fig. 2A) and from the H1G-associated allele in clonal DNA samples from the WI38VA13/2RA (ALT+) cell line (Fig. 2B). The 21 H1G-associated alleles mapped from the WI38VA13/2RA (ALT+) clones all share similar repeat interspersions that differ from the H1G-associated allele in the WI38 cell line [precursor to WI38VA13/2RA (ALT+) cell line]. Some of the H1G-associated alleles in the WI38VA13/2RA (ALT+) clones contain other changes that have only been seen once (e.g. clone 1) while other differences are shared by a subset of alleles (e.g. in clones 2, 11 and 18). In short, the mutations identified include changes of repeat types, deletions and insertions that distinguish individual or subsets of alleles and they must have occurred after activation of the ALT pathway.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 2. Comparison of MS32 MVR-maps from H1G-associated alleles in clones from the WI38VA13/2RA ALT+ cell line with progenitor alleles from the WI38 normal cell line. (A) Partial MS32 MVR maps from the WI38 normal cell line. The alleles contain very different interspersions of the sequence variant repeat units shown as e (green), E (blue), Y (red) and y (black) (27Go). The YYeYYeYEeYEYe motif (underlined) is present twice near the start of the repeat array of the H1G-associated allele. (B) Partial MVR maps of H1G-associated alleles from 21 clonal DNA samples of the WI38VA13/2RA (ALT+) cell line aligned to the derived consensus map. Differences from the consensus MVR map are marked in red. Grey lines have been introduced to improve alignment between the alleles. (C) The consensus MVR map from WI38VA13/2RA ALT+ clones is shown aligned to the progenitor allele in the WI38 normal cell line. (D) Comparison of the telomere repeat sequence with the MS32 and MS1 repeat sequences. Adjacent repeats are shown in alternating black and red text. Vertical lines show base identity between repeat arrays. Polymorphic bases in the MS32 and MS1 repeats are shown in italics. Y=C or T, R=A or G.

 
Comparison of the consensus H1G map from WI38VA13/2RA (ALT+) clones with the WI38 H1G allele shows a change of the first repeat (E to e), four insertions of nine, five, seven and two repeats and one deletion of 13 repeats (Fig. 2C). Partial triplication of the YYeYYeYEeYEYe motif in the WI38-H1G allele may account for the nine repeat insertion (Fig. 2C, underlined in red) but the origins of the other insertions are unknown. The timing of these mutations with respect to activation of the ALT pathway is uncertain but, given the instability of the MS32 minisatellite in ALT+ cells, it seems most likely they arose after activation of the ALT pathway.

In summary, the mutations identified in the MS32 minsatellite are not clustered towards one end of the array, as seen in the germline, nor are they all simple insertion or deletion events, as seen in the rare mutants found in normal somatic cells (20Go). Overall, the mutant alleles in the WI38VA13/2RA (ALT+) clones appear to be composites that have arisen from sequential mutations at the MS32 locus, consistent with the high MS32 mutation rate in the ALT+ cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Low level instability at minisatellite loci in tumour DNA samples was first noted through analysis of DNA fingerprints (28Go). A more extensive analysis of colorectal and gastric adenocarcinomas showed instability at five single minisatellite loci, including MS32 and the maximum mutation rate at MS32 was estimated at 4x10–6 per cell division (29Go). Although not tested, the majority of tumours in that study will have expressed telomerase, as seen in other surveys of colon and gastric tumours (30Go). Thus, the low frequency of MS32 instability we detected in telomerase+ cell lines and tumours is consistent with these observations. In contrast, the extraordinary instability that we detected at MS32 in ALT+ cell lines, ~2000-fold greater than seen in normal somatic DNA or tumours, has not been detected before and it indicates that activation of the ALT pathway destabilizes this minisatellite in cell lines and in a significant proportion of ALT+ soft tissue sarcomas. However, it does not have a universal destabilizing effect on all minisatellites. Modest instability seen in DNA fingerprints of an ALT-expressing cell line and subclones from it corroborate our observation (31Go).

Our analysis suggests that the MS32 mutation mechanism in ALT+ cells differs from that of the germline where frequent complex inter-allelic exchanges dominate (18Go). In normal somatic cells, the rare MS32 mutations most likely arise from simple intra-allelic events arising from slippage during replication or unequal sister-chromatid exchange. It is intriguing then that ALT+ cells appear to have elevated levels of inter-telomeric exchange that may arise from sister-chromatid exchange (32Go,33Go). However, curiously, activation of the ALT pathway does not destabilize all the GC-rich minisatellites investigated, although they all mutate by recombination mechanisms in the germline. Activation of the ALT pathway may relax a general repression of recombination-like processes at telomeres but the coincident effect on other loci in the genome (such as MS32) may depend on diverse factors (e.g. chromatin status). The MS31A, CEB1, MS205 and B6.7 minisatellites differ from the MS32 minisatellite, as they are all located in proterminal regions towards the ends of human autosomes. Proterminal regions of chromosomes are proficient at homologous recombination during meiosis and they are rich in hypervariable minisatellites (34Go). This raises a question as to whether minisatellites located at interstitial sites in the genome are more accessible to proteins utilized by the ALT pathway compared with minisatellites in proterminal regions. To begin to address this question, we have used small pool PCR to investigate instability at the MS1 minisatellite (D1S7) in eight ALT+ cell lines (WI38VA13/2RA, U2-OS, IIICF/a2, AT13LA, G292, SUSM1, W-V, JFCF6-T.1J/1D). The MS1 minisatellite comprises a tandem repeat array based on a 9 bp repeat unit (17Go,35Go) and it is located at an interstitial site on the short arm of chromosome 1 (1p35.2). The MS1 minisatellite did not show a highly elevated level of instability in ALT+ cell lines [average mutation frequency across the eight ALT+ cell lines=0.0023 (5/2205), data not shown], indicating that a simple relationship between minisatellite location and instability in ALT+ cells does not explain our observations. Alternatively, the MS32 minisatellite may have specific properties (as yet unidentified) that facilitate instability when the ALT pathway is active. However, similarity between the sequence of the MS32 repeat unit and telomere repeats is an unlikely explanation as there is less similarity between the MS32 repeat and telomere repeats than between telomere repeats and the MS1 repeat sequence (Fig. 2D).

Our survey of MS32 instability in sarcomas revealed that a subset of ALT+ tumours but none of the telomerase+ tumours showed excessive MS32 instability. MS32 instability in some ALT+ tumours may be an indicator of greater genome instability or of a higher level of ALT activity and therefore greater telomere length maintenance. Either way, MS32 instability in ALT+ tumours may be a useful diagnostic marker and further investigation of this locus in other mesenchyme-derived tumours will be revealing. Furthermore, analysis of the MS32 minisatellite will undoubtedly increase our understanding of the pathway that underlies telomere length maintenance in ALT+ cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell lines and DNA samples
The WI38 normal (ALT– telomerase–), HT1080 (tumour-derived, telomerase+), NT2D1 (tumour-derived, telomerase+), WI38VA13/2RA (ALT+) cell lines were obtained from the European Collection of Cell Cultures (ECACC). The Saos-2 (tumour-derived, ALT+) and U2-OS (tumour-derived, ALT+) cell lines were a gift from P. Salomoni. The cell lines IIICF/a2 precrisis (ALT– telomerase–), IIICF/a2 (ALT+), JFCF6-T.5K precrisis (ALT– telomerase–), JFCF6-T.1J/11E (ALT+) have been described before (3Go). All cell lines were grown and manipulated using standard techniques. DNA was extracted from cells grown to confluence in 80 cm2 flasks, using standard techniques.

Clonal DNA samples from the WI38 (normal), WI38VA13/2RA (ALT+), IIICF/a2(ALT+), JFCF6-T.1J/11E (ALT+), HT1080 (telomerase+) and NT2D1 (telomerase+) cell lines have been described before (3Go). Other DNA samples from cell lines AT13LA (ALT+), ATBR44neo (ALT+), G292 (tumour-derived cell line, ALT+), GM847DM (ALT+), SUSM1 (ALT+), W-V (ALT+), Met5A (telomerase+), GM02096 (ALT– telomerase–), JFCF6-T.1R (ALT+), JFCF6-T.1D (ALT+), JFCF6-T.1J/1D (ALT+), JFCF6-T.IJ/5H (ALT+), JFCF6-T.1F (telomerase+), IIICF-E6E7/C4 (ALT+), IIICF-T/A6 (ALT+) were provided by Reddel. The status of the ALT+ cell lines used had been determined previously by the absence of telomerase activity using the Telomere Repeat Amplification Protocol (TRAPeze, Intergen), and detection of characteristic ALT+ telomere length heterogeneity by TRF analysis (1Go,12Go). Henson and Pollock provided DNA samples from 18 adult sarcomas (15 soft tissue sarcomas and three osteosarcomas) from patients at the MD Anderson Cancer Centre, Houston, TX, USA (22Go).

Cloning ALT+ cell lines to determine the MS32 mutation rate per cell division
The WI38VA13/2RA (ALT+) and the IIICF/a2 (ALT+) immortal cell lines were cloned by dilution of a single-cell suspension in growth medium to 8 cells/ml. Aliquots (100 µl) were dispensed into each well of a 96-well plate, giving an average cell count of 0.8 cells per well. Colonies that arose from a single cell (determined by visual inspection) were transferred to 25 cm2 flasks to allow further expansion. Total cell counts were obtained for each clone when the cells were harvested for DNA extraction.

PCR amplification of the MS32 minisatellite in bulk and clonal DNA samples
PCR across the MS32 minisatellite was conducted with universal primers MS32B and MS32E that amplify all alleles and anneal 380 and 75 bp upstream and downstream from the repeat array, respectively (20Go,36Go). Allele-specific amplification was conducted from the H1C/G SNP using either the H1C or H1G primer in place of the MS32B universal primer. PCR reactions (total volume, 10 µl) included 50 ng genomic DNA in 1x PCR buffer (37Go) with flanking primers (1 µM) and 0.1 U/µl Taq DNA polymerase (ABgene). The PCRs were cycled at 96°C for 1 min and then 20 times at 96°C for 20 s, 62°C for 30 s, 70°C for 7 min. Very long MS32 alleles were amplified under long range PCR conditions by the addition of Tris base (12 mM), the Taq DNA polymerase was reduced to 0.07 U/µl and Pfu DNA polymerase added at 0.015 U/µl (Stratagene). The PCR products were resolved in 0.8–1.0% LE agarose gels (BioWhittaker) and transferred to Hybond Nfp (Amersham Pharmacia Biotech) for Southern blot hybridization to a 32P-labelled MS32 tandem repeat probe (17Go).

PCR amplification of MS32 minisatellite molecules from small aliquots of genomic DNA (small-pool PCR)
Amplification of small aliquots of genomic DNA was achieved as described previously (18Go) from either high molecular weight genomic DNA or genomic DNA that had been digested with the MboI restriction endonuclease (NEB). The DNA was then diluted to 4 µg/ml in 5 mM Tris–HCl (pH 7.5) with 1 ng/µl of high molecular weight salmon sperm DNA as a carrier. The DNA samples were further diluted to the desired concentration and finally into the PCR mix containing the PCR primers MS32B and MS32E (0.2 µM), 1x PCR buffer, Tris base (12 mM), Taq (0.07 U/µl), Pfu (0.015 U/µl) and 1 ng/µl salmon sperm DNA. Multiple 10 µl PCR reactions were then cycled 23 times at 96°C for 20 s, 62°C for 30 s, 70°C for 10 min and then once at 56°C for 1 min, 70°C for 10 min. The amplified products were resolved in an agarose gel and detected as described earlier.

PCR for Poisson analysis of single MS32 molecules was conducted as for small-pool PCR but the DNA was diluted further such that each of 40 reactions contained an estimated 0.5 molecules. The number of positive PCRs were used to estimate the number of amplifiable molecules present in the small pool reactions, as described elsewhere (18Go).

PCR amplification of other minisatellites in bulk and clonal DNA samples
The MS205 minisatellite (D16S309) was amplified from 50 ng of clonal DNA samples using universal PCR primers MS205A and MS205B (38Go) at 1 µM. The PCRs were cycled at 96°C for 1 min, then 26 times at 96°C for 1 min, 67°C for 1 min, 70°C for 5 min. The MS31A minisatellite (D7GoS21Go) was similarly amplified with the MS31A and MS31B primers cycled at 96°C for 30 s and then 22 times at 96°C for 20 s, 68°C for 40 s, 70°C for 5 min (23Go). Amplicons from these minisatellites were detected by Southern blot hybridization to a radioactively labelled MS205 or MS31A repeat probe. The CEB1 (D2S90) minisatellite was amplified by the Ceb1f and Ceb21r primers (0.4 µM) from 25 ng genomic DNA and cycled at 96°C for 2 min and 31 cycles at 96°C for 30 s, 64°C for 30 s, 72°C for 5 min (24Go). The B6.7 minisatellite was amplified from 25 ng genomic DNA in the presence of the 67A and 67B primers (0.4 µM) for 31 cycles (26Go). Products from the CEB1 and B6.7 minisatellites were visualized directly in an agarose gels.

MS32 MVR–PCR analysis
MVR-PCR was conducted as described before (27Go). The reactions included the TAG primer and a flanking primer, H1C or H1G at 0.2 µM. Each reaction also included 1–2 nM of one of the four MS32 sequence variant detector primers TAG-CG, TAG-CA, TAG-TA, TAG-TG that detect e, E, Y and y repeat types, respectively. The PCR products were resolved in a 1.2% agarose gel and analysed by Southern blot hybridization as described earlier.


   ACKNOWLEDGEMENTS
 
We are most grateful to Katie Bryan for her enthusiastic contribution to the work, to Rita Neumann for expert technical advice, to Celia May, Ingrid Berg and to all in Labs G18 and G19 in the Department of Genetics for helpful discussions. We thank Paolo Salomoni for kindly donating cell lines and Dihua Yu and Jonathon Hannay for the sarcoma samples. Funding for this work came from grants awarded to N.J.R. by the MRC (UK).

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Bryan, T.M., Englezou, A., Gupta, J., Bacchetti, S. and Reddel, R.R. (1995) Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J., 14, 4240–4248.[ISI][Medline]

  2. Murnane, J.P., Sabatier, L., Marder, B.A. and Morgan, W.F. (1994) Telomere dynamics in an immortal human cell-line. EMBO J., 13, 4953–4962.[ISI][Medline]

  3. Varley, H., Pickett, H.A., Foxon, J.L., Reddel, R.R. and Royle, N.J. (2002) Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells. Nat. Genet., 30, 301–305.[CrossRef][ISI][Medline]

  4. Roth, C.W., Kobeski, F., Walter, M.F. and Biessmann, H. (1997) Chromosome end elongation by recombination in the mosquito Anopheles gambiae. Mol. Cell. Biol., 17, 5176–5183.[Abstract]

  5. Rosen, M. and Edstrom, J.E. (2002) Chromosome ends in Chironomus tentans do not have long single-stranded overhangs characterizing canonical telomeres. Chromosome Res., 10, 21–31.[CrossRef][ISI][Medline]

  6. Lundblad, V. and Blackburn, E.H. (1993) An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell, 73, 347–360.[CrossRef][ISI][Medline]

  7. Lundblad, V. (2002) Telomere maintenance without telomerase. Oncogene, 21, 522–531.[CrossRef][ISI][Medline]

  8. Le, S., Moore, J.K., Haber, J.E. and Greider, C.W. (1999) RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase. Genetics, 152, 143–152.[Abstract/Free Full Text]

  9. Chen, Q., Ijpma, A. and Greider, C.W. (2001) Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events. Mol. Cell. Biol., 21, 1819–1827.[Abstract/Free Full Text]

  10. Teng, C.S., Chang, J., McCowan, B. and Zakian, A.V. (2000) Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process. Mol. Cell, 6, 947–952.[CrossRef][ISI][Medline]

  11. Salomoni, P. and Pandolfi, P.P. (2002) The role of PML in tumor suppression. Cell, 108, 165–170.[CrossRef][ISI][Medline]

  12. Yeager, T.R., Neumann, A.A., Englezou, A., Huschtscha, L.I., Noble, J.R. and Reddel, R.R. (1999) Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res., 59, 4175–4179.[Abstract/Free Full Text]

  13. Henson, J.D., Neumann, A.A., Yeager, T.R. and Reddel, R.R. (2002) Alternative lengthening of telomeres in mammalian cells. Oncogene, 21, 598–610.[CrossRef][ISI][Medline]

  14. Nabetani, A., Yokoyama, O. and Ishikawa, F. (2004) Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body. J. Biol. Chem., 279, 25849–25857.[Abstract/Free Full Text]

  15. Dunham, M.A., Neumann, A.A., Fasching, C.L. and Reddel, R.R. (2000) Telomere maintenance by recombination in human cells. Nat. Genet., 26, 447–450.[CrossRef][ISI][Medline]

  16. Bechter, O.E., Zou, Y., Shay, J.W. and Wright, W.E. (2003) Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency. EMBO Rep., 4, 1138–1143.[CrossRef][ISI][Medline]

  17. Wong, Z., Wilson, V., Patel, I., Povey, S. and Jeffreys, A.J. (1987) Characterization of a panel of highly variable minisatellites cloned from human DNA. Ann. Hum. Genet., 51, 269–288.[ISI][Medline]

  18. Jeffreys, A.J., Tamaki, K., MacLeod, A., Monckton, D.G., Neil, D.L. and Armour, J.A. (1994) Complex gene conversion events in germline mutation at human minisatellites. Nat. Genet., 6, 136–145.[CrossRef][ISI][Medline]

  19. Jeffreys, A.J., Murray, J. and Neumann, R. (1998) High-resolution mapping of crossovers in human sperm defines a minisatellite-associated recombination hotspot. Mol. Cell., 2, 267–273.[CrossRef][ISI][Medline]

  20. Jeffreys, A.J. and Neumann, R. (1997) Somatic mutation processes at a human minisatellite. Hum. Mol. Genet., 6, 129–136.[Abstract/Free Full Text]

  21. Jeffreys, A.J., Tamaki, K., Macleod, A., Monckton, D.G., Neil, D.L. and Armour, J. (1994) Complex gene conversion events in germline mutation at human minisatellites. Nat. Genet., 6, 136–145.[CrossRef][ISI][Medline]

  22. Henson, J.D., Hannay, J.A., McCarthy, S.W., Royds, J.A., Yeager, T.R., Robinson, R.A., Wharton, S.B., Jellinek, D.A., Arbuckle, S.M., Yoo, J. et al. (2005) A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin. Cancer Res., 11, 217–225.[Abstract/Free Full Text]

  23. Neil, D.L. and Jeffreys, A.J. (1993) Digital DNA typing at a 2nd hypervariable locus by minisatellite variant repeat mapping. Hum. Mol. Genet., 2, 1129–1135.[Free Full Text]

  24. Buard, J. and Vergnaud, G. (1994) Complex recombination events at the hypermutable minisatellite CEB1 (D2S90). EMBO J., 13, 3203–3210.[ISI][Medline]

  25. May, C.A., Jeffreys, A.J. and Armour, J.A.L. (1996) Mutation rate heterogeneity and the generation of allele diversity at the human minisatellite MS205 (D16S309). Hum. Mol. Genet., 5, 1823–1833.[Abstract/Free Full Text]

  26. Tamaki, K., May, C.A., Dubrova, Y.E. and Jeffreys, A.J. (1999) Extremely complex repeat shuffling during germline mutation at human minisatellite B6.7. Hum. Mol. Genet., 8, 879–888.[Abstract/Free Full Text]

  27. Tamaki, K., Monckton, D.G., Macleod, A., Allen, M. and Jeffreys, A.J. (1993) 4-state MVR-PCR-increased discrimination of digital dna typing by simultaneous analysis of 2 polymorphic sites within minisatellite variant repeats at D1S8. Hum. Mol. Genet., 2, 1629–1632.[Abstract/Free Full Text]

  28. Thein, S.L., Jeffreys, A.J., Gooi, H.C., Cotter, F., Flint, J., O'Connor, N.T., Weatherall, D.J. and Wainscoat, J.S. (1987) Detection of somatic changes in human cancer DNA by DNA fingerprint analysis. Br. J. Cancer, 55, 353–356.[ISI][Medline]

  29. Armour, J.A., Patel, I., Thein, S.L., Fey, M.F. and Jeffreys, A.J. (1989) Analysis of somatic mutations at human minisatellite loci in tumors and cell lines. Genomics, 4, 328–334.[CrossRef][ISI][Medline]

  30. Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P., 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.[Abstract/Free Full Text]

  31. Tsutsui, T., Kumakura, S., Tamura, Y., Tsutsui, T.W., Sekiguchi, M., Higuchi, T. and Barrett, J.C. (2003) Immortal, telomerase-negative cell lines derived from a Li–Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities. Carcinogenesis, 24, 953–965.[Abstract/Free Full Text]

  32. Londono-Vallejo, J.A., Der-Sarkissian, H., Cazes, L., Bacchetti, S. and Reddel, R.R. (2004) Alternative lengthening of telomeres is characterized by high rates of telomeric exchange. Cancer Res., 64, 2324–2327.[Abstract/Free Full Text]

  33. Bailey, S.M., Brenneman, M.A. and Goodwin, E.H. (2004) Frequent recombination in telomeric DNA may extend the proliferative life of telomerase-negative cells. Nucleic Acids Res., 32, 3743–3751.[Abstract/Free Full Text]

  34. Royle, N.J. (1995) The proterminal regions and telomeres of human chromosomes. Adv. Genet., 32, 273–315.[ISI][Medline]

  35. Jeffreys, A.J., Royle, N.J., Wilson, V. and Wong, Z. (1988) Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature, 332, 278–281.[CrossRef][Medline]

  36. Jeffreys, A.J., Neumann, R. and Wilson, V. (1990) Repeat unit sequence variation in minisatellites—a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis. Cell, 60, 473–485.[CrossRef][ISI][Medline]

  37. Jeffreys, A.J., MacLeod, A., Tamaki, K., Neil, D.L. and Monckton, D.G. (1991) Minisatellite repeat coding as a digital approach to DNA typing. Nature, 354, 204–209.[CrossRef][Medline]

  38. Armour, J.A.L., Harris, P.C. and Jeffreys, A.J. (1993) Allelic diversity at minisatellite MS205 (D16S309): evidence for polarized variability. Hum. Mol. Genet., 2, 1137–1145.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
A. Muntoni and R. R. Reddel
The first molecular details of ALT in human tumor cells
Hum. Mol. Genet., October 15, 2005; 14(suppl_2): R191 - R196.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
14/13/1785    most recent
ddi185v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Jeyapalan, J. N.
Right arrow Articles by Royle, N. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeyapalan, J. N.
Right arrow Articles by Royle, N. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?