Clinical Chemistry 45: 1718-1724, 1999;
(Clinical Chemistry. 1999;45:1718-1724.)
© 1999 American Association for Clinical Chemistry, Inc.
Simple, Rapid, Quantitative, and Sensitive Detection of Telomere Repeats in Cell Lysate by a Hybridization Protection Assay
Yasuhiro Nakamura1,
Minoru Hirose2,
Hajime Matsuo1,
Naohiro Tsuyama1,
Keiichi Kamisango2 and
Toshinori Ide1,a
1
Department of Cellular and Molecular Biology, Hiroshima University School of Medicine, Kasumi 1-2-3, Hiroshima City, Hiroshima 734-8551, Japan.
2
Diagnostic Science Laboratories, Chugai Diagnostic
Science Company Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171-8545, Japan.
a Author for correspondence. Fax 81-82-257-5294; e-mail tide{at}pharm.hiroshima-u.ac.jp
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Abstract
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Background: Detection of telomere repeats by Southern
hybridization of genomic DNA is time consuming, and the reading of a
mean terminal restriction fragment (TRF) length from a smear pattern of
an autoradiogram can be inaccurate. We developed a hybridization
protection assay (HPA) for telomere repeats.
Methods: We heated 5 µL of DNA solution or 10 µL of cell or
tissue lysate at 95 °C for 5 min, mixed it with 100 µL of
hybridization solution containing 3 x 106 relative
light units of acridinium ester-labeled probe, and incubated the
mixture for 20 min at 60 °C. We then added 300 µL of selection
buffer and incubated the mixture for 10 min at 60 °C to
differentially hydrolyze unhybridized probe. Chemiluminescence was
measured for 2 s per tube.
Results: The amount of telomere repeats was assayed by HPA within
linearity from 10 to 3000 ng of purified genomic DNA or from 1000 to
100 000 cell equivalents of lysate. To normalize the amount of DNA in
lysate, the amount of Alu sequence was measured by HPA.
A ratio of telomere to Alu (TA ratio) = 0.01
corresponded to ~2 kbp of mean TRF length determined by Southern
blotting in cultured fibroblast and colorectal tissue samples. The TA
ratio decreased from 0.06 to 0.02 with increasing division age from 30
to 90 population doubling levels of cultured human fetal fibroblasts.
The assay required ~45 min from collection of cell or tissue samples.
Conclusions: The amount of telomere repeats was quantitatively
measured by HPA in 10 ng of sheared genomic DNA or in the lysate of
1000 cells. This method is simple, rapid, quantitative, sensitive, and
applicable to the measurement of telomere repeats in clinical samples
such as needle biopsy specimen or as few as 1000 cells in body fluid or
washings.
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Introduction
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Telomeres are a specialized structure at the end of eukaryotic
chromosomes. Telomeric DNA generally consists of a tandemly repeated
G-rich sequence oriented 5' to 3' toward the end of the telomere
repeat. Because this sequence is evolutionally conserved, with some
exceptions, from unicellular organisms such as yeast and ciliates to
mammals, the telomeric repeat sequence appears critical for telomere
function in eukaryotes (1)(2).
Telomere DNA of human somatic cells shortens at each cell division,
which determines, as a mitotic clock, a finite proliferative capacity
of human somatic cells (3)(4)(5)(6). Telomerase is required for
such cells to proliferate indefinitely, maintaining telomere length, as
the germ line and most cancer cells. A causal relationship between
telomere shortening and cellular senescence has been established by
studies that showed that transfection of the human telomerase reverse
transcriptase gene (hTERT) into various human mortal somatic
cells leads to elongation of telomere length and extension of the in
vitro replicative life span (7)(8).
To examine telomere length of human genomic DNA, a majority of reports
has applied terminal restriction fragment
(TRF)1
length estimation by Southern blotting of genomic DNA digested
with restriction enzyme. This technique has advanced our knowledge of
telomere metabolism and provided important data on the shortening of
TRFs with cellular senescence of human somatic cells (3)(4)(5)(6)
and the elongation of TRFs after introduction of telomerase gene into
telomerase negative cells (7)(8).
TRF length estimated by Southern blotting does, however, have
disadvantages: (a) TRF does not indicate pure telomere
repeat length but includes various unknown lengths of subtelomic
sequences; (b) to estimate TRF length, genomic DNA should be
purified as intact (unsheared) as possible; (c) the mean TRF
length estimated by reading smear patterns of autoradiograms may be
inaccurate; and (d) Southern blotting is time consuming.
Recently we reported the application of a hybridization protection
assay (HPA) with an acridinium ester (AE)-labeled probe to quantify
telomerase reaction products (telomere repeat DNA)
(9)(10). In the present study, we applied HPA to
measure telomere repeats in genomic DNA. This method cannot measure
telomere repeats in individual cells or chromosomes, but it has
advantages when compared with Southern blotting: it is simple, rapid,
sensitive, quantitative, and reproducible; and it measures the number
of telomere repeats, rather than TRF with subtelomic sequence, in
purified and sheared genomic DNA as well as in unpurified DNA in cell
or tissue lysate.
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Materials and Methods
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hpa
The HPA procedure for quantifying telomere repeats in this study
was based on the method described previously to measure telomerase
activity (9). Five microliters of DNA solution was heat
denatured for 5 min at 95 °C. The volume of DNA solution could be
increased up to 20 µL without affecting the result. One hundred
microliters of AE-labeled probe [5'-CCC TAA CCC TAA CCC TAA CTC TGC
TCG AC-3', where indicates the AE position; emission, 3 x
106 relative light units (rlu)] in
hybridization buffer [0.1 mol/L lithium succinate buffer, pH 4.7,
containing 200 g/L lithium lauryl sulfate, 1.2 mol/L lithium chloride,
20 mmol/L EDTA, and 20 mmol/L
ethyleneglycol-bis-(ß-aminoethylether)-N,N,N',N'-tetraacetic
acid (EGTA)] was added into each reaction tube and incubated for 20
min at 60 °C. We added 300 µL of selection buffer (0.6 mol/L
sodium tetraborate buffer, pH 8.5, containing 50 mL/L Triton X-100) to
differentially hydrolyze unhybridized probe during incubation for 10
min at 60 °C. Chemiluminescence was measured for 2 s per tube
by a luminometer (Leader 1; Gen-Probe). Alu sequence
was also measured by HPA, using a probe: 5'-TGT AAT CCC AGC ACT TTG
GGA GGC-3', where indicates the AE position.
synthetic oligodeoxyribonucleotides
Synthetic oligodeoxynucleotides, (5'-GGT
TAG-3')4G and (5'-GGT
TAG-3')20 and their opposite strands were
purchased from Sawady Technology. Longer telomere repeats, ~1 and 2
kbp length, were synthesized by PCR, using a synthetic 120-mer. For the
Alu sequence, 5'-GCC TCC CAA AGT GCT GGG ATT ACA-3' and its
opposite strand were purchased from Sawady Technology. AE labeling was
performed by us at Chugai Diagnostic Science Company, Ltd.
preparation of genomic dna and cell lysate
Genomic DNA was purified from cultured cells or resected frozen
human colorectal tissue samples as described previously
(11). Lysate of cultured cells was prepared for direct
measurement of telomere repeats as follows. Cultured cells (~5
x 106) were scraped, collected into a 1-mL tube,
and pelleted by centrifugation. The pellet was dissolved in 100 µL of
hybridization buffer (0.1 mol/L lithium succinate buffer, pH 4.7,
containing 200 g/L lithium lauryl sulfate, 1.2 mol/L lithium chloride,
20 mmol/L EDTA, and 20 mmol/L EGTA). Released genomic DNA was
extensively sheared by pipetting (50 times). The tissue sample was
powdered in liquid nitrogen, and an aliquot (2–3 mg) was dissolved in
100 µL of hybridization buffer.
southern blotting
Southern blotting to measure TRF length was performed for
HinfI-digested genomic DNA by using
32P-labeled (TTAGGG)4 probe
as described previously (11).
cell culture
Normal human fibroblasts, TIG-3 (12), were cultured and
used at different population doubling levels (PDLs) as described
(13). TIG-3 had a maximal proliferative life span of ~80
PDLs. SVts9-3 and SVts9-4 were mortal cell lines
(telomerase activity negative) with maximum PDLs of ~100–110, which
were derived from TIG-3 after transfection with SV40 tsT
antigen (13). SVts8 was a immortal cell line
derived from TIG-3 after transfection with SV40 tsT antigen
(13) and was continuously cultured at 34 °C over 400
PDLs.
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Results
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quantification of telomere repeats by hpa
We first examined basic studies to quantify synthetic
oligonucleotides of telomere repeats by HPA probe. Because it was
confirmed, as shown later, that Escherichia coli DNA did not
give any positive signal, 5 µg of E. coli DNA was added as
a carrier in each assay. As shown in Fig. 1
, the detection limit for the single-stranded 25-mer,
(GGT TAG)4G, and the 120-mer, (GGT
TAG)20, was 1 pg, and the assay was linear up to
300 pg regardless of the size of telomere repeats. The emission, in
rlu, was always higher, as confirmed by three repeated
experiments, for the same amount (in picograms) of 25-mer vs 120-mer.
This may be the because 25-mer DNA was theoretically hybridized with
one probe molecule, whereas the 120-mer DNA was hybridized with four
probe molecules, i.e., the 120-mer DNA molecule was fivefold larger (by
weight) than the 25-mer, whereas it was fourfold larger in the number
of bindable probe molecules than the 25-mer. As expected, the emission
in rlu of the double-stranded 25-mer and 120-mer was one-half of that
obtained from the same weight (picograms) of the single-stranded 25-
and 120-mers (data not shown). The discrepancy between the weight of
the telomere repeats and the hybridizable probe number (which
corresponded to rlu) was negligible when the longer telomere repeats
were assayed using longer telomere repeat DNAs. Fig. 1
shows the
results for double-stranded DNA containing telomere repeats of ~1 kbp
and 2 kbp, which were prepared by preparative PCR amplification using
120-mer double-stranded molecules.
Genomic DNA from the cultured human cell line SVts8 was
examined by AE-labeled probe (Fig. 2
). Because the size of genomic DNA might affect the results, DNA
was mechanically sheared or digested with restriction enzyme before
assay (Fig. 2A
). No significant difference was observed between genomic
DNAs of unsheared and sheared by extensive pipetting (Fig. 2B
).
HinfI-digested DNA appeared to give a slightly higher signal
(Fig. 2B
), but this was insignificant and repeated experiments gave the
same results as unsheared DNA samples. Unsheared DNA also was viscous
and inappropriate for quantitative handling; therefore, we used
mechanically sheared DNA for further analysis. We also examined the
effect of the hybridization temperature used with the AE-labeled probe.
As seen in Fig. 2B
, hybridization was better at 60 °C, which was the
optimal temperature set for the standard procedure to measure
telomerase products that were small double-stranded DNA (9).
Because 95 °C was the optimal denaturation temperature for the
established procedure for detecting telomerase products (9),
higher denaturation temperatures might be required for large genomic
DNA. However, a denaturation temperature of 100 °C was not better
than 95 °C (data not shown), and we set the denaturation temperature
at 95 °C. Telomeric repeats in human genomic DNA were
detected at concentrations of 0.01 µg or less, and the assay
was linear up to 3 µg (Fig. 3
). The emission (in rlu) of DNA from TIG-3 at 39 PDLs was always
higher than that from SVts8, which was consistent with data
that the former had longer TRFs (Fig. 4
). E. coli DNA did not give any positive signal
(Fig. 3
).
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Figure 2. Assay of telomere repeats in genomic DNA.
Genomic DNA purified from SVts8 cells was unsheared,
sheared by extensive pipetting, or digested with HinfI.
(A), size of DNA was estimated by agarose gel
electrophoresis (7 g/L agarose gel; 25 V for 20 h). Numbers on the
left are the numbers of base pairs. (B),
telomere repeats were assayed by AE-labeled probe at different
hybridization temperatures in sheared and unsheared genomic DNA. Values
are the means of two measurements.
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Figure 3. Sensitivity and linearity of HPA for telomere
repeats of genomic DNA.
Telomere repeats were measured by HPA for different amounts of purified
genomic DNA (sheared) from normal human fibroblasts (TIG-3 at 39 PDLs;
•), SV40-transformed immortal clone (SVts8 over 300
PDLs;  ), and E. coli ( ). Each point represents the
mean (bars, SE) of four to six measurements.
Mechanically sheared and HinfI-digested DNA gave the
same rlu values, which were used together to calculate the mean.
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Figure 4. Telomere shortening of cultured human fibroblasts with
division age.
Genomic DNA from normal human fibroblasts (TIG-3), an
SV40-transformed mortal clone from TIG-3 (SVts9-3), and an
SV40-transformed immortal clone (SVts8) at different PDLs
was assayed for TRF length by Southern blotting (A) and
for telomere repeats by HPA (B). For Southern blotting
(A), DNA was digested with HinfI, and 2
µg was electrophoresed (7 g/L agarose gel; 25 V for 20 h).
Numbers on the left represent the numbers of base pairs. For
the HPA assay (B), 1 µg of sheared genomic DNA was
used. •, normal TIG-3; , SV40-transformed mortal clone
(SVts9-3);  , SV40-transformed immortal clone
(SVts8). Each point represents the mean of two
measurements.
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quantification of telomere repeats in genomic dna samples
Telomere shortening was examined for normal human fibroblasts and
their SV40 transformants. As presented in Fig. 4A
, the TRF length
decreased according to cell division age in both the normal human
fibroblast cell line, TIG-3, and its SV40 transformants. The mean TRF
length by Southern blotting of normal TIG-3 cells was 8.4 kbp at 36
PDLs, 6.2 kbp at 62 PDLs, 5.9 kbp at 78 PDLs, and 5.8 kbp at 83 PDLs,
at which TIG-3 cells senesced. SVts9-3 was a
SV40-transformed mortal cell line with an extended proliferative life
span up to ~100 PDLs. The mean TRF length by Southern blotting of
SVts9-3 was 6.7 kbp at 53 PDLs, 5.6 kbp at 80 PDLs, 4.4 kbp
at 93 PDLs, and 4.3 kbp at 99 PDLs. An immortalized clone,
SVts8, proliferated over 300 PDLs and had a TRF of ~6.6
kbp. The TRF of immortal SVts8 did not change with
division number. Corresponding to the above data, a decrease in the
amount of telomere repeats was clearly detected by HPA according to
cell division age in both normal and SV40-transformed cells (Fig. 4B
).
Genomic DNA from colorectal tissue samples was also examined. Fig. 5
A presents several examples of TRF length of genomic DNA from
colorectal tissues of either tumor (T) or non-tumor (NT) tissue.
Colorectal DNAs showed a rough correlation (correlation
coefficient = 0.62) between emission in rlu by HPA and mean TRF
length by Southern blotting except for several points, which were high
by HPA and low by Southern blotting (Fig. 5B
). These samples out of
correlation were degraded DNA, as confirmed by gel electrophoresis,
probably attributable to shearing during the purification process of
genomic DNA. The open circles in Fig. 5B
were results from TIG-3 and
its SV40 transformants at different PDLs, which also showed a rough
correlation between rlu and TRF length.
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Figure 5. TRF length by Southern blotting and telomere repeat by HPA
in genomic DNA from colorectal tissue samples.
(A), TRF length by Southern blotting. DNA was digested
with HinfI, and 2 µg was electrophoresed (7 g/L
agarose gel; 25 V for 20 h) for Southern blotting.
T and N are tumor and non-tumor sample
pairs, respectively, from the same patient. Numbers on left
represent the number of base pairs. (B), relationship
between telomere repeats measured by HPA and TRF length measured by
Southern blotting in genomic DNA from colorectal tissues (•) and
cultured fibroblasts (). For HPA, 1 µg of sheared DNA was used for
each assay.
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quantification of telomere repeats in cell and tissue lysates
For direct assay of telomere repeats by HPA, the cell pellet was
lysed with dilution buffer for the AE-labeled probe, which contained a
detergent, lithium lauryl sulfate, and high salt (see Materials
and Methods) and could lyse cells and tissues. Released DNA was
sheared by extensive pipetting. This lysate was used directly for assay
of telomere repeats. The Alu sequence determined by
Alu-specific AE-labeled probe could be used to normalize
amount of DNA (Fig. 6
A) because the amount of Alu sequence per genomic DNA
was constant in both cultured cells and colorectal tissues in all
samples we examined (data not shown). In Fig. 6B
, the
Alu sequence and telomere repeats were assayed in serially
diluted cell lysate. Telomere repeats in lysate from 101
cells could be detected. Because cell lysate contained mRNA that had
Alu sequences, the emission in rlu of the Alu
sequence in cell lysate might be higher than that in purified genomic
DNA. If so, the ratio of the emission of the telomere repeats to the
emission of the Alu repeats was lower in cell lysate than
that in purified genomic DNA. However, the ratio of emission of the
telomere repeats to that of the Alu sequence was ~0.03 in
lysate from SVts8 cells (Fig. 7
A, ). This ratio was almost equal within experimental error
to that obtained in purified genomic DNA from SVts8 cells
(Fig. 6B
). This indicates that the presence of mRNA did not alter the
rlu value of the Alu sequence. Fig. 7A
shows the rlu ratio
of telomere repeats to Alu repeats determined by HPA in cell
lysate of TIG-3 and its transformant cell lines. The rlu ratio
decreased according to cell division age. These data do not show
changes in telomere repeat length at the individual chromosome level
directly but gave information of telomere reduction according to cell
division age, as shown in Fig. 4
. Fig. 7B
shows three samples of
colorectal tissue in which the relative amount of telomere repeats
determined by HPA (rlu ratio, 0.042 for sample a, 0.018 for sample b,
0.048 for sample c) was well correlated with mean the TRF length by
Southern blotting (8.2 kbp for sample a, 4.1 kbp for sample b, 8.8 kbp
for sample c). Data from cultured fibroblasts and from colorectal
tissue samples showed that a ratio 0.01 for the emission (in rlu) of
telomere repeats to that of Alu repeats corresponded to ~2
kbp of mean TRF length measured by Southern blotting under our assay
conditions.
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Figure 6. Measurement of telomere and Alu sequences
by HPA.
(A), Alu sequence was measured by HPA in
purified genomic DNA from TIG-3 at 36 PDLs. (B),
Alu and telomere sequences were measured by HPA in
serially diluted lysates corresponding to the different number of
SVts8 cells. , Alu sequence; •,
telomere repeats. Each point represents the mean (bars,
SE) of four to six measurements.
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Figure 7. Amount of telomere repeats in extracts from cells and
tissues.
(A), lysates from 1 x 104, 3 x
104, and 1 x 105 cells were used, each in
triplicate, for HPA assay of telomere repeats, and lysates from 1
x 103, 3 x 103, and 1 x
104 cells were used, each in triplicate, for HPA assay of
Alu repeats. Because these values were within the linear
range of the assay, all values were normalized to 1 x
104 cells for calculation of the rlu ratio.
(B), lysates from three colorectal tissue samples
(a and b, colorectal carcinoma;
c, healthy colorectal tissue) were serially
diluted and assayed in triplicate for telomere repeats and for
Alu sequence by HPA. The TRF lengths by Southern
blotting of genomic DNA from these samples were 8.2 kbp for
a, 4.1 kbp for b, and 8.8 kbp for
c. Bars, SE.
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The HPA assay required ~45 min from collection of cell or tissue
samples. This procedure was applicable to biopsied or resected human
soft tissue samples for clinical examinations.
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Discussion
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Southern blotting has been widely used to measure mean TRF length.
This method is effective for obtaining a rough idea of mean TRF length
and the distribution pattern of TRF length among cell populations.
However, TRF length measurement by Southern blotting has several
intrinsic problems. Because TRF includes various unknown lengths of
subtelomic sequences, the pure telomere repeat length is not known. In
addition, the mean TRF of genomic DNA does not give information at the
individual cell or individual chromosome level. TRF length measurement
by Southern blotting has additional technical problems: (a)
Genomic DNA must be purified as intact (unsheared) as possible from
tissue and cells. (b) It is sometimes difficult to handle
viscous solutions containing high-molecular weight genomic DNA.
(c) It is a time-consuming procedure, involving genomic DNA
purification, digestion with restriction enzymes, gel electrophoresis,
blotting, hybridization, and autoradiography. (d)
Measurement of the mean TRF length by quantitative densitometry of the
smear pattern of an autoradiogram may not be accurate.
The HPA method, presented here to measure the amount of telomere
repeats, still has the disadvantage of not giving information at the
individual cell or individual chromosome level. Moreover, neither the
mean TRF size nor variation of TRF in the given cell population was
directly measured by this method. However, information could be
obtained by HPA on telomere shortening with increasing division age of
cultured fibroblasts and on variations of the amount of telomere among
different tissue samples. Results obtained by HPA showed a good
agreement with the results of mean TRF length measured by Southern
blotting. In addition, the HPA method has great advantages compared
with Southern blotting: (a) The amount of telomere repeats
measured by HPA does not include subtelomic sequences. (b)
Intact (unsheared) genomic DNA is not required; rather, sheared DNA is
recommended. (c) The HPA method is easy to handle, simple,
rapid, sensitive, and quantitative. (d) The amount of
telomere repeats is measured directly in cell and tissue lysates.
Direct assay of cell lysate requires the normalization of amounts of
DNA, which was achieved through calculating the rlu ratio of telomere
repeats to Alu sequence. Data from cultured fibroblasts and
from colorectal tissue samples showed that a rlu ratio of 0.01
corresponded to ~2 kbp of mean TRF length under our assay conditions.
The HPA procedure is convenient for clinical examination of telomere
repeats in a small number of cells in washings or body fluid or in a
small portion of human tissue obtained by endoscopy or needle-biopsy.
Other methods have been published to measure telomere repeat size, and
each method has its own advantages and disadvantages. Telomere staining
by fluorescence in situ hybridization with telomere peptide
nucleic acid-probe has the advantage of giving the amount of telomere
repeats at each chromosome end (14) but has the disadvantage
of requiring a complicated process with special equipment for
quantitative measurement; it also is hard to examine a large number of
cells and tissues with this method. Southern hybridization of DNA
trapped on a filter by slot-blot makes it possible to estimate telomere
amounts from whole cells, using standardization by the ratio of
telomere to centromere DNA content (15). This method does
not require purification of DNA, but it is time consuming for Southern
hybridization and is less accurate for quantification of the slot-blot
pattern. Flow cytometry of cells stained with fluorescent telomere
probes reveals the amount of telomere repeats at the individual cell
level with large number of cells, but it requires special equipment and
is not suitable for solid tissues (16). It is good to have
several different techniques to measure telomere repeats for various
purposes, and HPA can be an additional method. Researchers in various
fields can select the appropriate method according to their own
experimental and clinical purposes.
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Acknowledgments
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This work was supported, in part, by a grant-in-aid for
scientific research on priority areas (Cancer Research) from the
Ministry of Education, Science, Sports, and Culture of Japan and
grants-in-aid for scientific research from the Ministry of Education,
Science, Sports, and Culture of Japan.
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Footnotes
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1 Nonstandard abbreviations: TRF, terminal restriction fragment; HPA, hybridization protection assay; AE, acridinium ester; EGTA, ethyleneglycol-bis-(ß-aminoethylether)-N,N,N',N'-tetraacetic acid; rlu, relative light unit(s); and PDL, population doubling level. 
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The following articles in journals at HighWire Press have cited this article:
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R. M. Cawthon
Telomere measurement by quantitative PCR
Nucleic Acids Res.,
May 15, 2002;
30(10):
e47 - e47.
[Abstract]
[Full Text]
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Abstract
Introduction
