Telomere-dependent and telomere-independent origins of endogenous DNA damage in tumor cells
Abstract
Human tumors and cultured cells contain elevated levels of endogenous DNA damage resulting from telomere dysfunction, replication and transcription errors, reactive oxygen species, and genome instability. However, the contribution of telomere-associated versus telomere-independent endogenous DNA lesions to this damage has never been examined. In this study, we characterized the relative amounts of these two types of DNA damage in five tumor cell lines by noting whether γ-H2AX foci, generally considered to mark DNA double-strand breaks (DSBs), were on chromosome arms or at chromosome ends. We found that while the numbers of non-telomeric DSBs were remarkably similar in these cultures, considerable variation was detected in the level of telomeric damage. The distinct heterogeneity in the numbers of γ-H2AX foci in these tumor cell lines was found to be due to foci associated with uncapped telomeres, and the amount of total telomeric damage also appeared to inversely correlate with the telomerase activity present in these cells. These results indicate that damaged telomeres are the major factor accounting for the variability in the amount of DNA DSB damage in tumor cells. This characterization of DNA damage in tumor cells helps clarify the contribution of non-telomeric DSBs and damaged telomeres to major genomic alterations.
Introduction
Accumulation
of DNA damage is a hallmark of genome instability and is associated with both
aging and cancer [1-3]. Mice
deficient in proteins involved in DNA damage sensing and repair exhibit severe
deficiencies in these pathways leading to accelerated aging and oncogenic
transformation [4]. Many
progeria (premature aging) syndromes in humans are caused by mutations in
genes encoding proteins involved in DNA repair
and are associated with increased incidence of cancer [5,6].
One
major type of DNA lesion leading genomic instability is DNA double-strand
damage, which includes both telomere-independent DNA double-stand breaks (DSBs) and damaged telomeres (see schematic
in Figure 1). Telomere-independent DSBs, which localize at chromosome arms, can
be induced by a variety of agents including ionizing radiation, radiomimetic drugs, reactive oxygen
species, metabolic errors during replication and transcription, and deficient
DNA repair [7]. Telomeric damage is chromosome end-specific and includes two
types of lesions, DNA double-strand ends which are the consequence of telomere
dysfunction, and DNA DSBs at telomeres.
Figure 1. Types of endogenous DNA double-strand damage marked by γ-H2AX foci.
The endogenous DNA double-strand damage that induces H2AX phosphorylation includes both non-telomeric
DNA double-stand breaks (DSBs) located at chromatid arms and damaged telomeres.
Telomeric damage is a chromosome end-specific damage which includes two types of lesions:
1) DNA double-strand ends which are generally the consequence of telomere dysfunction,
though this type of damage can be also present at long telomeres when the telomere loop is open, and
2) DNA DSBs at telomeres.
Immediately
upon DNA double-strand damage formation, hundreds of histone H2AX molecules are
phosphorylated at the break site to form γ-H2AX foci. This
characteristic makes γ-H2AX foci a sensitive marker for DSB damage. An
important finding, made possible by the use of antibodies to γ-H2AX, is that cells that have not been subjected to deliberate damage
still contain endogenous DSB damage. This endogenous DNA DSB damage is present
at low levels in early passage primary cells, but it increases in human and
mouse cells during in vivo aging and in vitro cellular senescence
[3,8,9]. Increased and variable levels of DNA DSBs have also been found in
premalignant lesions, tumor cell lines and tumors of different origins [2,10-13]. The
endogenous γ-H2AX foci contain DNA DSB repair factors such as
53BP1, MRE11, RAD50, and NBS1, indicating that DNA DSB repair is being
attempted at these sites [3,9].
The existence of non-telomeric DNA DSBs and telomeres-associated
endogenous DNA double-strand damage creates confusion about which type of
damage is present. The confusion can be clarified by determining the location
of the γ-H2AX foci on the chromosomes. When this
type of analysis was performed on human and mouse senescent cells, both were
found to contain similar levels of total endogenous DNA DSB damage, but
differing contributions from non-telomeric DSBs and damaged telomeres. This
comparison of human and mouse cells suggested that both telomere-independent
and telomere-associated damage may be similarly involved in the signaling to
induce cellular senescence and organismal aging [14].
In
the present study we performed this analysis on five tumor cell lines to
clarify the relative contribution of telomeric damage to the high level of
endogenous DNA damage in tumors. We report that the numbers of non-telomeric
DNA DSBs, as measured by γ-H2AX foci present at chromosome arms, were
remarkably similar across all cultures studied. However, the numbers of
γ-H2AX foci associated with telomeres varied considerably and correlated
inversely with telomerase activity. These results indicate that human tumor
cells contain substantial and variable numbers of dysfunctional telomeres,
which account for most of the variation in the number of γ-H2AX foci in
different human tumor lines.
Results
Distribution
of γ-H2AX foci in proliferating tumor cell cultures
In contrast to senescent cells, which
contain similar numbers of endogenous γ-H2AX foci
irrespective of origin [14], malignant
cells have higher DSB levels which vary greatly in different cultures and
tumors [10,12,13]. We
performed parallel analyses of γ-H2AX foci in undamaged cultures of five
tumor cell lines of different origins, HeLa, SiHa, and SW756 (cervical
carcinomas); HCT116 (colon carcinoma) and M059K (glioblastoma) (Figure 2).
Endogenous γ-H2AX levels in these cultures have been shown earlier
to vary widely, from an average of 1.1 γ-H2AX foci per cell
in M059K cells to as high as 46 foci per cell in SW756 cells [10,13].
Additionally, comparison of DNA damage in 6 intact cervical carcinoma cell
lines showed great variability in γ-H2AX focal numbers,
indicating that endogenous DNA damage is independent of tumor origin [10]. In this
study we counted γ-H2AX foci in interphase in large cell populations
(400 - 600 cells) of the five lines, and found an average of 6.6 -10.6 foci per cell (Figure 2A, B). Cultures of the same tumor line yielded average
numbers of γ-H2AX foci per cell that varied by over two-fold in
three independent experiments, indicating that focal numbers are dependent on
culture conditions (Figure 2B). In addition, in these three experiments, the
standard deviations were often larger than the average values for the number of
γ-H2AX foci per cell, indicating a large amount of
heterogeneity in the population. The cause of these large standard deviations
may be explained by data shown in Figure 2C. In each tumor line, while the
majority of the cells contained less than 10 foci per cell, there was a substantial
fraction of cells that contained larger numbers of γ-H2AX foci, up to about 50 per cell, creating a long tail in the
distribution and leading to large standard deviations from the average.
Figure 2. Endogenous γ-H2AX foci in interphase cells of five human tumor cell lines. (A) Images of endogenous γ-H2AX foci (green) in untreated HeLa, HCT116,
M059K, SiHa and SW756 cells. DAPI staining (blue) indicates DNA. (B)
Average numbers of γ-H2AX foci per cell in three
independent experiments (Expt 1-3) with high SDs (n is at least 70 cells
counted in each experiment), and average of averages from these experiments
(n=3). (C) Fractions of cells in the five tumor cell populations
with the noted numbers of γ-H2AX
foci.
The
counts we present here are different from published data for these cell lines.
We account for this discrepancy by possible bias caused by a great disparity in
the number of γ-H2AX foci in a cell population, in the focal sizes
and intensities (Figure 2A), and by variations in the cells' proliferative
status, as well as their checkpoint status and expression of p53 or other
proteins involved in genomic stability that could have changed due to genetic
drift over time. Therefore, since counting γ-H2AX foci in interphase tumor cells can provide
only limited information, studies in metaphase cells were performed to
allow visualization of truly informative foci by avoiding at least some of
these problems, such as proliferative status and focal variability.
Origins
of endogenous γ-H2AX foci in metaphase tumor cells
Yu
et al. reported that tumor cell cultures exhibited large numbers of endogenous
γ-H2AX foci per cell, sometimes equivalent to several Gy of ionizing
radiation. Strikingly however, they found no difference in tail moments when these cultures were identically irradiated
and the cells were analyzed by the comet assay [12]. This
discrepancy suggests the hypothesis that a substantial fraction of the
endogenous γ-H2AX foci might be marking uncapped telomeres rather
than DSBs. Since the damage is at the end of the DNA, the comet or any other
DNA fragmentation assay would not detect it. To examine this notion, we
analyzed metaphases of five tumor cell lines for γ-H2AX and
telomeric DNA FISH signals to score the numbers of telomere-associated and
telomere-independent γ-H2AX foci
(Figure 3). This procedure permits the localization of γ-H2AX foci to either the chromatid arms, corresponding to DNA DSBs of
non-telomeric origin, or to the ends of the chromatids, corresponding to either
DSB-damaged telomeres (FISH-positive terminal foci), or double-strand ends at
critically short telomeres lacking detectable telomere repeats (FISH-negative
foci) (Figure 3A, B).
Figure 3. Distribution of γ-H2AX foci on metaphases of human tumor cells. (A) Metaphase spread of HCT116 cells stained for γ-H2AX (green) and telomeric DNA (red). (B)
Scoring of γ-H2AX foci as along chromatid
arms (Arms) or on chromatid ends (Ends). (C) The numbers of γ-H2AX foci in metaphases from five tumor cell
lines as noted. Foci are noted as non-telomeric (Arms, gray), telomeric
(Ends, blue), and total (black). (D) Telomeric γ-H2AX foci with (yellow) and without (green)
telomere FISH signal in the five tumor lines. At least 10 metaphases were
screened per data point in independent experiments. Error bars signify
standard errors. (E) Numbers of telomeric (open circles) and
non-telomeric (cross hatches) foci vs. the total numbers of γ-H2AX foci on the metaphase spreads of the five
tumor cell lines. The data from all five tumor cell lines was pooled for
this analysis. (F) Numbers of FISH negative (open squares) and FISH
positive telomeric (filled triangle) γ-H2AX
foci vs. total telomeric foci in all checked metaphases of the five tumor
cell lines. (G) Reverse correlation of the numbers of γ-H2AX foci and telomerase activity in the five
tumor cell lines. TPG is a total product generated corresponding to 600
molecules of telomerase substrate primers extended with at least four
telomere repeats [28].
When
the distribution of γ-H2AX foci on metaphase spreads was analyzed, the
total numbers per cell varied similarly to the average number of foci found in
the interphase nuclei (Figure 3C, black bars). Strikingly, the numbers of γ-H2AX foci along the chromosome arms were found to be similar in all
cell lines (Figure 3C, gray bars). In fact, in four of the cell lines the
numbers were the same within the standard error, with an average of 2.6 foci
per cell. Only HCT116 exhibited a different number of γ-H2AX foci on chromatid arms, 4.7 per cell. These results suggest that
the number of DNA DSBs may have fairly constant values among tumor lines. In
contrast, the numbers of telomeric γ-H2AX foci were more variable among the five
lines (Figure 3C, blue bars), suggesting that the differences in endogenous γ-H2AX focal numbers are primarily due to variations
in the number of damaged telomeres. When the
damaged telomeres containing γ-H2AX foci were classified
as to whether they were FISH positive or negative, the majority were found to
be FISH negative, confirming that telomeres were critically short (Figure 3D).
We next analyzed the metaphase spread data to discern the
distribution of telomeric and non-telomeric γ-H2AX foci in the cells with increasing numbers of total
foci (Figure 3E). This analysis demonstrates that in cells that contain more
than the average number of γ-H2AX foci, the increase is almost completely due to telomeric foci.
This result indicates that tumor cells maintain a fairy constant level of
non-telomeric DNA DSBs irrespective of the total DNA damage, and it is damaged
telomeres that become more plentiful in these cells. Similar analysis of the distribution of FISH-negative and FISH-positive
telomeric γ-H2AX foci
indicates that among the total telomeric foci per metaphase, critically short
telomeres account for disparities (Figure 3F).
A defining characteristic of cancer cells is the presence
of telomerase, which permits these cells to divide indefinitely [15,16]. Since telomeres are maintained
by telomerase, which catalyzes the addition of telomeric DNA repeats to the
chromosome ends [17,18], we asked whether the average
telomerase activity correlated with the average numbers of γ-H2AX foci in the five studied
tumor lines. We found an inverse relationship between the numbers of γ-H2AX foci and telomerase activity
(Figure 3G). These results indicate that the level of telomerase in a tumor
cell line is a major determinant of the average number of γ-H2AX foci.
Discussion
The
purpose of this study was to determine how much of the DNA double-strand damage
in tumor cells is actually due to damaged telomeres. The results clearly show
that damaged telomeres make up the majority of DNA double-strand damage in
tumor cells, and that cells with more foci contained more damaged telomeres,
while the numbers of telomere-independent DSBs remained fairly constant
throughout the population. The numbers of endogenous telomeric γ-H2AX foci in metaphases correlated inversely with telomerase activity
in these cell lines, confirming the importance of telomerase in malignant
phenotypes. These data parallel our recently published findings for senescent
cells which also contain elevated γ-H2AX foci compared
to actively growing low population doubling cultures, which in humans have
mainly telomere-associated origins [14]. Telomere
shortening and consequent telomere dysfunction or uncapping are associated with
many human diseases including aging and cancer, and have received a great deal
of attention (reviewed in [19,20]).
Genomic alterations observed in human cancers can be caused by inappropriate
DNA repair taking place at dysfunctional telomeres leading to loss of
heterozygosity, chromosomal rearrangements, aneuploidy, and repression of DNA
damage checkpoints [21]. Shorter
telomeres have been associated with increased cancer risk [22]. Differences
in telomere-associated DNA damage in different tumor cell lines can be
explained partly by the fact that these cell lines have been derived from
different individuals, thus telomere lengths are affected by the cellular
activity of telomerase, the cells' history of cell division and environmental
factors. Additionally, as the tumor lines were isolated many years ago, they
may have changed due to genetic drift. Finally, telomere length is
tissue-specific, and age-dependent [23,24], and
there is considerable heterogeneity between humans [25].
Telomerase expression is one of the most
clearly distinguishable characteristics between malignant and primary healthy
cells [15] which makes
it a suitable target for cancer therapy. Inhibiting telomerase activity in
tumor cells may increase the number of damaged telomeres and thereby limit
proliferation. Many telomerase inhibitors are now going through clinical trials
[26]. However,
previously there was no tool to analyze whether tumors show different
sensitivity for telomerase inhibitors and to control this sensitivity. Here we
show that each tumor cell line has a signature amount of telomere-associated
DNA damage. Therefore, telomerase inhibitors or telomere maintenance-targeting
drugs could affect different tumors with differing success, and analysis of telomere-associated
γ-H2AX focal numbers in primary tumors treated with telomerase-based drugs
could be used to monitor the drug efficiency. In addition, many cancer drugs
act by introducing sufficient excess DNA damage into a tumor cell to prevent further
proliferation. The procedure presented here enables researchers to determine
the extent of the two types of DNA double-strand damage, both of which are
relevant to cancer treatment, and provides useful information for developing
tailor-made cancer therapy.
Methods
Cell cultures.
HeLa, SiHa and SW756 (cervical carcinomas), HCT116
(colon carcinoma), and M059K (glioblastoma) cell lines were obtained from ATCC
(Manassas, VA) and grown in D-MEM medium containing 10% fetal bovine serum.
Cells were maintained in a humidified incubator at 37ºC, 5% CO2
and 20% O2.
Immunocytochemistry.
Cell cultures were plated on Labtek II slides (Nalge
Nunc International, Naperville, IL). After the cultures reached 80% confluency,
they were fixed with 2% paraformaldehyde for 20 min. Then the cells were washed
4 times with PBS, permeabilized with pre-chilled 70% ethanol at -20ºC and
stored overnight at 4ºC. PBS was replaced with PBS containing 0.5%
Tween-20 and 0.1% Triton X-100 (Bio-Rad Laboratories, Hercules, CA) for
blocking and antibody incubations. The samples were stained with primary mouse
monoclonal anti-γ-H2AX antibody (Abcam Inc., Cambridge, MA) followed by
secondary Alexa-488-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR).
Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole-dihydrochroride).
Images were acquired with the BD Pathway Bioimager and processed with
Attovision software (Becton Dickinson Biosciences, San LoseJose, CA). γ-H2AX foci were counted by eye in three independent
experiments, in a total of 400-600 cell nuclei.
Immunocytochemistry
and FISH.
Metaphase spreads were
prepared as described previously [27]. The slides
were stained with mouse monoclonal anti-γ-H2AX antibody
followed by Alexa-488-conjugated anti-mouse IgG. The staining with both γ-H2AX and telomere FISH was performed according to the telomere FISH
kit (DakoCytomation, Glostrup, Denmark) protocol with some modifications.
Briefly, the γ-H2AX stained cells were fixed with 50 mM ethylene
glycol-bis (succinic acid N-hydroxy-succinimide ester) (Sigma, St. Louis, MO). The hybridization was performed according to the kit protocol. DAPI was
used for visualization of DNA. The signal was detected with Olympus fluorescent
microscope (Olympus America Inc. Melville, NY).
Telomerase
assay
. Telomerase activity in tumor cell lines was analyzed
using the TRAPeze Telomerase Detection Kit (Chemicon International a division
of Serologicals Co., Temecula, CA). Cell extracts, prepared according to the
manufacturer's instructions, were assayed for telomerase activity in 50 μL
reactions provided with the TRAPeze Telomerase Detection Kit with the exception
of Platinum Taq DNA polymerase (Invitrogen, Eugene, OR). The reaction mixtures
were size-fractionated by electrophoresis in a 10% non-denaturating
polyacrylamide gel and stained with SYBR Green 1 dye (Sigma). The gels were
photographed using the Typhoon 8600 system (Amersham Pharmacia Biotechnology, Piscataway, NJ).
Acknowledgments
We
thank Jennifer Dickey, NCI, for critical reading of the manuscript. This work
was funded by the Intramural Research Program of the National Cancer Institute,
Center for Cancer Research, NIH.
Conflicts of Interest
The
authors in this manuscript have no conflict of interests to declare.
References
-
1.
Friedberg
EC
How nucleotide excision repair protects against cancer.
Nat Rev Cancer.
2001;
1:
22
-33.
[PubMed]
.
-
2.
Bartkova
J
, Horejsi
Z
, Koed
K
, Kramer
A
, Tort
F
, Zieger
K
, Guldberg
P
, Sehested
M
, Nesland
JM
, Lukas
C
, Orntoft
T
, Lukas
J
and Bartek
J.
DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.
Nature.
2005;
434:
864
-870.
[PubMed]
.
-
3.
Sedelnikova
OA
, Horikawa
I
, Redon
C
, Nakamura
A
, Zimonjic
DB
, Popescu
NC
and Bonner
WM.
Delayed kinetics of DNA double-strand break processing in normal and pathological aging.
Aging Cell.
2008;
7:
89
-100.
[PubMed]
.
-
4.
Finkel
T
, Serrano
M
and Blasco
MA.
The common biology of cancer and ageing.
Nature.
2007;
448:
767
-774.
[PubMed]
.
-
5.
Hickson
ID
RecQ helicases: caretakers of the genome.
Nat Rev Cancer.
2003;
3:
169
-178.
[PubMed]
.
-
6.
Karanjawala
ZE
and Lieber
MR.
DNA damage and aging.
Mech Ageing Dev.
2004;
125:
405
-416.
[PubMed]
.
-
7.
Bonner
WM
, Redon
CE
, Dickey
JS
, Nakamura
AJ
, Sedelnikova
OA
, Solier
S
and Pommier
Y.
gammaH2AX and cancer.
Nat Rev Cancer.
2008;
8:
957
-967.
[PubMed]
.
-
8.
d'Adda
di Fagagna F
, Reaper
PM
, Clay-Farrace
L
, Fiegler
H
, Carr
P
, Von
Zglinicki T
, Saretzki
G
, Carter
NP
and Jackson
SP.
A DNA damage checkpoint response in telomere-initiated senescence.
Nature.
2003;
426:
194
-198.
[PubMed]
.
-
9.
Sedelnikova
OA
, Horikawa
I
, Zimonjic
DB
, Popescu
NC
, Bonner
WM
and Barrett
JC.
Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks.
Nat Cell Biol.
2004;
6:
168
-170.
[PubMed]
.
-
10.
Banath
JP
, Macphail
SH
and Olive
PL.
Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.
Cancer Res.
2004;
64:
7144
-7149.
[PubMed]
.
-
11.
Gorgoulis
VG
, Vassiliou
LV
, Karakaidos
P
, Zacharatos
P
, Kotsinas
A
, Liloglou
T
, Venere
M
, Ditullio
RA Jr
, Kastrinakis
NG
, Levy
B
, Kletsas
D
, Yoneta
A
, Herlyn
M
, Kittas
C
and Halazonetis
TD.
Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.
Nature.
2005;
434:
907
-913.
[PubMed]
.
-
12.
Yu
T
, MacPhail
SH
, Banath
JP
, Klokov
D
and Olive
PL.
Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability.
DNA Repair (Amst).
2006;
5:
935
-946.
[PubMed]
.
-
13.
Sedelnikova
OA
and Bonner
WM.
GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence.
Cell Cycle.
2006;
5:
2909
-2913.
[PubMed]
.
-
14.
Nakamura
AJ
, Chiang
YJ
, Hathcock
KS
, Horikawa
I
, Sedelnikova
OA
, Hodes
RJ
and Bonner
WM.
Both telomeric and non-telomeric DNA damage are determinants of mammalian cellular senescence.
Epigenetics Chromatin.
2008;
1:
6
[PubMed]
.
-
15.
Prescott
JC
and Blackburn
EH.
Telomerase: Dr Jekyll or Mr Hyde.
Curr Opin Genet Dev.
1999;
9:
368
-373.
[PubMed]
.
-
16.
Blagosklonny
MV
Cell immortality and hallmarks of cancer.
Cell Cycle.
2003;
2:
296
-299.
[PubMed]
.
-
17.
Greider
CW
and Blackburn
EH.
Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.
Cell.
1985;
43:
405
-413.
[PubMed]
.
-
18.
Nakamura
TM
, Morin
GB
, Chapman
KB
, Weinrich
SL
, Andrews
WH
, Lingner
J
, Harley
CB
and Cech
TR.
Telomerase catalytic subunit homologs from fission yeast and human.
Science.
1997;
277:
955
-959.
[PubMed]
.
-
19.
Garcia
CK
, Wright
WE
and Shay
JW.
Human diseases of telomerase dysfunction: insights into tissue aging.
Nucleic Acids Res.
2007;
35:
7406
-7416.
[PubMed]
.
-
20.
Campisi
J
, Kim
SH
, Lim
CS
and Rubio
M.
Cellular senescence, cancer and aging: the telomere connection.
Exp Gerontol.
2001;
36:
1619
-1637.
[PubMed]
.
-
21.
De
Lange T
Telomere-related genome instability in cancer.
Cold Spring Harb Symp Quant Biol.
2005;
70:
197
-204.
[PubMed]
.
-
22.
Risques
RA
, Vaughan
TL
, Li
X
, Odze
RD
, Blount
PL
, Ayub
K
, Gallaher
JL
, Reid
BJ
and Rabinovitch
PS.
Leukocyte telomere length predicts cancer risk in Barrett's esophagus.
Cancer Epidemiol Biomarkers Prev.
2007;
16:
2649
-2655.
[PubMed]
.
-
23.
Hastie
ND
, Dempster
M
, Dunlop
MG
, Thompson
AM
, Green
DK
and Allshire
RC.
Telomere reduction in human colorectal carcinoma and with ageing.
Nature.
1990;
346:
866
-868.
[PubMed]
.
-
24.
Lindsey
J
, McGill
NI
, Lindsey
LA
, Green
DK
and Cooke
HJ.
In vivo loss of telomeric repeats with age in humans.
Mutat Res.
1991;
256:
45
-48.
[PubMed]
.
-
25.
Risques
RA
, Lai
LA
, Brentnall
TA
, Li
L
, Feng
Z
, Gallaher
J
, Mandelson
MT
, Potter
JD
, Bronner
MP
and Rabinovitch
PS.
Ulcerative colitis is a disease of accelerated colon aging: evidence from telomere attrition and DNA damage.
Gastroenterology.
2008;
135:
410
-418.
[PubMed]
.
-
26.
Harley
CB
Telomerase and cancer therapeutics.
Nat Rev Cancer.
2008;
8:
167
-179.
[PubMed]
.
-
27.
Nakamura
A
, Sedelnikova
OA
, Redon
C
, Pilch
DR
, Sinogeeva
NI
, Shroff
R
, Lichten
M
and Bonner
WM.
Techniques for gamma-H2AX detection.
Methods Enzymol.
2006;
409:
236
-250.
[PubMed]
.
-
28.
Kim
NW
and Wu
F.
Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP).
Nucleic Acids Res.
1997;
25:
2595
-2597.
[PubMed]
.