From the Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854
Received for publication, December 18, 2002, and in revised form, January 20, 2003
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ABSTRACT |
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Single-stranded DNA has been speculated to be the
initial signal in the DNA damage signaling pathway. We showed that
introduction of single-stranded DNA with diverse sequences into
mammalian cells induced DNA damage as well as apoptosis signals. Like
DNA damaging agents, single-stranded DNA up-regulated p53 and activated
the nuclear kinase ataxia telangiectasia mutant (ATM) as evidenced by
phosphorylation of histone 2AX, an endogenous ATM substrate. Single-stranded DNA also triggered apoptosis as evidenced by the formation of caspase-dependent chromosomal DNA strand
breaks, cytochrome c release, and increase in reactive
oxygen species production. Moreover, single-stranded DNA-induced
apoptosis was reduced significantly in p53 null cells and in cells
treated with ATM small interfering RNA. These results suggest
that single-stranded DNA may act upstream of ATM/p53 in DNA damage signaling.
DNA damage plays an important role in neuron degeneration,
carcinogenesis, aging, as well as cell killing by chemotherapeutic agents (1-3). At the cellular level, DNA damage induces complicated cellular responses including checkpoint activation and apoptotic cell
death (4, 5). Ataxia telangiectasia mutant
(ATM)1 and p53 are two key
regulatory molecules in DNA damage signaling and apoptosis. The tumor
suppressor p53 is known to mediate G1 cell cycle arrest and
apoptosis (6). p53, which is a transcription activator, is up-regulated
during DNA damage and transactivates a number of genes such as
gadd45, p21, and bax involved
in cell cycle arrest and apoptosis (7, 8). While multiple mechanisms, including modification of p53 and Mdm2, are involved in up-regulation of p53 upon DNA damage (9, 10), the immediate signal following DNA
damage remains unclear.
The nuclear kinase, ATM, and its related kinase ATR are critically
involved in integrating the initial DNA damage signals to cell cycle
checkpoints and apoptosis (1, 2). Activation of ATM/ATR by DNA damage
has been widely demonstrated (1, 11). For example, ionizing radiation
and etoposide have been shown to induce ATM-dependent
apoptosis (12), while camptothecin and UV have been shown to
induce ATR-dependent apoptosis (13, 14). Oxidative
stress-induced neuron degeneration has also been shown to depend on ATM
(15). It is generally believed that ATM mediates a signal from DNA
double-strand breaks, whereas ATR mediates a signal from replication
fork arrest (1, 16).
Activation of the ATM kinase, which is evidenced by phosphorylation of
the ATM target proteins, has been demonstrated in cells treated with
DNA damage agents, especially those that produce DNA double-strand
breaks (17, 18). ATM phosphorylates a large number of proteins in
response to DNA damage, e.g. Ser-139 of histone 2AX (H2AX)
(19), Thr-68 of Chk2 (20), and Ser-15 of p53 (21-23). While ATM kinase
is considered to be one of the earliest regulatory molecules in DNA
damage signaling, the initial DNA damage signal that triggers ATM
activation remains unclear.
Single-stranded DNA, which activates the RecA protease activity toward
LexA, is known to be the initial signal for activating the SOS
repair response in bacteria (24). Single-stranded DNA has also been
speculated to act as the initial signal for DNA damage responses in
eukaryotic cells (16). In the current study, we have investigated the
effect of single-stranded oligonucleotides on DNA damage signaling and
apoptosis. We showed that transfection with synthetic
oligodeoxyribonucleotides (oligos) as short as 5-mer up-regulated p53
and activated ATM. Moreover, single-stranded oligos induced
ATM/p53-dependent apoptosis. Our results thus suggest that
single-stranded DNA may act as a signal upstream of ATM/p53 in DNA
damage and apoptosis signaling.
Materials--
Tissue culture cells used in the current study
include BALB/c 3T3, NIH 3T3, p53 (wild type), and
p53 Cell Culture and Transfection of
Oligodeoxynucleotides--
BALB/c 3T3 cells were cultured to
semiconfluence in Dulbecco's modified Eagle's medium (DMEM)
containing 10% bovine serum at 37 °C in an environment containing
5% CO2. Oligos (200 nM or as indicated) or
oligos complexed with 10 µg Cellfectin (Invitrogen) were mixed
with 2 ml of serum-free DMEM according to the supplier's protocol and
then added to cells. Cells were harvested 4 h after transfection.
The following single-stranded oligodeoxynucleotides were used: NT36
(5'-AAG AGG TGG TGG AGG AGG TGG TGG AGG AGG TGG AGG-3'), NT27 (5'-TTG
AAT TCC TAG TTT CCC AGA TAC AGT-3'), NT12 (5'-TCG GTA ACG GG-3'), NT18
(5'-TTA GGG TTA GGG TTA GGG-3'), THIONT5 (a phosphorothioate oligo of
the sequence 5'-CGTTA-3'), NT8F (5'-GCCACTGC-3'), and NT8R
(5'-GCAGTGGC-3').
Alkaline Single Cell Gel Electrophoresis (SCGE)
Assay--
Genomic DNA strand breaks were analyzed by alkaline SCGE as
described previously (25). Briefly, pelleted cells (104/ml)
were resuspended in 0.7% low melting point agarose (SeaKem Gold®
from BioWhittaker Molecular Application, Rockland, ME) in phosphate-buffered saline (PBS) at 37 °C. After transfection, cells
(3000/sample) in 150 µl of liquefied 0.7% agarose were applied on
top of a very thin agarose layer on a frosted glass slide. Cells were
then lysed by treatment with an alkaline lysis solution (1%
N-lauryl sarcosine, 1% Triton X-100, 2.5 M
NaCl, 100 mM EDTA, and 10 mM Tris, pH 10.5) for
1 h, followed by incubation in a DNA-denaturing solution (0.3 M NaOH and 1 mM EDTA, pH 11.5) for 20 min.
Slides were subjected to electrophoresis at 1.7 V/cm for 10 min (DNA
SubCellTM gel electrophoresis apparatus from
Bio-Rad) using the DNA-denaturing solution as the gel electrophoresis
buffer. After electrophoresis, DNA was stained by submerging the slides
into a DNA staining solution containing 1× SybrGold® (Molecular
Probes, Eugene, OR). Nuclei in random and non-overlapping fields were
visualized using epifluorescent illumination on a Zeiss Axioplan
microscope. A comet tail length that was more than twice the diameter
of the nuclei was considered as comet-positive. Comet-positive nuclei
(damaged DNA) and intact nuclei (undamaged nuclei) were determined by
analyzing, random microscopic field, at least 100 nuclei for each
experiment. Each experiment was repeated three times. The percent of
comet-positive nuclei was calculated and plotted.
Preparation of Cytoplasmic Fraction--
BABL/c 3T3 cells were
harvested and lysed in a modified buffer containing 100 mM
Tris, pH 7.4, 10 mM MgCl2, 10 mM
CaCl2, 1 mM dithiothreitol, 100 µM EGTA, 0.5% Nonidet P-40, and 10 µg/ml each of
leupeptin, pepstatin, and aprotinin (26). The cytoplasmic fraction was
separated from the mitochondrial and nuclear fractions by
centrifugation at 12,000 × g for 10 min at 4 °C.
Supernatants were mixed with the Lammelli SDS sample buffer and
analyzed by Western blotting.
Measurement of Reactive Oxygen Species (ROS) Production--
ROS
production was measured using flow cytometry as described previously
(27). Briefly, BALB/c 3T3 cells were grown to semiconfluence and
transfected with the oligo:Cellfectin (10 µg) complex as described above. After 4 h, 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes) was added to the medium to a
final concentration of 20 µM. Cultures were then
incubated again for 40 min at 37 °C to allow accumulation of DCFH.
Cells were washed with PBS, trypsinized, and suspended in ice-cold PBS. Cellular fluorescence from a sample of 15,000 cells was analyzed using
a Coulter EPICS Profile II flow cytometer (Coulter Electronics, Miami,
FL). Fluorescence, excited at 488 nm, was detected using a 525 ± 20 band pass filter. Cellular debris was excluded from analysis by
gating based on forward angle light scatter. Histograms were analyzed
using EPICS Workstation Software (version 4).
Apoptotic Chromatin Condensation and Caspase Activation
Assays--
After transfection, cells were harvested and washed twice
with 1× PBS buffer. Hoechst 33342 (0.5 µM in PBS) and
CaspACE (10 µM in PBS) were then added to the cells.
Cells were then incubated for 20 min at 37 °C. Both chromatin
condensation and caspase activation were examined by fluorescence microscopy.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide
(MTT) Assay--
BALB/c 3T3 cells were cultured to semiconfluence.
Cells were transfected with indicated oligo (200 nM):Cellfectin (10 µg) complexes as described above and
incubated for 4 h. Cell viability was evaluated using a
modification of the MTT reduction assay (28). Briefly, a sterile
solution of 0.1 mg/ml MTT in DMEM was added to each well in a multiwell
plate and incubated for 4 h. The MTT color complex was then
solubilized in dimethyl sulfoxide (Me2SO), the
solution transferred to a 96-well plate, and transmission evaluated at
570 nm. Data are expressed as percent of control (no oligo and no Cellfectin).
Western Blotting--
Transfected or control cells were lysed in
Lammelli SDS sample buffer. Proteins were separated by SDS-PAGE and
transferred onto nylon membrane. Western blotting was performed
according to the ECL protocol provided by the suppliers (Amersham
Biosciences) using specific antibodies.
Knock Down of ATM in BALB/c 3T3 Cells with ATM
siRNA--
A pair of complementary RNA primers of 21 nucleotides
(Dharmacon Research, Inc.) corresponding to the 5'-non-coding region of
the ATM cDNA (5'-AAU GUC UUU GAG UAG UAU GdTdT-3' and 5'-CAU ACU
ACU CAA AGA CAU UdTdT-3') was annealed to form the ATM siRNA (a
19-nucleotide duplex stem with 2-nucleotide overhangs on either side)
according to the instructions provided by the Dharmacon Research, Inc.
145 ng of annealed double-stranded ATM siRNA or sense strand of ATM RNA
oligo was then complexed with 10 µg of Cellfectin. The RNA:Cellfectin
complex was used to transfect BALB/c 3T3 fibroblasts as described
above. After 4 h, cells were replenished with fresh DMEM
containing 10% bovine serum and further incubated for another 24 h. Cells were then transfected again with indicated oligo:Cellfectin
complexes. After 4-6 h, nuclear DNA damage or cytochrome c
release was determined by comet assay or immunocytochemistry, respectively.
Single-stranded DNA Induces Apoptotic Nuclease-mediated Chromosomal
DNA Cleavage--
To test whether single-stranded DNA could induce
apoptosis, apoptotic nuclease-mediated DNA cleavage in the chromosomal
DNA was monitored by an alkaline SCGE assay. Different single-stranded oligos were complexed with Cellfectin and used to transfect BALB/c 3T3
cells. Transfected cells were then analyzed for apoptotic nuclease-mediated DNA strand breaks in genomic DNA using the alkaline SCGE assay. As shown in Fig.
1A, oligos with different
sequences and sizes (from 5 to 36 nucleotides) were found to induce
genomic DNA strand breaks. The deoxyribonucleoside triphosphates
(dNTPs), on the other hand, were unable to induce genomic DNA strand
breaks above the background level (Fig. 1A). The THIONT5,
which is a 5-mer oligo with phosphorothioate linkages, was found to be
as active as other oligos in inducing DNA strand breaks (Fig.
1A). We have also tested different transfection agents,
including Oligofectamine, Cellfectin, Lipofectamin, and FuGENE, all of
which are effective in mediating the effect of oligos. These results
suggest that single-stranded oligos can indeed activate a nuclease (see
below for the involvement of an apoptotic nuclease) in treated
cells.
The oligo-induced chromosomal DNA cleavage showed dose dependence from
0 to 200 nM (Fig. 1B). A maximum of 60% of
cells was shown to exhibit DNA cleavage (Fig. 1A). Above 200 nM, the oligo was unable to increase the level of DNA
cleavage. The oligo-induced chromosomal DNA cleavage were found to be
time-dependent (Fig. 1C). DNA strand breaks were
detectable within 1 h of treatment and found to increase with time
(Fig. 1C). The oligo-induced DNA strand breaks were shown to
depend on the single-strandedness of the oligo. As shown in Fig.
1D, the double-stranded oligo was much less effective than
the single-stranded oligo in inducing DNA strand breaks. However, at
higher concentrations of double-stranded oligo, DNA cleavage was also
detectable (data not shown).
We have studied a possible involvement of apoptotic nuclease in
oligo-induced cleavage in the genomic DNA. Treatment of BALB/c 3T3
cells with the caspase 3 inhibitor Z-DEVD-FMK was found to inhibit
oligo-induced cleavage in the genomic DNA (Fig.
2A), suggesting that a
caspase-activated apoptotic nuclease(s) is involved in oligo-induced
chromosomal DNA cleavage.
The fact that oligos can induce an apoptotic nuclease in treated cells
has prompted us to examine whether oligos can induce (apoptotic) cell
death. As shown in Fig. 2B, oligos indeed induced cell death
in BALB/c 3T3 cells as evidenced by the MTT assay.
Oligos Induce Other Apoptotic Cell Death Markers--
To test
whether oligo-induced cell death is due to apoptosis, we have measured
the expression of various apoptotic markers in oligo-transfected cells.
As shown in Fig. 3A,
transfection of BALB/c 3T3 cells with different oligos caused release
of cytochrome c from the mitochondria into the cytoplasm
(Fig. 3A). Transfection of oligos also caused increased
production of ROS as evidenced by increased fluorescence of oxidized
DCFH (Fig. 3B). In addition, the NT8R oligo was shown to
induce both chromatin condensation (Fig. 3C, upper
panel) and caspase activation (Fig. 3C, lower panel) in BALB/c 3T3 cells. About equal numbers of cells (50%) were shown to undergo chromatin condensation and caspase activation (Fig. 3D). As a positive control, camptothecin, which is a
topoisomerase I-specific inhibitor, was shown to induce chromatin
condensation and caspase activation in about equal numbers of cells
(80%). In the aggregate, these results strongly suggest that oligos
can induce apoptotic cell death. Thus, it seems likely that
oligo-induced DNA strand breaks, which are blocked by a caspase 3 inhibitor (Fig. 2A), are due to activation of an apoptotic
nuclease(s).
Oligos Induce Up-regulation of p53 and Activate ATM
Kinase--
Oligos could mimic a DNA damage signal(s) to trigger
apoptosis. One of the most important cellular responses to DNA damage is up-regulation of p53 (29, 30). We have thus tested whether oligos
up-regulate p53 in BALB/c 3T3 cells. As shown in Fig.
4A, both oligos, NT36 and
NT27, up-regulated p53 in BALB/c 3T3 cells.
Activation of the nuclear kinase ATM is another important cellular
response to many DNA damaging agents (18, 31). H2AX is a target protein
of the ATM kinase and is known to be phosphorylated at Ser-139 by ATM
(19). Using an antibody specific for the phosphorylated peptide epitope
of H2AX, we have measured the amount of phosphorylated H2AX in BALB/c
3T3 cells treated with different oligos. As shown in Fig.
4B, NT36 and NT27 oligos stimulated phosphorylation of H2AX
in BALB/c 3T3 cells. The increase in the amount of phosphorylated H2AX
was not due to an overall elevation of histone H2A (Fig. 4B,
lower panel). Phosphorylation of H2AX at Ser-139 suggests that the ATM kinase is activated by oligos. Our results thus suggest that oligos can mimic a DNA damage signal(s) to both up-regulate p53
and activate ATM kinase.
Single-stranded DNA-induced Apoptosis (SIA) Is p53- and
ATM-dependent--
DNA damage is known to induce
ATM/p53-dependent apoptosis (32). As shown in Fig.
5A, SIA as monitored by the
alkaline SCGE assay was found to be significantly reduced (about 80%
reduction) in p53
To study the role of ATM in SIA, ATM siRNA was employed to knock down
the expression of ATM in BALB/c 3T3 cells (33, 34). As shown in Fig.
6A, ATM siRNA significantly
reduced (about 60%) SIA in BALB/c 3T3 cells as monitored by the
alkaline SCGE assay. As a control, the corresponding ATM sense strand
RNA did not affect SIA (Fig. 6A). Western blot analysis
indicated that the ATM protein level was reduced more than 3-fold in
ATM siRNA-treated, but not the corresponding ATM sense-strand
RNA-treated, BALB/c 3T3 cells (Fig. 6A). We have also
monitored the effect of ATM siRNA on cytochrome c release
from mitochondria in BALB/c 3T3 cells treated with different oligos. As
shown in Fig. 6B, oligo-induced release of cytochrome c from the mitochondria was greatly reduced in BALB/c 3T3
cells treated with ATM siRNA. These results suggest that oligos act upstream of ATM in SIA. Taken together, our results suggest that oligos
may mimic a DNA damage signal(s) to induce
ATM/p53-dependent apoptosis.
DNA damage is known to trigger a myriad of cellular responses
including cell cycle arrest and apoptosis (35, 36). While many key
regulatory proteins (e.g. ATM and p53) have been identified, the immediate signal following DNA damage remains unclear (37). A
number of studies have demonstrated that introduction of fragmented or
single-stranded DNA into cells can influence the cell cycle and induce
cell death, suggesting a potential role of DNA ends or single-stranded
DNA in DNA damage signaling. For example, transfection with randomly
fragmented herring sperm DNA induces apoptosis (38). Nuclear injection
of linearized plasmid DNA, circular DNA with a large gap, or
single-stranded circular phagemid induces a p53-dependent G1 arrest (39). Transfection of a plasmid containing the
telomeric sequence was found to activate p53 (40). Surprisingly, a CpG sequence has previously been reported to be required for induction of
apoptosis in T lymphocyte cell lines (41).
Our studies have shown that single-stranded oligos are more effective
than duplex forms in inducing apoptosis. In our system, all oligos
tested induce apoptosis. In addition to the oligos shown in Fig. 1, we
have tested over a dozen other oligos, all of which induce apoptosis.
We did not observe a CpG sequence requirement. However, some oligos
appear to be more effective than others in inducing apoptosis. Oligos
containing the human telomeric G-tail sequence or a stretch of Gs are
much more effective in inducing apoptosis.2 Indeed, a recent
report has shown that poly(G) motif-containing oligos can induce
apoptosis in prostate cancer cells (42). The molecular basis for
the observed sequence specificity is unclear and is currently under investigation.
Oligos have been used extensively in antisense research (43). SIA could
potentially be explained by an antisense effect. However, two
observations argue against the antisense explanation for apoptosis
induction. First, we have shown that oligos as short as a 5-mer can
induce apoptosis. It is unlikely that a 5-mer antisense oligo can form
a stable RNA/DNA hybrid (44). Second, all sequences used in this study
induce apoptosis. These sequences are not designed to be antisense
oligos. It seems highly unlikely that all these sequences act as
antisense oligos.
The oligos could potentially activate apoptosis by acting on
cytoplasmic or mitochondrial apoptosis modulators. However, our studies
have suggested that oligos act in the nucleus. First, oligos activate
the nuclear kinase ATM as evidenced by phosphorylation of the ATM
target protein H2AX in the nucleus. Second, like many DNA damaging
agents, oligos up-regulate p53. Third, like DNA damage-induced apoptosis, SIA was found to be ATM- and p53-dependent. The
similarity between SIA and DNA damage-induced apoptosis suggests that
oligos may mimic a DNA damage signal in the nucleus to activate
ATM/p53. Activated ATM/p53 signals are transduced to the mitochondria
leading to the activation of caspase pathway and apoptotic cell death.
The molecular mechanism by which oligos activate ATM is unclear. One
possibility is that oligos directly activate ATM kinase, which then
triggers DNA damage responses. Consistent with this hypothesis,
single-stranded DNA and linear DNA have been shown to activate
replication protein A (RPA) phosphorylation by the ATM
immunocomplex (45). However, it is unclear whether the role of
single-stranded DNA is for direct ATM kinase activation or for binding
to the ATM substrate RPA. Alternatively, oligos may act by
titrating single-stranded DNA binding proteins (e.g.
RPA) (46, 47). Such a titration may cause deprotection of
single-stranded regions in the genome (e.g. replication
forks), which in turn could trigger DNA damage responses. Regardless of
the mechanism, our results suggest that the single-stranded DNA must
act upstream of ATM/p53 in DNA damage signaling. As shown in Fig.
7, a schematic model for the role of
single-stranded DNA in DNA damage and apoptotic signaling is presented.
In this model, DNA damage is first converted into single-stranded DNA
by a nuclease. The single-stranded DNA then activates
ATM/p53-dependent DNA damage and apoptotic signaling. A
double-strand break in the chromosomal DNA or a telomere end may not
activate ATM/p53-dependent DNA damage and apoptotic
signaling unless the end is invaded by exonuclease/helicase or
de-protected (e.g. inactivation of telomere protecting
proteins) to produce single-stranded DNA. This is in contrast to the
DNA-PK signaling pathway in which the ends of a DNA double-strand break
may be sufficient to signal (48). Similarly, DNA adducts
(e.g. UV and carcinogenic DNA adducts) may not signal
through this pathway unless they are processed by nuclease/helicase to
expose single-stranded DNA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mouse embryo fibroblasts (MEF) (obtained from Dr.
Baoji Li, Institute of Molecular and Cell Biology, The National
University of Singapore, Singapore), HCT116 and HCT-116
p53
/
(obtained from Dr. Bert Vogelstein, Johns Hopkins
Medical School, Baltimore, MD), and HeLa cells. BALB/c 3T3 (p53 wild
type) and BALB/c 3T3(10)1 (p53 mutated) were obtained from Dr. Arnold
Levine, The Rockefeller University, New York. Antibodies against a
synthetic peptide consisting of the last nine amino acids (KATQASQEY)
of H2AX with phospho-Ser-139 were obtained from Dr. D. Chen (Lawrence Berkeley National Laboratory). Antibodies against histone 2A, cytochrome c, p53, ATM, and actin were purchased from Santa
Cruz Biotechnology, Inc. The caspase 3 inhibitor (Z-DEVD-FMK) was
purchased from Calbiochem. Oligos were synthesized by Integrated DNA
Technologies, Inc. CaspACE (FITC-VAD-FMK) was purchased from Promega.
Cellfectin®, Lipofectin, and Oligofectamine were purchased
from Invitrogen. FuGENE was purchased from Roche Diagnostics Corporation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Oligos induce nuclear DNA strand breaks in
BALB/c 3T3 cells. BALB/c 3T3 cells were cultured to
semiconfluency. Each oligo was complexed with 10 µg of Cellfectin and
then transfected into BALB/c 3T3 cells as described under
"Experimental Procedures." Nuclear DNA strand breaks were monitored
after 4 h using an alkaline SCGE assay. A, induction of
nuclear DNA strand breaks by different oligos. Different oligos (NT36,
NT27, THIONT5, NT18, or NT12, 200 nM each) were transfected
into BALB/c 3T3 cells, and nuclear DNA strand breaks were determined by
alkaline SCGE assay. Representative nuclei with intact DNA (no comet)
and broken strands (comet) are shown. B,
dose-dependent increase of nuclear DNA strand breaks
induced by oligos. The NT36 oligo was used in this experiment. The
concentration of the NT36 oligo for each reaction is indicated at the
bottom of the figure in nM. C,
time-dependent increase of oligo-induced nuclear DNA strand
breaks. The THIONT5 oligo (1 µM) was used, and the
alkaline SCGE assays were performed after 1, 2, and 3 h of
transfection. D, single-stranded, but not double-stranded,
oligos induce nuclear DNA strand breaks. NT8F, NT8R, and annealed
NT8F:NT8R duplex oligos (1 µM each) were transfected into
BALB/c 3T3 cells, and nuclear DNA strand breaks were monitored by
alkaline SCGE assay. Each experiment was repeated three times showing
similar results.
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Fig. 2.
Oligos induce caspase-dependent
DNA strand breaks and cell killing in BALB/c 3T3 cells.
A, oligo-induced caspase 3-dependent DNA strand
breaks. The caspase 3 inhibitor, Z-DEVD-FMK (50 µM), was
added together with each oligo:Cellfectin complexes to BALB/c 3T3 cells
for 4 h. Nuclear DNA strand breaks were monitored by alkaline SCGE
assay. B, oligos kill BALB/c 3T3 cells. A 200 nM
concentration of each oligo was used to transfect BALB/c 3T3 cells.
Cell survival was determined by MTT assay as described under
"Experimental Procedures."
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Fig. 3.
Oligos induce apoptosis in BALB/c 3T3
cells. BALB/c 3T3 cells were treated with DMEM (control),
Cellfectin or Cellfectin complexed with a mixture of four dNTPs (50 nM each), NT36 (200 nM), NT27 (200 nM), or NT8R (1 µM) for 4-6 h (see
"Experimental Procedures" for naming). Various apoptosis end points
were then measured with or without postincubation. A, oligos
induce cytochrome c release from the mitochondria. Cells
were harvested 4 h post-transfection. Cells were then lysed and
fractionated into cytoplasmic and nuclear/mitochondrial fractions.
Proteins in the cytoplasmic fraction were separated by SDS-PAGE and
immunoblotted with anti-cytochrome c antibodies. Equal
loading of protein was confirmed by Western blotting using actin
antibodies. B, oligos induce increased production of ROS.
BALB/c 3T3 cells were treated with DCFH-DA (20 µM) after
transfection with either the NT36 or the NT27 oligo for 4 h.
Production of ROS was measured by flow cytometry as described under
"Experimental Procedures." C, oligos induce both
chromatin condensation and caspase activation. BALB/c 3T3 cells were
transfected with NT8R (1 µM):Cellfectin complexes and
incubated for 6 h. Cells were washed twice with the complete DMEM
medium and then incubated with the complete DMEM medium at 37 °C for
another 6 h. Hoechst 33342 (top panels) and
FITC-VAD-FMK (bottom panels) were used to assess chromatin condensation
and caspase activation, respectively, as described under
"Experimental Procedures." The micrographs in the top
and bottom panels are taken from different fields.
D, quantification of data presented in C.
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Fig. 4.
Oligos up-regulate p53 and activate ATM
kinase. A, transfection of oligos up-regulates p53.
BALB/c 3T3 cells were transfected with Cellfectin complexed with dNTPs
(50 nM each), the NT36 oligo (200 nM), the NT27
oligo (200 nM), or the THIONT5 (200 nM) as
described under "Experimental Procedures." After 4 h, cells
were harvested and lysed in Lammelli SDS sample buffer. Proteins were
separated by SDS-PAGE, transferred to nylon membrane, and Western
blotted with anti-p53 antibodies. Equal loading of protein was
confirmed by Western blotting using actin antibodies. Arrows
show the positions of p53 and actin. B, oligos induce
phosphorylation of H2AX. BALB/c 3T3 cells were grown to semiconfluence
and transfected with Cellfectin complexed with dNTPs (50 nM
each), the NT36 oligo (200 nM), or the NT27 oligo (200 nM). After 4 h, cells were harvested and lysed in
Lammelli SDS sample buffer. Proteins were separated by SDS-PAGE,
transferred to nylon membrane, and Western blotted using antibodies
against a phosphopeptide derived from H2AX. Equal loading of H2A
protein was confirmed by Western blotting using anti-H2A antibodies.
Arrows mark the positions of H2AX and H2A.
/
MEF as compared with wild type MEF
(Fig. 5A). Similar results were obtained using two other
isogenic pairs of p53 cell lines, HCT116/HCT116p53
/
and
BALB/c 3T3 (p53 wild type)/BALB/c 3T3(10)1 (p53 mutated) (Fig. 5,
B and C). These results indicate that oligos act
upstream of p53 in SIA.
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Fig. 5.
Oligo-induced nuclear DNA strand breaks is
p53-dependent. Three independent pairs of cell lines
with wild type and mutant p53 were used in this study. Cells were
transfected with different oligos (as indicated). A, MEF
p53+/+ and two independent MEF
(p53 /
) from p53
/
mice.
Cellfectin was abbreviated as cellF in this figure.
B, human colorectal cancer cell lines HCT116
p53+/+ and p53
/
. C, BALB/c 3T3
(p53 wild type) and BALB/c 3T3(10)1 (p53 mutated). The experiments were
repeated three times showing similar results.
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Fig. 6.
SIA is ATM-dependent.
A, ATM siRNA was used to knock down ATM kinase in
BALB/c 3T3 cells as described under "Experimental Procedures." The
level of ATM in BALB/c 3T3 cells was determined by immunoblotting using
ATM antibodies. Equal loading of protein was determined by
immunoblotting using actin antibodies (Santa Cruz Biotechnology).
B, treatment of ATM siRNA inhibits oligo-induced nuclear DNA
strand breaks. Cells were transfected with either ATM sense RNA or ATM
siRNA (145 ng). 24 h later, cells were again transfected with
Cellfectin complexed with dNTPs (siRNA), NT36, or THIONT5 oligos.
4 h after transfection, cells were harvested and nuclear DNA
strand breaks were monitored by alkaline SCGE assay. The ATM protein
level in ATM siRNA-treated cells was determined by immunoblotting with
anti-ATM antibodies (A). C, ATM siRNA abolishes
oligo-induced cytochrome c release from mitochondria. BALB/c
3T3 cells were treated with ATM siRNA to knock down ATM expression as
described in A. 24 h later, cells were transfected with
different oligos (NT36 and NT27). 6 h after oligo transfection,
cells were fixed and immunostained using cytochrome c
antibodies followed by treatment with the FITC-conjugated anti-rabbit
IgG secondary antibodies. Oligo-induced cytochrome c release
from mitochondria was then examined using a fluorescent microscope. The
release of cytochrome c (green fluorescence) is
evidenced by transition from punctuated staining in the mitochondria
(the micrograph labeled siRNA/NT36) to the smooth distribution of
cytochrome c in the cytoplasm and punctated distribution in
the nucleus (the micrograph labeled sense RNA/NT36). The red
fluorescence is due to the DNA stain (propidium iodide). The number of
cells exhibiting cytochrome c release was plotted for
different treatment conditions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 7.
A proposed model for the role of
single-stranded DNA in DNA damage/apoptotic signaling. DNA damage
in the form of DNA adducts (A) and a double-strand break
(B) is first processed by nuclease/helicase to produce
single-stranded DNA (e.g. a single-stranded gap or
single-stranded oligos). The single-stranded DNA then activates
ATM/p53-dependent DNA damage/apoptotic signaling.
Transfection of oligos to induce DNA damage/apoptosis signals might
provide a very useful system to study the molecular mechanism of
apoptotic signal transduction pathways. The potency of oligos in
inducing apoptosis also suggests their potential use as anticancer agents. Clearly, more studies are necessary to establish the molecular mechanism of SIA and the potential application of oligos in cancer therapy. It should be noted that oligos have already been successfully used to treat tumors in animals (49). However, the antitumor activity
of the oligos has been attributed to the antisense effect (50). It
remains to be determined whether SIA may play a role in the antitumor
activity of oligos.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. B. Li for providing
p53/
MEF, Dr. A. Levine for providing BALB/c 3T3(10)1
(p53 mutated), Dr. B. Vogelstein for providing HCT116 and HCT-116
(p53
/
) cell lines, and Dr. D. Chen for providing
anti-phosphohistone H2AX antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported by New Jersey Commission on Cancer Research Grant 01-41-CCR-S-1 (to A. N. K.) and National Institutes of Health Grants GM27731 and CA39662 (to L. F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ
08854. Tel.: 732-235-0106; Fax: 732-235-4073; E-mail: nurekasa@umdnj.edu.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M212915200
2 H. Qi, T.-K. Li, A. Nur-E-Kamal, and L. F. Liu, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: ATM, ataxia telangiectasia mutant; ATR, ATM and Rad3-related; oligo(s), oligodeoxyribonucleotide(s); Z-DEVD-FMK, Z-Asp-Glu-Val-Asp-fluoromethylketone; FITC-VAD-FMK, fluorescein isothiocyanate-Val-Ala-Asp-fluoromethylketone; DMEM, Dulbecco's modified Eagle's medium; SCGE, single cell gel electrophoresis; PBS, phosphate-buffered saline; ROS, reactive oxygen species; DCFH-DA, 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; H2AX, histone 2AX; SIA, single-stranded DNA-induced apoptosis; MEF, mouse embryo fibroblasts.
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