Involvement of Caveolin-1 in Repair of DNA Damage
through Both Homologous Recombination and Non-
Homologous End Joining
Hua Zhu2*, Jingyin Yue3, Zui Pan4, Hao Wu2, Yan Cheng1, Huimei Lu3, Xingcong Ren1, Ming Yao2,
Zhiyuan Shen3, Jin-Ming Yang1*
1Department of Pharmacology and The Penn State Hershey Cancer Institute, The Pennsylvania State University College of Medicine, and Milton S. Hershey Medical
Center, Hershey, Pennsylvania, United States of America, 2Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of
New Jersey, New Brunswick, New Jersey, United States of America, 3Department of Radiation Oncology, Robert Wood Johnson Medical School, University of Medicine
and Dentistry of New Jersey, New Brunswick, New Jersey, United States of America, 4Department of Physiology and Biophysics, Robert Wood Johnson Medical School,
University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey, United States of America
Background: Caveolin-1 (Cav-1), the major component of caveolae, is a 21–24 kDa integral membrane protein that interacts
with a number of signaling molecules. By acting as a scaffolding protein, Cav-1 plays crucial roles in the regulation of various
physiologic and patho-physiologic processes including oncogenic transformation and tumorigenesis, and tumor invasion
Methodology/Principal Findings: In the present study we sought to explore the role of Cav-1 in response to DNA damage and
the mechanism involved. We found that the level of Cav-1 was up-regulated rapidly in cells treated with ionizing radiation. The
up-regulation of Cav-1following DNA damage occurred only in cells expressing endogenous Cav-1, and was associated with the
activation of DNA damage response pathways. Furthermore, we demonstrated that the expression of Cav-1 protected cells
against DNA damage through modulating the activities of both the homologous recombination (HR) and non-homologous end
joining (NHEJ) repair systems, as evidenced by the inhibitory effects of the Cav-1-targeted siRNA on cell survival, HR frequency,
phosphorylation of DNA-dependent protein kinase (DNA-PK), and nuclear translocation of epidermal growth factor receptor
(EGFR) following DNA damage, and by the stimulatory effect of the forced expression of Cav-1 on NHEJ frequency.
Conclusion/Significance: Our results indicate that Cav-1 may play a critical role in sensing genotoxic stress and in
orchestrating the response of cells to DNA damage through regulating the important molecules involved in maintaining
Citation: Zhu H, Yue J, Pan Z, Wu H, Cheng Y, et al. (2010) Involvement of Caveolin-1 in Repair of DNA Damage through Both Homologous Recombination and
Non-Homologous End Joining. PLoS ONE 5(8): e12055. doi:10.1371/journal.pone.0012055
Editor: Mark R. Cookson, National Institutes of Health, United States of America
Received March 10, 2010; Accepted July 12, 2010; Published August 6, 2010
Copyright: ? 2010 Zhu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from the National Cancer Institute CA109371 (Yang), CA066077 (Yang) and CA137738 (Shen). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (JMY); firstname.lastname@example.org (HZ)
Caveolin-1 (Cav-1), a major structural protein of caveolae, is
involved in many physiologic and patho-physiologic processes
such as cardiovascular diseases, neurological disorders, and
cancers. Although accumulating evidence indicate that expression
of Cav-1 is altered in a stage-dependent manner during
progression of various types of cancers [1,2,3,4], the precise roles
of Cav-1 in cancer development, progression, and treatment
remain to be fully defined. Based on its location at chromosome 7
(7q31.1), which is frequently deleted in human malignancies ,
Cav-1 is believed to be a tumor suppressor. Indeed, Cav-1 was
found to be down-regulated in many types of cancers including
breast cancer , colon cancer , lung cancer [8,9,10], ovarian
cancer [11,12], sarcomas , and thyroid cancer . Forced
expression of Cav-1 inhibits tumor growth and induces apoptosis
of tumor cells [15,16]. Additionally, a mutation in Cav-1 at codon
132 (P132L) was found in 16% of the primary human breast
cancer cases , and interbreeding Cav12/2
MMTVPyMT mice (mouse mammary tumor virus-Polyoma
middle T antigen) accelerated onset of mammary tumors in their
On the other hand, up-regulation of Cav-1 has been observed in
highly metastatic human cancers, and is associated with poor
clinical prognosis [10,19,20,21,22,23,24,25,26,27,28,29,30] and
with resistance to therapy [31,32]. These observations indicate
that re-expression of Cav-1 at advanced stages of cancer may play
a pro-survival role that protects tumor cells against various stresses
such as micro-environmental and therapeutic stress. Recently, it
was demonstrated that expression of Cav-1 promotes survival of
cancer cells following treatment with ionizing radiation (IR)
[33,34], further supporting Cav-1 as a stress protector in
PLoS ONE | www.plosone.org1 August 2010 | Volume 5 | Issue 8 | e12055
malignant cells. The protective effect of Cav-1 on IR-treated cells
also suggests that this signaling-modulating molecule may play an
important role in repair of damaged DNA. The main DNA
damage caused by IR is double strand break (DSB), which can be
repaired by two major pathways: homologous recombination (HR)
and non-homologous end joining (NHEJ). HR pathway can
accurately repair DSB via exchange of genetic material between
two similar or identical strands of DNA; NHEJ is a repairing
process in which the break ends are directly ligated without the
need for a homologous template and thus is error-prone. As
damage of DNA not only causes neoplasm but is also utilized in
therapeutic interventions such as radiotherapy and chemotherapy,
and as Cav-1 is differentially expressed during tumor progression,
understanding the role of Cav-1 in DNA DSB repair and the
underlying mechanism(s) may help further decipher the signaling
pathways involved in tumor initiation and progression, and help
develop new approaches to the prevention and treatment of
cancers. We report here that the up-regulation of Cav-1 protein in
response to DNA damage plays an important role in activating
DNA repair signaling cascade and in promoting repair of DSB
through both HR and NHEJ, thus contributing to maintenance of
Genotoxic stress induces a transcriptionally independent
up-regulation of Cav-1
Expression of Cav-1 was reported to be elevated in cells exposed
to IR [33,34]. As shown in Fig. 1A, treatment with IR stimulated
the expression of Cav-1 protein in MDA-MB-468 cells. The DNA
damage-induced Cav-1 up-regulation also occurred in other cell
lines (both tumor cells and non-tumor cells) expressing endogenous
Cav-1 such as NCI/ADR-RES, T98G and MCF-10A, but not in
cell lines (MCF-7 and PC-3) that do not express endogenous Cav-1
(Fig. 1B), and did not appear to result from altered transcription of
the Cav-1 gene, because IR did not affect the level of Cav-1
mRNA in MDA-MB-468 and A549 cells with or without silencing
of Cav-1, as determined by qRT-PCR (Fig. 2). With the use of
these cell lines containing different status of p53, it appeared that
IR – induced alteration of Cav-1 was independent of p53 status.
Expression of Cav-1 is associated with DNA damage
To further define the roles Cav-1 in DNA damage response, we
examined the effects of Cav-1 on signaling pathways that
Figure 1. Treatment with IR stimulates the expression of Cav-1 protein. (A) MDA-MB-468 cells were irradiated (5 Gy) for the indicated period
of time, and then the treated cells were collected for Western blot analysis of Cav-1. b-actin was used as a loading control. Expression of Cav-1 and b-
actin were quantified using imageJ software, and Cav-1 level was normalized to that of b-actin. The normalized Cav-1 at the zero time point was
arbitrarily set as 1. Bar represent mean 6 S.D. of three separate experiments. (B) MCF-7, NCI/ADR-RES, PC-3, T98G and MCF-10A cells were treated or
untreated with 5 Gy ionizing radiation, and Cav-1 expression was analyzed by Western blot. Results shown are the representative of three identical
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participate in DNA repair. siRNAs were utilized to inhibit Cav-1
expression. To avoid ‘‘off-target’’ effects of siRNA, we used two
Cav-1-targeted siRNA sequences that both knocked down Cav-1
expression (Fig. 3A). We found that IR - induced accumulation of
single-strand DNA (ssDNA) was increased in the cells transfected
with the Cav-1-targeted siRNA, as compared to the cells
transfected with a non - targeting RNA (Fig. 3B). The levels of
c-H2AX, the phosphorylated form of H2AX (at Ser139) associated
with DSB, were also significantly higher in MDA-MB-468 cells
with silencing of Cav-1 expression than in cells treated with a non-
targeting RNA following IR (Fig. 3C), another evidence for
defective DNA repair caused by loss of Cav-1. These observations
suggest that Cav-1 defect may impair DNA damage repair.
Furthermore, we investigated whether suppression of Cav-1
resulted in impairment of DNA damage signaling. As shown in
Fig. 4A, the activity of ATM, a kinase that is activated by DNA
damage signals and phosphorylates a series of downstream targets
such as CHK2, was lower in the cells with silencing of Cav-1 than
that in the control cells following IR, as demonstrated by
decreased levels of the phospho-ATM (Ser1981) and phospho-
CHK2 (Thr68). Treatment of cells with inhibitors (okadaic acid
and calyculin A) of PP2A, a phosphatase that decreases ATM
phosphorylation, augmented the IR-induced phosphorylation of
ATM (Fig. 4B), indicating the involvement of PP2A in the
regulation of ATM activity in response to DNA damage.
Moreover, our co-immunoprecipitation experiments demonstrat-
ed an increased physical association between Cav-1 with PP2A
following IR (Fig. 4C). These results suggest that in response to
DNA damage, Cav-1 plays an essential role in activating the
ATM-initiated repair pathway by sequestering and inhibiting the
function of PP2A. Also, using immunofluorescent microscopy we
observed that knockdown of Cav-1 by siRNA reduced both the
spontaneous and IR-induced foci formation of BRCA1, a DNA
repair protein whose expression is controlled by Cav-1 (Fig. 5).
The reduction of BRCA1 foci did not appear to be a consequence
of changes in cell cycle, as the silencing of Cav-1 had no effect on
cell cycle distribution (Fig. 6). These observations provide
additional evidence that depletion of Cav-1 weakens the ability
of cells to repair damaged DNA.
Expression of Cav-1 is required for HR repair of damaged
To begin to explore the mechanism by which Cav-1 regulates
DNA repair, we first tested whether silencing of Cav-1 expression
by siRNA altered the frequency of HR, one of the major pathways
involved in DSB repair. We used HT1080 cell line and an HR
reporter system developed by Brenneman et al . HT1080 cell
line carries a single integrated copy of a puro direct repeat HR
substrate. One of the puro repeats is driven by the PGK promoter,
but is inactive due to the insertion of an I-SceI recognition site; the
second allele codes the wild-type protein, but lacks a promoter
(Fig. 7). Introduction of an I-SceI expression vector into HT1080
cells creates DSBs at the I-SceI site, and only repair of these DSBs
by HR can produce a functional puro that confers puromycin
resistance. Fig. 8A demonstrates that similar to other Cav-1-
expressing cell lines, HT1080 cells showed an increased expression
of Cav-1 following IR. To determine the effect of Cav-1 on HR,
HT1080 cells were transfected with an I-SceI expression vector,
selected with puromycin, and then treated with a Cav-1 siRNA or
non-targeting RNA. In HT1080 cells, the silencing effect of Cav-1
siRNA could last until day 6 after transfection (Fig. 8B), which is
within the timeframe required assaying HR. Fig. 8C shows that
there was an equal level of I-SceI expression in Cav-1 knockdown
and control cells, but the Cav-1 knockdown cells had significantly
lower level of HR after I-SceI expression.
Expression of Cav-1 promotes NHEJ in response to DNA
NHEJ is another major pathway for mammalian cells to repair
DSB . As shown above in Fig. 6, silencing of Cav-1 reduced
the foci formation of BRCA1. As BRCA1 is a protein know to be
involved in DSB signal transduction and may regulate both HR
and NHEJ, we next wanted to know if Cav-1 is involved in the
regulation of this important DNA repair pathway. The phosphor-
ylation of DNA-PK, one of the necessary components of the NHEJ
pathway, was used as a read-out of this repair system. We found
that although exposure of MDA-MB-468 cells to IR markedly
induced DNA-PK phosphorylation at Ser2056, suppression of Cav-
1 expression by siRNA effectively inhibited the IR- stimulated
Figure 2. IR has no effect on expression of Cav-1 mRNA. MDA-MB-468 and A549 cells were transfected with a non-targeting RNA or siRNA
against Cav-1. Twenty-four hours later, cells were treated with 5 Gy radiation for the indicated period of time. To determine Cav-1 mRNA, total RNAs
were extracted from the cells and quantitative real-time RT-PCR was performed. Cav-1 mRNA level was normalized to b-actin mRNA. The Cav-1 mRNA
level of the cells treated with the non-targeting RNA and without IR treatment was arbitrarily set as 1. Results shown are the representative of three
similar experiments; each bar represents mean 6 SD of quadruplicate determinations.
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phosphorylation of this critical DSB repair factor (Fig. 9A),
suggesting that Cav-1 is involved in controlling the activity of the
NHEJ pathway. The results of the NHEJ assay, which measures
overall frequency of NHEJ , demonstrated that introduction of
Cav-1 into HEK293 cells (Fig. 9B, left panel) significantly
increased the NHEJ frequency as compared to the transfection
with a control empty vector (a 40% increase was observed) (Fig. 9B,
right panel). To further analyze how Cav-1 regulates the
phosphorylation of DNA-PK, we examined the effect of Cav-1
on IR-induced nuclear translocation of EGFR, which is known to
interact with DNA-PK and promote its phosphorylation . To
further examine the localization of Cav-1 and EGFR in response
to IR treatment, we immunostained the cells with Cav-1 (in green)
and EGFR (in red) before and after IR treatment. As shown in
Fig. 9C, Cav-1 and EGFR were located on plasma membrane
before IR treatment, but co-translocations of Cav-1 and EGFR in
the nuclei were observed 1 h following IR, as visualized by
confocal microscopy. By contrast, the nuclear co-translocation of
these two proteins was barely seen in the cells with silencing of
Cav-1 (Fig. 9C). The similar intensity of Cav-1 staining in the cells
transfected with a Cav-1 siRNA or a non-targeting RNA was likely
due to the high affinity of the Cav-1 antibody and the high
sensitivity of the immunofluorescence detection method. Physical
association between Cav-1 and EGFR also increased following IR
(Fig. 9D). These results suggest that Cav-1 can control NHEJ
through modulating the activity of DNA-PK via Cav-1-mediated
nuclear translocation of EGFR.
Silencing of Cav-1 expression increases sensitivity of
cancer cells to genotoxic stresses
To assess the functional significance of the up-regulation of Cav-
1 in response to DNA damage, we examined the effect of silencing
of Cav-1 expression on survival of the cells treated with IR, using a
colony formation assay. Fig. 10 shows that IR caused a
significantly more killing in the cells with loss of Cav-1 than in
the control cells, further supporting a role of Cav-1 in protecting
cells against genotoxic stress.
Loss of the putative tumor suppressor, Cav-1, is believed to be
one of the causes for development of several types of cancers, but
evidence also show that overexpression or re-expression of Cav-1
in advanced stages of the disease may contribute to tumor
Figure 3. Silencing of Cav-1 expression by siRNA increases the IR-induced accumulation of ssDNA and c-H2AX. (A) MDA-MB-468 cells
were transfected with a non-targeting RNA (NT) or either Cav-1-targeted siRNA sequence 1 or sequence 2. At the indicated time following
transfection, the cells were collected for Western blot analysis of Cav-1. b-actin was used as a loading control. (B) MDA-MB-468 cells were transfected
with a Cav-1 siRNA or a non-targeting RNA, followed by IR (5 Gy). The cells were collected at the indicated time points and fixed for
immunofluorescent detection of ssDNA. The signals of ssDNA and total DNA were quantified using imageJ software, and ssDNA signal was
normalized to total DNA signal at each time point. The results shown were mean6S.E. of five similar experiments. * p,0.05; ** p,0.01. (C) MDA-MB-
468 cells were transfected with a Cav-1-targeted siRNA or a non-targeting control (NT). Forty-eight hours later, the transfected cells were irradiated (5
Gy) for the indicated period of time followed by Western blot analysis of c-H2AX. Levels of c-H2AX and H2AX were quantified using imageJ software.
c-H2AX/H2AX ratios of untreated samples (zero time) were arbitrarily set at 100 as controls, and the treated samples were normalized to the controls.
Results shown are the representative of three similar experiments; each point represents mean 6 SD of triplicate determinations. * p,0.05, ** p,0.01.
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progression. Yet, how loss of Cav-1 facilitates tumorigenesis and
how re-induction of Cav-1 promotes tumor progression remain an
open question. It has been reported that expression of Cav-1
favors cancer cell proliferation by regulating survival pathways
such as Rac, Erk and PtdIns 3-kinase  and inhibits
detachment-induced apoptosis (anoikis) either through suppressing
p53 activation  or up-regulating the transcription of the IGF-I
receptor gene . We previously demonstrated that Cav-1-
regulated calcium homeostasis plays a role in growth and survival
of breast cancer cells . In the present study, we sought to
determine the functional significance of Cav-1 up-regulation
caused by treatments with DNA damaging agents, a phenomenon
that was also observed by others [33,34,43]. Our study reveals a
new function of Cav-1 as a possible sensor and mediator in the
DNA damage response/repair process. We show that expression
of Cav-1 can be rapidly up-regulated by DNA damaging agents
such as IR (Fig. 1), and that the up-regulation of Cav-1 protein
plays a critical role in activating the DNA repair signaling cascade,
since depletion of Cav-1 expression by siRNA impairs the cells’
ability to repair DNA, as evidenced by increased accumulation of
g-H2AX (Fig. 3C) and ssDNA (Fig. 3B), reduced phosphorylation
of ATM at Ser1981and CHK2 at Thr68(Fig. 4A), and decreased
formation of BRCA1 foci (Fig. 5). Moreover, our study reveals for
the first time that Cav-1 is able to regulate both HR and NHEJ
pathways, two major mechanisms responsible for repair of DNA
DSB. This conclusion is supported by use of two assays specific for
detecting HR and NHEJ. In the current study, the repair of DSBs
induced by the I-SceI endonuclease is monitored using artificial
chromosome-integrated reporters, namely HT1080-1885 for HR
pathway (Fig. 8) and EJ5-GFP for NHEJ pathway (Fig. 9). Each
individual reporter is designed such that repair of I-SceI-induced
DSBs by a specific pathway restores a puromycin resistance or a
GFP expression cassette. In each of the reporter-containing cell
lines, the activation of the reporter is confirmed to be dependent
upon expression of I-SceI. The restoration of puromycin resistance
in HT1080-1885 can only be achieved by HR repair of I-SceI
induced DSB using downstream homologue as template. For EJ5-
GFP cells, a promoter is separated from a GFP coding cassette by
a puro gene that is flanked by two I-SceI sites in the same
orientation. Once the puro gene is excised by NHEJ repair of the
two I-SceI-induced DSBs, the promoter is joined to the rest of the
expression cassette, leading to restoration of the GFP+ gene.
Figure 4. Cav-1-mediated inhibition of PP2A is responsible for the IR-induced accumulation of phospho-ATM. (A) MDA-MB-468 cells
were transfected with a Cav-1 siRNA or a non-targeting RNA, followed by IR (5Gy) for the indicated period of time. The treated cells were collected for
Western blot analysis of phospho-ATM, total ATM, phospho-CHK2, and tubulin. (B) MDA-MB-468 cells were irradiated (5Gy) for the indicated period of
time in the absence or presence of the PP2A inhibitors, okadaic acid or calyculin A. The treated cells were collected for Western blot analysis of
phospho-ATM and total ATM. Tubulin was used as a loading control. In order to show changes of phosphor-ATM, the results of two exposures were
included. LT: 1 min exposure; ST: 10 sec exposure. (C) MDA-MB-468 cells were irradiated (5 Gy) for the indicated period of time, followed by
immunoprecipitation and immunoblotting with the indicated antibodies. The results shown are the representative of three similar experiments.
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We demonstrated that IR induced the expression of Cav-1
(Fig. 1), a phenomenon previously reported by others [33,34,43],
(Fig. 1), but we also found that the increased expression of Cav-1
protein by IR does not appear to result from activation of Cav-1
transcription, as the mRNA level of Cav-1 was not affected by the
treatments (Fig. 2). The exact mechanism in which Cav-1
increased after DNA damage remains to be elucidated. Our
observation on the roles of Cav-1 in activating DNA repair
signaling (Fig. 3, 4, and 5) may explain the pro-survival function of
Cav-1 in IR-treated cells, as shown by us (Fig. 10) and others
[33,43,44]. Notably, we found that Cav-1 could be up-regulated
within 30 min following IR treatment (Fig. 1), earlier than the
24 h shown by Cordes et al , suggesting that Cav-1 may act as
a sensor and early mediator in response to DNA damage.
In this study we have begun to elucidate the mechanisms by
which Cav-1 regulates DNA repair. We demonstrated that Cav-1
participates in both HR and NHEJ repair pathways. The effect of
Cav-1 on HR was demonstrated by the experiments showing that
silencing of Cav-1 expression decreased HR frequency (Fig. 8).
The role of Cav-1 in HR might be related to, at least in part, its
effect on the accumulation of BRCA1 foci in nuclei after DNA
damage (Fig. 5), which was verified by cell cycle analysis showing
that knockdown of Cav-1 did not alter cell cycle distribution, a
factor known to affect the foci formation of BRCA1 protein .
Reciprocal regulation of the expression of Cav-1 and BRCA1 has
been reported [46,47], but whether this is associated with the Cav-
1-mediated BRCA1 nuclear accumulation remains to be clarified.
The role of Cav-1 in NHEJ was supported by our observation that
suppression of Cav-1 by siRNA dramatically inhibited the IR-
activated phosphorylation (Ser2056) of DNA-PK (Fig. 9A), one of
the key executers in the NHEJ system, and by the GFP-based
chromosomal reporter assay showing that the frequency of NHEJ
was significantly higher in HEK293 cells transfected with a Cav-1
expression vector than in the cells transfected with a control vector
(Fig. 9B). The mechanism of these effects might involve the Cav-1-
mediated nuclear translocation of EGFR, an activator of DNA-PK
, as Cav-1-targeted siRNA also inhibited the co-translocation
of Cav-1 and EGFR following IR treatment (Fig. 9C and D).
Therefore, it is likely that the signaling – modulating molecule,
Cav-1, may facilitate DNA repair via multiple pathways. How
precisely Cav-1 regulates HR and NHEJ and whether Cav-1 is
involved in other DNA repair pathways remain to be studied.
Figure 5. Silencing of Cav-1 expression decreases the IR-induced formation of BRCA1 foci. MDA-MB-468 cells were transfected with a
Cav-1 siRNA or a non-targeting RNA. Forty-eight hours later, the cells were irradiated (5 Gy), and fixed for immunostaining with a BRCA1 antibody.
BRCA1 foci were shown in green. DAPI was used for nucleus staining.
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Our results may provide a possible explanation for the differential
expression of Cav-1 at various stages of tumor progression. As
genome instability triggered by endogenous or exogenous DNA
of Cav-1 at early stages of cancer development may cause a defect in
DNA damage response leading to genomic alteration and oncogenic
transformation. However, re-expression of Cav-1 at later stages of
cancer may provide a protective mechanism for cancer cells to
survive various harsh conditions such as DNA damage. In fact, the
protective effects of Cav-1 against mechanical shearing damage,
hypoxia, and nutrient depletion, the stresses that are considered the
causes for death of tumor cells during their migration and metastasis,
have been reported recently [48,49,50,51,52]. Thus, re-expression of
Cav-1 at advanced stages of cancer may provide a survival
mechanism for tumor cells, and targeting Cav-1 may represent a
new stratagem for cancer treatment.
Figure 6. The effect of silencing of Cav-1 expression on cell cycle distribution. MDA-MB-468 cells with or without silencing of Cav-1 were
fixed for cell cycle analysis by FACS at the indicated time following IR treatment. The results shown are the representative of three similar
experiments; each bar represents the mean6S.D. of triplicate determinations.
Figure 7. The schematic illustration of HR reporter assay.
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Taken together, our study reveals a novel function for Cav-1 in
repairing DNA, which involves both HR and NHEJ, and suggests
that Cav-1 may play a critical role in orchestrating the response of
cells to DNA damage and in mediating DNA repair.
Materials and Methods
MDA-MB-468 (human breast cancer cell), MCF-7 (human
breast cancer cell), MCF-10A (human mammary epithelial cell),
PC-3 (human prostate cancer cell) and T98G (human glioma cell)
lines were purchased from American Type Culture Collection
(Rockville, MD). NCI/ADR-RES line (ovarian cancer cell line)
(previously named MCF-7/AdrR) was provided by Dr. Kenneth
Cowan of the Eppley Institute for Research in Cancer (Omaha,
NE). HT1080 (human fibrosarcoma cell) line was obtained from
Dr. Mark Brenneman (Rutgers University, Piscataway, NJ). MCF-
7, PC-3 and NCI/ADR-RES cell lines were maintained in RPMI
1640 medium (Invitrogen Life Technologies, Gaithersburg, MD);
MDA-MB-468 and HT1080 cell lines in Dulbecco’s modified
Eagle’s medium (Invitrogen Life Technologies); T98G in Ham’s F-
10/DMEM (10:1) medium (Invitrogen Life Technologies); and
MCF-10A in DMEM/F12 (Invitrogen Life Technologies) supple-
mented with 5% donor horse serum, 20 ng/ml epidermal growth
factor, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone and 100 ng/
ml cholera toxin (Sigma, St. Louis, MO). All the culture media
contained 100 units/ml penicillin and 100 mg/ml streptomycin
(Invitrogen Life Technologies, Gaithersburg, MD); all the cell lines
were cultured and grown in a 5% CO2- humidified incubator at
Cells in exponential phase of growth were plated in 60-mm cell
culture plates at 16106cells/plate and incubated for overnight,
and then transfected with 100 nM of Cav-1 siRNA or a non-target
RNA (Dharmacon, Inc, Lafayette, CO) using Lipofectamine 2000
and OPTI-MEM I reduced serum medium (Invitrogen Life
Technologies, Gaithersburg, MD), according to the manufactur-
er’s protocol. Silencing effects of siRNA were examined by
Western blot and real-time RT-PCR.
Western blot analysis
Cells were washed twice with PBS containing a Protease
Inhibitor Cocktail (Pierce Biotechnology Inc., Rockford, IL) and
lysed with CelLyticTMMT Cell Lysis Reagent (Sigma-Aldrich, St.
Louis, MO). Lysates were transferred to 1.5-ml eppendorf tubes
Figure 8. Silencing of Cav-1 expression reduces the DSB repair by HR. (A) HT-1080 cells were irradiated (5 Gy) for the indicated period of
time, and then cell lysates were prepared for Western blot analysis of Cav-1 and c-H2AX. b-actin was used as a loading control. (B) To determine the
turnover of the silencing effect of Cav-1 siRNA in HT-1080 cells, we performed Western blot of analysis of Cav-1 at the indicated time after siRNA
transfection. (C) HT-1080 cells were transfected with a non-targeting RNA or Cav-1 siRNA. Twenty-four hours after transfection, the cells were
transfected with an HA tagged I-SceI endonuclease expressing vector (HA-I-SceI) or empty vector (HA) by electroporation, followed by Western blot
analysis of Cav-1 and HA-I-SceI (upper panels), and by puromycin screening for HR frequency (lower panels). HR frequency was calculated as follows:
the average numbers of colonies/dish were divided by the plating efficiency of transfection and divided by 85,000 (the total number of cells plated).
The results shown are the representative of three similar experiments; each bar represents the mean6S.D. of triplicate determinations.
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and clarified by centrifugation at 16,0006g for 25 min at 4uC.
Equal amounts of cell lysates (25 mg proteins) were resolved by
SDS-PAGE, and then transferred to nitrocellulose. The mem-
branes were incubated in 5% nonfat milk in TBST (Tris-buffered
saline plus 0.1% Tween 20) at room temperature for 1 h, followed
by immunoblotting with the respective antibodies. Detection of
proteins by enzymed-linked chemiluminescence was performed
according to the manufacturer’s protocol (ECL; Pierce Biotech-
nology Inc., Rockford, IL). Quantification of protein bands was
performed using the ImageJ software (http://rsb.info.nih.gov/ij).
The antibodies used and dilution ratio were: mouse anti-b-actin
(AC-74), anti-tubulin (DM1A) antibodies (1:5000; Sigma-Aldrich,
St. Louis, MO); mouse anti-BRCA1(AB-1) antibody (1:500;
Calbiochem, La Jolla, CA); mouse anti-Cav-1 (Z034) (1:2000;
Zymed Laboratories, San Francisco, CA, USA); rabbit anti-EGFR
(1:1000; Cat. No. SC-03, Santa Cruz Biotech, Santa Cruz, CA);
rabbit anti-H2AX (ab2893), anti-DNA PKcs (ab32566) and anti-
DNA PKcs (phospho S2056) (ab18192) antibodies (1:2000, 1:1000
and 1:500; Abcam, Cambridge, UK); mouse anti-H2AX (phospho
S139) (JBW301) antibody (1:2000; Upstate, Chicago, IL); mouse
anti-PP2A-C (1D6) antibody (1:1000; Chemicon International,
Chandlers Ford, UK); rabbit anti-ATM (D2E2), anti-ATM
(phospho S1981) (10H11.E12)and anti-CHK2 (phospho T68)
(C13C1) (1:2000, 1:1000 and 1:500; Cell Signaling Technology,
Quantitative real-time RT-PCR
Total RNAs from cells were extracted with TriZol Reagent
(Invitrogen Life Technologies, Gaithersburg, MD) following the
manufacturer’s instruction. First strand cDNA synthesis and
amplification were performed using Omniscript RT Kit (QIA-
GEN Valencia, CA). The following human CAV1 primers were
used: forward: 59-CAC ATC TGG GCA GTT GTA CC-39;
reverse: 59-CAC AGA CGG TGT GGA CGT AG-39 . The
b-actin primers, designed by our laboratory , were as follows:
forward: 59-GCC AAC ACA GTG CTG TCT GG-39; reverse 59-
GCT CAG GAG GAG CAA TGA TCT TG-39. SYBR Green
quantitative PCR amplifications were performed on the Strata-
gene 3005P Real-TimePCR system. Reactions were carried out in
a 25-ml volume containing 12.5 ml of 2 SYBR Green PCR Master
Figure 9. Expression of Cav-1 expression contributes to the activity of the NHEJ repair pathways. (A) MDA-MB-468 cells with or without
silencing of Cav-1 were irradiated (5 Gy) for the indicated periods of time, and then subjected to Western blot analysis of phosphor-DNA-PKcs and
total DNA-PKcs. (B) Left panel: HEK 293 cells containing a GFP-based chromosomal reporter, EJ5-GFP, were transfected with a caveolin-1 expression
vector or a control empty vector. Thirty-six hours later, the cells were transfected with an HA tagged I-SceI endonuclease expression vector or an
empty vector. Expressions of Cav-1 and HA-I-SceI were determined by Western blot. Right panel: Seventy-two hours following transfection with the
HA-I-I-SceI plasmid, percentage of EGFP expressing cells, which represents the frequency of NHEJ, were determined by flow cytometry. The results
shown are the mean 6 S.E. from three identical experiments. (C) MDA-MB-468 cells with or without silencing of Cav-1were irradiated (5 Gy) for the
indicated periods of time, and then fixed for immunostaining with Cav-1 and EGFR antibodies. Cav-1 staining was shown in green and EGFR staining
in red. DAPI was used for nucleus staining. (D) MDA-MB-468 cells were irradiated (5 Gy) for the indicated periods of time. At the end of IR, cells lysates
were prepared and subjected to immunoprecipitation and immunoblotting with either Cav-1 or EGFR antibodies as indicated. The light chain and
heavy chains were used as loading controls.
Caveolin-1 DNA Damage Repair
PLoS ONE | www.plosone.org9August 2010 | Volume 5 | Issue 8 | e12055
Mix (Bio-Rad). The thermal profile for the real-time PCR was
95uC for 3 min followed by 40 cycles of 95uC for 20 s, 59uC for
30 s, and 70uC for 30 s. The DCt data were collected
automatically. The average DCt of each group was calculated by
the following formula: DCt=average CAV1 gene Ct-average of
HK (housekeeping) gene’ Ct. DDCt was calculated by DDCt=DCt
of non-target control group - DCtof the siRNA transfection group.
The fold-change for CAV1 expression level was calculated using
Cells were cultured on glass slides and stained as described
previously . Briefly, cells were treated with 5 Gy of IR, and
then fixed in PBS containing 4% paraformaldehyde (EMD
Chemicals, Merck Corporation, San Diego, CA) at room
temperature for 20 min. Fixed cells were rinsed with PBS and
with 25 mM NH4Cl in PBS for 10 min to quench free aldehyde
groups. The cells were permeabilized by incubation in freshly-
prepared 0.1% Triton X-100/PBS for 15 min. The cells were pre-
incubated for 1 h in PBS containing 3% bovine serum albumin
(BSA) and incubated for 2 h in diluted antibodies in PBS
containing 3% BSA. Following washing three times, the cells
were incubated for 1 h in diluted flourescence–labeled secondary
antibodies. After washing with PBS, immuno-stained cells were
examined with a Zeiss LSM 510 Meta laser scanning confocal
microscope or ApoTome Microscope (Carl Zeiss Ltd, Germany).
Primay antibodies and dilution ratio were: rabbit anti-Cav-1
antibody (1:1000; ab2910, Abcam, Cambridge, UK); mouse anti-
BRCA1 antibody (1:50; Calbiochem, La Jolla, CA); sheep anti-
EGFR (1E4) antibody (1:400; Upstate, Chicago, IL); mouse anti-
PP2A-C antibody (1:100; Chemicon International, Chandlers
Ford, UK); mouse anti-H2AX (phospho S139) antibody (1:2000;
Upstate, Chicago, IL). All secondary antibodies were purchased
from Molecular Probes (Eugene, OR) and used at a dilution of
Cells were washed with PBS, collected, and lysed with RIPA
buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS,
50 mM Tris pH 8.0) containing Protease Inhibitor Cocktail
(Pierce Biotechnology Inc., Rockford, IL). The lysates were
sonicated and centrifuged at 16,0006g for 25 min at 4uC. The
supernatant was pre-cleared with protein A/G agarose (1:25
dilution), and immunoprecipitation was carried out using the
respective antibodies. The immuno-complexes were washed four
times with RIPA buffer, and proteins were eluted with 2X SDS
sample buffer by boiling for 5 min. Ten ml precipitated proteins
were resolved on SDS–PAGE and subjected to Western blot
analysis using the respective antibodies.
Immunofluorescent detection of ssDNA
Detection of ssDNA was carried out as reported . Briefly,
cells were grown on cover slips for overnight, and then incubated
in medium containing 30 mmol/l BrdU (Bromodeoxyuridien,
Sigma) for 24 h in the dark. To visualize ssDNA, the cells were
fixed with methanol at 220uC for 10 min and then incubated in
blocking solution (2% bovine serum albumin in PBS) at room
temperature for 30 min, followed by incubation with an anti-BrdU
Figure 10. Silencing of Cav-1 expression sensitizes cells to IR and bleomycin. MDA-MB-468 cells with or without silencing of Cav-1 were
treated with varying doses of c-radiation, and colony formation assay was performed to compare cell survival. Results shown are the representative of
three similar experiments; each point represents mean 6 SD of quadruplicate determinations of the experiment.
Caveolin-1 DNA Damage Repair
PLoS ONE | www.plosone.org10 August 2010 | Volume 5 | Issue 8 | e12055
antibody (Becton Dickinson, Franklin Lakes, NJ, USA). The cells
were washed four times with PBS and then incubated with
rhodamine red-X- conjugated goat anti-mouse IgG (Jackson
ImmunoResearch Laboratories, Inc.) for 30 min at room
temperature in the dark. The cells were counterstained with
DAPI in blue for total DNA. Fluorescent images were taken using
Carl Zeiss fluorescent microscope (Axiovert-200M) equipped with
a Carl Zeiss digital camera (AxioCam MRC). The nuclear areas
were selected using the ImageJ v1.37 software (http://rsb.info.nih.
gov/ij/) and fluorescent signals from ssDNA and total DNA were
integrated. The ratio of ssDNA signal intensity to total DNA
represents the relative level of ssDNA in the nucleus. For each
experiment, at least 30 cells were analyzed. Data were reported as
mean 6 SE from five independent experiments. Statistical
significance of fluorescent signal intensity and ratio of signal
intensity in cells with or without silencing of Cav-1 were analyzed
by two-tailed t-test.
Human HT1080 cells were transfected with either a Cav-1
siRNA or a non-targeting RNA. Two days after transfection, cells
were trypsinized and resuspended in gene pulser electroporation
buffer. Four mg of an I-SceI expression vector pCMV(3_NLS) HA-
I-SceI or an empty vector were introduced into 3.56105cells by
electroporation. The cells were then seeded at 85,000 cells/100-
mm culture dish for puromycin selection. The seeded cell cultures
were re-fed with fresh medium containing 1 mg/ml puromycin on
day 2 following electroporation, and the puromycin - containing
medium was changed on days 6, 10, 12, and 14 days. At the end of
selection, cells were fixed and stained, and colonies with 50 or
more cells were counted.
NHEJ frequency assay
The frequency of total NHEJ was determined using a GFP-
based chromosomal reporter, EJ5-GFP, as previously described by
Bennardo et al. . Briefly, the transformed human embryonic
kidney HEK293 cells containing a GFP-based chromosomal
reporter, EJ5-GFP, were transfected with a caveolin-1 expression
vector or a control empty vector. Thirty-six hours later, the cells
were transfected with an HA tagged I-SceI endonuclease
expression vector or a control empty vector. Seventy-two hours
following transfection with the HA-I-I-SceI plasmid, percentage of
EGFP expressing cells, which represents the frequency of NHEJ,
were determined by flow cytometry.
Analysis of cell cycle distribution
Cells were trypsinized, washed, and fixed with 80% ethanol for
1 hour. Following treatment of cells with RNase and propidium
iodide (Sigma, St. Louis, MO), cell cycle distribution (5,000 cells)
was analyzed by fluorescence activated cell sorting (FACS) using a
flow cytometer (Coulter Cytomics FC, Beckman Coulter, Miami,
Colony formation assay
The numbers of cells to be plated for each IR dose and drug
concentration were determined by a pilot experiment in order to
yield 50–150 surviving colonies/100-mm plate. For IR tests, cells
were plated and incubated for 18 h at 37uC, and then were
irradiated with a Cs-137 c-irradiator (Nordion Inc., Canada). Two
weeks after IR treatment, colonies were fixed with methanol and
stained with 1% crystal violet. To calculate the survival fraction,
number of colonies was normalized to the number of cells plated.
For drug tests, cells in 60 - mm plates were treated with bleomycin
(EMD Chemicals, Merck Corporation, San Diego, CA) for 2 h,
and then the drug was washed off with drug-free medium. The
survival fractions were calculated as described above.
Student’s t-test was used to determine the degree of significance.
We are grateful to Dr. Jeremy Stark for providing us with the EJ5-GFP
expressing HEK293 cells and other reagents for NHEJ assay. We thank
Jeremy Haakenson for helpful discussion of this work.
Conceived and designed the experiments: HZ ZS JMY. Performed the
experiments: HZ JY ZP HW YC MY. Analyzed the data: HZ JY ZP HW
YC HL XR ZS JMY. Wrote the paper: HZ HL XR JMY.
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