Download Suppression of RAD21 gene expression decreases cell growth and

Molecular Cancer Therapeutics
Suppression of RAD21 gene expression decreases cell
growth and enhances cytotoxicity of etoposide and
bleomycin in human breast cancer cells
Josephine M. Atienza,1 Richard B. Roth,1
Caridad Rosette,1 Kevin J. Smylie,1
Stefan Kammerer,1 Joachim Rehbock,2
Jonas Ekblom,1 and Mikhail F. Denissenko1
1
Sequenom, Inc., San Diego, California and 2Frauenärzte
Rosenstrasse, Munich, Germany
Abstract
A genome-wide case-control association study done in our
laboratory has identified a single nucleotide polymorphism
located in RAD21 as being significantly associated with
breast cancer susceptibility. RAD21 is believed to function
in sister chromatid alignment as part of the cohesin complex
and also in double-strand break (DSB) repair. Following our
initial finding, expression studies revealed a 1.25- to 2.5fold increased expression of this gene in several human
breast cancer cell lines as compared with normal breast
tissue. To determine whether suppression of RAD21
expression influences cellular proliferation, RNA interference technology was used in breast cancer cell lines MCF-7
and T-47D. Proliferation of cells treated with RAD21specific small inhibitory RNA (siRNA) was significantly
reduced as compared with mock-transfected cells and cells
transfected with a control siRNA (Lamin A/C). This
inhibition of proliferation correlated with a significant
reduction in the expression of RAD21 mRNA and with an
increased level of apoptosis. Moreover, MCF-7 cell sensitivity to two DNA-damaging chemotherapeutic agents,
etoposide and bleomycin, was increased after inhibition of
RAD21 expression with a dose reduction factor 50 (DRF50)
of 1.42 and 3.71, respectively. At the highest concentrations of etoposide and bleomycin administered, cells
transfected with a single siRNA duplex targeted against
RAD21 showed 57% and 60% survival as compared with
control cells, respectively. Based on these findings, we
conclude that RAD21 is a novel target for developing cancer
therapeutics that can potentially enhance the antitumor
activity of chemotherapeutic agents acting via induction of
DNA damage. [Mol Cancer Ther 2005;4(3):361 – 8]
Received 9/10/04; revised 12/21/04; accepted 1/5/05.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Mikhail F. Denissenko, Sequenom, Inc., 3595 John
Hopkins Court, San Diego, CA 92121. Phone: 858-202-9000;
Fax: 858-202-9001. E-mail: [email protected]
Copyright C 2005 American Association for Cancer Research.
Introduction
The RAD21 gene codes for a human homologue of
Saccharomyces pombe Rad21 protein. The current knowledge
about this protein points to a role in modulation of cell
growth and in cell defense against DNA damage, both
processes being central to carcinogenesis. Several DNA
repair genes including rad21 were initially identified in the
fission yeast S. pombe as radiation-sensitive mutants (1, 2).
Specifically, Rad21 has been implicated in homologous
recombination – mediated double-strand break (DSB) repair, and is unique among the radiation response genes in
that it also plays a role in cell cycle regulation (3, 4). Yeast
Rad21 and its mammalian homologue were subsequently
identified as components of a conserved cohesin complex
(5, 6), which is believed to function in aligning sister
chromatids during the early stages of cellular division.
Deletion of RAD21/Scc1/Mcd1 in mammalian cells leads
to abnormal separation of sister chromatids during
interphase and improper alignment during metaphase.
These cells also incur increased levels of spontaneous
chromosomal breaks and ionizing radiation – induced chromosomal aberrations, probably due to a reduction in DSB
repair efficiency (7). These observations suggest that
RAD21 and the cohesin complex not only mediate the
alignment of chromosomes in preparation for segregation
into daughter cells during mitosis but also facilitate the
repair of DNA damage incurred during DNA replication
by holding sister chromatids together.
DNA damage is a consequence of exposure to both
exogenous and endogenous agents, resulting in a diverse
array of DNA modifications. Exogenous agents such as
ionizing radiation and radiomimetic chemicals and endogenous agents such as oxygen radicals can all induce DSBs.
In addition, certain cellular processes including replication,
meiosis, and V(D)J recombination also give rise to DSBs.
DNA DSBs are among the most deleterious of DNA
modifications, often resulting in mutagenesis or cytotoxicity (8). DSBs can contribute to tumorigenesis through the
introduction of mutations or chromosomal aberrations
leading to abnormal regulation of oncogenes or loss of
tumor suppressor genes (9). Defects in DSB repair and
increased levels of DSBs have both been linked to the
development of human tumors (9). Several intricate and
well-regulated DSB repair mechanisms have evolved to
counteract these damages. DSBs can be repaired by
homologous recombination or by nonhomologous end
joining.
Many common anticancer agents introduce DSBs in
DNA. This therapeutic option takes advantage of the
inefficient DNA-damage checkpoints in cancerous cells
and, therefore, their compromised ability to efficiently
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362 RAD21 Suppression Inhibits Growth of Breast Cancer Cells
repair the damage caused by the drugs before cell division,
resulting in increased cytotoxicity. Disabling the cellular
processing and repair of DSBs potentiates cell sensitivity to
traditional chemotherapeutic agents (10 – 12). Because human RAD21 is believed to play a dual role in the cell, both
in modulation of sister chromatid alignment during cell
cycle and through repair of DNA DSBs, it presents a good
target candidate for adjuvant gene-specific approaches that
enhance sensitivity of tumor cells to anticancer agents.
In a large-scale breast cancer case-control study, we
obtained evidence that RAD21 is a susceptibility gene for
breast cancer (13). In this article we investigated the role of
RAD21 in cancer cell proliferation by analyzing its
expression in a number of human breast tumor cell lines
and also exploring the utility of silencing its expression in
these cells. Here we present the first report showing that
inhibition of RAD21 gene expression in breast cancer cells
results in decreased cellular proliferation, increased apoptosis, and increased cell killing after exposure to chemotherapeutic agents.
Materials and Methods
Cell Lines and Culture Conditions
MCF-7, T-47D, Hs 578Bst, ZR-75-1, HCC1937, Au-565, Hs
578, BT-474, HCC1395, HCC1428, HCC1500, and SK-BR-3
were acquired from American Type Culture Collection
(Manassas, VA). Human mammary epithelial cells were
generously provided by S. Bates (City of Hope Medical
Center, Duarte, CA). MCF-7 and T-47D were maintained
in MEME (American Type Culture Collection) and RPMI
(American Type Culture Collection) media, respectively,
supplemented with 10% fetal bovine serum (Omega
Scientific, Tarzana, CA) and 10 Ag/mL insulin (Invitrogen,
Carlsbad, CA). Cells were fed every 5 days and subcultured
when confluent.
RNA, Reverse Transcription ^ PCR, and Quantitative
Gene Expression Analysis
Human normal breast tissue total RNA was purchased
from Ambion (Austin, TX) and used for cDNA synthesis.
To assess cellular expression of RAD21 mRNA, 50 106
cells were collected and total RNA extracted with a TRIzol
reagent (Invitrogen, Carlsbad, CA). Reverse transcription
was done with a SuperScript II kit (Invitrogen) using a
mixture of oligodeoxythymidylate and 18S rRNA – specific
primers and aliquots taken for PCR. RAD21 message was
detected using the following PCR primers: 5V-CAATGCCAACCATGACTGAT (forward) and 5V-CGGTGTAAGACAGCGTGTAAA (reverse). Amplification (30 cycles) was
done at annealing temperature of 60jC. For small inhibitory RNA (siRNA) experiments, cells were harvested on
day 2 post transfection with siRNA, and total RNA was
extracted using standard methods.
Levels of transcripts were assessed using competitive
reverse transcription – PCR and mass spectrometry (Quantitative Gene Expression by MassARRAY assay, Sequenom,
San Diego, CA). The competitive PCR step of Quantitative
Gene Expression by MassARRAY includes a synthetic
competitor oligonucleotide that differs at one base position
from the cDNA target. This competitor is used to calibrate
the assay and quantitate the genes of interest at an absolute
level by matrix-assisted laser desorption-ionization time-offlight mass spectrometry (MALDI-TOF) mass spectrometry. The principle of Quantitative Gene Expression by
MassARRAY assay was described elsewhere (14). Levels of
gene-specific mRNA were normalized against levels of 18S
rRNA by dividing the observed RAD21 transcript concentration by the observed concentration of 18S rRNA for each
respective sample. The following oligonucleotides were
used: 5V-ACGTTGGATGATATGGATGAGGATGATAATGTATC-3V (forward PCR primer), 5V-ACGTTGGATGCAGTCATGGTTGGCATTG-3V (reverse PCR
primer), 5V-GTTCAACGGGATCCACTGAAT-3V (MassEXTEND primer), 5V-CAGTCATGGTTGGCATTGGTTCAACGGGATCCACTGAATCAGGACTATCAGGCCCACCCATTGATACATTATCATCCTCATCCATAT-3V
(cDNA competitor). Analysis included triplicate experiments with quadruplicate spotting of reaction products
onto MALDI-TOF chips. Data shown represent means of
three experimental measurements.
Small Inhibitory RNA Design
siRNA duplexes were designed according to the guidelines of Elbashir et al. (15). The following siRNA against
RAD21 were used: siRad21_272, 5V-AAGCCCAUGUGUUCGAGUGUA-3V; siRad21_1175 5V-AAGAGUUGGAUAGCAAGACAA-3V, and scrambled siRad21_1175
siRNA, siRad21_1175s 5V-AAGACAGAUACGAUGAUGAGA-3V. The sequence of Lamin A/C control siRNA
duplex was 5V-CUGGACUUCCAGAAGAACA-3V. Ready to
use synthetic siRNA duplexes, including a fluorescent
transfection control Cy3-modified Luciferase (GL2) siRNA,
were purchased from Dharmacon (Lafayette, CO) and,
upon dilution in water, were directly used in transfection
experiments.
Cell Proliferation and Apoptosis Assays
MCF-7 and T-47D cells were plated in six-well plates
at 3.5 105 and 2.5 105 cell/mL concentrations,
respectively. Twenty-four hours after plating (day 0), cells
were transfected with siRNA (40 nmol/L) using Lipofectamine 2000 (Invitrogen) as suggested by the manufacturer.
On the next day (day 1) cells were trypsinized and plated
in triplicates in 96-well dishes at concentration 1 104
cells/well. Cell proliferation was measured using WST-1
assay kit (Roche Diagnostics, Indianapolis, IN) at designated time points, and relative proliferation calculated
by normalizing to day 1 values. Experiments were done at
least thrice. Apoptosis was measured on day 3 using the
Vybrant Apoptosis Assay Kit #3 (Molecular Probes,
Eugene, OR) as directed by the manufacturer.
Clonogenic Survival Assay
Cell survival was measured based on colony formation.
MCF-7 cells were transfected with siRad21_272. Two days
after transfection, cells were exposed to varying concentrations of etoposide and bleomycin (both from Sigma, St.
Louis, MO) for 2 hours at the concentrations indicated.
Cells were then trypsinized, washed, and replated at 200
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Molecular Cancer Therapeutics
cells per 10-cm dishes. Cells were grown for 11 to 12 days
(four population doublings), fixed, and stained with crystal
violet to detect colonies. Colonies of >50 cells were counted.
Survival of transfected cells was quantitated as a fraction of
surviving control (untreated) cells and plotted on a log
scale as a function of dose. Increased sensitivity was
measured as dose reduction factor (DRF), the factor by
which the dose of radiation or drug is reduced in the
presence of the sensitizing agent to achieve the same level
of cell killing in the absence of the sensitizing agent (10).
DRF was calculated at the dose that produced 50% cell
survival (50% clonogenic cell killing).
Statistical Analysis
Clonogenic survival curves for MCF-7 cells treated
with etoposide and bleomycin were generated by plotting
the average fraction of surviving clones F SD on a log
scale versus the compound concentration. The average
fraction of surviving clones was calculated relative to
control (untreated) cells using triplicate samples from
three independent experiments. DRF values were determined from the fitted curve generated using XLfit 4.0
software package. To determine the significance of
differences in sensitivities of transfected and control cells
to etoposide and bleomycin, Student’s t test was done on
values for compound concentrations that gave 50%
survival of clones. P values of <0.05 were considered
significant.
Results
Using DNA samples from clinically diagnosed breast
cancer cases and matched controls, a genome-wide study
consisting of 25,494 single nucleotide polymorphisms was
conducted in our laboratory to discover single nucleotide
polymorphisms associated with breast cancer (13). The
single nucleotide polymorphisms were selected to be gene
based, that is, located within 10 kb of 15,995 LocusLink
annotated genes, and to cover the genome at a median
spacing of about 40 kb. The study identified a C-to-G single
nucleotide polymorphism in intron 1 of the RAD21 gene
(rs1374297) as being strongly associated with breast cancer
susceptibility. The frequency of the G allele was increased
in cases (14.8%) compared with controls (7.5%), resulting in
a P value of 0.0003 and an odds ratio of 2.1. The analysis of
a total of 27 single nucleotide polymorphisms across the
gene in case and control samples pointed toward intron 1
as the region of highest significance (data not shown).
Therefore, we speculate that the observed disease association might be due to a variation in a regulatory element in
intron 1.
Gene expression analysis by van’t Veer et al. has
previously shown that RAD21 transcript levels are
increased in breast cancer tissue as compared with normal
breast tissue, and that this elevated level of RAD21
expression is also associated with a poor prognosis for
breast cancer (16). Prompted by these findings, we
quantitated RAD21 transcript levels in 11 tumorigenic
breast cancer cell lines, two immortalized breast cell lines
(Hs 578Bst and hMEC), and in normal breast tissue, using a
Quantitative Gene Expression analysis procedure that
combines competitive PCR and MALDI-TOF mass spectrometry (14). A comparison of normalized RAD21 levels
across all cell lines and tissue tested revealed that RAD21
mRNA expression is lower in normal and immortalized
breast cancer cell lines as compared with 9 out of 11
tumorigenic breast cancer lines (Table 1).
Null mutations of rad21 in yeast and vertebrate DT40
cells result in a loss of proliferative capacity, underscoring
the importance of Rad21 in mitosis. To determine whether
RAD21 plays a role in modulating proliferation of breast
cancer cells, we used RNA interference technology to
silence expression of this gene. Optimal cellular transfection conditions were selected for both cell lines before
transfection of RAD21-specific siRNAs. Various concentrations of Lipofectamine 2000 and Cy3-modified Luciferase
(GL2) siRNA were tested to determine the optimal
combination that resulted in the highest transfection
efficiency. Cy3-modified GL2 siRNA was used to monitor
transfection efficiency by counting the percentage of Cy3
fluorescent cells from three to five microscope fields. From
these preliminary experiments, we selected a working
transfection concentration of 40 nmol/L siRNA. This condition routinely produced a >95% transfection efficiency in
cells cultured in six-well plates. Other transfection
conditions may be optimal for 24-, 48-, or 96-well tissue
culture plates. Multiple siRNA were designed according
to the guidelines of Elbashir et al. (15) and BLAST verified
to ensure specificity for RAD21 mRNA. These duplexes
were then transiently transfected into breast cancer
cells and cellular proliferation (viability) was analyzed on
Table 1. Relative levels of RAD21 expression in normal breast
tissue, immortalized breast cell lines, and tumorigenic breast
cancer cell lines
Tissue or cell line*
Normal breast
Hs 578Bst
hMEC
ZR-75-1
HCC1937
T-47D
Au-565
MCF-7
Hs 578
BT-474
HCC1395
HCC1428
HCC1500
SK-BR-3
Expression levelc
3.5
1.0
1.3
2.1
3.5
3.8
4.0
5.1
5.2
6.9
7.9
8.7
8.9
9.8
F
F
F
F
F
F
F
F
F
F
F
F
F
F
1.1
0.3
0.5
0.8
1.2
0.9
0.5
0.7
1.1
2.3
1.3
1.5
1.7
3.7
*RAD21 cDNA levels were estimated in normal breast tissue, immortalized
breast cell lines Hs 578Bst and hMEC, and breast cancer cell lines ZR-75-1,
HCC1937, T-47D, Au-565, MCF-7, Hs 578, BT-474, HCC1395, HCC1428,
HCC1500, and SK-BR-3 using competitive RT-PCR and MALDI-TOF mass
spectrometry (MassARRAY).
cExpression levels were normalized to the levels of 18S rRNA. Data shown
are means F SD.
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364 RAD21 Suppression Inhibits Growth of Breast Cancer Cells
days 1, 2, 4, and 6 post transfection. Two siRNA duplexes,
siRad21_1175 and siRad21_272, effectively inhibited
proliferation in both MCF-7 and T-47D cells (Fig. 1A
and B). MCF-7 cells treated with siRad21_272 and
siRad21_1175 showed 60% and 34% survival as compared
with control cells treated with Lipofectamine 2000
alone, respectively. The effect in T-47D cells was more
pronounced: these cells treated with siRad21_272 and
siRad21_1175 showed 32% and 19% survival as compared
with control, respectively. Because no decrease in proliferation was observed in cells transfected with siRNA targeted
against Lamin A/C compared with cells mock-transfected
with Lipofectamine 2000 alone, we believe that the effect
of these siRNA duplexes was likely due to the specific
inhibition of RAD21. Furthermore, scrambled siRNA,
siRad21_1175S, designed by randomly shuffling the
siRad21_1175 nucleotide sequence showed no inhibitory
effect on cell proliferation (data not shown).
To confirm that gene expression was indeed inhibited
and that the siRNA-driven inhibition of cell growth was
specific to the reduction of RAD21 expression, we did a
quantitation experiment using competitive PCR and
MALDI-TOF mass spectrometry. Although the strongest
inhibition of cellular proliferation was observed on day 6,
total RNA for quantitation of gene expression was isolated
from cells on day 2 post transfection. This earlier time point
was used in order to detect the changes in RAD21 message
that were more likely due to siRad21-specific inhibition and
less likely due to general mRNA degradation as a result
of pronounced cell death. We have determined the utility
of early RNA isolation time points on days 1 or 2 in
preliminary experiments using a number of unrelated
siRNA species (data not shown). Consistent with the
results of the proliferation assay, transfection of both
siRad21_1175 and siRad21_272 led to suppression of
RAD21 expression in both cell lines (Fig. 1C). Cells
Figure 1. Cell proliferation and RAD21 gene expression levels in MCF-7 and T-47D cells transfected with siRNA specific to RAD21 . siRNA specific to
RAD21 [siRad21_1175 (x) and siRad21_272 (n)] were transfected in breast cancer cell lines MCF-7 (A) and T-47D (B) and proliferation measured using a
WST-1 assay on days 1, 2, 4, and 6 post transfection. Lipofectamine 2000 alone () and siRNA against Lamin A/C (E) were used as controls. Values were
normalized to day 1. C, RAD21 mRNA and 18S rRNA levels in cells transfected with RAD21 siRNA. RAD21 expression levels were normalized to the levels
of 18s rRNA. Total RNA was isolated on day 2 posttransfection, and quantitative expression analysis by competitive reverse transcription – PCR and
MALDI-TOF mass spectrometry was done using primers specific for RAD21 mRNA. Data shown represent means of three experimental measurements,
FSD. D, agarose gel electrophoresis of total RNA extracted from siRNA-transfected cells; lane numbering corresponds to that in C.
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Molecular Cancer Therapeutics
Figure 2. Apoptosis of MCF-7 and T-47D cells transfected with siRNA specific to RAD21 . MCF-7 (A) and T-47D (B) cells were transfected with
siRad21_1175 and apoptosis assessed on day 3 posttransfection using Annexin-V conjugated to FITC and propidium iodide staining. Several fields were
examined and representative fields are shown.
transfected with siRad21_1175 showed lower levels of
RAD21 mRNA than cells transfected with siRad21_272,
which correlates with the relative decreases in proliferation
observed in cells transfected with these siRNAs. This effect
is not due to changes in extracted RNA as evidenced in
Fig. 1D.
To determine whether inhibition of breast cancer cell
proliferation upon treatment with RAD21-specific siRNA
coincided with the induction of programmed cell death,
cells were stained with propidium iodide and Annexin
V-FITC 72 hours post transfection and examined by
phase contrast and fluorescence microscopy. Both MCF-7
and T-47D cell lines transfected with siRad21_1175 and
siRad21_272 contained higher numbers of cells undergoing apoptosis as compared with cells transfected with
control siLamin A/C siRNA and Lipofectamine 2000
alone (Fig. 2 and data not shown). The increased Annexin
V staining was inversely correlated to the level of
proliferation of cells transfected with RAD21 siRNA. We
have also done a quantitative assessment of the number of
Annexin V – stained apoptotic cells at 72 hours post
transfection in MCF-7 and T-47D cells transfected with
siRad21_1175 and siLamin A/C. The respective values
(percentage of Annexin V stained cells) in these two
samples were 63.6 F 5.1 and 0.02 F 0.01 for MCF-7 cells,
and 90.8 F 8.9 and 12.3 F 3.3 for T-47D cells. These results
suggest that the decrease in proliferation of siRNAtransfected cells could be due to the induction of
apoptosis.
Next we investigated the effect of down-regulating
endogenous RAD21 expression on cell killing by known
anticancer drugs that induce DSBs. We reasoned that
even a partial down-regulation of RAD21 mRNA level
would impair DSB repair and thus render cells more
sensitive to DSB-generating agents. To assess cell killing
under partial RAD21 silencing conditions, we did
clonogenic assays in cells that were initially transfected
with RAD21-specific siRNA and subsequently exposed to
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varying concentrations of model drugs that induce DNA
DSBs, etoposide and bleomycin. In these experiments, we
used siRad21_272, which had a less pronounced inhibitory effect on cell proliferation than siRad21_1175. On
day 2 posttransfection, MCF-7 cells were treated with
etoposide or bleomycin. This time point was chosen
based on the above results in which we observed no
difference in proliferation between siRad21-transfected
and control cells (Fig. 1A) and, at the same time, a marked
decrease in RAD21 message in siRad21-transfected but not
control cells (Fig. 1). After 11 to 12 days, surviving
colonies were counted to determine cellular sensitivity.
Cells transfected with siRad21_272 showed increased
sensitivity to etoposide (Fig. 3A) compared with control
cells transfected with Lamin A/C siRNA or Lipofectamine 2000 alone. This increased sensitivity corresponded
to a DRF50 of 1.42. Interestingly, this modest increase in
DRF was accompanied by a substantial decrease in cell
survival upon treatment with etoposide. At the highest
etoposide concentration, cells transfected with siRad21_272
showed 57% survival as compared with cells transfected
with Lamin A/C siRNA or Lipofectamine 2000. In cells
treated with bleomycin, a more significant increase in
DRF (DRF50 of 3.71) was measured (Fig. 3B). At the
highest concentration of bleomycin, cells transfected with
siRad21_272 showed 60% survival as compared with
control. The increase in cell killing is specific to downregulation of RAD21 gene expression because the
siRad21-transfected cells showed a higher sensitivity
compared with control cells over a wide range of
concentration of DSB-inducing drugs. These results
confirm that RAD21 gene – specific approach augments
breast cancer cell killing by DNA-damaging chemotherapeutic agents.
Discussion
Development of somatic malignancy is characterized by a
series of changes in processes that regulate cell growth,
differentiation, apoptosis, and DNA repair (multistage
carcinogenesis). Genetic or epigenetic alterations in one or
more genes involved in these processes can result in the
loss of coordinated regulation of these cellular events and
lead to a phenotype of growth advantage. DNA repair
mechanisms protect the integrity of the genome and
provide the main defense against these genetic alterations.
Several lines of evidence suggest that cancer cells may
include and/or acquire deficiencies in genes controlling
DNA repair. It is known that certain genetic syndromes
accompanied by predisposition to cancer are due to
mutations in genes involved in DNA repair (9, 17). For
example, ataxia telangiectasia is due to a mutation in ATM,
a gene involved in S-phase checkpoint response to DSB,
and is characterized by a predisposition to lymphoid
malignancies (18). Li-Fraumeni syndrome, which is accompanied by a high incidence of breast cancer, is caused by a
mutation in TP53 (19), a gene encoding a key protein in the
transduction of DNA repair signals. Familial breast cancer
Figure 3. Clonogenic survival of MCF-7 cells transfected with RAD21 specific siRNA after exposure to etoposide and bleomycin. MCF-7 cells
were transfected with RAD21 -specific siRNA followed by exposure to
varying concentrations of etoposide (A) and bleomycin (B). Dosedependent clonogenic survival was quantitated by colony formation.
Curve fitting was done using Xlfit 4.0. Comparison of sensitivity among
cells transfected with RAD21 siRNA (siRad21_272; .), with Lipofectamine 2000 alone (5), and with siRNA to Lamin A/C (4) at different
concentrations of chemotherapeutic agents. P values of 0.014 and
0.0002 were calculated for etoposide and bleomycin concentrations that
gave 50% survival of clones, respectively. Dashed lines, drug concentration that gives 50% survival of clones.
genes BRCA1 and BRCA2 function in DNA repair through
interactions with Rad51 (20). Furthermore, cytologic examinations of breast cancer cells by several groups have
revealed a very high frequency of chromosomal rearrangements (21, 22), a likely consequence of DNA repair
dysregulation.
In this article, we investigate a possible link between
the DNA repair gene RAD21 and proliferation of tumor
cells. Our initial discovery of a single nucleotide
polymorphism in RAD21 associated with increased
breast cancer risk stimulated the present study. We
subsequently showed that RAD21 mRNA levels are
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higher in multiple breast cancer cell lines compared with
normal tissue and immortalized breast cell lines. Consistent with these findings, recent microarray gene
expression analyses in human primary breast tumors
identified elevated RAD21 expression as associated with
poor prognosis for breast cancer (16). It is therefore
plausible that an elevated level of RAD21 expression is
indicative of a malignant cell growth phenotype. We went
on to show that down regulation of RAD21 expression in
breast cancer cell lines using targeted siRNA duplexes
results in significant loss of cell proliferation and viability.
This effect was gene specific and coincided with an
induction of programmed cell death. These findings
establish RAD21 as a potential factor for modulating
growth of breast cancer cells.
Ionizing radiation and surgery are most commonly used
to treat various cancers. Exposure of S. pombe rad21
mutants to g-irradiation results in the accumulation of
DSBs (4). In vertebrate cells, loss of Rad21 results in an
increase in spontaneously occurring chromosomal breaks
in addition to enhanced ionizing radiation – induced
chromosomal breaks (7). The accumulation of DSBs
may be explained by the role of Rad21 as a component
of the cohesin complex (5, 6). This complex facilitates
sister chromatid cohesion during mitosis and homologous recombination – mediated DSB repair (23). In cells
lacking functional RAD21, sister chromatids are loosely
held together, thus reducing the efficiency of homologous
recombination – mediated DSB repair. In addition to
ionizing radiation, tumors are frequently treated with
chemotherapeutic drugs that induce DSBs. Here we
report that down-regulation of RAD21 in MCF-7 breast
tumor cells increases cellular sensitivity to two of such
drugs, etoposide and bleomycin. The topoisomerase II
inhibitor, etoposide, causes DSBs by stabilizing the DNAtopoisomerase complex, and the radiomimetic antibiotic,
bleomycin, generates DSBs presumably via binding to
iron and activating oxygen in the vicinity of DNA. DRF
values for etoposide calculated in our experiments are
similar to DRF values (1.1 – 1.4) calculated upon siRNA
down-regulation of the DNA repair genes ATM and
DNA-PK in irradiated prostate cancer cell lines (10).
These modest DRF values may be attributed to the fact
that not all surviving cells were transfected and therefore
did not have decreased RAD21 transcript levels (10). It is
likely that a more detailed dosage/exposure regimen
would determine conditions that achieve more pronounced cytotoxic effects. The observed sensitivity of
RAD21 siRNA-transfected MCF-7 cells to etoposide is
consistent with the role of RAD21 in replication and
mitotic chromatid separation, processes critically dependent on topoisomerase activity (24). The high sensitivity
of transfected cells to bleomycin is an interesting
observation that may have implications for anticancer
therapy. This effect may be attributed to the role of
RAD21 as a DNA DSB repair protein active toward metal
ion- and oxygen-dependent oxidative damage induced by
bleomycin. This damage may result in the generation of
numerous DSBs at sites other than the sites of replication.
The exact molecular basis for eukaryotic cell resistance to
bleomycin remains unclear. Our results indicate that
RAD21 may be an important factor mediating this
resistance.
In summary, we have shown that down-regulation of
RAD21 gene expression in breast cancer cell lines leads to a
marked decrease in cell growth. We also found that this
treatment enhances the cytotoxicity of two chemotherapeutic drugs, most likely by reducing the efficiency of DSB
repair. Altogether, these data suggest that RAD21 might be
a new drug target in breast cancer and that RAD21 gene –
specific therapy may be studied as an adjuvant option
augmenting the antitumor activity of traditional chemotherapeutic and radiation treatments.
Acknowledgments
We thank Dr. John Nestor for helpful discussions and critical reading of the
manuscript.
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