[CANCER RESEARCH 64, 1834 –1842, March 1, 2004]
Silencing Expression of the Clusterin/Apolipoprotein J Gene in Human Cancer
Cells Using Small Interfering RNA Induces Spontaneous Apoptosis, Reduced
Growth Ability, and Cell Sensitization to Genotoxic and Oxidative Stress
Ioannis P. Trougakos,1 Alan So,2 Burkhard Jansen,2 Martin E. Gleave,2 and Efstathios S. Gonos1
1
Laboratory of Molecular and Cellular Aging, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece, and 2Prostate Centre
at Vancouver General Hospital, Division of Urology, University of British Columbia, Vancouver, British Columbia, Canada
ABSTRACT
Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological
processes and is differentially expressed in many severe physiological
disturbances, including tumor formation and in vivo cancer progression.
Despite extensive efforts, clarification of CLU’s biological role has been
exceptionally difficult and its precise function remains elusive. Short RNA
duplexes, referred to as small interfering RNAs (siRNAs), provide a new
approach for the elucidation of gene function in human cells. Here, we
describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to
siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular
growth and higher rates of spontaneous endogenous apoptosis. Moreover,
CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H2O2,
respectively. These effects were more pronounced in cell lines that express
high endogenous steady-state levels of the CLU protein and occur through
hyperactivation of the cellular apoptotic machinery. Overall, our results
reveal that, in the distinct cellular contexts of the osteosarcoma and
prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific
siRNA oligonucleotides that can potently silence CLU gene expression may
thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.
INTRODUCTION
Clusterin/Apolipoprotein J (CLU) in humans is constitutively secreted by several cell types, and it is found in all human body fluids
analyzed thus far (1). In human plasma, CLU exists in high-density
lipoprotein particles that contain apolipoprotein A-I and cholesteryl
ester transfer protein (2). Nevertheless, CLU, apart from functioning
as an apolipoprotein, is also implicated in additional intra- or extracellular processes. The primary translation product of the CLU gene is
a polypeptide of 449 amino acids, where the first 22 amino acids
represent the classical hydrophobic secretory signal sequence. Maturation of the primary translation molecule include disulfide bonding,
conversion to a high-mannose endoplasmic reticulum-associated form
of ⬃60 kDa, extensive additional N-linked glycozylation, and finally,
proteolytic cleavage in the trans-Golgi compartments that result in the
mature secreted heterodimeric CLU protein form of ⬃70 – 80 kDa (3).
Interestingly, another CLU protein form of ⬃55 kDa, designated as
nuclear (n)-CLU55 (4), has been recently described in MCF-7 cells.
According to a recent study (5), this protein form originates, after
Received 8/26/03; revised 12/5/03; accepted 12/29/03.
Grant support: European Union Full-Cost Grants “Cellage” QLK6-CT-2001-00616
and “Functionage” QLK6-CT-2001-00310 (to E. S. Gonos).
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: Efstathios S. Gonos, Laboratory of Molecular and Cellular
Aging, Institute of Biological Research and Biotechnology, National Hellenic Research
Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece. Phone: 30-2107273756; Fax: 30-210-7273677; E-mail: [email protected].
apoptosis induction, from a 49-kDa primary product (c-CLU49) that is
translated from an alternatively spliced CLU transcript.
Many diverse physiological functions have been attributed to CLU,
including tissue remodeling, membrane recycling, lipid transportation,
cell-cell or cell-substratum interactions, and sperm maturation (6, 7).
In addition, it was recently proposed that CLU functions as an
extracellular chaperone that stabilizes stressed proteins in a foldingcompetent state (8). Another prominent feature of the CLU protein is
its up-regulation in many severe physiological disturbance states,
including kidney degenerative diseases and several neurodegenerative
conditions (7, 9). CLU also exerts a significant role during tumorigenesis and progression of several human cancers. During in vivo
progression to carcinogenesis, higher CLU levels were seen in prostate and kidney carcinomas when compared with normal tissues (10,
11), whereas CLU is also up-regulated in anaplastic large-cell lymphomas (12), in ovarian cancer (13), and during seminal vesicle
carcinogenesis progression (14). Importantly, in breast tumor cells
where epithelial normal cells were always negative for CLU expression, CLU protein accumulation correlates with the aggressiveness of
a given breast tumor (15).
From all of the functions attributed to the CLU protein thus far, its
implication in apoptosis has been primarily investigated. Despite
extensive efforts, the role of CLU during apoptosis remains largely an
enigma, the main cause being the intriguingly distinct and usually
opposing functions proposed in an array of various cell types and
tissues, including cancer cells (3) and CLU knockout mice (16, 17).
According to some studies, CLU overexpression in various cell lines
either could not confer any protection to apoptosis-inducing agents or
exerts a proapoptotic function (4, 17–20). We have cloned CLU as a
senescence-induced gene (21), and we have shown that CLU is also
overexpressed in human diploid fibroblasts exposed to various types
of stress (21–23), and it accumulates in the human serum during
diabetes type II and atherosclerosis (24). Considering that CLU overexpression in HDFs protects cells from oxidative stress (23), its
overexpression in senescent cells is unlikely to be related to a proapoptotic function (3). In addition, CLU gene overexpression in
human androgen-dependent LNCaP prostate cells rendered them
highly resistant to androgen ablation in vivo (25) and to treatment with
chemotherapeutic agents (26), whereas antisense oligonucleotides targeting CLU resulted in significant increase of chemosensitivity in
prostate cancer (27).
Given the direct involvement of the CLU protein in human carcinogenesis, as well as the fact that a therapeutic (OGX-011) inhibiting
CLU is under development for treating human prostate cancer and
other CLU-expressing malignancies (28), we sought to investigate the
effect of altering CLU gene expression and to provide conclusive
evidence regarding its function during apoptosis. We specifically
knocked down the secreted (s)-CLU protein form in three osteosarcoma (OS) cell lines and in a prostate cancer cell line by using small
interfering RNAs (siRNAs) and provide evidence that CLU knockdown induces growth retardation that is accompanied by higher rates
of spontaneous endogenous apoptosis. Moreover, CLU knockdown
1834
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
resulted in significant sensitization to both genotoxic and oxidative
stress-inducing agents. Our data suggests that cell sensitization may
be directly related to activation of the cellular apoptotic machinery.
MATERIALS AND METHODS
Cell Lines and Culture Conditions. OS cell lines (KH OS, Sa OS, and
U-2 OS) and PC-3 cells were purchased from the American Tissue Culture
Collection. OS cells were cultured in DMEM (Life Technologies, Inc., San
Diego, CA) supplemented with 10% FCS, 2 mM glutamine, and 1% nonessential amino acids (complete medium) and PC-3 cells in DMEM supplemented with 5% heat-inactivated FCS.
Identification of Suitable CLU cDNA Sequences to be Targeted by
siRNA–RNA Oligonucleotide Synthesis. The human CLU cDNA was
scanned to identify sequences of the AA(N19)UU type that fulfill the required
criteria for siRNA (29). Two such sequences were found 433 (Cl-I) and 664
(Cl-II) nucleotides downstream of the CLU gene transcription initiation codon.
Two additional oligonucleotides used targeted a region 1620 nucleotides
downstream of the CLU gene transcription initiation codon (Cl-III) and the
transcription initiation site (Cl-V; Table 1). BLAST analysis showed no
homology with other known human genes. Selected RNA oligos were synthesized by Dharmacon Research, Inc. (Lafayette, CO). The Scramble-I (Sc-I) and
Scramble-II (Sc-II) oligonucleotides used were purchased from Dharmacon.
siRNA Transfection. siRNA transfection of the Cl-I, Cl-II, and scrambled
RNA duplexes in exponentially growing OS cells was performed as described
previously (29, 30). Briefly, cells were seeded the day before siRNA transfection in 24-well plates containing 500 ␮l of complete medium and were
⬃40 –50% confluent during transfection. For the transfection mixture, 100 nM
siRNA duplex/well were used. The RNA duplex was diluted in Opti-MEM I
(Life Technologies, Inc.) serum-free medium, and transfection efficiency was
enhanced by using the Oligofectamine reagent (Invitrogen-Life Technologies
Inc., San Diego, CA). When cells were treated with both Cl-I and Cl-II
oligonucleotides, 100 nM of each siRNA duplex were used. Cell treatment with
the siRNA oligonucleotides lasted for 2–3 days. In PC-3 cells, Lipofectin
(Invitrogen) was used. PC-3 cells were treated with 10, 50, or 100 nM of the
RNA duplexes after preincubation with 4 ␮g/ml Oligofectamine reagent in
serum-free Opti-MEM I for 20 min. Four h after starting the incubation, the
medium containing the RNA duplexes and Lipofectin was replaced with
standard medium. Cells were treated once on day 1 and then harvested 48 h
after treatment on day 3.
RNA Analysis—Immunoblotting Analysis—Immunofluorescence Antigen Staining. For RNA analysis, samples were transferred to nylon membranes (Amersham Life Science, Arlington Heights, IL) and hybridized with a
human CLU or glyceraldehyde-3-phosphate dehydrogenase probe (27). Density of bands was calibrated by densitometric analysis.
For immunoblotting analysis, cell monolayers were directly lysed in reducing or nonreducing Laemmli buffer, and samples were adjusted by Bradford
(Bio-Rad, Hercules, CA) assay. Loading was verified with an antiactin- or an
antivinculin-specific antibody. To detect phosphorylated double-stranded
RNA-dependent protein kinase, cells were lysed in a buffer containing 50 mM
Tris-HCL (pH 8.0), 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium
deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and a mixture of phosphatase and protease inhibitors (P2850/P5726 and P8340, respectively; Sigma
Chemical Co., St. Louis, MO). Protein fractionation by SDS/PAGE followed
by immunoblotting analysis was done as described previously (22). The
antihuman CLU polyclonal antibodies used were the sc-6419 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) that mainly cross-reacts with the cytoplasmic CLU and s-CLU protein forms (see figures) and the sc-8354 (Santa
Cruz Biotechnology, Inc.) that mainly binds to a putative n-CLU protein form
(5). Antibodies against actin, Stat1, phosphorylated double-stranded RNAdependent protein kinase (binds a phosphorylated-activated form of PKR),
poly(ADP-ribose) polymerase, bcl-2, pan-p53 (detects both wild type and
mutant p53), p21, bax, and Ku70 were purchased from Santa Cruz Biotechnology, Inc. The antibodies against OAS-1, MECL-1(␤2i), and vinculin were
purchased from Orbigen (Orbigen, Inc., San Diego), AFFINITI (AFFINITI
Research Products Ltd., Exeter, United Kingdom) and Sigma, respectively,
whereas the antibody against LMP7 (␤5i) was a generous gift from Dr. Klavs
Hendil.
CLU immunofluorescence labeling of siRNA-treated or control cells was
done as previously described (22) by using the sc-6419 antibody. Images of the
mounted coverslips were taken by using a Leica TCS SP-1 Confocal Laser
Scanning Microscope.
Clonogenic and Survival Assays of siRNA-Treated Cells. To analyze the
relative growth rate of the siRNA treated cells, 5 ⫻ 103 cells were seeded and
after treatment for 70 h, the total cell number was counted by using a Coulter
Counter (Coulter Counter Z series). For the clonogenic assays, cells were
washed with complete medium, trypsinized, and 5 ⫻ 103 cells were reseeded
in 6-well plates. Total cell number as well as the number and size of the
methylene blue-stained colonies were then recorded after 5–9 days of growth
(depending on the cell line used; see “Results”) in complete medium. Finally,
for the survival assays, cells treated with the siRNA oligonucleotides for 70 h
were either: (a) seeded in 6-well plates containing complete medium, allowed
to recover for 24 h, treated with 0.35 ␮M doxorubicin (DXR; Sigma) for 24 h
and subcultured in complete medium for another period of 72 h before
counting, or (b) DXR was added directly to the transfection medium to a final
concentration of 0.35 ␮M, cells were incubated in the drug-containing transfection medium for 24 h, washed, allowed to recover for 72 h in complete
medium, and counted.
Cell Proliferation–DNA Synthesis Analysis. For the quantitative determination of the DNA synthesis rate, the Cell Proliferation ELISA BrdUrd
colorimetric immunoassay method (Roche, Inc., Basel, Switzerland) was used.
Briefly, 5 ⫻ 103 cells were seeded in sextuplicates in 96-well plates and treated
with either the Sc-I oligonucleotide or the Cl-I CLU-specific oligonucleotide
for 60 h. Cells were then labeled for 3 h with bromodeoxyuridine, and DNA
synthesis was measured with the ELISA BrdUrd assay according to the
manufacturer’s instructions.
Quantitative Analysis of Endogenous Cellular Apoptosis or Apoptosis
after Cell Treatment with DXR or H2O2–Cell Death Detection by Terminal Deoxynucleotidyl Transferase-Mediated Nick End Labeling (TUNEL)
Assay. To assay the endogenous spontaneous cellular apoptosis, cells were
treated with either the Sc-I or the Cl-I siRNA duplexes for 60 h. Subsequently,
the cytoplasmic histone-associated DNA fragments, which are indicative of
ongoing apoptosis, were quantitatively measured by using the Cell Death
Detection ELISAPLUS photometric enzyme-immunoassay method (Roche,
Inc.) according to the manufacturer’s instructions.
To assay cell sensitization and the extent of apoptosis induction after DNA
damage or oxidative stress, an equal number of siRNA-treated cells were
subcultured in complete medium containing either: (a) 0.35 or 1 ␮M DXR for
24 h or (b) 400 ␮M H2O2 for 3 h followed by a recovery period in complete
medium for 21 h. Apoptosis induction was then quantified by using the
ELISAPLUS death assay.
To detect DNA fragmentation in cell monolayers after DXR treatment,
Table 1 Sequence and specific characteristics of the Cl-I, Cl-II, Cl-III, and Cl-V oligonucleotidesa
Cl-I
Cl-II
Targeted region
Sense small interfering RNA (siRNA)
Antisense siRNA
5⬘
5⬘
5⬘
AAccagagctcgcccttctacTT
ccagagcucgcccuucuacdTdT
guagaagggcgagcucuggdTdT
Targeted region
Sense siRNA
Antisense siRNA
5⬘
5⬘
5⬘
AActaattcaataaaactgtcTT
cuaauucaauaaaacugucdTdT
gacaguuuuauugaauuagdTdT
3⬘
3⬘
3⬘
5⬘
5⬘
5⬘
AAgtcccgcatcgtccgcagcTT
gucccgcaucguccgcagcdTdT
gcugcggacgaugcgggacdTdT
3⬘
3⬘
3⬘
5⬘
5⬘
5⬘
GCatgatgaagactctgctgcTG
augaugaagacucugcugc
gcagcagagucuucaucau
Cl-III
a
3⬘
3⬘
3⬘
Cl-V
The Cl-I, Cl-II, Cl-III and Cl-V CLU-specific siRNA oligonucleotides have a GC/AT ratio content of 57/43, 67/33, 24/76, and 48/52%, respectively.
1835
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
3⬘
3⬘
3⬘
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
TUNEL was performed by using the in situ Cell Death Detection kit (Roche,
Inc.) that relies on fluorescent labeling of DNA strand breaks. Briefly, equal
cells number were seeded in duplicates in 6-well plates, allowed to attach
overnight on coverslips, and treated with 0.35 or 1 ␮M DXR for 24 h.
Subsequently, cells were either washed and DNA fragmentation was detected
by TUNEL according to the manufacturer’s instructions or allowed to grow for
additional 24 h in drug-free medium before the application of the TUNEL
reaction. Positive apoptotic nuclei were recorded by fluorescence microscopy.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay.
The combined effects of siRNA plus Paclitaxel (Sigma) on PC-3 cells were
assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described previously (27). Briefly, 3 ⫻ 103 cells were plated in
sextuplicates in 96-well plates, allowed to attach overnight, treated with 50 nM
of either Cl-III, or Cl-V or Sc-I control oligonucleotides for 4 h and allowed
to recover for 48 h. Cells were then exposed to various concentrations of
Paclitaxel, ranging from 0 to 500 nM, for 48 h before assaying cell survival
with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
RESULTS
OS Cells as a Model System to Study CLU Function in Human
Cancer Cells. We hypothesized that the diverse functions attributed
to CLU during apoptosis in different cellular systems and tissues
could reflect specific intrinsic properties of the cell lines used. Therefore, an ideal in vitro model system to study CLU function should
fulfill the following criteria. Firstly, the selected cell lines should be
closely related and they should preferentially originate from the same
tissue. Secondly, they should be genetically well characterized and
they should express different steady-state endogenous CLU protein
levels. Finally, in the selected cell lines, CLU protein should accumulate at a distinct rate after cell exposure to apoptosis-inducing
agents.
After a survey of several human cell lines and subsequent analysis
of the various CLU protein forms in these cells, we concluded that
three OS cell lines, namely U-2 OS, KH OS, and Sa OS, fulfill these
criteria. As it can been seen in Fig. 1A, the selected OS cell lines
Fig. 1. A, steady-state endogenous levels of the clusterin (CLU) protein (top panel) in
OS cell lines, as revealed by immunoblotting analysis of whole cell lysates under
nonreducing conditions. The Sa OS and KH OS cells express significantly lower levels of
CLU protein as compared with U-2 OS cells. The OS cell lines used are distinct, as far as
the status of several oncogenes or tumor suppressor proteins (e.g., p53, middle panel) is
concerned. B, CLU protein kinetics in Sa OS (top panel), KH OS (middle panel), and U-2
OS cells (bottom panel) after cell treatment with 0.35 ␮M doxorubicin (DXR) for 24 h.
Although CLU protein is induced in all three cell lines after drug treatment, its upregulation is more intense in the KH OS and Sa OS cells as compared with U-2 OS cells.
Secreted (s)-CLU, the mature secreted protein form of ⬃75 kDa; cytoplasmic (c)-CLU,
the intracellular precursor protein form of ⬃60 kDa. Protein loading was verified by actin
(bottom panels). Molecular weight markers are indicated on the right of each blot.
express different endogenous CLU protein amounts, with the U-2 OS
being the cell line with the highest CLU protein content (Fig. 1A, top
panel). Moreover, these cells are well characterized regarding their
genetic background because for instance the p53 gene is deleted in Sa
OS cells, mutated in KH OS, but it is wild type in U-2 OS cells (Ref.
31; Fig. 1A, middle panel). Finally, we found that the steady endogenous CLU protein levels negatively correlate with the intensity
of CLU protein accumulation after cell exposure to various DNA
damage-inducing agents (Fig. 1B). The highest rate of CLU accumulation
after cell exposure to DXR was found in KH OS cells that express the
lowest endogenous CLU protein levels. In contrast, a moderate induction
(up to 2-fold) of the CLU protein can been seen in the U-2 OS cells that
express high endogenous levels of the CLU protein.
Efficient Silencing of the CLU Gene Expression in OS Cells by
Using siRNA. Treatment of the three OS cell lines with the Cl-I or
the Cl-II siRNA oligonucleotides was quite effective and resulted in
significant knock down of the cellular CLU protein levels (Fig. 2A–
2C2). The Cl-I oligonucleotide appeared to be slightly more effective
than Cl-II in silencing the CLU gene. No CLU gene silencing was seen
in the presence of the control Sc-I or the Sc-II (data not shown)
oligonucleotides or in the absence of RNA duplexes from the transfection medium.
Next we addressed the issue whether the CLU-specific siRNA
oligonucleotides could also inhibit the accumulation of the CLU
protein after cellular exposure to DXR. As shown in Fig. 2, D and E,
exposure of the KH OS and U-2 OS cells to DXR for 24 h in the
presence of the Cl-I, Cl-II or a mixture of both the Cl-I, Cl-II
oligonucleotides effectively abolished CLU protein accumulation. It is
thus evident that in the presence of the Cl-I or the Cl-II oligonucleotides, the cellular CLU protein cannot be induced after cell exposure
to apoptosis-inducing agents.
Phenotypic Effects in OS Cells after the Silencing of CLU Gene
Expression by siRNA. The effects of silencing of the CLU gene
expression in OS cells were studied by direct counting of the cells
following siRNA by recording both cellular morphology and phenotype, as well as by clonogenic assays. CLU knockdown in KH OS and
Sa OS cells did not result in any visible change on phenotype.
However, the CLU knock down cells were found to be significantly
growth retarded as compared with their control counterparts (Fig. 3A).
In contrast at the U-2 OS cells, which express higher endogenous
amounts of the CLU protein, the effects of CLU knockdown were
more pronounced. U-2 OS-treated cells lost their firm adherence to
plastic and acquired a rounding morphology (Fig. 3B). This phenotype
was accompanied by severe growth retardation (Fig. 3A). To study
whether a combination of both Cl-I and Cl-II RNA duplexes would be
more effective in inhibiting cell growth, we treated cells with both
these oligonucleotides. As shown in Fig. 3A for the KH OS and U-2
OS cells, only a slight increase in growth retardation was observed as
compared with the Cl-I-treated cells. Finally, to distinguish between
the cytostatic and cytotoxic effects of CLU protein elimination, we
directly assayed CLU knock down KH OS and U-2 OS cells for DNA
synthesis and spontaneous endogenous apoptosis. CLU knock down
cells showed a reduced DNA synthesis rate (Fig. 3C) and higher levels
of spontaneous endogenous apoptosis (Fig. 3D) as compared with
their sibling controls. Effects were again more pronounced in U-2 OS
cells. In summary, these results suggest that the reduced number of
CLU knock down cells is due to both a reduced rate of cell proliferation and an increased level of spontaneous apoptosis.
As it was reported recently that transfection of siRNAs or DNA
vectors that express shRNAs from RNA Pol III promoters can trigger
an IFN response (32, 33), we assayed our Sa OS and U-2 OS
siRNA-transfected cells lines for the expression levels of a number of
IFN-type I and type II genes. These include the type I viral-stimulated
1836
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
the vectors, although reduced the level of the target gene by ⬃80%,
did not induce the IFN system (32). Conclusively, the phenotypic
effects of CLU knockdown in OS cells are highly specific and are not
related to the activation of any off-target IFN-inducible gene.
As recent studies have demonstrated that gene silencing is sustained
for more than seven cellular doublings (29), the effect of CLU
knockdown in plating efficiency and growth after siRNA was subsequently studied by clonogenic assays. KH OS and U-2 OS cells were
selected for these assays because they represent two extreme opposite
cases as far as the endogenous CLU amount and the intensity of CLU
accumulation during stress are concerned. CLU knock down KH OS
cells when plated were firmly attached to the plastic (⬎90%) and only
a few of the attached cells showed an abnormal morphology. However, the growth potential of the adherent cells was impaired as found
5 days postplating after analyzing the total colony number and size of
the formed colonies (Fig. 4A1– 4A3). CLU knock down U-2 OS cells
were poorly attached to the plastic after trypsinization (⬃70%), and
Fig. 2. A–C1, effective silencing of the clusterin (CLU) gene expression in OS cells
after small interfering RNA (siRNA) treatment for 60 h as revealed by whole-cell lysate
immunoblotting analysis (reducing conditions) of the CLU protein endogenous levels in
Sa OS (A), KH OS (B), and U-2 OS (C1) cells. C2, confocal laser scanning microscopy
after CLU immunofluorescence localization in U-2 OS cells treated for 48 h with siRNA.
Cl-II or Cl-I siRNA-treated cells show minimal levels of immunofluorescence. D and E,
whole-cell lysate immunoblotting analysis of CLU protein levels in KH OS (D) and U-2
OS (E) cells after siRNA for 70 h and subsequent treatment with 0.35 ␮M doxorubicin
(DXR) for 24 h in the presence of the indicated siRNA oligonucleotides. In the presence
of the specific CLU siRNA oligonucleotides, the DXR-treated OS cells cannot accumulate
the CLU protein. Con-I, mock-transfected cells; Sc-I, cells treated with the Scramble-I
oligonucleotide; Cl-II, Cl-I, cells treated with the CLU oligonucleotides Cl-II or Cl-I,
respectively; Cl-I/II cells treated with a mixture of both CL-I and Cl-II oligonucleotides;
secreted (s)-CLU, the ␣-, ␤- chains (⬃40 kDa) of the secreted CLU protein form after
reduction of the mature protein; cytoplasmic (c)-CLU, the intracellular did not reducedprecursor CLU protein form of ⬃60 kDa. Protein loading was verified by actin (bottom
panels in A, B, C1, D, and E). Molecular weight markers are indicated on the right of each
blot. All assays were repeated at least three times. Bars in C2, 10 ␮M.
proteins OAS-1, Stat1, and phosphorylated double-stranded RNAdependent protein kinase as well as the type II ␤-subunits of the 20S
immunoproteasome LMP7 and MECL-I (34, 35). As can been seen in
Fig. 3E, none of the IFN-type I or -type II-inducible genes were
up-regulated. Similarly, no activation of the IFN-type I pathway was
found in four normal human cell lines after prolonged exposure to
siRNA duplexes (see supplementary figure). These results are consistent with the reported genome-wide expression-profiling studies
(36, 37) and with the observation that in human lung fibroblasts,
although DNA vectors that express shRNAs can trigger the IFN
response, transfection of the same cells with chemically synthesized
siRNA duplexes corresponding to the shRNAs species produced by
Fig. 3. A, relative growth rate of Sa OS, KH OS, and U-2 OS cells after small
interfering RNA (siRNA)-mediated clusterin (CLU) gene expression silencing. Cells were
treated with the indicated siRNAs for 70 h, and the total cell number was counted. A
significant reduction of the cell number that is more pronounced in the U-2 OS cells can
be seen in all CLU knocked down cells. Only a slight enhancement in growth retardation
is observed when a combination of Cl-I and Cl-II oligonucleotides (Cl-I/II) was used.
Growth of mock-transfected (Con-I) cells was set at 100%. B, morphological alterations
in U-2 OS cells treated with the indicated siRNA oligonucleotides for three days. Note the
rounding and shrinkage observed in the CLU knock down cells (arrow). C and D,
endogenous DNA synthesis levels and spontaneous apoptosis in CLU knock down KH OS
and U-2 OS cells, as estimated by using a Cell Proliferation ELISA BrdUrd colorimetric
immunoassay (C) and a Cell Death Detection ELISA photometric enzyme-immunoassay
(D), respectively. CLU knockdown is accompanied by reduced DNA synthesis (C) and an
enhanced rate of endogenous spontaneous cellular apoptosis (D) in both cell lines. The
effects are significantly pronounced in U-2 OS that express higher endogenous CLU
levels. E, CLU knockdown by chemically synthesized siRNA duplexes does not induce an
IFN response in OS cells. Sa OS and U-2 OS cells were treated with 100 nM of the
indicated siRNA duplexes for 48 h. Subsequent immunoblotting analysis revealed that the
presence of the siRNAs studied did not induce IFN type I (OAS-1, Stat1, and P-PKR) or
type II (LMP7 and MECL-1)-stimulated genes. All assays were performed in triplicates
and have been repeated two (C–E) or three (A) times. Each data point represents the mean
of the independent experiments and bars denote SD; ⴱ indicates differences from their
respective controls at P ⬍ 0.01; n.a., not assayed; abbreviations are as in Fig. 2. Bars in
B, 15 ␮M.
1837
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
by DXR in OS cell lines rely on both p53-dependent and p53independent mechanisms.
Next, we followed two complementary approaches to study the
effect of CLU knockdown in OS cells exposed to DXR (Fig. 5, C1 and
C2). siRNA-treated cells were either replated in complete medium and
Fig. 4. Reduced clonogenic potential of clusterin (CLU) knocked down OS cells. A1,
after CLU small interfering RNA (siRNA) for 70 h, KH OS cells were seeded in 6-well
plates, and cells were counted after 5 days of growth in complete medium. A comparative
presentation of the observed colonies number and size is shown in (A2) and (A3),
respectively. B1, corresponding analysis in U-2 OS cells. B2, representative colonies size,
9 days postplating, after a clonogenic assay of CLU siRNA-treated U-2 OS cells. The
growth potential of CLU knocked down U-2 OS cells was severely affected. Cell number
of mock-transfected cells (Con-I), after normalization for the adherence efficiency during
replating of the CLU knock down cells (see text), was set at 100%; assays were done in
triplicates and were repeated three times. Data points represent the mean of the independent experiments; bars denote SD; ⴱ indicates differences from their respective controls at
P ⬍ 0.01. Abbreviations are as in Fig. 2. Bars in A3 and B2, 15 ␮M.
most of the adherent cells appeared quite abnormal in shape. Cells
showed an extremely low proliferation potential (Fig. 4B1), and after
9 days in culture only, some small colonies could be seen (Fig. 4B2).
Sustained Silencing of CLU Gene Expression in OS Cells by
siRNA Results in Significant Sensitization to Apoptosis Induced
by Genotoxic and Oxidative Stress. Before CLU functional assays,
we analyzed the DXR effects in OS cells because the drug-related
reported effects vary in different cell types (38). As the DXR plasma
concentration in treated patients fall into a range of 1–2 ␮M and
decline into a range of 0.025– 0.25 ␮M within 1 h (39), cells were
treated with 0.35 and 1 ␮M of DXR. To analyze the extent of the
DXR-mediated cell death, we scored apoptosis by TUNEL. A representative analysis in KH OS cells is shown in Fig. 5A. Attached cells
after drug treatment for 24 h underwent significant morphological
changes as compared with nontreated control cells (Fig. 5A, a1). At
this time, cells exhibit an enlarged and flattened morphology that is
reminiscent of senescence, whereas a significant number of them are
TUNEL positive (Fig. 5A, a2 and a3). Ongoing apoptosis is also
apparent 24 h later (Fig. 5A, a4 and a5), verifying that apoptosis is a
dynamic process that continues even after DXR removal from the
medium. In agreement with the results obtained by TUNEL, DXR
treatment was accompanied by poly(ADP-ribose) polymerase cleavage; the antiapoptotic protein bcl-2 showed no altered expression in
drug-treated KH OS and U-2 OS cells (Fig. 5B). After DXR treatment,
accumulation of p53 protein and its downstream effectors related to
either growth arrest (p21) or apoptosis (bax) was found only in U-2
OS cells, indicating that the cytostatic and cytotoxic effects mediated
Fig. 5. Effect of doxorubicin (DXR) treatment in OS cells and cell sensitization to
DNA damage and oxidative stress after small interfering RNA (siRNA)-mediated clusterin (CLU) knock down. A, confocal micrographs demonstrating in situ identification of
DNA fragmentation by terminal deoxynucleotidyl transferase-mediated nick end labeling
(TUNEL) assay in control (a1) and 0.35 ␮M (a2 and a4) or 1 ␮M (a3 and a5) DXR-treated
KH OS cells. Cells were treated with DXR for either 24 h before TUNEL (a2 and a3) or
for 24 h followed by 24 h recovery in DXR-free medium (a4 and a5). DXR-treated cells
exhibit an enlarged and flattened morphology. Arrows indicate TUNEL-positive cells. B,
immunoblotting analysis of the indicated proteins in nontreated (C), 0.35 ␮M DXR
exposed for 24 h (24), and 0.35 ␮M DXR exposed for 24 h followed by postincubation in
drug-free medium for additional 24 h (24⫹24) KH OS (left panel) and U-2 OS (right
panel) cells. Arrow at poly(ADP-ribose) polymerase (PARP) immunoblots indicates the
noncleaved 115 kDa form and arrowhead the cleaved apoptosis-related 85 kDa protein
fragment. Molecular weight markers are shown at the right of each blot. (C1 and C2, f)
2 ⫻ 104 KH OS (C1) or U-2 OS (C2) cells were seeded in 24-well plates in complete
medium, siRNA treated for 70 h, and allowed to recover for 24 h. Cells were then exposed
to 0.35 ␮M DXR for 24 h, subcultured in complete medium for 72 h, and counted. (C1 and
C2, 䡺) KH OS (C1) or U-2 OS (C2) cells were siRNA treated for 70 h, and DXR was
added directly to the transfection medium at a final concentration of 0.35 ␮M. Cells were
then incubated in the drug-containing transfection medium for additional 24 h, washed,
allowed to recover in complete medium for 72 h, and counted. The combined chemotherapeutic drug treatment and CLU siRNA is more effective in promoting OS cells death.
Survival of control cells (Con-I) was set at 100%. D1 and D2, enrichment of the
cytoplasmic histone-associated-DNA fragments that are indicative of an ongoing apoptosis in KH OS (D1) and U-2 OS (D2) cells after siRNA with the indicated siRNA
oligonucleotides for 60 h and postcultivation in siRNA-free medium containing 0.35 or 1
␮M DXR for 24 h. E1 and E2, enrichment of the cytoplasmic histone-associated-DNA
fragments in KH OS (E1) and U-2 OS (E2) cells after siRNA with the indicated siRNA
oligonucleotides for 60 h, exposure to 400 ␮M H2O2 in a siRNA-free medium for 3 h, and
postcultivation in normal complete medium for 21 h. CLU protein knock down results in
significant cell sensitization to both genotoxic and oxidative stress. Samples were analyzed in duplicates, and data points represent the mean of three independent experiments;
bars denote SD; ⴱ indicates differences from their respective controls at P ⬍ 0.01 and ⴱⴱ
differences from dark bars (in C1 and C2) at P ⬍ 0.05. Abbreviations are as in Fig. 2. Bars
in (a1–a5), 20 ␮M.
1838
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
were subsequently exposed to 0.35 ␮M DXR for 24 h, or they were
exposed to 0.35 ␮M DXR in the presence of the Cl-II or Cl-I oligonucleotides. In both cases, viable cells were counted 3 days after DXR
treatment. CLU knock down KH OS cells appeared more sensitive to
DXR than their control counterparts (Fig. 5C1, f), whereas DXR
treatment appeared significantly more effective when it was combined
with the presence of the CLU-specific siRNA oligonucleotides in the
medium (Fig. 5C1, 䡺). Similarly, CLU knock down Sa OS cells were
more sensitive to the DXR treatment (data not shown). In U-2 OS
cells, both strategies appeared very effective, and CLU knock down
cells were significantly more sensitive to the drug as compared with
controls (Fig. 5C2). When DXR treatment was performed in the
presence of the CLU-specific siRNA oligonucleotides, the CLU
knocked down cells were significantly more sensitive to the drug (Fig.
5C2, 䡺), and massive apoptosis was observed. Finally, when U-2 OS
cells were treated with both the Cl-I and Cl-II oligonucleotides, cells
were almost eliminated (data not shown).
To understand the mechanism of cell sensitization after CLU knockdown, we directly assayed the intensity of apoptosis induction right after
cell exposure to agents inducing genotoxic (DXR) or oxidative stress
(H2O2). As shown in Fig. 5, D and E, exposure of the CLU knock down
cells to either DXR or H2O2 resulted in a significantly higher rate of
apoptosis in both KH OS and U-2 OS cells. This observation suggests
that CLU directly affects or interacts with the cellular machinery involved
in apoptosis by providing cytoprotective signals.
OS Cells Sensitization to Genotoxic and Oxidative Stress Because of CLU Gene Silencing Is Related to Activation of the
Cellular Apoptotic Machinery. By analyzing the expression levels
of other recently identified CLU protein forms, to our surprise, we
found that the Cl-I and Cl-II oligonucleotides did not exert any
significant effect on the putative 55 kDa (n-CLU55) CLU protein form
in both KH OS and U-2 OS cells; only a minor effect on the 49 kDa
c-CLU49 protein form level was detected (Fig. 6, A and B), although
the binding sites of the Cl-I or the Cl-II oligonucleotides are common
between the s-CLU and n-CLU mRNAs (5). We assume that the
explanation of this effect relies on our observation that the n-CLU
protein is extremely stable (unpublished data). Thus, CL-I and CL-II
oligonucleotides specifically knocked down the s-CLU protein form.
We then assayed the expression levels of several proteins involved
in regulating apoptosis in human cells. As it can be seen in Fig. 6, A
and B, CLU knockdown in both KH OS and U-2 OS cells resulted in
the down-regulation of the antiapoptotic molecule bcl-2. No effect
was detected on levels of Ku70, a protein implicated in DNA damage
repair and signaling that, moreover, binds n-CLU (4). Interestingly, in
U-2 OS cells, which bear a functional p53 molecule, CLU knockdown
apart from bcl-2 down-regulation is also accompanied by p53 accumulation and up-regulation of both its downstream proapoptotic effector, bax, and the cyclin-dependent kinase inhibitor, p21 (Fig. 6B).
Moreover, CLU knock down U-2 OS cells when exposed to DXR
showed a more intense and robust accumulation of the p53 protein
(Fig. 6C) as compared with the Sc-I-treated cells. We suggest that
sensitization of OS cells after CLU knockdown largely depends on the
activation of the cellular proapoptotic machinery.
Effects of CLU Gene Silencing in PC-3 Prostate Cancer Cells.
Having established the significant effects of CLU knockdown in OS
cells, we then applied CLU siRNA in PC-3 human prostate cancer
cells. PC-3 cells are p53 null (40) and express relatively low endogenous amounts of the s-CLU protein form similar to the Sa OS cells.
In these cells, apart from using the Cl-I and Cl-II oligonucleotides, we
also tested two additional CLU-specific siRNA oligonucleotides (ClIII, Cl-V; Cl-V targets the s-CLU transcription initiation site). Usage
of the Cl-I and Cl-II siRNA oligonucleotides in PC-3 cells resulted in
similar effects to those described for OS cell lines (data not shown).
As can been seen in Fig. 7A–C, both the Cl-III and Cl-V oligonucleotides are quite effective in silencing CLU mRNA and protein expression in a sequence-dependent but dose-independent manner. More
specifically, treatment of the PC-3 cells, for 1 day, with 10, 50, and
100 nM of either Cl-III or Cl-V siRNA oligonucleotides severely
reduced CLU mRNA levels ranging from 60 to 98% (Fig. 7, A and B).
This effect on mRNA levels was also evident at the protein level (Fig.
7C). Additionally and in agreement with findings in the U-2 OS cells,
CLU knockdown in PC-3 cells resulted in significant morphological
changes that resembled an ongoing apoptosis (Fig. 7D).
To determine whether treatment of PC-3 cells with CLU siRNA
oligonucleotides could enhance the cytotoxic effects of chemotherapeutic drugs, PC-3 cells were treated first with the Cl-III, Cl-V or the
Sc-I siRNA oligonucleotides and then incubated with medium containing various concentrations of paclitaxel for 2 days. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were
then performed to determine cell viability. As shown in Fig. 8, CLU
siRNA treatment significantly enhanced chemosensitivity of paclitaxel in a dose-dependent manner reducing the IC50 (the concentration
that reduces cell viability by 50%) of paclitaxel by ⬎90%, whereas
the scrambled siRNA had no effect.
DISCUSSION
Fig. 6. A and B, effect of clusterin (CLU) knock down [small interfering RNA (siRNA)
treatment for 60 h] in the steady-state levels of proteins related to the cellular anti- or
proapoptotic machinery in KH OS (A) and U-2 OS (B) cells. The CLU-specific siRNA
oligos used do not affect the levels of the putative nuclear (n)-CLU protein form of 55 kDa
(n-CLU55); a minor effect can be seen at the cytoplasmic protein form of 49 kDa
(c-CLU49). CLU knockdown induces down-regulation of the bcl-2 protein in both cell
lines that, in p53WT U-2 OS cells, is accompanied with p53, Bax, and p21 accumulation.
C, U-2 OS cells treated with Sc-I or Cl-I, Cl-I/II oligonucleotides for 70 h were exposed
to 0.35 ␮M doxorubicin (DXR) for 24 h, and cell lysates were analyzed for p53
accumulation. CLU knock down cells show a more intense and robust p53 accumulation
after DNA damage by DXR, as compared with controls.
CLU is an enigmatic molecule, and despite extensive studies, it remains unclear whether its functions reflect multifunctionality or alternatively they mask a common function. In particular, CLU’s implication in
cell death is still debatable because according to the relatively few studies
where the effects of experimental manipulation of CLU expression on
cell death and survival were analyzed, CLU reportedly relates to both a
proapoptotic and an antiapoptotic function (see “Introduction”). The
ability of 21-nt duplexes of siRNA to direct sequence-specific degradation of mRNA (29, 30) and thereby induce specific gene silencing in
mammalian cells has raised the possibility that siRNA can be used to
investigate gene function in a high-throughput fashion or to modulate
gene expression in human diseases. Our presented results clearly dem-
1839
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
Fig. 7. Sequence-specific clusterin (CLU) gene silencing by siRNA in PC-3 tumor
cells. A, PC-3 cells were treated with the shown concentrations of the indicated siRNA
oligonucleotides for 1 day. CLU and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA levels were then assayed by RNA blot analysis. B, quantitative analysis
of the CLU mRNA levels shown in (A) after normalization to GAPDH mRNA levels.
Each point represents the mean of triplicate analyses; bars represent SD; ⴱ differ from
control at P ⬍ 0.001. C, immunoblotting analysis (reducing conditions) of the secreted
(s)-CLU and cytoplasmic (c)-CLU protein forms levels in cells treated with the indicated
siRNA oligonucleotides as in (A). CLU siRNA is PC-3 cells is particularly effective.
Vinculin immunoblotting was used to estimate protein loading (bottom panels). D,
morphological changes two days after a daily treatment with 20 nM of the indicated siRNA
oligonucleotides. Note the rounding and shrinkage of PC-3 cells treated with the Cl-V
oligonucleotide (arrows) that are morphological changes suggestive of apoptosis. Cl-III,
Cl-V cells treated with the Cl-III or the Cl-V CLU siRNA oligonucleotides, respectively;
abbreviations are as in Fig. 2; bars in D, 30 ␮M.
onstrate that CLU mRNA is amenable to specific siRNA-induced degradation in vitro and that the secreted CLU protein form is an essential
molecule for the cellular homeostasis of the OS and prostate cancer cells
assayed. Moreover, it is evident that the primary function of CLU in all
distinct genetic backgrounds of these cancer cells is antiapoptotic. The
effects of CLU gene expression silencing are mainly related to the
endogenous CLU protein levels expressed by a given cell line and are
particularly highlighted in U-2 OS that express the highest CLU endogenous amounts among the cell lines assayed in this study. In U-2 OS
cells, CLU knockdown resulted in significant growth retardation, higher
rates of endogenous apoptosis, decreased plating efficiency (that may
indicate a reduced metastatic potential), and significant sensitization to
apoptosis-inducing agents. Interestingly, it was recently shown that CLU
over-expression into human renal cell carcinoma cells enhances their
metastatic potential (41). The proposed essential role of CLU for cellular
homeostasis is clearly supported by the fact that CLU is associated with
cells surviving programmed cell death during development (42, 43) and,
in addition, it is highly expressed in an array of specialized cell types of
adult human tissues and almost in all epithelial boundary cells and several
nonepithelial secretory cell types (44). In agreement with the presented
data, we previously demonstrated a cytoprotective role of the secreted
CLU protein form in HDFs during cellular senescence (22, 23). Moreover, we have found that CLU antisense oligonucleotides chemosensitize
human PC-3 prostate cancer cells (27), bladder cancer cells (45) and
human renal cell CAKI-2 tumors (46) both in vitro and in vivo and
showed that CLU overexpression in prostate cancer xenograft models
results in acquisition of a chemoresistant phenotype (26). Similarly,
according to a series of studies in CLU⫺/⫺ mice, it has been shown that:
(a) CLU has anti-inflammatory properties and limits the severity of
murine autoimmune myocarditis (47); (b) protects against ischemic brain
damage and reduces postischemic cell mortality in the brain (16); (c) has
a protective effect against heat-shock-mediated apoptosis (48); and (d)
exerts a protective role against chronic glomerular kidney disease (49).
Interestingly, no effects have been previously reported on cellular growth,
DNA synthesis or spontaneous endogenous apoptosis in CLU⫺/⫺ mouse
embryonic fibroblasts (16, 17) or in human cancer cells where CLU
levels were reduced by using antisense oligonucleotides (27). Our unpublished comparative analysis shows that siRNA treatment is significantly more effective in knocking down the CLU protein levels than
antisense oligonucleotides. Regarding differences between analysis in
CLU⫺/⫺ mouse embryonic fibroblasts and our in vitro data, it is plausible
CLU knockdown to result distinct phenotypes in normal and in cancer
cells. Additionally, considering that CLU overexpression in SV40immortalized human prostate epithelial cells slows down cell cycle progression, thus suggesting that CLU is a putative tumor suppressor gene
(50), whereas CLU exhibits elevated levels during human colon cancer
(51), it appears that only certain types of cancer cells have acquired
dependence on CLU to survive and proliferate.
Our data indicate that CLU may also exert a significant function
intracellularly. Apart from the exclusively intracellularly localized
CLU precursor high mannose form of ⬃60 kDa, in the rat ventral
prostate model, various CLU cytoplasmic protein-forms were found to
be related either to surviving or to dying tissue (19). Additionally,
tumor necrosis factor ␣ treatment of L929 cells induces the appearance of two novel cytoplasmic CLU forms exerting a cytoprotective
function (52). In contrast, it was shown recently that certain intracel-
Fig. 8. Effect of paclitaxel treatment on clusterin (CLU) knock down PC-3 cells growth
and apoptosis. Cells were treated with 50 nM of the Cl-III, Cl-V or the Sc-I siRNA
oligonucleotides for 1 day. Two days after siRNA treatment, cells were exposed to the
indicated concentrations of paclitaxel for 48 h, and cell viability was determined by an in
vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Each data point
represents the mean of three independent experiments; bars denote SD; ⴱ indicate
differences from control at P ⬍ 0.01.
1840
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
lular CLU protein forms produced by either a truncated CLU mRNA
(5) or by altered posttranslational modification and intracellular trafficking of the CLU protein form transcribed from the full-length
conventional transcript (53) are related to cell death. Similarly, it was
shown that forced expression of intracellular CLU fragments initiates
the formation of protein aggregates and cell death (54). These CLU
protein forms, however, are seemingly strictly related to cell exposure
to apoptosis-inducing agents and therefore should not be directly
related to the main CLU function under physiological conditions.
In support to our suggestion of an intracellular cytoprotective CLU
function, the observed reduced plating efficiency and growth of the
CLU knock down cancer cells is most likely related to a decreased
ability of cells to adhere to a substrate. CLU has been reportedly
implicated in cell-substrate interactions. More specifically, it has been
shown that in LLC-PK1 renal epithelial cells CLU promoted cell
aggregation and cell adhesion in a specific and dose-dependent manner (55), whereas in vascular smooth muscle cells, exogenously added
CLU promoted cell migration (56). The inhibition of cell adhesion
observed in CLU knock down cells may be partially related to growth
inhibition as indicated by the observed up-regulation of p21 to cellular
sensitization to apoptosis, as well as to the observed decreased levels
of bcl-2, the p53 stabilization, and the accumulation of Bax in U-2 OS
cells. Considering that bcl-2 down-regulation occurs in both KH OS
and U-2 OS cells, we suggest that it does not depend on p53 status and
occurs upstream of p53 stabilization. Interestingly, down-regulation
of both bcl-2 and bcl-xL in glioblastoma cell lines resulted in spontaneous cell death (57) and CLU levels are positively related to the
antiapoptotic molecule bcl-xL levels in human melanoma cell lines
(Hoeller et al., personal communication). Detachment of epithelial
cells from the extracellular matrix results in a form of apoptosis often
referred to as anoikis. Interestingly, the bcl-2 protein expression is
higher in rapidly adhering keratinocytes and nearly absent in slowly
adhering cells (58), whereas detachment from the extracellular matrix
or disruption of the cytoskeleton results in a significant down-regulation of bcl-xL in nonmalignant rat and human intestinal epithelial
cells (59). Moreover, loss of endogenous p53 activity in normal
epithelial cells transfected with dominant-negative mutated p53 inhibited anoikis, thus demonstrating the involvement of p53-dependent
processes in anoikis-mediated cell death (60). Our data in U-2 OS
cells are in line with a novel recently identified proapoptotic function
of p53 that does not require activation by genotoxic agents and
appears to be constitutively suppressed by bcl-2. Silencing of bcl-2 in
colorectal cancer cells by siRNA induced massive p53-dependent
apoptosis that requires as essential apoptotic mediators bax and
caspase-2 proteins (61).
In summary, considering that: (a) the CLU gene is differentially
expressed during carcinogenesis and tumor progression, (b) treatment
of cancer cells with chemotherapeutic drugs results in CLU gene
induction, and (c) the primary function of the 60- and 75-kDa CLU
protein forms is cytoprotective, we suggest that CLU is a target for
therapeutic inhibition in cancer. The siRNA oligonucleotides used in
this study are potent tools for modulating the CLU gene expression
and they may ultimately develop into attractive antitumor therapeutics.
ACKNOWLEDGMENTS
We thank Dr. Klavs Hendil for LMP7 antibody, Dr. Rebecca Matsas and
Prof. Lukas Margaritis for the use of microscopy facilities, and Dr. Alexandros
Lavdas for his kind help regarding confocal microscope operation.
REFERENCES
1. Jenne, D. E., and Tschopp, J. Clusterin: the intriguing guises of a widely expressed
glycoprotein. Trends Biochem. Sci., 17: 154 –159, 1992.
2. De Silva, H. V., Harmony, J. A. K., Stuart, W. D., Gil, C. M., and Robbins, J.
Apolipoprotein J: structure and tissue distribution. Biochemistry, 29: 5380 –5389,
1990.
3. Trougakos, I. P., and Gonos, E. S. Clusterin/Apolipoprotein J in human aging and
cancer. Int. J. Biochem. Cell Biol., 34: 1430 –1448, 2002.
4. Yang, C-R., Leskov, K., Hosley-Eberlein, K., Criswell, T., Pink, J. J., Kinsella, T. J.,
and Boothman, D. A. Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein
that signals cell death. Proc. Natl. Acad. Sci. USA, 97: 5907–5912, 2000.
5. Leskov, K. S., Klokov, D. Y., Li, J. Kinsella, T. J., and Boothman, D. A. Synthesis
and functional analysis of nuclear clusterin: a cell death protein. J. Biol. Chem., 278:
11590 –11600, 2003.
6. Rosenberg, M. E., and Silkensen, J. Clusterin: physiologic and pathophysiologic
considerations. Int. J. Biochem. Cell Biol., 27: 633– 645, 1995.
7. Koch-Brandt, C., and Morgans, C. Clusterin: a role in cell survival in the face of
apoptosis? Prog. Mol. Subcell. Biol., 16: 130 –149, 1996.
8. Poon, S., Easterbrook-Smith, S. B., Rybchyn, M. S., Carver, J. A., and Wilson, M. R.
Clusterin is an ATP-independent chaperone with very broad substrate specificity that
stabilizes stressed proteins in a folding-competent state. Biochemistry, 39: 15953–
15960, 2000.
9. Calero, M., Rostagno, A., Matsubara, E., Zlokovic, B., Frangione, B., and Ghiso, J.
Apolipoprotein J (clusterin) and Alzheimer’s disease. Microsc. Res. Tech., 50:
305–315, 2000.
10. Parczyk, K., Pilarsky, C., Rachel, U., and Koch-Brand, C. Gp80 (clusterin: TRPM-2)
mRNA levels is enhanced in human renal clear cell carcinomas. J. Cancer Res. Clin.
Oncol., 120: 186 –188, 1994.
11. Steinberg, J., Oyasu, R., Lang, S., Sintich, S., Rademaker, A., Lee, C., Kozlowski,
J. M., and Sensibar, J. A. Intracellular levels of SGP-2 (clusterin) correlate with tumor
grade in prostate cancer. Clin. Cancer Res., 3: 1707–1711, 1997.
12. Wellmann, A., Thieblemont, C., Pittaluga, S., Sakai, A., Jaffe, E. S., Siebert, P., and
Raffeld, M. Detection of differentially expressed genes in lymphomas using cDNA
arrays: identification of clusterin as a new diagnostic marker for anaplastic large-cell
lymphomas. Blood, 96: 398 – 404, 2000.
13. Hough, C. D., Cho, K. R., Zonderman, A. B., Schwartz, D. R., and Morin, P. J.
Coordinately up-regulated genes in ovarian cancer. Cancer Res., 61: 3869 –3876,
2001.
14. Ouyang, X. S., Wang, X., Lee, D. T., Tsao, S. W., and Wong, Y. C. Up-regulation of
TRPM-2, MMP-7 and ID-1 during sex hormone-induced prostate carcinogenesis in
the Noble rat. Carcinogenesis (Lond.), 22: 965–973, 2001.
15. Redondo, M., Villar, E., Torres-Muñoz, J., Tellez, T., Morell, M., and Petito, C.
Overexpression of clusterin in human breast carcinoma. Am. J. Pathol., 157: 393–
399, 2000.
16. Wehrli, P., Charnay, Y., Vallet, P., Zhu, G., Harmony, J., Aronow, B., Tschopp, J.,
Bouras, C., Viard,-Leveugle, I., French, L. E., and Giannakopoulos, P. Inhibition of
post-ischemic brain injury by clusterin overexpression. Nat. Med., 7: 977–978, 2001.
17. Han, B. H., DeMattos, R. B., Dugan, L. L., Kim-Han, J. S., Brendza, R. P., Fryer,
J. D., Kierson, M., Cirrito, J., Quick, K., Harmony, J. A., Aronow, B. J., and
Holtzman, D. M. Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia. Nat. Med., 7: 338 –343, 2001.
18. Buttyan, R., Olsson, C. A., Pintar, J., Chang, C., Bandyk, M., Ng, P. Y., and Sawczyk,
I. S. Induction of the TRPM-2 gene in cells undergoing programmed cell death. Mol.
Cell. Biol., 9: 3473–3481, 1989.
19. Lakins J., Bennett, S. A. L., Chen, J-H., Arnold, J. M., Morrissey, C., Wong, P.,
Sullivan, J. O’., and Tenniswood, M. Clustering biogenesis is altered during apoptosis
in the regressing rat ventral prostate. J. Biol. Chem., 273: 27887–27895, 1998.
20. Kalka, K., Ahmad, N., Criswell, T., Boothman, D., and Mukhtar, H. Up-regulation of
clusterin during phthalocyanine 4 photodynamic therapy-mediated apoptosis of tumor
cells and ablation of mouse skin tumors. Cancer Res., 60: 5984 –5987, 2000.
21. Gonos, E. S., Derventzi, A., Kveiborg, M., Agiostratidou, G., Kassem, M., Clark,
B. F. C., Jat, P. S., and Rattan, S. I. S. Cloning and identification of genes that
associate with mammalian senescence. Exp. Cell Res., 240: 66 –74, 1998.
22. Petropoulou, C., Trougakos, I. P., Kolettas, E., Toussaint, O., and Gonos, E. S.
Clusterin/Apolipoprotein J is a novel biomarker of cellular senescence that does not
affect the proliferative capacity of human diploid fibroblasts. FEBS Lett., 509:
287–297, 2001.
23. Dumont, P., Chainiaux, F., Eliaers, F., Petropoulou, C., Remacle, J., Koch-Brandt, C.,
Gonos, E. S., and Toussaint, O. Overexpression of apolipoprotein J in human
fibroblasts protects against cytotoxicity and premature senescence induce by ethanol
and tert-butylhydroperoxide. Cell Stress Chaperones, 7: 23–35, 2002.
24. Trougakos, I. P., Poulakou, M., Stathatos, M., Chalikia, 〈., Melidonis, A., and Gonos,
E. S. Serum levels of the senescence biomarker clusterin/apolipoprotein J increase
significantly in diabetes type II and during development of coronary heart disease or
at myocardial infarction. Exp. Gerontol., 37: 1175–1187, 2002.
25. Miyake, H., Nelson, C., Rennie, P. S., and Gleave, M. E. Testosterone-repressed
prostate message-2 is an antiapoptotic gene involved in progression to androgen
independence in prostate cancer. Cancer Res., 60: 170 –176, 2000.
26. Miyake, H., Nelson, C., Rennie, P. S., and Gleave, M. E. Acquisition of chemoresistant phenotype by overexpression of the antiapoptotic gene testosterone-repressed
prostate message-2 in prostate cancer xenograft models. Cancer Res., 60: 2547–2554,
2000.
27. Miyake, H., Chi, K. N., and Gleave, M. E. Antisense TRPM-2 oligodeoxynucleotides
chemosensitize human androgen-independent PC-3 prostate cancer cells both in vitro
and in vivo. Clin. Cancer Res., 6: 1655–1663, 2000.
28. Zellweger, T., Miyake, H., Cooper, S., Chi, K., Conklin, B. S., Monia, B., and Gleave,
M. E. Antitumor activity of antisense clusterin oligonucleotides is improved in vitro
and in vivo by incorporation of 2⬘-o-(2-methoxy) ethyl chemistry. J. Pharmacol. Exp.
Ther., 298: 934 –940, 2001.
1841
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
CELL SENSITIZATION BY CLU GENE EXPRESSION SILENCING
29. Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T., and Weber, K. Identification of
essential genes in cultured mammalian cells using small interfering RNAs. J. Cell
Sci., 114: 4557– 4565, 2001.
30. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P., and Sharp, P. A. Targeted
mRNA degradation by double-stranded RNA in vitro. Genes Dev., 13: 3191–3197,
1999.
31. Isfort, R. J., Cody, D. B., Lovell, G., and Doersen, C. J. Analysis of oncogenes, tumor
suppressor genes, autocrine growth-factor production, and differentiation state of
human osteosarcoma cell lines. Mol. Carcinog., 14: 170 –178, 1995.
32. Bridge, A. J., Pebernard, S., Ducraux, A., Nicoulaz, A-L., and Iggo, R. Induction of
an interferon response by RNAi vectors in mammalian cells. Nat. Genet., 34:
263–263, 2003.
33. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., and Williams, B. R. G.
Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol., 5:
834 – 839, 2003.
34. Samuel, C. E. Antiviral actions of interferons. Clin. Microbiol. Rev., 14: 778 – 809,
2001.
35. Kloetzel, P-M. Antigen processing by the proteasome. Nat. Rev. Mol. Cell. Biol., 2:
179 –187, 2001.
36. Chi, J-T., Chang, H. Y., Wang, N. N., Chang, D. S. Dunphy, N., and O’Brown, P.
Genome-wide view of gene silencing by small interfering RNAs. Proc. Natl. Acad.
Sci. USA, 100: 6343– 6346, 2003.
37. Semizarov, D., Frost, L., Sarthy, A., Kroeger, P., Halbert, D. N., and Fesik, S. W.
Specificity of short interfering RNA determined through gene expression signatures.
Proc. Natl. Acad. Sci. USA, 100: 6347– 6352, 2003.
38. Gewirtz, D. A. A critical evaluation of the mechanisms of action proposed for the
antitumor effects of the anthracycline antibiotics Adriamycin and daunorubicin.
Biochem. Pharmacol., 57: 727–741, 1999.
39. Muller, C., Chatelut, E., Gualano, V., De Forni, M., Huguet, F., Attal, M., Canal, P.,
and Laurent, G. Cellular pharmacokinetics of doxorubicin in patients with chronic
lymphocytic leukemia: comparison of bolus administration and continuous infusion.
Cancer Chemother. Pharmacol., 32: 379 –384, 1993.
40. Rohlff, C., Blagosklonny, M. V., Kyle, E., Kesari, A., Kim, I. Y., Zelner, D. J.,
Hakim, F., Trepel, J., and Bergan, R. C. Prostate cancer cell growth inhibition by
tamoxifen is associated with inhibition of protein kinase C and induction of p21(waf1/
cip1). Prostate, 37: 51–59, 1998.
41. Miyake, H., Gleave, M. E., Arakawa, S., Kamidono, S., and Hara, I. Introducing the
clusterin gene into human renal cell carcinoma cells enhances their metastatic
potential. J. Urol., 167: 2203–2208, 2002.
42. Garden, G. A., Bothwell, M., and Rubel, E. W. Lack of correspondence between
mRNA expression for a putative cell death molecule (SGP-2) and neuronal cell death
in the central nervous system. J. Neurobiol., 22: 590 – 604, 1991.
43. O’Bryan, M. K., Cheema, S. S., Bartlett, P. F., Murphy, B. F., and Pearse, M. J.
Clusterin levels increase during neuronal development. J. Neurobiol., 24: 421– 432,
1993.
44. Aronow, B. J., Lund, S. D., Brown, T. L., Harmony, J. A. K., and Witte, D. P.
Apolipoprotein J expression at fluid-tissue interfaces: potential role in barrier cytoprotection. Proc. Natl. Acad. Sci. USA, 90: 725–729, 1993.
45. Miyake, H., Hara, I., Kamidono, S., and Gleave, M. E. Synergistic chemosensitization
and inhibition of tumour growth and metastasis by the antisense oligodeoxynucleotide
targeting clusterin gene in a human bladder cancer model. Clin. Can. Res., 7:
4245– 4252, 2001.
46. Zellweger, T., Miyake, H., July, L., Akbari, M., Kiyama, S., and Gleave, M. E.
Chemosensitization of a human renal cell cancer model by antisense TRPM-2
oligodeoxynucleotides both in vitro and in vivo. Neoplasia, 3: 1– 8, 2001.
47. McLaughlin, L., Zhu, G., Mistry, M., Ley-Ebert, C., Stuart, W. D., Florio, C. J.,
Groen, P. A., Witt, S. A., Kimball, T. R., Witte, D. P., Harmony, J. A. K., and
Aronow, B. J. Apolipoprotein J/Clusterin limits the severity of murine autoimmune
myocarditis. J. Clin. Investig., 106: 1105–1113, 2000.
48. Bailey, R. W., Aronow, B., Harmony, J. A., and Griswold, M. D. Heat shock-initiated
apoptosis is accelerated and removal of damaged cells is delayed in the testis of
clusterin/ApoJ knock-out mice. Biol. Reprod., 66: 1042–1053, 2002.
49. Rosenberg, M. E., Girton, R., Finkel, D., Chmielewski, D., Barie, A., III, Witte, D. P.,
Zhu, G., Bissler, J. J., Harmony, J. A. K., and Aronow, B. Apolipoprotein J/Clusterin
prevents a progressive glomerulopathy of aging. Mol. Cell. Biol., 22: 1893–1902,
2002.
50. Bettuzzi, S., Scorcioni, F., Astancolle, S., Davalli, P., Scaltriti, M., and Corti, A.
Clusterin (SGP-2) transient overexpression decreases proliferation rate of SV40immortalized human prostate epithelial cells by slowing down cell cycle progression.
Oncogene, 21: 4328 – 4334, 2002.
51. Chen, X., Halberg, R. B., Ehrhardt, W. M., Torrealba, J., and Dove, W. F. Clusterin
as a biomarker in murine and human intestinal neoplasia. Proc. Natl. Acad. Sci. USA,
100: 9530 –9535, 2003.
52. Humphreys, D., Hochgrebe, T. T., Easterbrook-Smith, S. B., Tenniswood, M. P., and
Wilson, M. R. Effects of clusterin overexpression on TNF␣- and TGF␤-mediated
death of L929 cells. Biochemistry, 36: 15233–15243, 1997.
53. O’Sullivan, J., Whyte, L., Drake, J., and Tenniswood, M. Alterations in the posttranslational modification and intracellular trafficking of clusterin in MCF-7 cells
during apoptosis. Cell Death Differ., 10: 914 –927, 2003.
54. Debure, L., Vayssiere, J. L., Rincheval, V., Loison, F., Le Drean, Y., and Michel, D.
Intracellular clusterin causes juxtanuclear aggregate formation and mitochondrial
alteration. J. Cell Sci., 116: 3109 –3121, 2003.
55. Silkensen, J. R., Skubitz, K. M., Skubitz, A. P. N., Chmielewaki, D. H., Manivel,
J. C., Dvergsten, J. A., and Rosenberg, M. E. Clusterin promotes the aggregation and
adhesion of renal porcine epithelial cells. J. Clin. Investig., 96: 2646 –2653, 1995.
56. Millis, A. J. T., Luciani, M., McCue, H. M., Rosenberg, M. E., and Moulson, C. L.
Clusterin regulates vascular smooth muscle cell nodule formation and migration.
J. Cell. Physiol., 186: 210 –219, 2001.
57. Jiang, Z., Zheng, X., and Rich, K. M. Down-regulation of Bcl-2 and Bcl-xL expression with bispecific antisense treatment in glioblastoma cell lines induce cell death.
J. Neurochem., 84: 273–281, 2003.
58. Tiberio, R., Marconi, A., Fila, C., Fumelli, C., Pignatti, M., Krajewski, S., Giannetti,
A., Reed, J. C., and Pincelli, C. Keratinocytes enriched for stem cells are protected
from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner. FEBS
Lett., 524: 139 –144, 2002.
59. Rosen, K., Rak, J., Leung, T., Dean, N. M., Kerbel, R. S., and Filmus, J. Activated
Ras prevents down-regulation of Bcl-X(L) triggered by detachment from the extracellular matrix. A mechanism of Ras-induced resistance to anoikis in intestinal
epithelial cells. J. Cell Biol., 149: 447– 456, 2000.
60. Vitale, M., Di Matola, T., Bifulco, M., Casamassima, A., Fenzi, G., and Rossi, G.
Apoptosis induced by denied adhesion to extracellular matrix (anoikis) in thyroid
epithelial cells is p53 dependent but fails to correlate with modulation of p53
expression. FEBS Lett., 462: 57– 60, 1999.
61. Jiang, M., and Milner, J. Bcl-2 constitutively suppresses p53-dependent apoptosis in
colorectal cancer cells. Genes Dev., 17: 832– 837, 2003.
1842
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
Silencing Expression of the Clusterin/Apolipoprotein J Gene
in Human Cancer Cells Using Small Interfering RNA Induces
Spontaneous Apoptosis, Reduced Growth Ability, and Cell
Sensitization to Genotoxic and Oxidative Stress
Ioannis P. Trougakos, Alan So, Burkhard Jansen, et al.
Cancer Res 2004;64:1834-1842.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/64/5/1834
Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2004/04/06/64.5.1834.DC1
This article cites 58 articles, 25 of which you can access for free at:
http://cancerres.aacrjournals.org/content/64/5/1834.full#ref-list-1
This article has been cited by 30 HighWire-hosted articles. Access the articles at:
http://cancerres.aacrjournals.org/content/64/5/1834.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.