[CANCER RESEARCH 64, 1431–1435, February 15, 2004]
Small Interfering Double-Stranded RNAs as Therapeutic Molecules to Restore
Chemosensitivity to Thymidylate Synthase Inhibitor Compounds
John C. Schmitz, Tian-min Chen, and Edward Chu
Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VACT Cancer Center, VACT Healthcare System, West Haven,
Connecticut
ABSTRACT
RNA interference is a post-transcriptional mechanism by which double-stranded RNA specifically silence expression of a corresponding gene.
Small interfering double-stranded RNA (siRNA) of 21–23 nucleotides can
induce the process of RNA interference. Studies from our laboratory have
shown that translation of thymidylate synthase (TS) mRNA is controlled
by its own protein end-product TS in a negative autoregulatory manner.
Disruption of this process gives rise to increased synthesis of TS and leads
to the development of cellular drug resistance to TS-targeted compounds.
As a strategy to inhibit TS expression at the mRNA level, siRNAs were
designed to target nucleotides 1058 –1077 on human TS mRNA. Transfection of TS1058 siRNA into human colon cancer RKO cells resulted in
a dose-dependent inhibition of TS expression with an IC50 value of 10 pM
but had no effect on the expression of ␣-tubulin or topoisomerase I.
Inhibition of TS expression by TS1058 was maximal at 48 h and remained
suppressed for up to 5 days. Pretreatment of RKO cells with TS1058
siRNA suppressed TS protein induction following exposure to raltitrexed.
In addition, TS1058 restored chemosensitivity of the resistant RKOHTStet cell line to various TS inhibitor compounds. On treatment with
TS1058, IC50 values for raltitrexed, 1843U89, and 5-fluoro-2ⴕ-deoxyuridine decreased by ⬃15–16-fold. These studies suggest that TS-targeted
siRNAs are effective inhibitors of TS expression and may have therapeutic
potential by themselves or as chemosensitizers in combination with TS
inhibitor compounds.
INTRODUCTION
RNA interference (RNAi) is a novel regulatory process in which
double-stranded RNA (dsRNA) induces the specific degradation of its
target mRNA (1–3). This evolutionarily conserved process maintains
the genomic integrity of cellular organisms by silencing transposons
and guarding against exogenous viral infection. Recent studies have
shown involvement of the RNAi machinery with cellular processes
such as chromosomal separation, histone methylation, and association
with the fragile X protein (4 – 6). Fire et al. (7), who showed that
dsRNA injected into Caenorhabditis elegans resulted in specific
interference of cellular gene expression, first coined the term “RNAi.”
The inhibitory effect was much greater than that observed with
injection of either individual RNA strand. RNAi and closely related
mechanisms, such as cosuppression and quelling, have been observed
in fungi, plants, Drosophila, and mammals (1). The molecular mechanism involves cleavage of the dsRNA by an RNase III-like endonuclease to small 21–25 nucleotide (nt) dsRNAs (8 –10). These small
interfering dsRNAs (siRNAs) are bound in an endonuclease RNAinduced silencing complex, and the antisense strand then is used as a
guide to target complementary mRNAs for degradation (11).
RNAi gained rapid acceptance as a tool for studying gene function
in C. elegans and Drosophila. Application of RNAi in mammalian
systems was limited initially because of nonspecific effects of long
dsRNAs (12–14). However, since the discovery that siRNAs can be
introduced into mammalian cells, thereby bypassing the nonspecific
effects associated with dsRNAs, this mechanism has become widely
used to investigate the regulation of gene expression (15). In addition,
siRNAs may be developed as novel agents that target critical signaling
pathways involved in human disease.
Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP by the reduced folate
dTMP and dihydrofolate (16). Once synthesized, dTMP then is additionally metabolized intracellularly to the dTTP triphosphate form, an
essential precursor for DNA synthesis. Although dTMP also can be
formed through the salvage pathway, as catalyzed by thymidine
kinase, the TS-catalyzed reaction provides for the sole intracellular de
novo source of dTMP. Given its central role in DNA biosynthesis and
given that inhibition of this reaction results in cessation of cellular
proliferation and growth, TS represents an important target for cancer
chemotherapy (17–19).
In addition to its catalytic activity, TS functions as an RNA binding
protein (20, 21). Studies from our laboratory have shown that TS
protein binds with high affinity to two cis-acting sequences on its own
mRNA. Binding of TS to either of these elements results in translational repression. This interaction between TS protein and its mRNA
represents an efficient mechanism to control the cellular levels of TS.
Disruption of this normal autoregulatory process results in an acute
induction of TS and the rapid development of resistance in response
to exposure to TS inhibitors such as 5-fluorouracil and the antifolate
analogue raltitrexed. In support of this model, in vitro and in vivo
studies have shown acute induction of TS protein with no corresponding change in TS mRNA levels on treatment with various TS inhibitors (22–24).
In the present study, we demonstrate that siRNAs effectively induce
the process of RNAi in human colon cancer RKO cells. Treatment
with natural 2⬘-OH siRNAs directed against nt 1058 –1077 on human
TS mRNA specifically and effectively inhibits the expression of TS
mRNA and TS protein in RKO cells. In addition, siRNA treatment
suppressed TS protein induction following exposure to the TS inhibitor compound raltitrexed. We demonstrate that treatment with siRNAs restored chemosensitivity to resistant, TS-overexpressing, RKOHTStet cells against several clinically relevant TS anticancer agents.
The potential therapeutic application of siRNAs as single agents and
in combination with TS inhibitor compounds for the treatment of
human cancers is discussed.
MATERIALS AND METHODS
siRNA Synthesis. siRNA duplexes were designed to target sequences on
human TS mRNA corresponding to nt 1058 –1077 (5⬘-GGAUAUUGUCAGUCUUUAGG-3⬘). The selected sequence was screened in a BLAST search
Received 4/30/03; revised 11/19/03; accepted 12/11/03.
against all of the known human genes to verify that only human TS mRNA was
Grant support: National Cancer Institute (CA75712 and CA16359 to E. Chu) and the
targeted. siRNA duplexes were obtained from Dharmacon (Lafayette, CO).
Department of Veterans Affairs (VA Merit Review to E. Chu).
Each RNA contained two additional 2⬘-deoxythymidine nts on the 3⬘ end. In
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
addition, a mismatch siRNA, TS1058M10, and two control siRNA duplexes,
18 U.S.C. Section 1734 solely to indicate this fact.
GL2 and SCRII, were obtained from Dharmacon.
Requests for reprints: John C. Schmitz, VACT Healthcare System, Cancer Center–
Cell Culture. The human colon cancer cell lines RKO and RKO-HTStet
IIID, 950 Campbell Avenue, West Haven, CT 06516. Phone: 203-937-3421; Fax: 203were maintained in 75-cm2 tissue culture flasks (BD Bioscience, San Jose, CA)
937-3803; E-mail: [email protected].
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siRNA AND CHEMOSENSITIVITY TO TS INHIBITORS
in growth medium containing RPMI (1640; Invitrogen, Carlsbad, CA) with
10% dialyzed fetal bovine serum (Invitrogen).
A stably transfected, tetracycline-inducible RKO subline, which overexpresses human TS protein, was established by transfection with plasmid
pUHD172–1neo using lipofectin. This plasmid constitutively expresses the Tet
activator protein. Cells were grown in RPMI 1640 medium containing 10%
dialyzed fetal bovine serum and 600 ␮g/ml geneticin. After 3 weeks of growth,
colonies were selected individually and expanded into cell lines. The fulllength human TS cDNA (nt 1–1524) was isolated from the plasmid pcEHTS
by PCR amplification and cloned into pTRE2hyg, a tetracycline-responsive
plasmid (Clontech, Palo Alto, CA). The plasmid pTRE2hyg-HTS then was
transfected into RKO cells expressing the Tet activator protein by Eufectin 7
(JBL Scientific, San Luis Obispo, CA). Colonies were selected in 600 ␮g/ml
geneticin and 300 ␮g/ml hygromycin. Each clone was treated with 1 ␮g/ml
doxycycline for 24 h. Clones with elevated levels of induced TS protein
expression, as determined by Western blot analysis, then were selected, and the
clone that yielded the highest level of induced TS expression was termed
RKO-HTStet.
siRNA Transfection. Human colon cancer RKO cells were plated in
25-cm2 flasks in 3 ml RPMI 1640 at a density of 1.5 ⫻ 105 cells per flask.
siRNA duplexes were complexed with the cationic lipid Oligofectamine (Invitrogen) in OPTI-MEM medium, as described by the manufacturer’s protocol.
In brief, 4 ␮l of Oligofectamine were added to 21 ␮l OPTI-MEM medium and
allowed to sit at room temperature for 5 min. siRNA duplexes were added to
275 ␮l OPTI-MEM medium. Diluted Oligofectamine was added to the diluted
siRNA and incubated for 15 min at room temperature. Aliquots (300 ␮l) then
were added to the T25 flasks. siRNA concentrations cited herein are in a final
total volume of 3.3 ml. After 48 h, cells were trypsinized and washed twice
with ice-cold PBS. Cell pellets were stored at ⫺80°C for later use.
For cell proliferation assays, RKO-HTStet cells were plated in 12-well
plates in 1 ml RPMI 1640 at a density of 1 ⫻ 104 cells/well. Oligofectamine
(1 ␮l) was added to 14 ␮l OPTI-MEM and allowed to sit for 5 min at room
temperature. This solution then was added to the diluted siRNA (85 ␮l) and
allowed to incubate for 15 min before addition into the wells.
Western Immunoblot Analysis. RKO cells were plated, transfected, and
harvested as described previously. Cell pellets were resuspended in cell lysis
buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 1% Igepal (Sigma-Aldrich Co.,
Milwaukee, WI), 0.5% deoxycholic acid, and 0.1% SDS] containing freshly
added phenylmethylsulfonyl fluoride and protease inhibitor mixture (Sigma,
St. Louis, MO). Suspensions were incubated at 4°C for 20 min and centrifuged
for 10 min at 4°C. Protein concentration was determined using the DC protein
assay (Bio-Rad, Hercules, CA). Equivalent amounts of protein (30 ␮g) from
each cell lysate were resolved on SDS-PAGE (10 –12.5% acrylamide) using
the method of Laemmli (25). Gels were electroblotted onto nitrocellulose
membranes (0.2 ␮m; Bio-Rad), and membranes then were incubated in blocking solution (1⫻ PBS, 0.1% Tween-20, and 5% nonfat dry milk powder) for
2 h at room temperature. Membranes were incubated for 1 h with primary
antibodies at the following dilutions: anti-TS106 monoclonal antibody,
1:2,000; anti-␣-tubulin monoclonal antibody, 1:30,000 (Amersham, Piscataway, NJ); and antitopoisomerase I monoclonal antibody, 1:2,000 (a gift from
Dr. Yung-chi Cheng, Yale University, New Haven, CT). After five 10-min
washes in 1⫻ PBS and 0.1% Tween-20, membranes were incubated with a
dilution of 1:2,000 of horseradish peroxidase-conjugated secondary antibody
(IgG goat antimouse; Bio-Rad) for 1 h at room temperature. After an additional
five 10-min 1⫻ PBS and 0.1% Tween-20 washes, membranes were processed
by the enhanced chemiluminescence method (SuperSignal West Pico substrate; Pierce, Rockford, IL), and protein bands were visualized by autoradiography. Quantitation of signal intensities was performed using densitometry on
a Hewlett-Packard ScanJet 5370C (Palo Alto, CA) with NIH image 1.62
software.
Cell Proliferation Assays. RKO-HTStet cells were plated in 12-well plates
in the presence or absence of 1 ␮g/ml doxycycline. siRNA-Oligofectamine
complexes then were added to the wells on the following day. After a 24-h
incubation, TS inhibitor compounds, including raltitrexed (ZD1694),
1843U89, and 5-fluoro-2⬘-deoxyuridine (FdUrd), were added to the wells at
the indicated concentrations. After an additional 72 h, cells were trypsinized,
and the cell number was determined by a Coulter counter (Beckman Coulter,
Fullerton, CA) and by the trypan blue dye exclusion method.
RESULTS
We investigated the ability of siRNAs to inhibit the expression of
TS in RKO cells. As seen in Fig. 1, TS1058 siRNA, which targets the
3⬘-UTR immediately downstream of the translational stop site (nt
1058 –1077), inhibited the expression of TS protein in a dose-dependent manner with 50% inhibition being observed at 10 pM and
maximal inhibition (⬎95%) at 300 pM (Fig. 1, Lanes 4 and 7,
respectively). The expression of two control proteins, ␣-tubulin and
topoisomerase I, was unaffected by TS1058 siRNA treatment. Treatment with a control GL2 siRNA, at a concentration of 10 nM, had
absolutely no effect on levels of TS or the control proteins (Fig. 1,
Lane 10). Expression of TS protein remained unchanged by GL2
concentrations up to 100 nM (data not shown). We also investigated
the effect of TS1058 siRNA on TS enzyme activity using the TS
catalytic assay and the FdUMP radioenzymatic binding assay (26).
The reduction in TS protein expression, as determined by Western
blot analysis, corresponded exactly with inhibition of TS enzyme
activity and FdUMP binding activity (Fig. 1).
Previous studies from our laboratory had shown that binding of TS
protein to its own TS mRNA resulted in translational repression (20,
27). Disruption of this normal autoregulatory process gave rise to an
acute induction of TS and the acute development of resistance in
response to exposure to TS inhibitors such as 5-fluorouracil and
raltitrexed (ZD1694; Refs. 23 and 26). In attempt to overcome this
resistance mechanism, we investigated whether TS-targeted siRNA
duplexes could prevent this induction of TS protein. As seen in Fig.
2A, RKO cells were treated for 24 h with ZD1694, TS1058 siRNA, or
a combination of these two molecules. Treatment of RKO cells with
2 nM ZD1694 for 24 h resulted in a more than twofold induction of TS
protein (Fig. 2B). This induction was prevented when RKO cells were
pretreated with 1 nM TS1058 siRNA for 24 h before treatment with
ZD1694. However, when ZD1694 was administered first, followed by
TS1058 siRNA, TS protein remained elevated at levels observed with
ZD1694 treatment alone. Additionally, when RKO cells were treated
with TS1058 siRNA and ZD1694 simultaneously during the second
24-h period, induction of TS protein still was observed, albeit at a
slightly lower level than that observed with ZD1694 treatment alone.
However, when RKO cells were incubated together with ZD1694 and
TS1058 siRNA during the first 24-h period, TS protein levels decreased by nearly 70% compared with control, untreated levels. As an
important control, treatment of RKO cells with the control GL2
siRNA (1 nM) for 24 h before the addition of ZD1694 was unable to
abrogate the induction of TS protein.
Fig. 1. Western blot analysis of RKO cells after treatment with a thymidylate synthase
(TS)-targeted small interfering double-stranded RNA (siRNA). Cells were incubated in the
absence (Lane 1) or presence (Lanes 2–10) of siRNA:Oligofectamine complexes for 48 h
and then harvested and processed for Western blot analysis as described in “Materials and
Methods.” Lanes 2–9 correspond to treatment with Oligofectamine complexes containing
0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, and 10 nM TS1058 siRNA, respectively. Lane 10
corresponds to 10 nM GL2 siRNA. TS catalytic enzyme activity and FdUMP binding
assays were performed as described previously (26). Assay values are based on untreated
RKO cells, which represent 100%.
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siRNA AND CHEMOSENSITIVITY TO TS INHIBITORS
We next performed a time course experiment to determine the
effect of siRNA treatment on levels of TS protein following the 48-h
point. As seen in Fig. 2C, the expression of TS protein remained
elevated when RKO cells were treated only with ZD1694. However,
Fig. 2. Effect of TS1058 in combination with ZD1694 on induction of thymidylate
synthase (TS) protein. A, schematic of experimental design: cells were incubated with
either 1 nM TS1058 small interfering double-stranded RNA (siRNA; TS), 1 nM GL2
siRNA, or 2 nM ZD1694 (ZD) for a 24-h period. The number in parenthesis indicates
whether cells were incubated during the first 24-h (1) or the second 24-h period (2).
Culture medium was replaced after the first 24-h incubation. B, cells were harvested after
48 h and processed for Western blot analysis as described in “Materials and Methods.” C,
RKO cells that were treated with ZD1694(2) (䡺), ZD1694(1) (3), TS1058(2) (‚), or
TS1058(2) ⫹ ZD1694(2) (E) were harvested at the following times after the 48-h
incubation: 0, 12, 24, and 36 h. TS protein levels were determined by Western blot
analysis. TS protein induction values represent the mean ⫾ SE from densitometric
analysis of Western blot analyses from four experiments. TS protein content in untreated
RKO cells was set to 1. ⴱP ⱕ 0.005 versus ZD(2)
TS levels rapidly decreased during the next 12-h period when RKO
cells were treated with either ZD1694 being administered first before
the TS1058 siRNA or when ZD1694 and TS1058 siRNA were administered concomitantly. Taken together, these results suggest that
TS-targeted siRNAs are able to prevent the induction of TS protein in
response to exposure to TS inhibitor compounds.
The activity of TS1058 siRNA was investigated subsequently in a
tetracycline-inducible, TS-overexpressing cell line, RKO-HTStet.
This particular cell line had been established after stable transfection
of the human TS cDNA under the control of a tetracycline-inducible
promoter into parent RKO cells. Following treatment of RKO-HTStet
cells with 1 ␮g/ml doxycycline for 24 h, levels of TS protein, as
determined by Western immunoblot analysis, were 15-fold higher
compared with parent RKO cells (Fig. 3A, Lane 3 versus Lane 2).
Transfection of RKO-HTStet cells with 1 nM TS1058 siRNA resulted
in a 50% reduction in TS protein expression (Fig. 3A, Lane 4). On
treatment with 10 nM TS1058, levels of TS protein returned to the
same baseline levels observed in noninduced cells (Fig. 3A, Lane 6
versus Lane 2). In contrast, the control GL2 siRNA had no effect on
TS expression (Fig. 3A, Lane 7).
Fig. 3. Effect of TS1058 siRNA on thymidylate synthase (TS) protein expression and
cellular proliferation in RKO-HTStet cells. A, cells were incubated in the absence (Lanes
1–3) or presence (Lanes 4 –7) of small interfering double-stranded RNA (siRNA):Oligofectamine complexes for 48 h and then harvested and processed for Western blot analysis.
Lane 1 contains extracts from untreated parental RKO cells. Lane 2 contains extracts from
RKO-HTStet cells without doxycycline. Lanes 3–7 contain extracts from RKO-HTStet
cells treated with 1 ␮g/ml doxycycline. Lanes 4 –7 are treated with Oligofectamine
complexes containing 1, 3, and 10 nM TS1058 siRNA, respectively. Lane 7 corresponds
to treatment with 10 nM GL2 siRNA. B, cells were treated with 1 ␮g/ml doxycycline. On
the next day, cells were incubated with Oligofectamine in the absence or presence of
siRNA (1 nM TS1058 or 1 nM GL2). After 24 h, various concentrations of TS inhibitor
compounds [(B), ZD1694; (D), 1843U89; and (E), FdUrd] were added to the cells. After
an additional 72 h, cell number was determined, and drug concentrations that inhibit 50%
cellular proliferation (IC50) were estimated. C, in the presence of 1 ␮g/ml doxycycline,
RKO-HTStet cells were treated with various concentrations of TS1058. After 24 h,
various concentrations of ZD1694 were added to the cells. After an additional 72 h, cell
number was determined, and IC50 values were calculated. Values represent the
mean ⫾ SD from three to five experiments.
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siRNA AND CHEMOSENSITIVITY TO TS INHIBITORS
To determine the effect of TS1058 siRNA on the growth and
proliferation of RKO-HTStet cells, a series of cell proliferation experiments was performed. In the absence of doxycycline, RKOHTStet cells were treated with TS1058 for 4 days at a concentration
range of 0.01–100 nM. Cell growth was suppressed by only 25% on
exposure to 1 nM TS1058 siRNA (data not shown). Treatment with
higher concentrations of TS1058 siRNA (up to 100 nM) resulted in
only slightly higher growth inhibition (32%). To rule out the possibility that the lipid transfection reagent Oligofectamine might limit the
ability to transfect higher TS1058 siRNA concentrations, additional
experiments were performed in which increasing amounts of Oligofectamine were used for transfection. No additional inhibition of cell
growth was observed. Of note, the growth-inhibitory effects of
TS1058 siRNA were reversed completely when 10 ␮M thymidine was
added. This finding suggested that the growth-inhibitory effects of
TS1058 siRNA resulted from inhibition of thymidylate biosynthesis.
As an important control, treatment of RKO-HTStet cells with the
control GL2 siRNA had no effect on cell growth. In addition, when
RKO-HTStet cells were incubated with doxycycline (1 ␮g/ml), which
induced levels of TS protein by 15-fold, treatment with TS1058 had
absolutely no effect on the growth of RKO-HTStet cells.
To determine whether treatment with TS1058 siRNA could restore
chemosensitivity to agents that directly targeted TS, a series of growth
experiments was performed. As seen in Fig. 3B, treatment of RKOHTStet cells with the quinazoline antifolate analogue ZD1694, in the
absence of doxycycline, resulted in an IC50 value of 2.8 nM. On
induction of TS protein with doxycycline, the IC50 value was increased significantly to a value of 22.3 nM, which was nearly eightfold
higher than that observed in noninduced cells. Treatment of doxycycline-induced RKO-HTStet cells with 1 nM TS1058 for 24 h before
addition of ZD1694 was able to restore sensitivity to the antifolate
analogue. The resulting IC50 was decreased by 16-fold to a value of
1 nM. Simultaneous treatment with TS1058 siRNA and ZD1694
resulted in a threefold lower IC50 value (8 nM). In contrast, treatment
with GL2 siRNA did not alter the ZD1694 IC50 value. To provide
additional evidence that the reduction in ZD1694 IC50 values was
caused by a specific inhibition of TS expression, we tested the effect
of a mutant TS1058 siRNA with a G:C inversion at the 10th nt
(TS1058M10). This mutant siRNA did not inhibit the expression of
TS protein in either parent RKO or doxycycline-induced RKO-HTStet
cells nor did it inhibit the growth of either cell line. When doxycycline-induced RKO-HTStet cells were pretreated with this mutant
siRNA for 24 h and then treated with ZD1694, no abrogation of the
ZD1694-mediated induction of TS protein was observed (data not
shown). We next investigated the dose-dependent effect with regard to
the efficacy of the TS1058 siRNA to restore chemosensitivity to
ZD1694. As seen in Fig. 3C, siRNA concentrations as low as 10 pM
were effective at reducing the level of resistance to ZD1694 by nearly
twofold. Treatment with siRNA concentrations ⬎1 nM had little
additional impact on restoring sensitivity to ZD1694.
In addition to ZD1694, we investigated the activity of two other TS
inhibitor compounds, 1843U89 and FdUrd, using the doxycyclineinducible RKO-HTStet cell line as our model system. On induction of
TS protein with doxycycline, the sensitivity of RKO-HTStet cells to
each inhibitor compound was decreased by sixfold and eightfold,
respectively (Fig. 3, D and E). As in the case of ZD1694, pretreatment
with TS1058 siRNA for 24 h completely restored sensitivity to each
of these compounds. In contrast, pretreatment with the GL2 siRNA
was completely ineffective in reducing the level of cellular drug
resistance to 1843U89 and FdUrd.
DISCUSSION
In this report, we investigated the effect of siRNAs on expression
of TS protein and TS mRNA in human colon cancer RKO cells. Our
studies reveal that siRNAs significantly inhibit expression of TS
protein for an extended period. This effect on TS appears to be
specific because other important cellular proteins, including ␣-tubulin
and topoisomerase I, were unaffected by TS-directed siRNA treatment. Additionally, TS1058 siRNA was able to inhibit RKO cellular
proliferation cells by 30%. This limited effect of cell growth was
surprising initially given the significant degree of down-regulation of
TS protein (⬎95%). However, previous studies by Keyomarsi and
Moran had suggested that ⬎98% inhibition of TS enzyme activity was
required for TS enzyme inhibition to translate into inhibition of cell
growth and proliferation (28). Similar observations were reported with
antisense DNA molecules targeting TS mRNA, whereby oligodeoxyribonucleotides effectively inhibited TS mRNA levels but had
little to no effect on cell proliferation (29). Our laboratory had shown
previously that modified antisense oligoribonucleotides (ORNs) inhibited the same RKO cells with IC50 values in the 200-nM range (30).
However, control sense and mismatched ORNs, which had no effect
on TS protein expression, resulted in similar IC50 values as the
antisense ORN. This result suggested that the effect on inhibition of
cell growth was not caused entirely by specific suppression of TS
expression.
One of the major obstacles in cancer therapy is the development of
cellular resistance to cancer chemotherapy. In vitro, in vivo, and
clinical studies have shown that exposure to TS inhibitor compounds
results in acute induction of TS expression (22–24, 31). Our laboratory and others have proposed that this induction process leads to the
development of cellular drug resistance to TS inhibitors. Two wellestablished regulatory processes that mediate the acute induction of
TS expression are increased translation of TS mRNA and enhanced
stability of the TS protein (32). Our observation that the induction of
TS protein was not abrogated when RKO cells were first treated with
ZD1694 followed by the TS-targeted siRNA suggests that TS induction may be caused by stabilization of TS protein. Furthermore, levels
of TS protein were induced after 24 h of treatment with the combination of ZD1694 and TS1058 siRNA. However, when cells were
treated with this combination during the first 24 h, TS protein levels
decreased by nearly 70% compared with control levels. This finding
suggests that the inability of the TS-targeted siRNA to block the
ZD1694-mediated induction of TS protein may be caused by a timing
effect as to when cells are treated with the siRNA and TS inhibitor.
Under these conditions, the final expression of TS appears to be the
net result of two competing cellular events: the first is the acute
induction of TS protein by ZD1694, whereas the second is the active
degradation of TS mRNA by TS1058 siRNA. To investigate this issue
further, we conducted a time course experiment to determine the
effect on TS protein levels after the 48-h point. Our results demonstrate that regardless of whether ZD1694 was administered first followed by the siRNA or administered together with the siRNA, the
expression of TS decreased rapidly after an additional 12 h. This result
then would suggest that the TS protein is not stabilized under these
specific conditions. Thus, our findings show that TS1058 siRNA can
prevent induction of TS protein in RKO cells following incubation
with raltitrexed. Furthermore, they provide strong evidence that siRNAs can be used to abrogate effectively this acute resistance mechanism.
Our laboratory and others have shown that tumors overexpressing
TS protein, either acutely or chronically, are resistant to TS inhibitor
compounds (33, 34). We have shown that siRNAs may provide a
novel approach to circumvent such resistance mechanisms. Treatment
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siRNA AND CHEMOSENSITIVITY TO TS INHIBITORS
of TS-overexpressing RKO cells with siRNA concentrations as low as
10 pM can reduce IC50 values by twofold, whereas a concentration of
1 nM TS1058 siRNA restored chemosensitivity to three different TS
anticancer agents (ZD1694, 1843U89, and FdUrd) by 15-fold.
In the present report, we show that siRNAs directed at TS mRNA
specifically and potently repressed expression of TS mRNA and TS
protein in human colon cancer RKO cells via the process of RNAi.
This approach may prevent and/or overcome the acute induction of TS
and the subsequent development of cellular resistance observed with
TS inhibitor compounds now being used in the clinical setting. In this
regard, the use of siRNA molecules may have therapeutic promise as
a strategy to be used alone or in combination with other established
TS inhibitor compounds. Moreover, these studies may provide new
insights as to how siRNAs directed at other critical signaling pathways may be developed as novel therapeutic molecules for the treatment of human cancer.
ACKNOWLEDGMENTS
We thank the Yale Cancer Center and the VACT Healthcare System for
their continued support of this research.
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Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2004 American Association for Cancer
Research.
Small Interfering Double-Stranded RNAs as Therapeutic
Molecules to Restore Chemosensitivity to Thymidylate
Synthase Inhibitor Compounds
John C. Schmitz, Tian-min Chen and Edward Chu
Cancer Res 2004;64:1431-1435.
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