Download Allele-specific cancer cell killing in vitro and in vivo

Cancer Gene Therapy (2009) 16, 532–538
r
2009 Nature Publishing Group All rights reserved 0929-1903/09 $32.00
www.nature.com/cgt
ORIGINAL ARTICLE
Allele-specific cancer cell killing in vitro and in vivo targeting a
single-nucleotide polymorphism in POLR2A
ORF Mook, F Baas, MB de Wissel and K Fluiter
Department of Neurogenetics, Academic Medical Center, Amsterdam, The Netherlands
Cancer is one of the diseases for which RNA interference is a potential therapeutic approach. Genes involved in the promotion or
maintenance of tumor growth are obvious targets for RNAi. RNAi is also considered an attractive additional approach to
conventional chemotherapy for cancer treatment. Moreover, siRNAs have shown a high specificity for their molecular target
mRNAs as they can selectively inhibit cancer-promoting genes that differ by a point mutation. Loss of heterozygosity (LOH) reduces
genes to hemizygosity in cancer cells and presents an absolute difference between normal and cancer cells. The regions of LOH are
usually much larger than the tumor suppressor gene, which is lost, and has been shown to contain genes that are essential for cell
survival. Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man. SNPs in essential genes
that are frequently affected by LOH can be used as a target for a therapy against cancer cells with LOH. We have designed siRNAs
against the gene of the large subunit of RNA polymerase II (POLR2A), a gene located in close proximity to the tumor suppressor
gene p53, which frequently shows LOH in cancer cells. It is shown in vitro that siRNA can selectively inhibit POLR2A expression
dependent on its genotype. Furthermore, cancer cell proliferation and tumor growth inhibition in nude mice was genotype
dependent. We conclude that siRNA can be used for genotype-specific inhibition of tumor growth targeting an SNP in POLR2A
in vivo.
Cancer Gene Therapy (2009) 16, 532–538; doi:10.1038/cgt.2008.104; published online 23 January 2009
Keywords: RNA interference; siRNA; cancer; single nucleotide polymorphism; POLR2A.
Introduction
RNA interference (RNAi)1–3 was first discovered in
Caenorhabditis elegans as a potent gene-silencing mechanism.4 The inhibition of specific gene expression by means
of RNAi has also been achieved in mammalian cells by
directly introducing short dsRNA (siRNA) into cells,
which avoids a response evoked by long dsRNA.5 Cancer
is clearly an important potential target for RNAi-based
therapies.6 Target molecules usually represent genes that
have been shown previously to be relevant or rate limiting
for tumor growth, including growth factors and receptors
as well as antiapoptotic or downstream signal transduction proteins in cancer cells.7 Often these targeted
molecules are not unique for cancer cells thereby limiting
the therapeutic window or these molecules appear to be
redundant leading to resistance. Therefore, targeting of
molecules that are unique in cancer cells may lead to more
specific therapeutic interventions.
Correspondence: Dr ORF Mook, Department of Neurogenetics,
Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam,
The Netherlands.
E-mail: [email protected]
Received 22 August 2008; revised 13 October 2008; accepted 25
November 2008; published online 23 January 2009
One of the differences between cancer cells and normal
cells is that cancer cells have lost large segments of DNA.
The loss of large chromosomal regions, or even whole
chromosomes, is an early event in the clonal evolution of
cancers. Loss of heterozygosity (LOH) can involve 420%
of the total genome in certain cancers.8 This irreversible
difference between normal and tumor cells forms the basis
of an approach for anticancer drug development, called
allele-specific inhibition (ASI)9 (reviewed in10). We have
previously identified a large number of single-nucleotide
polymorphisms (SNPs) in genes that are essential for cell
survival and are localized in chromosomal regions often
involved in LOH in various cancer types.11 Inhibition of
the hemizygous essential gene left in the cancer cells will
lead to specific cancer cell death. The heterozygous
normal cells will loose maximally half of the expression
of the gene by this approach and cell survival is not
affected. Classic antisense approaches have shown proof
of principle of ASI in vitro9,11 and in vivo.12 However,
antisense-based approaches have some specific limitations
that can likely be overcome by the use of siRNA.
Over the past few years, several studies have reported
the selective silencing of mutant alleles resulting from
single-nucleotide point mutations by RNAi.2,13–17 In line
with that, systematic analysis of a single-nucleotidemismatched target showed that when the character and
the position of the mismatch is carefully considered, a
Allele-specific inhibition of tumor growth
ORF Mook et al
siRNA
si10C
si10T
si7C
si7T
si10CM9
si10TM9
siGFP
Antisense sequense 50 -30
GUCUUCGCCGUAGCGCAGCdTdG
GUCUUCGCCAUAGCGCAGCdTdG
UUCGCCGUAGCGCAGCUGCdAdC
UUCGCCAUAGCGCAGCUGCdAdC
GUCUUCGCAGUAGCGCAGCdTdG
GUCUUCGCAAUAGCGCAGCdTdG
GAUGAACUUCAGGGUCAGCdTdT
single mismatch can render an siRNA ineffective.18,19
Therefore, the sequence specificity and the high success
rate of finding a potent siRNA made it an attractive
molecule for ASI. In our approach we used siRNA to
target an essential gene (POLR2A) that is present in only
one copy in the cancer cell but present as a heterozygous
gene in normal cells. Heterozygosity due to a SNP in
POLR2A protects normal cells whereas hemizygosity for
POLR2A it is lethal for cancer cells.
In this study we show in vitro that siRNAs can
discriminate between two allelic forms of POLR2A that
differ by a single nucleotide in the target sequence.
Systemic delivery of unformulated siRNA resulted in
genotype-specific growth inhibition in vivo.
533
POLR2A
Table 1 Different siRNAs targeting POLR2A
3’
5’
C
G
3’
si10C
5’
Pos. 10
3’
5’
U
A
3’
si10T
3’
C
G
Pos. 7
si7C
3’
U
A
Pos. 7
si7T
3’
C G
G A
Pos. 10/9
siRNA synthesis
Six siRNA duplexes (Proligo, Paris, France) targeted to
the POLR2A C/T SNP at position 2673 relative to the
start codon were designed (Table 1; Figure 1). A siRNA
targeting eGFP (siGFP; Table 1) was purchased from
MedProbe (Lund, Sweden) and was used as a control for
in vivo experiments. In all siRNAs, a two DNA nucleotide
overhang was added at the 30 end to stabilize the duplex.
Sense and antisense strands were chemically synthesized
and obtained annealed.
si10CM9
3’
Cell lines
The pancreatic cancer cell line MiaPaca II (T genotype)
and the prostate cancer cell line 15PC3 (C genotype) were
maintained at 37 1C and 5% CO2 by serial passage in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine,
100 U ml1 penicillin and 100 mg ml1 streptomycin.
Transfections
Transfections were performed in six-well culture plates
with Lipofectamine 2000 as transfection agent. For
transfections the siRNA concentrations ranged between
5 and 100 nM. Total RNA was isolated 24 h posttransfection. Protein samples were 72 h posttransfection.
Northern blots
Cells were harvested in Trizol (Gibco, Breda, The
Netherlands) after removing the growth medium 24 h
posttransfection. The RNA isolation was according to the
manufacturer’s procedure. RNA was denatured using
glyoxal and separated on 1% agarose gels, following
U G
A A
3’
Pos. 10/9
mRNA
5’
5’
Materials and methods
mRNA
5’
5’
3’
mRNA
5’
5’
3’
mRNA
5’
Pos. 10
5’
3’
mRNA
mRNA
si10TM9
5’
Figure 1 Schematic representation of siRNAs and the relative
position of the single-nucleotide polymorphism (SNP) in large subunit
of RNA polymerase II (POLR2A) that were used in this study.
standard protocols. RNA was subsequently transferred to
Hybond-N þ membrane (Amersham, Roosendoal, The
Netherlands) in 20 SSC. Following transfer the RNA
was UV crosslinked and the membrane was baked for 4 h
at 80 1C. To detect POLR2A and 28S mRNA, we used the
probes previously described.20 Hybridizations and posthybridization washes were according to Church and
Gilbert.21 POLR2A mRNA levels were quantified using
Aida software version 3.44 (Raytest, Tilburg, The
Netherlands) and normalized to the levels of 28S mRNA.
Western blots
Cell extracts were prepared in lysis buffer (PBS; 1%
Triton X-100, 0.01% sodium azide), were subjected
to SDS–polycrylamide gel (6%) electrophoresis
(SDS–PAGE), and the resolved proteins were transferred
electrophoretically to polyvinylidene fluoride (PVDF)
membranes (Invitrogen, Breda, The Netherlands). POLR2A was detected with a mouse-anti-POLR2A monoclonal antibody (clone 8WG16; RDI). Elongation factor2a (rabbit-anti-eEF2a; Cell Signaling, Damvers, MA) was
used as loading control. Chemiluminescent detection was
performed on a LAS3000 (Fuji, Tokyo, Japan) in
accordance with the manufacturer’s instructions. POLR2A protein levels were quantified using Aida software
Cancer Gene Therapy
Allele-specific inhibition of tumor growth
ORF Mook et al
534
15PC3
1.50
1.50
0.50
0.75
0.50
si
10
C
M
oc
k
0.00
si
7T
0.00
si
7C
0.25
si
10
T
si
10
C
M
9
si
10
TM
9
0.25
M
oc
k
0.75
1.00
si
7T
1.00
1.25
si
7C
1.25
si
10
T
si
10
C
M
9
si
10
TM
9
POLAR2A/28S
1.75
si
10
C
POLAR2A/28S
MiaPaca II
1.75
Figure 2 Quantification of densitometric analysis of northern blots of MiaPaca II cells (a) and 15PC3 cells (b) transfected with indicated siRNA
targeting large subunit of RNA polymerase II (POLR2A) at concentrations of 25 nM. Bar graphs show the quantification of three independent
experiments (mean±s.e.m.) of POLR2A over 28S ribosomal RNA. Ratios are normalized to mock transfection.
version 3.44 (Raytest) and normalized to the levels of
eEF2a.
In vivo model
Female NMRI nu/nu mice (8- to 10-weeks old; Charles
River, Maastricht, The Netherlands) were injected
subcutaneously with both 106 MiaPaca II (right flank)
and 106 15PC3 cells (left flank) cells. The cells were
injected within 1 h after harvesting by trypsin treatment.
Before injection the cells were washed with phosphatebuffered saline (PBS), counted with a hemocytometer
and subsequently mixed with Matrigel on ice (Collaborative Biomedical Products, Bedford, MA). At 1 week after
tumor cell injection, unformulated si10C, si10T, si10CM9,
si10TM9, siGFP (0.15 mg kg1) or saline was administered via low volume (0.2 ml) tail vein injections22,23 twice
a week for 3 weeks. From the start of the treatment until
killing, tumor growth was recorded three times a week.
Tumor volumes were calculated according Meyer et al.24
Data points were fitted with GraphPad Prism using linear
regression in order to calculate the tumor growth rates
(GraphPad Prism Software Inc., San Diego, CA). Growth
rates of tumors in siRNA-treated animals were normalized to the growth rates of tumors in saline treated
animals. Aspartate aminotransferase (ASAT) and alanine
aminotransferase (ALAT) levels in serum were determined using standard diagnostic procedures with the
appropriate kits (Roche Diagnostics, Mannheim, Germany). For each treatment, five mice per group were used.
All animal experiments were conducted under the
institutional guidelines and according to the law; they
were sanctioned by the animal ethics committee.
Results
In vitro genotype-specific inhibition of POLR2A mRNA
and decreased POLR2A protein levels is dependent on
the relative position of the SNP
To test the sequence specificity of siRNA we used the
human cancer lines MiaPaca II and 15PC3. These cell
Cancer Gene Therapy
lines differ in sequence of POLR2A at position c.2673.
As POLR2A encodes an essential gene in mammalian
cells inhibition of gene expression would result in
diminished cell growth. POLR2A mRNA levels,
protein levels and growth inhibition were used as readout
for sequence specificity. In MiaPaca II cells (T genotype),
POLR2A mRNA was downregulated by si10T. Its
allelic variant si10C did not show downregulation of
POLR2A mRNA. Introduction of one mismatch at ninth
position of the siRNA targeting the T genotype
(si10TM9) completely abrogated its ability to downregulate POLR2A. POLR2A expression in MiaPaca II
cells was also not affected by a siRNA targeting
the C genotype with an additional mismatch at ninth
position (si10CM9), having two mismatches with this
genotype (Figure 2a). We observed sequence-specific
POLR2A mRNA knockdown with siRNA at concentration as high as 100 nM (data not shown). Shifting
the position of the siRNA so that the SNP was opposite
to the seventh position in the siRNA (si7T) resulted in
loss of sequence specificity. Both si7T and si7C downregulated POLR2A independent of the c.2673 genotype
(Figure 2a). This confirms previous findings that showed
that ASI is dependent on the relative position of the
mismatch.
Transfection of the same set of siRNAs into 15PC3
cells (C genotype) showed similar results. si10C
effectively downregulated POLR2A mRNA (Figure 2b).
The siRNAs si10T, si10CM9 and si10TM9 were not
effective, not even at a concentration of 100 nM, whereas
si7C and si7T were both effective in POLR2A mRNA
knockdown at all the concentrations used (Figure 2b).
These results show that in in vitro experiments siRNA
can discriminate between two genotypes that differ
only in one nucleotide. Discrimination was observed
over a range of siRNA concentrations up to 20 times
the effective concentration of 5 nM. However, discrimination was dependent on the relative position of the
SNP. POLR2A protein levels in MiaPaca II and 15PC3
cells (Figure 3) correlated with POLR2A mRNA
levels.
Allele-specific inhibition of tumor growth
ORF Mook et al
Effect of siRNAs targeting POLR2A on cell growth
As POLR2A is an essential component of the transcriptional machinery, downregulation is cytotoxic. Therefore
we can easily study whether allele-specific siRNA can
mediate specific target knockdown while leaving its allelic
variant that differs only for one nucleotide, unaffected.
To test the siRNA specificity in a toxicity assay, MiaPaca
II and 15PC3 cells were transfected with the same siRNAs
against POLR2A and cell survival was observed for 4
days. At 1-day posttranfection there were no obvious
differences between the transfections in either cell line,
indicating similar seeding and recovery of the cells after
plating them. At 5 days posttransfection, MiaPaca II cells
(T-genotype) that were transfected with siRNAs that had
no effect on POLR2A expression in this cell line (si10C,
si10CM9, si10TM9) or mock transfection had grown to
confluence (Figure 4). Transfection with siRNAs that
knocked down POLR2A in this cell line (si10T, si7C and
si7T) resulted in cell death and subsequent detachment
from their surface (Figure 4). Likewise, 15PC3 cells
(C genotype) transfected with siRNAs that had no effect
on POLR2A expression in this cell line (si10T, si10CM9,
si10TM9) grew at similar rates as mock transfected cells
and all grew confluent in 5 days (Figure 4). siRNAs that
MiaPaca II
15PC3
si7T
Mock
si7C
si10TM9
si10T
si10CM9
si10C
si7T
Mock
si7C
si10TM9
si10T
si10CM9
si10C
POLR2A
eEF2α
Figure 3 Representative western blot of three independent
experiments of MiaPaca II and 15PC3 cells transfected with the
indicated siRNA targeting the large subunit of RNA polymerase II
(POLR2A) at a concentration of 25 nM.
effectively lowered POLR2A mRNA and protein in
15PC3 cells (si10C, si7C and si7T) also resulted in cell
death and detachment from the surface (Figure 4). This
demonstrates that the fully matched siRNAs si10C and
si10T targeting POLR2A were toxic but more importantly, for siRNAs carrying a single mismatch at position
9 or 10, no toxicity was observed in this time frame.
Therefore, si10C and si10T are potential candidates for
genotype-specific drugs.
Genotype-specific tumor growth inhibition
Since the final goal of our approach is the development of
an allele-specific cancer drug we tested whether siRNA
directed against POLR2A could inhibit growth of tumor
xenografts dependent on their genotype. Growth of
MiaPaca II xenografts was inhibited in mice treated with
systemically administered si10C, si10T and si10TM9. In
contrast to the in vitro experiments, a single mismatch
of a siRNA with its target mRNA was toxic in vivo
(Figure 5a). Animals treated with siRNA with 2
mismatches (si10CM9) and siRNA targeting eGFP
showed similar growth rates of the MiaPaca II xenograft
as in the saline treated animals (Figure 5a). Introduction
of a second mismatch with the T-genotype was necessary
to obtain genotype-specific growth inhibition. Likewise,
in 15PC3 xenografts tumor growth was inhibited by
si10C, si10T and si10CM9 (Figure 5b). No growth
inhibition was observed with si10TM9 which has two
mismatches with the C-genotype and siRNA targeting
eGFP (Figure 5b). This demonstrates that also in 15PC3
xenografts two mismatches were needed to prevent tumor
growth inhibition and again a single mismatch is not
sufficient to render a siRNA ineffective in vivo. Therefore,
si10CM9 and si10TM9 showed ASI of tumor growth
(Figures 5a and b). Serum ASAT and ALAT levels, as
determined after killing of the animals, did not differ
significantly between the treatment groups indicating that
Figure 4 Representative images of cultured MiaPaca II and 15PC3 cells tranfected for 5 days with the indicated siRNA targeting large subunit
of RNA polymerase II (POLR2A) at a concentration of 25 nM.
Cancer Gene Therapy
535
Allele-specific inhibition of tumor growth
ORF Mook et al
536
ASI
ASI
0.2
Relative growth rate
1.0
0.8
0.6
0.4
0.2
)
P1
0
10
+
(p
(p
M
9
M
M
M
1
)
9)
(p
at
ch
m
G
FP
2
M
M
1
M
M
2
si
+
9
(p
M
1
in
)
P1
0
10
(p
M
M
M
1
)
9)
(p
at
ch
m
G
FP
si
e
in
Sa
l
e
0.0
0.0
Sa
l
Relative growth rate
0.4
si10TM9
0.6
1.2
si10T
0.8
si10C
si10CM9
1.0
si10C
si10TM9
si10T
1.2
si10CM9
15PC3
MiaPaca II
Figure 5 In vivo growth inhibition due to siRNA administration (a, b). Selective tumor growth inhibition was observed in MiaPaca II xenografts
(a) and 15PC3 xenografts (b). MM indicates the number of mismatches with the particular large subunit of RNA polymerase II (POLR2A)
genotype of the tumor. Bar graphs show the mean±s.e.m. of the relative tumor growth rates (n ¼ 5). Allele-specific inhibition (ASI) indicates
siRNAs that show genotype-specific tumor growth inhibition.
there was no liver toxicity due to administration of the
siRNAs (data not shown).
Discussion
Allele specific inhibition is a promising approach for the
treatment of various diseases including cancer.10,13,14,25
Proof of principle for ASI has been shown previously
using antisense oligonucleotides (ODNs)9,20 and siRNA16,17 in vitro. In vivo, tumor growth could be inhibited
allele specifically using antisense oligonucleotides however only with a very small therapeutic window.12 ASI
mediated by intravenously administered chemically
synthesized siRNA in vivo has not been shown before.
Here we show that ASI by siRNA can be applied both
in vitro and in vivo. We show in two genotypically
different cells lines that proper base pairing at the ninth
and tenth position of the antisense strand of siRNA and
its target RNA is important for its function because a
mismatch at these positions rendered siRNA directed
against POLR2A inactive. This is in line with the
observation that RNAi is lost upon introduction of
central mismatches (positions 9–11).13–18,25–27 The catalytic site is at that region and apparently loss of proper
base pairing at those specific positions results in loss of
RNAi. This was strengthened by the finding that the same
mismatches (C:A and G:U) at the seventh position in the
AS strand were tolerated.
However, it has been shown recently that besides the
position, the nature of the mismatch is also involved in
target recognition.18,19 Unfortunately, these rules do not
apply the same to different targets. For example, a C:A
Cancer Gene Therapy
mismatch at the tenth position did not affect gene
silencing,26 resulted in target knockdown of only approximately 25%18 or as shown in our study was not tolerated
at all. If this also applies for a G:U mismatch at the tenth
position was not investigated in this particular studies.
In our study, the introduction of a mismatch at the
ninth position resulted in a G:A mismatch. In general,
bulky purine–purine mismatches, A:A and G:A, are much
less tolerated and therefore render an siRNA inactive.18
Specifically, mutated K-RASV12 was discriminated from
its wild-type variant based on a A:G mismatach at
position 9.2 This is in agreement with our study that
showed loss of RNAi upon the introduction of a G:A
mismatch at the ninth position. There are a limited
number of exceptions to the central region; C:U mismatch
at the sixteenth position,28 G:G mismatch at the
seventeenth position for CEACAM6 siRNA22 and a
C:T mismatch at the seventh position29 and studied
systematically.18,19
We next tested if siRNA could inhibit growth of
xenografted tumors in nude mice in a genotype (allele)specific manner. It has recently been shown by us23 and
others that low dosages of unmodified siRNA can
mediate RNAi in vivo.22,30,31 We show siRNA mediated
genotype (allele)-specific inhibition of tumor growth.
However, siRNAs carrying one mismatch, either at the
ninth or tenth position, with its target retained activity in
vivo.
Combination of these two mismatches was necessary to
render the siRNA ineffective and achieve ASI. It is
unclear why in vitro selected genotype-specific siRNA lost
its specificity in vivo but the observed effect in vivo must be
sequence dependent as siRNA with two mismatches
Allele-specific inhibition of tumor growth
ORF Mook et al
targeting POLR2A and siRNA targeting GFP did not
inhibit tumor growth whereas fully matched or one
mismatched siRNAs targeting POLR2A did inhibit
tumor growth.
Mouse and human POLR2A are highly homologous
but differ at two bases from the human sequence in the
targeted region of the mRNA. Thus, depending of the
human genotype, two or three mismatches are present.
These mismatches are sufficient prevent POLR2A knockdown in mouse tissues. Xenografts are composed of
human cancer cells and mouse stromal cells. Stromal
POLR2A is not inhibited and cannot be discriminated
from human POLR2A on western or northern blots. It is
therefore not possible to determine the human POLR2A
levels in xenografts reliably.
In conclusion, siRNA-mediated ASI is a potential
therapeutic application to inhibit tumor growth in vivo.
Abbreviations
ASI, allele-specific inhibition; DMEM, Dulbecco’s modified Eagle’s medium; eEF2a, elongation factor-2a; LOH,
loss of heterozygosity; ODNs, antisense oligonucleotides;
POLR2A, large subunit of RNA polymerase II; PVDF,
polyvinylidene fluoride; RNAi, RNA interference;
RPA70, replication protein A, 70-kDa subunit; SNPs,
single nucleotide polymorphisms; SDS–PAGE, SDS–
polycrylamide gel electrophoresis.
Acknowledgements
This work was supported by the Dutch Cancer Society,
project number 2003-2968 and the Stichting Kindergeneeskundig Kanker Onderzoek.
References
1 Leung RK, Whittaker PA. RNA interference: from gene
silencing to gene-specific therapeutics. Pharmacol Ther 2005;
107: 222–239.
2 Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference.
Cancer Cell 2002; 2: 243–247.
3 Zhang Z, Jiang G, Yang F, Wang J. Knockdown of mutant
K-ras expression by adenovirus-mediated siRNA inhibits the
in vitro and in vivo growth of lung cancer cells. Cancer Biol
Ther 2006; 5: 1481–1486.
4 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE,
Mello CC. Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature 1998; 391:
806–811.
5 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K,
Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells. Nature 2001; 411:
494–498.
6 Kim DH, Rossi JJ. Strategies for silencing human disease
using RNA interference. Nat Rev Genet 2007; 8: 173–184.
7 Aigner A. Applications of RNA interference: current state
and prospects for siRNA-based strategies in vivo. Appl
Microbiol Biotechnol 2007; 76: 9–21.
8 Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities
in human cancers. Nature 1998; 396: 643–649.
9 Basilion JP, Schievella AR, Burns E, Rioux P, Olson JC,
Monia BP et al. Selective killing of cancer cells based on loss
of heterozygosity and normal variation in the human
genome: a new paradigm for anticancer drug therapy. Mol
Pharmacol 1999; 56: 359–369.
10 Fluiter K, Housman D, Ten Asbroek AL, Baas F. Killing
cancer by targeting genes that cancer cells have lost: allelespecific inhibition, a novel approach to the treatment of
genetic disorders. Cell Mol Life Sci 2003; 60: 834–843.
11 Ten Asbroek AL, Olsen J, Housman D, Baas F, Stanton Jr
V. Genetic variation in mRNA coding sequences of highly
conserved genes. Physiol Genomics 2001; 5: 113–118.
12 Fluiter K, ten Asbroek AL, van Groenigen M, Nooij M,
Aalders MC, Baas F. Tumor genotype-specific growth
inhibition in vivo by antisense oligonucleotides against a
polymorphic site of the large subunit of human RNA
polymerase II. Cancer Res 2002; 62: 2024–2028.
13 Abdelgany A, Wood M, Beeson D. Allele-specific silencing
of a pathogenic mutant acetylcholine receptor subunit by
RNA interference. Hum Mol Genet 2003; 12: 2637–2644.
14 Ding H, Schwarz DS, Keene A, Affar el B, Fenton L, Xia X
et al. Selective silencing by RNAi of a dominant allele that causes
amyotrophic lateral sclerosis. Aging Cell 2003; 2: 209–217.
15 Martinez LA, Naguibneva I, Lehrmann H, Vervisch A, Tchenio
T, Lozano G et al. Synthetic small inhibiting RNAs: efficient
tools to inactivate oncogenic mutations and restore p53
pathways. Proc Natl Acad Sci USA 2002; 99: 14849–14854.
16 Miller VM, Gouvion CM, Davidson BL, Paulson HL.
Targeting Alzheimer’s disease genes with RNA interference:
an efficient strategy for silencing mutant alleles. Nucleic
Acids Res 2004; 32: 661–668.
17 Miller VM, Xia H, Marrs GL, Gouvion CM, Lee G,
Davidson BL et al. Allele-specific silencing of dominant
disease genes. Proc Natl Acad Sci USA 2003; 100: 7195–7200.
18 Du Q, Thonberg H, Wang J, Wahlestedt C, Liang Z. A
systematic analysis of the silencing effects of an active siRNA
at all single-nucleotide mismatched target sites. Nucleic Acids
Res 2005; 33: 1671–1677.
19 Schwarz DS, Ding H, Kennington L, Moore JT, Schelter J,
Burchard J et al. Designing siRNA that distinguish between
genes that differ by a single nucleotide. PLoS Genet 2006; 2:
1307–1318.
20 ten Asbroek AL, Fluiter K, van Groenigen M, Nooij M,
Baas F. Polymorphisms in the large subunit of human RNA
polymerase II as target for allele-specific inhibition. Nucleic
Acids Res 2000; 28: 1133–1138.
21 Church GM, Gilbert W. Genomic sequencing. Proc Natl
Acad Sci USA 1984; 81: 1991–1995.
22 Duxbury MS, Matros E, Ito H, Zinner MJ, Ashley SW,
Whang EE. Systemic siRNA-mediated gene silencing: a new
approach to targeted therapy of cancer. Ann Surg 2004; 240:
667–674; discussion 675–676.
23 Mook OR, Baas F, de Wissel MB, Fluiter K. Evaluation of
locked nucleic acid-modified small interfering RNA in vitro
and in vivo. Mol Cancer Ther 2007; 6: 833–843.
24 Meyer T, Regenass U, Fabbro D, Alteri E, Rosel J, Muller
M et al. A derivative of staurosporine (CGP 41 251) shows
selectivity for protein kinase C inhibition and in vitro antiproliferative as well as in vivo anti-tumor activity. Int J
Cancer 1989; 43: 851–856.
Cancer Gene Therapy
537
Allele-specific inhibition of tumor growth
ORF Mook et al
538
25 Kurosawa T, Igarashi S, Nishizawa M, Onodera O. Selective
silencing of a mutant transthyretin allele by small interfering
RNAs. Biochem Biophys Res Commun 2005; 337: 1012–1018.
26 Dykxhoorn DM, Schlehuber LD, London IM, Lieberman J.
Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single
nucleotide polymorphism. Proc Natl Acad Sci USA 2006;
103: 5953–5958.
27 Li Y, Yokota T, Matsumura R, Taira K, Mizusawa H.
Sequence-dependent and independent inhibition specific for
mutant ataxin-3 by small interfering RNA. Ann Neurol 2004;
56: 124–129.
28 Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE.
EphA2: a determinant of malignant cellular behavior and a
Cancer Gene Therapy
potential therapeutic target in pancreatic adenocarcinoma.
Oncogene 2004; 23: 1448–1456.
29 Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE.
RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma
chemosensitivity to gemcitabine. Oncogene 2004; 23:
1539–1548.
30 Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L,
Shim H. Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res 2005; 65: 967–971.
31 Ocker M, Neureiter D, Lueders M, Zopf S, Ganslmayer M,
Hahn EG et al. Variants of bcl-2 specific siRNA for
silencing antiapoptotic bcl-2 in pancreatic cancer. Gut 2005;
54: 1298–1308.