Optimization of radiation controlled gene expression by adenoviral vectors in vitro.
ABSTRACT The radiation-inducible EGR-1-promoter has been used in different gene therapy approaches in order to enhance and locally restrict therapeutic efficacy. The aim of this study was to reduce nonspecific gene expression in the absence of irradiation (IR) in an adenoviral vector. Rat rhabdomyosarcoma R1H tumor cells were infected with adenoviral vectors expressing either EGFP or HSV-TK under control of the murine EGR-1 promoter/enhancer. Cells were irradiated at 0-6 Gy. Gene expression was determined by FACS-analysis (EGFP), or crystal violet staining (HSV-TK). The bovine growth hormone polyadenylation signal (BGH pA) was used as insulating sequence and was introduced upstream or upstream and downstream of the expression cassette. Infected R1H cells displayed IR dose-dependent EGFP expression. Cells treated with IR, AdEGR.TK and ganciclovir displayed a survival of 17.3% (6 Gy). However, significant gene expression was observed in the absence of IR with EGR.TK and EGR.EGFP constructs. Introduction of BGHpA upstream or upstream and downstream of expression cassette resulted in decreased nonspecific cytotoxicity by a factor of 1.6-2.3 with minor influence on the induced level of cytotoxicity. Introduction of insulating sequences in adenoviral vectors might allow tighter temporospatial control of gene expression by the radiation-inducible EGR-1 promoter.
- SourceAvailable from: Laure Marignol[Show abstract] [Hide abstract]
ABSTRACT: Significant evidence has accumulated indicating that certain genes are induced by ionising radiation. An implication of this observation is that their promoter regions include radiation-responsive sequences. These sequences have been isolated in the promoter of several genes including Erg-1, p21/WAF-1, GADD45alpha and t-PA. The mechanism by which radiation induces gene expression remains unclear but involves putative binding sites for selected transcription factors and/or p53. Consensus CC(A/T)6GG sequences have been localized in the Erg-1 promoter and are referred to as serum response elements or CArG elements. The tandem combination of CArG elements has been shown to improve gene expression levels, with a 9-copy motif conferring maximum inducibility. The response of these genes to ionising radiation appears to follow a sigmoid relationship with time and dose. Therapeutic induction of suicide genes and significant cytotoxicity can be achieved at clinically relevant x-rays doses both in vitro and in vivo but was found to be cell-type dependent. Radiation-inducible gene therapy can be potentially enhanced by exploiting hypoxia through the inclusion of hypoxia-response element motifs in the expression cassette, the use of the anaerobic bacteria or the use of neutron irradiation. These results are encouraging and provide significant evidence that gene therapy targeted to the radiation field is a reasonably attractive therapeutic option and could help overcome hypoxic radioresistant tumors.Cancer biology & therapy 06/2007; 6(7):1005-12. · 3.29 Impact Factor
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ABSTRACT: Tumor microenvironment is composed of different cell types including immune cells. Far from acting to eradicate cancer cells, these bone marrow-derived components could be involved in carcinogenesis and/or tumor invasion and metastasis. Here, we describe an alternative approach to treat solid tumors based on the genetic modification of hematopoietic stem and progenitor cells with lentiviral vectors. To achieve transgene expression in derivative tumor infiltrating leukocytes and to try to decrease systemic toxicity, we used the stress inducible human HSP70B promoter. Functionality of the promoter was characterized in vitro using hyperthermia. Antitumor efficacy was assessed by ex vivo genetic modification of lineage-negative cells with lentiviral vectors encoding the dominant-negative mutant of the human transforming growth factor-β receptor II (TβRIIDN) driven by the HSP70B promoter, and reinfusion of cells into recipient mice. Subsequently, syngeneic GL261 glioma cells were subcutaneously injected into bone marrow-transplanted mice. As a result, a massive antitumor response was observed in mice harboring TβRIIDN under the HSP70B promoter, without the need of any external source of stress. In summary, this study shows that stem cell-based gene therapy in combination with spatial and temporal control of transgene expression in derivative tumor-infiltrating cells represents an alternative strategy for the development of novel antitumor therapies.Cancer gene therapy 03/2012; 19(5):352-7. · 3.13 Impact Factor
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ABSTRACT: Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating reactive oxygen intermediates, which have been reported to activate the early growth response-1 (Egr-1) promoter through specific cis-acting sequences, termed CArG elements. The aim of this study was to construct an adenoviral vector containing CArG elements cloned upstream of the cDNA for human wt-p53, and to observe the effect of this vector on human non-small cell lung cancer (NSCLC) xenografts in athymic nude mice when combined with cisplatin treatment. The adenoviral vector AdEgr-p53 was generated by inserting CArG elements upstream of human wt-p53 cDNA. Two human NSCLC cell lines of varying p53 gene status, A549 (containing wild-type p53) and H358 (containing an internal homozygous deletion of the p53 gene) were used for in vitro and in vivo experiments. Wt-p53 production in cultured tumor cells and xenografts treated with the combination of AdEgr-p53 and cisplatin were detected by enzyme-linked immunosorbent assays. The antitumor responses in nude mice with the A549 or H358 xenografts following treatment with AdEgr-p53 and cisplatin were observed. We found that p53 was produced in tumor cells and xenografts treated with a combination of AdEgr-p53 and cisplatin. Furthermore, the Egr-1 promoter is induced by cisplatin, and this induction is mediated in part through the CArG elements. There was an enhanced antitumor response without an increase in toxicity following treatment with AdEgr-p53 and cisplatin, compared with either agent alone. Cisplatin-inducible p53 gene therapy may provide a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents. This is a novel treatment for human NSCLC.Cancer Science 11/2005; 96(10):706-12. · 3.48 Impact Factor
Optimization of radiation controlled gene expression by
adenoviral vectors in vitro
Martina Anton,1Iman EO Gomaa,1,2,3Tobias von Lukowicz,1,2Michael Molls,2Bernd
Gansbacher,1and Florian Wu ¨rschmidt2
1Institut fu¨r Experimentelle Onkologie & Therapieforschung, Klinikum rechts der Isar, TU Mu¨nchen, Munich,
Germany;2Klinik und Poliklinik fu ¨r Strahlentherapie & Radiologische Onkologie, Klinikum rechts der Isar,
TU Mu¨nchen, Munich, Germany; and3National Center for Radiation Research and Technology, Egyptian
Atomic Energy Authority, Cairo, Egypt.
The radiation-inducible EGR-1-promoter has been used in different gene therapy approaches in order to enhance and locally restrict
therapeutic efficacy. The aim of this study was to reduce nonspecific gene expression in the absence of irradiation (IR) in an
adenoviral vector. Rat rhabdomyosarcoma R1H tumor cells were infected with adenoviral vectors expressing either EGFP or HSV-
TK under control of the murine EGR-1 promoter/enhancer. Cells were irradiated at 0–6Gy. Gene expression was determined by
FACS-analysis (EGFP), or crystal violet staining (HSV-TK). The bovine growth hormone polyadenylation signal (BGH pA) was used
as insulating sequence and was introduced upstream or upstream and downstream of the expression cassette. Infected R1H cells
displayed IR dose-dependent EGFP expression. Cells treated with IR, AdEGR.TK and ganciclovir displayed a survival of 17.3%
(6Gy). However, significant gene expression was observed in the absence of IR with EGR.TK and EGR.EGFP constructs.
Introduction of BGHpA upstream or upstream and downstream of expression cassette resulted in decreased nonspecific cytotoxicity
by a factor of 1.6–2.3 with minor influence on the induced level of cytotoxicity. Introduction of insulating sequences in adenoviral
vectors might allow tighter temporospatial control of gene expression by the radiation-inducible EGR-1 promoter.
Cancer Gene Therapy (2005) 12, 640–646. doi:10.1038/sj.cgt.7700829
Published online 1 April 2005
Keywords: adenoviral vectors; EGR-1 promoter; insulating sequences; radiation
genes that regulate growth, sensitize tumor cells to
prodrugs, and induce antitumor immune response.1
Retroviral and adenoviral vectors have been chosen as
effective delivery vehicles for therapeutic genes. However,
they have limited effectiveness due to lack of specificity
and potential toxicity. Radiation can enhance gene
therapy by both the killing effect of irradiation (IR) and
the targeting potential of radiation to regulate gene
transcription.2One of the genes activated by radiation is
the early growth response gene Egr-1 (synonyms are
NGFI-A, zif268, TIS8, krox24),3which encodes a 533-
amino-acid phosphoprotein transcription factor. The
promoter sequence contains a transcription factor binding
site designated the serum responsive element. This motif
with itsconsensus sequence
‘‘CArG’’ box, within the promoter region was identified
as the radiation-inducible DNA element4targeted by
everal strategies of gene therapy of malignant tumors
are currently under investigation, including transfer of
reactive oxygen intermediates.5The usage of a radiation-
inducible promoter/enhancer is particularly interesting as
it opens the possibility to put gene expression under the
spatial and temporal control of radiation.2The feasibility
of this new approach has been demonstrated in different
Interference of viral promoters has been observed when
using adenoviral vectors in combination with tissue
specific promoters.12,13This interference may affect the
tissue specificity or inducibility of heterologous promo-
ters.12Most Ad vectors contain viral regulatory sequences
in their left end (nt 1–341) and in the E2 and E4
promoters that have been shown to interfere with tissue-
specific expression from a heterologous promoter. The
E1A enhancer cannot be removed from Ad vectors as it
overlaps with sequences required for packaging of viral
In order to overcome the problem of nonspecific gene
expression, sequences from the bovine growth hormone
polyadenylation signal (BGH pA) or the chicken b-globin
gene, respectively, have been used to restore tissue
selectivity of promoters in an adenoviral background.12,13
However, promoter/enhancer interference may not be
the only obstacle to tight regulation by the EGR-1
promoter; another one might be intrinsic ‘‘leakiness’’ of
Received November 17, 2004.
Address correspondence and reprint requests to: Dr Martina Anton,
Institut fu ¨r Experimentelle Onkologie & Therapieforschung, Klinikum
rechts der Isar, Ismaninger Str. 22, D-81675 Mu ¨nchen, Germany.
Cancer Gene Therapy (2005) 12, 640–646
All rights reserved 0929-1903/05 $30.00
r2005 Nature Publishing Group
transcription. The transcriptional control of the Egr-1
promoter is not clearly understood. It has been found to
be leaky in the context of many other vector systems and
the relative contribution of interference from viral
promoters remains to be defined.
We are interested in the efficacy and specificity of
radiation-inducibility of the EGR-1 promoter, with low
doses of IR. We here report on in vitro experiments in
rhabdomyosarcoma cells of the rat, which were trans-
duced with the Herpes simplex virus type 1 thymidine
kinase (HSV-TK) gene under control of the radiation-
inducible EGR-1 promoter by an adenoviral vector and
treated with ganciclovir (GCV) and IR. The influence of
insulators on nonspecific gene expression in the absence
of IR and inducibility with small doses of IR was
investigated. We show that EGR-1 can be induced by
small doses of IR and nonspecific gene expression in the
absence of IR can be reduced significantly without
compromising radiation-inducibility of the promoter.
Materials and methods
Cells, tissue culture and viral infections
The experiments were performed on rhabdomyosarcoma
R1H tumor cells of the rat. The tumor was derived from
the rhabdomyosarcoma R-1.14Originally the R-1 tumor
was derived from the BA 1112 tumor, which appeared
spontaneously in 1962 in the musculature of a Wistar rat
that had been irradiated 8 months before.15Professor
Jung, UKE Hamburg, kindly provided the R1H in 1999.
Adenoviral vector packaging 293 cells (Microbix,
Toronto, Canada) were cultivated in MEM medium
supplemented with glutamine and 10% fetal calf serum
(FCS). Vector amplification was in MEM medium with
glutamine and 5% heat inactivated horse serum. R1H
rhabdomyosarcoma cells were routinely cultivated in
DMEM medium supplemented with glutamine and 10%
FCS. During infection and IR experiments serum con-
centration was reduced to 5% FCS.
All restriction enzymes were obtained from Roche
Biochemicals or New England Biolabs. Standard DNA
manipulations were according to Sambrook et al.16
Adenoviral shuttle vectors consist of the left end of the
Ad genome. pMA41 was derived by digestion of pMA917
with BamHI and subsequent religation. The resulting
vector has the E1 region substituted by the human CMV
promoter, a multiple cloning site and an SV40 poly-
Control plasmids containing the CMV promoter were
obtained by insertion of the XbaI fragment of pEGFP
(Clontech), carrying the EGFP cDNA into the correspond-
ing site of pMA41 to yield pSW9 or an EcoRI–HindIII
fragment carrying the HSV-TK cDNA to yield pSW15.
The murine EGR-1 promoter containing XbaI–SalI
fragment of pE42518was subcloned into plasmid pDE1-
sp1A19XbaI–SalI to yield pTvL1. The SV40 polyadenyla-
tion signal was added by joining the ScaI–SalI fragment
containing the SV40 pA of pMA41 to the ScaI–XhoI
fragment of pTvL1 containing the EGR promoter
sequence. The resulting plasmid was called pTvL2. The
EGFP containing XbaI fragment of pEGFP was blunt
ended with Klenow enzyme and cloned into the EcoRV
site of pTvL2, to yield pTvL3. The HSV-TK cDNA was
subcloned from pSW15 by EcoRI–HindIII digest and
inserted accordingly into pTvL2 to obtain pTvL4.
The BGH pA was used as insulating sequence and was
obtained from pRc/RSV (Invitrogen, Groningen, Nether-
lands). The XhoI fragment containing the BGH pA was
inserted into the unique XhoI site 50of the Egr1-promoter
of pTvL3 to yield pTvL6. Additionally the BGH pA
containing BamHI fragment of pRc/RSV was inserted
into the BglII site 30of the SV40 pA in an antiparallel
orientation (to prevent possible influence of down stream
vector sequences) after partial digestion of pTvL6. The
resulting plasmid was pTvL7. pIEOG1 containing the
HSV-TK gene under control of the Egr1-promoter and an
50insulating sequence was constructed by substituting the
EGFP carrying BglII fragment of pTvL6 by the HSV-TK
pIEOG2 the BamHI–HindIII fragment of pSW15 was
introduced into BglII–HindIII cut pTvL7 to replace
EGFP by HSV-TK.
Integrity and orientation of inserts and insulating
sequences were verified by DNA sequencing.
Adenoviral vector construction by cotransfection of
shuttle vectors (pSW9, pSW15, pTvL3, pTvL4, pIEOG1,
pBHG10 into 293 cells was performed according to Hitt
and yielded AdCMV.EGFP (from pSW9),
pTvL3), AdEGR.TK (from pTvL4), AdI-EGR.TK (from
pIEOG1) and AdI-EGR.TK-I (from pIEOG2), respec-
tively. Identity of Ad vectors was confirmed by restriction
enzyme digests. High titer stocks were prepared as
described20and plaque-forming units per ml (pfu) were
A schematic presentation of the left ends of replication
defective Ad vectors carrying the HSV-TK expression
cassette, and insertions of insulating sequences is given in
Infection and IR experiments
Cells were irradiated in vitro under ambient conditions
with 6MeV photons of a linear accelerator or 70kV X-
rays. Single doses of 0Gy (control) to 6Gy were given.
Cells were kept at room temperature during IR.
For infection and IR experiments using EGFP expres-
sing Ad, 105cells were seeded in six-well plates. At 15–
20hours later, cells were infected at an multiplicity of
infection (MOI) of 25, respectively, in 500ml PBS2þ/well
for 30minutes at 371C, subsequently fed with medium
containing 5% FCS and irradiated 3hours later.
For infection and IR experiments using TK expressing
Ad, 7.5?103cells were seeded in 24-well dishes. The next
day infections were performed at an MOI of 10 in a total
Radiation controlled gene expression
M Anton et al
Cancer Gene Therapy
volume of 200ml PBS2þper well. A measure of 1ml
medium containing GCV (Cymeven, Roche Pharma,
Grenzach-Whylen) at 1mg/ml was added and 3hours
postinfection cells were irradiated. Additional doses of
GCV were given 48 and 96hours later where appropriate
and staining of cells was performed by adding a solution
of 3% formaldehyde, 5% glacial acetic acid, 50% ethanol,
1.15% crystal violet for 10minutes and extensive washing
5 days postinfection/IR. Stained cells were counted at
200-fold magnification. For each sample, 10 randomly
chosen fields were counted and the nonirradiated,
uninfected sample was set to 100%.
For FACS analysis, cells were plated in triplicates in six
wells, infected and irradiated as described above. EGFP
expression was monitored 15 or 20hours post-IR by
FACS analysis of trypsinized cells with a fluorescence
activated cell sorter (Becton, Dickinson FACS Vantage,
Heidelberg, Germany) using Argon Laser beam (Spectra
– Physics) of excitation energy 40mW at 488nm and the
CellQuest Software. In order to detect low levels of gene
expression, cellular autofluorescence was subtracted by
two-color compensation: two band-pass filters in the
green (530/30nm; FL1) and red (630/22nm; FL3)
channels were used to measure logarithmic amplification
of the FL1 and FL3.
In the SSC vs. FSC plot mock treated parental cells
were used to define a cell population (R1). In the FL1 vs.
FL3 plot, a polygonal region (R3) was defined as gate R3
so as to exclude any nonexpressing cells. All cells that
were part of R3 and R1 were used for quantification and
are expressed as % gated.
Northern blot analysis
Total RNA of 2?106
nonirradiated cells was prepared using the RNeasy Mini
Kit andQIAshredder (Qiagen,
infected and irradiated or
according to the manufacturer’s instructions. RNA was
separated on denaturing agarose gel, transferred to
GeneScreen hybridization transfer membrane (Perkin
Elmer Life Sciences, Zaventem, Belgium). The HSV-TK-
containing EcoRI–HindIII fragment of pSW15 was
labeled with a32P-dCTP (ICN; Germany) using the
Prime-It II Random Primer labeling Kit (Stratagene,
Germany). Loading control was with an 18S-coding
DNA fragment. Membranes were exposed to FLA-2000
PhosphoImager (raytest, Straubenhardt, Germany). Ex-
pression was quantitated using the AIDA software
(raytest, Straubenhardt, Germany). HSV-TK expression
was normalized to the respective 18S signal. The ratio
of irradiated vs. nonirradiated HSV-TK samples was
Data were pooled from two to three independent
experiments. Results are presented as means and SD.
Statistical analyses were performed using unpaired t-test.
A P-value o.05 was considered significant.
EGFP expression under control of CMV or EGR-1
Representative examples of FACS analyses are shown in
Figure 2a. In Figure 2b, EGFP expression of R1H cells,
infected with AdEGR.EGFP, AdCMV.EGFP (control
virus) or uninfected control (PBS) is shown after no IR or
6Gy IR. EGFP expression was found in 59.9%
(SD71.3%) of AdCMV.EGFP infected cells (control
virus), which increased significantly to 71.2% (71.8%)
after 6Gy IR (P¼.0008). Nonspecific EGFP expression
was observed in unirradiated, AdEGR.EGFP infected
tumor cells with 14.1% (70.6%) of cells showing EGFP
expression increasing significantly to 29.4% (70.3%)
after 6Gy IR (Po.0001).
AdEGR.EGFP infected R1H cells demonstrated radia-
tion dose-dependent reporter gene expression with 7.2%
at 0Gy, 11.7% at 2Gy, 15.1% at 4Gy and 17.6% at 6Gy,
respectively, 15hours after IR. This results in a 1.6-, 2.1-
and 2.5-fold induction of EGFP expression at 2, 4 and
6Gy, respectively (Fig 3).
Radiation inducible expression of HSV-TK and cell
Egr-1 driven expression of HSV-TK was analyzed by
Northern blot at early times after infection and IR. R1H,
infected with AdEGR.TK, showed an induction of gene
expression after IR with 6Gy of 1.8-fold over the
nonirradiated control (Fig 4). In contrast, CMV-promo-
ter driven expression of HSV-TK showed a 1.2-fold
AdEGR.TK and GCV was also compared to cell killing
delivered by the constitutive human cytomegalovirus
immediate early promoter (CMV) (Fig 5). After treatment
Figure 1 Structure of the left end of replication defective Ad vectors
carrying HSV-TK under control of the EGR promoter and insertions
of insulating sequences. Open box: HSV-TK cDNA; black arrow:
EGR promoter, gray box: SV40 pA; hatched arrows: insulating
sequences preventing transcriptional activation from the extreme left
end of the Ad genome (leftward pointing arrow) or possible influence
of the E2/E4 regions (rightward pointing arrow); C: packaging signal;
ITR: left end inverted terminal repeat.
Radiation controlled gene expression
M Anton et al
Cancer Gene Therapy
with AdCMV.TK only, cell survival decreased to 13.2%
(74.1%) as compared to the uninfected control. A
significant decrease to 5.2% (72.1%) was achieved after
IR with 6Gy (Po.0001). Under the same conditions,
when using the AdEGR.TK, a survival of 17.3%
(75.3%) was achieved after IR as compared to 33.1%
(712.9%) of nonirradiated sample (Fig 5).
Introduction of insulating sequences to reduce
background expression in the absence of IR
The BGHpA was chosen as insulating sequence and was
either introduced upstream of the EGR promoter or
upstream and downstream of the EGR/HSV-TK/SV40pA
expression cassette to avoid transcriptional activation
from E1A enhancer sequences (upstream) or E2/E4
promoter/enhancer sequences (downstream) (Fig 1).
Introduction of an insulating sequence upstream of the
EGR-promoter resulted in an induction of HSV-TK
mRNA after IR with 6Gy as compared to nonirradiated
cells by a factor of 2.3 and a factor of 3.0 after
introduction of two insulating sequences at early times
after infection (Fig 4).
Insertion of an insulator upstream of the expression
cassette reduced the nonspecific cytotoxicity by a factor of
1.6 in nonirradiated samples (54.1713.4% surviving cells)
0 Gy 6 Gy
Figure 2 EGFP expression of R1H cells infected with AdEGR.
EGFP, control virus (AdCMV.EGFP) or uninfected control 20hours
post-irradiation. (a) FACS-analysis obtained after correction for
autofluorescence. One representative sample is shown for each
combination. (b) Quantification of EGFP expression. PBS: unin-
fected control; CMV: infection with constitutively EGFP expressing
Ad (AdCMV.EGFP) (MOI 25); EGR: infection with AdEGR.EGFP
(MOI 25). 0Gy: nonirradiated sample; 6Gy: irradiated sample. The
numbers of EGFP-positive cells by FACS were expressed as percen-
tage of gated cells. Values represent means of three samples and SD.
Figure 3 Radiation dose dependence of EGR driven EGFP
expression (fold increase as compared to nonirradiated tumor cells).
AdEGR.EGFP infected R1H cells (MOI 25) were irradiated at the
indicated doses. At 15hours later EGFP expression was quantitated
by FACS-analysis as described in Figure 2b.
Figure 4 Radiation controlled gene expression after infection of R1H cells with AdEGR-TK. Representative Northern blot analysis of R1H cells,
infected with MOI 25 of either AdCMV.TK (CMV), AdEGR.TK (EGR), AdI-EGR.TK, containing one insulator 50of the expression cassette
(I-EGR), AdI-EGR.TK-I flanked by insulators (I-EGR-I), or uninfected (PBS). Samples were either nonirradiated (‘‘0’’) or irradiated at 6Gy (‘‘6’’)
3hours postinfection. RNA was collected 6hours postirradiation. Hybridization was with an HSV-TK probe (top). 18S served as loading control; %
TK: Quantification of TK expression. The signal intensity is expressed as % of the intensity of the corresponding 18S signal.
Radiation controlled gene expression
M Anton et al
Cancer Gene Therapy
as compared to the parental vector carrying no insulator
(33.1712.9%) as shown in Figure 5. Inducibility of the
EGR-promoter by IR with 6Gy was not impaired with
17.375.3% without insulator). Additional introduction
of a downstream insulator further decreased nonspecific
gene expression resulting in 75.8% (718.0%) surviving
cells in the absence of IR (decrease of a factor of 2.3
compared to no insulator). The inducibility of the
promoter was modestly but significantly decreased
(21.973.3% surviving cells after 6Gy IR as compared
to Egr 6Gy IR; P¼.0014). However, the relative
difference in surviving tumor cells between nonirradiated
and irradiated cells was highest with the introduction of
The combination of the radiation-inducible AdEGR.TK
construct and IR resulted in a significant increase in cell
kill as compared to IR alone and was surprisingly strong
after induction by IR (Fig 5). Gene expression, as
determined by reporter gene expression, was increased
in a radiation dose-dependent manner (Fig 3). However,
the EGR promoter/enhancer was rather strongly induced
in the absence of IR (Figs 2b and 5). This leakiness of the
expression cassette limits the efficacy of temporal and
spatial gene targeting by IR, at least in the R1H tumor
cell line. However, introduction of insulating sequences
reduced this leakiness, while retaining the inducibility of
the system (Fig 5).
Several investigators used the radiation-inducible EGR
promoter to control gene expression in a variety of tumor
cells in vitro and in vivo and with different vector systems.
Hallahan et al2linked the EGR promoter to the gene
encoding TNF-a and transduced the human squamous
carcinoma cell line SQ-20B growing in nude mice with a
replication-deficient adenovirus. Compared to 50Gy
alone (given in 5Gy fractions four times weekly), the
combination of Ad5.Egr-TNF and 50Gy produced a
higher growth delay of tumors. Injection of Ad5.Egr-
TNF alone with 2?108pfu for 2 weeks twice weekly
induced a similar tumor growth reduction up to 4 weeks
after initiation of treatment as the combination of
Ad5.Egr-TNF and 50Gy. This indicates TNF-induction
in the absence of radiation. No increased soft tissue
toxicity was detected in the combined treated animals in
comparison to IR alone. Seung et al21applied the same
EGR-TNFa construct and transfected a murine fibrosar-
coma cell line using cationic liposomes. They demon-
strated a significant increase in growth delay with the
combination of intratumoral injection of the EGR-TNFa
and a single dose of 20Gy IR as compared to IR or EGR-
TNFa alone up to about 3 weeks after treatment. The
effect was more pronounced in larger than in small
tumors. A significant increase in the number of thrombo-
tic vessels resulting in necrotic regions was present in the
combined treated tumors.10A hepatocellular carcinoma
was transfected with EGR-TK and became highly
sensitive to GCV after IR but not without IR.7TK
mRNA expression was found 3hours after IR and
reached a maximum 12 hours after IR. However, a 40%
growth suppression of hepatoma cells transfected with
EGR-TK and exposed to GCV was observed in the
absence of IR. In vivo, a significant growth delay could be
achieved with EGR-TK and GCV without IR as
compared to EGR-TK (stably) transfected and irradiated
(20Gy single dose) tumors. In combining gene therapy
and IR tumors regressed almost completely after 3 weeks.
As was observed in our in vitro experiments, the
nonspecific induction of TK-gene expression in vivo is
also considerable. An increase of about three-fold in lacZ
transgene activity was observed in 9L glioma cells infected
by a replication-deficient adenovirus and irradiated with 2
in vitro. IR of intracerebral 9L tumors
transfected with AdEGR/lacZ also increased b-galactosi-
All these studies provide evidence that vectors contain-
ing the radiation-inducible EGR promoter can be used to
control gene expression by small doses of IR. However, it
seems that its activation is not as tightly regulated by IR
as needed and background expression of transgenes in the
absence of IR might be a problem. The background
expression might be either due to intrinsic leakiness of the
promoter or due to the presence of viral enhancer
sequences inducing transgene expression.
A series of experimental results addressing the optimi-
zation of radiation-inducible gene expression using the
CArG elements of the EGR-1 promoter have been
published. For example Scott et al22constructed synthetic
promoters carrying four, six, nine and 12 tandem-repeat
copies of the prototype CArG sequence. MCF-7 cells
were transfected by reporter plasmids containing the
Figure 5 Influence of insulating sequences on radiation inducible,
HSV-TK mediated cell killing of R1H cells. Cells were either
nonirradiated (white bars) or irradiated (black bars), uninfected
(PBS) or infected at MOI10 with AdCMV.TK (CMV) or AdEGR.TK
(EGR) or AdI-EGR.TK, respectively, containing one insulator 50of
the expression cassette (I-EGR) or AdI-EGR.TK-I, containing an
additional insulator 30of the expression cassette (I-EGR-I). All
samples were treated with 1mg/ml GCV. At 5 days postirradiation,
cells were stained with crystal violet and counted. Values represent
means and SD of three independent experiments (except AdI-
EGR.TK-I, which was performed twice).
Radiation controlled gene expression
M Anton et al
Cancer Gene Therapy
synthetic promoters. They could demonstrate that the
synthetic promoters showed less nonspecific gene induc-
tion with the enhancer carrying nine CArG sequences
resulting in least nonspecific expression and most effective
IR-inducible gene expression. The introduction of spacer
elements varying the spatial arrangement of CArG
elements, however, had only a minor influence on the
A molecular switching device based on the Cre/loxP
switsch system and a synthetic radiation-responsive
enhancer was constructed by the same group.23MCF-7
cells were stably transfected with the molecular switching
device, then transfected with HSVtk-coding sequence and
irradiated with doses as low as 1Gy. The cell growth
inhibition was equivalent to 3Gy IR alone.
Another group used synthetic elements of the EGR-1
enhancer combined with a minimal CMV-promoter to
drive reporter gene expression in stably transfected head
and neck carcinoma cell lines. However, this construct
revealed only weak gene induction and was marked by
high background expression.24
Vassaux et al13demonstrated that the introduction of
an insulating sequence from the BGH pA restored tissue
specificity of the ERBB2 promoter to ERBB2 expressing
cells in an adenoviral vector. Steinwaerder and Lieber12
observed an influence of both upstream and downstream
sequences. Our results demonstrate a major influence of
the upstream E1 enhancer sequence, as well as down-
stream promoter/enhancer elements of adenoviral vectors.
These seem to override the radiation inducibility of the
EGR promoter/enhancer in producing background ex-
pression in the absence of IR. Insertion of BGH pA
restored the radiation inducibility of EGR-carrying
adenoviral vectors. Although treatment with a vector
carrying two insulators and IR reduced cell killing
slightly, but significantly, the overall difference in cell
killing (6 vs. 0Gy) was highest after introduction of two
insulators. Thus, we here demonstrate for the first time
that radiation inducibility of EGR promoter/enhancer
can be restored by BGH pA as insulating sequences in
The remaining gene expression in the absence of IR is
most probably due to an intrinsic leakiness of the EGR
promoter. The EGR promoter can be induced by a
variety of extracellular stimuli, for example, hypoxia,
cytokines and growth factors.25
Buvoli et al26most recently described the reduction of
gene expression of the a-myosin heavy chain promoter
downstream of a BGH pA in an adenoviral vector in vivo.
Whether the introduction of BGH pA as insulating
sequences in vivo will allow or abolish gene expression
in our system will have to be determined.
Furthermore, we found that the CMV promoter
consistently shows significant radiation inducibility. This
has been reported previously in hepatocellular carcinoma
cells7and cholangiocarcinoma cells in vitro and in vivo.27
In conclusion, combining insulating sequences with the
EGR promoter allows tighter regulation of gene expres-
sion than the CMV promoter, and thus potential
protection of the surrounding nonirradiated tissue.
MOI, multiplicity of infection; CArG Box, CC(A/T)6GG
like sequence; GCV, ganciclovir; IR, irradiation; Egr-1,
early growth response gene; BGH, bovine growth
The skillful technical assistance of S Wegerer and B Essien
is greatly acknowledged. We thank P Wendt for FACS
Analysis. Plasmid pE425 was kindly provided by VP
Sukhatme. This work was supported by Deutsche
Forschungsgemeinschaft (grant number WU 335/1-1).
IEOG was supported by a DAAD PhD scholarship.
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