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Advances in Brief |
Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 [Q. H., J. K. H., F. L., L. Z., R. B., J. L., J. B. L., M. W. D., C-Y. L.], and Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115 [J. B. L.]
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ABSTRACT |
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genes and then heated (42°C, 30 min), expression was at
least 13,600- or 6.8 x 105-fold over
background, respectively. Intralesion injection of the
interleukin-12-carrying adenovirus vector in a mouse melanoma tumor
model caused significant tumor growth delay only with hyperthermia
treatment. Our results therefore support heat-induced gene expression
as a feasible approach for targeted cancer gene therapy. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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,4
IL-2, and IL-12. Although these cytokines possess potent tumoricidal
activities, their utility in practice has been severely limited by
systemic toxicity (1, 2, 3)
. A tightly controlled mechanism
for gene induction will be very useful in expanding the current
applications of these potent therapeutic genes because localized
production of potent cytokines is less likely to generate systemic
toxicity. A significant amount of effort has been put into developing new systems for inducible gene expression. Some strategies have focused on induction systems that require a chemical signal. Elegant examples include the tetracycline-inducible or -suppressible gene expression systems (Tet-On and Tet-Off), which are based on the bacterial tetracycline operon. The activation or silencing of the system can be controlled by the binding of tetracycline to two different forms of tet repressors that respond oppositely to tetracycline due to allosteric changes that alter their DNA binding properties. The Tet-Off system is silent in mammalian cells when tetracycline is present in the cells at concentrations of 5–50 ng/ml. However, gene expression is activated from 2–5 orders of magnitude when tetracycline is withdrawn from the cells (4 , 5) . On the other hand, the Tet-On system is only active when tetracycline is present in the cells. Up to 1000-fold induction can achieved with the addition of tetracycline to the culturing medium (6) . Other inducible gene expression systems have used the strategy of disease-specific gene activation. For example, therapeutic "suicide" genes have been placed under the control of tumor-specific gene promoters so that they will be expressed only in tumor cells. An example is CEA, which is selectively expressed in a variety of cancers (7 , 8) . Chimeric genes that contain 5' sequences of CEA controlling the coding region of the Escherichia coli cytosine deaminase gene have been used to selectively sensitize CEA-overexpressing cancer cells to the cytotoxic effect of 5-fluorocytosine. The latter is a prodrug that is converted to the active drug, 5-fluorouracil, by the bacterial cytosine deaminase gene. This approach has been successful in some experimental applications. However, one major obstacle in this approach is the requirement to identify tumor cell-specific promoters for each individual tumor. A further potential impediment to these systems is the lack of a means to switch off gene expression when necessary.
External physical agents possess distinct advantages in controlling
gene expression both spatially and temporally. Ionizing radiation has
been used successfully to activate TNF- gene expression with
some spatial and temporal control (9, 10, 11)
. In this study,
we describe an inducible gene expression system in which thermal energy
is used to activate gene expression. The approach is based on using the
promoter for the so-called "hsps" that are conserved across
different species from bacteria to man. It has been shown that
promoters of some of the hsps can activate gene expression several
thousand fold in response to moderate hyperthermia (12)
.
By use of a 400-bp hsp70 promoter, a reporter gene GFP, or cytokine
genes TNF- and IL-12 engineered into recombinant adenovirus vectors,
we demonstrate that moderate hyperthermia (39°C-43°C) for
relatively short time periods (20–60 min) can activate gene expression
efficiently. This is consistent with an early report that demonstrated
the in vitro feasibility of this approach (13)
.
Additional experiments were carried out to examine the effectiveness of
this approach in vivo.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Adenovirus Preparation.
The construction of the adeonvirus was carried out according to
established protocols (15)
using a set of commercially
available plasmids (Microbix, Toronto, Canada). Briefly, the EGFP gene,
a human TNF- gene, and a murine IL-12 gene were cloned separately
into plasmid p
Esp1A under the control of the hsp70B promoter
(12
, 16) . The EGFP gene was excised from the pEGFP-N1
plasmid (Clontech, Palo, Alto, CA). The TNF- gene was derived from
plasmid pE4 (American Type Culture Collection), whereas the murine
IL-12 gene cassette was derived from a plasmid that was obtained from
the National Gene Vector Laboratory (University of Michigan, Ann Arbor,
MI). The two subunits of IL-12, p40 and p35, were connected using the
internal ribosome entry site sequence (17)
so that both
would be transcribed under the control of the same promoter.
Recombinant adenovirus was obtained by cotransfection of pEsp1A and
pBHG10 into 293 cells following a published procedure
(18)
. After plaque purification, the virus was then
amplified in 293 cells to a titer of 0.5–3 x 1011pfu/ml.
FACS Analysis.
FACS analysis and sorting were performed at the Flow Cytometry Shared
Resource at the Comprehensive Cancer Center of Duke University Medical
Center. For analysis, a FACScan apparatus from Becton Dickinson was
used. For sorting, a FACScan Star Plus was used. In either case, a
cooled argon blue laser was used for the excitation of the samples at
488 nm.
Western Blot Analysis.
Western blot analysis of the GFP level was carried out according to a
previously published procedure (19)
. After lysis of the
cells directly in the Petri dish in a buffer consisting of 50
mM Tris-HCl (pH 7.4), 250 mM NaCl, 0.5% NP40,
50 mM NaF, 1 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 1 mM
bezamide, and 10 µg/ml trypsin inhibitor (all from Sigma), the lysed
cells were centrifuged at 16,000 x g for 5
min, and the supernatants were analyzed. About 60 µg of protein were
loaded into each lane in a 12% polyacrylamide gel, electrophoresed,
and blotted onto polyvinylidene difluoride (Millipore) membrane. A
polyclonal antibody against GFP from Clontech was used as the primary
antibody. The signal was then developed with the enhanced
chemiluminescence system from the Amersham Corp.
Fluorescence Microscopy.
Visualization of GFP expression was carried out on a Zeiss Axioskope
equipped with a color camera and a silicon-enhanced tube camera. A
computer equipped with a frame grabber was connected to the camera to
capture images on line. To visualize GFP-transfected cells,
epifluorescence (xenon arc source and FITC filter) with or without
concomitant transillumination (with a 40-W tungsten source) was
used.
Measurement of Cytokine Levels.
Commercially available ELISA kits (R&D Systems, Minneapolis, MN) were
used for cytokine measurements. For in vitro experiments,
4T1 cells at 70% confluence were infected with recombinant adenoviral
vectors at a MOI of 25. They were then heated at 42°C for 30 min.
Supernatants were collected 48 h later. For in vivo
measurements, AdhTNF- (1 x 108
pfu/tumor) and AdhspmIL12 (3 x 108 pfu/tumor ) were injected into B16F10
melanoma tumors grown in C57BL/6 mice (Charles River
Laboratories, Wilmington, MA). The injection was carried out using a
30-gauge needle when the tumors reached 5–7 mm in diameter. The
injection volume was 50 µl. Heating of the tumor was carried out by
immersing the tumor-bearing leg in a circulated water bath for 40 min,
24 h after the injection. Tumors were excised 24 h after
heating and homogenized in PBS for measurement.
Tumor Growth Delay Study.
Tumors were formed by injecting 1 x 106 cells s.c. into the hind leg of C57BL/6 mice.
The tumors grew to a diameter of 5–7 mm in 7 days. At this time, viral
vectors were injected intratumorally using a 30-gauge needle at a
dosage of 1 x 109 pfu/tumor in a
volume of 50 µl. Viral dilutions were carried out from the stock in
Opti-MEM (Life Technologies, Inc.). Twenty-four h after intratumoral
viral injection, tumors were heated to 42.5°C in a water bath for 40
min by immersing the tumor-bearing leg in the water bath. Seven days
after the initial viral injection, a second injection was carried out
with a viral dose of 3 x 108
pfu/tumor in a volume of 50 µl. The second heating was carried out
24 h later at the same temperature and with the same duration.
Measurement of the tumor was started on the day of the first viral
injection. The longest and shortest dimensions of tumors were measured
by use of a caliper. The tumor volume (assuming that tumors took the
shape of a ellipsoid) was calculated using the formula:
V = (
/6) x W2 x L, where
L = length and W = width.
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Results |
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, promoter activation started at 39°C and peaked at around
42°C-43°C (gene induction at 43°C is similar to that at 42°C).
At 41°C-43°C, 500-1000-fold GFP induction was observed by FACS
analysis. However, at 44°C, GFP expression dropped significantly. A
careful examination indicated that most cells died within hours of this
thermal exposure, which explains the reduced expression at the higher
temperature. The heat inducibility of this promoter was further proven
by showing significant heat-induced GFP induction in the B16F10
melanoma cell line (data not shown). Western blot analysis of gene
expression indicates that heat treatment caused a 27-fold induction of
the reporter GFP protein (Fig. 1B
, using densitometry
analysis). The discrepancy between FACS analysis (500–1000-fold
increase) and Western blot analysis (27-fold increase) probably
reflected the nonlinear relationship between the amount of GFP protein
and its fluorescence intensity.
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. As early as 3 h after hyperthermia
treatment, GFP expression started to increase, and peak expression was
seen at around 18–24 h. GFP expression dropped back to background
levels in 72 h. Similar kinetics was seen in the B16F10 melanoma
cells.
Adenovirus-mediated, Heat-induced Cytokine Gene Expression
in Vitro and in Vivo.
To examine the efficacy of the heat-inducible gene therapy approach in
experimental tumor models, two recombinant adenovirus vectors encoding
the mouse IL-12 gene and the human TNF- gene, respectively, under
the control of the hsp70B promoter (AdhspmIL12 and AdhspTNF) were
constructed and produced. The viruses were then used to infect 4T1
mouse mammary adenocarcinoma cells at a MOI of 25. ELISA test kits were
used to measure TNF- and IL-12 concentrations in the supernatants.
Table 1
shows some typical results. Heat treatment caused the induction of the
TNF- and IL-12 at >6.8 x 105
and >13,600-fold over background, respectively, in infected cells. An
equally important fact is that the nonheated control showed cytokine
levels that were below the detection limits of the cytokines, similar
to that observed for noninfected control cells. This demonstrates the
very low leakiness for the promoters. For measurement of heat-inducible
gene induction in vivo, the viral vectors were injected
intratumorally (see "Material and Methods" for details) into B16F10
melanomas grown in the hind leg of C57BL/6 mice. Tumors were excised
from animals that had been injected with the virus with and without
heat treatment. They were then homogenized in PBS. The cytokine levels
were measured using ELISA kits. As shown in Table 1
, with
AdhhspTNF-, there was an 835-fold induction, whereas with
AdhspmIL12, there was a 33-fold induction. For both of these viral
vectors, the levels of the cytokines in the tumor without heat
treatment were comparable similar to those in control mice that had
been injected with an adenoviral vector encoding a GFP virus,
demonstrating the very low leakiness of the promoter in
vivo. The lower induction levels in vivo are perhaps
a reflection of the inability of the injected virus to reach the
bulk of the tumor. Indeed, fluorescence microscopy
examination of a AdhspEGFP-injected and heated tumor that had been
excised and sectioned indicates that only a small fraction (less than
5%) of the tumor mass was infected by the virus. Taken together, these
results clearly demonstrate that the heat-inducible gene expression
system functions well in vivo and in vitro.
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, AdhspmIL12 injections in combination with moderate hyperthermia
treatment (two heat treatments, 40 min each, 42.5°C, 7 days
apart) significantly inhibited tumor growth, whereas the tumors
of animals infected with the virus alone (without heat treatment) grew
at the same rate as those of controls injected with an irrelevant
control virus (AdhspGFP, which encoded heat-inducible GFP gene
expression), with or without heat treatment. Immunohistological
analysis indicates the existence of a central necrotic area devoid of
tumor vessels in the group with a significant growth delay. This
necrotic area is absent among tumors from the other groups. This is
consistent with other reports that IL-12 has antiangiogenic effects in
addition to its immunostimulatory functions (20, 21, 22)
. In
summary, these results confirm in vivo that the
heat-inducible IL-12 adenovirus is effective in controlling the growth
of the B16F10 melanoma in C57BL/6 mice.
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Discussion |
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Ionizing radiation and hyperthermia each have advantages and
disadvantages as physical agents. The main advantage of ionizing
radiation is the precision with which the diseased area can be
targeted. The disadvantage of radiation is the current lack of
promoters that can respond to ionizing radiation sufficiently and
consistently. Published data suggest that available radiation-inducible
promoters are relatively leaky, and a 20-Gy single dose of
-irradiation induced only a 9-fold increase in reporter gene
expression in vitro (11)
. This radiation dose
is not one that could be used typically in day-to-day practice of
radiation therapy and thus may not be very practical. On the other
hand, the hyperthermia-based approach shown herein has the advantage of
low expression without heat and high inducibility. The hsp promoters
can activate gene expression by hundreds to several thousand fold over
background at temperatures that are readily achievable in the clinic.
However, the spatial resolution of hyperthermia may be less than that
of radiation (centimeters versus millimeters).
The high inducibility and low leakiness of the heat-inducible gene
expression system is important for potent therapeutic genes such as
IL-12. Proteins encoded by such genes usually have significant systemic
toxicities (1, 2, 3)
. Therefore, most groups opted for local
delivery of the cytokine. Several groups have experimented with local,
intratumoral injection of cytokines in a similar melanoma model with
success (23
, 24)
. Other groups have reported experiments
with intratumoral injection of adenoviruses encoding the IL-12 gene
with success (25, 26, 27)
. However, some reports (27
, 28)
and our own results indicate that intratumorally injected
adenovirus can leak into systemic circulation and induce abnormally
high levels of cytokines, which leads to serious side effects. In
contrast, the use of AdhspIL12 induces minimal levels of IL-12 in the
serum, although the intratumoral level is substantial (Table 1)
.
In addition to high inducibility by heat, many hsps are selectively overexpressed in tumor cells due to various intrinsic microenvironmental stresses such as hypoxia or oxidative free radicals. Such stress exposures can potentially induce stress gene promoter-controlled therapeutic gene expression in the tumor mass without the application of hyperthermia. Indeed, selective expression of reporter and therapeutic gene expression has been reported using a stress gene promoter-based approach without the thermal exposure (29 , 30) . Preliminary evidence in our laboratory indicates that transfected cells express 1–3-fold more EGFP in vitro by transient exposure to 1% hypoxia. This level of increased GFP reporter gene expression was comparable with that seen in a previous report (29) , which showed a 0.5–3-fold increase under 1% hypoxia using different promoters. Because hypoxia has been clearly demonstrated to exist in human tumors (31 , 32) , these data suggest the potential for microenvironment-selective activation of heat-inducible promoters in tumors, albeit at a much lower level than that achievable by heat.
Hyperthermia has another advantage that makes it potentially suitable for diseases other than cancer. This is possible because no long-term side effects have been observed during 20 years of experimental cancer treatment with hyperthermia, particularly for temperatures in the range shown here to be most effective for heat-induced gene induction. On the other hand, ionizing radiation is a known carcinogen that can cause various acute and long-term biological effects. For this reason, a radiation-inducible gene therapy system is most likely to be limited to cancer therapy, where the main goal is the eradication of cancer cells, whereas a hyperthermia-induced gene expression approach may have other applications outside of cancer therapy.
There are also potential disadvantages of a hyperthermia-based gene therapy approach. The main obstacles include the difficulty in heating some areas in the body to the desired temperatures. However, extensive technological development in recent years is significantly improving the ability to heat more target areas in the body. Current technology allows cancers of the extremities and other sites including the ovary (33) , brain (34) , breast (35) , prostate (36) , and head and neck (37) to be heated to a temperature range that is adequate for heat-inducible gene expression. In fact, hyperthermia has already shown promise in Phase III clinical trials aimed at treating these types of cancers when combined with radiation (35 , 37) . But it has to be pointed out that this is not an approach that is applicable to all sites within the body. Another potential obstacle is that the heat shock response may be activated by other factors, such as fever and inflammation. At present, it is not known how these factors affect the heat-inducible gene therapy approach in vivo. Future experiments are necessary to answer these questions because they are important in the practical applications of this approach. In the worst case scenario, it may be necessary to control body temperature below 39°C using appropriate pharmaceutical agents.
In summary, the heat-inducible gene expression system described in this study provides a promising gene therapy approach that may have applications in the treatment of cancer and other diseases where local or transient therapeutic gene expression is desired.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute Grant
CA81512, a pilot grant from the Duke University SPORE in Breast Cancer,
a grant from the Celsion Corp. (to C-Y. L.), and National Cancer
Institute Grants CA40355 and CA42745 (to M. W. D.). Q. H. is a
Raychem/Duane/Roger/John Morris Fellow. F. L. is supported by Grant Lo
713/1-1 from the German Research Council (Deutsche
Forschungsgemeinschaft). 
2 Present address: No. 1 People’s Hospital,
Shanghai, People’s Republic of China.
3 To whom requests for reprints should be
addressed, at Box 3455, Department of Radiation Oncology, Duke
University Medical Center, Durham, NC 27710. Phone: (919) 681-4721;
Fax: (919) 684-8718; E-mail: cyli{at}radonc.duke.edu
4 The abbreviations used are: TNF, tumor necrosis
factor; GFP, green fluorescence protein; EGFP, enhanced GFP; IL,
interleukin; CEA, carcinoembryonic antigen; hsp, heat shock protein;
FACS, fluorescence-activated cell-sorting; MOI, multiplicity of
infection; pfu, plaque-forming unit(s).
Received 3/ 9/00. Accepted 5/18/00.
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REFERENCES |
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in IL-12 adverse effects. Clin. Immunol. Immunopathol., 83: 18-20, 1997.[Medline]
in mice. Cancer Immunol. Immunother., 45: 100-108, 1997.[Medline]
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), mice injected with
the murine IL-12 gene (
) alone, and mice injected with the murine
IL-12 gene with heat treatment (
). There were 10 animals in each
group. Error bars for all of the data points, SE.