Gene Therapy (1997) 4, 280–287
 1997 Stockton Press All rights reserved 0969-7128/97 $12.00
Gene therapy of experimental malignant mesothelioma
using adenovirus vectors encoding the HSVtk gene
MC Esandi 1, GD van Someren1, AJPE Vincent2, DW van Bekkum3, D Valerio1,31, A Bout1,3
and JL Noteboom1,4
1
Section Gene Therapy, Department of Medical Biochemistry, Leiden University, Rijswijk; 2Department of Neurosurgery, University
Hospital Rotterdam; and 3IntroGene BV, Rijswijk, The Netherlands
Replication-defective adenovirus vectors were generated
in which the gene of interest (lacZ, luciferase or HSV-tk) is
driven by the adenovirus major late promoter (MLP) or
the human cytomegalovirus immediate–early gene
promoter/enhancer (CMV). In vitro experiments with rat (II45) and human (MERO 25) mesothelioma cell lines
revealed that the CMV promoter was stronger than the
MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency
after tk gene transfer. Following administration of
IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG)
into the pleural cavity of Fischer rats with established
mesothelioma, a widespread distribution of infectious virus
particles through the thorax contents was demonstrated.
However, a relatively small proportion of tumor cells were
transduced. Nevertheless, a strong tumor growth inhibition
was observed following treatment with IG.Ad.CMV.TK
recombinant adenovirus and GCV. Separate groups of rats
inoculated on day 0 with 10 5 II-45 cells into the pleural
cavity, received 7 × 10 9 infectious particles of IG.Ad.
CMV.TK on day 1, day 2, day 4 or day 8. One day after
virus administration, 25 mg/kg GCV or PBS (controls) was
injected i.p. (intraperitoneally) twice daily. On day 15, all
animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting
an impressive bystander effect. In a survival study, animals
were treated 9 days after inoculation of 105 tumor cells with
IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days
in the controls to 33 days in the treated group. These
responses can be best explained by assuming continued
tk expression in or around the tumor tissue during GCV
treatment. Our results confirm and extend earlier findings
with the same model and demonstrate the potential of the
herpes simplex virus thymidine kinase suicide gene
therapy as a local treatment for malignant mesothelioma.
Keywords: suicide gene therapy; malignant mesothelioma; recombinant adenovirus; HSV-thymidine kinase gene;
promoter activity comparison
Introduction
Malignant mesothelioma (MM) is a cancer of the mesothelium most commonly occurring in the pleural cavity.
Its incidence is related to exposure to asbestos.1 MM has
a very poor prognosis. Despite intensive treatment with
surgery, radiation therapy, or chemotherapy, the average
survival time is only 18–24 months from diagnosis.2
Tumor growth is often limited to the thoracic cavity.
Most patients die of local extension of the disease rather
than of metastases. This growth pattern suggests that
MM may be a good candidate for local treatment.
One potentially useful strategy of local treatment is
transduction of tumor cells in vivo with ‘suicide’ genes.
The most widely explored of these ‘suicide’ genes is the
herpes simplex virus thymidine kinase (HSVtk) gene. In
the presence of the thymidine kinase, ganciclovir (GCV)
is phosphorylated to a toxic nucleotide analogue which
Correspondence: MC Esandi, Section Gene Therapy, Department of Medical Biochemistry, Leiden University, PO Box 3271, 2280 GG Rijswijk,
The Netherlands
4
Current address: Department of Clinical Oncology, Leiden University
Hospital, The Netherlands
Received 2 October 1996; accepted 19 November 1996
inhibits DNA replication. Rapidly dividing cells were
shown to be killed by this treatment while slowly replicating cells are less affected.3 Thus, this strategy is considered appropriate for the treatment of solid tumors that
are invading normal tissues constituted predominantly of
slowly or nondividing cells such as gliomas. In animal
tumors effective transfer of the HSVtk gene has been achieved with both recombinant adenovirus and recombinant
retrovirus. Retroviral vectors mediate stable integration
into the genome exclusively in dividing cells but this system has the limitation of low viral titers and low transduction efficiency.4 To overcome this limitation, murine
HSVtk retrovirus-producer cells have been injected
directly into the cerebrospinal fluid of animals with
malignant leptomeningeal neoplasia5 or into the tumor of
rats carrying gliomas in the brain.6,7 Since this last strategy has proven to be effective, trials in human patients
with gliomas have been initiated. In contrast to retrovirus, adenovirus vectors have been shown to be efficient
tools for local gene transfer in vivo. Obvious advantages
of recombinant adenovirus are that they have a broad
range of target cells and no requirement of integration
for expression.8 Efficient adenovirus-mediated gene
transfer of HSVtk in vivo has been described in transplantable animal tumors such as melanoma,9 glioma,7
Gene therapy for malignant mesothelioma
MC Esandi et al
leptomeningeal neoplasia,10 mesothelioma11 and in
human tumors growing in immunodeficient mice, eg
head and neck squamous cell cancer12 and hepatocellular carcinoma.13
In the current study we investigated the therapeutic
potential of two recombinant adenoviruses using the
adenovirus type 2 major late promotor (MLP) and the
cytomegalovirus immediate–early promoter (CMV)
respectively to drive the HSVtk gene. The CMV promoter
has been found to be stronger than the MLP promoter in
adenovirus harboring the p53 gene.14 We were interested
to know whether this difference would be reflected in
more efficient cell kill after expression of the tk gene and
treatment with GCV. The strength of the CMV promoter
and the MLP promoter were compared by constructing
vectors containing the luciferase gene and infecting
human and rat mesothelioma cell lines with these vectors. The adeno-tk vectors were employed in combination
with GCV to determine the cytotoxicity in vitro for both
rat and human mesothelioma cell lines. Although the
suicide effect was greater in vitro for the human cell line,
we have used the rat mesothelioma for our in vivo studies. The human mesothelioma requires transplantation in
immune deficient animals for in vivo studies. Since recent
studies have suggested that cells transduced in vivo by
adenovirus vectors are rapidly cleared by the immune
system,15 we preferred an immunocompetent animal
model to study the in vivo potential of the suicide system
because it mimics more closely the patient situation. The
most effective of the two viruses, IG.Ad.CMV.TK, was
used for treating mesotheliomas growing in the pleural
cavity of rats. To study the distribution of transduced
cells following intrapleural administration of recombinant adenovirus we applied an adenovirus vector encoding the lacZ gene under the control of the CMV promoter. We compared the results of the in vivo treatment
of rat mesothelioma with those reported by Elshami et
al11 who treated the same tumor with an adenovirus vector using a different promoter (RSV) to drive the HSVtk
gene. In our experience, in vivo treatment with the CMV
adenovirus was more tumoricidal than treatment with
the adeno-RSV virus as described by Elshami et al. 11 It is
not clear whether that difference has to be ascribed to a
more effective expression of the tk gene or to other factors, such as a faster growth rate of the mesothelioma in
our laboratory. Other differences between the effects of
the various viruses such as in vitro cytotoxicity and the
reactions in normal tissues following injection into the
pleural cavity are also discussed.
Results
In vitro studies
Luciferase expression after infection of human and rat
mesothelioma cells with either IG.Ad.MLP.luc or
IG.Ad.CMV.luc: Human and rat mesothelioma cell lines,
MERO 25 and II-45 respectively, were infected with
IG.Ad.MLP.luc and IG.Ad.CMV.luc (Figure 1). With both
recombinant vectors the luciferase activity measured in
human cells was 100-fold higher than the activity measured
in rat cells. These observations suggest higher susceptibility
of the human cells to infection by adenovirus vectors.
Furthermore, 10-fold higher expression was observed
281
Figure 1 Evaluation of luciferase activity after infection of human
(MERO 25) and rat (II-45) mesothelioma cells with IG.Ad.CMV.luc and
IG.Ad.MLP.luc. MERO 25 and II-45 cells were infected with
IG.Ad.MLP.luc and IG.Ad.CMV.luc at MOI: 1, 10, 100. After 72 h,
luciferase activity was measured in the lysates of the infected cells according to Materials and methods. (a) MERO 25 cells; (b) II-45 cells. l,
Ig.Ad.MLP.Luc; , IG.Ad.CMV.Luc.
in both the human and the rat cell line after infection with
the CMV vector than with the MLP vector, indicating a
stronger activity of the CMV promoter.
In vitro comparison of IG.Ad.MLP.TK and IG.Ad.CMV.TK
mediated tumor cell kill: GCV sensitivity of HSVtk-transduced mesothelioma cells was first tested in vitro. Human
(MERO 25) or rat (II-45) cells were infected with either
IG.Ad.MLP.TK or IG.Ad.CMV.TK at different multiplicities of infection (MOI). After 72 h of culture in the
presence or absence of GCV, a clonogenic assay was performed. The results, presented in Figure 2, demonstrate
that there is an inverse relation between MOI and cell
survival after GCV administration.
In addition, the CMV promoter appeared to be more
effective than the MLP promoter in driving HSVtkmediated GCV toxicity both in human and rat cell lines.
The human MERO 25 cells were killed by GCV after
transduction at a MOI of 1 with the IG.Ad.CMV.TK vector whereas a MOI of 10 was required for a similar
response with the IG.Ad.MLP.TK vector (Figure 2a and
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282
Figure 2 Comparison of in vitro efficacy of IG.Ad.MLP.TK and IG.Ad.CMV.TK transduction and GCV treatment. Human and rat mesothelioma cells
were infected with the recombinant virus (MOI: 0, 1, 3, 10, 100) and cultured in the presence or absence of GCV. After 4 days a clonogenic assay was
performed (see Materials and methods). (a) MERO 25 cells infected with IG.Ad.CMV.TK; (b) MERO 25 cells infected with IG.Ad.MLP.TK; (c) II-45
cells infected with IG.Ad.CMV.TK; (d) II-45 cells infected with IG.Ad.MLP.TK. (o: No colonies seen; bars, s.d.).
b). At the highest MOI (100) IG.Ad.MLP.TK induced only
limited GCV sensitivity in II-45 rat cells, while with
IG.Ad.CMV.TK more than 90% cell kill was seen at an
MOI of 30 and a weak response at an MOI of 10 (Figure
2c and d). These differences between the two promoters
are quantitatively similar to the difference of a factor of
10 observed following luciferase transductions (Figure 1).
The data in Figure 2 also reveal that in terms of MOI
the rat cells are less permissive to transduction by both
recombinant viruses than the human cells; by a factor of
100 in the case of the CMV vector. Using the MLP vector
this difference could not be calculated accurately because
of the weak response of the rat cells at an MOI of 100,
but it is definitely more than 30-fold (Figure 2, compare
a with c and b with d).
In the human mesothelioma cells both recombinant
viruses were cytotoxic in the absence of GCV. This effect
was less pronounced in the case of IG.Ad.MLP.TK.
Neither of the two viruses were cytotoxic for cultured
rat cells at the MOIs tested, which may reflect the lower
permissiveness of rat cells to adenovirus as compared
with human cells.
In vivo studies
Tumor growth rate: The tumor growth characteristics of
II-45 cells in F344 rats were determined by killing the ani-
mals 1, 4, 8, 13 and 15 days after inoculation of 105 tumor
cells into the pleural cavity. The tumor growth as measured by the weight of the total thoracic organs is shown
in Figure 3. After 8 days, small disseminated tumor nodules were visible in the pleural cavity but this was not
reflected by an increased weight of the thoracic organs.
By the first time-point at which an increase was recorded,
tumor growth was already apparent macroscopically
throughout the pleural cavity. At this point, day 13, the
tumor mass has attained an average weight of 2.4 g
which roughly represents 2.4 × 109 cells. Thus, during the
first 13 days the inoculum of 105 tumor cells has
expanded 24 000-fold. This expansion requires at least
between 14 and 15 cell doublings. Taking into account a
certain amount of tumor cell loss, the cell cycle time of
this tumor has to be less than 1 day. For this and the
following calculations we have disregarded the contribution of the stroma to the tumor weight, as the microscopical inspection of these tumors shows an overwhelming predominance of mesothelioma cells (Figure 4c).
Efficiency of in vivo gene transfer: To evaluate in vivo gene
transfer efficiency and the distribution of the recombinant
adenovirus through the thorax contents, 7 × 109 infectious
units of IG.Ad.CMV.lacZ were injected intrapleurally into
rats bearing 1- or 10-day-old malignant mesotheliomas.
Control animals received IG.Ad.CMV. TK. Three days after
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MC Esandi et al
283
Figure 3 Tumor growth presented as the weight of the contents of the
thoracic cavity. The growth of MM was evaluated by weighing the
thoracic contents of rats 1, 4, 8, 13, 15 days after injection of 105 II-45
cells in the pleural cavity, four animals per group. (k, mean; bars, s.d.).
virus injection, the rats were killed and the thoracic cavity
stained with X-gal solution to monitor lacZ expression. At
the time of death the tumors had been growing for 4 and
13 days, respectively. In the former group, macroscopical
tumors could not be found, but at day 13 the thoracic cavity
contained a large tumor mass. In both groups of rats the
parietal and visceral pleura were stained uniformly blue.
Microscopic inspection of paraffin sections showed a homogeneous distribution of blue cells in the mesothelium
(Figure 4a and b). In the day 13 group a patchy distribution
of blue cells mostly in the superficial cell layers of the
tumor tissue was observed (Figure 4c). The pleural surfaces
were normal, no inflammatory reaction of the mesothelium
was observed.
Treatment of malignant mesotheliomas in rats with
IG.Ad.CMV.TK/GCV
After confirmation of the susceptibility of HSVtk expressing mesothelioma cells to GCV exposure in vitro, we
investigated the effect of administration of the
IG.Ad.CMV.TK virus followed by treatment with GCV
in pleural mesotheliomas in rats. After inoculation of 105
II-45 cells into the right pleural cavity on day 0, groups
of four rats received a single injection of 7 × 109 infectious
particles at the site of tumor inoculation either on day 1,
2, 4 or 8, respectively. One day after the injection of the
virus, i.p. treatment with GCV was started until the animals were killed on day 15. The macroscopical appearance and the weight of the thoracic contents are listed in
Table 1. In the animals of groups 1, 2 and 3 macroscopic
tumor growth was not observed inside the thorax, but in
five out of 12 rats tumor was present in the scar of the
thoracotomy. This may be due to leakage of tumor cells
into the wound bed. Presently, we have replaced the thoracotomy by needle injection into the pleural cavity; this
procedure minimizes tumor growth in scar tissue. The
animals that were treated with IG.Ad.CMV.TK at day 8
Figure 4 In vivo IG.Ad.CMV.lacZ-mediated gene transfer into intrapleural MM. Microscopic picture of the b-galactosidase activity. Blue nuclear
staining indicates b-galactosidase activity. (a) Heart and pericardium
(HPS stained, × 400); (b) lung and pleura (HPS stained, × 400); (c) lacZ
expression in tumor tissue (HPS stained, × 200).
and subsequently received GCV, presented small tumor
deposits in several sites within the thoracic cavity. However, these tumor masses were much smaller than the
ones found in the control group receiving PBS, as is
reflected in the weight of the thoracic contents (groups 4
and 8 respectively, Table 1). These small tumor deposits
could not be dissected out accurately, so that the total
weight could not be estimated. An approximation is provided by subtracting the average thoracic content weight
of groups 1 to 3 (3.03 g) from that of group 4, which
yields 0.17 g. The treated animals did not show any clinical abnormalities at any time during the treatment. The
pleural surfaces of these animals were macroscopically
normal, but microscopic inspection revealed a mild
inflammatory reaction of the mesothelium with fibrosis
and infiltration by macrophages and lymphocytes (Figure
5). This reaction was seen in both the parietal and the
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284
Table 1 In vivo effect of the IG.Ad.CMV.TK/GCV treatment of II-45 mesotheliomas in Fisher rats
Group (n = 4)
IG.Ad.CMV.TK on day
Treatment
1
2
3
4
1
2
4
8
GCV
GCV
GCV
GCV
2.9
3.2
3.0
3.2
(±0.2)a
(±0.4)
(±0.4)
(±0.3)
1/4b
1/4b
3/4b
4/4
5
6
7
8
1
2
4
8
PBS
PBS
PBS
PBS
6.2
6.7
5.7
7.6
(±1.3)
(±1.1)
(±0.6)
(±0.5)
4/4
4/4
4/4
4/4
9 (No tumor)
2
GCV
2.8 (±0.4)
0/4
a
Weight (g) of thoracic
contents on day 15
Fraction of animals with
macroscopic tumor
Data are mean ± s.d., n = 4. bTumor only present in the scar of the thoracotomy.
trols. As a result of the treatment with GCV the symptom-free survival was significantly prolonged to 33 ± 1.8
days as compared to 19.2 ± 1.2 days (Figure 6, log rank
test P , 0.004). The tumor weight at the time of death as
calculated from comparison of the weights of the thoracic
contents with that of age-matched nontumor bearing animals was 2.9 ± 1.1 g in the GCV treated rats and 4.4 ±
0.8 g in the controls, the difference being not statistically significant.
Discussion
The in vitro data indicate that the CMV vectors
(IG.Ad.CMV.luc, IG.Ad.CMV.TK) are about 10-fold more
effective than the respective MLP vectors in the induction
of luciferase expression and in inducing sensitivity to
GCV-mediated toxicity. The latter suggests higher
expression levels of tk following infection with the CMV
virus. A similar difference was reported by Wills et al14
for vectors carrying the p53 gene with the CMV and the
MLP promoter, respectively.
The CMV virus, as well as the MLP virus, exhibited cytotoxicity in the absence of GCV for the human cell line.
Figure 5 Histologic section of the mesothelium after treatment with
IG.Ad.CMV.TK/GCV. (a) Heart and pericardium of a rat treated with
IG.Ad.CMV.TK on day 2, followed by treatment with GCV until death
on day 14 (HPS stained, × 400). (b) Heart and pericardium of a nontreated
animal (HPS stained, × 400).
visceral pleura as well as in the pericardium. In the animals treated with PBS the pleurae were completely infiltrated with tumor tissue, so that it could not be established whether the inflammation noted in the GCV
treated animals was caused by the recombinant virus or
the combination of GCV and recombinant virus.
Survival following treatment with IG.Ad.CMV.TK and
GCV
In a separate experiment two groups of eight rats each
received the recombinant virus 8 days after inoculation
of 105 tumor cells. Treatment with GCV was started the
following day in one group and continued for 14 days.
The other group was treated with PBS and served as con-
Figure 6 Survival curves of rats with established mesothelioma treated
with recombinant adenovirus and GCV. Rats (n = 16) were injected with
IG.Ad.CMV.TK 9 days after tumor implantation. Twenty-four hours later
25 mg/kg of GCV or 1 ml of PBS was administered i.p. twice a day for
14 days. Rats were killed when moribund or when dyspnea developed.
Log-rank statistical analysis revealed that the two survival curves were
different (P , 0.04).
Gene therapy for malignant mesothelioma
MC Esandi et al
Again, based on MOI the CMV virus was more toxic than
the MLP virus. The viruses were not toxic for rat cells but
this may well be due to the lower susceptibility of rat cells
to infection by adenovirus, which we found to be about
100-fold for all the various end points studied (Figures 1
and 2). We have observed a similar dose-dependent toxic
effect of adeno-tk virus in other human cell lines including:
glioma cells (U251), small cell lung carcinoma cells (GLC01), non-small cell lung carcinoma cells (A549) and melanoma cells (518 A2), but not in the rat glioma cell line 9L.16
This toxic effect seems to be related to the levels of
expression of the transgene in the infected cells; we also
observed cytotoxicity related to higher expression of the
lacZ gene after recombinant adenovirus infection of human
cells.16 It is important to know whether these cytotoxic
effects in vitro have relevance for the clinical use of adenoviral vectors for gene therapy. In the present study, we
observed a mild inflammation of the normal mesothelium
after treatment with IG.Ad.CMV.TK and GCV. This process
is not related to tumor growth because it was also observed
in animals without tumor that received the same treatment.
This reaction could be caused by overexpression of the tk
gene. More extensive toxicity studies are clearly needed to
elucidate whether this reaction is caused by the recombinant adenovirus, the GCV or the combination of both
agents. More relevant is whether this treatment-related
inflammation will occur in patients. The only way to investigate this issue is dose-finding studies in patients.
The results of the two in vivo experiments using suicide
gene therapy provide some data to evaluate the resulting
tumor inhibition in a quantitative way. Firstly, a theoretical growth curve can be constructed based on three measured tumor weights obtained at day 13: 2.4 g, day 15:
4.6 g (both from Figure 3: weight of thoracic organs of
tumor-bearing rats minus weight of thoracic organs of
rats 1 day after injection of tumor cells) and day 19: 4.4 g
(from survival experiment data, control group). These are
converted to number of tumor cells considering 1 g equal
to 109 cells. The other informative point is the inoculum
of 105 tumor cells at day 0. A Gompertz-fitted growth
curve17 was estimated using these points (Figure 7, curve
A). In the experiment described in Table 1 the rats of
Figure 7 Gompertz fitted growth curve for rat mesothelioma. Curve A
(—I—): estimated curve of tumor growth in nontreated animals. Curve
B (--k--): estimated curve of tumor growth in animals treated according
to Table 1, group 4.
group 4 were exposed to GCV at day 9. As was calculated
in the Results section, at day 15 tumor weight was 0.17
g that is equivalent to 1.7 × 108 tumor cells. Based on this
last point, a regrowth curve was constructed for the
treated rats of group 4, Table 1 (Figure 7, curve B). The
fraction of tumor cells killed by the treatment can be calculated assuming an immediate cytotoxic effect on day 9
followed by a regrowth at the same rate as represented
by the growth curve for nontreated tumors (Figure 7,
curve A) and comparing the number of cells for both
curves on day 9. By these means of comparison the result
of the treatment can be expressed as about 5 log cell kill.
The tumor growth curve of the treated animals in this
experiment has shifted 9.7 days (Figure 7, curve B).
Accordingly these animals were expected to die on day
29. If we compare this with the survival time of the rats
treated for 14 days with GCV (Figure 6), the latter animals survived for 33 days, that is 4 days longer than
expected. Since the only difference was the longer
exposure to GCV it has to be assumed that the tumor
response is determined by the duration of the GCV treatment. It is therefore worth investigating longer exposure
times than presently employed. Apart from this issue, the
tumor response in our survival experiment seems to significantly exceed expectations based on the distribution
of the transduced cells as demonstrated with the lacZ
recombinant virus. In that experiment only a few per cent
of the tumor cells were transduced. Apparently, the socalled bystander effect in this tumor is much greater than
so far assumed. It seems unlikely that the bystander effect
could persist beyond the few days of GCV treatment. By
that time the vast majority of transduced tumor cells
should have entered cell division with suicide as a consequence. Then, the only other source of thymidine kinase
to produce phosphorylated GCV is nondividing transduced cells. Such cells could be tumor cells that are
resting. Alternatively as we observed that a large proportion of normal mesothelium cells were also transduced in the lacZ experiment (Figure 4a and b), it is
tempting to speculate that these cells are an additional
and long-lasting source of toxic GCV metabolites that can
be transfered to nontransduced tumor cells. Another
hypothesis could be an immunological reaction against
the tumor. These issues are presently under investigation.
Our in vivo results largely confirm the data published
recently by Elshami et al11 on treatment of MM. However,
there are some differences. First, we found a longer survival time after treatment with IG.Ad.CMV.TK/GCV, 14
days against 8 days reported by Elshami et al.11 Moreover,
we did not observe differences in tumor weight at the time
of death in our survival study while Elshami et al reported
lower tumor weights in the treated animals. Both groups
of investigators used the same tumor model, similar techniques for implantation of the tumor cells and treated similar tumor sizes. However Elshami et al11 started the treatment 4 days after injection of 106 II-45 cells and in our case
we started 8 days after injection of 105 cells. Apparently,
the growth characteristic of the tumor is different between
the two groups. In our hands the tumor cell doubling time
is about 24 h while for Elshami et al it is longer, approximately 2 days. This discrepancy could explain why we did
not find a marked difference in tumor weight at the time
of death in our survival experiment. Both groups applied
similar recombinant adenovirus doses, but it is difficult to
compare them since the procedures for determining virus
285
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MC Esandi et al
286
titers were different. The recombinant adenovirus vectors
are also different: in our case the promoter that drives the
HSVtk gene is the CMV promoter and Elshami et al used
a construct with the RSV (Rous Sarcoma virus) promoter.
Moreover, we retained in our vector the E3 region whose
function in the wild-type virus is to evade an immune
response against infected cells. This may have caused
longer HSV-tk expression and may have resulted in more
effective therapy. We can not provide a conclusive explanation for the observed difference in outcome between the
two laboratories. Both the different vectors as well as the
faster tumor growth may have contributed to the higher
sensitivity of the tumor cells to the HSV-tk/GCV treatment
in our hands.
Clearly, the responses observed in the present experiments were obtained with tumor masses that are many
orders of magnitude smaller than those to be encountered in patients with MM. On the other hand, human
tumor cells seem to be more permissive to adeno-tk. In
analogy with the strategy successfully developed for conventional treatment modalities, it should be determined
if successive cycles of gene transfer/GCV improve the
response rate of larger tumors. In addition, new ways of
distributing the virus throughout the deeper layers of a
tumor, have to be developed.
Materials and methods
Cell lines and culture conditions
MERO 25, a human mesothelioma cell line, was kindly
provided by Dr M Versnel (University of Rotterdam, The
Netherlands). II-45, a cell line isolated from asbestosinduced rat mesothelioma18 was a gift from Dr Ch
Walker (Anderson Cancer Center, Houston, TX, USA).
Tumor cells were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum, nonessential amino acids, penicillin (100 IU/ml; Gibco,
Breda, The Netherlands) and streptomycin (50 mg/ml;
Gibco, Breda, The Netherlands). All cell lines were maintained at 37°C in a humidified atmosphere at 5% CO2.
Adenovirus vectors
The construction of IG.Ad.MLP.TK and IG.Ad.MLP.luc
have been described in detail elsewhere.7,10 IG.Ad.CMV.TK
was made from the pCMV.TK plasmid (Figure 8), in which
the HSV-tk expression is under the control of the CMV promoter and SV40 RNA splicing signals (180 bp) containing
splice donor and acceptor signals of the late viral genes 16s
and 19s. These sequences were isolated from pCMV
NLS/lacZ,19 obtained from Dr Fortunati (Erasmus University, Rotterdam, The Netherlands). The Escherichia coli lacZ
marker gene preceded by a nuclear location signal and the
luciferase gene were cloned in plasmids similar to
pCMV.TK called pCMV.LacZ and pCMV.luc respectively.
The adenovirus vectors IG.Ad.CMV.TK, IG.Ad.CMV.LacZ
and IG.Ad.CMV.luc were generated by cotransfecting 293
cells with SalI linearized plasmids and the large ClaI fragment of wild-type Ad5 DNA. Recombinant adenovirus
were plaque-purified twice, propagated and titrated according to standard procedures. The virus titers were determinated by end point cytopathogenic effect (CPE) assay.20
In vitro studies
To compare the efficiency of gene transfer in vitro, mesothelioma cell lines were infected with IG.Ad.MLP.luc or
Figure 8 Physical map of pCMV.TK, the plasmid used for the generation
of IG.Ad.CMV.TK. HSV-TK: herpes simplex virus thymidine kinase; CMV:
cytomegalovirus immediate–early gene promoter and enhancer; SD-SA: 180
bp region of the SV40 genome containing late viral protein gene 16s/19s
splice donor and acceptor signals; SV40: Simian virus 40 polyadenylation
sequence (nt 2533–2668 of the SV40 genome); BglII–ScaI fragment of adenovirus type 5: nt 3328–6092 of the adenovirus type 5 genome.
IG.Ad.CMV.luc. The MERO 25 human mesothelioma and
II-45 rat mesothelioma cells were plated in 24-well culture dishes (Costar Europe, Badhoevedorp, The
Netherlands) at a density of 104 cells per well. The cells
were infected in triplicate with IG.Ad.MLP.luc or
IG.Ad.CMV.luc at an MOI of 0, 1, 10 and 100. Lysates of
the infected cells were prepared 72 h after infection to
measure luciferase activity according to Fortunati et al.19
To study the suicide gene efficacy a clonogenic assay was
performed. MERO 25 and II-45 cells were seeded in 24-well
culture dishes (Costar), 104 cells per well. After 3 h, the cells
were infected in triplicate with IG.Ad.MLP.TK or
IG.Ad.CMV.TK at MOI of 0, 1, 3, 10, 30 and 100, respectively. The culture medium in each well was replaced with
medium 24 h later with or without 10 mm of GCV (Syntex
BV, Rijswijk, The Netherlands). After 72 h the cells in the
control wells (MOI 0, without GCV) were counted and
diluted in order to seed 200 cells in a 60 mm culture dish
(Costar). The same dilutions were made for the cells of the
other wells, thus identical volumes were placed in 60 mm
dishes and the cells were cultured using the same conditions as described previously. After 8–10 days, dishes
were fixed with methanol (70% solution) and stained with
a methylene blue solution; macroscopic colonies (more than
50 cells) were counted.
In vivo protocols
MM were established in syngeneic Fisher 344 rats (11week-old, males, weighing 250–360 g) by injecting 105 II45 cells suspended in 500 ml PBS into the pleural cavity
via thoracotomy between the eighth and ninth ribs at the
lateral side of the right thorax. To evaluate the tumor
growth rate, animals were killed 1, 4, 8, 13 or 15 days
after injection of the tumor cells, and the thoracic contents
(lungs, heart, mediastinum, trachea, and diaphragm)
including tumor tissue were weighed. The average
weight of the thoracic contents of age-matched control
rats were subtracted to arrive at an approximation of the
tumor mass. Four rats were used per group.
Gene therapy for malignant mesothelioma
MC Esandi et al
In vivo gene transfer using lacZ as gene marker was
evaluated as follows: on day 0, MM were established in
Fisher rats (n = 16) by injecting 105 II-45 cells according
to the procedure described above. On day 1 (n = 4) and
on day 10 (n = 4), the same procedure was used to administer 7 × 109 infectious particles of IG.Ad.CMV.LacZ in
700 ml of PBS. Control animals received an identical dose
of IG.Ad.CMV.TK. Rats were killed 3 days after virus
administration. The vascular system of the animals was
first perfused with 10 ml of PBS and subsequently with
10 ml of 0.2% gluteraldehyde (Sigma, Axel, Belgium). The
thoracic contents were removed and fixed in the 0.2%
gluteraldehyde solution for 1 h, washed three times in
PBS and stained with X-gal (5-bromo-4-chloro-3-indolylb-galactopyranoside) solution (Molecular Probes Europe,
Leiden, The Netherlands) for 3 h at 37°C, as described by
Bout et al21 Samples of lung, heart, mediastinum, diaphragm, intercostal muscle and tumors were embedded
in paraffin, 2 mm sections were made and stained with
hematoxylin, pholin and saphrane (HPS).
For the in vivo treatment of MM, 7 × 109 infectious particles of IG.Ad.CMV.TK were introduced into the pleural
cavity of Fisher rats (n = 32) on day 1, 2, 4 or 8 after
tumor cell implantation on day 0. Administration of GCV
(50 mg/kg/day) or PBS i.p. twice per day was started 24
h after the virus infection and continued until the animals
were killed. Another group (n = 4) without tumor was
treated with IG.Ad.CMV.TK on day 1 and received the
GCV treatment for 13 days. At day 15, all rats were killed
and inspected for macroscopic tumor growth in the thoracic cavity. The total thoracic contents and the diaphragm including tumors were weighed. Histologic sections were made of the thoracic organs at the midway
transverse level to include parts of the lungs, heart and
mediastinum. The diaphragm was cut separately. The
tissues were embedded in paraffin, 2 mm sections were
made and stained with HPS.
For the survival study, 8 days after intrapleural implantation of 105 II-45 tumor cells on day 0, Fisher rats (n =
16) were injected with 7 × 109 infectious particles of IG.Ad.
CMV.TK. On day 9, eight rats received 50 mg/kg per day
GCV and another eight rats were injected with PBS for 14
days. The rats were observed daily and killed when showing dyspnea, which was accompanied by lethargy and
weight loss. Thoracic contents were dissected and weighed.
The symptom-free period is presented in a survival curve.
The log rank test was applied for statistical analysis of
symptom-free latency data. Gompertz-fitted curves were
calculated according to Simpson-Herren et al.17
Acknowledgements
This work was supported by the Foundation ‘Banco del
Sud’ (MCE), The Netherlands Organization for Scientific
Research and Het Preventie Fonds. The authors wish to
thank HMJPM Brok, J van der Brugge and J de Vast for
expert technical assistance, J Jansen (TNO-CSD, The
Netherlands) who kindly performed the Gompertz-fitted
curves constructions and calculations and Dr C Zurcher
(Division of Health Research, TNO, The Netherlands) for
evaluation of the histological preparations.
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