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Gene Therapy (2000) 7, 224–231
 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00
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ACQUIRED DISEASES
RESEARCH ARTICLE
Secreted human ␤-glucuronidase: a novel tool for
gene-directed enzyme prodrug therapy
D Weyel1, H-H Sedlacek2, R Müller1 and S Brüsselbach1
1
Institute of Molecular Biology and Tumor Research (IMT), Philipps-University Marburg; and 2Hoechst Marion Roussel
Deutschland, Marburg, Germany
A major problem of tumor gene therapy is the low transduction efficiency of the currently available vectors. One way to
circumvent this problem is the delivery of therapeutic genes
encoding intracellular enzymes for the conversion of a prodrug to a cytotoxic drug which can then spread to neighboring non-transduced cells (bystander effect). One possibility
to improve the bystander effect could be the extracellular
conversion of a hydrophilic prodrug to a lipophilic, cell-permeable cytotoxic drug. Toward this end, we have used a
secreted form of the normally lysosomal human ␤-glucuronidase (s-␤Gluc) to establish an extracellular cytotoxic effector
system that converts an inactivated glucuronidated derivative of doxorubicin (HMR 1826) to the cytotoxic drug. We
demonstrate that s-␤Gluc-transduced tumor cells convert
HMR 1826 to doxorubicin which is taken up by both transduced and non-transduced cells. s-␤Gluc in combination
with HMR 1826 efficiently induces tumor cell killing both in
cell culture and in vivo. This effect is mediated through a
pronounced bystander effect of the generated cytotoxic
drug. Most notably, this gene therapeutic strategy is shown
to be clearly superior to conventional chemotherapy with
doxorubicin. Gene Therapy (2000) 7, 224–231.
Keywords: ␤-glucuronidase; prodrug; HMR 1826; doxorubicin; GDEPT
Introduction
A major limitation of conventional chemotherapy is its
lack of selectivity for tumor cells resulting in dose-limiting, toxic side-effects and the generation of multidrug
resistant tumor cells due to insufficient drug concentrations at the tumor site.1 One possibility to overcome
these problems is the use of non-toxic forms of cytotoxic
compounds (prodrugs) that are converted to the toxic
drug selectively at the tumor site. Strategies using tumorspecific antibodies to guide the enzyme to the tumor
(antibody-enzyme conjugates) have been replaced to a
large extent by gene-directed approaches. Unfortunately,
the vectors currently in use for gene therapy show a very
low transduction efficiency in vivo and lead to the
expression of the therapeutic gene in only a small fraction
of the target tissue. For this reason it is necessary to use
an effector mechanism which targets not only the transduced cell itself, but the neighboring cells as well
(bystander effect). Most cytotoxic effector systems available at present – such as the herpes simplex virus thymidine kinase (HSVtk),2 the E. coli cytosine deaminase (EcCD)3,4 and the E. coli nitroreductase (Ec-NR)5 – act intracellularly, ie convert the respective prodrug to the active
drug within the cell. One way to improve the bystander
effect could be the extracellular conversion of a prodrug,
which is easily taken up by surrounding cells, as exemplified by prokaryotic carboxypeptidase G2 displayed on
Correspondence: R Müller, Institute of Molecular Biology and Tumor
Research (IMT), Philipps-University Marburg, Emil-Mannkopff-Strasse
2, D-35033 Marburg, Germany
Received 16 June 1999; accepted 13 September 1999
the surface of the transduced cells.6 We have chosen the
human ␤-glucuronidase to establish an extracellular cytotoxic effector system, converting a hydrophilic non-cellpermeable prodrug (glucuronide prodrug of doxorubicin, HMR 1826) to the lipophilic, membrane-permeable
drug doxorubicin. ␤-Glucuronidase seems to be a particularly suitable candidate as a prodrug-converting
enzyme, because the endogenous enzyme is located in
lysosomes and therefore not available for prodrug conversion under normal circumstances. In addition, ␤-glucuronidase leaking out of cells is rapidly internalized via
the mannose-6-phosphate (M6P) receptor on the cell
surface as discussed in detail below.
The physiological function of ␤-glucuronidase is the
degradation of glucuronic acid containing glucosaminoglycans (like heparan sulfate, chondroitin sulfate, and
dermatan sulfate).7 In higher eukaryotes, the enzyme
undergoes a series of post-translational modifications.8
Individual subunits of 651 amino acids are synthesized
on membrane-bound ribosomes and translocated into the
endoplasmic reticulum (ER), where several post-translational events occur. These include signal sequence
cleavage, N-linked glycosylation, disulfide bond formation, and homotetramerization. In the Golgi complex, the
glycans are modified to expose M6P residues. In the
trans-Golgi network these M6P residues serve as ligands
for specific receptors, which transport lysosomal
enzymes to a prelysosomal compartment. After pHdependent dissociation of the receptor-enzyme complex,
the receptor relocates to the Golgi, and the enzyme stays
in the developing lysosome.
The active homotetramer has a Mr of 332 000. There are
two active sites located in a large cleft at the interface of
Extracellular prodrug conversion
D Weyel et al
two monomers. Each cleft has two catalytic centers,
which means that there are four catalytic centers per
tetramer.9 The pH-optimum of ␤-glucuronidase is
approximately 5.5 which corresponds to its natural acid
environment in lysosomes.
The prodrug used in this study, glucuronyl-doxorubicin [N-(4-␤-glucuronyl-3-nitrobenzyloxycarbonyl)doxorubicin], has been successfully used for an experimental monotherapy of human xenografts in mice.10 The
rational of this approach is the presence of large amounts
of endogenous ␤-glucuronidase in necrotic tumor areas.
A problem associated with this strategy obviously is the
fact that not all tumors contain necrotic areas, and that
such regions of the tumor are particularly poorly penetrated by chemicals. An initial destruction of cells at the
tumor edge due to the expression of transduced ␤-glucuronidase could therefore improve the penetration of the
prodrug into the tumor and result in an amplification of
the cleavage reaction by endogenous ␤-glucuronidase.
In the present study, we demonstrate that tumor cells
transduced with a secreted form of human ␤-glucuronidase (s-␤Gluc) convert HMR 1826 to doxorubicin, and that
the generated drug produces a strong bystander effect in
cell culture. We also show that the secreted ␤-glucuronidase in combination with HMR 1826 efficiently induces
tumor cell killing in a human xenograft model in nude
mice through a clear bystander effect, and that this gene
therapeutic strategy is superior to conventional chemotherapy with doxorubicin.
Results
Secretion of enzymatically active human
␤-glucuronidase
Human ␤-glucuronidase cDNA containing the signal
sequence from the human IgG1 heavy chain was cloned
into the pcDNA3 expression vector. Transiently transfected cells of the human choriocarcinoma cell line JEG3 were established and the expression of the construct
was verified using a monoclonal antibody specific for
human ␤-glucuronidase. The immunofluorescence analysis in Figure 1A shows a staining pattern that is typical
of proteins entering the secretory pathway, with a bright
staining around the nuclear membrane and vesicles distributed in the cytoplasm. Expression of ␤-glucuronidase
was confirmed by the immunoprecipitation (Figure 2a).
Analysis of the immunoprecipitate by denaturing polyacrylamide gel electrophoresis showed a strong band of
monomeric ␤-glucuronidase with the expected size of 83
kDa. In contrast, expression of endogenous ␤-glucuronidase was not detectable by immunofluorescence (data not
shown), and only very low amounts of ␤-glucuronidase
could be immunoprecipitated from untransfected cells
(Figure 2a).
For further biochemical studies and therapeutic experiments, stable clones were selected in G418-containing
medium from a pool of transfected cells. These clones
were analyzed for ␤-glucuronidase expression by immunofluorescence analysis and immuno-precipitation. Clone
JEG-3sG, which contains approximately 15% ␤-glucuronidase positive cells, was used for all subsequent experiments. Secretion of ␤-glucuronidase was verified by
pulse-chase labeling of cells with 35S-methionine followed by immunoprecipitation of the cell lysate and
supernatant. ␤-Glucuronidase was detected in the supernatant as early as 3 h after withdrawal of the radioactively labeled methionine, although the synthesized
enzyme was only partially secreted (Figure 2a). The reason for this lies presumably in the fact that secretion is
brought about by endosomal overflow (see Discussion).
In parallel the activity of lactate dehydrogenase (LDH) as
a cytosolic marker enzyme was determined to assess the
passive release of ␤-glucuronidase from dying cells. No
LDH activity was detectable in the culture medium during the course of the experiment (data not shown), indicating that the ␤-glucuronidase in the culture supernatant
was actively secreted. We will subsequently refer to this
secreted form of ␤-glucuronidase as s-␤Gluc.
The enzymatic activity of the recombinant s-␤Gluc was
demonstrated using two different approaches. JEG-3sG
cells were stained with the chromogenic substrate X-gluc
which is converted to a blue precipitate upon cleavage
(Figure 1B, bottom panel). In agreement with the
expression data obtained by immunofluorescence analysis (Figure 1A) 15% of the cells stained blue. The ‘negative’ cells in the transduced cell population showed a
similarly weak staining as the parental non-transduced
cells (Figure 1B, top panel), indicating that the staining
of the former cells results from the expression of
endogenous lysosomal ␤-glucuronidase. In addition, the
culture supernatant was analyzed for enzymatic activity
under different pH conditions using pNPG as the substrate (Figure 2b). As expected, this analysis showed a
pH-optimum around pH 5. Half-maximal ␤-glucuronidase activity was observed at a pH of approximately 6.
Under physiological pH conditions (pH approximately 7)
the enzymatic activity was about 20% of the maximum.
225
Conversion of a doxorubicin prodrug and toxicity in cell
culture
We next investigated whether s-␤Gluc is able to cleave
the glucuronide prodrug of doxorubicin, HMR 1826. In
addition, we addressed the question to which extent the
activated drug is taken up by cells not expressing the prodrug-converting enzyme in order to assess a potential
bystander effect. For this purpose, we made use of the
strong autofluorescence of doxorubicin with an emission
maximum at 600 nm.11 HMR 1826 is hydrophilic and cannot pass cell membranes. After cleavage of the prodrug,
the generated hydrophobic doxorubicin can cross cell
membranes, intercalates into DNA and thereby leads to
a red nuclear fluorescence. This experiment was carried
out with a transiently transfected cell population containing 2% s-␤Gluc expressing cells. Two days after transfection the cells were incubated with the prodrug for 24 h.
Transfected cells were visualized by indirect immunofluorescence to detect ␤-glucuronidase expression (green
fluorescence in Figure 1C, panel b). In comparison to
untransfected cells, where weak staining was observed
(Figure 1C, panel a), all the nuclei stained strongly positive for doxorubicin in the transfected dish (Figure 1C,
panel c) despite the low number of s-␤Gluc expressing
cells. The weak staining of untransfected cells (Figure 1C,
panel a) is due to a low level of generated doxorubicin
rather than autofluorescence of the cells (data not
shown).
Cytotoxic effects of the s-␤Gluc/HMR 1826 system in
cell culture were analyzed using the crystal violet assay.
As can be seen in Figure 3, the JEG-3sG clone containing
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D Weyel et al
226
A
C a
B
b
c
Figure 1 (A) Immunostaining of s-␤Gluc transfected JEG-3 cells. (B) Detection of enzyme activity with X-gluc as substrate. (C) Prodrug conversion
by JEG-3 cells transiently transfected with s-␤Gluc. Two days after transfection the cells were incubated with 10 ␮m prodrug for 24 h and formalinfixed. ␤-Glucuronidase expressing cells were detected by indirect immunofluorescence using a DTAF-labeled secondary antibody (green; panel b) and
prodrug conversion was visualized by the red nuclear staining of doxorubicin intercalated into DNA (panel c). Untransfected cells did not show any
prodrug conversion (panel a). Arrows point to s-␤Gluc expressing cells.
15% s-␤Gluc expressing cells was highly sensitive to the
prodrug (IC50 = 1.8 ␮m). This IC50 value is considerably
higher than that for doxorubicin (34 nm) which is due to
an incomplete conversion of the prodrug to doxorubicin
under the cell culture conditions used (eg dilution of
s-␤Gluc by the large volume of culture medium). In contrast, control JEG-3 cells (con JEG-3) were affected by the
prodrug only at high concentrations (IC50 ⬎ 8 ␮m).
In vivo functionality of the s-␤Gluc/HMR 1826 system
Since the s-␤Gluc/HMR 1826 system was functional in
cell culture we sought to study its effect in an in vivo
tumor model. In order to verify expression and enzymatic activity of s-␤Gluc in the tumor tissue, s-␤Gluc
expressing cells (JEG-3sG) and non-expressing control
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JEG-3 cells were injected subcutaneously (s.c.) into nu/nu
mice. When tumors had grown to a size of 100 mm2 the
mice were killed, and cryosections of the tumors were
prepared for enzyme histochemistry. In JEG-3sG xenografts ␤-glucuronidase activity could be demonstrated as
shown by the deposition of the water-insoluble red naphthol-neufuchsin dye complex (Figure 4a). Addition of an
excess of a saccharolactone (D-saccharic-1,4-lactone), a
competitive inhibitor of ␤-glucuronidase, completely
abrogated staining (data not shown). No enzymatic
activity could be detected in control JEG-3 xenografts
(Figure 4b). Tumor growth curves did not show any significant difference between s-␤Gluc expressing and control JEG-3 cells (Figure 5). The extent of staining in the
tumor tissue was compatible with the expression of
s-␤Gluc in 15% of the injected cell population (see above).
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D Weyel et al
227
Figure 3 Cytotoxic effect of s-␤Gluc mediated prodrug conversion in cell
culture. s-␤Gluc expressing cells (JEG-3sG) and non-expressing control
JEG-3 cells (con JEG-3) were incubated with either HMR 1826 or doxorubicin for 24 h in microtiter plates, followed by staining with crystal violet
and measuring the optical density at 540 nm 2 days later (as an indicator
of cell number). Specific cytotoxicity of the prodrug in ␤-glucuronidase
expressing cells relative to non-expressing control JEG-3 cells is evident.
The following IC50 values were calculated from the data: JEG-3sG cells +
prodrug, IC50 = 1.8 ␮m; JEG-3sG cells + doxorubicin, IC50 = 34 nm;
control JEG-3 cells + prodrug, IC50 ⬎ 8 ␮m; control JEG-3 cells + doxorubicin, IC50 = 28 nm.
Figure 2 (a) Secretion of s-␤Gluc. JEG-3 cells were stably transfected
with s-␤Gluc and labeled with 35S-methionine in a pulse-chase experiment. A clear enrichment of secreted ␤-glucuronidase (arrow) was seen
in the supernatant. M, marker proteins. Numbers at the top indicate hours
after removal of 35S-methionine. (b) pH-dependence of s-␤Gluc. The supernatant of ␤-glucuronidase secreting cells was incubated with 1 mm pNPG
at different pH-values and the optical density 415 nm was determined.
We then compared the sensitivity of established con
JEG-3 and JEG-3sG xenografts with HMR 1826 or doxorubicin. At a tumor size of 10–110 mm2 prodrug or drug
was injected into the tail vein and tumor growth was
monitored. Prodrug treatment (100 mg/kg) of mice
injected with con JEG-3 cells did not show any significant
difference in tumor growth in comparison with untreated
mice injected with con JEG-3 cells or JEG-3sG cells
(Figure 5a and b). In contrast, tumors established with
JEG-3sG cells immediately started to regress (Figure 5a
and b). This effect was independent of the tumor size at
the time of prodrug injection, since even the biggest
tumor (110 mm2) regressed and actually showed a transient complete response (Figure 5b). In four out of five
mice, tumor growth resumed after another 20 days, and
one mouse has remained tumor-free for 7+ months. We
also performed mixing experiments where equal numbers of JEG-3sG and con JEG-3 cells were coinjected. In
this case (ie approximately 7% transduced cells) a clear
therapeutic effect was also detectable, but no complete
tumor regression was seen (Figure 5a). In addition,
tumor-bearing mice were treated with doxorubicin (1.5
mg/kg), but only a marginal effect on tumor growth was
detectable (Figure 5a). Since the IC50 value of HMR 1826
is 270-fold higher than that of doxorubicin (see above),
this dose of doxorubicin would correspond to 740 mg/kg
of HMR 1826 with respect to toxicity. However, the prodrug showed a dramatic therapeutic effect already at 100
mg/kg (ie one-seventh of the calculated equitoxic dose)
confirming the superiority of the prodrug-converting
system.
Discussion
A major problem of conventional cancer chemotherapy is
the low tumor specificity of the currently available drugs.
Severe side-effects limit the applicable dose and consequently the achievable drug concentration in the tumor.
This in turn favors the acquisition of multidrug resistance
and recurrent tumor growth. One way to prevent such
an adjustment of the tumor cells to chemotherapy would
be the development of strategies that allow for high local
drug concentrations. In the present study, we demonstrate that the local expression of extracellularly active
human ␤-glucuronidase can be used to sensitize tumor
cells to a doxorubicin prodrug. Since the maximal tolerable dose of prodrug is at least 130-fold higher than that
of doxorubicin,10 considerably higher doxorubicin concentrations can be reached at the site of the tumor in the
absence of an increased unspecific toxicity.
A number of strategies have been developed in the
past to sensitize tumor cells to specific prodrugs. Most
cytotoxic effector systems available at present, such as
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D Weyel et al
228
a
b
Figure 4 Activity of ␤-glucuronidase in cryosections of tumors grown in
nude mice after s.c. inoculation of s-␤Gluc expressing cells (a) or nonexpressing control JEG-3 cells (b). ␤-Glucuronidase activity was visualized by the deposition of the water-insoluble red naphthol-neufuchsin
dye complex.
HSVtk/ganciclovir (GCV), Ec-CD/5-fluorocytosine (5FC) or Ec-NR/CB1954, convert a prodrug to an active
drug intracellularly. The distribution of the activated
drug beyond the transduced cell (bystander effect)
depends on gap junction,12,13 phagocytosis of apoptotic
bodies14 or diffusion.15 An extracellularly active prodrug
converting system could circumvent these problems
which lead to a limited bystander effect, at least in part.
To date one such system has been described.6 In this case,
bacterial carboxipeptidase G2 was endowed with a transmembrane domain to tether the enzyme to the cell surface. This enzyme was used in conjunction with prodrug
that is converted by carboxipeptidase G2 to a mustard
drug. This system was shown to exert a bystander effect
in vitro and to be therapeutically efficacious in an experimental tumor model. The s-␤Gluc/HMR 1826 system differs from the carboxipeptidase system in several potentially relevant aspects, including the use of a human and
thus non-immunogenic enzyme, the presence of an
amplification effect in necrotic areas of the tumor and
presumably the absence of enzyme leakage into the circulation. These aspects of the s-␤Gluc/HMR 1826 system
are discussed in detail below.
Human ␤-glucuronidase is normally a lysosomal
enzyme. Similar to secreted proteins all natural lysosomal
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proteins have a leader sequence, but bind in the ER to
the mannose-6-phoshate receptor which identifies these
proteins for translocation to the lysosomes. If the
expression of lysosomal proteins exceeds the capacity of
this mechanism (as determined by the numbers of mannose-6-phoshate receptors in the ER) the protein will be
secreted. This most likely applies to our system as well
and explains the incomplete secretion of s-␤Gluc in Figure 2a. In order to achieve an optimal translocation of
␤-glucuronidase to the ER we exchanged the endogenous
signal sequence with the highly efficient leader of IgG.
However, this sequence is removed from the protein
upon processing in the ER with the consequence that the
secreted s-␤Gluc is identical to wild-type enzyme.
In the s-␤Gluc/HMR 1826 system introduced in the
present study, a hydrophilic prodrug is cleaved in the
intercellular space, and hydrophobic doxorubicin can
enter the surrounding cells. In cell culture experiments,
where s-␤Gluc can diffuse freely in the supernatant, 2%
␤-glucuronidase-positive cells suffice to achieve a distribution of doxorubicin to all cells in the population, as can
be seen by the positive nuclear staining. In vivo the situation is of course different. Diffusion of secreted ␤-glucuronidase is restricted by tissue barriers and the secreted
enzyme is internalized via M6P-receptors on the cell surface. On the one hand these mechanisms limit the
bystander effect, but on the other hand they restrict the
activation of the prodrug to the site of the tumor. This
selectivity of prodrug activation is complemented by a
site-restricted action of the generated cytotoxic drug,
which is due to the specific chemical properties of the
compounds. HMR 1826 is hydrophilic and can therefore
hardly enter living cells which prevents its conversion by
the normally lysosomal ␤-glucuronidase. In contrast,
doxorubicin is lipophilic and is rapidly taken up by surrounding cells, which strongly limits its leakage into
the circulation.
That the s-␤Gluc/HMR 1826 system is site-selective is
indirectly shown in our in vivo experiments. Fifteen percent of s-␤Gluc expressing tumor cells were sufficient to
achieve a complete macroscopic tumor regression in all
mice treated with the prodrug showing the functionality
of the system. On the other hand, the high concentrations
of HMR 1826 that were applied systemically did not produce any visible toxic effects (unpublished observations).
There are a number of other aspects that distinguish
the s-␤Gluc system from other prodrug converting systems. First, the low pH-optimum of ␤-glucuronidase
might contribute to a preferential activation of the
enzyme at the site of a tumor, because pH levels in certain tumors are often low.16,17 At physiological pH ␤-glucuronidase activity is reduced, which limits prodrug conversion at distal sites, for example in case of the
inadvertent expression of the recombinant enzyme in
non-tumor cells.
Second, HMR 1826 can also be converted to doxorubicin by the endogenous ␤-glucuronidase when the
enzyme is released from the cells. This happens in tumors
containing necrotic areas where an efficient conversion of
the prodrug has been shown, which leads to the suggestion of using HMR 1826 for monotherapy of necrotic
tumors.10 A combination with a gene therapeutic
approach should potentiate the effect. The tumor margins
are normally well vascularized and therefore accessible
by the vectors used for gene therapy, but these cells are
Extracellular prodrug conversion
D Weyel et al
229
Figure 5 Effect of the ␤-Gluc/HMR 1826 system on tumor growth in vivo. s-␤Gluc expressing cells (JEG-3sG), non-expressing control cells (con JEG3) or a mixture (1:1) of both were injected s.c. into nude mice (106 cells per mouse). After 2 weeks tumors had reached a size of 20–110 mm2 and both
groups of mice were divided into three subgroups each, either receiving 100 mg/kg of prodrug (+PD), 1.5 mg/kg of drug or no treatment. Tumor growth
was recorded every 2–3 days. (a) Tumor size (area) plotted against time after prodrug/drug treatment. Values represent averages of five to eight mice.
One mouse of the JEG-3sG/HMR 1826 group showed a lasting complete response (see also b) and was therefore not included in the calculations beyond
day 15. (b) Graphs displaying the data for individual JEG-3sG mice and used for the calculation of average values in panel a (marked # or *).
not necrotic and therefore do not release endogenous
␤-glucuronidase. In contrast, the cells in the center of the
tumor are hardly accessible by vectors due to the lack of
intact blood vessels, but release ␤-glucuronidase owing
to their necrotic nature. This should lead to a scenario,
where large areas in the tumor contain extracellular
␤-glucuronidase available for prodrug cleavage.
Third, an important aspect is the fact that s-␤Gluc can
in principle be used in combination with any cytotoxic
drug that can be inactivated by glucuronidation.18,19 This
is relevant in view of the fact that even highly toxic compounds could be used that could normally not be used
as chemotherapeutics because of their small therapeutic
window. An important factor in this context is the ability
to kill non-proliferating cells. Tumors often contain a
fraction of ‘resting’ malignant cells, which are not eliminated by conventional radiation or chemotherapy. The
most widely used prodrug converting system, the
HSVtk/ganciclovir combination, lacks the ability to target
non-proliferating cells. Although doxorubicin has some
cell cycle independent cytotoxic effects it should be possible to use compounds for prodrug synthesis, which
work much more efficiently with non-cycling cells. In
addition, the use of non-conventional drugs could also
be important to circumvent common problems associated
with chemotherapy, such as multidrug resistance, p53
dependence or the expression of anti-apoptotic genes. In
this context it may be particularly appealing to employ
combinations of different prodrugs to achieve synergistic
effects with respect to tumor cell killing.
Fourth, immunologic aspects deserve some consideration. Previously established prodrug converting systems
use xenogenic enzymes with unique substrate specificities to avoid unspecific prodrug activation by
endogenous enzymes. However, foreign proteins provoke an immune response, which can limit the
expression of the transgene20,21 and the action of its encoded product. The use of a human enzyme in human
patients should circumvent this problem. On the other
hand, activation of the immune system might enhance
drug induced tumor regression. In the case of
HSVtk/GCV a bystander effect has been described,
which works in syngeneic mice only.22 Tumorigenicity
was shown to depend on the induction of heat shock proteins which have been suggested to function through the
cross-priming of antigen-presenting cells by antigens
released from tumor cells and may provide an immunostimulatory signal in vivo to break tolerance to tumor
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D Weyel et al
230
antigens.23 In agreement with this finding, the treatment
of HSVtk transduced brain tumors with GCV2,24 and
EcCD transduced colon carcinoma with 5-FC25 protected
mice against the rechallenge with untransfected tumor
cells. In the same way the ‘biochemical’ bystander effect
of the s-␤Gluc/HMR 1826 system might cooperate with
an ‘immunological’ component in syngeneic animals, a
question we will investigate in future experiments.
Materials and methods
Plasmid constructs
The human ␤-glucuronidase c-DNA26 was cloned into the
pcDNA3 expression vector, containing the neomycin
resistance gene. The IgG signal sequence (coding for the
first 19 amino acids)27 was cloned 5⬘ to the human ␤-glucuronidase into the first PstI restriction site. For cloning of
the signal sequence an oligonucleotide was synthesized,
containing a Kozak sequence in front of the start codon
to improve translation initiation. Plasmids were amplified in E. coli and purified according to a standard protocol (Qiagen, Hilden, Germany). Cloned oligonucleotide
sequence was verified by DNA sequencing.
Cell culture
The human choriocarcinoma cell line JEG-3 (obtained
from A Wellstein, Georgetown University, Washington
DC), was cultured in Dulbecco-Vogt modified Eagle’s
medium (DMEM; BioWhittaker, Verviers, Belgium),
supplemented with 10% fetal bovine serum (FBS;
BioWhittaker) and 2 mm l-glutamine (BioWhittaker).
Cells were passaged 1:8 by trypsinization (0.05% trypsin,
0.02% EDTA; BioWhittaker) and grown at 37°C in 5%
CO2. Cell culture experiments, where ␤-glucuronidase
activity was necessary (X-gluc staining, crystal violet
assays, see below), cells were cultured in DMEM pH 7.
JEG-3 cells were transfected at 70% confluence in six-well
plates using the calcium-phosphate method.28 One day
after transfection cells were diluted 1:3, and the next day
selection with 750 ␮g/ml G418 (Life Technology, Eggenstein, Germany) was started. Growing clones were
analyzed for ␤-glucuronidase expression by immunofluorescence.
Immunostaining
Cells were fixed with 3.7% formaldehyde and permeabilized with 0.2% Triton. Indirect immunostaining was performed using a monoclonal antibody against human
␤-glucuronidase (hybridoma supernatant 2118/157,
obtained from HMR, Marburg, Germany, diluted 1/10)
and a Cy3(red)- or DTAF(green)-labeled secondary antimouse antibody (Dianova, Hamburg, Germany).
Between each step cells were washed with PBS two to
three times.
Immunoprecipitation
JEG-3sG cells, grown on 6 cm dishes, were labeled for 3
h with 0.2 ␮Ci/ml 35S-methionine (Amersham Pharmacia
Biotech, Freiburg, Germany) in DMEM + 10% dialyzed
FBS. Afterwards the supernatant was disposed and cells
were further incubated with 2 ml DMEM + 10% FBS. At
the indicated time-points supernatant was collected and
cells were lysed in 700 ␮l RIPA buffer (10 mm Tris pH
8, 150 mm NaCl, 0.25% SDS, 1% sodium deoxycholate,
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1% Nonidet P40, 1 mm DTT, protease inhibitors: 0.5 mm
PMSF, 50 ␮g/ml Aprotinin, 50 ␮g/ml Leupeptin). For
immunoprecipitation 1/3 of supernatant, buffered with
RIPA, or 1/3 of cell lysate was incubated with the ␤-glucuronidase antibody (100 ␮l) for 1 h on ice. Immune complexes were precipitated with protein A sepharose
(Amersham Pharmacia Biotech) and washed five times
with RIPA buffer (without protease inhibitors). Proteins
were separated by SDS-PAGE (10%). The dried gel was
exposed for 3 days.
Enzymatic activity
X-gluc: Cells were fixed with 0.1% glutaraldehyde in PBS
and stained for 6 h with 0.08% X-gluc (5-bromo-4-chloro3-indolyl-␤-D-glucuronide-cyclohexylammonium
salt;
Boehringer Ingelheim, Ingelheim, Germany), 3 ␮m potassium ferricyanide, and 3 ␮m potassium ferrocyanide in
0.1 m acetate buffer pH 5. Afterwards the cells were fixed
with 3.7% formaldehyde and mounted in moviol
(Calbiochem, Bad Soden, Germany).
pNPG: Substrate reaction buffer were prepared by mixing 65 mm KH2PO4 and 65 mm Na2HPO4 in different
amounts to obtain buffers ranging from pH 5 to 8. One
mm pNPG (p-nitrophenyl-␤-D-glucuronide; Sigma,
Deisenhofen, Germany) was dissolved freshly before the
experiment. Supernatant of transfected cells was collected, centrifuged, mixed with equal amounts of substrate-buffer, and incubated for 12–24 h at 37°C. The reaction was stopped by adding 2.5 m 2-amino-2-methyl-1,3propandiol (Sigma) and the extinction was measured at
a wavelength of 415 nm. Controls were incubated in the
presence of the specific inhibitor D-saccharic-1,4-lactone
(Sigma) in phosphate buffer pH 5.
Prodrug conversion
Two days after transfection cell were incubated with 10
␮m prodrug [N-(4-␤-glucuronyl-3-nitrobenzyloxycarbonyl)doxorubicin] for 24 h (obtained from HMR). Expression
of transfected ␤-glucuronidase was detected by indirect
immunofluorescence with a DTAF labeled secondary
antibody (Dianova, Hamburg, Germany). Converted prodrug was seen as red autofluorescence of the nuclei, due
to DNA-intercalating doxorubicin.
Crystal violet assay
One day before drug application cells were seeded in 96well flat-bottomed microtiter plates (10 000 cells per well).
Cells were incubated for 24 h with doxorubicin (Sigma)
or prodrug (HMR), applied as serial 1:2 dilutions (100 ␮l
per well) ranging from 500 ␮m down to 238 pm. Three
days after drug application cell viability was assessed by
the crystal violet assay. After removal of medium
attached cells were incubated with 100 ␮l crystal violet
solution (0.5% crystal violet, Sigma, in 20% v/v methanol-H2O) for 15 min at room temperature. Unbound
crystal violet was removed and retained dye was
measured with an ELISA-reader at 540 nm.
In vivo studies
Nude mice (strain CD-1 nu/nu; five to eight animals per
group) were injected s.c. with 106 control JEG-3 or JEG3sG cells (in 300 ␮l PBS) using a 21 G needle. After 1–2
weeks tumors were visible. At a size of 20–110 mm2 pro-
Extracellular prodrug conversion
D Weyel et al
drug (100 mg/kg) or doxorubicin (1.5 mg/kg) was
injected i.v., and tumor growth was measured regularly.
Histology
␤-Glucuronidase activity was determined on cryosections
following the procedure described by Bosslet et al.29 Controls were incubated in the presence of 10 mm D-saccharic-1,4-lactone monohydrate (Sigma) in 0.1 m acetate
buffer pH 5.
Acknowledgements
We are grateful to Klaus Bosslet for useful discussions,
to Claudia Cybon and Tina Stroh for excellent technical
assistance, to A Wellstein for the JEG-3 cell line and to
M Krause for synthesis of oligonucleotides.
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