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Experimental Therapeutics |
Transgene S.A., 67082 Strasbourg Cedex, France [P. E., E. R., J. K., P. L., Y. P.]; Laboratoire de Génétique, Centre National de la Recherche Scientifique, UPR 9003, Institut de Recherche contre les Cancers de l’Appareil Digestif, Hôpital Civil, 67091 Strasbourg Cedex, France [F. E., R. J.]; and IntroGene BV, 2301 CA Leiden, the Netherlands [M. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the most intensely studied suicide genes is the HSV-TK2 gene (7) . Expression of HSV-TK in tumor cells allows the conversion of prodrugs such as GCV and acyclovir, two acyclic guanosine analogues, into their monophosphate forms, which are then further phosphorylated by cellular kinases into their di- and triphosphate derivatives. Intracellular accumulation of such triphosphate metabolites and their subsequent incorporation into nascent DNA strands inhibit mammalian DNA polymerases and ultimately lead to cell death (7) . Interestingly, neighboring tumor cells that do not express the HSV-TK gene were also shown to be actively killed in presence of GCV (8, 9) . This enhancement of the antitumor activity by a bystander killing effect was demonstrated to be mediated by cellular connexins that allow the transfer of the toxic metabolites from the transduced cells to neighboring cells (10–13) .
Another suicide gene that has recently received considerable attention is CDase. CDase, an enzyme present in fungi and bacteria but absent from mammalian cells, deaminates cytosine to uracil (14) . CDase can also deaminate the nontoxic prodrug 5-FC to its highly toxic derivative 5-FU. This metabolite is then converted by cellular enzymes into 5-FUTP and 5-fluoro-dUMP (5-FdUMP). 5-FUTP can be incorporated into RNA in place of UTP, resulting in the inhibition of the nuclear processing of the ribosomal and mRNAs, whereas 5-FdUMP irreversibly inhibits thymidylate synthase, preventing DNA synthesis. Interestingly, and in contrast to GCV and its derivatives, 5-FC and 5-FU can penetrate tumor cells by passive diffusion and expand the local toxic effect to neighboring cells, irrespective of the presence or absence of specific cellular connexins. The potential for using the bacterial Escherichia coli CDase gene (codA) to confer sensitivity to 5-FC has already been demonstrated in vitro and in vivo in various experimental tumor models (15–20) . However, relatively disappointing results were also obtained in cell lines derived from breast and pancreatic tumor cell lines (21) . Such cells were found to be relatively resistant to 5-FU as a consequence of possible defects in downstream cellular metabolism of 5-FU. Supporting this hypothesis, expression in mammalian cells of upp, the gene encoding the E. coli UPRTase, which catalyzes the conversion of uracil and 5-FU into UMP and 5-fluoroUMP (5-FUMP), respectively, was shown to greatly enhance the sensitivity of the cells to 5-FU (22) .
We had previously cloned and characterized the Saccharomyces cerevisiae FCY1 and FUR1 genes encoding the CDase and UPRTase enzymes (23–25) , respectively. In this work, we have investigated in vitro and in vivo whether the co-expression of these two yeast genes may cooperatively increase the chemosensitivity of tumor cells to 5-FC. Replication-deficient adenoviruses were selected for these studies, given their ability to efficiently transfer and express candidate therapeutic genes in a variety of dividing and postmitotic cell types, including tumor cells, in vitro and in vivo (26) . We report that transduction of tumor cells by adenoviruses expressing the FCY1 or the FUR1 genes increases their sensitivity to 5-FC and to 5-FU, respectively, and that co-expression of FCY1 and FUR1 further enhances the cells sensitivity to 5-FC by more than 10-fold when compared to the transfer of FCY1 alone. More intriguing, we describe a novel chimeric protein, designated FCU1, that displays both enzymatic activities. This chimeric protein was generated by fusing the FCY1 and FUR1 coding sequences in frame. Its exhibits an UPRTase activity similar to the one encoded by the native FUR1 gene and, unexpectedly, a CDase activity 100-fold more elevated than the one encoded by the FCY1 gene. In addition, we present data showing in vitro and in vivo that the FCU1/5-FC system is a more effective prodrug activation system than the HSV-TK/GCV and CDase/5-FC systems. These results suggest that this novel suicide gene may constitute an original and potent candidate therapeutic gene for cancer gene therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
Human colon cancer cell lines SW480 and LoVo, human breast cancer cell
line SK-BR-3, human pancreas cancer cell line PANC-1, human embryonal
kidney cell line 293 and murine melanoma cancer cell line B16F0 were
obtained from the ATCC (Manassas, VA). SW480, SK-BR-3, PANC-1,
293, and B16F0 cells were grown in DMEM supplemented with 10% fetal
bovine serum, and LoVo cell line was maintained in Ham’s F-12 medium
supplemented with 10% fetal bovine serum. Before use, all cell lines
were tested and found to be free of Mycoplasma infection.
Plasmid Constructions.
The FCY1 gene was modified from phagemid
pRS315-FCY1 (25)
by oligonucleotide-directed
mutagenesis. The 5' primer (5'-GTTAAAAGCTTCATAGGCCATGGTG-3') was
designed to introduce a Kozak sequence for mammalian cell expression,
as well as a HindIII site to facilitate cloning. The 3'
primer (5'-AGTAGAGAATTCAGCACGCTG-3') introduces an EcoRI
site after the stop codon. The resulting 490-bp
HindIII-EcoRI fragment carrying FCY1
was subcloned into the plasmid pRS306 (27)
, and the
integrity of the sequence was verified by sequencing. From plasmid
pRS306-FCY1, the fragment XhoI-XbaI
containing the FCY1 gene was cloned into the corresponding
sites of the mammalian expression vector pCI-neo (Promega,
Charbonnieres, France) giving rise to vector pCI-neoFCY1.
From plasmid
pCI-neoFUR1-105,3
the fragment EcoRI-NotI, containing the
FUR1 open-reading frame starting from the second natural
translation initiation codon (24)
, was inserted into the
pCI-neoFCY1 linearized by EcoRI-NotI,
generating the plasmid pCI-neoFCY1-FUR1 (Fig. 1A
). Single-stranded phagemid was prepared from vector
pCI-neoFCY1-FUR1, and the stop and start codons
of FCY1 and FUR1 were deleted, respectively, by
oligonucleotide-directed mutagenesis (primer,
5'-ATGGTTCCGAAGCCTCACCAATATCT-3'), joining in-frame the two enzymatic
moieties (Fig. 1A
). The resulting plasmid carrying the
fusion gene, designated FCU1, is called
pCI-neoFCU1.
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) were
generated by transfection into the 293 complementation cell line of the
viral genomes released from their respective plasmids by
PacI digestion. Virus propagation, purification, and
titration of IUs by indirect immunofluorescence of the viral DNA
binding protein were carried out as described previously
(29)
. Purified viruses were stored in 1
M sucrose, 10 mM Tris-HCl,
pH 8.5, 1 mM MgCl2, 150
mM NaCl, 0.005% Tween 80. The same methods were
used to generate the Ad-GFP and Ad-TK vectors, which express GFP and
the HSV-TK protein, respectively. Ad-null was used as a control (Fig. 1B
).
Adenovirus Infections in Vitro.
All infections were performed in suspension by a 30-min incubation of
cells with virus vectors dilutions in 100 µl of PBS supplemented with
2% FCS, 1% cations. Cells were then plated in complete fresh medium,
and analysis was performed at various times postinfection. To determine
the in vitro transduction efficiency, cells were infected
with Ad-GFP, and 24 h later, single-cell suspensions were analyzed
by flow cytometry using a FACScan instrument (Becton Dickinson).
Enzymatic Assays.
To evaluate the percentage of conversion of cytosine to uracil, of
uracil to UMP and of cytosine to UMP, SK-BR-3 human tumor cells
(5 x 106 cells) were transduced
in suspension by Ad-null, Ad-FCY1, Ad-FUR1, or Ad-FCU1 at a MOI of 100.
Twenty-four h later, the cells were resuspended in 30 µl of lysis
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5
mM EDTA, 1 mM DTT, 1% Triton X-100) and
incubated for 30 min at 4°C. Cellular debris were removed by
centrifugation, and 4 µl of cell lysate were combined with 2 µl of
reaction buffer [100 mM Tris-HCl, pH 7.5, 0.4
mM [14C]cytosine (0.02 µCi/µl)
for CDase activity; 100 mM Tris-HCl, pH 7.5, 10
mM MgCl2, 10 mM 5-PRPP,
0.4 mM [14C]uracil (0.02
µCi/µl) for UPRTase activity; and 100 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 10 mM
5-PRPP, 0.4 mM [14C]cytosine (0.02
µCi/µl) for CDase-UPRTase activities] and incubated at 37°C for
2 h. The enzymatic reactions were stopped by heating at 90°C for
2 min. Aliquots of each reaction (1 µl) were run on
polyethyleneimine-impregnated cellulose TLC plate (Schleicher &
Schuell, Dassel, Germany) and developed in a mixture of 1-butanol
(86%)/water (14%). Scanning of the radioactive spots was achieved
with a Molecular Dynamics 445 SI PhosphorImager.
For determination of the different specific enzymatic activities using 5-FC and 5-FU as substrates, SW480, SK-BR-3, and PANC-1 human tumor cells were infected in suspension with each adenovirus vector at a MOI of 20. Twenty-four h after infection, 10 µg of cell lysate proteins (determined by Bio-Rad protein assay) were incubated at 37°C for 20 min with a final concentration of 1 mM 5-FC ([3 H]5-FC at 0.01 µCi/µl) for CDase and CDase-UPRTase activities and 1 mM 5-FU ([14C]5-FU at 0.006 µCi/µl) for UPRTase activity. Aliquots of each reaction were analyzed by TLC as described above.
In Vitro Cell Sensitivity to 5-FC and 5-FU.
Human tumor cells were transduced in suspension by Ad-null, Ad-FCY1,
Ad-FUR1, or Ad-FCU1 at a MOI of 5. A total of 2 x 105 cells/well were plated in 6-well culture
dishes in 2 ml of medium containing various concentrations of 5-FC or
5-FU. Cells were then cultured at 37°C for 6 days, and the number of
viable cells were counted by trypan blue exclusion. Results are
expressed as the ratio between the number of viable cells in plates
containing the drugs versus the number of viable cells in
the corresponding drug-free controls.
In Vitro Evaluation of the Bystander Effect.
Human tumor cells were infected in suspension by Ad-null, Ad-FCY1,
Ad-FUR1, or Ad-FCU1 at a MOI of 20. After 48 h, cells were rinsed
extensively to remove the free virions, trypsinized, mixed with
uninfected cells at varying ratios, and plated in 6-well culture dishes
at a density of 2 x 105
cells/well. Cells were then cultured at 37°C for 6 days in the
presence of 1 mM 5-FC. The number of viable cells was
measured by trypan blue exclusion. To determine whether cell-to-cell
contact was needed for the observed bystander effect, human cells were
infected in suspension with Ad-null, Ad-FCY1, Ad-FUR1, or Ad-FCU1 at a
MOI of 20 and then plated in 12-well culture dishes
(106 cells/well) in 1 ml of serum-containing
medium. After 24 h, medium was aspirated, and cells were washed
with PBS to remove all free viruses. Cells were then incubated for
48 h in a medium containing 0.01, 0.1, or 1 mM 5-FC.
This supernatant was collected, diluted 1:5 with normal medium, and
transferred to monolayers of parental cells (2 x 105/well) that were incubated for 6 days. Viable
cells were then counted by trypan blue exclusion. To examine whether
newly synthesized 5-FU metabolites could passively diffuse into the
culture supernatant of transduced cells exposed to 5-FC, cells were
infected in suspension with the different vectors at a MOI of 20 and
plated in 12-well culture dish (106 cells/well).
After 24 h, 1 mM [3
H]5-FC with
an activity of 0.25 µCi/µl was added to the cultures. After an
additional 24–48 h, the concentrations of
[3
H]5-FC and [3
H]5-FU
in the media were measured using TLC as described above. The
data are expressed as the percentage of 5-FU in the media for various
incubation times with [3
H]5-FC.
Comparison of the FCU1/5-FC and HSV-TK/GCV Prodrug Activation
Systems.
Mouse and human tumor cells were infected in suspension by Ad-null,
Ad-FCU1, or Ad-TK (at a MOI of 50 for B16F0 and a MOI of 1 for SW480,
SK-BR-3 and PANC-1). A total of 2 x 104 cells/well were plated in 6-well culture
dishes in 2 ml of medium containing various concentrations of 5-FC or
GCV. Viable cells were counted on day 10 by trypan blue exclusion.
In Vivo Experiments.
The mouse B16F0 tumors were established by injecting 3 x 105 cells (in 100 µl PBS) s.c. into the
flanks of 6-week-old female immunocompetent B6D2 mice
(IFFA-CREDO, L’Arbresle, France). Eight days later, when tumors
became palpable, the mice were randomized in a blinded manner (10
mice/group) and were treated with the indicated adenovirus vectors. The
test vectors (at a dose of 5 x 108 IU) or the control vehicle used to suspend
the adenovirus (10 mM Tris-HCl, pH 7.5, 1 mM
MgCl2) were administrated to the tumors at days 8, 9, and 10 after
tumor implantation. From day 8 on, the animals were treated twice daily
with i.p. injections of 5-FC (500 mg/kg), GCV (50 mg/kg, maximum
tolerated dose), or saline (0.9% NaCl). The treatment was maintained
for 3 weeks. To evaluate the ability of Ad-FCU1/5-FC to suppress human
tumor growth in vivo, SW480 and LoVo cells were injected
s.c. into athymic BALB/c nu/nu mice (6-week-old female;
IFFA-CREDO). Each animal was implanted with 107
SW480 cells or 5 x 106 LoVo cells
suspended in 100 µl of PBS. When tumors had reached 30
mm3
, the mice were randomized in a blinded manner
and treated with the indicated vectors at a dose of
108 IU (SW480) or 5 x 108 IU (LoVo tumors). The vectors or vehicle were
directly injected into the tumor at days 15, 17, and 19
postimplantation for the SW480 tumors and at days 8, 10, and 12 for the
LoVo tumors. 5-FC (500 mg/kg) or saline was administered i.p. twice
daily for 2 weeks, starting at day 15 (SW480) or at day 8 (LoVo). Tumor
size was measured in three dimensions using calipers. Tumor volume were
calculated in mm3
using the formula
(
/6)(length x width x height).
Statistical Analyses.
Statistical analyses were performed using the nonparametric
Mann-Whitney U test and STATISTICA 5.1 software (StatSoft,
Inc.). A P < 0.05 was considered to be
statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The comparative analysis of the respective enzymatic
activities of FCU1, CDase, and UPRTase was first performed in
vitro in various human and mouse tumor cell lines. For that
purpose, the FCU1, FCY1, and FUR1
genes were cloned into recombinant E1/E3-deleted vectors derived from
the human adenovirus serotype 5 (Fig. 1B
). Although
recombinant adenoviruses constitute currently the most efficient
vectors for in vitro and in vivo expression of
therapeutic genes in tumor cells, wide variations in viral
susceptibility can be observed among various cell types. To first
determine the experimental conditions that allow an efficient
transduction of the human (SW480, SK-BR-3, PANC-1, and LoVo) and mouse
(B16F0) tumor cells selected for this study, the susceptibility of
these cells to virus transduction was analyzed using the
replication-deficient Ad-GFP (Fig. 1B
). These
experiments, summarized in Table 1
, showed that more than 90% of the human cell populations could be
transduced with Ad-GFP at a MOI of 20, whereas only 12% of the mouse
B16F0 melanoma cells were transduced at a MOI of 200.
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). Unexpectedly however, infection with Ad-FCU1 revealed a
higher CDase activity, with a conversion of the entire cytosine pool to
uracil (Fig. 2A
). In contrast to CDase, a low basal
endogenous UPRTase activity was detected in the SK-BR-3 cells
(conversion of 2% of the uracil molecules to UMP; Fig. 2B
),
but transduction with Ad-FUR1 or Ad-FCU1 further enhanced this UPRTase
activity, thus allowing the conversion of the entire uracil pool
to UMP. No difference in the UPRTase activities encoded by the
FUR1 and FCU1 genes was noticed, however (Fig. 2B
). Co-infection of SK-BR-3 cells with both Ad-FCY1 and
Ad-FUR1 vectors led to the direct conversion of 18% of the cytosine
molecules to UMP, confirming the ability of Ad-FCY1 and Ad-FUR1 to
simultaneously express the complementary CDase and UPRTase enzymatic
activities (Fig. 2C
). Under similar experimental conditions,
the chimeric FCU1 enzyme displayed a much more efficient combined
CDase/UPRTase activity, with a conversion of the entire cytosine pool
into UMP (Fig. 2C
), as a probable consequence of its higher
CDase activity (Fig. 2A
).
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, elevated CDase activities were found in cells
infected with either Ad-FCY1 or Ad-FCU1, whereas no CDase activity was
detectable in noninfected cells or in mock-infected cells (Table 2
). The CDase activity was, however, more than 100-fold higher in cells
infected with Ad-FCU1 than in cells infected with Ad-FCY1 (Table 2)
.
The reason for this enhanced CDase activity of FCU1 remains
unknown. We can nonetheless exclude variations in the efficiency of
cell transduction as a cause because identical experimental conditions
were used for all infections. In contrast, the UPRTase enzymatic
activity was similar in cells infected with Ad-FCU1 and Ad-FUR1, but
significantly higher than in mock-infected cells or in cells infected
with Ad-null or Ad-FCY1 (Table 2)
. Analysis of the direct conversion of
[3
H]5-FC to [3
H]5-FUMP,
indicative of the existence of a combined CDase-UPRTase activity,
confirmed that FCU1 does encode a bifunctional enzyme (Table 2)
. Moreover, this combined enzymatic activity was found to be 10-fold
higher in cells infected with Ad-FCU1 than in cells co-infected with
Ad-FCY1 and Ad-FUR1.
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Expression of FCU1 Enhances the Sensitivity of Human
Tumor Cells to 5-FC.
To determine whether the combined CDase/UPRTase activities and the
higher CDase activity would make of FCU1 a suitable suicide
gene for cancer gene therapy, the sensitivities to 5-FC of the human
SW480, SK-BR-3, and PANC-1 tumor cells were analyzed in
vitro after transduction with the vectors Ad-FCU1, Ad-FCY1, or
Ad-FUR1, either alone or in combination (Fig. 3
A, C, and E). These results demonstrate that
infection with Ad-FCY1 confers to these cells a higher sensitivity to
5-FC, which is further increased by 10-fold when the cells are
co-infected with Ad-FUR1 (Fig. 3)
. This observation supports the
hypothesis that the simultaneous expression of CDase and UPRTase can
lead to a cooperative effect, resulting in an increased sensitivity of
the target cells to 5-FC. In agreement with these results, infection
with Ad-FCU1 also led similarly to an enhanced sensitivity of the tumor
cells to 5-FC. However, cells transduced with Ad-FCU1 died at
concentrations of 5-FC that were at least 1000- and 100-fold lower than
the concentrations toxic for cells transduced with Ad-FCY1 or
co-transduced with Ad-FCY1 + Ad-FUR1, respectively (Fig. 3
,
A, C, and E). Consistent with the results from
the enzymatic analyses (Table 2)
, transduction with Ad-FUR1 or Ad-FCU1
increased similarly the sensitivity of the human SW480, SK-BR-3, and
PANC-1 cells to 5-FU by approximately 50-fold as compared to the
mock-infected cells or to cells infected with Ad-null (Fig. 3
, B,
D, and F). As expected, transduction with Ad-FCY1 did
not modify the the sensitivity of the cells to 5-FU.
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and
further demonstrate the bifunctionality of the FCU1 chimeric protein.
More importantly, these studies also show that FCU1 is much
more potent than FCY1, or even than the combination of
FCY1 and FUR1, in conferring a sensitivity to
5-FC to transduced tumor cells.
Bystander Killing Is More Effective with FCU1 than
with FCY1 or FCY1+FUR1.
Given the relatively poor efficiency of gene transfer generally
achieved in vivo, even with recombinant adenovirus vectors,
the major interest of most prodrug activation systems is their ability
to extend the prodrug-induced cytotoxicity beyond the tumor area that
was effectively transduced. To investigate the existence and the
potency of an eventual bystander killing associated with the expression
of FCU1, human tumor cell lines were infected with Ad-null,
Ad-FCY1, Ad-FUR1, and Ad-FCU1 and mixed at different ratios with
mock-infected cells. Cell viability was then measured after 6 days of
exposure to 1 mM of 5-FC. Consistent with
previous reports using the E. coli CDase (17, 20)
, these experiments show that expression of FCY1
in 10–20% of the cells is required to attain cytotoxicity in the
entire cell population (Fig. 4
). Similar results were obtained when cells were infected simultaneously
with FCY1 and FUR1. In contrast, expression of
FCU1 from as few as 1% transduced cells was sufficient to
provoke a near complete eradication of the mixed cell population (Fig. 4)
.
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). As expected, supernatants recovered from mock-infected cells or from
cells infected with Ad-null or Ad-FUR1 displayed no evident toxicity,
whereas supernatants from cells infected with Ad-FCY1 or Ad-FCY1 + Ad-FUR1 induced a major cytotoxicity in the treated cells.
Consistent with the stronger bystander effect described above for
FCU1, tumor cells were more efficiently killed when
supernatants were collected from cells transduced with Ad-FCU1 than
with Ad-FCY1 (Fig. 5)
. These results indicate that freely diffusible
and stable toxic metabolites derived from 5-FC mediate the bystander
effect. Analysis of the cell culture supernatant by TLC revealed a
progressive increase in the amount of 5-FU in the extracellular milieu
of cells transduced with Ad-FCY1 or Ad-FCU1 (Fig. 6
). Again, FCU1 was much more effective in transforming 5-FC in 5-FU.
Consistent with previous data from Hirschowitz et al.
(17)
describing an adenovirus vector encoding the
bacterial CDase, approximately 20% of the
3
H-labeled 5-FC was deaminated by the
FCY1-encoded CDase into 5-FU at 48 h postinfection. In
contrast, FCU1 converted close to 100% of the 5-FC into 5-FU during
the same period (Fig. 6)
.
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In Vitro Comparison of the Conditional Cytotoxicity
of FCU1 and HSV-TK.
Tumor cells infected with Ad-FCU1 or Ad-TK under similar experimental
conditions were compared for their sensitivity toward 5-FC and GCV,
respectively (Table 3
). The IC50 (the concentration of prodrug that
inhibits cell growth by 50% as compared to the untreated control
cells) were determined for 5-FC and GCV, and the therapeutic indexes
were calculated as the ratio of the IC50 of cells
infected with Ad-null to the IC50 of cells
infected with Ad-FCU1 or Ad-TK. In all human cell lines tested in this
study, the therapeutic index of the FCU1/5-FC regimen was
higher than that of the TK/GCV combination. A similar
observation was made for the mouse B16F0 melanoma cells, despite a
reduced therapeutic index for both suicide genes, because of the lower
susceptibility of murine cells to adenovirus infection (Table 1)
. These
results suggest that the FCU1/5-FC prodrug activation system
is more efficient at killing tumor cells in vitro than the
TK/GCV system.
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) or in immunodeficient BALB/c nude mice implanted with two different
human colorectal tumor models (SW480 and LoVo cells; Fig. 8
). All test vectors were directly injected into the tumors as indicated
in "Materials and Methods," with a concomitant i.p. administration
of the appropriate prodrug (5-FC or GCV). The comparative analysis of
Ad-FCU1 and Ad-TK in the B16F0 tumor model (Fig. 7)
, an aggressive,
poorly immunogenic tumor cell line, confirmed the higher potency of
FCU1. A more sustained control of tumor growth was achieved in mice
treated with Ad-FCU1/5-FC than in animals treated with Ad-TK/GCV. As
expected, no significant inhibition of tumor growth was observed in any
the control groups (Fig. 7)
. Given the poor susceptibility of mouse
cells to adenovirus infection, similar in vivo gene transfer
experiments were performed in BALB/c nude mice implanted with the more
susceptible human SW480 and LoVo colon cancer cells. In both cases,
administration of Ad-FCU1/5-FC resulted in a statistically significant
suppression of tumor growth, whereas no modification in tumor growth
was observed in the control groups (Fig. 8)
. At 4 weeks after delivery
of Ad-FCU1/5-FC, the SW480 and LoVo tumors were 60–65 and 50–55%
smaller, respectively, compared to the controls (all controls averaged;
Fig. 8
). In parallel, control experiments were also performed to
determine the in vivo antitumor effect of 5-FU in
nontransduced tumors derived from SW480 and LoVo cells. Despite the
administration of doses of 5-FU that were at the maximum tolerated
concentrations (i.p. injection of 10 mg of 5-FU/kg twice daily during 3
weeks), no statistically significant inhibition of tumor growth was
observed (data not shown). This result supports the notion that a local
production of high concentrations of 5-FC derivatives is necessary to
achieve a significant antitumor effect in absence of noticeable
toxicity.
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DISCUSSION |
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To exert its cytotoxic effect, the 5-FU produced by the yeast CDase must subsequently be converted by cellular enzymes into one of its active metabolites, 5-fluoroUTP (5-FUTP) or 5-fluoro-dUMP (5-FdUMP). 5-FUTP can be incorporated into the cellular RNAs, whereas 5-FdUMP is a potent inhibitor of the cellular thymidylate synthase, a key enzyme in the de novo synthesis of dTMP, which is a precursor for DNA synthesis. The conversion of 5-FU to 5-FUMP can be directly catalyzed by orotate phosphoribosyltransferase using 5-PRPP as the cosubstrate or indirectly in a sequence of reactions in which 5-FU is first converted to 5-fluorouridine by a pyrimidine nucleoside phosphorylase using ribose-1-phosphate as the cosubstrate and then phosphorylated to 5-FUMP by uridine kinase (33) . 5-FU can also be converted by a cellular pyrimidine nucleoside phosphorylase into 2'deoxy-5-fluorouridine, with deoxy-Rib-1-P as the cosubstrate, and then by a cellular thymidine kinase to 5-FUMP (33) . However, certain cancer cells may become relatively resistant to 5-FU, because of a poorly efficient conversion of 5-FU to its toxic metabolites (21) . The existence in E. coli and S. cerevisiae of genes (upp and FUR1, respectively) encoding an enzyme, UPRTase, that can actively convert uracil to UMP, suggests that transfer into tumor cells of these genes should in principle induce a more efficient conversion of 5-FU to 5-FUMP, thus restoring the cells sensitivity to 5-FU. This hypothesis is supported by our demonstration that the adenovirus-mediated transduction of FUR1 into tumor cells enhances their sensitivity to 5-FU by 50-fold. These results are consistent with a recent report from Kanai et al. (22) showing that expression of the bacterial UPRTase gene similarly enhances the cells sensitivity to 5-FU. These results support the notion that in some tumor cells the conversion of 5-FU to 5-FUMP may be a rate-limiting step that can be bypassed by the expression of the FUR1 or upp genes. Interestingly, we also demonstrate in this report that FUR1 gene transfer can substantially augment the sensitivity of tumor cells to 5-FC when such cells do simultaneously express FCY1.
These demonstrations of the cooperativity between the FCY1- and FUR1-encoded enzymes prompted us to investigate whether a direct channeling of 5-FC to 5-FUMP could be achieved through the generation of a bifunctional protein that combines the two enzymatic activities in one single polypeptide. This fusion protein, designated FCU1, was constructed by fusing in frame the FCY1 and FUR1 coding sequences and was demonstrated in vitro to exhibit both enzymatic activities. However, although the UPRTase activity of FCU1 was comparable to that encoded by the parental FUR1 gene, the CDase activity was found to be 100-fold higher than the activity encoded by the parental FCY1 gene. These unexpected results were confirmed by the 1000-fold and 100-fold higher in vitro sensitivity to 5-FC of the FCU1-transduced tumor cells as compared to cells transduced with FCY1 or with both FCY1 and FUR1, respectively. The mechanism whereby the enzymatic activity of the CDase moiety was improved in FCU1 is currently unknown, but future studies using purified CDase, UPRTase and FCU1 proteins are under consideration to better characterize the properties of these proteins. The construction of a similar fusion protein combining the CDase and UPRTase enzymatic activities of the bacterial codA and upp genes was recently reported (34) . Interestingly, although this chimeric bacterial protein also exhibited both enzymatic activities, their efficiencies were comparable to the activities of the proteins encoded by the native codA and upp genes (34) .
Because one of the major limitation of current cancer gene therapy strategies is the poor efficiency of in vivo gene transfer, successful application of a suicide gene will rely on the potency of its bystander killing effect. A direct consequence of the increased CDase activity of FCU1 is the very rapid intracellular conversion of 5-FC to 5-FU and the release of 5-FU into the extracellular milieu. This very fast kinetics of production and release of 5-FU and the diffusible nature of the metabolite (35) may both contribute to the observed powerful bystander effect induced by FCU1, because bystander killing was more effective in cells transduced with FCU1 than in cells expressing FCY1 alone or in combination with FUR1. FCU1 was also more effective than HSV-TK both in vitro and in implanted mouse tumor models. This observation confirms and extends previous studies showing that the bacterial CDase/5-FC system was more potent than the HSV-TK/GCV system (36, 37) . As discussed above, a significant disadvantage of the HSV-TK suicide gene as compared to FCU1 is the requirement for direct cell-to-cell contact and active transport of the toxic GCV metabolites to neighboring cells through the gap junctions (10–13) . An increased expression level of HSV-TK does not necessarily correlate with more efficient bystander killing because the transport of GCV may become limiting (37) . In contrast to GCV, diffusible metabolites such as 5-FU should exert a more potent tumoricidal effect. Any increase in enzymatic CDase activity, as is the case with FCU1, should directly translate into higher local and more toxic concentrations of 5-FU in the tumor area. Another important advantage of the FCU1/5-FC system is its impact on both dividing and nondividing cancer cells. Although sensitization to GCV mediated by HSV-TK expression occurs only when the target cells are in the S-phase of the cell cycle (7) , the incorporation of 5-FU into RNA is independent of the cell cycle (38) .
Confirming the in vitro properties of FCU1, adenovirus-mediated transfer of FCU1 to tumors derived from mouse melanoma cells or from human colon carcinoma cells could also effectively suppress in vivo the growth of the tumors upon systemic administration of 5-FC to the animals. These results are encouraging because such human tumor models were shown to be relatively resistant to 5-FU treatment, and adenovirus injection was found in these experimental conditions to transduce only a few percent of the implanted tumor cells (data not shown).
Thus, the unique combination of an enhanced CDase activity, the
presence of an UPRTase activity, and the powerful bystander killing
effect mediated by diffusible metabolites makes FCU1 a
promising novel candidate for cancer gene therapy applications, even
for 5-FU-resistant target tumors. Moreover, FCU1 may also be
considered for combinations with other therapeutic genes, such as genes
encoding immunomodulatory molecules (4–6)
. Similarly,
5-FU being a radiosensitizer (39)
, the overall antitumor
effect of the FCU1/5-FC treatment may be further increased
by radiotherapy, as suggested by recent studies showing a better
response to radiation when associated with CDase/5-FC gene transfer
(40, 41) . Indeed, our ongoing animal studies have
confirmed that co-transfer of FCU1 with either the
interleukin 2 or IFN-
cytokine genes further improves the in
vivo antitumor response in immunocompetent mice implanted with the
B16F0 melanoma tumor model.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Transgene S.A., 11 rue de Molsheim, 67082 Strasbourg
Cedex, France. Fax: 33-388-22-58-07; E-mail: erbs{at}transgene.fr 
2 The abbreviations used are:
HSV-TK, herpes simplex virus type-1 thymidine kinase;
5-FC, 5-fluorocytosine; CDase, cytosine deaminase; 5-FU,
5-fluorouracil; UPRTase, uracil phosphoribosyltransferase; 5-FUMP,
5-fluorouridine-5'-monophosphate; GCV, ganciclovir; 5-PRPP,
5-phosphoribosyl-1-pyrophosphate; MOI, multiplicity of infection; GFP,
green fluorescent protein; Ad-FCU1, adenovirus expressing
FCU1; Ad-FCY1, adenovirus expressing
FCY1; Ad-FUR1, adenovirus expressing
FUR1; Ad-GFP, adenovirus expressing GFP; Ad-TK,
adenovirus expressing HSV-TK; Ad-null, adenovirus carrying no
expression cassette; CMV, cytomegalovirus; IU, infectious unit(s).
3 P. Erbs, F. Exinger, and R. Jund, manuscript in
preparation.
Received 11/29/99. Accepted 5/16/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-fetoprotein producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene. Cancer Res, 57: 461-465, 1997.This article has been cited by other articles:
|
|
 |
|
  Z. Li, Y. Fortin, and S.-H. Shen Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries Nucleic Acids Res., January 1, 2009; 37(1): e8 - e8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Kucerova, V. Altanerova, M. Matuskova, S. Tyciakova, and C. Altaner Adipose Tissue-Derived Human Mesenchymal Stem Cells Mediated Prodrug Cancer Gene Therapy Cancer Res., July 1, 2007; 67(13): 6304 - 6313. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Ramnaraine, W. E. Mathews, J. M. Donohue, C. M. Lynch, M. J. Goblirsch, and D. R. Clohisy Osteoclasts Direct Bystander Killing of Bone Cancer. Cancer Res., November 15, 2006; 66(22): 10929 - 10935. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. M. Braks, B. Franke-Fayard, H. Kroeze, C. J. Janse, and A. P. Waters Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium Nucleic Acids Res., March 14, 2006; 34(5): e39 - e39. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Acres, M. Gantzer, C. Remy, N. Futin, N. Accart, O. Chaloin, J. Hoebeke, J.-M. Balloul, and S. Paul Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity Cancer Res., October 15, 2005; 65(20): 9536 - 9546. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Porosnicu, A. Mian, and G. N. Barber The Oncolytic Effect of Recombinant Vesicular Stomatitis Virus Is Enhanced by Expression of the Fusion Cytosine Deaminase/Uracil Phosphoribosyltransferase Suicide Gene Cancer Res., December 1, 2003; 63(23): 8366 - 8376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Miller, C. R. Williams, D. J. Buchsbaum, and G. Y. Gillespie Intratumoral 5-Fluorouracil Produced by Cytosine Deaminase/5-Fluorocytosine Gene Therapy Is Effective for Experimental Human Glioblastomas Cancer Res., February 1, 2002; 62(3): 773 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Nanda, R. Vogels, M. Havenga, C. J. Avezaat, A. Bout, and P. S. Smitt Treatment of Malignant Gliomas with a Replicating Adenoviral Vector Expressing Herpes Simplex Virus-Thymidine Kinase Cancer Res., December 1, 2001; 61(24): 8743 - 8750. [Abstract] [Full Text] [PDF]
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, mock-infected; 
, Ad-null; 
;
Ad-FUR1; 
, Ad-FCY1; 
, Ad-FCY1 + Ad-FUR1; 
, Ad-FCU1)
were exposed to various concentrations of 5-FC (A, C,
and E) or 5-FU (B, D, and
F), and cell survival was measured 6 days later as
described in "Materials and Methods." Results are expressed as the
percentage of surviving cells in the presence and absence of the drug.
Values are represented as mean ± SD of four individual
determinations.
