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Experimental Therapeutics |
CRC Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TA, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The minimal 628-bp PSA and hKLK2 promoters showed only
low-level expression in either PSA-positive or PSA-negative cells and
showed no increase with the addition of androgen. Tandem duplication of
the PSA promoter slightly increased expression in PSA-positive LNCaP
cells. The addition of CMV enhancer sequences upstream of a single PSA
or hKLK2 promoter substantially but nonspecifically
increased luciferase expression in all cell lines tested. However,
placing a 1455-bp PSA enhancer sequence upstream of either the PSA or
hKLK2 promoters increased expression 20-fold in the
PSA-positive cell line LNCaP but not in the PSA-negative lines. Tandem
duplication of the PSA enhancer increased expression to 
50-fold
higher than either promoter alone while retaining tissue-specific
control. The level of expression was reduced by the addition of a third
copy of the PSA enhancer. Expression from all enhancer constructs was
increased 100-fold above basal levels when induced with the androgen
dihydrotestosterone, with the PSA-based constructs consistently
exhibiting roughly twice the level of expression of the
hKLK2-based constructs at all androgen concentrations.
Adenovirus vectors were produced in which either enhanced green
fluorescent protein or nitroreductase could be expressed from the
optimized PSA double enhancer-promoter construct and evaluated in LNCaP
cells and the bladder-derived line EJ. Control vectors with the CMV
promoter gave good levels of expression in both cell lines, whereas the
PSA constructs only produced detectable levels of protein in the LNCaP
cells as assessed by fluorescence of enhanced green fluorescent protein
or by Western blotting of nitroreductase. LNCaP but not EJ cells were
selectively sensitized to the prodrug CB1954 following infection with
Ad-PSAEEP-NR. The PSA-based nitroreductase virus produced
comparable amounts of nitroreductase and sensitization to CB1954
approaching that of the CMV-driven virus.
Plasmid and adenovirus constructs combining PSA enhancer and promoter sequences demonstrate selective expression of linked genes in PSA-positive cells. The expression is induced by androgen and gives therapeutically relevant levels of effector proteins.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gene therapy approaches to prostate cancer are attractive because they hold the possibility of selectively targeting therapy to affected tissue, thereby avoiding toxicities associated with treatments such as cytotoxic chemotherapy, which has thus far proved of limited benefit in prostate cancer. Because the prostate is a nonessential organ, treatment can be targeted with tissue-specific rather than tumor-specific promoters. A number of proteins are known to be expressed in a tissue-specific manner, including prostate-specific membrane antigen and the kallikreins PSA and hKLK2, and the regulatory DNA sequences controlling these genes are therefore suitable candidates for driving tissue-specific expression of therapeutic genes.
PSA is a protein expressed exclusively by benign, hyperplastic, and malignant prostatic epithelium (1, 2, 3) . Rising levels in serum are indicative of prostate disease (Ref. 4 ; benign hyperplasia or malignant carcinoma), and this has allowed PSA to be used as a diagnostic marker. Expression of the related kallikrein hKLK2 gene encoding human kallikrein-2 protein (hK2), which shows considerable sequence homology with PSA (5 , 6) , is also restricted to prostate tissue (3) . Expression of both PSA and hKLK2 is up-regulated by androgen (3 , 7 , 8) . Detection of hK2 has also been shown to be related to disease both on immunohistology and in serum (9 , 10) . The restriction of PSA expression exclusively to prostate tissue has led to investigations of the PSA gene regulation (11) for the purposes of targeting prostate cancer therapy. Initial reports suggested that the immediate 5' promoter region was sufficient to target expression to prostate tissue (11) , and this was further supported by subsequent studies aimed at testing targeted expression constructs for gene therapy vectors (12) . Subsequent research suggested that an upstream enhancer was required for tissue-specific expression (13) . The present study aimed to further examine the control of PSA expression, to resolve apparent discrepancies between these papers, and to optimize possible promoter constructs for use in gene therapy vectors. In addition, we set out to investigate the possible use of the hKLK2 promoter for prostate gene therapy. A range of therapeutic options is available with tissue-specific control of gene therapy vector expression. These include expression of suicide genes, prodrug activating enzymes (virus- or gene-directed enzyme prodrug therapy), immunomodulatory proteins, and gene replacement therapy (for reviews, see Refs. 14 or 15 ). The present study reports the construction and evaluation of a prostate tissue-specific promoter and its incorporation into plasmid reporter constructs. The optimal control sequence was subsequently validated in an adenovirus vector in which it showed appropriate regulation of either EGFP or the prodrug activating enzyme nitroreductase. Expression of nitroreductase selectively sensitized the PSA-positive cell line LNCaP to the prodrug CB1954.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of PSA Reporter Vectors for Transfection Studies.
All vector maps are shown in Fig. 1
.
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The PSA promoter was subcloned as an Asp718-HinDIII fragment into the pGL3-basic luciferase expression vector (Promega) to give plasmid pPSA-GL3. A second copy of the PSA promoter introducing an Asp718 site just upstream of the TATA box was obtained by PCR with primers primerGL2 (Promega) and 5'-CCGGTACCTTCATTCCCCAGGACTC-3'. The PCR product was digested with Asp718 and inserted into the Asp718 site of pPSA-GL3; a clone with the two promoters in tandem was selected.
hKLK2-Luciferase Construct.
The hKLK2 promoter was amplified by PCR from the same
LNCaP genomic DNA as for the PSA promoter using 5' primer
5'-CCGGTACCTGTAATCTATCACTTTGGGCA-3', which introduced an
Asp718 site, and 3' primer
5'-CCAAGCTTCCACGGCCAGGTGGTGA-3', which introduced a
HinDIII site.
CMV-based Constructs.
The CMV immediate early gene control region derived from plasmid pLNCX
(kindly donated by Dusty Miller; Ref. 17
) was inserted
between the SmaI and HinDIII sites of plasmid
pGL3-basic (Promega) to give plasmid pCMV-GL3. An Asp718
fragment containing the CMV enhancer was obtained by PCR amplification
with primers RVprimer3 (Promega) and
5'-CCGGTACCTCCCACCGTACATGCC-3', which introduced an
Asp718 site just upstream of the TATA box. The PCR product
was cut with Asp718 and inserted into the Asp718
site upstream of the PSA promoter in pPSA-GL3 in the forward
orientation to generate pCMVEPSA-GL3.
PSA and hKLK2 Enhancer-Promoter Constructs.
A DNA fragment containing sequences from -5322 to -3869 of the PSA gene
flanked by Asp718 sites was prepared from LNCaP genomic DNA
by PCR with primers 5'-CCGGTACCTCTAGAAATCTAGCTGATATAGTG-3'
and 5'-CCGGTACCAACGTTGAGACTGTCCTGCAGAC-3'. This was cloned
into the Asp718 site upstream of the PSA promoter in
pPSA-GL3; to generate pPSAEP-GL3, three
independent clones were evaluated in preliminary transfection assays
with similar results, and one clone was selected for further
studies. Second and third copies of the PSA enhancer were
inserted as further Asp718 fragments to generate
pPSAEEP-GL3 and
pPSAEEEP-GL3. Equivalent single and double
enhancer constructs were made using the hKLK2 promoter and
PSA enhancer in a similar fashion to give constructs
phKLK2EP and
phKLK2EEP.
Evaluation of Expression of Luciferase Reporter Constructs in
Prostate and Nonprostate Cell Lines.
Cells were washed once in PBS, trypsinized, and then
washed. Then, 5 x 106
cells were resuspended in 500 µl of RPMI and placed in a 1-ml
electroporation cuvette with an electrode spacing of 4 mm with 10 µg
of the relevant luciferase reporter plasmid and 1 µg pCMV-ßgal DNA
as an internal control for transfection efficiency. The cells
were pulsed at 125 µF and 0.45 kV using a Bio-Rad Gene Pulser,
transferred to 10 ml of medium in a 25-cm2 tissue
culture flask, and incubated for 48 h. Cells were washed in PBS
and then lysed in 500 µl of lysis buffer [100 mM HEPES
(pH 8), 2 mM magnesium chloride, 5 mM DTT, 2%
Triton X-100]. Luciferase activity was determined by the addition of
100 µl of luciferase assay reagent [20 mM glycylglycine
(pH 7.9), 5 mM magnesium chloride, 0.1 mM EDTA,
33 µM DTT, 270 µM CoA, 470 µM
beetle luciferin, and 530 µM ATP] to 100 µl of lysate
and by the measurement of light emission for 10 s in a Berthold
LB9501 luminometer. The luciferase activity in each sample was then
normalized for variations in transfection efficiency by measuring the
level of ß-galactosidase expression from the cotransfected
pCMV-ßgal plasmid. After incubating the residual cell lysate at
50°C for 1 h to inactivate endogenous ß-galactosidase, a
50-µl aliquot was mixed with 200 µl of substrate (K2048–1,
Clontech) and incubated for a further 1 h at room
temperature. ß-galactosidase activity was determined by measuring
light emission for 5 s in the luminometer.
Results were controlled for transfection efficiency in LNCaP cells by dividing the luciferase expression from the test construct by the ß-galactosidase expression from the cotransfected pCMV-ßgal plasmid. Parallel transfections were also carried out using pCMV-GL3 and pCMV-ßgal, thus allowing activity of the promoter constructs to be expressed as a percentage of the CMV promoter activity. This double control was necessary because preliminary experiments had shown that expression from the CMV promoter was itself influenced by androgen, and the additional control removed any differences arising between androgen-sensitive and -insensitive cell lines from this effect. All plasmid transfection results are the mean of six experimental measurements.
Construction of Adenovirus Vectors.
The self-complementary oligonucleotide
5'-GTACGGATCCTTCGAAGGATCC-3' containing BamHI and
BstBI sites was inserted into the Asp718 site
upstream of the two PSA enhancers after partial digestion of
pPSAEEP-GL3. The PSA sequences were then excised
with BstBI and HinDIII and inserted between these
sites upstream of the nitroreductase gene in a derivative of
pPS1017A5/pxLNC-NR (17)
, replacing the CMV promoter. A
PSAEEP-EGFP cassette was assembled in a similar
manner by insertion into a similar plasmid with the EGFP gene
(Clontech) in place of NR. The PSAEEP-NR and
PSAEEP-EGFP cassettes were then excised with
BamHI and inserted individually into the BamHI
site of the adenovirus transfer vector pSW115A5, a derivative of
pPS971C5 (18)
in which the EcoRI site at the
left end of the adenovirus sequence has been converted to a
SwaI site. The resulting plasmids with the
PSAEEP-NR and PSAEEP-EGFP
cassettes in the rightward orientation relative to adenovirus sequences
were linearized with SwaI and separately cotransfected into
293 cells with pPS1092B1. The latter is a plasmid that contains an
adenovirus type 5 sequence from nucleotide 3328 to the right end of the
genome, with a deletion between the XbaI sites in the E3
region replaced with a oligonucleotide containing BstI and
PacI sites; it was linearized before transfection at a
SwaI site inserted at the right end of the viral genome. The
Ad-CMV-ntr virus is described
elsewhere.4
For the rescue of adenovirus constructs, plasmid DNA containing the target sequences was transfected into 293 80% confluent cells using the calcium phosphate method, and the cells were harvested when a complete viral cytopathic effect was apparent (10–14 days). The cells were subjected to three cycles of freezing and thawing, and the virus was separated from cell debris by centrifugation at 900 x g for 10 min. The virus stock was amplified by serial passage in 293 cells and used at the fourth passage. Virus was banded in a caesium chloride/glycerol gradient (19) , and the titer was determined by plaque assay on 293 cells.
Assessment of EGFP Expression.
Cells were plated out in 6-well plates at
105/well and infected with adenovirus at MOIs 1,
10, and 30/cell 24 h later. Twelve to twenty-four hours after
infection, cells were trypsinized and plated onto
poly-L-lysine-treated slides and incubated for a further
24 h prior to fixation in acetone-methanol and visualization under
phase-contrast microscopy. Experiments were carried out in triplicate,
and results shown are the mean of three experiments.
Prodrug Activation Assays.
Cells were grown until 70–80% confluent and then infected with virus
at MOIs 1, 10, or 30. After 24 h of incubation, cells were
harvested by trypsinization, and an aliquot was saved for subsequent
Western blot analysis using standard methodology using a sheep
polyclonal antinitroreductase antibody (Cobra Therapeutics; Ref.
20
). Remaining cells were plated at 5 x 104/well on 96-well plates and incubated for a
further 24 h prior to the addition of the prodrug CB1954. Cell
survival was quantified 72 h after the addition of the prodrug
using alamarBlue (Serotec) by fluorimetry. Experiments were carried out
in triplicate, and results shown are the mean of three experiments.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The PSA promoter constructs derived from the LNCaP cell line and the
normal donor LCL were examined. The level of expression was expressed
as a percentage of that from a control pCMV-GL3 plasmid normalized
against cotransfected p-CMV-ßgal as described above. Expression from
both PSA and the hKLK2 promoters was detectable but very
low, 0.1–0.3% of the level of expression from CMV, and there
were no significant differences between the expression from the PSA and
hKLK2 promoters in LNCaP cells and the other cell types. A
construct with a tandem repeat of the PSA promoter sequence upstream of
the TATA box showed at best a marginal increase in the expression in
LNCaP cells (data not shown). The PSA promoter derived from the LCL
clone was chosen for further study.
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, the level of expression was increased 100-1000-fold over that obtained
with the promoters alone; however, there was no apparent
tissue-specificity because similar high levels of expression were
observed in all tested cell lines.
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). A single enhancer copy caused a substantial increase in expression in
LNCaP cells but not in other nonprostate cell types. The double and
triple enhancers further increased tissue-specific expression with the
double enhancer giving higher levels than the triple enhancer,
50-fold higher than that seen with the promoter alone.
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). Both promoter constructs showed no detectable response to androgen.
The enhancer constructs all gave increased expression with androgen,
the PSA enhancer-PSA promoter constructs giving approximately double
the level of expression at each androgen concentration compared to the
PSA enhancer-hKLK2 promoter constructs. This shows that the
despite the low level of activity of the minimal PSA and
hKLK2 promoters, the PSA promoter contributed additional
activity to the enhancer constructs compared to the closely homologous
hKLK2 promoter. On the basis of these experiments, the
double enhancer-PSA promoter construct was incorporated into a series
of adenovirus constructs for further study.
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) and by Western blotting to detect nitroreductase following infection
with the respective viruses (Fig. 7
). The Ad-CMV-EGFP virus gave high levels of expression in both the
LNCaP and EJ cell lines. In contrast, the
Ad-PSAEEP-EGFP vector only expressed detectable
levels of EGFP in the LNCaP line with no detectable expression in the
bladder-cancer-derived EJ line. A lower level of expression was seen
with Ad-PSAEEP-EGFP than with Ad-CMV-EGFP, which
is consistent with the difference in expression seen with transient
transfection of the plasmid constructs where the
pPSAEEP-luc construct only possesses around
4–5% of the activity of the pCMV-based control (Fig. 3
).
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shows levels of nitroreductase assessed by Western blotting
following viral infection. As with the EGFP viruses, tissue-specific
expression is retained with Ad-PSAEEP-NR. The
control Ad-CMV-ntr virus gave expression of nitroreductase in both cell
types. Ad-PSAEEP-NR gave a comparable level of
nitroreductase protein expression to the Ad-CMV-ntr construct in LNCaP
cells but little or no expression in the EJ bladder cancer cell line.
To test the selectivity and efficacy of target cell sensitization, the
nitroreductase viruses were also evaluated in prodrug sensitization
assays (Fig. 8
). The LNCaP but not the EJ cells were found to be unusually sensitive
to prodrug alone. Nonetheless, the experiment showed that additional
CB1954-dependent cytotoxicity was observed in the LNCaP cells upon
infection with either Ad-CMV-ntr or Ad-PSAEEP-NR
(Fig. 8, a and b
). As anticipated from the
Western blots, the potency of the two viruses was comparable in LNCaP
cells. In contrast, although infection with the Ad-CMV-ntr virus
sensitized the bladder cancer EJ line to CB1954 in a dose-dependent
manner, cells infected with Ad-PSAEEP-NR remained
fully resistant to the prodrug, thereby demonstrating the tissue
specificity of this construct.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Earlier reports with the minimal 5' PSA promoter suggested that the
region from -539 to +12 from the gene was sufficient to give
tissue-specific regulation of gene expression (11)
. We
cloned the 5' promoters of PSA and the closely homologous kallikrein
hKLK2 for our initial investigations aimed at developing
promoter constructs for our gene therapy program. The PSA promoter
cloned from two distinct sources differed from the published sequence:
the sequence variations seen in the LCL-derived PSA clones were
detected in the LNCaP clone and also in a third clone obtained from
another research group (data not shown); an additional base change was
seen in the LNCaP-derived clone. Because the LCL cells were derived
from a nonprostate tissue in a normal donor, it was believed that the
differences were likely to be normal variants rather than of any
functional significance. However, in our hands, as shown in Fig. 2
, neither the minimal 5' promoter nor the promoter of its close
homologue hKLK2 gave tissue-specific regulation of
expression. In addition, absolute levels of expression compared to the
CMV promoter-driven control vectors were very low and were insufficient
to produce immunologically detectable levels of the coimmunostimulatory
molecule CD80 (B7.1; data not shown). The use of a tandem repeat of the
minimal promoter resulted in only a minimal, nonsignificant increase in
expression (data not shown). These results contrast with those of Pang
and coworkers (12)
who reported results with a similar
620-bp PSA promoter termed PCPSA and showed tissue-specific regulation
and androgen inducibility with this promoter in LNCaP but not in
PSA-negative cell lines both prostatic and nonprostatic in origin.
In an attempt to increase the level of expression, a CMV enhancer was
introduced upstream of the PSA and hKLK2 promoters. This did
give substantial increases in expression, but neither construct showed
tissue specificity in our panel of cell lines (Fig. 3
), which again
contrasted to the results reported by Pang et al.
(12)
for their CMV-PSA construct. We are unclear as
to how this discrepancy arose because we think it is unlikely that the
sequence changes listed above would totally abrogate tissue-specific
regulation from our promoter construct (see also Fig. 5
and discussion
below). However, the PCPSA construct included the CMV TATA
sequence upstream of the PSA promoter, which might have resulted in
some transcription initiation upstream of the PSA promoter. In our
attempt to avoid the possibility of upstream initiation, we excluded
the CMV TATA sequence from our construct.
While the early stages of this work were in progress, Schuur et
al. (13)
described the location of enhancer sequences
in the DNA upstream of the PSA gene. We therefore cloned this sequence
and introduced one, two, or three copies of the 5' to the PSA promoter
and one or two copies of the 5' to the hKLK2 promoter. The
single enhancer, as predicted, gave increased expression in a
tissue-specific manner (Fig. 4
). The addition of further tandem repeats
of this enhancer gave additional tissue-specific increases in
expression, with two enhancers being the optimal configuration (Fig. 4
). Comparison of the PSA and hKLK2 promoters with the PSA
enhancers showed that, despite the low activity of the minimal PSA
promoter (Fig. 2
), it did contribute additional activity to the
enhancers compared to the closely homologous hKLK2 promoter
(Fig. 5
). On the basis of these data, the tandem PSA enhancer-PSA
promoter configuration PSAEEP was chosen for
further study. All promoter-enhancer constructs showed androgen
inducibility (Fig. 5
). Interestingly, the PSA-based constructs had
approximately twice the activity of the hKLK2-based
homologues, a ratio reflected in the relative concentrations of mRNA
for the two genes in vivo (3)
.
A variety of gene therapy strategies are possible, including the introduction of genes encoding "suicide enzymes," which activate a prodrug, and immune modulatory proteins, such as GM-CSF or CD80 and replacement tumor suppressor gene function, e.g., p53 (14 , 15) . Possible delivery systems for gene therapy include viruses, in particular, adenoviruses, adenoassociated viruses, and retroviruses, and nonviral systems, such as liposomes, as well as direct injection of naked DNA. We have chosen an adenovirus delivery system because of the high yields of virus obtainable compared to retroviruses and the high infection efficiencies possible with adenoviruses. In addition, adenoviruses do not integrate into the host genome, lessening concerns about insertional mutagenesis.
We have evaluated the PSAEEP transcription
regulatory construct in two adenoviruses, one encoding the reporter
protein EGFP and the other encoding the prodrug activating enzyme
nitroreductase. The test virus Ad-PSAEEP-NR
contained only 7 bp of bacterial DNA 3' to the nitroreductase open
reading frame, compared to 81 bp in the Ad-CMV-ntr virus. We had
recently observed that, in a retroviral vector, nitroreductase was
expressed more efficiently from a construct with the shorter 3'
sequence (21)
. Seeking to maximize expression from the
prostate-specific enhancer/promoter construct, we therefore used this
more efficient version of the nitroreductase gene (which we abbreviated
to NR to distinguish it from the original, unmodified version, ntr) in
the construction of Ad-PSAEEP-NR. The
differential expression seen with plasmid transfection is retained in
the Ad-EGFP constructs in which the Ad-CMV-EGFP virus produced
detectable green cells at MOI = 1 compared to
MOI = 30 for the Ad-PSAEEP-EGFP
virus (Fig. 6
). In contrast, infection with the modified
Ad-PSAEEP-NR resulted in similar expression to
Ad-CMV-ntr as assessed by Western blotting (Fig. 7
) and sensitization
to prodrug (Fig. 8
). This effect is consistent with an 20-fold
improvement in the expression of the nitroreductase protein with the
shorter 3' bacterial untranslated sequence while retaining tissue
specificity. The nitroreductase/CB1954 system has been shown to produce
bystander lysis in a variety of cell systems (21
, 22)
. We
anticipate that this would further amplify the effect of nitroreductase
expression in PSA-positive cells in vivo.
The virus constructs were assessed using the bladder line EJ as the
specificity control. This line was chosen because it is derived from
transitional cell epithelium that comprises one of the cell types in
the urethra and bladder and which may be dose-limiting in clinical
studies. High infection efficiency was noted with the Ad-CMV virus in
both cell lines (Fig. 6, a and b
). In contrast,
the LNCaP line is difficult to transfect using standard laboratory
techniques, and transfection efficiencies of 0.1–10% were obtained
with a variety of nonviral transfection techniques (data not shown). In
addition, virus infection appeared to be directly toxic to the LNCaP
line but not to the EJ line (Fig. 8
), which may prove to be
therapeutically useful for a more generalized feature of
prostate tissue. Direct toxicity resulting in objective tumor responses
(including a complete pathological response) has been reported with the
injection of replication-deficient adenovirus encoding a marker protein
(ß-galactosidase) in one clinical study (23)
, although
it is possible that this effect may be immune-mediated rather than due
to direct viral cytotoxicity (24)
. Additional cell killing
was seen with the addition of prodrug to EJ cells infected with
Ad-CMV-ntr and to LNCaP infected with either virus (Fig. 8
), which is
consistent with the expression of nitroreductase seen on Western
blotting (Fig. 7
). Studies are under way to investigate both these
effects in other prostate cell lines and in cultured primary prostate
tissue.
The lack of PSA-positive cell lines other than LNCaP has limited the evaluation of the construct; as a result, the expression seen may be restricted to this cell type rather than to prostate tissue in general. However, given the consistent expression of PSA on both benign and malignant prostate tissue and the androgen inducibility of the promoter construct, it seems likely that the promoter will behave in a similar fashion in other PSA-positive cells.
We are planning to evaluate the Ad-PSAEEP-NR
virus in clinical trials. The simplest strategy uses direct virus
injection into the prostate gland in patients with locally recurrent
disease. A trial using HSV-tk plus ganciclovir by local intratumoral
injection has demonstrated activity both in animal models and in
locally recurrent prostate cancers (25)
. The
Ad-PSAEEP-NR virus gave equivalent enhancement of
CB1954 toxicity to the viral promoter-driven Ad-CMV-ntr (Fig. 8
), but
it would be anticipated to have less local toxicity to nonprostate
tissue, for example the urethra. Virus cleared from the prostate may
also infect other tissues, leading to the potential for toxicity. Early
clinical studies in mesothelioma with the nontissue-specific Rous
sarcoma virus promoter-enhancer showed transient elevation of liver
enzymes following prodrug therapy (26)
, which is
consistent with the infection of hepatocytes and consequent
sensitization to prodrug. This effect should be abrogated by the use of
a tissue-specific promoter such as PSAEEP while
retaining high-level prodrug expression in the target tissue. Phase I
trials are presently underway in our institute to establish the maximal
tolerated dose of the prodrug CB1954, thereby paving the way for
clinical trials with direct injection of the PSA promoter-driven virus.
In conclusion, an expression vector based on elements of the PSA gene
regulatory sequences has been developed and shown to be tightly
regulated in a panel of cells from tissues of various origins.
Evaluation of the construct in an adenovirus vector has shown high
infection rates in LNCaP together with the tissue-specific production
of functional protein in therapeutically relevant quantities, thereby
providing a solid platform for launching clinical studies.
ACKNOWLEDGMENTS
We thank Karin Gonzalez for technical support.
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FOOTNOTES |
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1 Supported by a grant from the United Birmingham
Hospitals Endowment Fund and by a donation from the Kinver Rotary Club,
Shropshire, United Kingdom. 
2 To whom requests for reprints should be
addressed, at CRC Institute for Cancer Studies, University of
Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TA, United
Kingdom. Phone: 44-121-414-4097; Fax: 44-121-414-3700; E-mail: n.d.james{at}bham.ac.uk
3 The abbreviations used are: PSA,
prostate-specific antigen; EGFP, enhanced green fluorescent protein;
LCL, lymphoblastoid cell line; MOI, multiplicity of infection; CMV,
cytomegalovirus.
4 Weedon et al. submitted
for publication.
Received 7/12/99. Accepted 11/11/99.
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REFERENCES |
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E. L. Davies, J. D. Ramsden, H. Cocks, P. F. Searle, J. C. Watkinson, H. Ueno, A. Logan, J. A. Franklyn, V. Mautner, and M. C. Eggo Adenovirus-Mediated Expression of Dominant Negative Fibroblast Growth Factor (FGF) Receptor 1 in Thyroid Cells Blocks FGF Effects and Reduces Goitrogenesis in Mice J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4472 - 4480. [Abstract] [Full Text] [PDF]
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 W.-S. Cheng, V. Giandomenico, I. Pastan, and M. Essand Characterization of the Androgen-Regulated Prostate-Specific T Cell Receptor {gamma}-Chain Alternate Reading Frame Protein (TARP) Promoter Endocrinology, August 1, 2003; 144(8): 3433 - 3440. [Abstract] [Full Text] [PDF]
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M. Messina, D. M. T. Yu, G. W. Both, P. L. Molloy, and B. G. Robinson Calcitonin-Specific Transcription and Splicing Targets Gene-Directed Enzyme Prodrug Therapy to Medullary Thyroid Carcinoma Cells J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1310 - 1318. [Abstract] [Full Text] [PDF]
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 S. P. Balk, Y.-J. Ko, and G. J. Bubley Biology of Prostate-Specific Antigen J. Clin. Oncol., January 15, 2003; 21(2): 383 - 391. [Abstract] [Full Text] [PDF]
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 G. M. Anlezark, T. Vaughan, E. Fashola-Stone, N. Paul Michael, H. Murdoch, M. A. Sims, S. Stubbs, S. Wigley, and N. P. Minton Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954 Microbiology, January 1, 2002; 148(1): 297 - 306. [Abstract] [Full Text] [PDF]
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X. Xie, X. Zhao, Y. Liu, J. Zhang, R. J. Matusik, K. M. Slawin, and D. M. Spencer Adenovirus-mediated Tissue-targeted Expression of a Caspase-9-based Artificial Death Switch for the Treatment of Prostate Cancer Cancer Res., September 1, 2001; 61(18): 6795 - 6804. [Abstract] [Full Text] [PDF]
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L. J. DeGroot and R. Zhang CLINICAL REVIEW 131: Gene Therapy for Thyroid Cancer: Where Do We Stand? J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 2923 - 2928. [Full Text] [PDF]
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G. M. Yousef and E. P. Diamandis The New Human Tissue Kallikrein Gene Family: Structure, Function, and Association to Disease Endocr. Rev., April 1, 2001; 22(2): 184 - 204. [Abstract] [Full Text]
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S. Suzuki, T. Tadakuma, T. Asano, and M. Hayakawa Coexpression of the Partial Androgen Receptor Enhances the Efficacy of Prostate-specific Antigen Promoter-driven Suicide Gene Therapy for Prostate Cancer Cells at Low Testosterone Concentrations Cancer Res., February 1, 2001; 61(4): 1276 - 1279. [Abstract] [Full Text]
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X. Li, M. Marani, J. Yu, B. Nan, J. A. Roth, S. Kagawa, B. Fang, L. Denner, and M. Marcelli Adenovirus-mediated Bax Overexpression for the Induction of Therapeutic Apoptosis in Prostate Cancer Cancer Res., January 1, 2001; 61(1): 186 - 191. [Abstract] [Full Text]
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 X. Zheng, M. Johansson, and A. Karlsson Retroviral Transduction of Cancer Cell Lines with the Gene Encoding Drosophila melanogaster Multisubstrate Deoxyribonucleoside Kinase J. Biol. Chem., December 8, 2000; 275(50): 39125 - 39129. [Abstract] [Full Text] [PDF]
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