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Thyroid Study Unit, Department of Medicine (K.M., K.M., R.Z., L.J.D.), Kovler Viral Oncology Laboratory (R.L.), University of Chicago, Chicago, Illinois 60637; and the Department of Geriatrics (T.T., T.N.), Shinshu University School of Medicine, Matsumoto, Japan
Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, MC 3090, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.
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Introduction |
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An attractive approach for treating MTC is use of gene therapy, based on the HSVtk/GCV system (3). In this system, a virus is used to transduce tumor cells with the Herpes Simplex Virus thymidine kinase gene (HSVtk), followed by exposure of cells to the antiviral drug ganciclovir (GCV). GCV is an acyclic nucleoside analog that is not normally metabolized by mammalian cellular thymidine kinase. However, HSVTK is able to convert the nontoxic drug GCV to GCV-monophosphate. Endogenous cellular kinase is able to convert the GCV-monophosphate to GCV-triphosphate. This purine analog, GCV-triphosphate, competes with normal nucleotides and acts as a DNA chain terminator, leading to cell death.
Over the past decade, several methods have been developed for delivering genes to mammalian cells (4, 5). Recombinant adenovirus vectors have certain advantages. They can be produced in very large quantities and high titers. Secondly, adenovirus has no significant potential for integrating into genomic DNA. Thirdly, high-efficiency gene transfer and expression can be obtained in a wide range of tissues. Fourthly, we can choose the promoter that drives the expression of the transduced gene. Several groups have reported adenovirus-mediated gene therapy using HSVtk expression driven by strong nonspecific promoters (6, 7, 8, 9, 10, 11, 12), or promoters with restricted expression (13, 14, 15, 16, 17).
Calcitonin (CT) expression is thought to be largely restricted by promoter (18, 19, 20, 21, 22) and splicing (23, 24) specificity to C cells, MTC and a few ectopic hormone-producing tumors. Specificity of CT secretion is so high that CT is used as a tumor marker for MTC.
The CALC-I gene provides one of the first described and best studied examples of tissue-specific alternative RNA processing (23, 24). In thyroid C-cells or medullary thyroid carcinoma, the main gene product is calcitonin messenger RNA (mRNA), which contains exons 1 to 4. In contrast, in particular neural cells, exons 1 to 3 of the CALC-I pre-mRNA are spliced to exons 5 and 6 to form Calcitonin Gene-Related Peptide (CGRP). CGRP is also expressed in MTC cells and C-cells to a certain extent (25, 26).
To treat medullary thyroid carcinoma, we have developed a
replication-defective adenovirus vector, AdDCTtk, which contains a
human CALC-I minigene and HSVtk gene (Fig. 1
). The 1.5-kb human CALC-I promoter (CT
promoter) drives expression of message from the construct including
portions of exon 1, exon 3, intron 3, exon 4, intron 4, and exon 5
(27). HSVTK is fused to a portion of calcitonin coded in the exon 4
(28). Therefore, even though our adenovirus vector, AdDCTtk, is
able to infect and transfer the HSVtk gene to most cells
indiscriminately, the expression of the HSVtk gene should be restricted
to C-cells and medullary carcinoma cells. If tumor-specific expression
is achieved, toxicity in normal surrounding cells could be reduced
in vivo (29). We also developed the adenovirus vector
carrying the HSVtk gene under the control of the human cytomegalovirus
(CMV) promoter (AdCMVtk) to compare the killing effect (Fig. 1).
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with 10% FBS,
100 U/ml penicillin, and 100 µg/ml streptomycin. 293 cells were grown
in MEM with heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml
streptomycin. WAG/Rij rats were maintained at the Carlson Biocontainment Suite under standard conditions, according to the Guidelines of the Animal Resource Center.
Plasmid construction and recombinant adenovirus preparation
pCTGH1 and pGEM4CT9 (19, 21) were kindly provided by Dr. R.
F. Gagel (University of Texas). pSP64dI3I4 (30) was kindly
provided by Dr. P. D. Baas (Utrecht University). pBS-tk was kind
gift of Dr. S. L. Woo (Baylor College of Medicine). pGEMTK was
kindly provided by Dr. B. Roizman (University of Chicago). pJM17 was
kindly provided by Dr. S. Refetoff (University of Chicago).
The plasmid, p
DCTkozak, including the CALC-I promoter and the CALC-I
minigene splicing cassette, was constructed using segments of the gene
from pCTGH1, pGEM4CT9, pSP64dI3I4 and pE1SP1B (Microbix Biosystems),
by multiple conventional cloning steps (detailed information will be
provided upon request to the corresponding author). The artificial
initiation start site with Kozak sequence (31) was located in exon 3.
pDCTkozak and pJM17 were cotransfected into 293 cells to generate a
recombinant adenovirus vector, AdDCT (Fig. 1
). The virus stock was
prepared as previously described (2).
AdDCTtk (Fig. 1) is similar to AdDCT, but it includes the HSVtk gene.
The initiation start site of the HSVtk gene, ATG, in pGEMTK, was
mutated to ACT. This HSVtk gene with its polyadenylation signal
replaced the ScaI-NsiI fragment in exon 4 of
pDCTkozak to make pDCTktk. pDCTktk was used to make an
adenovirus vector, AdDCTtk.
For AdCTlacZ (Fig. 1), a fragment of pCMVsportßgal (Life Technologies, Inc.) including the lacZ gene was generated by
digestion with NotI, and was ligated to the
BssHII and NsiI site of pDCTkozak, after both
ends were blunt-ended by T4 DNA polymerase, to
make pDCTß. pDCTß and pBHG10 (Microbix Biosystems) were used
to make AdCTlacZ.
For AdCMVtk, the HindIII-BamHI fragment of pBS-tk including the HSVtk gene and polyA signal was inserted into the multiple cloning site of pCA14 (Microbix Biosystems) to make pCA14TK. pCA14TK and pJM17 were used to make AdCMVtk.
AdCMVlacZ was prepared as previously described (2).
Adenoviral transduction of lacZ gene driven by calcitonin or CMV
promoter
The cells were plated in 24-well plates at a density of 2.5
x 104 (1 x 105 for
TT cells) per well 24 h before infection. The cells were then
infected with either AdCTLacZ or AdCMVLacZ at a multiplicity of
infection (moi) of 0 to 100 plaque forming units (pfu) per cell. Two
days later (3 days for TT cells), ß-galactosidase activity was
measured by color reaction using Chlorophenol
red-ß-D-galactopyranoside (CPRG) as substrate as
previously described with minor modification (32). Briefly, the cells
were washed with PBS. Fifty microliters of cell lysis buffer
(Promega Corp.) was added and incubated for 15 min at room
temperature. Twenty microliters of cell lysate (15 µl for TT cells)
was mixed with 280 µl of CPRG solution (165 µg of CPRG (Roche Molecular Biochemicals, Indianapolis, IN) in 0.1
M sodium-phosphate buffer, pH 7.5. with 1 mM
MgCl2, 45 mM ß-mercaptoethanol) and
incubated at room temperature for 60 min (15 min for TT). After adding
700 µl of 1 M sodium bicarbonate, 200 µl (100 µl for
TT cells) of sample mixture was transfer into a 96-well plate. The
color reaction was quantitated by an automatic plate reader at 570 nm
with reference filter of 650 nm.
GCV sensitivity of cell lines infected with AdDCTtk or
AdCMVtk
Cells were plated in 96-well plates at a density of 1.25 x
103 (1.25 x 104 cells
for TT) per well 24 h before infection. Cells were infected with
either AdDCT, AdDCTtk or AdCMVtk at 0 to 100 moi. After incubation for
24 h, the cells were incubated with the complete medium containing
various concentrations of ganciclovir sodium (GCV) (CYTOVENE IV,
Roche Molecular Biochemicals). GCV conditioned medium was
changed each 36 h. 4 days later, viability of the cells was
measured by the MTT assay system (CellTiter 96 NonRadioactive Cell
Proliferation Assay, Promega Corp.) according to the
protocol from the manufacturer. Briefly, the medium was changed just
before assay. Fifteen microliters of dye solution was added to each
well and incubated for 2 h at 37 C in a tissue culture incubator.
One hundred microliters of stop solution was added. After overnight
incubation at 37 C, the color reaction was quantitated using an
automatic plate reader at 570 nm with reference filter of 650 nm.
Percentage of cell survival was calculated as the fraction of surviving
cells compared with noninfected cells incubated without GCV taken as
100%.
For the time course study, cells were prepared and infected with
AdDCTtk or AdCMVtk at 40 moi as described above. GCV conditioned medium
was changed each 48 h (72 h for TT cells). MTT assay was performed
on the days shown in Fig. 4 after cells were treated with GCV.
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Primers used for PCR amplification:
Ex4TKA: 5'-GTGTAGATGTTCGCGATTGT-3'
Ex3ATGS: 5'-ATGGTGCAGGACTATGTGCA-3'
Ex3ATGSin: 5'-ATGAAGGCCAGTGAGCTGGA-3'
Ex4TKAin: 5'-GGTACGTAGACGATATCGTC-3'
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), or the
CMV promoter (AdCMVlacZ). Expression of ß-galactosidase driven by the
CALC-I promoter or CMV promoter was evaluated in rMTC (medullary
thyroid carcinoma cell line from rat), TT (human medullary thyroid
carcinoma), T98G (human glioblastoma cells), Cos1 (monkey kidney
cells), HepG2 (human hepatocellular carcinoma cells), and HeLa (human
cervix carcinoma cells) cells, using AdCTLacZ or AdCMVLacZ (Fig. 2). Each cell line was infected with
either AdCTLacZ or AdCMVLacZ at 0 to 100 moi. The activity of
ß-galactosidase was measured by color reaction with CPRG.
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Medullary thyroid carcinoma cells infected with AdDCTtk are
sensitive to GCV
As shown Fig. 1, we developed replication-defective adenovirus
vectors. AdDCT includes the human CT minigene under the control of the
CALC-I promoter. AdDCTtk is similar to AdDCT, but the HSVtk gene is
inserted into exon 4 to form a fusion protein. AdCMVtk carries the
HSVtk gene under the control of the CMV promoter.
To assess the sensitivity of cell lines to GCV, rMTC, TT, T98G, Cos1, HepG2, and HeLa cells were infected with either AdDCTtk, AdCMVtk or AdDCT at moi of 0 to 100, and exposed to GCV at various concentrations. The viable cells were estimated by tetrazolium (MTT) assay.
rMTC cells infected with the control vector, AdDCT, showed a similar
dose-response curve to GCV as did noninfected cells. rMTC cells
infected with either AdDCTtk or AdCMVtk were rendered sensitive to GCV.
The killing effect depended on both GCV concentration and virus titer
(Fig. 3, rMTC). The 50% growth
inhibition concentrations (IC50) of GCV with
AdDCTtk at moi of 20, 40 and 100 were 36, 1.1 and 0.13 µmol/liter,
respectively. The IC50s with AdCMVtk were 0.78,
0.34, and 0.077 µmol/liter.
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, TT). The IC50 of
GCV with AdDCTtk at 40 moi was 0.6 µmol/liter.
The viability of T98G cells infected with AdDCTtk was not affected by
GCV, and these cells behaved similarly to noninfected or AdDCT-infected
cells. In contrast T98G cells were sensitive to GCV after infection
with AdCMVtk (Fig. 3, T98G). The IC50s of T98G
cells with AdCMVtk at moi of 20, 40, and 100 were 8.4, 7.4, and 3.7
µmol/liter.
AdDCTtk at 40 moi and more than 10 µmol/liter of GCV had a small
effect to reduce viability of Cos1 cells. These effects were much less
than with AdCMVtk at the same moi and concentration of GCV (Fig. 3, Cos).
HepG2 cells infected with either AdCMVtk or with AdDCTtk were also
sensitive to GCV (Fig. 3, HepG2).
Overexpression of the HSVtk gene had a toxic effect on HeLa cells
(Fig. 3, HeLa). The percent survival of HeLa cells infected with AdDCT,
AdDCTtk, or AdCMVtk at a moi of 100 without GCV was 84.6 ± 1.6,
63.4 ± 6.7 or 9.0 ± 1.9%, respectively. Although HeLa
cells infected with AdCMVtk at 20 moi were very sensitive to GCV,
AdDCTtk at 20 moi only showed a killing effect on HeLa cells with more
than 1 µmol/liter GCV (the IC50 being 0.26 and
50 µmol/liter, respectively).
Similar data were obtained by trypan blue exclusion cell count (data not shown).
The long-term killing effects of the HSVtk/GCV system were assessed
(Fig. 4). The cytotoxic effects were
observed in all cell lines infected with AdCMVtk at 40 moi and treated
with 1 µmol/liter GCV.
Seventy-five percent of rMTC cells infected with AdDCTtk (moi of 40) were killed after 5 days incubation with 1 µmol/liter GCV. AdDCTtk and GCV treatment also inhibited the growth of TT cells and HepG2 cells. Twenty to 30% of HeLa cells were killed by this treatment after 5 days. No killing was observed using T98G and Cos1 cells.
Detection of spliced mRNA by RT-PCR
To detect the spliced mRNA from the CT-HSVtk chimeric gene, we
performed semiquantitive RT-PCR(Fig. 5, upper panel).
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, lower panel).
RT-PCR products from noninfected rMTC cells did not show any bands
(Fig. 5, lane 1). PCR products using DNaseI treated RNA from
AdDCTtk-infected rMTC cells as template without RT also did not show
any bands (Fig. 5, lane 2). We could not detect any PCR products from
noninfected T98G, Cos1, HepG2, HeLa or TT cells, nor from DNaseI
treated RNA of AdDCTtk-infected cells without RT (data not shown). The
primers complimentary to exon 3 and the HSVtk gene inserted into exon 4
allowed specific detection of minigene transcripts in the background of
endogenous gene expression. Analysis of RNA products from rMTC cells
infected with AdDCTtk revealed both precursor and mRNA of exon 3
spliced to exon 4/HSVtk chimeric gene (Fig. 5, lane 3). No transcripts
or very faint bands could be detected with RNA from T98G cells infected
with AdDCTtk (Fig. 5, lane 4). Weaker spliced mRNA compared with
precursor was detected in extracts from Cos1 cells (Fig. 5, lane 5).
Both precursor and spliced mRNA were detected in extracts from HeLa,
HepG2, and TT cells (Fig. 5, lanes 6, 7, and 8).
PCR amplification of serial diluted plasmids showed linear
amplification from 250 to 250 x 16-6 pg.
The amplification reached a plateau and PCR products derived from the
first primers were amplified when more than 250 pg of plasmid was used
(Fig. 5, lower panel).
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Discussion |
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To increase selectivity, the HSVtk gene was inserted into exon 4 of our calcitonin minigene. In the context of a tissue specific alternative splicing mechanism, CT producing cells such as rMTC can express HSVTK transduced by AdDCTtk as demonstrated by RT-PCR analysis, and are killed after administration of GCV. The possibility of virus-directed enzyme/prodrug therapy targeting metastatic brain tumors by alternative splicing control has been suggested (34). However, to our knowledge, there are no previous reports using a tumor-selective alternative splicing based on an adenovirus vector to kill tumor cells at the cell line level.
The CALC-I promoter has a greater function in rMTC cells than the CMV promoter, as demonstrated by the lacZ transduction study. However cell killing by AdDCTtk was not as strong as by AdCMVtk, as demonstrated by MTT assay. This suggests that some primary transcripts might be processed not only to calcitonin inclusion mRNA, but also by the calcitonin exclusion pathway. To evaluate this possibility, we are constructing an adenovirus vector in which the CALC-I promoter directly controls the HSVtk gene without the CALC-I minigene cassette. It is also possible that the enzyme activity of the fusion protein, HSVTK with the attached portion of CT, is not as strong as the native enzyme, or that the fusion protein is not as stable as the native HSVTK.
TT cells were also rendered sensitive to GCV after infection with AdDCTtk or AdCMVtk. It took longer than 7 days incubation to kill 50% of cells as demonstrated by the time course study. It has been thought that the HSVtk/GCV system affects only dividing cells such as tumor cells or metabolically active tissues such as liver (29). It is reasonable that very slowly growing cells, such as TT cells, need longer exposure to GCV to achieve effective cytotoxicity.
Even though the CALC-I promoter drives the fusion gene to some extent in Cos1 cells, they only weakly express HSVTK. We think this might be because in addition to low function of the promoter, exon 4 is largely excluded by the splicing mechanism. It will be useful to quantitate mRNA of exon 3 spliced to exon 5. However AdDCTtk includes only the untranslated region of CGRP in exon 5 without the downstream poly A signal, making mRNA of exon 3 spliced to exon 5 unstable and difficult to quantitate.
Selection of exon 4 or exon 5 by the splicing mechanism is not a simple
on/off switch. Instead, different ratios of these exons are used by
certain cell lines or tissues (36, 37). Thus a high titer of AdDCTtk
and a high concentration of GCV could show some killing effect on Cos1
cells. Although HeLa cells have been used for a model showing the
splicing pattern of calcitonin "inclusion" (35, 36, 37, 38), Baas and
colleagues have reported that deletion of intron 4 increased splicing
of exon 3 to exon 5 in HeLa cells (30). Later, an intron enhancer which
increased use of the CT-specific terminal exon was identified (39, 40).
The backbone of our AdDCTtk vector is based on a human calcitonin
minigene provided by Baas and colleague, and this splicing enhancer
region is deleted in AdDCTtk (Fig. 1) (30). Therefore, we anticipated
that HeLa cells infected with AdDCTtk would not be killed effectively,
whereas cells infected with AdCMVtk would be killed. The killing effect
of AdDCTtk was weaker than the effect of AdCMVtk, even though large
amounts of CT/HSVtk spliced mRNA were detected by RT-PCR analysis. We
are not able to fully explain the discrepancy. Possibly deletion of
portions of exon 4 (41, 42), 5 and 6 (43), intron 3 and 4 (30, 39, 40),
or insertion of the HSVtk gene with its polyadenylation signal (44, 45), affects the natural splicing pathways in certain cell lines. We
think that the vector could be improved by using these splicing
regulating elements of the CALC-I gene. We are making an adenovirus
vector carrying the HSVtk gene with the same CALC-I minigene cassette
as AdDCTtk driven by the CMV promoter, to clarify the benefits of the
splicing system. In addition we are constructing a vector which
includes the enhancer elements, that augment CT inclusion present in
exon 4 and intron 4.
HepG2 cells were rendered as sensitive to GCV after infection with AdDCTtk as with AdCMVtk. Recently, HSVTK/GCV treatment were reported to cause severe liver damage (29). We also observed similar sensitivity to GCV after infection with AdDCTtk when using GH3 (rat pituitary adenoma) cells and Neuro2a (mouse glioma) cells (data not shown). The possibility of production of CT in the pituitary and the liver at a very low level has been reported (46, 47, 48). However, serum calcitonin serves as a tumor marker for MTC. After surgical excision of MTC tumors, calcitonin level dramatically falls. These clinical findings suggest that in patients the MTC tumor is the main tissue in which calcitonin is produced and that the CALC-I gene is strongly activated. Our system could be applied to such CT producing MTC cells. It is also reported that liver and other organs might have the ability to choose a CT splicing pattern, as suggested by transgenic mice expressing the rat calcitonin/CGRP (49). It is not certain that results from these established tumor cell lines reflect the events in normal tissues. Thus we are testing the effects of the vector and GCV in an animal model in vivo. We have observed that the human CT promoter is much less active than the CMV promoter in rat liver, by measurement of ß-galactosidase activity after injection of AdCTlacZ or AdCMVlacZ via the tail vein (data not shown).
Virus-directed HSVtk/GCV systems are now applied in clinical trials. Initially, nonspecific promoters such as the CMV promoter were chosen to get high expression of HSVTK in the tumors. More recently, restricted expression systems have been sought for targeting certain tumors in experimental animal models. The specificity of AdDCTtk for MTC was not complete, but rather partial at the cell line level. However, we think it is clinically meaningful to reduce the side effects of viral gene therapy on normal surrounding tissues.
We have reported that the transduction of interleukin-2 abrogated the tumorigenecity of MTC cells in animal model (2). Recently, the combination of the suicide and cytokine gene therapy has been tested for certain tumor models and shown stronger effects on tumor growth than the single gene therapy (50). These reports encouraged us to test the animal model.
In conclusion, the CALC-I promoter confers partially selective expression of the reporter gene by an adenovirus vector. The HSVtk gene inserted into exon 4 of the CALC-I minigene cassette can be expressed through the splicing pathway, although regulation of splicing might be abnormal and room remains for improvement of our vector. MTC cells can be killed effectively by the combination of GCV and AdDCTtk vector. We believe that AdDCTtk will be useful for treatment of medullary thyroid carcinoma. Our vector provides the possibility of a new approach to achieve relative tumor selectivity in gene therapy by using promoter specificity and cell-specific alternative splicing patterns.
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Acknowledgments |
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Footnotes |
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Received December 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein-producing
human hepatoma cells by adenovirus-mediated transfer of the herpes
simplex virus thymidine kinase gene. Hepatology 23:1359–1368[Medline]
-CGRP and
ß-CGRP neuroendocrine genes. J Biol Chem 263:5–8This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
GCCATGG). Portions
of intron 3 and intron 4 are deleted as indicated
(triangle) (see Ref. 30). AdDCTtk is similar to
AdDCT, but the HSVtk gene with poly A signal is inserted into
ScaI and NsiI sites in exon 4, following
5 amino acids of CT, to produce the CT-HSVTK fusion protein. AdCTlacZ
includes the LacZ gene downstream, with the same promoter and exon 1 of
AdDCT. AdCMVtk includes the CMV promoter and the HSVtk gene
followed by the poly A signal. Fragment length, some restriction sites,
and position of poly A signal (PA) are indicated.
