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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 3 1310-1318
Copyright © 2003 by The Endocrine Society

Calcitonin-Specific Transcription and Splicing Targets Gene-Directed Enzyme Prodrug Therapy to Medullary Thyroid Carcinoma Cells

Marinella Messina, Denise M. T. Yu, Gerald W. Both, Peter L. Molloy and Bruce G. Robinson

Department of Cancer Genetics (M.M., D.M.T.Y., B.G.R.), Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, New South Wales, Australia 2065; Department of Medicine (B.G.R.), University of Sydney, Sydney, Australia; and Commonwealth Scientific and Industrial Research Organization Molecular Science (G.W.B., P.L.M.), Sydney, New South Wales, Australia 1670

Address all correspondence and requests for reprints to: Prof. Bruce G. Robinson, Cancer Gentics Unit, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales, Australia 2065. E-mail: bgr{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recurrent and metastatic medullary thyroid carcinoma (MTC) remains difficult to treat due to its limited responsiveness to chemotherapy, radiotherapy, and imaging. To investigate an alternative therapeutic approach, we examined the feasibility of targeting gene-directed enzyme/prodrug therapy delivered by adenoviral vectors to MTC. We previously described a modified human calcitonin (CT)/CT gene-related peptide promoter that produced increased expression while maintaining specificity for MTC cells. In this study, we introduced an additional level of specificity by using cell-specific splicing and examined whether the selectivity of the gene-directed enzyme/prodrug therapy for MTC was enhanced when both the promoter and splicing features were combined in a single transcription unit. Two replication-defective adenoviruses were constructed that expressed the Escherichia coli purine nucleoside phosphorylase (PNP) gene under the transcriptional control of a modified T2 promoter (Ad.T2-PNP) or the T2 promoter in combination with a CT minigene cassette in which the PNP gene was imbedded within the CT gene exon 4 (Ad.T2-CT/PNP). The specificity of PNP expression by Ad.T2-PNP, Ad.T2-CT/PNP, and control viruses in the MTC cell line, TT, and in a panel of non-MTC cell lines was evaluated. The highest level of PNP gene expression and the most effective cell killing in the presence of prodrug occurred in TT cells infected with Ad.T2-PNP, followed by Ad.T2-CT/PNP. Infection of most non-MTC cell lines, even with high multiplicities of Ad.T2-PNP, produced only low-level PNP expression that resulted in minimal cell killing in the presence of prodrug. High-level expression of PNP and effective cell killing was observed with both adenoviral gene constructs. The highest level of cell specificity was achieved with the combined use of promoter and splicing regulation in the Ad.T2-CT/PNP virus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEDULLARY THYROID CARCINOMA (MTC), which comprises 5–10% of all carcinomas in the thyroid gland, may be either sporadic or familial. The familial form is part of the multiple endocrine neoplasia type 2 syndrome. MTC arises from the parafollicular C cells of the thyroid gland that secrete the polypeptide hormone, calcitonin (CT), a highly sensitive and specific serum marker of persistent or recurrent disease. The primary form of treatment for MTC is total thyroidectomy, however recurrent and metastatic disease are difficult to manage. Metastases to distant sites including lung, liver, and bone usually occur early. Consequently, any form of effective therapy with curative intent must be systemic. Because MTC has poor responses to both chemotherapy and radiotherapy, gene therapy may offer an alternative form of treatment for this tumor type (1).

The expression in tumor cells of genes that encode prodrug-activating enzymes which are able to generate a highly toxic metabolite is an approach that has been considered for MTC (2, 3, 4, 5). The effectiveness of these approaches can be enhanced by a bystander effect in which surrounding nontransduced tumor cells are also killed. Thus, the tumor may be eliminated even if only a small percentage of the tumor cell population expresses the therapeutic gene. Purine nucleoside phosphorylase (PNP) catalyzes the conversion of adenosine analogs such as 6-methylpurine-2-deoxyriboside (6-MPDR) and fludarabine to 6-methylpurine (6-MeP) and 2-fluoroadenine, respectively. These toxic compounds are membrane permeable and readily diffuse, resulting in a potent bystander effect. Furthermore, because they inhibit DNA, RNA, and protein synthesis, both quiescent and proliferating tumor cells are killed (6). A significant advantage of gene therapy approaches for cancer treatment is the potential to restrict the expression of therapeutic genes to the target cell population, thus avoiding possible adverse systemic effects. Tumor-specific expression of the prodrug-activating gene can be achieved through tumor-selective gene transcription or targeting of the delivery vector to surface receptors on tumor cells, and these strategies may be complementary. Vector-targeting technology is progressing, but specific transcriptional targeting remains an important component of approaches to in vivo cancer therapy. Transcriptional targeting approaches have used tissue-specific promoters to restrict the expression of a transgene to tissues such as prostate (7, 8), melanoma (9), hepatocellular carcinoma (10), and lung carcinoma (11), or to restrict viral replication genes (12, 13, 14, 15, 16) to target cancer tissues.

Our initial studies for targeting potential therapeutic genes to MTC used the tissue-specific promoter of the CT/CT gene-related peptide (CGRP) gene to direct the expression of a transgene in MTC cells (1). However, transcription from the native promoter was relatively weak compared with constitutive viral promoters, and only partial specificity was retained (1, 17). We therefore developed a modified CT/CGRP promoter in which a duplicated upstream enhancer was linked to a minimal CT/CGRP promoter, with resultant enhanced activity and specificity for expression in MTC cells. However, in neuronal cells, the CT/CGRP promoter also drives expression of an alternate CGRP transcript, and our modified CT promoter thus has the potential to be active within these cells. In vivo, CT expression is also restricted to C cells and MTC by RNA splicing specificities (18, 19). Alternate transcriptional splicing of the CT/CGRP gene is shown in Fig. 1Go. CT mRNA, which is composed of exons 1 to 4, is the main gene product in thyroid C cells and MTC. In neuronal cells, the same primary transcript is processed by splicing of exon 3 to exons 5 and 6, leading to the production of CGRP mRNA (Fig. 1Go; Refs. 20 and 21).



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  		Figure 1. Schematic diagram of the CT/CGRP gene and its alternative RNA processing in thyroid and neuronal cells.

 
To target therapeutic gene expression to MTC, we have developed a strategy that uses both the promoter specificity and additional specificity based upon C cell-specific splicing of the CT/CGRP gene to prevent the expression of transgenes in nontarget, particularly neuronal cells. Recombinant replication-deficient adenovirus type 5 vectors were developed to contain a transgene cassette incorporating CT-specific exons and introns, including the PNP gene within exon 4, to allow for selective expression of this gene in human MTC cells. Adenoviral vectors have been used previously for effective gene transfer to MTC tumors (3, 4, 22, 23). Here, we have demonstrated that an adenovirus vector harboring the PNP gene linked to the T2 promoter targeted PNP gene expression predominantly to MTC cells, producing high enzyme levels that allowed efficient and preferential cell killing in the presence of prodrug. Expression in non-MTC (including neuronal) cells was very much reduced. Selectivity of transgene expression was further enhanced by placing the PNP gene within exon 4, such that it was included by the splicing mechanism in MTC cell CT/CGRP transcripts and excluded in non-MTC cells. Under these conditions, high-level PNP expression was detected in TT cells with only minor activity in one other cell type.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction

The CT minigene cassettes (Fig. 2Go) were assembled in multiple steps by PCR amplification of individual exons and introns followed by ligation into one transcriptional unit using a modified pGEM4Z (Promega Corp., Madison, WI) vector backbone. The pGEMCT-9 plasmid, kindly provided by Dr. R. F. Gagel (University of Texas, Austin, TX), containing the human genomic CT/CGRP gene was used as the major template. Plasmid pSP64dI3I4, kindly provided by Dr. P. D. Baas (Utrecht University, Utrecht, The Netherlands), containing CT exon 3 with a deletion was also used as a template. The Escherichia coli PNP gene was amplified from a PNP plasmid (24). All PCR amplifications used sense (forward) and antisense (reverse) primers incorporating restriction enzyme sequences (Table 1Go) that allowed 5' to 3' directional cloning and were performed with the Pfu DNA polymerase (Promega Corp.). The identity of all PCR fragments and resulting clone inserts was confirmed by direct sequencing.



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  		Figure 2. Diagrammatic representation of transgenes harbored in replication-deficient recombinant adenoviruses. All transgenes were inserted into the E1 region of the adenovirus type 5 genome by homologous recombination. A and B, The PNP gene expression under control of the constitutive RSV.LTR (R) promoter or the modified CT/CGRP promoter TSE2.CP1 (T2), respectively. The T2 promoter contains tandem repeat of the tissue-specific enhancer (TSE) upstream of the basal promoter and exon 1 of the CT/CGRP gene. Both transgenes terminate with pA signal from bovine GH. C and D, Transgenes are similarly driven by the constitutive R or T2 promoter, respectively, and contain the PNP gene inserted at nucleotide 12 within exon 4 of a CT minigene. The CT minigene comprises exons 3, 4, and 5, intron 3 containing a 796-bp deletion (triangle), and intron 4 (thin lines), together with all recognized CT-specific splicing enhancers (not shown). The minigenes terminate with the bovine GH (pA) signal, and a second pA used for CT-specific splicing is also present in exon 4. The sole initiation codon in the PNP gene is indicated by the arrow.

 

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  		Table 1. Primers used for cloning and RT-PCR

 
The 662-bp DNA containing CT exon 3 and the 5' portion of intron 3 (nucleotides 4108 to 4271) and a polyadenylation (pA) sequence from bovine GH (pA) were cloned stepwise by PCR amplification, digestion with restriction enzymes incorporated within the primers, and ligation into HindIII/SpeI restriction sites of pBluescriptKS (Stratagene, La Jolla, CA) vector to generate an intermediate plasmid pCT3.KS. The ATG codons at nucleotides +4195–97 and at +4203–05 within exon 3 were mutated to ATC and ATT, respectively, by oligonucleotide site-directed PCR amplification, using primers that each carried a nucleotide change (Table 1Go).

The remaining (1429-bp) region of intron 3, containing a 796-bp deletion, exon 4, and a portion of exon 5 were cloned in between the exon 3, 5' intron 3, and the pA region in multiple steps. This cassette was then subcloned into a modified pGEM4Z (Promega Corp.) vector, pGEM6, to generate the plasmid pCT/mg. Details of vector modifications and cloning strategy are available on request.

To generate the CT/PNP minigene, the PNP gene was PCR amplified as a 753-bp BglII/BamHI fragment, digested, and inserted into the BglII restriction site of exon 4, upstream of splice enhancer elements (25). A Kozak consensus sequence (26) was incorporated within the forward BglII primer, upstream of the native PNP initiation codon.

Constructs that do not contain the CT minigene (Fig. 2Go, A and B) were generated by PCR amplification of the PNP gene, digested with primer restriction sites, and cloned into HindIII/BamHI site of pGEM6 vector. The pA was subcloned downstream of the PNP gene as a 240-bp BamHI/SpeI fragment, obtained from intermediate plasmid pCT3.KS, generating plasmid pPNPpA.

Recombinant adenoviruses

To generate a recombinant replication-deficient Ad5 virus, the PNPpA gene was initially subcloned into an adenovirus shuttle vector, pXCX3 (27), in two steps. The tissue-specific T2 promoter or the constitutive RSV.LTR promoter were subcloned from plasmids TSE2.CP1.GL3 (1) or pRSV.CAT (28), respectively, as a KpnI/HindIII fragment followed by subcloning of the PNPpA cassette downstream as a HindIII/SpeI fragment. These shuttle plasmids together with adenoviral packaging vector, pJM17, were cotransfected into human HEK293 cells (Microbix Biosystems, Toronto, Canada) using the calcium phosphate coprecipitation method, and the Ad.T2-PNP and Ad.R-PNP viruses were rescued (29). To rescue Ad.R-CT/PNP and Ad.T2-CT/PNP viruses, a shuttle plasmid was generated by first subcloning promoters T2 or RSV.LTR upstream of the CT/PNP gene cassette at KpnI/HindIII sites, followed by subcloning the adenovirus 5' (left) E1 region (nucleotide 1–468) upstream of both promoters at EcoRI/KpnI sites. The 3' (right) E1 (nucleotide 503-2950) region was cloned downstream of CT/PNP gene at SpeI/ClaI sites. These shuttle vectors were cotransfected with plasmid pJM17 into human HEK293 cells as described above.

Cells were harvested when an extensive cytopathic effect was evident, and the presence and integrity of the appropriate transgenes was confirmed by Southern analysis using gene-specific probes. Viruses were plaque-purified by two subsequent passages in HEK293 cells and confirmed as correct. Stocks were expanded in HEK293 cells and purified by cesium chloride gradient ultracentrifugation and desalting through NAP25 columns. The viral particle number was determined spectrophotometrically at the absorbance of 260nm (Mittereder). Infectious titers were determined by a limiting dilution TCID50 assay.

Cell culture

All cells were maintained at 37 C in a 5% CO2 incubator and grown in DMEM or RPMI 1640 (Life Technologies, Inc., Invitrogen, Grand Island, NY), supplemented with 500 U penicillin, 500 ng/µl streptomycin, and 10% fetal bovine serum. The human cell lines TT (MTC), T98G (glioblastoma), and HepG2 (hepatoma) were obtained from the American Type Culture Collection. The HeLa (cervical carcinoma) cell line was kindly provided by Dr. Pat Delhanty (Kolling Institute, Sydney, Australia).

PNP enzyme assay

Cells were plated at 1 x 104 cells per well in a 96-well plate. The following day, cells were infected with recombinant adenovirus at multiplicity of infection (MOI) of 10 or 100 TCID50 per cell and incubated at 37 C for 4 d. Cells were then washed with 50 mM phosphate, subjected to three freeze-thaw cycles in 50 mM phosphate, and centrifuged to obtain a clarified cell lysate. Lysate (25 µl) was mixed with 25 µl prodrug 6-MPDR (5 mM; Ref.30) prepared in 0.01 M Tris (pH 7.2) and incubated at 37 C for 1 and 2 h. Reactions were terminated by boiling for 5 min and diluted to 100 µl with 50 mM phosphate buffer, then stored at -70 C before HPLC analysis. Samples (50 µl) were injected onto a Waters HPLC system using a reverse phase Millipore C18 column (Millipore Corp., Bedford, MA) with a mobile phase of 50 mM ammonium dihydrogen phosphate and 5–10% acetonitrile (flow rate, 1.5 ml/min). 6-MeP and 6-MPDR were detected at 254 nm, eluting at 3.33 and 5.03 min, respectively. All experiments were performed in triplicate, and each experiment was repeated at least twice.

Sensitivity of cells to fludarabine

Cells were plated in 96-well plates at a predetermined density (as indicated below) that did not result in deterioration of the monolayer over a 7-d period. TT cells were plated at density of 1 x 105 cells/well; HeLa, TPC-1, and CHO were plated at 3 x 103 cells/well, T98G at 1 x 103, and HepG2 at 1 x 104 cells/well. Twenty-four hours later, cells were washed in serum-free medium and infected with recombinant adenovirus in serum-free medium at MOIs of 5–120. Cells were incubated for 4 h at 37 C in complete media containing 5 µM fludarabine, a concentration that was not cytotoxic to any of the cell lines under the culture conditions. Incubation at 37 C was continued for 5 d. The viability of cells was measured using the MTS cell proliferation assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay, Promega Corp.). Medium was removed, and 20 µl MTS/PMS solution in serum-free medium was added to wells and incubated at 37 C for 1 h. The color reaction was quantified immediately on a microplate reader at 490 nm. Absorbances due to medium alone were subtracted. The viability of infected cells treated with prodrug was expressed as a percentage of control uninfected cells incubated with fludarabine. All experiments were performed in triplicate and repeated. The two-sample t test was used for all statistical calculations (Microsoft Excel, Microsoft Corp., Redmond, WA).

RT-PCR

All cell lines were plated in six-well plates at a density of 5 x 105 cells/ml, except for TT cells that were seeded at 1 x 106 cells/ml. The following day, cells were infected with recombinant adenovirus at an MOI of 50. Total RNA was isolated from infected and uninfected cells at 48 h post infection using Tri Reagent RNA/DNA/Protein Isolation Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. All extracts were frozen and maintained at -70 C until use. To eliminate any possible genomic DNA contamination, the RNA was treated with 50 U of DNase I (Promega Corp.) according to the manufacturer’s procedure. cDNA was synthesized from 1.5 µg RNA using oligo dT (Promega Corp.) and Superscript II RNaseH-reverse transcriptase (Invitrogen) in a 20-µl total volume, according to instructions from Invitrogen.

cDNA (2 µl) was used as a template in a PCR that was performed in 1x AmpliTaq buffer (Perkin-Elmer Corp., Norwalk, CT) with 0.5 mM MgCl2, 50 µM deoxynucleoside triphosphate mix, 0.4 µM of each exon-specific and PNP gene-specific forward and reverse primer, and 1 U AmpliTaq polymerase in a 25-µl total volume. The reaction was denatured at 95 C for 2 min, followed by 25 cycles of denaturation at 95 C for 30 sec, annealing at 58 C for 30 sec, extension at 72 C for 30 sec, with a final extension at 72 C for 5 min. One tenth of the PCR products was separated on 1–1.5% agarose gels and stained with ethidium bromide.

RNA integrity and loading equivalence were confirmed in all samples by RT-PCR of the housekeeping gene, ß-glucuronidase, using GUSB3 and GUSB5 gene-specific primers (31). All primers for RT-PCR are listed in Table 1Go.

Sequencing

All positive PCR samples were confirmed further by direct sequencing using the ABI Prism BigDye Terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA). Purified cycle sequencing products were electrophoresed on 4.8% PAGE Plus gels (Amresco, Cleveland, OH) and analyzed on a 377L automated DNA sequencer (PE Applied Biosystems; data not shown).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of MTC-specific replication-deficient adenovirus

We previously described the construction of the TSE2.CP1 (T2) promoter derived from the CT/CGRP promoter (1) that exhibited high level expression and enhanced tissue specificity for MTC cells compared with a constitutive viral promoter, RSV.LTR (R).

Two replication-deficient adenoviruses were generated that contained the PNP transgene linked to either the RSV promoter (Ad.R-PNP) or the T2 promoter (Ad.T2-PNP; Fig. 2Go, A and B). To further enhance selective expression, the PNP coding sequence was inserted within exon 4 of a CT minigene derived from the CT/CGRP gene such that CT-specific splicing of the transcript would include the PNP sequence. This CT/PNP chimera was similarly linked to either the RSV promoter or the T2 promoter and introduced into an E1-deleted adenovirus type 5 to generate Ad.R-CT/PNP and Ad.T2-CT/PNP, respectively (Fig. 2Go, C and D). The CT/PNP minigene included CT-specific exons and introns and all identified enhancer regulatory sequences required for CT-specific splicing. To avoid secretion of expressed gene, exon 2 was excluded because this encodes a signal peptide. Intron 3 contains a deletion that has been shown not to affect CT-specific splicing (25, 32, 33). The E. coli PNP gene with its own initiation codon was placed within the 5' region of exon 4, upstream from two splicing enhancer elements that are required for the inclusion of exon 4 in a location-independent manner (25).

Splicing pattern of infected cell lines with Ad.R-CT/PNP and Ad.T2-CT/PNP

To determine whether cell lines infected with Ad.R-CT/PNP and Ad.T2-CT/PNP were capable of splicing the chimeric minigene transgene to produce mRNA encoding PNP, RT-PCR analysis was performed on RNA extracted from TT and a range of non-MTC cells infected with Ad.R-CT/PNP or Ad.T2-CT/PNP.

To detect mRNA products derived from CT-specific splicing, PCR amplification used a primer that spanned the exon 3/4 junction (34.F) with a downstream primer in the PNP gene within exon 4 (PNP.R2). Similarly, primers complementary to exon 3 (3.F2) and exon 5 (5.R) were used to identify CGRP-specific mRNA, which excludes exon 4. The latter primer pair would also detect nonspliced mRNA.

Control RT-PCR analyses with CT-specific primers 34.F and PNP.R2 produced no product in the absence of RT enzyme (data not shown) or from mock infected TT, HeLa, T98G, or HepG2 cells (Fig. 3AGo, lanes 1c–4c). RNA from HeLa cells, which model CT-specific splicing; T98G cells, which model CGRP-specific splicing; an MTC cell line, TT; and the hepatocellular cell line, HepG2, infected with the Ad.R-CT/PNP, all produced a 400-bp product that varied in intensity (Fig. 3AGo, lanes 1a, 2a, 3a, and 4a). Sequence analysis confirmed that this product contained exon 3, exon 4, and the PNP gene. In contrast, no transcripts were detected in HeLa or T98G cells infected with Ad.T2-CT/PNP (Fig. 3AGo, lanes 2b and 3b). Infection with this tissue-specific virus produced a large amount of PCR product from infected TT cells and less product from HepG2 infected cells (Fig. 3AGo, lanes 1b and 4b, respectively).



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  		Figure 3. Splicing genotypes of Ad.R-CT/PNP and Ad.T2-CT/PNP. Total RNA was extracted from cells infected with Ad.R-CT/PNP or Ad.T2-CT/PNP (MOI 50) or mock infected for analysis. RT-PCR products from infected TT cells are shown in lanes 1a and 1b, and from non-MTC cells, HeLa, in lanes 2a and 2b; T98G, lanes 3a and 3b; and HepG2, lanes 4a and 4b. Mock transduced TT, HeLa, T98G, and HepG2 cells are shown in lanes 1c, 2c, 3c and 4c, respectively. Primer pairs are indicated at the left of each panel, and molecular weight marker lanes M1 and M2 contain the 1-kb DNA ladder (MBI Fermantas) and puc19/Hpa II (Bresatec) fragments, respectively. Panel A shows resulting RT-PCR products using primers located at the junction of the exon 3 and exon 4 (CT specific) of the CT/CGRP gene and complementary to the PNP gene; panel B shows resulting RT-PCR products using primers located in exon 3 (3.F2) and exon 5 (5.R; CGRP specific). C, RNA integrity and recovery was confirmed with primers to the housekeeping gene ß-glucoronidase. D, Schematic diagram of the CT/PNP minigene construct. Potential splicing pathways and the relative position of the primers (arrows) used in RT-PCR are indicated. Asterisk indicates the location of a cryptic splice site within intron 4 used for a third alternate splicing.

 
Using primers for exons 3 (3.F2) and 5 (5.R), a CGRP mRNA product of 155 bp that excluded exon 4 was detected in mock-infected TT cells (Fig. 3BGo, lane 1c) that produce endogenous CGRP. The same product was detected in Ad.R-CT/PNP or Ad.T2-CT/PNP-infected TT cells (Fig. 3BGo, lanes 1a and 1b). A small amount of 155-bp product was also detected in neuronal cell T98G infected with the same two viruses (Fig. 3BGo, lanes 3a and 3b). No CGRP mRNA was detected in any other non-MTC cell lines infected with Ad.R-CT/PNP or Ad.T2-CT/PNP (Fig. 3BGo, lanes 2a, 2b, 4a, and 4b). However, two larger products were produced in the non-MTC cell lines (HeLa, HepG2, and T98G) infected with Ad.R-CT/PNP. These represent unspliced heteronuclear precursor mRNA of approximately 1800 bp and an alternative spliced mRNA PCR product of approximately 1500 bp (Fig. 3BGo, lanes 2a, 3a, and 4a). Sequence analysis of the latter product showed that it contained exon 3, the PNP sequence within exon 4, the intron 4 enhancer region (to nucleotide 5359), and exon 5. This spliced product was not seen in TT cells (Fig. 3BGo, lane 1a). In addition, a small amount of alternatively spliced product was also observed in HepG2 cells infected with the Ad.T2-CT/PNP (Fig. 3BGo, lane 4b). The inclusion of PNP within this alternated mRNA was verified by analysis of cDNA from all non-MTC cell lines infected with Ad.R-CT/PNP using additional primers within PNP and exon 5 (data not shown).

Selective PNP expression by Ad.T2-PNP and Ad.T2-CT/PNP in the MTC cell line, TT

PNP expression levels were evaluated in the human MTC cell line, TT, and in non-MTC cells, HeLa, T98G, TPC-1, HepG2, and CHO. Cells were infected with one of the four recombinant adenoviruses at a MOI of 10 or 100. Lysates were assayed for PNP activity using 6-MPDR as a substrate.

Expression of PNP was observed in all cell lines infected with Ad.R-PNP (control) at MOI 10 (Fig. 4AGo) following infection with Ad.T2-PNP. At MOI 10, the highest PNP expression was seen in TT cells with low-level expression in HeLa and the liver carcinoma cell line, HepG2. At the MOI 100, low-level expression was also observed in T98G, TPC-1, and CHO cells. However, in all cell lines PNP expression was significantly reduced (P < 0.005), compared with the TT cell line. (The actual level has not been determined because the T2 promoter activity in TT cells was beyond the linear range of the assay.)



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  		Figure 4. Comparison of PNP gene expression in the MTC cell line, TT, and non-MTC cells following infection with recombinant adenoviruses. Cell lines were infected at MOI 10 (top) or MOI 100 (middle) with Ad.R-PNP, Ad.T2-PNP, Ad.R-CT/PNP, Ad.T2-CT/PNP, or Ad (empty vector). After 4 d, cell lysates were assayed for PNP activity by the conversion of the substrate 6-MPDR to 6-MeP, plotted as a percentage. The maximum conversion under the assay conditions is approximately 70–80%. Bottom, TT cells were infected with the viruses indicated, and the assay was repeated using half the amount of lysate to provide a better comparison between viruses. Experiments were performed in triplicate, and data are presented as mean ± SD (P < 0.005).

 
TT cells infected with Ad.T2-PNP expressed high levels of PNP that were above the assay limit. To better determine the relative levels of expression for individual viruses, half volumes of the infected TT cell lysates were assayed for 1 h (Fig. 3CGo). Relative to Ad.R-PNP infection at MOI 10, PNP activity from Ad.T2-PNP infected cells remained significantly greater. Insertion of the PNP gene within a CT minigene (Ad.T2-CT/PNP) reduced the enzyme activity in TT cells compared with Ad.T2-PNP, but the level of expression remained substantially higher than that seen when the constitutive RSV promoter was used in conjunction with splicing (Ad.R-CT/PNP).

In various cell lines, significant PNP expression was still detected with MOI 100 of Ad.R-CT/PNP (constitutive promoter). However, at MOI 10, PNP expression was not detected in T98G and TPC-1 cells. In HeLa and CHO cells, which have been used to model CT-specific splicing, PNP expression was increased 4- to 6- and 2.5- to 3.5-fold, respectively, compared with infected T98G cells. In contrast, infection of these cell lines with the highly specific Ad.T2-CT/PNP virus in which the tissue-specific promoter and splicing are combined produced no PNP expression at MOI 10 or 100. Although HepG2 cells showed some PNP activity at MOI 10 (in the more reliable range of the assay), this level was at least 10-fold lower than that induced by the Ad.T2-CT/PNP in TT cells.

Cytotoxicity of the PNP/fludarabine system in MTC and non-MTC cell lines

To determine the relative abilities of recombinant adenoviruses carrying specific or constitutively expressed gene cassettes to render MTC cells sensitive to gene-directed enzyme prodrug therapy (GDEPT)-directed cell killing, MTC and non-MTC cells were infected with each of the four viruses at MOIs of 5–120 per cell. The following day, infected cells were exposed to 5 µM fludarabine for 5 d, and cell viability was then determined and expressed as a percentage of the viability of fludarabine-treated uninfected cells.

Infection with the control virus Ad.R-PNP rendered all cell lines sensitive to fludarabine with approximately 50–75% cell killing at the highest viral dose (Fig. 5AGo). With the MTC-specific virus Ad.T2-PNP, infected TT cells showed the highest level of sensitivity to fludarabine, with only 50% of cells remaining viable after 5 d at the lowest MOI of 5. There was no reduction in viability at this MOI for non-MTC cells (Fig. 5BGo). At MOI 100, when all TT cells were killed, the viability of HeLa, T98G, or TPC-1 cell lines was reduced by only 20–30%. HepG2 cells showed an intermediate level of sensitivity to Ad.T2-PNP, remaining 50% viable at MOI 100.



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  		Figure 5. Sensitivity of recombinant adenoviral-infected cells to fludarabine. In vitro cell killing by E. coli PNP following fludarabine treatment of the MTC cell line, TT, and non-MTC cell lines, HeLa, T98G, HepG2, and TPC-1. Cells were transduced with 5–120 MOI of either Ad.R-PNP (A), Ad.T2-PNP (B), Ad.R-CT/PNP (C), or Ad.T2-CT/PNP (D) and treated with 5 µM fludarabine for 5 d. Cell viability was measured on d 5 using the MTS cell proliferation assay and normalized to uninfected fludarabine-treated cells. Experiments were performed in quadruplicate and repeated three times. Data are presented as mean ± SD.

 
Infection with constitutively expressing Ad.R-CT/PNP sensitized all cell lines to fludarabine to varying degrees. T98G and TPC-1 cells were largely resistant to killing, with only approximately 10% cell killing observed at the highest viral dose. Killing of HepG2 cells and the HeLa cell line, which has been used to model CT splicing, was similar to TT cells with only 50–45% viability at the highest viral dose. The highly specific Ad.T2-CT/PNP virus had little or no effect on the viability of HeLa, T98G, or TPC-1 cells in the presence of prodrug (Fig. 5DGo). However, 50% of HepG2 cells remained viable at the highest dose, which was approximately 10% improvement over the use of Ad.T2-PNP (Fig. 5BGo). TT cells infected with Ad.T2-CT/PNP were the most sensitive to fludarabine over the range of viral inputs and were completely killed in the presence of prodrug at MOI 100 (Fig. 5Go, B and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have designed a GDEPT strategy using transcriptional regulatory sequences from the CT/CGRP gene to target the expression of the bacterial enzyme PNP to MTC cells. Enzymatic activation of the prodrug fludarabine then resulted in selective toxicity in the target cells. Previously, we demonstrated that modification of the native CT/CGRP promoter led to improved promoter activity and specificity of gene expression in the MTC cell line, TT (1). In this report, we added a second level of specificity to the PNP transgene by using the alternative splicing mechanism of the CT/CGRP gene to favor the CT processing pathway, thus further restricting expression of the transgene to C cells. To achieve this, we constructed a CT minigene cassette containing only CT-specific exons and introns. These included all known constitutive and tissue-specific regulatory regions for CT-specific alternative splicing, including enhancer sequences located in exon 4 and intron sequences located in introns 3 and 4 (25, 33, 34, 35, 36, 37, 38, 39, 40). The E. coli PNP gene was placed at nucleotide 12 of exon 4 such that CT-selective splicing incorporated the PNP sequences. Infection of the MTC cell line, TT, with a replication-deficient adenovirus carrying either the T2/PNP or the dual specificity T2/CT/PNP cassette resulted in selective high-level expression. Although the addition of splicing specificity to the minigene cassette reduced the level of PNP expression approximately 5-fold in TT cells, this did not significantly reduce cell killing in the in vitro assay.

Differential splicing of the CT/CGRP gene was previously exploited for targeting the HSVtk GDEPT system to MTC cells (17). However the CT minigene contained deletions in both exon 4 and the intron 4 enhancer, produced a CT/HSVtk fusion protein, and was driven by the less active native CT/CGRP promoter. High prodrug levels and high viral inputs (100 MOI) were required to achieve killing of TT cells, and at this level the selectivity was reduced. Furthermore, because the HSVtk GDEPT system is effective only in dividing cells, whereas the PNP GDEPT system affects both dividing and nondividing cells (30), the latter may be more effective in slow growing tumors such as MTC. Indeed, the HSVtk/GCV system has shown an inability to inhibit MTC tumor growth due to an inefficient bystander effect (41). In comparative studies with both systems, PNP/6-MPDR was shown to be more potent at tumor cell killing in vitro (24, 42).

The PNP gene at nucleotide 12 of exon 4 was positioned upstream of two site-independent enhancer regions located between nucleotides 67–88 and 117–146 (25). However, placement of PNP at this position within exon 4 may interfere with the formation of stable RNA secondary structures that could play a role in proper splice site selection/recognition (43) and lead to less efficient CT splicing of the CT/PNP minigene in vitro. The reduced level of PNP produced by Ad.T2-CT/PNP was reflected in the higher viability of infected TT cells in the presence of fludarabine compared with infection with the Ad.T2-PNP, even when the latter was used at low MOI. However, this difference was negated at higher infectious dose when TT cells were killed with each of the tissue-specific viruses.

Infection of HeLa and CHO cells that predominantly use the CT splicing pathway (39, 44, 45) with the constitutive virus Ad.R-CT/PNP resulted in PNP production and subsequent cell killing in the presence of fludarabine. Analysis of these infected cell lines by RT-PCR revealed that they were able to process the CT/PNP minigene to incorporate PNP sequences via the CT splicing pathway and an alternate pathway that activated a cryptic splice site within the intron 4 enhancer allowing splicing to exon 5. The alternate splicing event and similar mRNA species have been described previously in HeLa cell lines transfected with other CT minigenes and are probably due to a constitutive pathway (46, 47). Similar mRNAs were detected at even lower levels in the CGRP-processing cell line, T98G, and in papillary TPC-1 cell lines infected with the constitutive Ad.R-CT/PNP. It is possible that the alternate splicing event accounted for the small amount of PNP expression observed in these cell lines. This was sufficient to cause a small loss of viability (~10%) in the presence of fludarabine at highest viral input. The activation of the cryptic splice site within the intron 4 enhancer also occurred in HepG2 cells infected with Ad.R-CT/PNP only.

Activation of a second cryptic splice site located within exon 4 of the CT/CGRP gene leads to the production of another mRNA species containing exon 3, the 5’ portion of exon 4 and exon 5 sequences, and has been reported to occur in transfected HeLa cells, in TT cells MTC, normal thyroid (48, 49), and normal and tumoral liver (50). This second cryptic splice site was retained in our CT/PNP transgene downstream of the PNP gene but was only activated in HepG2 cells infected with (50 MOI) Ad.R-CT/PNP, thus producing a PNP-containing transcript (data not shown). Inclusion and exclusion of exon 4 in CT/CGRP splicing is not an all-or-none event but occurs in different ratios in different cell lines (18, 51, 52). The inclusion of exon 4 was the predominant phenotype observed in most cell types when the CT/CGRP gene was constitutively expressed in transgenic mice (53). In Ad.R-CT/PNP-infected T98G cells, expression of the CT/PNP minigene also excluded the PNP gene, suggesting that the CGRP splicing pathway was activated, resulting in the elimination of PNP sequences from the CT/PNP transcript.

In designing the CT/PNP minigene, we retained these cryptic sites to preserve the sequences within exon 4 and, in particular, the intron 4 enhancer that is essential for CT-specific splicing, which is the pathway most strongly activated in MTC tumors. Neither of the alternate PNP mRNA species nor a transcript containing both the PNP and CGRP exon 5 sequences was produced by the expression of the CT/PNP transgene in TT-infected cells, although TT cells produce equivalent endogenous CT and CGRP mRNAs. This confirms the efficient activation of the CT pathway from the CT/PNP minigene in MTC.

In the non-MTC cell lines (HeLa, TPC-1, and neuronal T98G) infected with Ad.T2-PNP, some PNP was produced at higher viral doses (100 MOI), although the levels were significantly less compared with the MTC cell line, TT. Nevertheless, the small amount of PNP activity was sufficient to cause 20–30% cell killing in the presence of fludarabine. In contrast, infection with the dual specificity virus, Ad.T2-CT/PNP, resulted in the continuing viability of HeLa, TPC-1, and T98G cells, whereas TT cells were very effectively killed in the presence of prodrug.

PNP gene expression was also observed when HepG2 cells were infected with the tissue-specific virus, Ad.T2-PNP. Thus, the T2 promoter was active in these cells, and correct processing of the CT minigene occurred. However, we observed a 5-fold reduction in PNP expression when HepG2 cells were infected with the Ad.T2-CT/PNP virus, indicating that the addition of splicing specificity reduced the overall level of PNP expression. The PNP gene expression in HepG2 cells correlated with fludarabine-induced cytotoxicity that was observed with Ad.T2-PNP and Ad.T2-CT/PNP viruses. TT cells infected with Ad.T2-CT/PNP exhibited significantly increased (11-fold) PNP expression compared with HepG2 cells at MOI 25. An MOI of 120 was required to achieve the same degree of cell killing (50%) in HepG2 cells. In vivo studies using the T2 promoter showed no liver toxicity or transgene expression in nontarget organs when recombinant adenovirus constructs were injected iv (54). Thus, the specificity of the Ad.T2-CT/PNP vector should provide an even greater margin of safety in the clinic.

In summary, we have developed two recombinant adenoviral vectors that can transcriptionally target the expression of functional PNP to MTC cell line, TT. Expression levels from both T2-PNP and T2-CT/PNP transgenes were sufficient to selectively eradicate TT cells in vitro in the presence of the clinically approved prodrug, fludarabine. We expect that the novel combination of T2 transcriptional regulatory sequences and CT splicing coupled with the efficient PNP GDEPT system may produce improved outcomes when tested in vivo.


   Footnotes
 
This study was supported by grants from the University of Sydney Cancer Research Fund and The Elizabeth Rosenthal Trust.

Abbreviations: CGRP, CT gene-related peptide; CT, calcitonin; CT/PNP, CT minigene with PNP gene; GDEPT, gene-directed enzyme prodrug therapy; 6-MeP, 6-methylpurine; MOI, multiplicity of infection; 6-MPDR, 6-methylpurine-2-deoxyriboside; MTC, medullary thyroid carcinoma; pA, polyadenylation; PNP, purine nucleoside phosphorylase; RSV.LTR, rous sarcoma virus long terminal repeat; T2, modified CT/CGRP promoter containing tandem dual TSE; TCID50, 50% tissue culture infectious dose; TSE, tissue-specific enhancer.

Plasmids: pCT3.KS, pBluescript vector containing CT exon 3 sequence; pCT/mg, modified pGEM4Z vector containing CT minigene; pRSV.CAT, pBluescript vector containing rous sarcoma virus promoter driving the chloramphenicol acetyl transferase gene; pXCX3, shuttle plasmid containing the left end of adenoviral genome; pJM17, plasmid containing the adenoviral genome; TSE2.CP1.GL3, Luciferase vector containing the modified CT/CGRP promoter, adenoviral constructs; Ad.R-PNP, adenovirus containing PNP gene driven by rous sarcoma virus promoter; Ad.R-CT/PNP, adenovirus containing CT/PNP minigene driven by rous sarcoma virus promoter; Ad.T2-PNP, adenovirus containing PNP gene driven by CT/CGRP modified promoter (T2); and Ad.T2-CT/PNP, adenovirus containing CT/PNP minigene CT/CGRP modified promoter (T2).

Received September 27, 2002.

Accepted November 21, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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