Materials and methods

Chemicals and antibodies

Polibrene and Puromycin were from Sigma (St. Louis, MO). GCV was from Roche (Mannheim, Germany) and Cisplatin (CDDP) was from Bristol-Myers-Squibb (New York, NY) and all were prepared immediately before use. Antibody against Cyclin B1 was from Dako (Glostrup, Denmark). Antibody against E1a was from Oncogene Science (Boston, MA) (M73 Clone). Cy3 conjugated rabbit IgG anti-mouse was from Jackson Immunoresearch Laboratories (West Grove, PA).

Cell lines

NIH-3T3 fibroblast cell line (kindly provided by Dr S. Gutkind, NIDCR, NIH, Bethesda, MD) was routinely grown in DMEM (GibcoBRL, Rockville, MD) containing 10% newborn calf serum (BioWhittaker Europe, Verviers, Belgium). Murine carcinoma MSC11-A5 (ATCC, Manassas, VA), GP293 (Clontech, Palo Alto, CA), human fibroblasts IMR90 (ATCC), human cervix carcinoma HeLa (ATCC), and human carcinoma MDA-MB-435 Lung-2 (kindly provided by Dr A. Fabra, Barcelona, Spain) cell lines were grown in DMEM (GibcoBRL) containing 10% fetal calf serum (BioWhittaker Europe). All the cell lines were grown at 37°C and 5% CO₂.

Generation of TK and E1A-IRES-TK constructs

DNA plasmids were constructed using standard procedures. PLPCX E1A 12s was kindly provided by Dr Silvio Gutkind; thymidine kinase (TK) gene and IRES sequences were kindly provided by Dr Marta Izquierdo (CBM, CSIC, Madrid, Spain). To generate E1a-IRES-TK, IRES-TK was extracted from pSXLC TK as an EcoR1 fragment and cloned into the EcoR1 site of PLPC E1a12s. To generate PLPC TK, the TK gene was extracted from pSXLC IRES TK as a HindIII–XhoI fragment and then cloned into PLPCX vector (Clontech).

Transfection and retroviral infection

NIH 3T3 fibroblasts, MSC11-A5, IMR90, HeLa, and MDA-MB-435Lung-2 cell lines were infected with the pLPC GFP, pLPC E1a, pLPC TK, or pLPC E1a-TK retroviruses (Clontech). Briefly, GP293 cells were transfected when they were 50–80% confluent with pLPC E1a, pLPC TK, or E1a-TK retroviruses, in 100-mm dishes using the calcium phosphate method. Supernatant was removed 2 days after transfection and used to infect the different cell lines for 6 hours in the presence of Polibrene (Sigma). Two days later, cells were selected with the appropriate dose of Puromycin. Before experiments, antibiotic-containing medium was replaced with regular medium.

RNA extraction

Total cellular RNA was extracted with TriReagent TM (Molecular Research Center, Cincinnati, OH) following the manufacturer's instructions. Samples were then treated with RQ1 RNAse-free DNAse (Promega, Madison, WI) following manufacturer's instructions.

Reverse transcription–polymerase chain reaction (RT-PCR)

Two hundred nanograms of RNA were reverse transcripted using Moloney murine leukemia virus reverse transcriptase (MMLV, Promega). Briefly, samples were preincubated 5' at 25°C in the reaction mix with oligo-dT primer and then incubated 1 hour at 37°C, followed by a 15-minute period at 70°C to denature the enzyme. PCR conditions were: 94°C for 5 minutes followed by 25 cycles of 94°C for 15 hours, 66.7°C for 20 hours, 72°C for 20 hours, and a final extension period of 10 minutes at 72°C. Primers used were: HSV1TK-F: 5'-atgacaagcgcccagataacaatggg-3' and HSV1TK-R: 5'-cagataccgcaccgtattggcaagc-3' to amplify a 387-bp cDNA sequence of the herpes simplex virus 1 thymidine kinase gene, and G6DP-F: 5'-ATCCTGAGGGAAGAGTTGTACCAGGG-3', G6DP-R: 5'-ATCCTGAGGGAAGAGTTGTACCAGGG-3' to amplify a 473-bp cDNA sequence of the Mus musculus glucose 6-phosphate dehydrogenase gene.

DIG-labeled probe synthesis

PCR probes were made employing DIG-11-dUTP (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer's instructions. The primers used were: HSV1TK-F and HSV1TK-R for TK probe and G6PD-F and G6PD-R for G6PD probe. After synthesis, the PCR products were agarose gel purified with QIAquick gel extraction kit (Qiagen, Hilden, Germany).

Southern blot

For Southern blot, PCR products were identified by 1.5% agarose gel electrophoresis and ethidium bromide staining, denatured, and transferred to a nylon membrane, Nytran 0.45 (Schleicher & Schuell, Dassel, Germany). DNA was then fixed to the membrane by UV cross-linking and hybridized with 25 ng/mL of appropriate DIG-labeled probe. Chemiluminescent detection was performed with DIG Luminescent Detection Kit (Roche) following manufacturer's instructions. Densitometry analysis was performed using Scion Image software (Scion, MD).

Real-time quantitative PCR

PCR product from G6PD amplification was cloned and serial dilutions from 10⁸ to 10² was made to create an external standard curve. Lightcycler-FastStar DNA Master SYBR Green I (Roche) was used to amplify 2 L of cDNA samples (400 ng RNA) and 2 L of each plasmid dilution from the external standard. Procedures were performed following manufacturer's indications in a Lightcycler Instrument (Roche). Primers used were G6PD-F, G6PD-R, and HSV1TK-F, HSV1TK-R. Reaction conditions: 95°C for 10 minutes followed by 40 cycles of 95°C for 10 hours, 66°C for 5 hours, and 72°C for 19 hours. Second derivate maximum method was used to analyze the data. TK expression from each sample was normalized with respect to its G6PD expression.

Viability experiments

Cells were seeded in 24-well plates (10⁴ cells/well) and GCV (20 g/mL) and/or cisplatin (0.5–1 g/mL) were added the following day. Media was changed every 2 days for fresh media and GCV or cisplatin. For viability assays, we used the crystal violet method.^22,24 Briefly, cells were washed once with PBS and then fixed in glutaraldehyde 1% for 20 min, washed twice in PBS, and stained with crystal violet (0.1%) for 20 min, and then washed with abundant deionized water. Colorant was recovered by 1% acetic acid and OD was evaluated at 590 nm.

Replating assays

Cells were seeded in 24-well plates (10⁴ cells/well) and GCV (20 g/mL) was added the following day. Seventy-two hours later, cells were replated to six-well plates, and allowed to grow without GCV for 2 or 5 days. The crystal violet method was used to evaluate cell viability.

Western blot and immunocytochemistry for cyclin B1 and E1A

Cells were treated and collected in lysis buffer (25 mM HEPES, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid, 20 mM B-glycerophosphate) in the presence of protease inhibitors (20 g/mL leupeptin, 20 g/mL aprotinin, 1 mM PMSF). Then, 20 g was subjected to 10% SDS-PAGE, blotted onto nitrocellulose membranes and probed against Cyclin B1 (dilution 1:1000) or E1a (1:300). Antibody detection was achieved by enhanced chemiluminescence (Amersham, Piscataway, NJ), according to the manufacturer's protocol.

For immunocytochemistry, cells were washed twice with PBS, fixed with 2% paraformaldehyde in PBS for 20 minutes, and then treated with 100 mM glycine in PBS for 20 minutes. Cells were then permeabilized with -20°C methanol for 6 minutes, and then blocked with 1% BSA in PBS for 30 minutes. Cells were incubated with a 1:250 dilution of E1a or Cyclin B1 antibodies, washed and incubated with Cy3-conjugated anti-mouse antibodies and then examined using a Leica DMLB fluorescence microscope.

Flow cytometry analysis

For cell cycle analysis, cells were collected and fixed with 70% ethanol in PBS. Propidium iodide was added (20 g/mL) and samples were treated with RNAse (20 U/mL). Samples were analyzed by a EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL).

Statistics

Repeated measures ANOVA analysis was performed to study the within-subject effect (viability measures at the different time points) at the different levels of the between-subject effect (transfected genes), and the interacting effect (timetransfected genes). Software SPSS 10.0 (SPSS, Chicago, IL) was used.

Results

Expression and biological effects of E1A-TK and TK constructions

First, retroviruses were produced carrying the 12SE1a or TK genes or the 12S E1a linked to TK by IRES sequences. In all cases, expression was under a CMV promoter. Then the NIH3T3 cells and MSC11-A5 carcinoma cell lines were infected. As E1a protein expression increases the percentage of cells in the S-phase of the cell cycle, flow cytometry of the cell lines was performed (Fig 1). Immunohistochemistry and Western blotting assessed the expression of E1a protein. The effects of the HSVTK-GCV system were tested by adding GCV in various doses and evaluating cell viability by the crystal violet method 3–7 days after treatment. After selection of puromycin-resistant clones, we assessed whether the concomitant expression of E1a protein increased the effects of the HSVTK-GSV system. The experiments were performed in both nonconfluent and confluent cultures. All the infections were repeated twice, and the puromycin-resistant cells were pooled together and used for the viability studies. As shown in Figure 1a, by Western blot, E1a-expressing cells showed high levels of E1a protein. When flow cytometry was used, E1a-expressing cells displayed a higher percentage of cells in the S-G2M phase of the cell cycle without apparent apoptosis, even in confluence cultures as shown in Figure 1b.

Figure 1.

E1a protein and cell cycle analysis of E1a-expressing cells. a: Western Blot analysis for E1a expression in MSC11A5 and NIH-3T3 cell lines. As the figure shows, after infection with retrovirus E1a-TK, there was a high level of E1a protein in the E1a-expressing cells. b: Cell cycle analysis of E1a-expressing cells. To study the different cell cycle phases and the presence of apoptosis, we performed flow cytometry. b: Cell cycle profile of the MSC11A5 cell line. Experiments were performed in confluent cultures. Note that MSC11A5-E1a-expressing cells show a higher percentage of cells in S-G2M phase, compared to their control MSC11A5 cells, and there is no significant apoptotic cell population. Both graphs are represented with log scales. c: TK expression in the cells. The level of TK expression in the E1a-TK- and TK-expressing cells was evaluated by RT-PCR and Southern blot. As shown in c, there were no differences in the band detected in the several cell lines studied. Similarly, we studied by RT-PCR and Southern blot the level of G6PD and no differences were observed.

Full figure and legend (65K)

To study whether the expression of mRNA TK varied in the different cell lines, we performed RT-PCR and Southern blot of the PCR products. As shown in Figure 1c, we did not detect significant differences, indicating that the mRNA level of TK was similar in the E1a-TK and in the TK-expressing cells. To test the TK mRNA levels of the various murine cell lines, we analyzed simultaneously the level of a housekeeping gene, the murine G6PD, by RT-PCR and Southern blot. As shown in Figure 1c, with these methods, no differences of G6PD mRNA were observed in the several cell lines. Finally, we performed real-time quantitative PCR. With this technique, we did not detect less mRNA TK expression in the TK-expressing cells than in the E1a-TK-expressing cells (data not shown).

E1A-HSVTK-GCV system in murine cells

NIH3T3 cell fibroblasts were sensitive to TK-GCV and their sensitivity was increased in cells expressing the E1a protein. The E1a-TK effect was apparent from the first day after treatment with GCV in nonconfluent cultures of NIH-3T3 (Fig 2a). The parental cell lines, as well as cells infected with pLPC GFP and pLPC E1a, were treated with 20 g/mL GCV and no toxicity was observed compared to the untreated controls (data not shown). In confluent cultures, the differences between TK and E1a-TK-expressing cells were evident 3 days after adding CGV. Statistically, there were significant differences in cell viability in the E1a-TK-expressing cells versus TK-expressing cells (in confluent and nonconfluent cultures, P<.01). As shown in Figure 2b, over 60% of the E1a-TK cells were killed, whereas only about 15% of the TK-cells were killed after 3 days of treatment with GCV (Fig 2, a and b).

Figure 2.

E1a-HSVTK-GCV system in murine cells. Murine NIH3T3 TK and E1a-TK fibroblasts were sensitive to GCV. Both nonconfluent and confluent NIH-3T3 E1a-TK were more sensitive to treatment with GCV in the days following treatment with GCV (P<.01) (a and b). Murine carcinoma MSMSC11A5 TK and E1a-TK were also sensitive to GCV treatment. The MSC11A5 E1a-TK-expressing cells displayed a higher sensitivity to treatment with GCV similar to NIH-3T3 (P<.01) (c and d). Cells before treatment with GCV are taken as 100%. Results are the average of at least three independent experiments performed in triplicate. Bars represent the SD.

Full figure and legend (20K)

Subsequently, malignant murine cells with multiple genetic alterations, such as the MSC11A5 cell line, were studied. Using a similar approach, both nonconfluent and confluent cell cultures were studied. The E1a-TK-expressing cells were also more sensitive to treatment with GCV than the TK-expressing cells. As shown in Figure 2, c and d, MSC11A5-E1a-TK cells did not increase in number 2 days after treatment with GCV, and most cells were not viable after 4–5 days. Notably, the MSC11A5-E1a-TK-expressing cells were more sensitive than the MSC11A5-TK cells (in confluent and nonconfluent cultures, P<.01). In confluent cultures, the percentage of cell death evaluated by crystal violet was less evident than in the NIH3T3 cells. Because TK-expressing cells treated with GCV showed a larger size and could be arrested in G2-M cell cycle phase, as previously described,^25,26 we replated the cells in other wells. We replated the cells 24 hours after treatment with GCV and observed a marked decrease in cell density of the TK-expressing cells (data not shown). These results would indicate that the measurement of cell density obtained by staining with crystal violet is not representative of cell viability in the HSVTK-GCV system, because of the large size of the GCV treated cells, and clear differences in viability can only be obtained after replating experiments. In these experiments, as well, the E1a-TK-expressing cells were the most sensitive and showed the lowest percentage of cell density.

E1A-HSVTK-GCV system in human cells

Similarly, the E1a-TK and TK constructions were studied in normal human cells (IMR90 fibroblasts) and malignant human tumor cell lines (MDA-MB-435, breast carcinoma cells, HeLa cells, cervix carcinoma cells). As previously shown with NIH3T3, IMR90 human cells, in both confluent and nonconfluent cultures, were significantly sensitive to this system and the sensitivity to TK-GCV increased when there was concomitant expression of E1a protein (Fig 3, a and b). The IMR90-GFP and IMR90-E1a-expressing cells did not show any increase of lethality after addition of GCV (data not shown). Diverse results were obtained with the transformed human cell lines. In nonconfluent cultures, the MDA-MB-435 breast carcinoma cell line showed clear sensitivity to the HSVTK-GCV system and especially to the E1a-HSVTK-GCV system (P<.01) (Fig 3c). Statistically, there were significant differences in cell viability in the E1a-TK-expressing cells versus TK-expressing cells (in confluent and nonconfluent cultures, P<.01). In confluence, according to the crystal violet density, MDA-MB435 was apparently more resistant (Fig 3d). Similar results were obtained with HeLa cells (data not shown). Both cell lines also displayed larger nuclei and cytoplasm after treatment with GCV, and the decrease in cell viability could only be confirmed by studying the viability after replating the cells.

Figure 3.

E1a-HSVTK-GCV system in human cells. Human IMR90 TK and E1a-TK fibroblasts were sensitive to the TK-GCV system. As with murine cells, the nonconfluent (a) and confluent (b) E1a-TK-expressing cells showed a higher response to treatment with GCV 24 hours after the addition of GCV (P<.01). The human carcinoma MDA MB435 lung2 TK and E1a-TK were more resistant to treatment with GCV, according to the crystal violet method. Although in nonconfluence experiments (c) the density of MDA MB435 lung2 E1a-TK cells was much lower than the control MDA MB435 lung2 cells (P<.01), in confluence (d), there were no clear differences in cell density. Cells before treatment with GCV are taken as 100%. Results are the average of at least three independent experiments performed in triplicate. Bars represent the SD.

Full figure and legend (20K)

Concomitant treatment with GCV and cisplatin in E1a-TK-expressing cells

Interestingly, the addition of cisplatin into E1a-TK cells clearly increased the sensitivity to this anticancer agent. As shown in Figure 4, there was a clear increase in lethality in the E1a-expressing cells after 3 days with CDDP and GCV. This sensitivity was even higher than that observed in the cells that had not been treated with GCV, indicating that E1a expression can even more selectively enhance the sensitivity of cells expressing the HSVTK-GCV system. This chemosensitivity of the E1a-TK-expressing cells was assessed in normal human IMR90 fibroblasts and the malignant MSC11A5 carcinoma cell line. The sensitivity of MSC11A5 E1a-TK carcinoma cells to treatment with cisplatin or concomitant treatment with cisplatin and GCV was evident, with a marked loss of cell viability when both drugs were added (P<.01) (Fig 4). In the IMR90-E1a-TK cells, concomitant treatment with cisplatin and GCV also increased the sensitivity to these drugs (data not shown).

Figure 4.

Concomitant treatment with cisplatin and GCV in E1a-TK-expressing cells. The MSC11A5 TK and E1a-TK cell lines were sensitive to treatment with GCV (20 g/mL) and to cisplatin CDDP (0.5 g/mL). Concomitant treatment with both GCV and CDDP increased the lethality of TK- and E1a-TK-expressing cells (P<.01). Note that almost all E1a-TK-expressing cells were dead after 3 days of combined treatment. Histograms represent the relative survival of the TK and E1a-TK cell lines with respect to their untreated controls.

Full figure and legend (68K)

Cell cycle studies, cytological features, and cyclin B1 and E1a protein expression

Due to the fact that the HSVTK-GCV system induces an irreversible G2-M arrest,^25,26 the cells were studied by flow cytometry after treatment with GCV. As shown in Figure 5a, most E1a-TK-expressing cells were located in the G2-M phase of the cell cycle 24–48 hours after treatment with GCV. After 48–72 hours, most E1a-TK-expressing cells have a subdiploid DNA content, consisting of an apoptotic population (Fig 5).

Figure 5.

Cell cycle analysis and cyclin B1 expression after treatment with GCV. a: Cell cycle profile of NIH-3T3 E1a-TK cells after treatment with GCV. Note the high percentage of cells in the G2-M cell cycle phase 24 hours post-treatment with GCV. After 48–72 hours post-GCV, most E1a-TK-expressing cells were located previous to the G0-G1 peak, being considered as an apoptotic cell population. b: By immunoblotting, before treatment with GCV, the level of cyclin B1 was higher in the E1a-TK-expressing cells (3T3 E/T) than in the TK-expressing cells (3T3 TK). Twenty-four hours post-treatment with GCV, there was a high increase in the level of cyclin B1 in the NIH-3T3 E1a-TK (3T3 E/T) and less in the NIH-3T3-TK cells (3T3 TK).

Full figure and legend (45K)

As cyclin B1 is expressed mainly in the G2 cell cycle phase, we analyzed the levels of cyclin B1 protein by immunocytochemistry and Western blot. As shown in Figure 5b, the levels of cyclin B1 were clearly increased in E1a-expressing cells and in cells with the HSVTK-GCV system after treatment with GCV. In E1a-expressing cells, there is a higher level of cyclin B1 expression due to the cell cycle changes induced by adenovirus E1a. Moreover, the increase was higher in E1a-TK-expressing cells than in TK-expressing cells during the first 48 hours. This increase of cyclin B1 protein is consistent with a marked arrest in the G2-M cell cycle phase of treated cells.²⁶

Cytologically, the E1a-TK- and TK-expressing cells were much larger, with bigger cytoplasm and vesicular nuclei (Fig 6). Importantly, most of the cells did not grow after replating in other wells. These results indicate that GCV-induced cell cycle arrest and that morphological changes are irreversible and lead to the death of the cells.

Figure 6.

Cytological features of GCV-treated cells. After treatment with GCV, E1a-TK-expressing cells showed a striking enlargement of the nuclei and cytoplasm. Note that at the same magnification (100), the E1a-TK cells treated with GCV show large cytoplasms compared to the same cells without GCV. Crystal violet staining. a: NIH-3T3 E1a-TK cells without GCV. b: NIH-3T3 E1a-TK cells after 3 days of treatment with GCV. c: MDA-MB435 E1a-TK cells without GCV. d: MDA-MB435 E1a-TK cells with GCV.

Full figure and legend (231K)

Discussion

This paper shows that concomitant expression of adenovirus E1a protein and the HSVTK suicide gene clearly enhances the cytotoxic effect of the HSVTK-GCV system and increases the sensitivity to treatment with anticancer agents such as cisplatin. It is known that the percentage of cells in S-phase, mutations in the TK gene, expression of DNA polymerase and other, as yet not well characterized, cellular mechanisms can provoke resistance to the HSVTK-GCV system.^{7,8,9,10,11,12,13} For that reason, we took advantage of the E1a protein as an inducer of DNA synthesis and cell proliferation and as a great inducer of sensitivity to treatment with DNA-damaging agents. Both adenovirus E1a gene properties can complement the antitumor efficacy of the HSVTK-GCV system and, we show that the concomitant expression of adenovirus E1a protein clearly increases the efficiency of the suicidal gene TK. Therefore, we propose the use of vectors carrying E1a or other cell cycle inducer genes together with the HSVTK-GCV system for improving the antitumor efficacy.

The concomitant expression of adenovirus E1a protein also has important advantages because it can act as a tumor suppressor gene and the E1a region is complete in most of the conditional replicative adenoviruses developed so far. In fact, adenovirus E1a gene has already been used in clinical trials of breast and ovarian carcinomas and E1a expression can exert an antitumor effect by down-regulation of neu promoter, increasing the sensitivity to TNF, and other mechanisms that are not well defined.^{19,20,21,22,23} Moreover, E1a expression induces great sensitivity to treatment with DNA-damaging agents, driving cells to apoptosis, by p53-dependent and -independent pathways.^20,22 Interestingly, TK expression has also been associated with up-regulation of p53 protein and CD95 TNF receptor.²⁷ In our model, with the E1a-HSVTK-GCV system, the sensitivity to cisplatin of TK-resistant cells increased clearly favoring the cytotoxic and antitumor effects of the E1a and TK proteins.

The expression of adenovirus E1a protein as an S-phase inducer to enhance the HSVTK-GCV system is effective in both normal and tumor cells. With this retrovirus-mediated E1a-TK expression, we demonstrated that the TK effect can clearly be enhanced. In fact, G2-M cell cycle arrest is evident in confluent cells and the increase of cyclin B1 is also indicative of this arrest. Moreover, E1a protein has been shown to provoke endoreduplication of DNA and drive cells to apoptosis from cells in G2 cell cycle phase.²⁸ The TK system has also been studied with replicative-conditional adenovirus trying to enhance the effects of both antitumoral strategies. In fact, Wildner and Morris³ and Wildner et al²⁹ introduced HSVTK in the Ad1520 and other mutant adenoviruses to increase the antitumor effects of this conditional replication adenovirus. The concomitant expression of TK gene in these vectors does not seem to increase the oncolytic effects or survival of mice. Similar results have recently been obtained by Lambright et al³⁰ using an E3-deleted adenoviral vector containing the HSVTK-suicide gene driven by the endogenous E3 promoter. These results may indicate that conditional replication adenoviruses carrying the HSVTK-GCV system do not work efficiently and could discourage those projects based on these ideas. Nevertheless, there are important points to consider; first, they did not test whether the tumors studied were resistant to the HSVTK-GCV system and, second, a clear and real limiting factor is the schedule of GCV treatment. Obviously, after adding GCV, the infected cells die or are arrested and may prevent more virus replication and spread throughout the tumor cells. Importantly, our system is based on a retrovirus that aims to combine adenovirus E1a and the suicide gene TK effects without expressing 19K or 55K E1b proteins which can block the apoptotic and antitumor effects mediated by E1a. Adenovirus carrying an E1a-TK construction is underway with specific tumor and tissue promoters. In our system, combining E1a and TK, we show that both antitumor effects mediated by E1a and TK are augmented and can be complementary. The schedule of treatment with GCV and antitumor drugs such as cisplatin have to be made to determine the most effective results, combining the cytotoxic effects of the HSVTK-GCV system and the sensitivity to DNA-damaging agents induced by the E1a protein and the TK system.

To summarize, this paper shows that concomitant expression of adenovirus E1a protein with the HSVTK-GCV system, by a retroviral system, significantly enhances the cytotoxic properties of the suicide gene TK. We propose that vectors carrying the HSV TK transcriptionally coupled by IRES to adenovirus E1a or other cell cycle inducer genes can significantly enhance the effects of the TK suicide gene in human malignant tumors. Moreover, the expression of E1a protein can increase the response to concomitant treatment with anticancer agents and the presence of a suicide gene in the vector is an important mechanism in the prevention of theoretical side effects.

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Acknowledgements

We thank Marta Izquierdo for providing the IRES-TK plasmid and Ramón Alemany and Silvio Gutkind for advice, support, and critical reading of the manuscript. We thank Martin Hadley-Adams for preparing the manuscript and Ruth Valero for technical work. This work was supported by grants from: the "Fondo Español de Investigaciones Sanitarias," FIS 99/0504; the Comunidad de Madrid, CAM 98/08.1/2, Aventis; and the Areces Foundation. V.F.S. is supported by a postdoctoral fellowship from "Consejeria de Educación (CAM)" with participation of "Fondo Social Europeo." C.P.C. is supported by a predoctoral fellowship from the "Fondo Español de Investigaciones Sanitarias."