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Cancer Biol Ther. 2016; 17(12): 1221–1230.
Published online 2016 September 19. doi:  10.1080/15384047.2016.1235655
PMCID: PMC5199163

Autoregulated expression of p53 from an adenoviral vector confers superior tumor inhibition in a model of prostate carcinoma gene therapy

ABSTRACT

Alternative treatments for cancer using gene therapy approaches have shown promising results and some have even reached the marketplace. Even so, additional improvements are needed, such as employing a strategically chosen promoter to drive expression of the transgene in the target cell. Previously, we described viral vectors where high-level transgene expression was achieved using a p53-responsive promoter. Here we present an adenoviral vector (AdPGp53) where p53 is employed to regulate its own expression and which outperforms a traditional vector when tested in a model of gene therapy for prostate cancer. The functionality of AdPGp53 and AdCMVp53 were compared in human prostate carcinoma cell lines. AdPGp53 conferred greatly enhanced levels of p53 protein and induction of the p53 target gene, p21, as well as superior cell killing by a mechanism consistent with apoptosis. DU145 cells were susceptible to induction of death with AdPGp53, yet PC3 cells were quite resistant. Though AdCMVp53 was shown to be reliable, extremely high-level expression of p53 offered by AdPGp53 was necessary for tumor suppressor activity in PC3 and DU145. In situ gene therapy experiments revealed tumor inhibition and increased overall survival in response to AdPGp53, but not AdCMVp53. Upon histologic examination, only AdPGp53 treatment was correlated with the detection of both p53 and TUNEL-positive cells. This study points to the importance of improved vector performance for gene therapy of prostate cancer.

KEYWORDS: Adenovirus, apoptosis, gene therapy, prostate cancer, p53

Introduction

Prostate carcinoma's importance is easily understood since 28% of new cancer cases are located in the prostate and it is the second leading cause of death from cancer in men.1 Despite an initial positive response in 90% of prostate carcinomas (PCa), the majority of cases progress to hormone resistance.2 Patients who develop only a localized tumor present a 5-year survival rate of almost 100%, but the rate is only 31% for patients with metastatic disease.3

Among all gene therapy clinical trials, 64% treated cancer and adenoviral vectors are the most common vehicle (www.abedia.com/wiley/). Treatment of prostate carcinoma with gene therapy has been attempted using either non-replicating or oncolytic adenoviral vectors expressing different transgenes, such as decorin, fusion protein of prostate-specific antigen and CD40 ligand, and have exhibited antitumor activity.4-6 In addition, gene therapy with p53 has yielded some positive results in the treatment of prostate cancer,7-10 yet this strategy has not been extensively explored.

Adenovirus expressing p53 under the control of a constitutive promoter, such as CMV or RSV, induces tumor regression and increases survival in animal models without adverse effects on normal tissue11,12 and in clinical trials the treatment of non-small cell lung carcinoma was shown to be safe and to present antitumor activity.13,14 In China, an adenoviral vector expressing p53 was approved for clinical use in 2003 and marketed under the name “Gendicine.” The treatment of thousands of patients using AdRSVp53 virus preparations that meet international standards and with no serious adverse events has been reported.15,16 Even so, improvements to the delivery system may enhance the efficacy and range of gene therapy strategies involving p53.

Our group has inserted a p53-responsive element in viral vectors such that transgene expression is then controlled by p53. Initially the p53-responsive element (referred to here as PG) was incorporated in a retroviral system in which the retroviral LTR (Long Terminal Repeat) was modified with the insertion of the PG element, creating the pCLPG vector. This vector was shown to offer p53-dependent expression that was 7-times greater than the expression from the parental vector.17 When the p53 cDNA was inserted downstream of the p53-responsive PG promoter, an autoregulatory, positive feedback mechanism was established, providing high levels of p53 expression and activity.18

Next we transferred the PG platform to adenoviral vectors and the PG promoter was shown to direct transgene expression at levels 5-times greater than the commonly used CMV promoter.19 We also demonstrated enhanced induction of cell death in a mouse melanoma model when combined, but not individual, transfer of the p19Arf and interferon-β genes was performed using these p53-responsive adenoviral vectors20 and that this combination promotes an important immune response.21

In this report we have evaluated the potential of an adenoviral vector expressing p53 under the control of a p53-responsive promoter (AdPGp53) in comparison to the more typical AdCMVp53 vector where expression is constitutive. We show that AdPGp53 expresses high levels of p53 protein necessary for the reduction of proliferation and increase in cell death in prostate cancer cell lines. The high levels of p53 expression mediated by the AdPGp53 vector were correlated with reduced tumor volume and increased overall survival in a xenograft mouse model of in situ gene therapy.

Results

Comparison of p53 expression mediated by PG and CMV promoters

The PCa cell lines DU145 and PC3 (mutant p53 and p53-null, respectively) were transduced with adenoviral vectors expressing p53 under control of the p53-responsive PG element (AdPGp53) or the constitutive CMV promoter (AdCMVp53, see Fig. S1 for vector maps). Cells were harvested 24, 48 and 72 hours after transduction and the expression of p53 protein analyzed by western blot. AdPGp53 conferred much higher levels of p53 as well as distinct kinetics of protein accumulation as compared to AdCMVp53 in both cell lines (Fig. 1). In DU145, p53 expression from AdPGp53 achieves its maximum levels after 48 hours and decreases after 72 hours, possibly due to cell death at this time point. Also, the CDK inhibitor p21 (CDKN1a), a downstream target gene in the p53 pathway,22 was induced more readily in the presence of the AdPGp53 vector at time points that correlate with the onset of cell death, as shown in the following assays. Expression from the AdPGp53 vector was also confirmed by immunofluorescence in PC3 cells (Fig. S2A).

Figure 1.
Detection of p53 protein in PCa cell lines transduced with adenoviral vectors. (A) PC3 cells were transduced with a MOI of 1000 with AdPGp53 (PG) or AdCMVp53 (CMV) and incubated for 24, 48 or 72 hours before total cellular protein was collected ...

Cell cycle alterations and apoptosis mediated by p53 expression

Since the AdPGp53 vector conferred such high levels of p53 expression, we verified its impact on proliferation and viability in DU145 and PC3 cells. As seen in Fig. 2, viability and proliferation of DU145 cells was markedly reduced in the presence of the AdPGp53 vector, but not AdCMVp53. Cell cycle analysis revealed accumulation of hypodiploid (Sub-G1) cells only when DU145 was treated with AdPGp53. In addition, accumulation of Annexin-V/PI positive cells was directly correlated with AdPGp53 treatment, indicating a cell death mechanism consistent with apoptosis. In contrast, the impact of AdPGp53 transduction of PC3 cells was revealed only when a high MOI was applied, yet some reduction in proliferation and induction of cell death was observed, as seen in Fig. 3. In either cell line, the kinetics of cell death was consistent with that of protein expression, including p21.

Figure 2.
Functional assays reveal the impact of adenovirus-mediated gene transfer in DU145 cells. (A) Cell viability was measured using the MTT assay 72 hours post transduction with different MOIs (50, 100, 250, 500 or 1000) represented by the triangle. ...
Figure 3.
Functional assays reveal the impact of adenovirus-mediated gene transfer in PC3 cells. Legend as per Fig. 2, except that the different MOIs used in panel A were 100, 500, 1000, 2500 and 5000 and a MOI of 5000 was used in panels B through D.

Transduction efficiency explains the difference in AdPGp53 performance in PCa cell lines

The outstanding performance of the AdPGp53 vector as compared to AdCMVp53 may be due to differences in the virus preparations, relative promoter activity or transduction efficiency. By transducing HEK293 cells and staining for expression of the adenoviral hexon protein, we show that the viral preparations are actually quite equivalent in terms of infectivity (Fig. S2B). The functionality of the CMV promoter was confirmed upon transduction of H1299 cells with AdCMVp53 and detection of p53 protein, revealing constitutive expression as expected (Fig. S3). Viability and cell cycle were also impacted by AdCMVp53 in H1299 cells (Fig. S4), suggesting a cell type dependent response to treatment. These assays show that the AdCMVp53 vector preparation was quite reliable in terms of transgene expression and function in H1299 cells, yet performance was inadequate in the PCa cell lines in question.

We next explored whether transduction efficiency could explain the differences in transgene expression level. The expression of mRNA under the control of the CMV promoter was quite similar between DU145 and H1299, yet appeared to be reduced in PC3 cells (Fig. S5A). However, when transduction efficiency is taken into consideration, the activity of the CMV promoter was similar among all cell lines tested (Fig. S5B and C). These assays indicate the proper functioning of the CMV promoter and that transduction efficiency was severely reduced in PC3 cells.

We measured PG promoter activity among the PC3, DU145 and H1299 cell lines. In this assay, the cells were co-transduced with 2 adenoviral vectors, one encoding luciferase under the control of the PG promoter and the other used to supply p53 directed by the PG or CMV promoter (Fig. S6). As measured via luciferase activity, the performance of the PG promoter in PC3 or DU145 cells was superior when p53 was supplied by the AdPGp53 vector as compared to AdCMVp53. In H1299 cells, activation of the p53-responsive promoter was more easily accomplished when p53 was supplied by the AdCMVp53 vector as compared to AdPGp53. Thus, the AdPGLuc vector served as an alternative read out of p53 activity in the cell lines studied here. Consistent with our other findings, cell specific differences played a role in vector performance, yet the AdPGp53 vector was superior in terms of transgene expression and function when applied in the PCa cell lines and represents an advance for our gene transfer approach. However, transduction efficiency seemed to limit the response of PC3 cells to AdPGp53 gene transfer.

In order to better study AdPGp53 performance, we developed a PC3 cell line expressing the human coxsackie-adenovirus receptor, hCAR, thus facilitating virus-cell interaction. Indeed, the PC3-hCAR cells were transduced more readily as compared to the parental PC3 cell line (Fig. S7). Upon transduction with the p53-expressing adenoviral vectors, PC3-hCAR showed higher levels of p53 protein compared to the parental cell line at the same MOI (Fig. S8). In order to produce equivalent levels of p53 expression, a 4-fold higher MOI of AdPGp53 was applied to PC3 as compared to PC3-hCAR (MOI 1000 vs. 250, respectively; Fig. S8). PC3-hCAR transduced with AdPGp53 showed greater accumulation of hypodiploid cells (Fig. S9) as compared to the assay using the parental cell line (Fig. 3). In PC3-hCAR cells, the AdPGp53 vector conferred an increased effect even when using a 10-fold lower concentration of virus, confirming the importance of transduction efficiency. The AdCMVp53 vector did not induce cell death in the PC3-hCAR cells even when an elevated MOI was used. In all, AdPGp53 was effective in killing cells resistant to treatment with the traditional CMV vector, as was the case with the PC3 and DU145 PCa cell lines.

Xenograft mouse model of PCa treated with adenoviral vectors

Even though PC3 cells were resistant to cell death mediated by over-expression of p53, in vivo factors may influence the outcome of the gene therapy approach and reveal effects not seen in the tissue culture assays. Athymic male nude mice were inoculated subcutaneously with 2×106 PC3 cells (parental, non-modified) and when tumors were palpable the mice were treated with intratumoral injections of AdPGLuc, AdCMVp53 or AdPGp53. Tumors were measured on alternate days and mice were sacrificed when tumors reached a tumor volume of 1000 mm3. AdPGp53 showed a significant reduction in tumor volume and increased overall survival compared to both AdPGLuc and AdCMVp53, median survival (sub-maximal tumor volume) was 26.5, 36 and 62 days for AdPGLuc, AdCMVp53 and AdPGp53 respectively (Fig. 4A and B). Analysis of p53 expression in histologic sections indicates stronger expression mediated by AdPGp53 compared to AdCMVp53 (Fig. 5). AdPGp53 gene therapy was also associated with increased TUNEL staining, indicating cell death possibly due to apoptosis (Fig. 6). Despite the resilience of PC3 cells to treatment in the in vitro assays, in situ gene therapy with AdPGp53, but not AdCMVp53, reduced tumor progression and increased overall survival.

Figure 4.
Xenograft model of in situ gene therapy. Male athymic nude mice were subcutaneously inoculated with 2×106 PC3 cells and, once tumors were palpable, treated intratumorally with 6 doses (indicated by arrows) of 1×109 TU of AdPGLuc, AdCMVp53 ...
Figure 5.
p53 expression in tumor cells after in situ gene therapy. As per Fig. 4, s.c. PC3 tumors were treated with adenoviral gene therapy and 48 hours after the last injection the tumors were removed and analyzed by immunofluorescence. Tumor ...
Figure 6.
Apoptosis detected in tumor cells after in situ gene therapy. As per Fig. 4, s.c. PC3 tumors were treated with adenoviral gene therapy and 48 hours after the last injection the tumors were removed and analyzed using the In situ cell death ...

Discussion and conclusions

Prostate carcinoma is one of the most frequent cancers and development of new strategies for its treatment is a serious public health issue. The results of preclinical studies show that restoration of p53 gene function can activate apoptosis, thus destroying cancerous cells. Restoration of wild-type p53 function in tumors can be achieved using an adenoviral vector for the transfer of the wild type p53 cDNA. Innumerous studies have shown that Adp53 triggers a dramatic tumor regression response in various cancers in both the laboratory and clinical settings. Adenoviral vectors expressing p53 under control of a constitutive promoter have been approved for clinical use for head and neck carcinoma,16 yet application of p53 gene therapy for PCa has not been fully explored.

In this report we have compared the performance of 2 adenoviral vectors that differ only in the promoter, the constitutive CMV promoter versus the p53-responsive PG promoter. We have observed that p53 expression in PCa cell lines is higher after transduction with AdPGp53 compared to AdCMVp53 and that DU145 cells were especially susceptible to the tumor suppressor activities offered by the AdPGp53 vector. The in vivo assay revealed an important advantage when the AdPGp53 vector was employed for in situ gene therapy of s.c. PC3 tumors, where progression was retarded and overall survival was increased. In this way, the AdPGp53 vector was superior to the traditional AdCMVp53 gene transfer strategy when tested in PCa cell lines.

Several interesting aspects of the gene transfer approach were revealed in the in vitro assays. We show here that the level of p53 expression was not predictive of cellular response, probably due to cell specific differences that were not explored in this study. The DU145 cells were especially sensitive to the treatment with AdPGp53 and, despite high level p53 expression, PC3 cells were quite resistant to p53 in the in vitro assays. Introduction of hCAR in PC3 cells increased the efficacy of the AdPGp53 adenoviral vector, indicating that inferior transduction efficiency may explain the differences when PC3 cells are compared to DU145 or H1299. Since the transduction efficiency of PC3 cells was quite low, orders of magnitude below DU145 or H1299, there is still room for improving the gene transfer approach.

The sensitivity of H1299 cells to AdCMVp53 treatment has been well documented,23,24 though we were surprised to find that these cells were less susceptible to AdPGp53 treatment at the time points tested. The kinetics of p53 expression from the viral vectors may have played a role in the cellular responses, including accumulation of p21 protein which correlated with the onset of cell death in our study. Expression from AdPGp53 followed slower kinetics as compared to AdCMVp53. We can only postulate that a gradual increase in p53 concentration associated with the AdPGp53 vector may lead to a distinct cellular response as compared to a sudden burst of p53 expression, as seen with AdCMVp53. Even so, the slower kinetics from AdPGp53 does not seem to be a detriment since DU145 cells were efficiently killed when treated with this vector.

In the 1990s, adenoviral vectors expressing p53 under control of the CMV promoter were applied in murine models of prostate cancer. Some studies have shown that expression of p53 can reduce tumor volume,7,8 while others showed that constitutive expression of p53 did not have a greater effect when compared to the control group. Furthermore, p21 gene transfer was shown to significantly reduce the tumor volume and increase animal survival.25

In the clinical setting, use of adenovirus expressing p53 under the control of the CMV promoter administered by intratumoral injection was shown to be safe, conferred p53 expression and induced apoptosis in the patients' prostate cancer. However, the survival curve of patients treated with this adenovirus and prostatectomy showed no difference with patients at the same stage treated with chemotherapy and prostatectomy.26 To date, gene therapy approaches targeting p53 in prostate cancer have not been fully explored. We propose that improved vectors as well as combining gene therapy with other agents, such as chemotherapy, may warrant further study.

Our xenograft mouse model shows that even in the more resistant cell line (PC3), the overexpression of p53 provided by AdPGp53 is able to reduce tumor volume and increase survival. Strikingly, this in vivo assay revealed induction of apoptosis in the presence of the AdPGp53 vector, an event that was observed in vitro, but only at high MOI. The context of the tumor microenvironment as well as the duration of the study may have presented conditions that favored inhibition of tumor progression by AdPGp53 in vivo. For example, though not tested directly, the introduction of exogenous p53 may lead to the recruitment of NK cells or inhibition of angiogenesis,27,28 effects that would not be revealed in vitro. Our data with AdCMVp53 are in accordance with other publications where a similar vector did not increase animal survival or decrease tumor volume.29 Our attempts to establish s.c. tumors with the DU145 cells were not successful and, therefore, we were not able to perform an in vivo test with this cell line. Even so, our data indicates that AdPGp53 with positive feedback regulation of transgene expression provides an advantage as compared to the traditional vector when tested in vivo. Admittedly, this treatment was not associated with complete inhibition of tumor progression. This issue is the subject of ongoing studies where improvements to the vector as well as simultaneous treatment the chemotherapeutics are being explored.

In our previous studies we revealed additional uses for the p53-responsive promoter in a variety of viral vector platforms. For example, we have used retroviruses as reporter constructs of p53 activity.17,18,30 We have employed the PG-vectors to transfer genes such as p19Arf30 and interferon-β20 and have observed striking anti-tumor activity, including an important protective immune response.21 We have also constructed an adenoassociated virus, AAVPG,31 to transfer VEGF-A165 in a model of cardiac hypertrophy, resulting in marked functional improvement using ‘on-demand’ transgene expression in response to physiologic stress.32 Though the concept of p53-responsive vectors was initiated specifically for the transfer of p53 and treatment of cancer, we have found many avenues for the application of these interesting and innovative gene transfer tools.

In summary, using a p53-responsive promoter to drive expression of the p53 cDNA established an autoregulatory mechanism that resulted in drastically elevated protein levels as compared to the use of a constitutive promoter (CMV). The benefit of this vector was clearly seen in DU145 cells in vitro and in PC3 cells in vivo. Further development of our approach is currently underway. Though much remains to be examined, we propose that AdPGp53 warrants further investigation as a potential gene therapy agent.

Material and methods

Cell lines

Human PCa cell lines DU145 (heterozygous p53 mutant, P223L, V274F) and PC3 (p53 null) as well as H1299 human lung cancer (p53-null) and HEK293 (human embryonic kidney) cell lines were cultivated in DMEM (Life Technologies, # 12100-046) with 10% fetal calf serum (Life Technologies, #12657), supplemented with antibiotic-antimycotic (Life Technologies, # 15240-062) and maintained at 37°C and 5% CO2 atmosphere. PC3-hCAR is derived from PC3 cell line transduced with pLXSN-hCAR33 and selected with 1 mg/ml of G418 (Life Technologies, #11811-031).

Construction of adenoviral vectors

AdCMVp53 was constructed by first isolating the cDNA for wild-type human p53 from pCLp5334 and inserting this into a modified form of pENTR2B (Life Technologies, #A10463) followed by recombination with pAdCMV-V5dest (Life Technologies, #V493-20).

AdPGp53 was obtained by transferring a sequence encoding the p53-responsive promoter (PGTxβ or simply PG), wild type human p53 cDNA and the SV40 late polyadenylation signal from pAdenovatorPGp53 (BES, unpublished data) to pENTR/D-TOPO (Life Technologies, #K2400-20), generating pENTR/TOPO-PGp53, which by recombination was transferred to the pAd/PL-DEST (Life Technologies, #V494-20), generating pAdPGp53. The construction of AdPGLuc followed the similar procedure as per AdPGp53 and has been described previously.20 The AdCMVLacZ vector was obtained commercially (Life Technologies).

Virus production

Virus particles were obtained after transfection of linearized plasmids into the HEK293 producer cells and further rounds of amplification until virus was harvested from 25 15-cm dishes. Viral particles were purified by 2 rounds of cesium chloride density gradient ultracentrifugation, the first consisted of 1.2 g/ml and 1.4 g/ml of cesium chloride for 90 min at 4 °C and 23,000 rpm (rotor SW28, Beckman, Brea, CA, USA) and the second of 1.33 g/ml for 18 hours at 4 °C and 46,000rpm (rotor 70Ti, Beckman), followed by 4 rounds of dialysis against dialysis buffer (10 mM tris-HCl pH8.0, 2 mM MgCl2 and 5% sucrose). The titer (expressed as Transducing Units per milliliter, TU/ml) was determined using the Adeno-X Rapid Titer Kit (Clontech, #632250).

Western-blot

One-hundred thousand DU145 or PC3 cell lines were seeded in 6-well plates and transduced with adenovirus with different Multiplicities of Infection (MOIs) ranging from 10–1000. Proteins were extracted, quantified and loaded in 12% SDS-PAGE, transferred onto nitrocellulose and incubated with rabbit anti-p53 (Santa Cruz Biotechnology, #SC6243), rabbit anti-p21 (Santa Cruz Biotechnology, SC397) or mouse monoclonal anti-β-actin (A5316, Sigma-Aldrich, St. Louis, MO, USA) antibodies. The Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Lifesciences, #RPN2232) was used for chemiluminescent detection in an Image Quant LAS 4000 (GE Healthcare Lifesciences).

Cell viability (MTT)

In each well of 96-well plates, 2.5 × 103 cells (DU145 or PC3) were seeded and the plate incubated at 37°C and 5% CO2 for 16 hours. In quadruplicate, the cells were then transduced with adenoviral vectors and incubated for periods of 24 to 96 hours. At the end of the experimental period, MTT (Life Technologies, # M6494) solution (3 - (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, 5 mg / ml) was added to each well and incubated at 37 °C and 5% CO2 for 4 hours, then solubilizing buffer (20% SDS in 50% DMF/2% acetic acid, pH adjusted to 4.7) was added and allowed to incubate at 37 °C for 16 hours. The plates were read on a spectrophotometer at a wavelength of 570 nm.

Cell cycle analysis

In 6-well plates, 1 × 105 cells (DU145 or PC3) were seeded per well and the plate incubated for 16 hours. The cells were then transduced with adenoviral vectors and incubated for a period of up to 72 hours. At the end of the experimental period, the cells were collected, fixed with 70% ethanol and centrifuged at 4 °C for 5 minutes at 400g, washed with PBS and centrifuged again. The supernatant was discarded and the cells treated with 5µl of RNAse (10 mg/ml, Life Technologies, #12091-021), 1µl of propidium iodide (10 mg/ml, Sigma-aldrich, #P4170) and 0.5 µl of Triton X-100 for 30 minutes at 37°C. After this period, cells were centrifuged and the pellet resuspended in 0.5 ml of PBS. The emitted PtdIns fluorescence was measured by means of flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA) and analyzed using Cell Quest software (Becton Dickinson).

Apoptosis analysis

Prostate cancer cell lines were transduced with the adenoviral vectors and after 72 h adherent and floating cells were collected and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) at a concentration of 1 × 106 cells/ml. From this cell suspension, 100 µl (1 × 105 cells) was transferred to a 5-ml tube and 5 µl of Annexin V- Alexa 488 conjugate (Life Technologies, #A13201) and 1 µl of propidium iodide (0.1 mg/ml) were added to the cells before incubating 15 minutes at room temperature in the dark. Detection of apoptotic cells was done by flow cytometry (FACSCalibur, Becton-Dickinson).

Immunofluorescence

For in vitro experiments, the PC3 and HEK293 cell lines were plated 1×104 cells per 13 mm round cover slip, transduced the next day then incubated 24 to 48 hours before fixation with 4% paraformaldehyde (PFA). For in vivo assays, tumors were fixed with PFA 4% for 4 hours and left in 30% sucrose solution for 16 hours at 4 °C. After this time the tumors were frozen in OCT-Tissue Tek and cut in 4 µm tissue sections using a cryostat and tissue sections were placed on silanized glass slides. P53 protein was detected with a rabbit anti-p53 antibody (Santa Cruz Biotechnology, #SC6253) and the adenoviral hexon protein with mouse anti-hexon antibody (Abcam, #Ab8251). The secondary antibodies were anti-rabbit conjugated with Alexa488 (Life Technologies, #A31627) and anti-mouse conjugated with Alexa 647 (Life Technologies, #A31625) and nuclear staining was performed with Hoechst 33258 (Life Technologies, #H1398) at a concentration of 20 mg/ml. The cells were analyzed by Confocal Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany).

Real-time PCR

PC3, DU145 or H1299 cells (1 × 105) were transduced with the adenoviral vectors and after 48 hours incubation total RNA was harvested using TRizol (Life Technologies, #10296010). cDNAs were generated from 1 μg of total RNA using MMLV reverse transcriptase (Life Technologies, #28025-013). Amplifications were carried out using SYBR Green I mix (Life Technologies, #4309155) on 7500 ABI (Applied Biosystems) using 0.36 µl of specific primers in the concentration of 12.5 µM. At the end of the PCR cycles, a melting curve was generated to identify the specificity of the PCR product. Expression levels of LacZ were analyzed using the ΔΔCt method and the absolute quantification of the number of genome copies, as measured by detection of the LacZ gene present in 10 ng of episomal DNA extracted by Hirt´s modified method and compared to a standard curve as described previously.35 Also, for normalization of each sample, the amount of hGAPDH cDNA was measured. The sequences of the primers are as follows: LacZf: 5´-TCGAAGTGACCAGCGAATAC-3´, LacZr: 5´-TACTGTGAGCCAGAGTTGGC-3´, GAPDHf: 5´-TGCACCACCAACTGCTTAGC-3´, GAPDHr: 5´-GGCATGGACTGTGGTCATGAG-3´.

Detection of β-galactosidase activity

In 6-well plates, 1×105 DU145, PC3 or H1299 cells were seeded and after 24 hours transduced with AdLacZ at different MOIs. After 48 hours of incubation, the cells were fixed (2% paraformaldehyde, 0.2% glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.3) for 5 min at 4 °C and stained (3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1.2 mM MgCl2 and 1 mg/mL X-gal in 100 mM sodium phosphate buffer, pH 7.3) for 16 hours at 37 °C.

Xenograft mouse model

Athymic male BALB/c nude mice, 8–12 weeks old, were used in the in vivo experiment. Animal experimentation was performed at the Gene Therapy Laboratory, Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of Sao Paulo. The experimental protocols, as described here, were approved by the Ethics in Animal Use Committee of the Institution. PC3 cells (2 × 106) were implanted subcutaneously in the flank of the mice and on alternate days the tumors were measured with calipers and the volume calculated according to the formula ½ x (major diameter) x (minor diameter),2 as per Tomayko and Reynolds.36 After 3 weeks the tumors reached 60 mm3, an appropriate size for the start of treatment. The dose of adenovirus injected intratumorally was 1 × 109 TU/25 µl PBS/injection and this treatment was repeated a total of 6 times at 48 hour intervals and the tumor growth was monitored over time. Animals were euthanized by CO2 inhalation when the tumor volume reached 1000 mm3. In all procedures the mice were anesthetized in a chamber with 4% isoflurane.

Tumor apoptosis analysis

Forty-eight hours after the last in situ adenovirus administration the animals were euthanized, the tumors removed, fixed with PFA 4% for 4 hours and left in 30% sucrose solution for 16 hours at 4°C. After this time the tumors were frozen in OCT-Tissue Tek and cut in 4 µm tissue sections using a cryostat. The tissue sections were subjected to the Tunel reaction (In situ cell death detection kit- fluorescein, Roche Applied Science, #11684795910) as directed by the manufacturer's protocol. The samples were also stained with Hoechst and observed under a fluorescence microscope (Evos, Life Technologies). Total and positive cells from at least 10 fields were counted using ImageJ software.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software version 5.0. Statistical significance was calculated using a 2-tailed Student t-test and were considered significant when p < 0.05.

Supplementary Material

Supplementary_data.zip:

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Roger Chammas (ICESP-FMUSP) and his staff for ongoing support and critical discussions, Paulo Roberto Del Valle (ICESP-FMUSP) for critical reading of the manuscript.

Funding

Financial support was received from the Sao Paulo Research Foundation, FAPESP (RET, 2011/21256-8; RBSS, 05/03543-9; BES, 2007/50210-0, 2013/25167-5).

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