Transgene expression by oncolytic adenoviruses is modulated by E1B19K deletion in a cell type-dependent manner

Virology 395 (2009) 243–254

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Virology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o

Transgene expression by oncolytic adenoviruses is modulated by E1B19K deletion in a cell type-dependent manner Stanimira Rohmer a, Christina Quirin a, Andrea Hesse b, Stefanie Sandmann a, Wibke Bayer c, Christel Herold-Mende d, Yosef S. Haviv e, Oliver Wildner c, Alexander H. Enk f, Dirk M. Nettelbeck a,⁎ a

Helmholtz-University Group Oncolytic Adenoviruses, German Cancer Research Center and Heidelberg University Hospital, Department of Dermatology, Heidelberg, Germany Department of Dermatology, University Hospital Erlangen, Germany Department of Molecular and Medical Virology, Institute of Microbiology and Hygiene, Ruhr-University Bochum, Bochum, Germany d Division of Neurosurgical Research, Department of Neurosurgery, Heidelberg University Hospital, Heidelberg, Germany e Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel f Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany b c

a r t i c l e

i n f o

Article history: Received 15 June 2009 Returned to author for revision 28 July 2009 Accepted 29 September 2009 Available online 25 October 2009 Keywords: Adenovirus Oncolysis E1B19K Arming Late expression Viral release Viral spread Apoptosis

a b s t r a c t Major strategies to increase oncolytic adenovirus efficacy, as required for clinical applications, are enhancing oncolysis by acceleration of virus release/spread and “arming” by insertion of therapeutic genes. We investigated whether these strategies can be effectively combined as it has been speculated that the arming approach rather benefits from delayed cell lysis and extended time for protein synthesis. We report that deleting adenoviral E1B19K results in an apoptosis-dependent early viral release and thus enhanced oncolysis in several tumor cells, but inhibits virus replication in others. In the former case, apoptosis induction and early cell lysis did not interfere with late transgene expression. Thus, transgene expression was dramatically increased over time due to better virus spread. In A549 cells, transgene expression by the E1B19K− virus at 5 days post-infection was higher than for the E1B19K+ virus at 10 days. These properties may be of critical importance in patients, where time for virus spread is limited. © 2009 Elsevier Inc. All rights reserved.

Introduction Oncolytic adenoviruses have emerged as promising agents for cancer therapy by tumor-restricted virus infection. Tumor-specificity of adenovirus replication has been achieved by both mutation of viral gene functions or transcriptional targeting of viral gene expression (Alemany et al., 2000; Nettelbeck, 2003). Clinical studies have revealed a favorable safety profile for a first generation of oncolytic adenoviruses (Reid et al., 2002; Ko et al., 2005). However, virus monotherapy has not resulted in significant therapeutic activity, although striking tumor responses were observed for individual patients. In consequence, improving the therapeutic efficacy of oncolytic adenoviruses is a key challenge of current research efforts. Two major strategies have been pursued toward this goal. First, increased oncolytic activity resulting from accelerated virus release

⁎ Corresponding author. Helmholtz University Group Oncolytic Adenoviruses, DKFZ (German Cancer Research Center) and Department of Dermatology, Heidelberg University Hospital, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Fax: +49 6221 42 4902. E-mail address: [email protected] (D.M. Nettelbeck). 0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2009.09.030

and spread has been assessed by mutating viral genes. Second, insertion of transgenes into the genome of oncolytic adenoviruses aims at the destruction of neighboring and distant, uninfected cancer cells by transgene-encoded therapeutic proteins or RNA (Hermiston and Kuhn, 2002). Hence, this strategy implements multimodality in one agent, called an “armed” oncolytic adenovirus that features both viral oncolysis and gene therapy. The goal of this study was to investigate whether these two approaches, accelerated lysis of tumor cells and efficient transgene expression by the same cell, are compatible. This would be an advantageous scenario because it combines improved virus spread with bystander killing. However, it is not clear as to whether early lysis of cancer cells or the underlying molecular mechanisms, such as apoptosis induction (see below), abort efficient expression of the transgene. Indeed, it was speculated previously that a longer lifespan of infected cells, achieved by attenuation rather than augmentation of viral cell lysis, might ensure efficient therapeutic protein synthesis by armed oncolytic adenoviruses (Hawkins and Hermiston, 2001). Adenoviruses with enhanced lytic activity have been generated by two approaches: targeted mutation and random mutagenesis followed by bioselection. This strategy is based on the hypothesis that

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adenovirus replication, cell killing and spread are sub-optimal in tumor cells, which are not the normal host cells of adenovirus infection. The most prominent virus mutants resulting from this work lack expression of functional anti-apoptotic E1B19K, which is thought to counteract pro-apoptotic activity of E1A thereby preventing premature death of the host cell during natural infection. Already in 1983 and 1984, Ad2 and Ad5 mutants that showed a large plaque phenotype in tumor cells, indicative of accelerated cell-to-cell spread, were characterized to be deficient for E1B19K (Chinnadurai, 1983; Takemori et al., 1984). More recently, in an effort to improve adenoviral oncolysis, Sauthoff and colleagues demonstrated for an E1B19K-deleted Ad5 mutant markedly increased cytotoxicity in tumor cells with minor effects on infectious virus particle production. Moreover, they reported for this mutant an earlier release of infectious viruses from tumor cells, large plaques and a better antitumor activity in vivo (Sauthoff et al., 2000; Harrison et al., 2001). Interestingly, E1B19K-deficient mutants were also obtained by selection of large plaque clones from randomly mutagenized Ad5 particles in lung cancer cells (Subramanian et al., 2006). Considering the markedly enhanced lytic activity and accelerated spread phenotype reported in these studies, the E1B19K-deleted adenovirus represents a lead virus mutant for applications in oncolysis. Furthermore, these results have shown that adenoviral replication and cell killing activity can indeed be improved in tumor cells by mutagenesis. The large genome size of adenoviruses allows for the insertion of therapeutic genes, such as suicide, immunoregulatory or siRNA genes, without loss of replication competency establishing so-called “armed” oncolytic adenoviruses (Hermiston and Kuhn, 2002). The outcome of this approach for combination therapy, however, critically depends on the site of transgene insertion into the virus genome and on the resulting mechanism of transgene expression. One established approach is the incorporation of the transgene into the late viral transcription unit by an internal ribosome entry site (IRES) (Sauthoff et al., 2002; Rivera et al., 2004b). Thereby, the viral gene expression machinery is exploited for transgene expression which occurs with late kinetics and thus is dependent on replication of the virus genome. This is considered to be advantageous for the expression of therapeutic proteins that might interfere with virus production if active at early virus replication. Also, replication dependence is thought to result in indirect tumor-selectivity of transgene expression when viruses are engineered to replicate in tumor cells, only. When aiming at enhanced lysis in the context of armed oncolytic adenoviruses, deleting E1B19K is especially advantageous because deletion of viral genes increases the capacity for transgene insertion. Moreover, the E1B19K deletion has been shown to reverse a reduction in host cell lysis resulting from the deletion of the E3 genes including ADP (Suzuki et al., 2002; Subramanian et al., 2006). Therefore, adenovirus E1B19K deletion mutants might ideally combine both, enhanced oncolysis and increased capacity for therapeutic gene insertion. However, early studies showed for E1B19K mutant adenoviruses increased cytocidal effects (cyt phenotype) and degradation of host cell and viral DNA (deg phenotype) during replication (Subramanian et al., 1984a, 1984b; Pilder et al., 1984; White et al., 1984; Takemori et al., 1984). This was later confirmed and described as E1A-dependent apoptosis induction (White et al., 1992; Sauthoff et al., 2000). How such cytopathic effects during virus replication might affect transgene expression by oncolytic adenoviruses is unclear. Considering the mentioned qualities of E1B19K deletion and late transgene expression, we generated oncolytic adenovirus Ad5/3.19K.IL that features both a deleted E1B19K gene and an insertion of the reporter gene luciferase via an IRES into the late transcription unit. We investigated how the E1B19K deletion affects cell lysis and spread in a panel of tumor cell lines and low passage tumor cell cultures and how therapeutic gene expression was modified. As we observed enhanced

as well as reduced lytic potential of the mutant, we also analyzed the mechanisms underlying these cell type-dependent differences. Results Spread of E1B19K-deleted, transgene-encoding oncolytic adenovirus in tumor cell cultures The genomes of recombinant adenoviruses used in this study are depicted in Fig. 1. To study the combination of early host cell lysis/ viral release with transgene expression, we generated the transgeneencoding oncolytic adenovirus Ad5/3.19K-.IL. This virus has the following features: (i) deletion of E1B19K, (ii) insertion of the reporter gene luciferase into the late fiber transcription unit using an IRES (Rivera et al., 2004b), (iii) Δ24 mutation of E1A for restriction of virus replication to tumor cells (Fueyo et al., 2000; Heise et al., 2000), (iv) a chimeric fiber with Ad5 tail and shaft and Ad3 knob domains facilitating viral entry into CAR-negative cells (Krasnykh et al., 1996; Rivera et al., 2004a) and (v) deletion of the E3 genes due to size limitations. Ad5/3.19K + .IL was generated as the matching control virus with wild-type E1B genomic locus. In a virus spread and cell killing assay, we confirmed for Ad5/3.19K-.IL, i.e. in the context of E3deletion, Δ24 mutation of E1A, transgene expression and chimeric fiber, the enhanced spread phenotype of the E1B19K mutation (Fig. 2). Indeed, this virus caused a 1000-fold higher cytotoxicity in A549 cells compared with Ad5/3.19K + .IL, resembling the previously reported phenotype of the E1B19K deletion mutant of Ad5 in these cells (Subramanian et al., 2006). We next infected a panel of melanoma and glioblastoma cell lines and low passage cultures to investigate virus spread. The results show that the phenotype of the E1B19K deletion depended on the individual cell line or culture (Fig. 2). In two melanoma cell lines (C8161 and SK-MEL-28) and in two of three low passage glioblastoma cultures (NCH82 and NCH468), virus spread of Ad5/3.19K-.IL was enhanced 10- to 100-fold compared with

Fig. 1. Schematic outline of the genomes of adenoviruses used in this study. The relevant genomic regions of the adenoviruses used in this study are shown. CMV, cytomegalovirus promoter; ΔE1/ΔE1B19K/ΔE3, deleted early genes; E1A/E1B19K/E3/ E4, early viral genes; E1AΔ24, E1A mutant featuring a deletion of the pocket proteinbinding conserved region two; IRES, internal ribosome entry site; LITR/RITR, left/right inverted terminal repeat; Luc, luciferase gene; pA, polyadenylation signal for transcription termination; ψ, packaging signal.

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Fig. 2. Spread-dependent cell killing of E1B19K+ or E1B19K− transgene-encoding oncolytic adenoviruses in different tumor cell cultures. Cell cultures were infected with Ad5/3.19K-.IL, Ad5/3.19K + .IL, replication-deficient Ad5/3Luc1 or wild-type Ad5 fiber containing Ad5Δ24E3− at indicated titers or were mock infected (framed wells). Virus spread-dependent cytotoxicity was visualized by crystal violet staining of surviving cells. Therefore, stainings were performed when cytotoxicity was observed for the most potent virus at 0.01 or 0.1 vp/cell for each cell type individually. A549, lung adenocarcinoma; C8161, SK-MEL-28, melanoma cell lines; PMelL, PMelA, low passage melanoma cultures; NCH82, NCH468, NCH89, low passage glioblastoma cultures; vp, viral particles.

Ad5/3.19K + .IL. However, in two low passage melanoma cultures (PMelL and PMelA) and one of three low passage glioblastoma cultures (NCH89), cell spread and lysis by Ad5/3.19K-.IL were attenuated up to 100-fold. Thus, the deletion of E1B19K can result in either an enhanced or reduced spread phenotype of (transgeneencoding) oncolytic adenoviruses dependent on the infected cell type. Apoptosis induction and cell type-dependent modulation of the adenovirus replication cycle by E1B19K deletion To further investigate the cause for the cell type-dependent differences in virus spread of Ad5/3.19K-.IL, we studied in more detail A549 and PMelL cells representing the respectively enhanced or reduced spread phenotype. We first analyzed how cells are affected by virus infection: Considering the anti-apoptotic activity of E1B19K, we hypothesized that the opposing phenotypes of the deletion mutant result from differences in apoptosis induction between the cell types. Therefore, we investigated cleavage of PARP and Pro-caspase 3, both characteristic for caspase activation, a hallmark of apoptosis induction. After infection with Ad5/3.19K + .IL, cleavage of PARP and Pro-caspase 3 was weak for A549 cells but emerged at 24 h for PMelL and was quite strong for these cells at 48 h (Fig. 3a). These results indicate that the melanoma cells showed a distinct apoptotic response to infection with E1B19K wild-type adenovirus. Importantly, for both cell types, the deletion of E1B19K resulted in strongly increased/accelerated cleavage

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of PARP and Pro-caspase 3 with marked cleavage observed starting at 24 h post-infection. These results reveal a similarly pro-apoptotic effect of the E1B19K deletion mutant in both cell types arguing that differences in apoptosis induction are not responsible for the different oncolysis phenotypes of Ad5/3.19K-.IL. Next, we determined virus cytotoxicity 48 h after high titer infection, i.e. before virus spread and re-infection could occur by crystal violet staining (Fig. 3b) and Annexin/PI staining (Fig. 3c) to investigate direct, i.e. spreadindependent cytotoxicity. In this setting, Ad5/3.19K-.IL showed higher cytotoxicity than Ad5/3.19K + .IL for both cell types. When monitoring cytopathic effects by crystal violet staining, this difference was more pronounced for A549 cells. In the Annexin/PI staining, increased numbers of total dead cells were observed after infection with Ad5/3.19K-.IL relative to Ad5/3.19K + .IL for both A549 and PMelL cells (for A549 cells 60% versus 20%; for PMelL cells 70% versus 30%). Furthermore, increased numbers of Annexin-positive/PI-negative cells are indicative for enhanced apoptosis induction confirming the results of PARP and Caspase 3 cleavage. Overall, these results indicate increased cytotoxicity of Ad5/3.19K-.IL in both A549 and PMelL cells during the first virus replication cycle. Thus, we hypothesized that reduced cell killing by the E1B19K mutant virus in low passage PMelL cells 14 days post-infection (Fig. 2) was due to reduced virus spread. This again might be a consequence of attenuated virus production and/or of a reduction or delay in virus release. To test this hypothesis, we more closely investigated virus replication in A549 and PMelL cells. Specifically, we aimed to delineate what step of the adenovirus replication cycle was affected by the E1B19K deletion. To this end, we performed virus burst assays determining infectious virus particles produced by A549 and PMelL cells 2 and 4 days post-infection in cells and supernatant separately (Fig. 4). For A549 cells, similar amounts of total infectious virus particles (supernatant + cells) were produced in Ad5/3.19K-.IL- and Ad5/3.19K + .IL-infected cells and no or a minimal increase in virus titers was observed from 2 to 4 days. However, virus release into the supernatant (ratio supernatant/cells) was markedly increased for the E1B19K mutant at 2 and even more so at 4 days post-infection, when viral release was more than 2 orders of magnitude higher than for the E1B19K wild-type virus. For PMelL cells, we observed a different picture: total infectious virus particle production was significantly lower for Ad5/3.19K-.IL compared with Ad5/3.19K + .IL at 4 days post-infection. For Ad5/3.19K + .IL, an increase in total infectious virus particles from 2 to 4 days post-infection was observed. The amount of infectious viral particles in the supernatant was also lower for Ad5/3.19K-.IL than for Ad5/3.19K + .IL, even though virus release was slightly higher. We observed that genome copy numbers and late viral gene expression for the E1B19K-deleted virus were 1.5- to 7.7-fold lower than for the E1B19K+ virus in PMelL cells, but not in A549 cells (not shown) indicating that in PMelL cells inhibition of virus replication initiated during early infection. We conclude that deletion of E1B19K interferes with adenovirus replication resulting in reduced production of infectious virus particles in PMelL cells, but not in A549 cells where the deletion rather strongly enhances virus release. Next we investigated whether these effects of the E1B19K deletion on virus replication and release are dependent on apoptosis induction. Therefore, we performed burst assays in the presence of a pan-caspase inhibitor (Fig. 5). For A549 and PMelL cells, we were able to completely block both UV- and Ad5/3.19K-.IL-induced PARP cleavage with Q-VD or Z-VAD, respectively (not shown). Q-VD was used for A549 cells because in these cells Z-VAD resulted in partial inhibition of PARP cleavage, only. By inhibition of caspase activity during infection of A549 cells, the enhanced release phenotype of Ad5/3.19K-.IL was almost completely reverted. Total infectious particle production for Ad5/3.19K-.IL and Ad5/3.19K+.IL and viral release of Ad5/3.19K + .IL, however, were not affected. In PMelL cells, inhibition of caspase activity during virus infection resulted in an approximately 10-fold

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Fig. 3. Apoptosis induction and spread-independent cytotoxicity of E1B19K+ or E1B19K− transgene-encoding oncolytic adenoviruses in A549 and PMelL cells. (a) A549 or PMelL cells were infected with Ad5/3.19K-.IL or Ad5/3.19K + .IL and were harvested at the indicated time post-infection. Control cultures were mock infected or were treated by UV irradiation for apoptosis induction. Immunoblots were performed to detect PARP or Pro-caspase 3 cleavage. Casp.3, caspase 3; PARP, Poly (ADP-ribose) polymerase. (b) A549 or PMelL cells were infected with Ad5/3.19K-.IL, Ad5/3.19K + .IL or Ad5/3Luc1 at indicated titers. Forty-eight hours post-infection, cytotoxicity was visualized by crystal violet staining of surviving cells. vp, viral particles. (c) A549 or PMelL cells were infected with Ad5/3.19K-.IL or Ad5/3.19K + .IL and were harvested 48 h post-infection. Control cultures were mock infected or were treated by UV irradiation for apoptosis induction. Cells were analyzed by Annexin/PI staining and flow cytometry. Data shown are the mean percentages of Annexin negative, PI positive (PI+), Annexin positive, PI negative (A+), double positive (A+/PI+) or double negative (A−/PI−) cells of three independent experiments. For mock-infected cells, the mean percentage of double negative cells was 95.0% for A549 and 88.8% for PMelL. For UV-irradiated cells, the mean percentage of Annexin positive, PI negative cells was 40.19% for A549 and 58.1% for PMelL.

increase in total virus particle production, but reduced virus release (ratio supernatant/cells) for both Ad5/3.19K-.IL and Ad5/3.19K + .IL. Furthermore, attenuation of infectious virus particle production by E1B19K deletion in these cells was partially reversed. These results argue that early apoptosis induction resulting from E1B19K deletion is required for increased release of Ad5/3.19K-.IL in A549 cells and at least contributes to the reduction of replication efficacy in PMelL cells. Modulation of the expression of transgenes inserted into oncolytic adenoviruses by E1B19K deletion Having demonstrated that the deletion of E1B19K in transgeneencoding oncolytic adenoviruses results in early apoptosis of infected cells and modulation of virus spread, we next investigated how transgene expression is affected. As expected from our previous work (Rivera et al., 2004b), expression of transgenes from the late transcription unit of oncolytic adenoviruses Ad5/3.19K-.IL and Ad5/ 3.19K + .IL was weaker than transgene expression directed from the CMV promoter of replication-deficient Ad5/3Luc1 early after infection

(Fig. 6a). However, transgene expression for oncolytic adenoviruses increased dramatically over time surpassing transgene expression of Ad5/3Luc1 at 14 h (A549) or 2 days (PMelL) post-infection. Importantly, transgene expression after infection with Ad5/3.19K-.IL and Ad5/3.19K + .IL was quite similar during the first day postinfection for both cell types, excluding detrimental effects of early apoptosis induction due to deletion of E1B19K. Transgene expression by Ad5/3.19K + .IL continuously increased until the last day of measurement in both cell types. In PMelL cells, transgene expression by Ad5/3.19K-.IL was lower than by Ad5/3.19K + .IL beginning at day 2 post-infection and from day 4 no further increase was observed. This result correlates with reduced virus replication and spread of this virus in PMelL cells. In A549 cells, transgene expression by Ad5/3.19K.IL clearly surpassed transgene expression by Ad5/3.19K + .IL at 4 days post-infection, correlating with increased virus spread. Beginning at 6 days post-infection, transgene expression by Ad5/3.19K-.IL dropped, parallel to increasing viral cell lysis and thus loss of cells, as observed microscopically. To address this limitation of the experiment and allow for a better comparison of transgene expression between Ad5/

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Fig. 4. Viral replication and release of E1B19K+ versus E1B19K− transgene-encoding oncolytic adenoviruses in A549 and PMelL cells. A549 or PMelL cells were infected with Ad5/3.19K-.IL, Ad5/3.19K + .IL or replication-deficient Ad5/3Luc1. Two or 4 days post-infection, supernatants (SN) and cells were harvested separately and titers of infectious viral particles were determined by TCID50 assay. Left panels show total infectious virus particles (cells + supernatants); center panels show infectious viral particles in the supernatant; right panels show the ratio of infectious viral particles in supernatants to infectious viral particles in cells to indicate virus release. Infections were performed in triplicates. Columns show mean titers, arrow bars show standard deviations. Numbers show fold titer differences and asterisks indicate statistical significant differences (p b 0.05).

3.19K-.IL and Ad5/3.19K + .IL in A549 cells, we performed infections with 10-fold virus dilutions and performed parallel cytotoxicity and reporter gene assays at 5 days and 10 days post-infection (Fig. 6b). For the replication-deficient virus, luciferase expression increased with increasing virus titers, but not between 5 and 10 days post-infection. For the replication-competent oncolytic viruses, we detected in general increasing luciferase expression with both increasing virus titers and progressing infection time. However, when cell lysis was evident by crystal violet staining, luciferase activities leveled or were

undetectable due to complete loss of cells for both transgeneencoding oncolytic adenoviruses. The former was observed for Ad5/ 3.19K-.IL at 1 vp/cell on day 5 and at 0.001 vp/cell on day 10, but for Ad5/3.19K+.IL only at the highest titer, 10 vp/cell, on day 10. Of note, transgene expression by Ad5/3.19K-.IL was superior to transgene expression by Ad5/3.19K + .IL by 2.5 orders of magnitude on day 5 (at 0.0001 to 0.1 vp/cell) and 4 orders of magnitude on day 10 (0.0001 vp/ cell), i.e. at virus titers for which loss of cell substrate was not a limiting factor. Indeed, at 5 days post-infection, transgene expression

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Fig. 5. Effect of caspase inhibition on viral replication and release of E1B19K+ or E1B19K− transgene-encoding oncolytic adenoviruses in A549 and PMelL cells. Cells were infected as described in Fig. 4 in the presence or absence of caspase inhibitors. Three days post-infection, supernatants and cells were harvested separately and infectious viral particles were determined. Left panels show total infectious virus particles (cells + supernatants); center panels show infectious viral particles in the supernatant; right panels show the ratio of infectious viral particles in supernatants to infectious viral particles in cells to indicate virus release. Infections were performed in triplicates. Columns show mean titers, arrow bars show standard deviations. Numbers show fold titer differences and asterisks indicate statistical significant differences (p b 0.05).

by Ad5/3.19K + .IL was already superior to transgene expression by Ad5/3.19K + .IL at 10 days post-infection. We conclude that in A549 cells the deletion of E1B19K did not interfere with transgene expression by Ad5/3.19K-.IL during the first replication cycle and resulted in a dramatically increased transgene expression beginning at 4 days post-infection due to accelerated virus spread. Apoptosis induction, virus replication and transgene expression in NCH468 and PMelA cells infected with E1B19K+ versus E1B19K− transgene-encoding oncolytic adenovirus We next investigated whether the properties of the E1B19Kdeleted, transgene-encoding adenovirus described above for A549 and

PMelL cells could also be observed in further cell cultures. Therefore, we studied NCH468 low passage glioblastoma cells and PMelA low passage melanoma cells (derived from a different patient than PMelL), as further representatives of cells supporting the respectively enhanced or reduced spread phenotype of the E1B19K-mutant adenovirus (Fig. 2). These cells were infected with Ad5/3.19K-.IL or Ad5/3.19K + .IL and were characterized for apoptosis induction, infectious particle production and transgene expression. In both NCH468 and PMelA cells, cleavage of Pro-caspase 3 (Fig. 7a) was not detectable or minimal 12 to 48 h after infection with Ad5/3.19K + .IL, but was detected beginning at 12 h after infection with Ad5/3.19K-.IL. Also PARP cleavage was stronger and earlier after infection with Ad5/3.19K-.IL in both cell cultures (not shown). This indicates that the

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Fig. 6. Transgene expression by E1B19K+ or E1B19K− transgene-encoding oncolytic adenoviruses in A549 and PMelL cells. (a) Cells were infected with Ad5/3.19K-.IL, Ad5/3.19K + . IL or replication-deficient Ad5/3Luc1 at 500 (A549) or 5000 (PMelL) vp/cell (left panels) or at 0.5 (A549) or 5 (PMelL) vp/cell (right panels). At indicated time points, cells were harvested and luciferase activity was determined. Infections were performed in triplicates. Shown are mean values. For clarity of presentation, standard deviations are not shown and were below 22% (A549, 6–22 h), 20% (A549, 2–10 days), 23% (PMelL, 6–22 h) or 33% (PMelL 2–10 days). (b) A549 cells were infected with Ad5/3.19K-.IL, Ad5/3.19K + .IL or Ad5CMVLuc at indicated titers for cytotoxicity assay and luciferase assay in parallel. At 5 (upper panels) and 10 days (lower panels), cells were stained by crystal violet to visualize cytotoxicity and luciferase activities were determined for cells infected in parallel. Infections for luciferase assay were performed in triplicates. Columns show mean titers, arrow bars show standard deviations. # indicates that luciferase activity was below the detection limit. Asterisks indicate statistical significant differences for comparisons between Ad5/3.19K-. IL and Ad5/3.19K + .IL with p b 0.05.

E1B19K deletion results in apoptosis induction after adenovirus infection in both NCH468 and PMelA cells, which correlates to what we observed for A549 and PMelL cells (Fig. 3). Infectious virus particle production (supernatant + cells) after infection with Ad5/3.19K-.IL was approximately 10-fold increased compared with Ad5/3.19K + .IL in NCH468 cells (Fig. 7b). This was different from A549 cells, in which both viruses produced similar amounts of infectious viral particles (Fig. 4). Importantly, virus release (ratio supernatant/cells) was more than two orders of magnitude higher for the E1B19K mutant virus in NCH468 cells (Figs 7b), which matches with the results for A549 cells (Fig. 4). In contrast, infectious viral particle production was severely reduced by deletion of E1B19K in PMelA cells. This reduction was even stronger than observed for PMelL cells (Fig. 4). Finally, we investigated transgene expression by oncolytic adenoviruses Ad5/3.19K-.IL and Ad5/3.19K + .IL after infection of NCH468 and PMelA cells. Therefore, we performed infections with 10-fold virus dilutions and cytotoxicity and reporter gene assays in parallel at 5 days and 10 days postinfection (Fig. 7c). This allowed us to judge the cellular status with respect to both cell lysis and transgene expression. In NCH468 cells, we principally detected for the replication-competent oncolytic viruses increasing luciferase expression with both increasing virus titers and

progressing infection time. However, luciferase activities leveled or were undetectable when cells were lost due to cell lysis (see crystal violet staining). This was observed for Ad5/3.19K-.IL at virus titers ≥1 vp/cell on day 5 and ≥0.01 vp/cell on day 10, but for Ad5/3.19K + .IL only at 1 vp/cell, on day 10. Transgene expression by Ad5/3.19K-.IL was superior to transgene expression by Ad5/3.19K + .IL by approximately 10-fold on day 5 (at 0.001 to 0.1 vp/cell) and approximately 50-fold on day 10 (0.001 vp/cell), i.e. at virus titers for which loss of cell substrate was not a limiting factor. These data demonstrate a strongly increased transgene expression of the E1B19K-deleted virus due to accelerated virus spread. This confirms our results for A549 cells (Fig. 6b), although the difference between Ad5/3.19K-.IL and Ad5/3.19K + .IL with respect to both transgene expression and oncolysis was smaller in NCH468 cells. In PMelA cells, transgene expression 5 and 10 days after infection with Ad5/3.19K-.IL was markedly attenuated compared with Ad5/ 3.19K + .IL-infected cells, which is in accord with the strongly reduced virus replication (Fig. 7b) and spread (crystal violet stainings in Fig. 7c) of Ad5/3.19K-.IL in PMelA cells. These properties match the results for PMelL cells (Figs. 4 and 6), although both virus replication and transgene expression were more severely attenuated by the E1B19K deletion in PMelA cells. We conclude that attenuated oncolysis of

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Ad5/3.19K-.IL in PMelA cells is due to reduced production of infectious virus particles and results in reduced transgene expression. In NCH468 cells, increased virus particle production and their accelerated release are the basis for the enhanced spread phenotype of Ad5/3.19K-.IL, which also results in strongly increased transgene expression. Overall, these results confirm our observations for the properties of Ad5/3.19K.IL in PMelL versus A549 cells. Discussion This study demonstrates that E1B19K deletion of transgeneencoding oncolytic adenoviruses in susceptible cell types not only

mediates accelerated viral release and spread, but also dramatically increases transgene expression from late viral transcription units. Both accelerating viral spread by early viral release/cell lysis and combining viral oncolysis with therapeutic gene expression have been reported to improve virotherapy in pre-clinical and clinical studies (Sauthoff et al., 2000; Harrison et al., 2001; Hermiston and Kuhn, 2002; Yan et al., 2003; Subramanian et al., 2006; Freytag et al., 2007; Gros et al., 2008). It has not been clear to date, however, as to whether a combination of both strategies is feasible. In this regard, three scenarios could have been envisioned: (i) attenuated viral replication resulting in an extended lifespan of infected cells allows for sufficient time for therapeutic protein synthesis whereas early lysis aborts

Fig. 7. Apoptosis induction, infectious virus particle production and transgene expression by E1B19K− versus E1B19K+ transgene-encoding oncolytic adenoviruses in NCH468 and PMelA cells. (a) NCH468 or PMelA cells were infected with Ad5/3.19K-.IL or Ad5/3.19K + .IL or were mock infected and harvested at indicated times post-infection. Immunoblots were performed to detect Pro-caspase 3 cleavage. (b) NCH468 or PMelA cells were infected with Ad5/3.19K-.IL, Ad5/3.19K + .IL or replication-deficient Ad5/3Luc1. Four days postinfection, supernatants (SN) and cells were harvested separately and titers of infectious viral particles were determined by TCID50 assay. Left panels show total infectious virus particles (cells + supernatant); center panels show infectious viral particles in the supernatant; right panels show the ratio of infectious viral particles in supernatants to infectious viral particles in cells to indicate virus release. Infections were performed in triplicates. Columns show mean titers, arrow bars show standard deviations. Numbers show fold titer differences and asterisks indicate statistical significant differences (p b 0.05). (c) NCH468 and PMelA cells were infected with Ad5/3.19K-.IL, Ad5/3.19K + .IL or Ad5CMVLuc at indicated titers for both cytotoxicity assay and luciferase assay. At 5 and 10 days post-infection, crystal violet stainings and luciferase assays were performed for cells infected in parallel. Infections for the luciferase assay were performed in triplicates. Columns show mean titers, arrow bars show standard deviations. # indicates that luciferase activity was below the detection limit. Asterisks indicate statistical significant differences for comparisons between Ad5/3.19K-.IL and Ad5/3.19K + .IL with p b 0.05.

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Fig. 7 (continued).

production of therapeutic proteins, (ii) transgene expression is efficient before early host cell lysis and viral release and, consequently, accelerated virus spread causes enhanced therapeutic protein expression from an increased number of infected cells, or (iii) a combination of both, i.e. early lysis and viral release reduces therapeutic protein expression by each infected cell, which is, however, compensated by increased virus spread. Our results show that the second scenario holds true if early viral release and enhanced spread by E1B19K deletion are combined with transgene expression from the late adenoviral transcription unit. The deletion of E1B19K expression has been the most widely investigated mutation for improving lytic potency of adenoviruses. Indeed, this mutant features a substantial increase in spreaddependent adenoviral cell killing activity (Sauthoff et al., 2000; Harrison et al., 2001; Subramanian et al., 2006). We show that lytic activity of transgene-encoding oncolytic adenoviruses can be increased by E1B19K deletion up to several orders of magnitude in different tumor cells. We further demonstrate that the induction of apoptosis, specifically caspase activation, is responsible for this outcome by causing accelerated release of infectious viral particles at least in A549 cells. This, however, was of concern to the “arming” approach because in addition to the reduced time for transgene expression by early lysis, early apoptosis induction might further interfere with transgene expression. Nevertheless, our results reveal that deleting E1B19K does not affect transgene expression and activity in infected cells that show the accelerated spread phenotype. Even more, accelerated virus spread resulted in dramatically increased

cumulative transgene expression in cultures of such cells. Specifically, both viral spread/oncolysis and transgene expression by the E1B19K mutation virus were dramatically increased at 10 days post-infection: for NCH468 cells approximately 100-fold (oncolysis) and 50-fold (transgene expression); for A549 cells approximately 10,000-fold and 1000-fold. Of note, in A549 cells, transgene expression by the E1B19K mutant virus was already at 5 days post-infection superior to transgene expression observed at 10 days post-infection for the matching E1B19K wild-type virus. These results were obtained even in the context of restricting transgene expression to late viral replication by insertion of the transgene into the late viral transcription unit. Transgene expression from late viral transcription units has been widely implemented using internal ribosome entry, as we did here, or alternative splicing (Fuerer and Iggo, 2004; Jin et al., 2005; Cascante et al., 2007). This strategy is considered advantageous because of the high efficiency of the expression of viral genes and because late transgene expression is dependent on replication of the viral genome and should in consequence be tumor-specific for oncolytic adenoviruses. Transgenes have also been inserted into early viral transcription units of oncolytic adenovirus genomes (Hawkins et al., 2001; Rivera et al., 2004b) or have been inserted as independent transcription unit including a promoter (Freytag et al., 1998; Wildner et al., 1999; for an overview of gene expression strategies in oncolytic adenoviruses, see also Nettelbeck, 2008). It remains to be determined how transgene expression in these formats is influenced by the E1B19K deletion. However, it can be speculated that earlier onset of transgene

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expression is even less sensitive than late transgene expression toward mutations that result in early apoptosis induction and/or viral release. Ad5 mutants other than the E1B19K deletion have been reported to cause an early viral release/enhanced spread phenotype in tumor cells, namely C-terminal truncations of the I-leader or of the E3/19k protein (Yan et al., 2003; Subramanian et al., 2006; Gros et al., 2008). For these mutants, the molecular basis of early viral release has been reported to be distinct from apoptosis induction. Rather, earlier onset of virus genome replication and thus early entry into late phase of the viral life cycle (truncated I-leader) or membrane permeabilization (truncated E3/19k) have been revealed. As our study demonstrates that early viral release resulting in enhanced viral spread per se does rather increase transgene expression, we speculate that also these mutations can improve transgene expression from “armed” oncolytic adenoviruses. The results of our study underline that the interaction of adenovirus infection with host cell apoptosis pathways is complex. Several adenoviral genes have pro- or anti-apoptotic activity when investigated individually and anti-apoptotic activity during early replication is thought to be required to prevent premature cell death and abortive virus replication in normal host cells. Recent studies have reported that adenoviral cell killing of cancer cells is not apoptotic, since (i) minimal or no caspase activation was detected after adenoviral infection and (ii) neither expression of proteins with anti-apoptotic activity nor treatment with caspase inhibitors blocked viral oncolysis (Abou El Hassan et al., 2004; Baird et al., 2008). We obtained similar results with the E1B19K wild-type virus for A549 cells. However, for low passage melanoma cells, we detected PARP and Pro-caspase 3 cleavage during late adenovirus infection. Furthermore, caspase inhibition during virus infection resulted in an approximately 10-fold increase in infectious virus particle production for PMelL cells. Thus, adenovirus infection indeed activates apoptosis in these cells that counteracts productive virus replication. This corresponds with the observation that deletion of E1B19K and concomitant increased apoptosis induction resulted in attenuated adenovirus replication as measured by infectious viral particle production in both low passage melanoma cultures investigated. However, caspase inhibition only partially reversed this attenuation, as shown in PMelL cells, and thus caspase-independent effects might also be involved. In contrast, deleting E1B19K did not interfere with productive adenovirus replication in A549 cells, even mediated increased infectious virus particle production in NCH468 cells and showed pro-release activity in both cell types. The dramatic increase in viral release was apoptosis dependent, as shown in A549 cells. Thus, the E1B19K deletion resulted in apoptosis induction in all cell types analyzed, which, however, had opposing effects on virus replication and oncolysis in A549 and NCH468 cells versus PMelL and PMelA cells. Clearly, it will be of interest to further investigate the mechanism of adenovirus release in cancer cells and how it is affected by activation of the apoptosis program. Our data are in accord with earlier studies investigating the phenotype of E1B19K mutant adenoviruses. These have shown that deletion of E1B19K results in cellular responses typical for apoptosis induction during adenovirus infection, but differently affects infectious virus particle production in different cell types. Thus, virus yields were reported to be reduced in KB cells, HeLa cells and monocytes but increased in WI38 fibroblasts and a panel of tumor cell lines (Subramanian et al., 1984a, 1984b; Pilder et al., 1984; White et al., 1986; Hu and Hsu, 1997; Liu et al., 2004). Others confirmed mildly reduced infectious virus particle production in HeLa cells, but observed that E1B19K deletion did not affect virus production in A549 and WI38 cells (Telling et al., 1994). Both apoptosis induction and attenuation of virus yields by E1B19K mutant adenoviruses in HeLa cells were shown to be counteracted by caspase inhibition (Chiou and White, 1998), which is similar to the properties we identified for the mutant virus in PMelL cells.

An important result of our study with respect to clinical applications of armed oncolytic adenoviruses is that the oncolysis phenotype of a mutant adenovirus can be cell type dependent. Indeed, opposing and drastic effects of the E1B19K mutant on viral spread were observed in different cancer cell types. Furthermore, cumulative transgene expression by oncolytic adenoviruses correlated with viral spread and in consequence was either reduced or increased, dependent on the cell type. In consequence, future applications of adenoviruses engineered for improved lytic activity should consider tumor type restrictions. For example, our results for low passage tumor cell cultures imply that the E1B19K mutant adenovirus is not advantageous for treatment of malignant melanoma, at least not for each patient. This is in accordance with previous observations (Schmitz et al., 2006). It remains to be shown whether the enhanced spread phenotypes of the I-leader and E3/19K mutant viruses are likewise cell type dependent and more specifically, whether they mediate enhanced virus spread in those cell types in which the E1B19K mutant was attenuated. In light of these results, approaches to identify viral mutants with increased cell killing potency by random mutagenesis and bioselection seem very attractive because they allow for the selection on the individual tumor type of interest (Yan et al., 2003; Subramanian et al., 2006; Gros et al., 2008). Conclusions We report that deletion of E1B19K in transgene-encoding oncolytic adenoviruses triggers apoptosis induction in infected cells resulting— for several, but not all tumor cells—in accelerated viral release without interfering with transgene expression. This was observed even with transgene expression restricted to late virus replication. In consequence, viral spread is accelerated causing remarkable increases in both oncolysis and cumulative transgene expression. Thus, we suggest deleting E1B19K, or inserting similar mutations that accelerate virus release, for optimization of armed oncolytic adenoviruses. However, as the release and spread phenotype of viral mutants can be cell type dependent, as shown here for the E1B19K deletion, improved efficacy of such mutant viruses needs to be confirmed in the specific tumor to be targeted. As the time window for viral oncolysis in patients is limited by mounting anti-viral immune responses, mutations of viral genes facilitating both accelerated virus spread and increased transgene expression have the potential to critically improve the efficacy of armed oncolytic adenoviruses in cancer therapy. Materials and methods Cell culture Human tumor cell lines A549 (lung adenocarcinoma), SK-MEL-28 (melanoma) and C8161 (melanoma, kindly provided by Danny Welch, Birmingham, AL), and low passage glioblastoma cells NCH82, NCH89 and NCH468 (Karcher et al., 2006) were cultivated in DMEM (Invitrogen, Karlsruhe, Germany). Two hundred ninety-three cells and low passage melanoma cells PMelL and PMelA (purified from skin metastases; Nettelbeck et al., 2004) were cultivated in RPMI1640 (Invitrogen). Media were supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA, Cölbe, Germany), 100 IU/ml penicillin and 100 μg/ml streptomycin (both Invitrogen). For low passage melanoma and glioblastoma cultures, 10 mM HEPES (Lonza, Cologne, Germany), 250 ng/ml Amphotericin (Cambrex, Verviers, Belgium) and 20 μg/ml gentamycin (Invitrogen) were added. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2. Recombinant adenoviruses For a schematic outline of the adenovirus genomes generated and used in this study, see Fig. 1. Ad5/3.19K + .IL was generated by

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replacing the Ad5 knob domain of AdΔfiberIL (Rivera et al., 2004b) with the knob domain of adenovirus serotype 3. For Ad5/3.19K-.IL, the E1B19K deletion was derived by PCR with oligonucleotides 19K5′ (5′-CGA GGA CTT GCT TAA CGA GC-3′) and 19K-3′ (5′-GGA CGG AAG ACA ACA GTA GC-3′) from Ad337 (kindly provided by Matthias Dobbelstein). Virus particles were produced by transfection of A549 cells with PacI-digested genome plasmids using Lipofectamine (Invitrogen). These viruses and Ad5Δ24E3- (Suzuki et al., 2002) were further amplified in A549 cells. The replication-deficient adenoviruses Ad5CMVLuc and Ad5/3Luc1 (with a chimeric fiber; Krasnykh et al., 1996) were amplified in 293 cells. Viruses were purified by 2 rounds of CsCl equilibrium density gradient ultracentrifugation. Verification of viral genomes and exclusion of wild-type contamination was performed by PCR. Physical particle concentration (viral particles (vp)/ml) was determined by OD260 reading; infectious viral particle titers were determined by TCID50 assay on 293 cells. Cytotoxicity assay For the determination of virus-mediated cytotoxicity (and spread), 5 × 104 tumor cells were seeded either in 48- or 24-well plates and were infected the next day in 200 or 250 μl of growth medium containing 2% FBS. Four hours post-infection, growth medium containing 10% FBS was added. When cell lysis was observed at the lowest titer (Fig. 2) or at indicated time post-infection (Figs. 3, 6 and 7), cells were fixed and stained with 1% crystat violet in 70% ethanol for 20 min, followed by washing with tap water to remove excess color. Plates were dried and images were captured with an EPSON Perfection V500 Photo scanner. Western Blot analysis For the determination of apoptosis induction, 6 × 105 cells were seeded in 10 cm dishes and were infected with the indicated viruses at 10.000 vp/cell or were mock infected in 5 ml growth medium containing 2% FBS. Two hours post-infection, growth medium containing 10% FBS was added. As a positive control for apoptosis induction, cells were irradiated with UVC at 80 kJ for PMelL (Stratalinker, Stratagene, Waldbronn/Karlsruhe, Germany) or UVB at 3 × 0.5 J/cm2 for A549 (Waldmann UV 181 BL, company, Villingen-Schwenningen, Germany). Cells were lysed 16 h (PMelL) or 26 h (A549) post-irradiation or at indicated time points postinfection in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP40 (Igepal), 1% sodium desoxycholate, 0.1% SDS, 1 mM PMSF, 20 mM sodium fluoride and 2 mM sodium orthovanadate. Antibody binding was visualized using enhanced chemiluminescence (Pierce ECL, Thermo Fisher Scientific, Bonn, Germany). PARP antibody was obtained from BD Biosciences, Heidelberg, Germany (cat. no.: 556494) and Caspase-3 antibody from Imgenex, San Diego, CA (cat. no.: IMG-144A). Flow cytometric analysis of cell death Cytofluorometric analysis of cell death by Annexin/propidium iodide (PI) staining was performed using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). Therefore, 6 × 105 cells were seeded in 10 cm dishes and were infected the next day with the indicated adenoviruses at 10.000 vp/cell or were mock infected in 5 ml growth medium containing 2% FBS. Two hours post-infection, growth medium containing 10% FBS was added. Control cells were irradiated for apoptosis induction as described above. Cells were harvested and stained 16 h (PMelL) or 26 h (A549) after irradiation 48 h post-infection following the manufacturer's protocol and were analyzed with a FACSort (BD Biosciences).

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Burst assay To determine yield and release of infectious viral particles, 5 × 104 cells were seeded in 24-well plates. The next day, cells were infected at 10 vp/cell in a volume of 250 μl growth medium containing 2% FBS. Two hours post-infection, the medium was removed and cells were washed twice with PBS to remove unbound viruses. Then 1 ml of growth medium containing 10% FBS was added in the presence or absence of the caspase-family inhibitors Z-VAD-FMK (BioVision, Mountain View, USA; cat. no.: 1010-100) or Q-VD-OPH (Imgenex; cat. no.: IMI-2309-1) at a concentration of 50 μM, which was replenished every 2 days. After 2, 3 or 4 days, supernatants and cells were harvested separately and viruses were released from cell pellets by three cycles of freeze-thaw. Infectious virus particles were determined by TCID50 assay on 293 cells. Luciferase assay For the determination of transgene expression kinetics, 5 × 104 cells were seeded in 24-well plates. The next day, cells were infected in a volume of 250 μl growth medium containing 2% FBS. Two hours post-infection, growth medium containing 10% FBS was added. Luciferase activity of cell lysates was determined at indicated time points post-infection using a luciferase assay system (Promega, Mannheim, Germany). Statistical analysis Differences between indicated groups were analyzed using the Student's t test. P values of b0.05 were considered statistically significant. Acknowledgments We thank David T. Curiel (UAB, Birmingham, AL), Detlef Dieckmann (Erlangen University Hospital, Germany), Matthias Dobbelstein (University of Göttingen, Germany), Bert Vogelstein (Johns Hopkins University, Baltimore, MD) and Danny Welch (UAB, Birmingham, AL) for research material and Mathias Leber for technical support. This work was supported by the Helmholtz Association of National Research Centers (Helmholtz-University Group Grant) and by grants from the Deutsche Forschungsgemeinschaft DFG (SFB 643, Teilprojekt A5), the Wilhelm Sander-Stiftung (grant 2003.118.1), the German-Israeli Foundation (I-817-38.11/2004), and by the Monika Kutzner-Stiftung to DMN. References Abou El Hassan, M.A., van der Meulen-Muileman, I., Abbas, S., Kruyt, F.A., 2004. Conditionally replicating adenoviruses kill tumor cells via a basic apoptotic machinery-independent mechanism that resembles necrosis-like programmed cell death. J. Virol. 78 (22), 12243–12251. Alemany, R., Balague, C., Curiel, D.T., 2000. Replicative adenoviruses for cancer therapy. Nat. Biotechnol. 18 (7), 723–727. Baird, S.K., Aerts, J.L., Eddaoudi, A., Lockley, M., Lemoine, N.R., McNeish, I.A., 2008. Oncolytic adenoviral mutants induce a novel mode of programmed cell death in ovarian cancer. Oncogene 27 (22), 3081–3090. Cascante, A., Abate-Daga, D., Garcia-Rodriguez, L., Gonzalez, J.R., Alemany, R., Fillat, C., 2007. GCV modulates the antitumoural efficacy of a replicative adenovirus expressing the Tat8-TK as a late gene in a pancreatic tumour model. Gene Ther. 14 (20), 1471–1480. Chinnadurai, G., 1983. Adenovirus 2 Ip+ locus codes for a 19 kd tumor antigen that plays an essential role in cell transformation. Cell 33 (3), 759–766. Chiou, S.K., White, E., 1998. Inhibition of ICE-like proteases inhibits apoptosis and increases virus production during adenovirus infection. Virology 244 (1), 108–118. Freytag, S.O., Rogulski, K.R., Paielli, D.L., Gilbert, J.D., Kim, J.H., 1998. A novel threepronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy. Hum. Gene Ther. 9 (9), 1323–1333. Freytag, S.O., Stricker, H., Peabody, J., Pegg, J., Paielli, D., Movsas, B., Barton, K.N., Brown, S.L., Lu, M., Kim, J.H., 2007. Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer. Molec. Ther. 15 (3), 636–642.

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