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Journal of Virology, April 2006, p. 3904-3911, Vol. 80, No. 8
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.8.3904-3911.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of the Recombinant Adenovirus Vector AdYB-1: Implications for Oncolytic Vector Development

Gabriel Glockzin,1,2 Klaus Mantwill,1 Karsten Jurchott,3 Alexandra Bernshausen,1 Axel Ladhoff,3 Hans-Dieter Royer,4 Bernd Gansbacher,1 and Per Sonne Holm1,5*

Institut fuer Experimentelle Onkologie und Therapieforschung, Technische Universitaet Muenchen, Klinikum rechts der Isar, Munich, Germany,1 Department of Surgery, University of Regensburg Medical Centre, Regensburg, Germany,2 Max-Delbrueck Centrum für Molekulare Medizin, Berlin, Germany,3 Center of Advanced European Studies and Research (CAESAR), Bonn, Germany,4 XVir Therapeutics GmbH, Munich, Germany5

Received 27 July 2005/ Accepted 18 January 2006


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ABSTRACT
 
Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection. However, we have previously reported that nuclear accumulation of the human transcription factor YB-1 by regulating the adenoviral E2 late promoter facilitates viral DNA replication of E1-deleted adenovirus vectors which are widely used for cancer gene therapy. Here we report the promotion of virus-mediated cell killing as a new function of the human transcription factor YB-1. In contrast to the E1A-deleted vector dl312 the first-generation adenovirus vector AdYB-1, which overexpresses YB-1 under cytomegalovirus promoter control, led to necrosis-like cell death, virus production, and viral release after infection of A549 and U2OS tumor cell lines. Our data suggest that the integration of YB-1 in oncolytic adenoviruses is a promising strategy for developing oncolytic vectors with enhanced potency against different malignancies.


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INTRODUCTION
 
Human adenoviruses have developed complex processes that regulate both the induction and the suppression of apoptosis. While E1A, E4orf4, and E3-11.6K act as proapoptotic proteins when tested individually, E1B-19K, E1B-55K in complex with E4orf6, and certain E3 proteins, which are encoded by early-transcribed genes, act as antiapoptotic proteins. In the early phase of infection the E1A-mediated host cell apoptosis is antagonized by E1B-55K, partly in cooperation with the E4orf6 gene product (33, 44, 55). Moreover, E1A is reported to induce apoptosis together with E4orf4 independently of p53 (29, 30, 31). This E1A function is suppressed by E1B-19K (34). However, in the literature, cell death induced by selective replicating adenoviruses is often referred to as apoptosis, which has been challenged in a recent study. The authors conclude in their studies that conditionally replicating adenoviruses die a necrosis-like programmed cell death (35).

Viral DNA replication occupies a central position in the adenovirus life cycle. Efficient viral DNA replication is a requirement for production of adenoviral particles, host cell lysis, and release of new viruses. It depends on the expression of the adenoviral E2 region, which encodes three essential proteins (45). E2 expression is controlled by two viral promoters, the E2 early promoter, which is E1A dependently activated by the human transcription factor E2F (23, 28), and the E2 late promoter, which takes control of the E2 expression about 6 h after infection (11, 48).

Recently, we could show that the E1/E3-deleted recombinant vector AdYB-1, which expresses the human transcription factor YB-1 under cytomegalovirus (CMV) promoter control, facilitates adenoviral replication. Overexpression of YB-1 leads to its nuclear accumulation and binding to the adenoviral E2 late promoter and in consequence promotes viral replication independently of E1A (15). Moreover, the significance of YB-1 for adenoviral replication was proven in another study using dl520 (14), an E1A-mutated adenovirus, which replicates in cells where YB-1 is located in the nucleus. These studies indicated that the early adenoviral gene products E1B-55K and E4orf6 are involved in translocation of YB-1 from the cytoplasm into the nucleus. Thus, the human cellular transcription factor YB-1 in conjunction with E1B-55K and E4orf6 plays an important role in adenoviral replication.

YB-1 is a member of a family of DNA-binding proteins which are characterized by a highly conserved nucleic acid recognition domain, the so-called cold shock domain, and specifically interacts with a sequence motif termed Y-box, which is characterized by the presence of an inverted 5'-CCAAT sequence (7). In addition to the regulation of transcription, YB-1 is a multifunctional protein that affects splicing, translational control, and repair of damaged DNA by interacting with several repair proteins (6, 17, 20, 22, 32). Several studies investigating cancer patient samples established a predictive value of overexpressed/nucleus-localized YB-1 for drug resistance and tumor progression in breast, ovarian, lung, synovial, and prostate cancer (2, 10, 18, 21, 24, 38, 39, 46, 47). Moreover, YB-1 interacts with p53 (40) and functions as a transcriptional repressor of the cell death-associated fas gene (25). The importance of YB-1 for cell growth and survival was demonstrated by inhibition using antisense strategies: adenocarcinoma, hepatoma, fibrosarcoma, and colon cancer cells cease proliferating and die (26).

In the present article we evaluated the influence of the transcription factor YB-1 on the mode of cell killing, production, and release of viral particles after infection of two different tumor cell lines with AdYB-1 in vitro. As a control vector we used the E1A-deleted adenovirus dl312, which is capable of replicating and producing viral particles in the utilized cancer cell lines depending on the multiplicity of infection (MOI) (37). Here we show that the overexpression of YB-1 leads to improved production and release of adenoviral particles without induction of apoptosis independently of E1A. Owing to its remarkable dual role in facilitating adenoviral replication and cell killing/viral release, YB-1 offers new strategies in developing novel oncolytic adenoviral vectors.


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MATERIALS AND METHODS
 
Cell lines and culture conditions. 293 cells were kindly provided by Frank Graham (McMaster University, Hamilton, Ontario, Canada). 293, U2OS (ATCC HTB-96, human osteosarcoma, p53+ Rb+), HeLa (ATCC CCL-2, human cervix carcinoma, p53+ Rb+, human papillomavirus E6/E7), and A549 (ATCC CCL-185, human lung carcinoma, p53+ Rb+) cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS). All media were supplemented with 200 µg/ml L-glutamine, 100 µg/ml penicillin, and 25 µg/ml streptomycin.

Recombinant adenovirus vectors. The E1/E3-deleted adenovirus vector AdYB-1 expresses the human transcription factor YB-1 under CMV promoter control as described by Holm et al. (15). The E1A mutant virus dl312 (19) and the E1/E3-deleted vector dl70-3 (4) have also been described earlier. The E1-deleted adenovirus vector AdCMVLacZ served as a control for electron microscopy. All vectors are based on human adenovirus type 5. Virus was purified by two consecutive cesium chloride gradient centrifugations and additionally dialyzed overnight. To exclude contamination of the E1A-deleted vectors by wild-type E1A, PCR with specific E1A gene primers was performed (E1A fw, 5'-ATGGCCGCCAGTCTTTTG-3'; E1A rev, 5'-GCCATGCAAGTTAAACATTATC-3'). PCR was performed with 1 min at 95°C, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min and a final extension at 72°C for 5 min. PCR with E2A primers was performed as a positive control (for primer sequences see below). Viral titers of AdYB-1, dl312, dl70-3, and AdCMVLacZ were determined by plaque assay using 293 cell monolayers.

Adenovirus infection. Subconfluent cells were infected with adenovirus at an MOI of 10 to 200 PFU/cell in Opti-MEM containing 2% FCS. The cells were incubated for 1 h at 37°C in a 5% CO2 atmosphere with brief agitation every 10 min. After incubation, the virus-containing infection medium was removed and replaced by Dulbecco's modified Eagle's medium supplemented with 10% FCS.

Reverse transcription-PCR (RT-PCR) and PCR analysis. Total RNA was prepared from cultured cell lines 72 h after infection. Two micrograms of total RNA was used for reverse transcription. A 1:20 dilution of the synthesized cDNA was applied to subsequent PCR. PCR was performed with 1 min at 95°C, followed by 30 cycles of 95°C for 45 s, 55°C for 45, and 72°C for 45 s and a final extension at 72°C for 5 min.

For detection of adenoviral death protein (ADP) and E1B-19K within the vector genome viral DNA was used and PCR was performed as described above. PCR products were separated in a 1% agarose gel and visualized with ethidium bromide. The primer sequences were as follows: 5'-ATGTCAGCATCTGACTTTGGCC-3' (ADP fw), 5'-ATCGAGGAATCATGTCTC-3' (ADP rev), 5'-CGTCTTCACCACCATGGAGA-3' (glyceraldehyde-3-phosphate dehydrogenase [GAPDH] fw), 5'-GGCCATCACGCCACAGTTT-3' (GAPDH rev), 5'-ATGGAGGCTTGGGAGTGTTTG-3' (E1B-19K fw), and 5'-TCATTCCCGAGGGTCCAG-3' (E1B-19K rev).

Southern blot analysis. Replication analysis was performed on A549 and U2OS cells infected with adenovirus vectors at different MOIs. Cells (5 x 105) were infected with indicated viruses. Virus DNA was isolated 72 h after infection. Viral replication was determined by Southern blot analysis using a specific 32P-labeled E2A probe synthesized by PCR (E2A fw, 5'-GTCGGAGATCAGATCCGCGT-3'; E2A rev, 5'-GGTCCTCGTCGTCTTCGCTT-3'). To differentiate between input virus DNA (infection) and newly produced virus DNA (replication), cells were infected with methylated adenoviruses produced on 293-PMT cells that were kindly provided by André Lieber (University of Washington, Seattle) as described earlier by Nelson and Kay (36). In contrast to the probe described by Nelson and Kay, we used the specific E2A cDNA probe mentioned above for Southern blot analysis.

Plaque assay for virus yields. Determination of virus yield after infection of A549 and U2OS cells at different MOIs was performed by plaque assay on 293 cell monolayers. Cells were scraped into the cell culture medium 72 h after infection and centrifuged at 3,000 rpm for 10 min. Virus particles were harvested from the cells by three cycles of freezing and thawing. After additional centrifugation at 3,000 rpm for 10 min, supernatant was used for the quantification of virus particles. To distinguish between intracellular and released viral particles, cells and cell culture medium were collected separately 5 days after infection and treated as mentioned above. Following this procedure the plaque assay was performed using 293 cells as indicator cells.

Crystal violet staining. Cell culture medium was removed 5 days after infection with AdYB-1 and dl312 at different MOIs, and cells were washed with phosphate-buffered saline (PBS) and stained with 1 ml of crystal violet staining solution (60% ethanol, 3.3% formalin, 4.3% acetic acid, 10 mg/ml crystal violet) per well. After 15 min of incubation, crystal violet staining solution was removed and cells were washed twice with PBS and air dried.

Immunofluorescence analysis. For immunofluorescence analysis, a polyclonal affinity-purified YB-1 rabbit antibody was used. Cells were grown on slides, fixed with acetone-methanol, and stained using a 1:200 dilution of anti-rabbit immunoglobulin G-fluorescein F(ab')2 fragment (Boehringer Mannheim, Germany) as described earlier by Bargou et al. (2). Cell nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI; Roth, Karlsruhe, Germany).

Analysis of apoptosis. Induction of apoptosis in cells infected with 50 PFU of AdYB-1/cell was measured using annexin V-FLUOS (Roche, Germany) following the manufacturer's instructions. Cells (5 x 105) were washed with PBS and centrifuged at 200 x g for 5 min. Cells were resuspended in 100 µl of annexin V-FLUOS labeling solution, diluted in incubation buffer containing propidium iodide (PI), and incubated for 15 min at room temperature. Fluorescence was analyzed by flow cytometry using filters for fluorescein and PI detection.

Electron microscopy. For ultrastructural analysis, confluent monolayers of infected HeLa and A549 cells were washed three times with 0.1 M sodium phosphate buffer (pH 7.2) and fixed with 2.5% glutaraldehyde in the same buffer for 30 min at room temperature. Cell layers were washed again with buffer and postfixed for 30 min with 1% osmium tetroxide. For further processing the fixed cells were scraped from the culture dishes; collected by centrifugation; embedded in low-melting-point agarose; and subsequently dehydrated, infiltrated, and embedded in Epon resin according to standard procedures. Finally thin sections were cut from resin blocks, mounted on 200-mesh copper grids, and stained with uranyl acetate and lead citrate. Sections were examined on a Zeiss EM10CR transmission electron microscope at 60 kV.


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RESULTS
 
Confirming replication capacity and ADP expression of dl312. To investigate the influence of E1B-55K and E1B-19K expression on the replication efficiency of dl312, Southern blot analysis was performed. The results as shown in Fig. 1a indicate that the E1A-deleted vector dl312 exhibits a higher replication capacity than the E1A/E1B-deleted adenovirus dl70-3 after infection of U2OS cells at different MOIs. Moreover, we investigated the presence of E1B-19K and ADP genes within the genome of dl312 and the expression of ADP in comparison to wild-type adenovirus type 5 (wt-Ad5) and AdYB-1. The E3-11.6K (ADP) region is detectable within the viral genomes of wt-Ad5 and dl312 (Fig. 1b). ADP-specific mRNA can be detected by RT-PCR after infection of A549 cells with wt-Ad5 and dl312, but not with AdYB-1 (Fig. 1c), suggesting ADP expression in dl312-infected cells.


Figure 1
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  		FIG. 1. Replication capacity and ADP expression of dl312. (a) Purified total DNA from U2OS cells infected with the indicated adenovirus vectors was digested with KpnI and separated on a 1% agarose gel. Southern blot analysis was performed using an E2A-specific DNA probe. Lanes 1 to 3, cells infected with dl70-3 at MOIs of 20, 100, and 500 PFU/cell, respectively; lane 4, 1-kb DNA ladder; lanes 5 to 7, cells infected with dl312 at MOIs of 20, 100, and 500 PFU/cell, respectively. (b) PCR analysis was performed using viral DNA as indicated and specific primers for E1B-19K and ADP. wt-Ad5 was used as a positive control. (c) RT-PCR was performed after total mRNA preparation from A549 cells 72 h after infection with the indicated vectors at an MOI of 100 PFU/cell (wt-Ad5, 10 PFU/cell). Noninfected cells were used as a negative control. Detection of GAPDH mRNA transcripts served as a control for reverse transcription and subsequent cDNA amplification.

Overexpression of YB-1 leads to nuclear localization. To investigate the localization of YB-1 in AdYB-1-infected HeLa cells, we performed immunofluorescence studies. Figure 2 shows that, in uninfected HeLa cells, YB-1 protein was predominantly located in the cytoplasm in the perinuclear space (control). In contrast, after infection with AdYB-1 with 50 PFU/cell, exogenous YB-1 accumulated in the nuclei of HeLa cells, where it was distributed in speckles.


Figure 2
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  		FIG. 2. Nuclear accumulation of YB-1 after infection with AdYB-1. The figure shows indirect immunofluorescence of HeLa cells after infection with AdYB-1 at an MOI of 50 PFU/cell. Cells were treated with a polyclonal rabbit anti-human YB-1 antibody. Nuclei were stained with DAPI. Arrows indicate YB-1 localization. Uninfected cells served as a control.

Replication analysis of AdYB-1 and dl312. We could previously show that AdYB-1 but not dl312 efficiently replicates in A549 cells at an MOI of 50 PFU/cell (15). It is established that E1A-independent replication of first-generation adenovirus vectors occurs in different tumor cells at high MOIs. For example mRNAs of the adenoviral early genes were not detectable in HeLa cells infected with the E1A-deleted adenovirus dl312 at an MOI of 20 PFU/cell in vitro. In contrast dl312 showed sufficient gene expression and strong viral DNA replication after infection at a high MOI (200 PFU/cell) (37). We therefore investigated the DNA replication of AdYB-1 in comparison to dl312 at low and high MOIs. To differentiate between adenovirus input DNA and DNA replication, we used methylated adenoviruses generated on 293-PMT cells as previously described by Nelson and Kay (36). Methylation of a XhoI recognition site prevents cleavage by the restriction enzyme XhoI. Therefore, two signals are detectable after viral DNA replication. The 8.0-kb signal represents the uncleaved material, and the 6.5-kb signal represents the newly produced DNA cleaved by the restriction enzyme XhoI. As shown in Fig. 3, Southern blot analysis with methylated adenovirus vectors revealed that there was no input DNA detectable 72 h after infection. Hence, every specific signal in the Southern blot analysis is caused by replicated DNA. Since DNA detected by Southern blot analysis originated from newly replicated virus 72 h after infection, the following studies were performed with unmethylated virus to avoid influence of methylation on viral DNA replication. Consistent with published data, we found DNA replication after infection of A549 with dl312 at an MOI of 50 PFU/cell. Similar results were obtained with U2OS cells, and replication occurred in a dose-dependent manner (Fig. 4). In contrast, we found DNA replication after infection of A549 and U2OS cells with the E1/E3-deleted adenovirus vector AdYB-1 at an MOI of 10 PFU/cell. The signal generated by AdYB-1 after infection with 50 PFU/cell is comparable with that with dl312 infection at an MOI of 200 PFU/cell. At high MOIs both vectors showed strong DNA replication.


Figure 3
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  		FIG. 3. Distinguishing input adenovirus from replicated virus by methylated AdYB-1. Total cellular DNA was isolated from A549 cells infected with AdYB-1 at MOIs of 10, 50, and 100 PFU/cell 72 h after infection and digested with XhoI. Adenovirus DNA replication was demonstrated by Southern blot analysis using a specific 32P-labeled E2A cDNA probe. Lane 1, wt-Ad5-infected A549 cells; lane 2, mixture of DNA isolated from wt-Ad5-infected 293 and 293-PMT cells; lanes 3 to 5, AdYB-1-infected A549 cells. The upper band represents the DNA of virus used for infection.


Figure 4
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  		FIG. 4. Replication analysis of AdYB-1 and dl312. Purified total DNA from A549 or U2OS cells infected with unmethylated adenovirus vectors was digested with KpnI and separated on a 1% agarose gel. Southern blot analysis was performed using an E2A-specific DNA probe. Lane 1, wt-Ad5-infected cells; lane 2, mock-infected cells; lanes 3 to 5, cells infected with AdYB-1 at MOIs of 10, 50, and 100 PFU/cell, respectively; lanes 6 to 8, cells infected with dl312 at MOIs of 10, 50, and 100 PFU/cell, respectively.

Induction of CPE. As shown in Fig. 5, AdYB-1 led to cytopathic effect (CPE), whereas dl312 did not affect cell growth even 8 days after infection. The evaluation of CPE by AdYB-1 and dl312 after infection at different MOIs revealed that viral DNA replication and production of virus particles did not positively correlate with virus-induced cell lysis. The results suggest that overexpression of YB-1 in the E1/E3-deleted adenovirus vector AdYB-1 caused CPE at low (50 PFU/cell) and high (200 PFU/cell) MOIs. After 5 days nearly all infected A549 cells were lysed. In contrast, cells infected with dl312 at an MOI of 50 PFU/cell, 100 PFU/cell, or 200 PFU/cell did not develop CPE (Fig. 6a). The crystal violet staining of A549 cells 5 days after infection with AdYB-1 and dl312 clearly displays the differences in lytic ability (Fig. 6b). These results indicate that overexpression of YB-1 plays an important role in adenoviral cell killing.


Figure 5
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  		FIG. 5. Induction of CPE in cells infected with AdYB-1 and dl312. CPE was evaluated by microscopy after infection of A549 with AdYB-1 and dl312 at an MOI of 100 PFU/cell on days 2, 4, 6, and 8. p.i., postinfection.


Figure 6
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  		FIG. 6. Dose-dependent induction of CPE in cells infected with AdYB-1 and dl312. CPE was evaluated by microscopy and crystal violet staining after infection of A549 cells with AdYB-1 and dl312 at a low MOI (50 PFU/cell) and a high MOI (200 PFU/cell). Noninfected cells were used as a negative control. (a) Microscopy pictures on days 2 and 5 postinfection (p.i.); (b) crystal violet staining on day 5 postinfection.

Generation of infectious particles. We next investigated the correlation between adenovirus DNA replication and production of infectious particles (defined as PFU) after infection with AdYB-1 and dl312 (Fig. 7a and b). The results, which were reproducible in a second independent experiment, demonstrate that AdYB-1 and dl312 produce comparable amounts of PFU after infection of tumor cells at high MOIs (200 and 400 PFU/cell). After infection at an MOI of 10 PFU/cell, dl312 produced no (A549) or very low (U2OS) amounts of infectious particles in contrast to AdYB-1. We saw the most impressive difference after infection at an MOI of 50 PFU/cell. Here AdYB-1 generated up to 100-fold-more infectious particles than dl312. As expected, the production of infectious particles corresponded to adenovirus DNA replication.


Figure 7
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  		FIG. 7. Production of virus particles in cells infected with AdYB-1 and dl312. A549 and U2OS cells were infected with wt-Ad5, AdYB-1, and dl312 at different MOIs. At 72 h after infection cells were isolated and viral particles were harvested by freezing and thawing. Then a plaque assay was performed on 293 cell monolayers. The analysis was repeated to confirm results. (a) A549 cells infected with wt-Ad5 (5 to 10 PFU/cell), AdYB-1, and dl312 as indicated; noninfected cells were measured as a negative control. (b) U2OS cells infected with wt-Ad5 (5 to 10 PFU/cell), AdYB-1, and dl312 as indicated; noninfected cells were used as negative controls.

Viral release. The results of the previous experiments suggest that there is no tight correlation between production of viral particles and CPE/cell killing after infection at a high MOI (Fig. 5, 6, and 7). We therefore isolated cells and cell culture medium separately for the plaque assay to differentiate between intracellular and released infectious particles after infection of A549 cells with AdYB-1 or dl312 at an MOI of 50 PFU/cell. Five days after infection signs of CPE were visible in cells infected with AdYB-1 but not in cells infected with dl312 (data not shown). The plaque assay revealed that AdYB-1 produced more infectious particles than dl312 5 days after infection (Fig. 8). Moreover, in contrast to the infection with dl312 the majority of newly produced viruses were released from AdYB-1-infected cells into the medium. These results provide an indication of the involvement of the human transcription factor YB-1 in virus-mediated cell killing and release of infectious particles.


Figure 8
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  		FIG. 8. Release of virus particles. To distinguish between intracellular and released viral particles, A549 cells infected with AdYB-1 and dl312 at an MOI of 100 PFU/cell and cell culture medium were isolated separately 5 days after infection. p.i., postinfection. Viral particles were harvested by freezing and thawing. Virus yield (PFU/ml) was determined by plaque assay on 293 cell monolayers.

Infection by AdYB-1 is not associated with apoptosis. Since AdYB-1-infected cells did not display the microscopic characteristics of apoptosis-like membrane blebbing, we performed flow cytometry of AdYB-1-infected A549 and U2OS cells using annexin V and PI staining, which represents a well-established early hallmark of programmed cell death. Cells treated with camptothecin served as a positive control for apoptosis. The fluorescence-activated cell sorting analysis showed that the number of annexin V- and PI-positive A549 cells increased after AdYB-1 infection. In addition, compared to cells treated with camptothecin only a minor portion of cells showed signs of apoptosis (Fig. 9). A similar outcome was obtained in infected U2OS cells (data not shown). Our results indicate that AdYB-1 triggers phosphatidylserine (PS) externalization accompanied with PI staining without induction of the classical apoptotic pathway.


Figure 9
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  		FIG. 9. Apoptosis detection assay after infection with AdYB-1. A549 cells (2 x 105) were infected with 50 PFU of AdYB-1/cell. As a positive control for apoptosis, uninfected cells were exposed for 48 h to 5 µM camptothecin. The majority of AdYB-1-infected cells were PI and annexin V positive. Whereas camptothecin-treated A549 cells were subject to apoptosis, only a minor portion of AdYB-1-infected cells displayed a similar effect.

Next, we asked whether signs of apoptosis were visible by transmission electron microscopy. We found by an inspection of approximately 500 AdYB-1-infected HeLa and A549 cells that less than 5% showed signs of apoptosis (Fig. 10a). However, characteristic morphological changes including nuclear morphology as marginal accumulation of fibrous material were visible in nearly all infected cells. In E1-minus AdLacZ-infected cells the cellular ultrastructure appeared normal (Fig. 10a and b). Interestingly, not all of the AdYB-1-infected cells were able to produce viral particles (Fig. 10b). This observation is consistent with our previous results (15).


Figure 10
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  		FIG. 10. Electron microscopy of adenovirus-infected cells. (a) HeLa cells. (A) Cells infected with E1-minus AdCMVLacZ replication-deficient adenovirus; (B) example of AdYB-1-infected cell displaying typical features of apoptosis (nuclear shrinkage, chromatin condensation, and membrane blebbing); (C) representative example of the majority of AdYB-1-infected cells displaying necrosis-like cell death (enlarged nucleus, no membrane blebbing). (b) A549 cells. (A) Noninfected cells (control); (B) cells infected with E1-minus AdCMVLacZ; (C) cells infected with wt-Ad5; (D) representative AdYB-1-infected cell displaying necrosis-like cell death (enlarged nucleus, no membrane blebbing) and production of virus particles; (E) example of an AdYB-1-infected cell without virus particle production.


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DISCUSSION
 
Replication-selective oncolytic adenoviruses represent a rapidly growing experimental therapeutic platform. However, critical parameters for antitumor efficacy with oncolytic adenoviruses are among other things efficient viral replication, cell killing, and distribution to the tumor site, which need to be overcome before virotherapy can fulfill its goals in the clinic.

In this study we demonstrate that overexpression of the human transcription factor YB-1 leading to its nuclear localization (Fig. 2) not only facilitates viral DNA replication of E1A-deleted adenovirus vectors but is also involved in the induction of cytopathic effect/cell killing and release of viral particles in AdYB-1-infected tumor cells. Thus, our data indicate that YB-1 directly supports virus-mediated necrosis-like cell killing, since signs of apoptotic hallmarks were missing. Nevertheless, the molecular mechanisms responsible for the accelerated cell lysis by YB-1 need to be further investigated.

For the generation of more effective oncolytic viruses it is essential to understand the mechanisms of cell killing. In this scenario, the occurrence of cell death early during infection would severely reduce virus production and thus limit the spread of virus to distant tumor cells. In our studies we used the adenoviral vectors AdYB-1 and dl312 which lack E1A. E1A is associated with caspase 3 activation and poly(ADP-ribose) polymerase cleavage (5). Since both vectors lack E1A expression, the differences in their lytic potential are not caused by E1A-mediated cell killing. Yet we cannot rule out that E1B-19K present in dl312 (Fig. 1b) plays a role in blocking dl312 in cell killing.

However, previously it has been reported that conditionally replicating adenoviruses kill tumor cells via an apoptosis-independent mechanism that resembles necrosis-like cell death, although PS externalization was detected (27, 35). Our experiments with AdYB-1 showed that AdYB-1-induced cell death also was associated with PS externalization without induction of the typical apoptotic pathway. Our results are also in line with our observation that the majority of AdYB-1-infected cells lacked electron microscopic signs of apoptosis such as chromatin condensation, membrane blebbing, and nuclear shrinkage. Instead we observed an increase in nuclear size rather than a nuclear shrinkage (Fig. 10a and b).

It has been reported that the E3-encoded ADP plays a critical role in release of adenoviruses from infected cells (51). Its expression is mainly controlled by the adenovirus major late promoter (MLP) at the late phase of infection. Its overexpression in an adenovirus vector leads to efficient cell killing and viral release (9, 13). In dl312 a functional E3 region is present, whereas the genome of AdYB-1 is E1/E3 deleted (Fig. 1b). Since E3-ADP gene expression is controlled by the MLP, it is probable that it is correlated with the expression of adenovirus late genes (45). The production of large yields of viral particles after infection of tumor cells with the E1A-deleted adenovirus vector dl312 at an MOI of ≥100 PFU/cell shows that MLP is active and that adenovirus late genes are efficiently expressed. Moreover, Bett et al. have shown that E3-ADP is expressed in cells infected with the adenovirus vector dl309, which contains an E3 deletion identical to that of dl312 (3). In consistency with these findings our results suggest that E3-ADP expression is unaffected by the alterations in dl312 (Fig. 1c), although we did not perform Western blot analysis. However, E3-ADP mRNA is not detectable in AdYB-1-infected cells either by RT-PCR (Fig. 1c) or by Northern blot analysis (unpublished data). Therefore, we could exclude that the AdYB-1-mediated cell lysis is a function of the adenoviral death protein. Furthermore, we assume that E3-ADP expression by dl312 is not sufficient for cell killing and release of newly produced viral particles.

The observation that dl312 infection led to cell killing of multidrug-resistant cancer cells (14), which are characterized by nuclear accumulation of YB-1 (2), indicates the involvement of this human transcription factor in adenovirus-mediated cell lysis. Infection with dl312 at high MOIs leads to diffuse accumulation of YB-1 in host cell nuclei (15), whereas infection with AdYB-1 caused a rather speckled distribution (Fig. 2), suggesting a colocalization with the nuclear viral inclusion bodies (15, 41, 42). Since localization of cellular YB-1 is unaffected by replication-deficient adenoviruses (15), we conclude that the speckled signals in the nucleus represent exogenous YB-1. The reason for the discrepancy between dl312- and AdYB-1-mediated distribution of YB-1 is still unknown. Since adenovirus is very complex and several proteins are multifunctional, such as E1A, E1B-55K, and E4orf6, we assume that there may exist additional viral and cellular proteins besides YB-1 which contribute to YB-1 translocation and distribution in the nucleus. In addition, it is tempting to speculate that E1A plays a pivotal role in YB-1 translocation by modification of YB-1. Our results indicate that overexpression and/or nuclear localization of YB-1 is necessary for promoting virus-mediated cell lysis. However, the involvement of viral inclusion bodies and their interaction with YB-1 in this process have to be further investigated.

Development of CPE/cell killing and release of viral particles are highly important functions of selectively replicating adenovirus vectors for cancer gene therapy. Different experiments using ADP-overexpressing vectors for infection of cancer cells showed that the oncolytic potential of selectively replicating vectors is significantly increased by virus release (52, 53, 56). In contrast to E3-ADP, YB-1 facilitates viral DNA replication, late gene expression, and production of viral particles (14, 15), which is a clear advantage over ADP.

We have shown recently that the adenovirus early genes E1B-55K and E4orf6 are involved in translocating YB-1 from the cytoplasm to the nucleus (15). It is well established that E1B-55K in a complex with E4orf6 relocates p53 to the cytoplasm for degradation (8, 33, 43, 44, 49, 50, 55). The inactivation of the cellular tumor suppressor protein by the two adenovirus gene products prevents p53-mediated cell cycle arrest and apoptosis in adenovirus-infected cells. In the late phase of infection E1B-55K and E4orf6 support the accumulation of late viral mRNA in the cytoplasm and mRNA shuttling from the nucleus to the cytoplasm for the production of viral proteins in infected host cells (1). Thus, E1B-55K and E4orf6 are important for the replication and particle formation of adenoviruses (12, 16, 54). It is obvious to examine AdYB-1 in combination with E1B-55K and E4orf6 expression.

In conclusion, our data suggest that overexpression of the human transcription factor YB-1 facilitates both viral DNA replication, necrosis-like cell killing, and release of viral particles. Considering the clinical application, the tumor-selective expression of YB-1 in oncolytic adenoviruses may be beneficial regarding replication, cell killing, and viral spread. Currently, we are addressing this issue.


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ACKNOWLEDGMENTS
 
We thank Hildegard Kalvelage and Savula Michailidou for excellent technical assistance.

This work was supported by grants from the Novartis Stiftung für Therapeutische Forschung and the Deutsche Forschungsgemeinschaft (DFG; Ho 1482/4-2) to P.S.H.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut fuer Experimentelle Onkologie und Therapieforschung, Technische Universitaet Muenchen, Klinikum rechts der Isar, Ismaninger Str. 22, 81675 Munich, Germany. Phone: 49-89-4140-4462. Fax: 49-89-4140-4476. E-mail: per.s.holm{at}lrz.tum.de. Back


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Journal of Virology, April 2006, p. 3904-3911, Vol. 80, No. 8
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.8.3904-3911.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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