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[Cancer Research 64, 322-328, January 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Multidrug-resistant Cancer Cells Facilitate E1-independent Adenoviral Replication

Impact for Cancer Gene Therapy

Per S. Holm1, Hermann Lage2, Stephan Bergmann46, Karsten Jürchott4, Gabriel Glockzin1, Alexandra Bernshausen1, Klaus Mantwill1, Axel Ladhoff2, Anke Wichert2, Joe S. Mymryk7, Thomas Ritter3, Manfred Dietel2, Bernd Gänsbacher1 and Hans-Dieter Royer458

1Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität München, Klinikum Rechts der Isar, München, Germany; 2Institut für Pathologie and 3Institut für Immunologie, Universitätsklinikum Charité, Humboldt-Universität Berlin, Berlin, Germany; 4Max-Delbrück Centrum für Molekulare Medizin, Berlin, Germany; 5Institut für Transplantationsdiagnostik und Zelltherapeutika and 6Institut für Humangenetik und Anthropologie, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany; 7Department of Biochemistry, McMaster University, London, Ontario, Canada; and 8Center of Advanced European Studies and Research (CAESAR), Bonn, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Resistance to chemotherapy is responsible for a failure of current treatment regimens in cancer patients. We have reported previously that the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug resistant-tumor phenotype. YB-1 predicts drug resistance and patient outcome in breast cancer. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In drug-resistant cancer cells and in adenovirus-infected cells YB-1 is found in the nucleus. Nuclear accumulation of YB-1 in adenovirus-infected cells is a function of the E1 region, and we have shown that YB-1 facilitates adenovirus replication. Here we report that E1A-deleted or mutant adenovirus vectors, such as Ad312 and Ad520, replicate efficiently in multidrug-resistant (MDR) cancer cells and induce an adenovirus cytopathic effect resulting in host cell lysis. Thus, replication-defective adenoviruses are a previously unrecognized vector system for a selective elimination of MDR cancer cells. Our work forms the basis for the development of novel oncolytic adenovirus vectors for the treatment of MDR malignant diseases in the clinical setting.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of clinical drug resistance is a major limitation for effective cancer chemotherapy. The classical multidrug-resistant (MDR) phenotype is associated with increased transcription and translation of the mdr1 gene, which encodes P-glycoprotein, a multifunctional drug transporter (1 , 2) . Certain environmental stresses, for instance chemotherapy, UV light irradiation (3 , 4) , and hyperthermia (5) , cause nuclear accumulation of Y-box protein (YB-1). Nuclear localization of the transcription factor YB-1 is associated with transcriptional activation of the human mdr1 gene (3 , 6) . Results from the literature suggest that YB-1 is involved in pleiotropic resistance to different classes of DNA-targeting drugs (7) . YB-1 interacts with p53 (8) and functions as transcriptional repressor of the cell death-associated fas gene (9) , indicating that YB-1 is involved in certain processes that control cell survival. Y-box proteins are characterized by a highly conserved nucleic acid recognition domain, the so-called cold shock domain, and are acting as transcriptional, translational, and developmental regulators (10, 11, 12) . Y-box proteins interact specifically with a sequence motif termed Y-box, which is characterized by the presence of an inverted 5'-CCAAT sequence.

Adenoviruses have attracted considerable attention for their use as vectors for gene therapy. First-generation, replication-deficient adenovirus gene therapy vectors have been constructed with a deletion of the E1 region consisting of the E1A and E1B genes. These viral early genes are required for efficient adenovirus replication (13, 14, 15) .

Because of differential splicing, the E1A region of human adenovirus type 5 encodes for two major proteins with a length of 289 and 243 amino acid residues (16 , 17) . The small E1A protein induces transcription of viral and cellular genes less efficiently than the large E1A protein (18) . Thus, E1A mutant adenoviruses, which produce just the small E1A protein, are replication-defective like E1A-deleted adenoviruses (19) . However, a number of reports demonstrated low-level viral DNA replication and formation of viral particles in cultured cells, and it was hypothesized that as yet unidentified viral or cellular proteins can substitute functionally for E1A in viral promoter regulation (15 , 20, 21, 22, 23) . Adenovirus DNA replication depends on viral replication factors, which are encoded by the E2 genes. Expression of the E2 genes is controlled by E2 early and late promoters during the viral life cycle (24) . Activity of the E2 early promoter is controlled by E1A and the host cell factor E2F (25) . In contrast, activity of the E2 late promoter does not depend on E1A or E2F. We have reported recently that an infection with adenovirus causes nuclear accumulation of YB-1 as a function of the E1 region, and in consequence E2 late promoter activation, which facilitates adenovirus replication (26) . Because YB-1 is mainly located in the nuclei of MDR tumor cells (3) , we investigated whether E1A-deleted adenoviruses (Ad312) and the E1A mutant adenovirus Ad520 (originally termed dl520; Ref. 27 ), which does not express the large 289 amino acid long E1A protein, can replicate in MDR cancer cells. Here we show that both the E1A-deleted and E1A mutant adenoviruses replicate efficiently in MDR cancer cells causing an adenovirus cytopathic effect (CPE) with concomitant cell lysis. Our findings demonstrate that replication-defective adenoviruses are a promising vector system for the treatment of MDR malignant diseases.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
Cells (293; Ref. 28 ) were kindly provided by Frank Graham (McMaster University). The MDR cell lines EPG85–257RDB, EPP85–181RDB (29 , 30) , MCF-7Adr, and HCT-15 (5) were maintained in L-15 medium with 10% FCS. HeLa, 293, U2OS, and A549 cells were maintained in DMEM containing 10% FCS. The cell lines HBL-100 and HBL-100/YB-1 were cultivated in RPMI 1640 with 10% FCS. All of the drug-resistant P-glycoprotein-positive cell lines were treated with 100 ng/ml daunoblastine every 4 weeks to ensure P-glycoprotein expression. All of the media were supplemented with L-glutamine (200 µg/ml), penicillin (100 µg/ml), and streptomycin (25 µg/ml).

Recombinant Adenovirus.
The viral titer of the E1A-deleted adenovirus Ad312 and Ad520 was determined by plaque assays using 293 cell monolayers. To exclude any contamination with wild-type adenoviruses with an intact E1A region in Ad312 virus stocks, PCR was performed using specific primers (26) . Subconfluent cells were infected by addition of 50–100 plaque-forming units/cell of adenoviral vectors to infection medium (OPTIMEM containing 2% FCS) after incubation for 2 h at 37°C in a 5% CO2 atmosphere with brief agitation every 15 min. After infection, the medium was replaced by medium containing 10% FCS.

Preparation of Nuclear Extracts and Western Blot Analysis.
Cells (107) were washed twice in ice cold PBS and permeabilized by incubation for 5 min in 3 ml of ice cold hypotonic lysis-buffer [10 mM Tris-Cl (pH 8.0) supplemented with Complete protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany)]. For the preparation of nuclear extracts, we followed our published procedure (31) . Ten µg of proteins per lane were subjected to electrophoresis on 10% SDS-PAGE. For immunoblotting, standard procedures were used. For the detection of P-glycoprotein we used the monoclonal antibody C219 (Roche Diagnostics). Western blots were developed with the enhanced chemiluminescence system (Amersham).

Electrophoretic Mobility Shift Assay (EMSA).
Preparation of nuclear extracts and conditions for EMSA were described previously (26 , 32) . To detect nuclear expression of YB-1, the following recognition oligonucleotide was used: mdr1 promoter (-86 to -67): TGAGGCTGA TTGGCTGGGCA (the Y-box is italicized). For competition experiments, unlabeled oligonucleotides from the Y-box of the mdr1 promoter and human cyclin E promoter (5) were used in 50-fold and 100-fold excess, respectively.

Determination of Viral Yields.
Cells (106) were infected with Ad312 at a multiplicity of infection (MOI) of 50–100. After infection, the medium was replaced with medium containing 10% FCS. At 72 h after infection, cells were scraped into the culture medium and centrifuged at 2500 rpm for 10 min. The cell pellets were extensively washed with PBS and centrifuged again. PBS was removed, and cells were suspended in 1 ml OPTIMEM. Virus was harvested by multiple cycles of freezing and thawing, and the supernatants were tested for virus production by a plaque assay in 293 cells.

Northern Blot Analysis.
Total RNA of infected cells was isolated using the Trizol system (Life Technologies, Inc.) according to the manufacturer’s instructions. Ten µg of total RNA were size fractionated on 1% agarose-formaldehyde gels, transferred to a nylon membrane (Amersham), and hybridized using a 32P-labeled E2A-cDNA probe as described previously (26) .

Viral DNA Analysis.
For viral replication analysis the cells were infected at a MOI of 10–50, and DNA was isolated 72 h after infection using the Qiagen Purification System (DNeasy Tissue kit) according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Two µg of DNA were digested with the restriction endonuclease Kpn I and size fractionated on 1% agarose gel, transferred to a nylon membrane, and hybridized using a 32P-labeled E2A-cDNA probe.

Generation of an E2A-specific cDNA Probe.
A radioactively labeled cDNA probe for the adenovirus E2A gene was generated by PCR using wild-type Ad5 DNA and specific primers for the E2A gene (33) . Primers used for the amplification of the cDNA were 5'-CCGGACAGGCCGCGTCA-3' and 5'-TGGTGCCGCGCACACCCA-3'. Cycling conditions were 30 cycles consisting of 60 s at 95°C, 60 s at 55°C, and 60 s at 72°C.

Electron Microscopy.
For ultrastructural analysis, confluent monolayers of infected 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 additional processing, the fixed cells were scraped from the culture dishes, collected by centrifugation, embedded in low melting 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.

Analysis of Adenovirus Cytotoxicity.
For determination of virus-mediated cytotoxicity cells were grown in six-well plates and then infected with either Ad312 or Ad520 at a MOI of 1–50 pfu/cell. One h after infection the medium was replaced. After 7 days, the cells were fixed and stained with 1% crystal violet in formaldehyde, followed by washing with water to remove excess of color. All of the experiments were performed in duplicate wells.

Analysis of in Vivo Adenovirus DNA Replication and Oncolytic Activity.
Xenografts were established in 8-week-old BALB/c-nu/nu mice by injecting 5 x 106 257RDB- or HeLa cells suspended in 100 µl of PBS s.c. When tumor volume reached between 200 and 500 mm3 mice were randomized into groups of 4 animals. The first group received Ad520 at a dose of 2.5 x 108 pfu in a volume of 50 µl daily for 3 days. As a control, a second group was treated with normal saline. Tumors were measured every 3–4 days in two dimensions by external caliper, and volume was estimated by the formula 4/3{pi} x a/2 x (b/2)2. After 30 days animals were sacrificed, and tumor DNA was isolated using a Qiagen purification system (DNeasy Tissue kit) according to the manufacturer’s instructions. To detect adenovirus replication, 2 µg of tumor DNA were digested with the restriction endonuclease Kpn I, and size fractionated on 1% agarose gels, transferred to a nylon membrane, and hybridized using a 32P-labeled adenovirus E2A-cDNA probe.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
P-Glycoprotein and YB-1 Expression in MDR Cancer Cells.
To detect P-glycoprotein expression in cancer-derived MDR cell lines we performed Western blot analysis using the monoclonal antibody C219. For this purpose, membrane extracts of several MDR and chemosensitive cell lines were isolated. As shown in Fig. 1Citation , the MDR cell lines 181RDB (pancreatic cancer), 257RDB (gastric cancer), and MCF-7Adr (breast cancer) produced large amounts of P-glycoprotein, whereas the drug-sensitive cell lines HeLa and U2OS did not synthesize any detectable P-glycoprotein. As a next step YB-1 expression was analyzed by an EMSA using nuclear extracts and a radiolabeled Y-box oligonucleotide from the mdr1 gene promoter as a probe (3) . Substantial amounts of YB-1 were detected in nuclear extracts of the three MDR cell lines (Fig. 2Citation , lanes 257RDB, 181RDB, and MCF-7Adr). In contrast, no or trace amounts of YB-1 were detected in drug-sensitive HeLa and U2OS cells, respectively (Fig. 2Citation , lanes HeLa and U2OS). The presence of YB-1 in retarded DNA:protein complexes was controlled by competition with a molar excess of unlabeled Y-box oligonucleotides from the mdr1 gene promoter and an unrelated promoter fragment from the cyclin E gene (Ref. 5 ; data not shown).



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  		Fig. 1. Immunoblot detection of P-glycoprotein with the monoclonal antibody C219. Each lane was loaded with 10 µg of membrane protein. The position of P-glycoprotein is indicated by an arrow.

 


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  		Fig. 2. EMSA for nuclear YB-1 expression in MDR cells. Nuclear extracts of cells were prepared as described in "Materials and Methods." Detection of YB-1 was performed by using a probe from the mdr1 promoter region spanning the YB-1-binding site. The complex of YB-1 with the mdr1 promoter fragment is indicated by an arrow.

 
Efficient Lysis of MDR Cancer Cells by a Replication-defective E1A-deleted Adenovirus Mutant.
MDR cells 181RDB, 257RDB, and chemosensitive control cells (U2OS and A549) were infected at a MOI of 50 with the E1A-deleted adenovirus mutant dl312 referred to as Ad312 throughout the text. To detect replicated adenovirus DNA in infected cells, total DNA was isolated, digested with the restriction endonuclease Kpn I, and processed for Southern hybridization (26) . Fig. 3Citation shows efficient adenovirus DNA replication in MDR pancreatic and gastric cancer cells (Fig. 3Citation , lanes 181RDB and 257RDB). In contrast, viral replication was barely detectable in chemosensitive A549 and undetectable in U2OS cells. To exclude the possibility that any difference in replication efficiency was attributable to a difference in infectivity, we performed an infectivity assay using Ad5CMVlacZ. The assay revealed that there was no difference in Ad5CMVlacZ infectivity for U2OS, A549, 181RDB, and 257RDB cells (data not shown). Next we examined whether Ad312 could induce CPEs in the MDR cell lines. Therefore, the drug-resistant and drug sensitive cells were infected at various MOIs (20–200 pfu/cell), and monitored for the appearance of an adenovirus CPE. As shown in Fig. 4Citation , induction of a CPE occurs 4–7 days after infection in the MDR cell lines (Fig. 4Citation , panels 181RDB and 257RDB). In contrast, drug-sensitive Ad312-infected HeLa and U2OS cells did not develop any sign of an adenovirus CPE. Thus, the E1A-deficient Ad312 vector has the potential to selectively eliminate MDR cancer cells.



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  		Fig. 3. MDR cells support E1A-independent adenoviral DNA replication. Purified total DNA isolated from Ad312-infected cells (50 pfu/cell) was digested with Kpn I and separated on a 0.8% agarose gel. Southern analysis was performed using a specific E2A-cDNA as probe.

 


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  		Fig. 4. Ad312 induces CPE in MDR cells indicating adenoviral replication. Cells were infected with 100 pfu/cell and analyzed for the appearance of a CPE 4–7 days after infection.

 
The Adenovirus E2 Genes Are Transcribed in Ad312-infected MDR Cancer Cells.
We have constructed a recombinant E1-minus adenovirus vector expressing YB-1 as a transgene (AdYB-1) and have shown that YB-1 activates transcription of the E2 genes through the E2 late promoter (26) . The E2 genes encode the three viral replication factors (34) . Next, we investigated whether nuclear localization of YB-1 in MDR cancer cells facilitates E2 gene transcription. To test this issue we analyzed E2 gene activity in Ad312-infected cells by Northern analysis using an E2A-cDNA as a probe (Fig. 5)Citation . As a control, HeLa cells were infected with Ad5 viruses (Fig. 5Citation , panel Ad5). It is recognizable that the E2 gene was not expressed in drug-sensitive Ad312-infected U2OS and HeLa cells (Fig. 5Citation , lanes U2OS and HeLa). In contrast, infected 257RDB and 181RDB cell expressed considerable amounts of E2A mRNA. In MCF-7Adr cells E2A mRNA levels were almost as high as in Ad5-infected HeLa cells. Note that the level of nuclear YB-1 protein was higher in MCF-7Adr cells than in 257RDB and 181RDB cells (Fig. 2)Citation . Thus, the nuclear levels of YB-1 in MDR cancer cells correlate directly with adenovirus E2 gene activity.



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  		Fig. 5. Induction of E2A gene expression in MDR cells. MDR and nonresistant cells were infected with 50 pfu/cell Ad312, total RNA were isolated 40 h after infection, and E2A gene expression was monitored by Northern blotting using a radioactively labeled E2A-cDNA probe. The position of the specific E2A signal is indicated by an arrow.

 
Efficient Ad312 Replication in Breast Epithelial Cells with Constitutive Nuclear Overexpression of YB-1.
To rule out a contribution of unrecognized transcription factors in E2 late promoter regulation in Ad312-infected MDR cancer cells, we investigated whether nuclear overexpression of YB-1 in chemosensitive cells permits viral replication. To this end, we used a cell line with constitutive high level expression of YB-1 (5) . High level expression of YB-1 in a normal diploid breast epithelial cell line (HBL-100) was achieved by transfection with a YB-1 cDNA under control of the cytomegalovirus promoter (5) . For detecting exogenous YB-1 protein, the YB-1 cDNA was fused to a cDNA encoding the V5-epitope (5) . The V5-epitope is derived from a paramyxovirus protein (35) . In HBL-100 cell clones overexpressing the V5 epitope-tagged YB-1 cDNA (HBL-100/YB-1), YB-1 protein was located predominantly in the perinuclear region and in the nucleus (data not shown). In HBL-100 parental cells YB-1 expression is low, and in these cells YB-1 is located in the cytoplasm in the perinuclear region (3) . To confirm nuclear localization of the V5-tagged YB-1 protein in HBL-100/YB-1 cells using a biochemical approach, we analyzed nuclear extracts by immunoblotting with a V5-specific antibody (Fig. 6)Citation . The V5 tag does not affect the function of YB-1, as it is able to specifically interact with the Y-box.9 In addition, the V5 tagged YB-1 has the capacity to transactivate a mdr1 promoter-driven reporter gene (5) . Next, we infected HBL-100/YB-1 and HBL-100 cells with Ad312, and monitored the cells for the appearance of an adenovirus CPE. Fig. 7Citation shows an adenovirus CPE in Ad312 infected HBL-100/YB-1 cells 4–6 days after infection. The infected HBL-100/YB-1 cells exhibited rounded morphology and loss of adherence, whereas the Ad312-infected HBL-100 cells appeared normal. To exclude a potential difference of infectivity between parental HBL-100 and HBL-100/YB-1 cells we performed an infectivity assay using a recombinant adenovirus expressing the lacZ gene. The assay revealed that stable YB-1 transfectants have the same infectivity as the parental HBL-100 cells.4 Thus, YB-1 is important for the completion of an adenovirus life cycle and the development of CPE.



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  		Fig. 6. Overexpression of V5-tagged YB-1 in nuclei of transfected HBL-100 cells. Thirty µg of nuclear extracts from HBL-100 cells and HBL-100/YB-1 cells were size fractionated on SDS-PAGE, transferred to nitrocellulose, and incubated with a monoclonal antibody directed against the 14 amino acid long V5 epitope. The position of V5-tagged YB-1 protein is indicated by an arrow.

 


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  		Fig. 7. YB-1 is important for adenoviral DNA replication. The appearance of a CPE in HBL-100/YB-1 cells indicates adenoviral DNA replication.

 
Production of Infectious Progeny Virus Particles in Ad312-infected MDR Cancer Cells.
To test whether infectious virus particles are generated in Ad312-infected MDR cancer cells, we determined the amount of infectious virus progeny particles in supernatants of infected cells by a plaque assay using 293 cells, which allows replication of E1A-minus adenoviruses. We considered it necessary to control for the presence of E1A-positive adenovirus contaminants in these experiments. To test for contaminating E1A-positive adenoviruses we used three different approaches: (a) E1A-specific PCR (PCR conditions and primer were published elsewhere; Ref. 26 ); (b) E1A-specific immunofluorescence; and (c) a plaque assay on A549 cells. We were unable to detect any E1A-positive contaminants by PCR, we did not detect E1A protein in infected cells, and we were unable to detect an adenovirus CPE even at high MOI.4 A low titer of infectious virus particles could be detected in supernatants of infected drug-sensitive HeLa, U2OS, SKOV3, and A549 cells. However, the Ad312 titers in supernatants of infected MDR cancer cells were 3–4 orders of magnitude higher (Fig. 8)Citation . This result indicates efficient Ad312 replication and progeny particle formation in MDR cancer cells (Fig. 8)Citation .



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  		Fig. 8. MDR cells affect progeny viral formation. MDR and nonresistant cells were infected with 50–100 pfu/cell Ad312, and virus yields were measured by plaque assay on 293 cells. Virus yields are expressed as average from two independent experiments performed in duplicate.

 
Identification of Adenovirus Progeny Particles in Ad312-infected MDR Cancer Cells by Electron Microscopy.
To evaluate our results by another method we examined progeny particle formation in Ad312-infected drug-resistant cancer cells by electron microscopy. As shown in Fig. 9Citation adenovirus particles could be detected in MCF-7Adr breast cancer and 181RDB pancreatic cancer cells. Typical crystalline arrays and randomly scattered individual particles are seen (Fig. 9, B and CCitation , arrows). In addition, characteristic morphological changes including nuclear morphology and marginal accumulation of fibrous material were only visible in the MDR cells (Fig. 9DCitation , arrows).



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  		Fig. 9. Progeny virus particle formation by E1-minus Ad5CMVlacZ in MDR cells. Typical crystalline arrays and randomly scattered individual particles are seen only in MDR-infected cells (B and C).

 
E1A-deleted and E1A Mutant Adenoviruses Induce a Strong CPE in MDR Cancer Cells.
To investigate whether Ad312 and Ad520 induce a CPE in MDR and drug-sensitive cancer cells, CPE assays were performed. As shown in Fig. 10Citation Ad312 and Ad520 caused complete cytolysis of infected MDR 257RDB cancer cells within 7 days. Ad520-infected 257RDB cells were completely lysed at a MOI of 2.5, whereas Ad312-infected 257RDB cells were lysed at a MOI of 30. In contrast, a CPE could not be detected in Ad520- and Ad312-infected drug-sensitive HeLa cells, even at a MOI of 50–100 pfu/cell (data not shown). Our results demonstrate that the E1A mutant adenovirus Ad520 is able to eliminate MDR cancer cells with a much higher efficiency than the E1A-deleted adenovirus Ad312.



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  		Fig. 10. Selective-cytotoxicity of Ad312 and Ad520 in MDR cells (257RDB) and nonresistant cells (HeLa). Cells were infected at indicated MOIs. Seven days after infection cells were stained with crystal violet.

 
Oncolytic Activity of Ad520 in Xenotransplanted MDR Tumors.
To evaluate a potential oncolytic activity of Ad520 we established a mouse xenograft model system using MDR 257RDB- and drug-sensitive HeLa cells. Once tumors had developed in the transplanted mice, they were infected with the E1A mutant adenovirus Ad520, and the tumor volume was monitored over time. The tumor volume data presented in Fig. 11ACitation show that there was a significant decrease in tumor volume in the Ad520-treated group of mice, which carried MDR 257RDB tumors (Fig. 11ACitation , filled rectangles). Four weeks after treatment the relative volume of 257RDB tumors had decreased 72.5% (average of 4 mice) compared with the saline treated group (Fig. 11ACitation , open rectangles). Thus, intratumorally delivered Ad520 displays oncolytic activity in the 257RDB mouse model. In contrast, Ad520 infection had no effect on tumor volumes in mice transplanted with drug-sensitive HeLa cells (Fig. 11ACitation , filled triangles).



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  		Fig. 11. A, inhibition of s.c. 257RDB tumor growth after treatment with Ad520. Tumor received three daily intratumoral injections of 2.5 x 108 pfu/50 µl dose. MDR tumor growth was inhibited significantly in animals treated with Ad520. Whereas nonresistant HeLa tumor growth show no effect after receiving Ad520. {triangleup}, HeLa treated with PBS; {blacktriangleup}, HeLa treated with Ad520; {square}, 257RDB treated with PBS; {blacksquare}, 257RDB treated with Ad520. B, replication of Ad520 in human xenografts after intratumoral administration. Viral replication as evidenced by Southern blot analysis using a specific E2A-cDNA as probe. Lane 1, positive control; Lanes 2–4, 257RDB tumor; Lanes 5–7, HeLa tumor.

 
To determine viral DNA replication of Ad520 in the transplanted tumors, DNA was isolated 30 days after treatment. Fig. 11BCitation shows that all three of the established 257RDB tumors treated with Ad520 allow efficient adenovirus DNA replication (Fig. 11BCitation , Lanes 2–4). In contrast, viral replication was not detectable in Ad520-treated tumors, which were derived from drug-sensitive HeLa cells (Fig. 11BCitation , Lane 5–7). The data show that the E1A mutant adenovirus Ad520 is able to efficiently replicate in MDR 257RDB tumors. These results explain, at least in part, how the oncolytic activity of Ad520 in MDR tumors is brought about.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The development of clinical drug resistance is a major problem for the therapy of malignant diseases. Although certain mechanisms that cause a MDR tumor phenotype are understood, a translation of this knowledge into successful therapeutic regimens in the clinic is still a major challenge. Recently, results obtained in pilot studies suggest that YB-1 could serve as a new tumor biological marker involved in determining chemotherapy resistance and disease progression in breast and lung cancer (36 , 37) . YB-1 nuclear overexpression occurs in many malignant diseases, for example in breast cancer (3) , non-small cell lung cancer (37) , osteosarcoma (6) , and ovarian and colon carcinoma (38 , 39) . Furthermore YB-1 mRNA is highly abundant in glioblastomas (40) and malignant melanomas (41) . In the present study we show that YB-1 is located in the nuclei of pancreatic-, gastric-, and mammary carcinoma cell lines displaying the classical MDR phenotype. In addition to P-glycoprotein, YB-1 controls the expression of the multidrug transporter MRP1 (5) , MRP2 (42) , and matrix metalloproteinase 2 (43) . YB-1 is involved in damage recognition (44) , it interacts with a repair enzyme (45) , binds p53 (8) , and plays a key role in cell proliferation (46) . Furthermore, nuclear accumulation of YB-1 occurs in response to environmental stresses such as genotoxic stress and UV light (47) . These data demonstrate that YB-1 is involved in several aspects of drug resistance, and it was shown that YB-1 is an important regulator of pleiotropic resistance (7) . Thus, YB-1 is a very promising target molecule for the therapy of clinical drug resistance in a variety of malignant diseases.

We have reported recently that YB-1 facilitates adenovirus replication in the absence of the E1 region (26) , and have shown that YB-1 induces transcription of the E2 genes through the E2 late promoter (26) . These findings suggested that replication-defective gene therapy adenovirus vectors can be developed for the treatment of MDR breast-, pancreatic-, and gastric cancer-derived cell lines. And high levels of nuclear YB-1 demonstrated efficient replication of an E1A-deleted (Ad312) and E1A-mutant adenovirus (Ad520) in drug-resistant cells. MDR cells strongly enhanced viral DNA replication and progeny particle formation with concomitant cell lysis. However, it is not understood at this stage how E2 gene expression levels are linked to the efficiencies of particle formation in the three drug-resistant cell lines that we investigated (Figs. 5Citation and 8Citation ). For example, E2A mRNA levels are very high in -infected MCF-7Adr cells; however, the yield of particles is lower than in the other drug-resistant cell types. It is likely that the MDR cancer cells that were used here have different genetic lesions, which are responsible for this phenomenon. Future experiments will clarify these issues. However, an infection of drug-sensitive cells had little or no effect. Given the fact that the human transcription factor YB-1 is located in the nucleus in MDR cells and our recent observation that YB-1 is a downstream effector of the E1-genes, the data demonstrate a correlation between the efficiency of adenovirus DNA replication in the absence of E1A and the presence of YB-1 in the nucleus. Because the E1B Mr 55,000 protein is involved in relocating YB-1 to the nucleus (26) , our results can also explain the difference in replication capacity of an E1B-attenuated adenovirus in drug-sensitive and drug-resistant cell lines (48) .

In search for a more potent oncolytic adenovirus we investigated whether the replication-defective mutant dl520 (Ad520), which produces just the smaller 243 amino acid long E1A protein (19) , is more effective in eliminating MDR cancer cells. Our data show that Ad520 is superior to Ad312 in eliminating MDR cancer cells in vitro. This suggests that the smaller E1A protein from the 12S mRNA contributes to viral replication probably by disrupting the pRb-E2F complex. In addition, E1B-55K and E4orf6 are important for viral replication and particle formation (49) . Several groups have reported (50 , 51) that the 12S E1A product was able to induce low-level expression of certain adenovirus genes e.g., E1B-55K, E4orf6, and E3ADP (adenoviral death protein). Future studies will investigate whether the 12S E1A gene product and/or E1B-55K and E4orf6 contribute to the enhanced efficiency of Ad520 to eliminate MDR cancer cells. In light of these in vitro results we investigated whether Ad520 is able to operate in an in vivo situation. Our results show that Ad520 has a strong oncolytic activity in xenotransplanted MDR tumors. On the basis of these data we think that E1A-defective adenoviruses can ultimately be used to treat MDR tumors in the clinical setting.

We are of course aware that it is now necessary to determine physiological expression patterns of YB-1 in the different tissues of the human body. In this context it is interesting to note that YB-1 mRNA levels are high in fetal tissues for example muscle, brain, lung, adrenal gland, heart, and liver (52) . However, in adult tissues, YB-1 is expressed at much lower levels e.g., adult kidney and breast epithelial cells (3) . To investigate this issue in detail we have begun a systematic analysis using an immunohistochemistry approach.

In cancer gene therapy, the failure to achieve sufficient tumor transduction led to the strategy to use replication-competent adenoviruses. Viral replication seems to be of benefit in the treatment of human cancer, because this may improve transgene delivery and cancer cell killing (53 , 54) . Our data suggest that in a therapeutic setting it is of advantage to induce nuclear accumulation of YB-1 to achieve a more efficient elimination of drug-resistant tumor cells in vivo. We have reported that heat shock leads to a rapid nuclear accumulation of YB-1 in colon cancer cells (5) , and it was shown that heat shock enhances the oncolytic effect of replicative adenoviruses (55) . It was reported that treatment of carcinoma cells with oncolytic adenoviruses in combination with DNA-damaging agents results in enhanced cell killing (56 , 57) . Therefore, it is a therapeutic option to combine adenoviral cancer therapy with other treatment modalities, which cause nuclear accumulation of YB-1 in vivo. Thus, our findings delineate a novel therapeutic approach for MDR malignant tumors.


   ACKNOWLEDGMENTS
 
We thank Thomas Shenk for Ad312. We thank Alexandra Krumnow and Hildegard Kalvelage for technical assistance.


   FOOTNOTES
 
Grant support: Supported by grants from the Berliner Krebsgesellschaft (to H-D. R.), Deutsche Forschungsgemeinschaft (DFG; Ho 1482/2-2; to P. S. H.), Deutsche Krebshilfe (10-1313-La3), and Novartis Stiftung für Therapeutische Forschung (to H. L.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Per S. Holm, Institute of Experimental Oncology, Technical University Munich, Klinikum Rechts der Isar, Ismaninger Str. 22, 81675 Munich, Germany. Phone: 49-89-41404452; Fax: 49-89-41404476; E-mail: per.s.holm{at}lrz.tum.de

9 Hans-Dieter Royer, unpublished observations. Back

Received 2/20/02. Revised 1/ 7/03. Accepted 1/29/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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