The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.Adenoviruses have developed efficient strategies to force infected cells into the S phase of the cell cycle (1). This process involves the adenoviral E1A and E1B proteins, which are the first viral proteins to be expressed after infection, and both are essential for viral replication (2, 3). Replication of adenovirus DNA depends directly on interactions between the host cell replication factors NFI, NFII, and NFIII (4) and the three viral replication proteins encoded by the E2 region. The adenovirus E2 transcription unit consists of the E2A and E2B genes, which encode precursor terminal protein pTP, DNA polymerase, and DBP, a multifunctional DNA-binding protein (5). E2 gene expression is driven from two promoters. At early times of infection, E2 gene transcription is under control of the E2 early promoter. At intermediate stages of infection, E2 gene expression is controlled by the E2 late promoter (6). E2 early promoter activity is regulated by adenovirus E1A protein, which controls the activity of the E2F transcription factor by targeting the tumor suppressor protein pRB (7,8). In contrast, activity of the E2 late promoter is repressed by E1A (9). The E2 late promoter is characterized by the presence of a TATA box, two SP1 recognition sites, and three CCAAT boxes. Two of the inverted CCAAT boxes are located at positions Ϫ72 and Ϫ135 relative to the E2 late cap site in a 157-bp sequence of the of the E2 late promoter, which is sufficient for efficient E2 gene transcription (10, 11).Inverted CCAAT boxes have been identified as sites for Y box proteins, which are highly conserved through evolution from prokaryotes to eukaryotes, and they can function as transcriptional, translational, and developmental regulators (12)(13)(14). In eukaryotes, increased expression of Y box proteins in somatic cells is associated with drug resistance and a malignant phenotype (15), and it was discussed that Y box proteins are involved in activating certain genes that are expressed in the S phase of the cell cycle (16). Recently, it has...

BackgroundThe brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. This study determines the expression of YB-1 in glioblastoma (GBM) specimen in vivo and in brain CSC lines. In addition, the capacity of Ad-Delo3-RGD, an YB-1 dependent oncolytic adenovirus, to eradicate CSC was evaluated both in vitro and in vivo.MethodsYB-1 expression was investigated by immunoblot and immuno-histochemistry. In vitro, viral replication as well as the capacity of Ad-Delo3-RGD to replicate in and, in consequence, to kill CSC was determined by real-time PCR and clonogenic dilution assays. In vivo, Ad-Delo3-RGD-mediated tumor growth inhibition was evaluated in an orthotopic mouse GBM model. Safety and specificity of Ad-Delo3-RGD were investigated in immortalized human astrocytes and by siRNA-mediated downregulation of YB-1.ResultsYB-1 is highly expressed in brain CSC lines and in GBM specimen. Efficient viral replication in and virus-mediated lysis of CSC was observed in vitro. Experiments addressing safety aspects of Ad-Delo3-RGD showed that (i) virus production in human astrocytes was significantly reduced compared to wild type adenovirus (Ad-WT) and (ii) knockdown of YB-1 significantly reduced virus replication. Mice harboring othotopic GBM developed from a temozolomide (TMZ)-resistant GBM derived CSC line which was intratumorally injected with Ad-Delo3-RGD survived significantly longer than mice receiving PBS-injections or TMZ treatment.ConclusionThe results of this study supported YB-1 based virotherapy as an attractive therapeutic strategy for GBM treatment which will be exploited further in multimodal treatment concepts.

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.