Agents for treating malignant diseases using the protein YB-1

The invention concerns agents for the treatment of malignant diseases using the protein YB-1. It makes it possible to produce an E1A-independent replication of adenoviruses in tumour cells in order to destroy these tumour cells, and to destroy tumour cells which contain the protein YB-1 in the nucleus by using E1A-defective adenoviruses.

DETAILED DESCRIPTION OF THE INVENTION 
 EXAMPLE 1 
 Nucleus Located YB-1 Allows for E1 Independent Adenoviral Replication The immortalized epithelial breast cell line HBL-100 was transfected using a vector (pcDNA6N5-HisB, Invitrogen) into which the cDNA of YB-1 was introduced, by using lipofectamine (Gibco). After selection with blasticidine (5 &mgr;l/ml) a stable cell line was isolated and established which over-expressed YB-1 in the nucleus. This newly established cell line was named HBL-100/YB-1. The experimental proof for the successful transfection is shown in FIG. 1 . More particularly, FIG. 1 shows a Western blot of a nuclear lysate of both HBL-100 (lane 1) and HBL-100/YB-1 (lane 2). YB-1 was detected by using a V5 antibody (Invitrogen). Only HBL-100/YB-1 over-expressed YB-1 in the nucleus. Subsequently, both cell line HBL-100 and HBL-100/YB-1 were transformed with a E1 deleted adenovirus (AdlacZ). This adenovirus is not capable of replicating due to a deletion in the E1 region. The infection was performed by using a multiplicity of infection of 100 for 1 hour at 37° C. After infection the infection medium (Optimen supplemented with 2% FCS) was removed and standardised growth medium added (DMEM with 10% FCS). 3 to 5 days post infection a cytopathic effect (CPE) characterised by a rounding-up of the cells, was observed for the infected HBL-100/YB-1 only as depicted in FIG. 2 d ). In contrast to this, HBL-100 cells also infected with the E1 deficient adenovirus (E1-minus AdlacZ) did not show such CPE ( FIG. 2 b )). Uninfected HBL-100 cells and HBL-100/YB-1 cells are depicted in FIG. 2 a ) and FIG. 2 c ), respectively. The CPE observed in connection with cell line HBL-100/YB-1 after infection with an E1-minus recombinant adenovirus proves viral replication within said cell line. This clearly shows that YB-1 allows for an E1-independent viral replication. 
 EXAMPLE 2 
 Oncolysis of Tumor Cells by zn Adenovirus Expressing YB-1 HeLa cells and SkOV3 tumour cells were infected with an E1-minus adenovirus (AdlacZ) and an adenovirus coding for and expressing YB-1 (AdYB1). The cells were infected with a multiplicity of infection of about 100 for 1 hour at 37° C. After infection the infection medium (Optimen with 2% FKS) was removed and conventional growth medium added (DMEM with 10% FKS). FIGS. 3 and 4 show the result of the experiments. AdlacZ infected cells did not show any cytopathic effect, i.e. rounding-up of the cells ( FIGS. 3, B and E), whereas those cells infected with AdYB-1 show a very pronounced cytopathic effect ( FIGS. 3, C and F). This means that the adenoviral vector AdYB-1 replicates and induces cell lysis. 
 EXAMPLE 3 
 Formation of Adenoviral Particles in Cell Lines Expressing YB-1 in the Nucleus This result as shown in example 3 was confirmed in another experiment. HeLa cells were infected with AdYB-1 and AdlacZ (MOI 50). 72 h after infection the cells were prepared for electromicroscopic analysis by standard procedures (2.5% glutaraldehyde embedded in Epon resin and stained with uranyl acetate). Microsections were analysed using a Zeiss EM10CR transmission electron microscope at 60 kV. Adenoviral particles are only detectable in AdYB-1 infected cells ( FIGS. 4, C and D at different magnification). HeLa cells not infected with AdlacZ (B) may not be discriminated from control cells which were not infected (A) using morphological criteria. This example as well as example 2 proves that AdYB-1 undergoes a complete viral life cycle once YB-1 is present in the nucleus of a cell which is infected by an E1-minus adenovirus coding for YB-1. The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.