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.

Elevated expression of the heparan sulphate proteoglycan glypican-3 (GPC3) was found on mRNA and protein levels in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV, which was established by in vitro selection against mitoxantrone. In order to elucidate a putative role of GPC3 in the drug-resistant phenotype, the mitoxantrone-resistant cell line EPG85-257RNOV was transfected with an expression vector construct carrying an anti-GPC3 hammerhead ribozyme. It could be demonstrated that in anti-GPC3 ribozymetransfected cell clones, the GPC3-specific mRNA and corresponding protein expression levels were decreased to levels that are similar to those observed in nonresistant, parental cells. The anti-GPC3 ribozyme-containing clones reduced the mitoxantrone resistance level up to 21% of the original resistance and the crossresistance against etoposide to 33% of the original value. This reversal of drug resistance was accompanied by an increased cellular mitoxantrone accumulation in the anti-GPC3 ribozymeexpressing cells. In conclusion, it was verified that GPC3 is involved in the cellular protection against mitoxantrone in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV. Oncogene (2004) 23, 945-955.

Breast cancer resistance protein ( BCRP ) is a recently identified new member of the superfamily of ATP -binding cassette transporters. BCRP is a``half transporter'' that may homo -or heterodimerize to form an active transport complex. A considerable overexpression of BCRP was reported from various atypical multidrug -resistant tumor cell lines, in particular from those which were established by treatment with mitoxantrone. Thus, BCRP represents a very interesting candidate molecule for reversal of a drug -resistant phenotype. Six hammerhead ribozymes directed against the BCRP -encoding mRNA were designed and tested for their ability to cleave their target molecule. The anti -BCRP ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. BCRP -encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using total RNA prepared from the atypical multidrug -resistant gastric carcinoma cell line EPG85 -257RNOV exhibiting a high BCRP mRNA expression level. One anti -BCRP ribozyme was found to show a very high endoribonucleolytic cleavage activity at physiologic pH and temperature. This ribozyme was characterized in a cell -free system with regard to its specific kinetic parameters using large target molecules.