Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, coinfection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by backrecombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, α[sub v]β[sub 5]- and α[sub v]β[sub 3]-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdVmediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was 'trans-complementation' of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in... [ABSTRACT FROM AUTHOR]