A dual specificity promoter system combining cell cycle-regulated and tissue-specific transcriptional control

The expression of both proliferation-associated and cell type-specific gene s is a hallmark of both cancer cells and tumor endothelial cells. The possi bility to combine both features in a single transcriptional control unit wo uld greatly increase the selectivity of vectors used for cancer gene therap y. Previous studies by our laboratory have shown that the transcription of several cell cycle genes is regulated by a novel cell cycle-regulated repre ssor, termed CDF-1. This repressor functions by blocking in resting cells t he transcriptional activation by specific factors binding to the upstream a ctivating sequence (UAS), most notably the CCAAT-box binding factor NF-Y/CB F Based on this work we have developed a dual specificity promoter system t hat combines cell type specificity with cell cycle regulation. A chimeric t ranscription factor (Gal4/NF-Y) consisting of the transactivation domain of NF;Band;the DNA-binding domain, of GaGAl4s expressed from a tissue-specifi c promoter. Gal4/NF-Y can bind to a second promoter consisting of a minimal cyclin A promoter with multiple Gal4 binding sites replacing the normal UA S. This leads to the tissue-specific expression of Gal4/NF-Y whose stimulat ory activity on the promoter is restrained in resting cells by the recruitm ent of the CDF-1 repressor to the promoter, the functionality of this syste m is demonstrated for the specific: transcriptional targeting of proliferat ing melanoma cells, where cell cycle regulation was >20-fold and cell type specificity was >50-fold.