Mitotic Spindle Directed Therapeutics for Early Stage Breast Carcinoma
Final rept. 31 Aug 2004-30 Aug 2005
MOUNT SINAI MEDICAL CENTER OF THE CITY OF NEW YORK
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For the proposed studies, we used a recombinant adenovirus that carries anti-stathmin ribozyme, Rz184, for inhibiting stathmin expression in breast cancer cells. We used a bicistronic expression vector, pAdCMV5- IRES-GFP, that contains an adenovirus origin of replication and 2.5 kilobases of human adenovirus type 5 AdS DNA for efficient homologous recombination. This vector also contains an internal ribosome entry site IRES element derived from the encephalomyocarditis virus that permits the translation of two open reading frames from the same messenger RNA. The IRES expression cassette of this vector is downstream from a unique cloning site to allow co-expression of a green fluorescent protein GFP selectable marker and the therapeutic gene i.e. the ribozyme. Both the selectable and the therapeutic gene are placed under the control of CMV5, a CMV promoter-enhancer modified for maximal expression. This arrangement allows high-level expression of the transgene and easy visualization andor selection of transduced cells. We also generated a control adenovirus that carries GFP but not the ribozyme. The schematic illustrations of the adenoviral vectors are shown in Fig. 1. We tested the activity of these recombinant adenoviruses in JC and SC 115 in vitro murine models of breast cancer.
- Anatomy and Physiology