Mucin-1 is expressed in a variety of colon carcinomas and Muc-1/DF3 promoters have been utilized to reduce systemic toxicity through specific gene expression. To overcome weak expression, which is much lower than the widely used cytomegalovirus-promoter (CMV), new adenoviral vectors containing a binary system of transgene amplification have been developed. The Muc-1/DF3 promoter was used to control the expression of a Gal4VP16 fusion protein. This vector also contained Gal4 binding sites enabling the fusion protein to act as a transactivator, inducing transgene expression within the same construct. Mucin-1 expression was analyzed in a variety of colon cancer cell lines. After infection with recombinant adenoviruses, transgene expression was quantified using the luciferase system. Integration of the Gal4VP16-binary resulted in an up to 250-fold increase of Muc-1/DF3-specific gene expression. In mucin-positive cell lines utilizing this amplified Muc-1/DF3 promoter, expression was up to 590-fold higher as compared to the CMV-promoter. Western blot detected the presence of Gal4VP16 in infected muc-1-positive but not-negative cell lines. These new adenoviral vectors combing highly efficient and specific transgene expression and will contribute to the safety and efficacy of experimental approaches in cancer gene therapy.
Mucin-1 is expressed in a variety of colon carcinomas and Muc-1/DF3 promoters have been utilized to reduce systemic toxicity through specific gene expression. To overcome weak expression, which is much lower than the widely used cytomegalovirus-promoter (CMV), new adenoviral vectors containing a binary system of transgene amplification have been developed. The Muc-1/DF3 promoter was used to control the expression of a Gal4VP16 fusion protein. This vector also contained Gal4 binding sites enabling the fusion protein to act as a transactivator, inducing transgene expression within the same construct. Mucin-1 expression was analyzed in a variety of colon cancer cell lines. After infection with recombinant adenoviruses, transgene expression was quantified using the luciferase system. Integration of the Gal4VP16-binary resulted in an up to 250-fold increase of Muc-1/DF3-specific gene expression. In mucin-positive cell lines utilizing this amplified Muc-1/DF3 promoter, expression was up to 590-fold higher as compared to the CMV-promoter. Western blot detected the presence of Gal4VP16 in infected muc-1-positive but not-negative cell lines. These new adenoviral vectors combing highly efficient and specific transgene expression and will contribute to the safety and efficacy of experimental approaches in cancer gene therapy.