Construction and characterization of recombinant adenoviruses expressing human BRCA1 or murine Brca1 genes

Recombinant adenoviruses expressing human BRCA1 (AdBRCA1), murine Brca1 (Ad Brca1), three clinically relevant human mutant BRCA1 proteins (t340, C61G, and 1853Stop), or a murine Brca1 C-terminal deletion mutant were constructe d and evaluated in vitro. These recombinants were capable of transducing hi gh-level transgene expression to a wide variety of cell lines in vitro. Thr ee independent methods were utilized to monitor cell growth following trans duction with these recombinants. High-level expression of either the human or mouse wild-type BRCA1 protein was incompatible with maximal levels of ce ll growth. AdBRCA1 transduction inhibited the outgrowth of several human br east and ovarian cell lines in colony formation assays. Flow cytometric ana lysis revealed an accumulation of the transduced cells in the G(0)/G(1) pha se of the cell cycle. This BRCA1-mediated accumulation of cells in G(0)/G(1 ) was accompanied by an increase in the cellular level of hypophosphorylate d pRB. Ad mutant BRCA1 t340, C61G, and 1853Stop viruses were impaired, to v arying degrees, in their ability to transduce a growth-arrested state to th e target cells. Using these same three criteria, overexpression of murine B rca1 by AdBrca1 was also capable of transducing a growth-arrested state to human cells. Deletion of the C-terminus of Brca1 diminished this activity. This panel of adenoviruses may be useful reagents as part of an approach to understand the function of BRCA1/Brca1 in normal breast and ovary and he I p to define the tu mor suppressor defect (s) conferred by clinical BRCA1 mu tations in breast and ovarian cell tumorigenesis.