Identification of the Role of MnSOD in EGFR-Positive Breast Cancer Development

In accordance with Task 1 of the Statement of Work, the HB4a cell line was characterized with respect to its requirements for normal cellular growth, and the conditions needed to support growth in soft agar. After determining the optimal conditions for supporting growth in soft agar, further effort was placed on optimizing the use of the human erbB2 expression vector. Because a large part of the experimental design included the use of single cell microinjection assays, there was a need to tag the erbB2 construct for ease of identification. Since erbB2 is a membrane protein, I was unwilling to attach a tag directly to the protein. Instead, an Ires-GFP element was placed downstream of erbB2 in the pcDNA background. Transient transfection and microinjection studies confirmed the expression from the Ires-GFP element in HB4a cells. Microinjection experiments were performed using both MnSOD and erbB2-Ires-GFP in HB4a cells to identify the role of MnSOD in mediating the proliferative response to erbB2. The results initially showed a MnSOD mediated inhibition of erbB2 induced cell cycle progression. However, a recent analysis of the dual expression of MnSOD and erbB2 indicated a significant level of promoter competition between the 2 constructs. Current efforts are focused on subcloning the MnSOD cDNA into an expression vector that has been shown to work well with the pcDNA promoter. Once this issue is resolved, I will repeat the analysis of cell cycle progression and continue with Tasks I and 2 as planned. The original document contains color images. All DTIC reproductions will be in black and white.