Figure 3.

AdRac1N17 infection increases Rac1 protein levels, inhibits growth, and decreases superoxide levels in tumorigenic pancreatic ductal epithelial cells that express K-ras.

A. Cells transduced with 100 MOI AdRac1N17 demonstrated increases in Rac1 immunoreactivity with increasing viral titer. No difference was seen with AdEmpty 100 MOI transfection compared with parental cells.

B. Plating efficiency was unchanged with AdRac1N17 infection in H6c7 cells but decreased in KrasT cells. Feeder cells were used by irradiating B1 mouse fibroblasts Twenty-four hours before clonogenic assay, irradiated cells were thawed and diluted in keratinocyte serum free media. Cells were then seeded 3 × 10⁵/ plate overnight until attached, transduced by AdRac1N17 or AdEmpty for 24 hours, recovered in full media without adenovirus for another 24 hours, and then trypsinized and plated for clonogenic survival. No significant changes were seen plating efficiency in H6c7 cells infected with the AdRac1N17mpty vector. However, KrasT cells infected with AdRac1N17 decreased plating efficiency. Each point represents the mean values, n = 3. *P < 0.05 vs.100 MOI AdEmpty.

C. Superoxide levels decreased in KrasT cells after infection with the AdN17Rac1 construct. Intracellular hydroethidine fluorescence decreased in KrasT cells but not in H6c7 cells infected with AdN17Rac1. Intracellular superoxide levels as measured by DHE decreased significantly 48 hours after infection with AdN17Rac1 100 MOI compared to KrasT cells infected with the AdEmpty vector. *p < 0.05 vs Adempty, means ± SEM, n=3.

D. Overexpression of AdN17Rac1 did not change immunoreactive protein for MnSOD or CuZnSOD in KrasT cells. KrasT cells were transfected with 100 multiplicity of infection (MOI) of AdEmpty as a negative control or 100 MOI of AdN17Rac1. After 48 hr of transfection, total cell lysate was prepared and immunoblotted for MnSOD and CuZnSOD.