Figure 3.

Oncogenic ras-mediated activation of SAF-1 is regulated via MEK/MAPK pathway. (A) Oncogenic pCMV-rasV12 transformed MCF-10A cells were either left untreated or incubated with DMSO (vehicle), MEK 1/2 inhibitor U0126 (20 μmol/L), MAP kinase inhibitors PD98059 (20 μmol/L), SB203580 (20 μmol/L), or SB202474 (20 μmol/L) for 24 h. Nuclear extracts (10 μg of protein) prepared from these cells, as indicated, were incubated with ³²P-labeled DNA containing SAF-1-binding element of the SAA promoter. Resulting DNA-protein complexes were fractionated in a 6% nondenaturing polyacrylamide gel. (B) MCF-10A or pCMV-rasV12-transformed MCF-10A cells were transfected with equal amount (1.0 μg) of wt SAF-CAT reporter plasmid. Cells were grown for an additional 24 h in absence or presence of 20 μmol/L each of U0126, PD98059, SB203580, or SB202474, as indicated. Relative CAT activity in ras-transformed cells was determined by comparing it to that in normal MCF-10A cells. Results represent an average of three separate experiments. *P < 0.05. (C) MCF-10A cells were transfected with equal amount (1.0 μg) of wt SAF-CAT reporter plasmid. In addition, some cells were cotransfected with either a wild-type SAF-1 expression plasmid, wt pcD-SAF-1 (0.5 μg) or a mutant SAF-1 expression plasmid, mut pcD-SAF-1(V71) (0.5 μg). Also, some cells were transfected with Ras expression plasmid, pCMVrasV12 (0.5 μg), as indicated. Relative CAT activity was determined by measuring CAT activity of cotransfected MCF-10A cells compared to that of SAF-CAT transfected MCF-10A cells. Results represent an average of three separate experiments. *P < 0.05. SAF-1, serum amyloid A activating factor 1; CAT, chroramphenicol acetyl transferase.