Up-regulation of cell cycle genes during 4-OHE₂ induced neoplastic transformation of mammary cells and their expressions are inhibited by ROS modulators.

<p>A) Fold change of PCNA transcripts in 4-OHE2 transformed cells treated with ROS modulators, or transformed cells transfected with Akt1 RNAi. B) Fold change of cell cycle genes in 4-OHE2 transformed cells overexpressing catalase. MCF-10A cells were seeded for transformation as described in the legend of Fig. 2. At the end of transformation period, cells were treated for additional 18 hours with vehicles or 4-OH-E2 (100 ng/ml). For inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells were transfected with 50 MOI adenovirus expressing catalase or MnSOD or treated with an antioxidant Ebselen (40 uM) or 10 mM NAC. Cells were first washed with cold PBS containing protease inhibitors, detached with trypsin, and RNA was isolated from 2.0×10⁶ cells. The TaqMan primers and probe recognizing PCNA, NRF1, PRC1, CDC2 and 18S were used in this study. Quantitative gene expression analysis was performed by TaqMan-based QRT−PCR on ABI 7700 (PE Applied Biosystems, Foster City, CA, USA). The fold change in gene expression was calculated using the {Delta} Ct method with 18S rRNA as the internal control. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05, significantly different from control. **P<0.05 indicates significantly different from 4-OH-E2, (P<0.05).