Optimization of ROS-response promoter activity by the addition of hypoxia response motifs

POLICASTRO L; IEZZI ME; WERBAJH S; CANZIANI G; PODHAJCER O

Buenos aires

Congreso; VIII Meeting of the Society for Free Radical Biology and Medicine-South American Group; 2013

Increased reactive oxygen species (ROS) production appears as a distinctive feature of cancer microenvironment. Our group developed a synthetic chimeric promoter, named E6(40)VE whose activity was largely dependent on variations in intracellular ROS levels. Transient expression of the thymidine kinase gene driven by E6(40)VE, followed by gancyclovir administration, inhibited established human colorectal cancer and melanoma cell growth in vitro and in vivo (Policastro et al, 2009, 2012). In this work we optimized the activity of E6(40)VE by adding a hypoxic response element (HRE). The 3 X HRE motif obtained from the VEGF promoter was placed upstream of E6(40)VE. The 3 XHRE motif was placed 6 or 30 bp upstream of E6(40)VE to obtain HRE(6)E6(40)VE and HRE(30)E6(40)VE, respectively. In normoxia the HRE(6)E6(40)VE promoter exhibited only 50% of the basal activity of E6(40)VE while HRE(30)E6(40)VE) activity was comparable to E6(40)VE. Several malignant cell lines were transiently tranfected with HRE(30)E6(40)VE. We observed 2-3 fold increased activity in hypoxia compared to normoxia in almost all cell lines. The highest effect was obtained in breast and pancreas cell lines. These studies open the possibility of using this novel chimeric promoter for therapeutic use.