Hypoxia-inducible expression of BAX: application in tumor-targeted gene therapy

Abstract

Object. The presence of hypoxic cells in human brain tumors contributes to the resistance of these tumors to radia- tion therapy. However, because normal tissues are not hypoxic, the presence of hypoxic cells also provides the poten- tial for designing cancer-specific gene therapy. Suicide genes can be expressed specifically in hypoxic conditions by hypoxia-responsive elements (HREs), which are activated through the transcriptional complex hypoxia-inducible fac- tor–1 (HIF-1). Methods. The authors have transfected the murine BAX–green fluorescent protein (GFP) fusion gene under the reg- ulation of three copies of HRE into U-87 MG and U-251 MG cells and selected stably transfected clones. Even though BAX was expressed under both oxic and anoxic conditions in these clones, cell survival assays demonstrated increased cell killing under anoxic as compared with oxic conditions. Cells obtained from most of these clones did not grow in vivo, or the tumors exhibited highly variable growth rates. However, cells obtained from the U-251 MG clone A pro- duced tumors that grew as well as tumors derived from parental cells, and examination of the tumor sections under flu- orescent microscopy revealed GFP expression in localized regions. Western blot analyses confirmed an increased BAX expression in these tumors. Analysis of the results suggests that HRE-regulated BAX can be a promising tool to tar- get hypoxic brain tumor cells. However, there are measurable levels of BAX-GFP expression in this three-copy HRE–mediated expression system under oxia, suggesting promoter leakage. In addition, most clones did not show sig- nificant induction of BAX-GFP under anoxia. Therefore, the parameters of this HRE-mediated expression system, including HRE copy number and the basal promoter, need to be optimized to produce preferential and predictable gene expression in hypoxic cells.