The NAD(P)H:Quinone Oxidoreductase (NQOI) (DT diaphorase) is a flavoprotein which catalyzes the two-electron reduction of quinones and azodyes and thus prevents the formation of free radicals and toxic oxygen metabolites that may be generated by the one-electron reductions catalyzed by Cytochrome P450 reductase. The NQOL gene is expressed at much higher levels in tumor cells compared to normal cells of their origin and its expression is induced by dioxin and phenolic antioxidants. Recently we have identified a 1.42 Kb DNA segment in the 5' flanking region of human NQOI gene responsible for high levels of expression of NQOI in tumor cells and a 1.72 Kb DNA segment essential for induction of NQ01 gene expression in response to dioxin. In addition we have isolated a second related human (NQO2) cDNA and its gene with flanking regions for further studies. We plan to elucidate the molecular mechanisms that control the basal level of expression in normal and tumor cells, induction in transcription due to xenobiotics, and tissue specific and developmental expression of NQOI and NQO2 genes. To localize the cis-acting DNA elements in the NQOL gene essential for increased expression in tumor cells and induction in expression due to dioxin and phenolic antioxidants, we will perform transfection experiments in Hep-G2 and Hepa- I cells with chimeric gene constructs in which varying segments of the NQOL gene 5' flank drive expression of a marker gene. These DNA elements will be precisely localized by creating further deletions, internal deletions and linker scanning mutations. Gel mobility shift assays, DNAse I footprinting, dimethyl sulfate methylation interference experiments will be performed in order to (I) determine the precise sequence motifs with which the regulatory proteins interact, (II) identify trans-acting protein factors interacting with cis-acting DNA elements and (III) demonstrate the interactions between different trans-acting proteins and with transcription factors that are essential for higher expression of NQOI gene in tumor cells and gene activation in response to dioxin and phenolic antioxidants. The cDNAs encoding the regulatory proteins identified to play important roles in the regulation of NQO1 gene expression will be cloned for further studies on the function of these proteins in the regulation of NQOI gene expression. We will also sequence the NQO2 gene and its flanking regions and study the induction of NQO2 gene expression by xenobiotics. In addition, we will investigate the tissue specific and developmental expression of NQOI and NQO2 genes in rat tissues by measuring the RNA and protein contents. If significant differences are observed, the promoter elements of NQOI and NQO2 genes will be analyzed in an in vitro transcription system with nuclear extracts from tissues at different stages of development.
| Gaballa, Ahmed; Newton, Gerald L; Antelmann, Haike et al. (2010) Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli. Proc Natl Acad Sci U S A 107:6482-6 |
