No abstract

The NAD(P)H:quinone oxidoreductase 1 (NQO1)*2 polymorphism is characterized by a single proline-to-serine amino acid substitution. Cell lines and tissues from organisms genotyped as homozygous for the NQO1*2 polymorphism are deficient in NQO1 activity. In studies with cells homozygous for the wild-type allele and cells homozygous for the mutant NQO1*2 allele, no difference in the half-life of NQO1 mRNA transcripts was observed. Similarly, in vitro transcription/translation studies showed that both wild-type and mutant NQO1 coding regions were transcribed and translated into full-length protein with equal efficiency. Protein turnover studies in NQO1 wild-type and mutant cell lines demonstrated that the half-life of wild-type NQO1 was greater than 18 h, whereas the half-life of mutant NQO1 was 1.2 h. Incubation of NQO1 mutant cell lines with proteasome inhibitors increased the amount of immunoreactive NQO1 protein, suggesting that mutant protein may be degraded via the proteasome pathway. Additional studies were performed using purified recombinant NQO1 wild-type and mutant proteins incubated in a rabbit reticulocyte lysate system. In these studies, no degradation of wild-type NQO1 protein was observed; however, mutant NQO1 protein was completely degraded in 2 h. Degradation of mutant NQO1 was inhibited by proteasome inhibitors and was ATP-dependent. Mutant NQO1 incubated in rabbit reticulocyte lysate with MG132 resulted in the accumulation of proteins with increased molecular masses that were immunoreactive for both NQO1 and ubiquitin. These data suggest that wild-type NQO1 persists in cells whereas mutant NQO1 is rapidly degraded via ubiquitination and proteasome degradation.

NAD(P)H:quinone oxidoreductase 1 (NQO1) has been proposed to stabilize p53 via a redox mechanism involving oxidation of NAD(P)H as a consequence of the catalytic activity of NQO1. We report that treatment of HCT-116 human colon carcinoma cells with the NQO1 inhibitor ES936 had no effect on the levels of p53 protein. ES936 is a mechanism-based inhibitor of NQO1 that irreversibly blocks the catalytic function of the enzyme. This suggests that a redox mechanism involving NQO1-mediated NAD(P)H oxidation is not responsible for the stabilization of p53. We also examined the ability of the NQO1 protein to associate with p53 using co-immunoprecipitation experiments. Results from these experiments demonstrated co-immunoprecipitation of NQO1 with p53 and vice versa. The association between p53 and NQO1 was not affected by treatment of HCT-116 cells with ES936, demonstrating that the association was not dependent on the catalytic activity of NQO1. A comparison of isogenic HCT-116 p53؉/؉ and HCT-116 p53؊/؊ cells demonstrated an interaction of NQO1 and p53 only in the p53؉/؉ cells. Experiments performed in an in vitro transcription/translation system utilizing rabbit reticulocyte lysates confirmed the interaction of NQO1 and p53. In these experiments a full-length p53 coding region was used to express p53 in the presence of recombinant NQO1 protein. An association of p53 and NQO1 was also observed in primary human keratinocytes and mammary epithelial cells. In studies where mdm-2 co-immunoprecipitated with p53, no association of mdm-2 with NQO1 was observed. These data demonstrate an association between p53 and NQO1 that may represent an alternate mechanism of p53 stabilization by NQO1 in a wide variety of human cell types.

The gonadotropin releasing hormone (GnRH) gene encodes a protein which plays a critical role in mammalian reproductive physiology. Its expression is predominantly restricted to the hypothalamus although it has also been described in the placenta. To begin to determine the promoter elements important for tissue specific expression and to examine the mechanisms of developmental and hormonal regulation of the rat GnRH (rGnRH) gene, we cloned the rGnRH gene from a rat liver genomic DNA library. The nucleotide sequence of greater than 3 kb of 5'-flanking region was determined. The transcriptional initiation site in rat hypothalamic tissue and a mouse hypothalamic cell line were mapped by primer extension analysis and found to be different. In addition, transient transfection studies demonstrated that multiple regions of the distal promoter are important for tissue specific and basal promoter activity in hypothalamic cells. Furthermore, in these cells a potent activation region resides between -3026 and -1031 bp and suppressor region between -1031 and -903 bp upstream of the transcriptional start site. We conclude that different portions of the 5'-flanking region, which are activating and suppressing in nature, are critical for hypothalamic expression of the rGnRH gene.