Lack of NQO1 induction in human tumor cells is not due to changes in the promoter region of the gene.
DT-diaphorase is a two-electron reducing enzyme that is an important detoxifying enzyme and activator of bioreductive antitumor agents. Expression of the DT-diaphorase gene, NQO1, appears to be transcriptionally regulated, and the gene is induced by a wide variety of compounds. We showed that 1,2-dithiole-3-thione can selectively increase DT-diaphorase activity in human and murine tumors, and that this enhanced the antitumor activity of bioreductive antitumor agents. However, we found that DT-diaphorase activity was not increased in some human tumor cell lines after treatment with the inducer, and this appeared to be due to a lack of increased transcription. To determine if this lack of increased transcription was due to a mutation in the promoter region of the NQO1 gene in these cells, we sequenced approximately 2000 bases of the NQO1 promoter region from non-induced cells and compared these with sequences from human HT29 colon cancer cells, which showed significant increases in NQO1 transcription, and the sequence reported for human liver cells in Genbank. Sequence analysis showed no changes in the sequences of the major transcriptional elements, XRE, CAT box, ARE, AP1 site or AP2 site, in the tumor cells compared with the Genbank sequences. The only major change was a deletion of a 20 base repeat region approximately 400 bases 5' to the XRE element in all the cells, including the HT29 cells, compared with the sequence reported in Genbank. There were also several insertions of a single base in various parts of the sequences which occurred in most, or all, of the cell lines compared with the reported sequence, and a small number of single base changes, insertions or deletions that occurred in a single cell line. However, these changes did not appear to correlate with differences in induced transcription of the NQO1 gene. These results suggest that the differences in transcription of the NQO1 gene after treatment with DT-diaphorase inducers was not due to alterations in the promoter region of the gene.
Begleiter
Asher
A
Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Manitoba R3E 0V9, Canada. begleit@cc.umanitoba.ca
Lange
Laurie
L
D004252:DNA Mutational Analysis; D004790:Enzyme Induction; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000378:metabolism; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D016660:NAD(P)H Dehydrogenase (Quinone)