Hypermethylation of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene in human hepatocellular carcinoma.
OBJECTIVE
NAD(P)H: quinone oxydoreductase 1 (NQO1) and glutathione S-transferase P1 (GSTP1) belong to phase II xenobiotic-metabolizing enzymes. GSTP1 inactivation via CpG island hypermethylation in hepatocellular carcinoma (HCC) was previously reported, but the involvement of NQO1 in HCC is not well known. In this study, we assessed the transcription and status of methylation of NQO1 gene in human hepatoma cells and primary human HCC tissues.


METHODS
NQO1 transcription and DNA hypermethylation in hepatoma cells with or without 5-aza-deoxycytidine (5-Aza-CdR) treatment were investigated by reverse-transcription PCR (RT-PCR), sodium bisulfite sequencing and methylation-specific PCR (MSP). The methylation status of NQO1 and GSTP1, and NQO1 mRNA in 44 HCC cases was also analyzed by MSP and real-time PCR, respectively.


RESULTS
NQO1 transcription was down-regulated and the CpG island DNA was hypermethylated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, NQO1 transcription was restored and CpG island DNA was demethylated in these cells. MSP analysis revealed that NQO1 hypermethylation occurred in 50.0% of HCC. All of the tumors that exhibited lesser amounts of NQO1 mRNA than corresponding non-tumorous tissues showed NQO1 hypermethylation.


CONCLUSIONS
NQO1 transcription might be inappropriately suppressed by promoter hypermethylation in a subset of HCC, as well as GSTP1 gene.
Tada
Motohisa
M
Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan.
Yokosuka
Osamu
O
Fukai
Kenichi
K
Chiba
Tetsuhiro
T
Imazeki
Fumio
F
Tokuhisa
Takeshi
T
Saisho
Hiromitsu
H
D000107:Acetylation; D000368:Aged; D000369:Aged, 80 and over; D000975:Antioxidants / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000502:physiology; D019175:DNA Methylation; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites / Q000494:pharmacology; D014158:Transcription, Genetic / Q000502:physiology
D000975:Antioxidants; D006657:Histones; D017382:Reactive Oxygen Species; D013447:Sulfites; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D005982:Glutathione Transferase; C009279:sodium bisulfite