Volume 42, Issue 4, Pages 511-519 (April 2005) Hypermethylation of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene in human hepatocellular carcinoma  Motohisa Tada, Osamu Yokosuka, Kenichi Fukai, Tetsuhiro Chiba, Fumio Imazeki, Takeshi Tokuhisa, Hiromitsu Saisho  Journal of Hepatology  Volume 42, Issue 4, Pages 511-519 (April 2005) DOI: 10.1016/j.jhep.2004.11.024 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 RT-PCR analysis of NQO1 expression in hepatoma cell lines. (A) Without treatment with 5-Aza-CdR, NQO1 mRNA expression was down-regulated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, NQO1 transcription was restored in Hep3B and HuH6 cells. (B) RNA samples were also amplified using β-actin gene primers as control. +, 5-Aza-CdR-treated; −, untreated. Journal of Hepatology 2005 42, 511-519DOI: (10.1016/j.jhep.2004.11.024) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Methylation status of 24 CpG dinucleotides located between −113 and +219 in the promoter and exon 1 of NQO1 gene. Bisulfite sequencing was performed to determine the methylation status of the 5′CpG island of NQO1 gene in each cell line indicated. Each circle indicates a CpG site in the primary DNA sequence, and each line of circles represents the analysis of a single cloned allele. Closed circles, methylated CpG dinucleotides; open circles, unmethylated CpG dinucleotides. The 5′CpG island of NQO1 was densely methylated in each clone of HuH6 cells and in five of 10 clones of Hep3B cells, while few nucleotides were methylated in HLE, HepG2 and PLC/PRF/5, and no CpG dinucleotide was methylated in HuH7. Journal of Hepatology 2005 42, 511-519DOI: (10.1016/j.jhep.2004.11.024) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 MSP analysis performed to assess the methylation status of the 5′CpG island of NQO1 in hepatoma cells. (A) Without treatment with 5-Aza-CdR. (B) Treatment with 5-Aza-CdR. MSP analysis revealed that the 5′CpG island of NQO1 was hypermethylated in Hep3B and HuH6 cells without treatment with 5-Aza-CdR. After treatment with 5-Aza-CdR, the 5′CpG island of NQO1 was demethylated in Hep3B and HuH6 cells. M, methylated; U, unmethylated. Journal of Hepatology 2005 42, 511-519DOI: (10.1016/j.jhep.2004.11.024) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 ChIP assay. Crosslinked chromatin from each cell line was incubated with acetylated histones H3 (A) and H4 (B). Immnoprecipitates from each antibody were aliquotted and analyzed by PCR with primer specific NQO1 promoter. Acetylated histone H3 and H4 were undetectable in Hep3B and HuH6 cells without treatment with trichostatin A, while they were abundant in HLE, HuH7, HepG2 and PLC/PRF/5 cells. But histones H3 and H4 were also deacetylated in Hep3B and HuH6 cells after treatment with trichostatin A. +, TSA-treated; −, untreated. Journal of Hepatology 2005 42, 511-519DOI: (10.1016/j.jhep.2004.11.024) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Representative results of MSP analysis of NQO1 and GSTP1 hypermethylation in primary human HCC tumors and corresponding non-tumorous liver tissues. (A) Analysis of NQO1 hypermethylation. (B) Analysis of GSTP1 hypermethylation. Case 1 and Case 3 were hypermethylated at NQO1 gene and Case 1 and Case 2 were hypermethylated at GSTP1 gene. Corresponding non-tumorous liver tissues were neither methylated at NQO1 gene nor GSTP1 gene in the three cases. M, methylation; U, unmethylation. Journal of Hepatology 2005 42, 511-519DOI: (10.1016/j.jhep.2004.11.024) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions