Methylation status of genes upregulated by demethylating agent 5-aza-2'-deoxycytidine in hepatocellular carcinoma.
OBJECTIVE
To determine the clinical significance of gene promoter methylation in hepatocellular carcinoma (HCC), we examined in clinical samples the methylation status of those promoters that showed elevated activity in hepatoma cell lines after 5-aza-2'-deoxycytidine treatment.


METHODS
Regarding the genes with promoter hypermethylation in the cell lines, their expression levels and methylation status in HCC and non-HCC tissues were assessed by semiquantitive RT-PCR and methylation-specific PCR. To confirm the result, the expression levels and methylation status in 16 additional HCC and non-HCC tissues were assessed.


RESULTS
The promoter regions of caveolin 1 (CAV1), cysteine and glycine-rich protein 1 (CSRP1), Kruppel-like factor 6 (KLF6), myosin (light polypeptide 9) (MYL9), and transgelin (TAGLN) were highly methylated in the cell lines. CAV1 and CSRP1 were methylated in HCC more frequently than in non-HCC. KLF6, MYL9, and TAGLN were fully methylated in both HCC and non-HCC. Using additional clinical samples, downregulation of CAV1 and CSRP1 was observed in 38 and 56%, respectively, of the 16 HCC samples and aberrant methylation of CAV1 and CSRP1 was observed in 56% of HCC in both cases.


CONCLUSIONS
CAV1 and CSRP1 were inactivated in HCC by aberrant methylation and they may serve as important biomarkers of malignancy.
Hirasawa
Yuichi
Y
Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Arai
Makoto
M
Imazeki
Fumio
F
Tada
Motohisa
M
Mikata
Rintaro
R
Fukai
Kenichi
K
Miyazaki
Masaru
M
Ochiai
Takenori
T
Saisho
Hiromitsu
H
Yokosuka
Osamu
O
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics; D051242:Caveolin 1 / Q000235:genetics; D045744:Cell Line, Tumor; D019175:DNA Methylation; D000077209:Decitabine; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D060588:LIM Domain Proteins; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics; D018994:Myosin Light Chains / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000964:Antimetabolites, Antineoplastic; C495540:CAV1 protein, human; C486181:CRIP1 protein, human; D002352:Carrier Proteins; D051242:Caveolin 1; C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; C051615:transgelin; D000077209:Decitabine; D001374:Azacitidine