title: Epigenetic silencing of glutaminase 2 in human liver and colon cancers.
abstract: BACKGROUND
  Glutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism. Gls2 has been reported to be downregulated in human hepatocellular carcinomas (HCC). However, the underlying mechanism responsible for its downregulation is still unclear. Here, we investigated Gls2 expression and its promoter methylation status in human liver and colon cancers.
  
  
  METHODS
  mRNA expression of Gls2 was determined in human liver and colon cancer cell lines and HCC tissues by real-time PCR and promoter methylation was analyzed by methylation-specific PCR (MSP) and validated by bisulfite genome sequencing (BGS). Cell growth was determined by colony formation assay and MTS assay. Statistical analysis was performed by Wilcoxon matched-pairs test or non-parametric t test.
  
  
  RESULTS
  First, we observed reduced Gls2 mRNA level in a selected group of liver and colon cancer cell lines and in the cancerous tissues from 20 HCC and 5 human colon cancer patients in comparison to their non-cancerous counter parts. Importantly, the lower level of Gls2 in cancer cells was closely correlated to its promoter hypermethylation; and chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza) increased Gls2 mRNA level in both liver and colon cancer cells, indicating that direct epigenetic silencing suppressed Gls2 expression by methylation. Next, we further examined this correlation in human HCC tissues, and 60% of primary liver tumor tissues had higher DNA methylation levels when compared with adjacent non-tumor tissues. Detailed methylation analysis of 23 CpG sites at a 300-bp promoter region by bisulfite genomic sequencing confirmed its methylation. Finally, we examined the biological function of Gls2 and found that restoring Gls2 expression in cancer cells significantly inhibited cancer cell growth and colony formation ability through induction of cell cycle arrest.
  
  
  CONCLUSIONS
  We provide evidence showing that epigenetic silencing of Gls2 via promoter hypermethylation is common in human liver and colon cancers and Gls2 appears to be a functional tumor suppressor involved in the liver and colon tumorigenesis.
authors: Jianbin
authors: J
authors: Zhang
authors: Cheng
authors: C
authors: Wang
authors: Mingquan
authors: M
authors: Chen
authors: Jianping
authors: J
authors: Cao
authors: Ying
authors: Y
authors: Zhong
authors: Liting
authors: L
authors: Chen
authors: Han-Ming
authors: HM
authors: Shen
authors: School of Public Health, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China. dxia@zju.edu.cn.
authors: Dajing
authors: D
authors: Xia
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003106:Colon / Q000201:enzymology; D003110:Colonic Neoplasms / Q000201:enzymology*; D019175:DNA Methylation; D015536:Down-Regulation; D004794:Enzyme Repression; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D005972:Glutaminase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology; D008113:Liver Neoplasms / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA
chemical_list: C586504:GLS2 protein, human; D005972:Glutaminase