Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promoter in human hepatocellular carcinoma

Abstract

Growth arrest DNA damage-inducible gene 45 beta (GADD45beta) has been known to regulate cell growth, apoptotic cell death, and cellular response to DNA damage. Down-regulation of GADD45beta has been verified to be specific in hepatocellular cancer (HCC) and consistent with the p53 mutant, and degree of malignancy of HCC. This observation was further confirmed by eight HCC cell lines and paired human normal and HCC tumor tissues by Northern blot and immunohistochemistry. To better understand the transcription regulation, we cloned and characterized the active promoter region of GADD45beta in luciferase-expressing vector. Using the luciferase assay, three nuclear factor-kappaB binding sites, one E2F-1 binding site, and one putative inhibition region were identified in the proximal promoter of GADD45beta from -865/+6. Of interest, no marked putative binding sites could be identified in the inhibition region between -520/-470, which corresponds to CpG-rich region. The demethylating agent 5-Aza-dC was used and demonstrated restoration of the GADD45beta expression in HepG2 in a dose-dependent manner. The methylation status in the promoter was further examined in one normal liver cell, eight HCC cell lines, eight HCC tissues, and five corresponding nonneoplastic liver tissues. Methylation-specific polymerase chain reaction and sequencing of the sodium bisulfite-treated DNA from HCC cell lines and HCC samples revealed a high percentage of hypermethylation of the CpG islands. Comparatively, the five nonneoplastic correspondent liver tissues demonstrated very low levels of methylation. To further understand the functional role of GADD45beta under-expression in HCC the GADD45beta cDNA constructed plasmid was transfected into HepG2 (p53 WT) and Hep3B (p53 null) cells. The transforming growth factor-beta was assayed by enzyme-linked immunosorbent assay, which revealed a decrease to 40% in transfectant of HepG2, but no significant change in Hep3B transfectant. Whereas, Hep3B co-transfected with p53 and GADD45beta demonstrated significantly reduced transforming growth factor-beta. The colony formation was further examined and revealed a decrease in HepG2-GADD45beta transfectant and Hep3B-p53/GADD45beta co-transfectant. These findings suggested that methylation might play a crucial role in the epigenetic regulation of GADD45beta in hepatocyte transformation that may be directed by p53 status. Thus, our results provided a deeper understanding of the molecular mechanism of GADD45beta down-regulation in HCC.

Figure 1

Analysis of GADD45β expression in HCC and nonneoplastic liver tissue. A: Northern blot was probed with a 222-bp PCR product that included GADD45β exon 3. The RT-PCR product was generated based on the GADD45β sequence AF078077 in GenBank. GAPDH was used as an internal control for RNA loading. RNAs were isolated from normal liver cell and HCC cells, eight HCC samples, and five matched nonneoplastic liver tissues. N, H, and Pt indicated nonneoplastic liver tissues, HCC tissues, and patient number, respectively. B: IHC study was used to confirm the diagnosis and expression level. Top: H&E-stained sample. The N indicates normal liver tissue and H indicates HCC tissue. Bottom: Stained for GADD45β and shows a diffuse yellowish tint, predominantly in cytoplasm of normal cells. The boundary between cancer tissue and noncancerous tissue was separated by fibrotic tissue. The bottom panel showed very low GADD45β expression in HCC tissue, compared with high GADD45β expression in normal liver tissue. Original magnifications, ×40.

Figure 2

Identification of GADD45β proximal promoter region. The diagram illustrates the location of putative binding sites for the putative transcription factors. Fragments deleting each binding site were cloned into the pGL3 luciferase reporter plasmid. Relative luciferase activity for each promoter fragment is shown. Putative transcriptional factor binding sites are shown as blank boxes. Experiments were performed in triplicates and the results are presented as the mean ± SD.

Figure 3

Induction of GADD45β expression after 5-Aza-dC treatment. Forty-eight hours after treatment by different doses of 5-Aza-dC, expression of GADD45β in HepG2 and CL-48 cells was analyzed by Northern blot. The experiments were performed in triplicates and are presented as the mean ± SD. The probe for Northern blot was generated as mentioned in Material and Methods. GAPDH was used as an internal control for RNA loading. ImageQuant version 5.0 was used for quantification.

Figure 4

Analysis of GADD45β promoter methylation in DNA from tissues and HepG2 cells. A: MSP analyses of methylation in DNA from HCC tissues, nonneoplastic liver tissues, and HepG2 cells. Top: The MSP results of nine cell lines. Middle: The MSP results of eight HCC samples. Bottom: Nonneoplastic liver tissues. The M indicates the hypermethylated PCR products and the U indicates unmethylated PCR products. B and C: Nucleotide sequencing of the third NF-κB and the putative inhibition region after sodium bisulfite treatment. •, complete methylation; □, partial methylation, ○, unmethylated.

Figure 5

Suppression of TGF-β and colony formation by p53-dependent GADD45β expression. A: The p53 expression was examined by Western blot. B: GADD45β expressing fluorescence green staining confirmed the transfection in HepG2 and Hep3B cells (Hep3B cell image not shown). C: The flow cytometry assay examined HepG2 and Hep3B without and with transfection of GADD45β was examined. D: The TGF-β examined by ELISA was described in the Materials and Methods. E: The colony formation assay was described in Materials and Methods. The number of colonies scored in the presence of pIRES2-EGFP vector was designated 100%. All of the experiments were performed at least three times with SD.