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Cell Biology International (2012) 36, 427–432 (Printed in Great Britain)
Promoter hypermethylation of DNA damage response genes in hepatocellular carcinoma
Zhiqin Li*1, Hongyan Zhang†1, Jianping Yang†, Tielai Hao‡ and Shenglei Li†2
*Department of Infection, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, Peoples Republic of China, †Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, Peoples Republic of China, and ‡Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, Peoples Republic of China


Aberrant methylation of promoter CpG islands is a major inactivation mechanism of tumour-related genes that play a crucial role in the progression of silencing in human cancers, including HCC (hepatocellular carcinoma). We have examined the promoter methylation status of five important DNA damage response genes in fresh-frozen HCC tissues and cell lines, as well as the possible correlation between methylation patterns and clinical features of the carcinoma. Promoter methylation status of RASSF1A (Ras association domain family 1), CHFR (checkpoint with forkhead and ring finger domains), GSTP1 (glutathione-S-transferase-pi gene), MGMT [O(6)-methylguanine-DNA methyltransferase] and hMLH1 (human mutL homologue 1) were examined by the MSP (methylation-specific PCR) in 70 HCC tissues and five HCC cell lines. The mRNA expression levels of these genes were measured by RT–PCR (reverse transcription–PCR). Methylation frequencies of these genes tested in HCC were 54 (78%) for RASSF1A, 30 (43%) for CHFR, 26 (38%) for GSTP1 and 22 (32%) for MGMT. No hypermethylation was detected for hMLH1 in any case of HCC or HCC cell lines. Moreover, promoter hypermethylation of RASSF1A, CHFR and GSTP1 in both HepG2 and SNU398 cells, and hypermethylation of MGMT in Huh7 cells, were detected. Treatment of three cell lines with 5Aza-dC (5-aza-20-deoxycytidine) restored or increased the expression of these genes, implicating aberrant DNA methylation in transcriptional silencing. Hypermethylation of RASSF1A and patient age were significantly associated. CHFR methylation status showed a statistically significant correlation with HCC progression. Methylation of the RASSF1A, CHFR, GSTP1 and MGMT genes seem therefore to play an important role in the pathogenesis of HCC. These epigenetic changes may have prognostic importance for patients with HCC.


Key words: DNA damage response genes, hepatocellular carcinoma (HCC), promoter hypermethylation

Abbreviations: 5Aza-dC, 5-aza-20-deoxycytidine, CHFR, checkpoint with forkhead and ring finger domains, GSTP1, glutathione-S-transferase-pi gene, HBV, hepatitis B virus, HCC, hepatocellular carcinoma, hMLH1, human mutL homologue 1, MGMT, O(6)-methylguanine-DNA methyltransferase, MSP, methylation-specific PCR, RASSF1A, Ras associatition domain family 1, RT–PCR, reverse transcription–PCR

1These authors contributed equally to this work.

2To whom correspondence should be addressed (email lslbljys@126.com).


1. Introduction

HCC (hepatocellular carcinoma) is the fifth most common cancer in the world (Parkin et al., 2005), the incidence rate is showing a dramatic increase in China and other countries over the last three decades (Wang et al., 2001; Davila et al., 2004; El-Serag 2004). Although the causative links of various factors, including HBV (hepatitis B virus) or HCV (hepatitis C virus) infections, dietary aflatoxin, the male gender and alcohol consumption, have been implicated in the aetiology of HCC based on epidemiologic grounds, the molecular mechanisms regarding HCC development and progression remain unclear.

Aberrant epigenetic silencing due to CpG island methylation has emerged recently as one of the pivotal genetic alterations in cancer development and progression. CpG islands are in the 0.5–2 kb regions that are rich in cytosine–guanine dinucleotides, and mostly found at the 5′ regulatory regions of genes, and 50–70% of human gene promoters are embedded in CpG islands (Vucic et al., 2008). A panel of DNA damage responses genes, including RASSF1A (Ras associatition domain family 1), CHFR (checkpoint with forkhead and ring finger domains), GSTP1 (glutathione-S-transferase-pi gene), MGMT [O(6)-methylguanine-DNA methyltransferase] and hMLH1 (human mutL homologue 1), have been reported in different types of tumour, which are involved in cell-cycle checkpoints, DNA repair, apoptosis, detoxification and signal transduction. These genes have been implicated by demonstrating a significant correlation between CpG island methylation and loss of mRNA or protein expression (Esteller, 2007). For example, as a new family of tumour suppressor genes, RASSF1A is lost in many tumour lines and primary tumours by promoter methylation and play an important role in carcinogenesis (Agathanggelou et al., 2001; Burbee et al., 2001; Pfeifer et al., 2002). Similarly, the G2/M checkpoint gene CHFR promoter hypermethylation appears to be a good molecular marker for predicting sensitivity to microtubule inhibitors in colon, lung and gastric cancer (Scolnick and Halazonetis, 2000; Satoh et al., 2003; De Jong et al., 2009). However, most of these studies have analysed only single gene methylation. It seems the methylation profile of these genes in HCC remains largely unknown.

We have examined the methylation status of multiple DNA damage responses genes (RASSF1A, CHFR, GSTP1, MGMT and hMLH1) in HCC tissues and cell lines by MSP (methylation-specific PCR) analysis. The correlation between the methylation status of these genes and the clinicopathologic features known to be important for the prognosis of cases was also examined.

2. Materials and methods

2.1. Tissue samples

Fresh samples of HCC tissues were obtained from 70 patients who underwent curative surgical resection at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). None of the patients have received preoperative treatment. Information about the patients’ demographics and lifestyle variables was obtained using an interviewer-administered questionnaire. Ethical approval for the study and agreement by all patients was obtained from the Ethics Committee of Zhengzhou University. Each subject signed an agreement of participation in the study that was approved by the First Affiliated Hospital of Zhengzhou University.

2.2. Cell lines and cell culture

Five human HCC cell lines (Hep3B, HepG2, Huh7, SNU398 and SNU449) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. All these cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS (fetal bovine serum; Hyclone) and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin) at 37°C in a humidified 5% CO2 in air incubator.

2.3. DNA preparation

Genomic DNA from the HCC tissues and cells was prepared by the proteinase-K method. HCC tissues or cells were digested using the proteinase–K method and subjected to classical DNA extraction using phenol/chloroform/isoamylalcohol and ethanol precipitation. After precipitation, DNA was resuspended in water and quantified by spectrophotometric analysis (260 nm). Finally, DNA was dissolved in low TE buffer and stored at −20°C.

2.4. MSP

One microgram of genomic DNA was diluted in H2O for a final volume of 50 μl and was bisulfite modified, as previously described. The final volume of bisulfite-treated DNA for each sample was 20 μl, and all bisulfite-treated DNA was stored at −20°C. MSP primers were designed according to genomic sequences skirting the presumed transcription start sites for all genes. Primer sequences were oligo-synthesized (IDT) to allow MSP to detect bisulfite-induced changes affecting unmethylated (U) and methylated (M) alleles. MSP primers sequence for all these genes and Tm (melting temperature) are summarized in Table 1. Each MSP reaction incorporated approximately 100 ng of bisulfite-treated DNA, 25 pmoles of each primer, 100 pmol dNTPs, 10×PCR buffer and 1 unit of JumpStart Red Taq polymerase (Sigma) in a final reaction volume of 25 μl. Cycle conditions were: 95°C×5 min; 35 cycles×(95°C×30 s, 60°C×30 s, 72°C×30 s); 72°C×5 min. MSP products were analysed using 2% agarose gels and an imaging system.


Table 1 Primer sequences and annealing temperatures used in MSP

Genes Primer Sequences Tm (°C)
RASSF1A U-F 5′-TTTGGTTGGAGTGTGTTAATGTG-3′ 58
U-R 5′-CAAACCCCACAAACTAAAAACAA-3′
M-F 5′-GTGTTAACGCGTTGCGTATC-3′ 59
M-R 5′-AACCCCGCGAACTAAAAACGA-3′
CHFR U-F 5′-GATTGTAGTTATTTTTGTGATTTGTAGGTGAT-3′ 56
U-R 5′-AACTAAAACAAAACCAAAAATAACCCACA-3′
M-F 5′-GTTATTTTCGTGATTCGTAGGCGAC-3′ 58
M-R 5′-CGAAACCGAAAATAACCCGCG-3′
GSTP1 U-F 5′-GATGTTTGGGGTGTAGTGGTTGTT-3′ 58
U-R 5′-CCACCCCAATACTAAATCACAACA-3′
M-F 5′-TTCGGGGTGTAGCGGTCGTC-3′ 60
M-R 5′-GCCCCAATACTAAATCACGACG-3′
MGMT U-F 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ 56
U-R 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′
M-F 5′-TTTCGACGTTCGTAGGTTTTCGC-3′ 59
M-R 5′-GCACTCTTCCGAAAACGAAACG-3′
hMLH1 U-F 5′-TTTTGATGTAGATGTTTTATTAGTGGGTTGT-3′ 56
U-R 5′-ACCACCTCATCATAACTACCCACA-3′
M-F 5′-ACGTAGACGTTTTATTAGGGTCGC-3′ 62
M-R 5′-CCTCATCGTAACTACCCGCG-3′



2.5. RNA extraction and RT–PCR (reverse transcription–PCR)

Total RNA was prepared with TRIzol® reagent (Life Technologies Inc.) according to the manufacturer's instruction. Four micrograms of total RNA were used to synthesize the first strand of cDNA using cDNA Synthesis Kit (Fermentas). β-Actin was used to ascertain the equal amount of cDNA in each reaction. A total of 30 cycles were performed with each cycle consisting of 30 s at 94°C, 35 s at 65°C and 40 s at 72°C with an initial denaturation of 5 min at 95°C and a final extension of 7 min at 72°C. The reaction products were separated on 2% agarose gel and analysed by an imaging system.

2.6. Treatment with 5Aza-dC (5-aza-20-deoxycytidine)

Three HCC cell lines (HepG2, Huh7 and SNU398) were treated with 5Aza-dC (Sigma–Aldrich) based on the hypermethylation of these four genes. Cells were exposed continuously to 5Aza-dC (5 μM) for 4 days. As we had previously observed that treatment of these cell lines with 5Aza-dC (5 μM) for 4 days results in the re-expression of genes silenced by aberrant methylation without evidence of excessive cell death, we used this time for the experiment.

2.7. Statistical analysis

Statistical analysis was performed with SPSS10.0 software. Correlation between methylation and clinicopathological features was assessed by the χ2 test. A value of P<0.05 was considered statistically significant.

3. Results

3.1. Methylation analysis in HCC tissues and cell lines

MSP was used to examine the methylation status of five DNA damage response genes in HCC tissues and cell lines. Representative examples of the MSP analysis are shown in Figure 1; among these genes, RASSFIA is the most frequently affected (54/70, 78%), followed by CHFR (30/70, 43%), GSTP1 (26/70, 38%) and MGMT (22/70, 32%). No hypermethylation was detected for hMLH1 in any HCC. The methylation profile of these genes in a panel of HCC cell lines (Hep3B, HepG2, Huh7, SNU398 and SNU449) analysed by MSP showed that RASSF1A, CHFR and GSTP1 were methylated in HepG2 and SNU398 cells. MGMT was methylated in Huh7. There was no evidence of methylation of these four genes in Hep3B and SUN449 cells.

3.2. RASSF1A, CHFR, GSTP1 and MGMT expression in HepG2, SNU398 and Huh7 cells after 5-Aza-dC treatment

To assess the possible correlation between these four genes expression and their promoter hypermethylation, the demethylation agent 5-Aza-dC was used in these three cell lines. RT–PCR showed that RASSF1A, CHFR, GSTP1 and MGMT expression were increased or restored after treatment with 5-Aza-dC (Figure 2). These results strongly implicated hypermethylation of these four gene promoters was closely related to the absence or decreased these genes expression. Concordantly, lack of methylation was associated with strong expression of these genes.

3.3. Methylation and clinicopathological correlations

We also investigated the correlation between methylation of RASSF1A, CHFR, GSTP1 and MGMT, and the clinicopathological features of the primary HCC, including age, gender, HBV, tumour size and pathological grade (Table 2). There was no significant association between the methylation of GSTP1 or MGMT and any of the clinicopathological features. However, there was a significant correlation between promoter hypermethylation of CHFR and pathological grade (P = 0.02). Meanwhile, methylation status of RASSF1A was significantly correlated with patient age (P = 0.015).


Table 2 Correlation between methylation status and clinicopathological parameters in 70 HCC tissues

Characteristics RASSF1A CHFR GSTP1 MGMT
U M U M U M U M


78%* P-value 43%* P-value 38%* P-value 32%* P-value
Sex
Male 16 19 NS 25 12 NS 33 10 NS 29 8 NS
Female 10 25 15 18 11 16 19 14
Age
<60 years 13 11 0.02 18 17 NS 29 12 NS 22 11 NS
≥60 years 13 33 22 13 15 14 26 11
HBV
+ 13 20 NS 16 16 NS 25 9 NS 28 12 NS
13 24 24 14 19 17 20 10
Size
≤3cm 12 22 NS 17 20 NS 16 14 NS 18 9 NS
>3cm 14 22 23 10 28 12 30 13
Pathological grade
I/II 9 23 NS 22 7 0.015 24 16 NS 27 8 NS
III/IV 17 21 18 23 20 10 21 14


Frequency of methylation. Abbreviations: M, methylation; U, unmethylated; NS, not significant.



4. Discussion

The present evaluation focused on genes involved in cellular responses to DNA damage, including genes involved in DNA repair, cell cycle control, apoptosis signalling and on possible correlations between promoter hypermethylation and clinicopathological parameters.

In HCC tissues, hypermethylation frequencies of the investigated genes ranged from 78% for RASSFIA down to 32% for MGMT, with functional loss of these genes in HCC tissues and cell lines being attributed to methylation-mediated gene silencing mechanisms. Since hypermethylation of RASSFIA was significantly associated with increasing age, whereas methylation of CHFR was associated with tumour pathological grade, it seems that CpG island hypermethylation is common in HCC. The high frequency of hypermethylation detected in RASSFIA, CHFR, GSTP1 and MGMT genes suggests that the inactivation of these tumour suppressor genes plays a pivotal role in HCC tumorigenesis.

Hypermethylation of the RASSF1A promoter occurs in HCC tissues (Yu et al., 2002; Zhong et al., 2003), as well as in the circulation of HCC patients (Chan et al., 2008). RASSF1A gene has two main variants (RASSF1A and RASSF1C), transcribed from distinct CpG island promoters. Functional analysis of RASSF1A indicates an involvement in apoptotic signalling, microtubule stabilization and mitotic progression. This agrees with other reports since we found similar frequencies for RASSFIA hypermethylation in HCC cell lines. It is noteworthy that 78% RASSF1A promoter methylation in HCC was detected, which is a little lower than that in previous reports (∼95%; Yu et al., 2002; Zhong et al., 2003). This discrepancy might be due to the sensitivity of MSP methods and different CpG sites.

The significant association between promoter hypermethylation and age is similar to the finding of Sugawara et al. (2007) that patients with a RASSF1A methylated hepatoblastoma were older in age than those with RASSF1A unmethylated tumour. A possible explanation is that the fraction of hepatocytes already methylated with age at the RASSF1A gene promoter might become more susceptible to oncogenic injuries (Di Gioia et al., 2006).

Another important finding in this study is that CHFR hypermethylation significantly correlated with the pathological grade of HCC. CHFR expression is silenced by CpG island hypermethylation in different tumour cell lines and primary tumours, the frequencies of CHFR methylation varying in different human primary tumour types: lung cancers (19%) (Mizuno et al., 2002), oesophageal cancers (30%) (Shibata et al., 2002) and colorectal cancers (∼37–40%) (Toyota et al., 2003). As shown in our results, high (43%) frequency of CHFR methylation in 70 HCC tissues, even in HCC cell lines (40%). These results indicate that the epigenetic inactivation of CHFR plays an important role in HCC aggressiveness.

An aberrant hypermethylation of GSTP1 and MGMT promoters and a subsequently induced lack of GSTP1 and MGMT mRNA expression were found in HCC cell lines. The detoxifying gene, GSTP1, may play an important role in protecting cells against damage induced by carcinogens, especially for liver cells (Rebbeck 1997). Loss of GSTP1 may increase the risk of DNA damage and mutation (Lee et al., 1994; Zhang et al., 2005), which might be related to the hypermethylation status of the GSTP1 promoter that we noted. The DNA repair gene, MGMT, is frequently inactivated in neoplasia (colorectal, lung and lymphomas) through hypermethylation silencing (Esteller et al., 1999; Soejima et al., 2005; Hibi et al., 2009a, 2009b). However, these two genes and clinicopathological features of the primary HCC were not significantly correlated.

In conclusion, the methylation status of multiple DNA damage responses genes in HCC tissues and cell lines has been established. Further longitudinal studies of DNA methylation are needed to investigate its potential usefulness as a molecular biomarker and clinical efficacy. This would be an opportunity to prevent the development and progression of HCC.

Author contribution

Zhiqin Li carried out the laboratory experiments, analysed the data, interpreted the results and wrote the paper; Hongyan Zhang and Jianping Yang provided study material and samples, data analysis and interpretation; Tielai Hao and Shenglei Li designed experiments, discussed analyses and approved the final paper.

Funding

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

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Received 26 November 2010/13 February 2011; accepted 25 August 2011

Published as Cell Biology International Immediate Publication 25 August 2011, doi:10.1042/CBI20100851


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


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