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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 349-355 |
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doi:10.1111/j.1745-7270.2006.00164.x |
Genetic and Epigenetic Alterations
of DLC-1, a Candidate Tumor Suppressor Gene, in Nasopharyngeal Carcinoma
Dan PENG, Cai-Ping REN*,
Hong-Mei YI, Liang ZHOU, Xu-Yu YANG, Hui LI, and Kai-Tai YAO*
Cancer
Research Institute, Xiangya School of Medicine, Central South University, Changsha
410078, China
Received: February
18, 2006
Accepted: March 6,
2006
*Corresponding
authors:
Cai-Ping REN: Tel,
86-731-2355066; Fax, 86-731-4360094; E-mail, [email protected]
Kai-Tai
YAO: Tel, 86-731-4805451; Fax, 86-731-4360094; E-mail, [email protected]
Abstract������� The DLC-1 gene, located at the
human chromosome region 8p22, behaves like a tumor suppressor gene and is
frequently deleted in diverse tumors. The deletion of 8p22 is not an uncommon
event in nasopharyngeal carcinoma (NPC), therefore we explored the expression
levels of the DLC-1 gene in NPCs and NPC cell lines by reverse
transcription-polymerase chain reaction. The results showed the mRNA level of DLC-1
was downregulated. To identify the mechanism of DLC-1 downregulation in
NPC, we investigated the methylation status of the DLC-1 gene using
methylation-specific PCR, and found that 79% (31 of 39) of the NPC tissues and
two DLC-1 nonexpressing NPC cell lines, 6-10B and 5-8F, were methylated
in the DLC-1 CpG island. Microsatellite PCR was also carried out, and
loss of heterozygosity was found at four microsatellite sites (D8S552, D8S1754,
D8S1790 and D8S549) covering the whole DLC-1 gene with ratios of 33% (4
of 12 informative cases), 18% (2 of 11), 5% (1 of 18), and 25% (3 of 12),
respectively. Taken together, our results suggest that DLC-1 might be an
NPC-related tumor suppressor gene affected by aberrant promoter methylation and
gene deletion.
Key words������� DLC-1; nasopharyngeal carcinoma; hypermethylation;
loss of heterozygosity
The tumorigenesis of nasopharyngeal carcinoma (NPC) is a multi-step process involving various factors, including Epstein-Barr virus infection and accumulation of epigenetic and genetic alterations. Because of its exceptionally high incidence in southern China, discovering the molecular basis of NPC is a priority in our research. Genome-wide microsatellite polymerase chain reaction (PCR) revealed high frequency of loss of heterozygosity (LOH) on chromosome 8p22, and published reports indicate 8p22 might be a promising region containing candidate tumor suppressor genes of NPC [1].
DLC-1 (deleted in liver cancer-1), a candidate tumor suppressor gene, was isolated from human hepatocellular carcinoma (HCC) by the PCR-based subtractive hybridization approach. DLC-1 shares high sequence similarity with rat p122RhoGAP [2], which is a GTPase-activating protein (GAP) for Rho family proteins [3]. Rho family proteins play essential roles in regulating diverse biological functions, including cytoskeletal organization, cell adhesion, and cell cycle progression [4-6], and are known to be involved in Ras-mediated oncogenic transformation [7]. A GAP serves as a negative regulator of Rho proteins by stimulating its intrinsic GTPase activities [8], thus it may function as a tumor suppressor by inactivating Rho proteins. Transfection of the DLC-1 cDNA into HCC cell lines with homozygous deletions of the gene caused a strong inhibition of cell growth [9]. In addition, transfection of the DLC-1 cDNA into non-small cell lung carcinoma cell lines caused a significant inhibition of cell proliferation and a decrease in colony formation in vitro, and abolished tumorigenicity of non-small cell lung carcinoma cell lines in nude mice in vivo, which clearly showed that DLC-1 might exert tumor suppressor activity [10].
The DLC-1 gene was found to be located at 8p22, a region of LOH in a number of human cancers such as liver, lung, breast, colon, prostate, and head and neck cancers [11-15]. Of note, DNA methylation of DLC-1 was found in lung, liver, gastric and primitive neuroectodermal tumors [10,16-18], which strongly suggests that hypermethylation in the DLC-1 promoter might perform an important role in the transcriptional silencing of the gene. In this study, our data indicate that genetic and epigenetic alterations are involved in the inactivation of DLC-1 in NPC.
Materials and Methods
Samples and cell lines
Forty-one poorly-differentiated NPC biopsies (T1-T41) of primary tumors and an additional 20 NPC biopsies with matched constitutional DNA from peripheral blood lymphocytes were obtained from NPC patients with consent before treatment at the Hunan Cancer Hospital (Changsha, China). The male to female ratio of the NPC patients was 2.73:1 (30:11), and the age range was 31-62 years (mean age, 48.15 years). In addition, 16 normal nasopharynx (NP) tissues were obtained from patients without NPC at Hunan Cancer Hospital. The tissues were studied simultaneously as controls under similar experimental conditions. The male to female ratio of the patients without NPC was 3:1 (12:4), and the age range was 26-69 years (mean age, 46.42 years). All the specimens were reviewed by an otorhinolaryngologic pathologist. Fresh NPC tissues or normal tissues were snap-frozen in liquid nitrogen and stored until required.
Four NPC cell lines were obtained: HNE1 and CNE2 were from the Cancer Research Institute, Xiangya School of Medicine, Central South University (Changsha, China); 5-8F and 6-10B were from the Cancer Center, Sun Yet-Sen University (Guangzhou, China). All were maintained in RPMI 1640 containing 10% fetal bovine serum at 37 �C in a humidified 5% CO2 atmosphere. Cells were harvested for total RNA and genomic DNA extraction at 70%-80% confluence.
RNA preparation and reverse
transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from NPC cell lines and cryopreserved NPC tissue samples or normal NP tissue samples and was extracted with TRIzol Reagent (Invitrogen, Carlsbad, USA). RNA was quantified at 260 nm by a spectrophotometer and RNA quality was assessed by visualization of clear 18S and 28S RNA bands after electrophoresis through agarose gels. cDNA was synthesized from total RNA using oligo(dT) as the primer with a commercially available reverse transcription system (Promega, Madison, USA). Reverse transcription was performed in a total volume of 20 ml containing 5 mM MgCl2, 1�buffer, 1 mM dNTPs, 20 U RNasin, 14.4 U AMV reverse enzyme, 1 pM oligo(dT), 2 mg RNA. The reaction was performed according to the instructions. A pair of primers was used to amplify the 299 bp region of DLC-1, and the primer sequences were as follows: 5'-AGCCAATTCTGGAACCAAAC-3' (forward) and 5'-GGAAGACCCCAAGAAACACA-3' (reverse). At the same time, a 550 bp fragment of glyceraldehyde phosphate dehydrogenase (GAPDH) was amplified as a control. Negative controls for PCR were run in reagent mixtures without RNA or reverse transcriptase. The PCR was terminated at the exponential phases: 32 cycles for DLC-1 and 24 cycles for GAPDH, including 1 cycle of hot start at 95 �C for 5 min, followed by amplification at 94 �C for 30 s, 58 �C for 30 s, and 72 �C for 30 s, and a final extension at 72 �C for 5 min.
DNA preparation
Genomic DNA was prepared from NPC cell line pellets and NPC tissues using an improved method of extracting high molecular weight DNA with phenol/chloroform, as described previously [19]. Briefly, lysis overnight at 37 �C in 500 ml salt/EDTA buffer, 25 ml 20% (W/V) sodium dodecylsulfate and 25 ml proteinase K solution (2 mg/ml), followed by phenol/chloroform extraction and stored at -20 �C.
Bisulfite modification of DNA
and methylation analysis
The sodium bisulfite reaction converts unmethylated cytosine in DNA to uracil while leaving the methylcytosine unchanged, so that methylated and unmethylated alleles can be distinguished by methylation-specific PCR (MSPCR) or sequencing. Genomic DNA from tumor samples was purified using the standard proteinase K digestion and phenol/chloroform extraction method, as described above. Then sodium bisulfite treatment was carried out using a protocol modified from Clark et al. [20]. Ten micrograms of genomic DNA was denatured with 0.3 M NaOH and mixed with 300 ml of 10 mM hydroquinone (Sigma, St. Louis, USA), 5.2 ml of 3.6 M NaHSO3 (pH 5.0; Sigma), covered with paraffin oil then deaminated in the dark for 4 h at 55 �C. Bisulfite-treated DNA was purified with purification columns (TaKaRa, Shiga, Japan). Subsequently purified DNA samples were desulfonated with 0.3 M NaOH at room temperature for 10 min, neutralized with ammonium acetate, ethanol precipitated, and resuspended in 20 ml of Tris-EDTA buffer.
Bisulfite-treated DNA was amplified by PCR with methylation-specific primer pairs described in previously published reports [17]. The primer pairs were able to discriminate between methylated and unmethylated alleles of the DLC-1 gene. MSPCR was carried out under the following conditions: hot start at 95 �C for 5 min, followed by 35 cycles of 94 �C for 30 s, 55 �C for 30 s, and 72 �C for 30 s, and a final extension at 72 �C for 10 min. The PCR reaction conditions for the unmethylated allele of the DLC-1 gene were the same as those for MSPCR.
Allelic loss analysis
To examine the allelic loss in the DLC-1 locus, we selected four microsatellite markers on chromosome 8p22, which covered a relatively wide chromosomal region of approximately 1.6 Mb encompassing the DLC-1 gene. Primers for amplification of microsatellite markers D8S549 (maps to 1.0 Mb upstream of DLC-1), D8S1754 (locates at DLC-1), D8S1790 (locates at DLC-1), and D8S552 (maps to 0.2 Mb downstream of DLC-1) are available through the genome database on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). The microsatellite markers were amplified by PCR from 50-100 ng DNA extracted from human NPC tissues and their matched blood samples which were used as controls. Reaction was initiated at 95 �C for 5 min, 28 cycles of 94 �C for 15 s, 58 �C for 15 s, and 72 �C for 15 s, followed by a final elongation at 72 �C for 5 min.
After amplification, 6-8 ml of the reaction mixture was mixed with 8 ml of loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol), heat denatured, chilled on ice, then electrophoresed on a 6% polyacrylamide gel containing 8 M urea. The DNA bands were visualized by silver staining. LOH was scored if one of the heterozygous alleles showed at least a 50% reduction in intensity in tumor DNA as compared with the matched blood DNA.
Grayscale scanning and
statistical analysis
RT-PCR products were separated through 1.5% agarose gel containing ethidium bromide. The sizes of the RT-PCR products were 299 bp for DLC-1 and 550 bp for GAPDH. The intensity of each band was measured by Image Master VDS (Pharmacia Biotech, Piscataway, USA), and analyzed by VDS software version 2.0 for band quantification. The expressions of DLC-1 in tumors and normal tissues were investigated after they were normalized by transforming them into two groups of ratios of the band intensity of DLC-1 over that of GAPDH of the same sample. Each RT-PCR reaction was carried out in triplicate.
Otherwise, the association of DLC-1 expression with histological
type of tissue, gender and node metastasis was examined by Mann-Whitney U-test.
The association of DLC-1 methylation with gender was analyzed by Fisher抯 exact test, and the
association of DLC-1 methylation with age was analyzed by Mann-Whitney U-test,
as appropriate. All statistical analysis was performed using spss version 10.0 statistical software for
Windows (SPSS, Chicago, USA). P<0.05 was regarded as statistically
significant.
Results
Downregulation of DLC-1
expression in NPC tissues and NPC cell lines
To evaluate the relationship between mRNA expression of DLC-1 and NPC, RT-PCR was carried out in 41 samples of NPC tissues and 16 normal NP tissues as well as four NPC cell lines. Our analysis revealed that all the normal NP tissues demonstrated stable DLC-1 gene expression, which is consistent with the earlier finding that DLC-1 was widely expressed in normal tissues [2]. The DLC-1 mRNA level was normalized against the housekeeping gene GAPDH (Fig. 1). DLC-1 mRNA was undetectable in 13 cases, and the overall DLC-1 expression in NPCs was reduced significantly (P=0.001) (Table 1). DLC-1 transcripts were nearly undetectable in HNE1, CNE2, 5-8F and 6-10B NPC cell lines. Statistical analysis revealed no significant difference in expression of DLC-1 between males and females or node metastasis and non-node metastasis (P=0.063 and P=0.057, respectively). We also ranked the DLC-1 expression and age, and no statistical correlation between expression and age was found using Spearman's rank coefficient analysis (P=0.370).
Methylation status of the DLC-1
CpG island in NPC tissues and NPC cell lines
To explore the potential role of CpG island methylation in the transcriptional silencing of the DLC-1 gene, we checked the methylation status in the NPC tissues by MSPCR, which can specifically amplify the methylated and unmethylated alleles, after chemically modifying the isolated DNA with sodium bisulfite [17]. Our MSPCR analysis showed that the DLC-1 CpG island was methylated in 79% (31 of 39) of the NPC tissues and two NPC cell lines (5-8F and 6-10B) with loss of DLC-1 expression. Methylated DLC-1 alleles were also observed in 30.7% (4 of 13) of the normal samples. A representative illustration of MSPCR is shown in Fig. 2(A). In tumorous samples, T1, T2 and T4 showed both the methylation-specific and unmethylation-specific bands, but T3 showed only the unmethylation-specific band. In non-tumorous samples, N1 and N2 showed only the unmethylation-specific band. A significant difference in age was found between the methylated and unmethylated groups (Mann-Whitney U-test, P=0.003) (Table 2). No relationship was found between methylation and gender in NPCs (Fisher抯 exact test, P=0.682) (Table 3).
Allelic deletion of DLC-1
in NPCs
Previous reports manifested that the deletion of the DLC-1 gene was associated with downregulation of DLC-1 in multiple tumors [2]. To determine whether the downregulation of DLC-1 in NPCs was due to genomic deletion of DLC-1, the allelic status of DLC-1 was investigated by microsatellite analysis. As shown in Fig. 3, D8S552 and D8S549 flank a 1.6 Mb region on chromosome 8p22 containing the DLC-1 locus, and D8S1754 and D8S1790 are intragenic markers mapped in the DLC-1 gene. The LOH frequencies for D8S552, D8S1754, D8S1790, and D8S549 were 33% (4 of 12 informative cases), 18% (2 of 11), 5% (1 of 18), and 25% (3 of 12), respectively. In total, 35% (7 of 20) of informative NPCs demonstrated LOH of at least one marker: case 6 displayed LOH at site D8S549; case 8 displayed LOH at site D8S552; case 10 displayed LOH at site D8S549; case 13 displayed LOH at site D8S1790; and case 20 displayed LOH at site D8S552. Cases 9 and 16 were likely to have lost one allele of DLC-1, for these two cases displayed LOH at both of the intragenic markers and the flanking markers. These findings indicated that allelic loss at the DLC-1 locus was not uncommon in NPCs.
Discussion
Since DLC-1 was isolated from human HCC by PCR-based subtractive hybridization approach, subsequent reports have shown that DLC-1 was associated with several kinds of tumors acting like a tumor suppressor gene [9,10,21-23]. In our study, RT-PCR of DLC-1 in NPC tissues and NPC cell lines showed manifest downregulated expression of DLC-1 mRNA compared with normal NP tissues, therefore DLC-1 might be a candidate NPC tumor suppressor gene. The loss of DLC-1 mRNA expression was not only observed in tumors with genomic deletions but also in tumors without homozygous deletion, such as HCCs and gastric cancer cells [16,17], suggesting that multiple mechanisms are responsible for inactivating DLC-1 in these tumors.
To find the reasons leading to the reduced level of DLC-1 mRNA, we carried out LOH studies to investigate the allelic status of DLC-1. While, our results showed that 35% (7 of 20) informative cases of NPC biopsies demonstrated LOH in at least one site, strongly suggesting that downregulation of DLC-1 expression might not be only due to genomic deletions compared with the more conspicuous downregulation of DLC-1 detected in NPCs and epigenetic mechanism remained investigated.
There is a growing realization that LOH at a given tumor suppressor gene locus is not a prerequisite for neoplasia, following the intensive comprehension of epigenetic alterations in tumorigenesis. Methylation of the CpG island is an alternative way of making genes inactive. Structurally, the 5'-upstream region from the start codon of the DLC-1 gene (GenBank accession No. AF404867) has a high G+C content (73%), which meets the criteria for a CpG island [Fig. 2(B)]. Recently, hypermethylation of the DLC-1 promoter region was reported in HCC, lung cancer and gastric cancer unceasingly [10,16-18,24]. MSPCR analysis in our study showed that the DLC-1 CpG island was methylated in 79% (31 of 39) of the NPC tissues. Confusingly, the theme at the center of this controversy is whether significance can be attached to the methylated promoter in NPCs, considering those CpG islands methylated in four samples of 13 normal tissues. General reasons for the methylation present in histologically normal samples have been provided by others [24], such as infiltrating tumor cells, epigenetic field defect, imprinting, or tissue-specific methylation. Notwithstanding, we want to discuss individual possible mechanisms according to our results.
Promoter hypermethylation can occur in conjunction with allelic loss and/or mutation and is regarded as an alternative form of "knockout" in bi-allelic inactivation. According to accepted knowledge, within the whole sequence of the CpG island of a gene, methylation taking place at transcription factor binding sites takes overwhelming responsibility in silencing a certain gene. Actually, Bais et al. [25] reported that complex high to low methylation levels are found in primary breast tumors and their normal counterparts at multiple regions within the promoter sequence of a potential tumor suppressor gene named CBFA2T3B. They revealed that only a few cell lines displayed clear hypermethylation in association with reduced expression of CBFA2T3B in breast cancer. A second-round real-time MSPCR (quantitating methylation levels) combined with real-time RT-PCR (examining mRNA levels), still revealed the strong correlation between reduced expression and "hypermethylation" at several certain regions out of a "basal" methylation existing in the promoter CpG island. For this reason, we selected those primers with amplification products residing within a consensus and predicted specificity protein (Sp1) binding site in MSPCR. No data has directly proved the existence of the Sp1 binding site in DLC-1 utilized in MSPCR by experiment, even though the potential transcription factor binding sites are delicately predicted, precisely designed and adopted by many researchers, including us. Methylation of DLC-1 found in normal NP tissues might be due to the undefined Sp1 binding site. However, further studies using DLC-1 promoter constructs are required to identify the functional roles of the Sp1 binding sites in DLC-1 silencing.
Furthermore, a more interesting finding in our research is that a significant difference was found in age between the methylated group and the unmethylated group (P=0.003). Higher age tends to correlate with higher frequency of methylation. The available explanation for this result comes from the role of the environment, particularly carcinogens, in causing epigenetic changes. Previously published reports showed that aberrant methylation of RASSF1A was associated with exposure to smoke and correlated to a long-term smoking habit [26,27]. It is not a rare phenomenon that hypermethylation of RASSF1A has been detected from cells in the sputum and bronchioloalveolar lavages of smokers [28], which might also provide us with an alternative way of studying the methylation appearing in some normal tissues in our study. Perhaps there is something associated with the methylation of DLC-1 in our living circumstances, even though there has been no report, until now, demanding intensive investigation. Andrew P. Feinberg hypothesized that genetic and epigenetic alterations might interact, in that epigenetic alterations might influence the effect of subsequent genetic insults during the course of cancer initiation and the probability of cancer development depends on the presence of epigenetic alterations after genetic alterations have occurred [29]. Thus, considering the environmental effects on epigenetic alterations, as we age, the number of epigenetic errors increase followed by the increasing probability of carcinogenic progression after genetic changes.
Lack of DLC-1 expression was detected in one medulloblastoma cell line [18], in which no genomic deletion, somatic mutation, or promoter hypermethylation was found. A similar observation was reported in two DLC-1-nonexpressing gastric cancer cell lines without detectable methylated alleles of DLC-1 [17]. In this study, the authors were able to reactivate DLC-1 expression by treating these cells with a histone deacetylase inhibitor [17]. Thus, there is another epigenetic mechanism mediating transcriptional silencing of DLC-1 at least in gastric cancer, which is histone deacetylation. These findings have led to the speculation that perturbation in the chromatin environment with histone deacetylation might contribute to transcriptional silencing of the DLC-1 gene in gastric cancer cells.
In summary, our results indicate that mRNA levels of DLC-1 were downregulated in NPC, and promoter hypermethylation and LOH of DLC-1 were commonly found in NPC tissues. The results suggest that both promoter hypermethylation and LOH of DLC-1 might have occurred in NPC, and they might take major responsibility for the downregulation of DLC-1 in NPC.
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