Original Paper
|
|
|
file on Synergy |
Acta Biochim Biophys
Sin 2006, 38: 349-355
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? 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? 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.
References
1 Shao JY, Wang HY, Huang XM, Feng QS, Huang P,
Feng BJ, Huang LX et al. Genome-wide allelotype analysis of sporadic
primary nasopharyngeal carcinoma from southern China. Int J Oncol 2000, 17:
1267–1275
2 Yuan BZ, Miller MJ, Keck CL, Zimonjic DB,
Thorgeirsson SS, Popescu NC. Cloning, characterization, and chromosomal
localization of a gene frequently deleted in human liver cancer (DLC-1)
homologous to rat RhoGAP. Cancer Res 1998, 58: 2196–2199
3 Homma Y, Emori Y. A dual functional signal
mediator showing RhoGAP and phospholipase C-delta stimulating activities. EMBO
J 1995, 14: 286–291
4 Hall A. Rho GTPases and the actin
cytoskeleton. Science 1998, 279: 509–514
5 Moon SY, Zheng Y. Rho GTPase-activating
proteins in cell regulation. Trends Cell Biol 2003, 13: 13–22
6 Etienne-Manneville S, Hall A. Rho GTPases in
cell biology. Nature 2002, 420: 629–635
7 Qiu RG, Chen J, McCormick F, Symons M. A role
for Rho in Ras transformation. Proc Natl Acad Sci USA 1995, 92: 11781–11785
8 Morii N, Kumagai N, Nur-E-Kamal MS, Narumiya
S, Maruta H. Rho GAP of 28 kDa (GAP2), but not of 190 kDa (p190), requires
Asp65 and Asp67 of rho GTPase for its activation. J Biol Chem 1993, 268: 27160–27163
9 Ng IO, Liang ZD, Cao L, Lee TK. DLC-1
is deleted in primary hepatocellular carcinoma and exerts inhibitory effects on
the proliferation of hepatoma cell lines with deleted DLC-1. Cancer Res
2000, 60: 6581–6584
10 Yuan BZ, Jefferson AM, Baldwin KT,
Thorgeirsson SS, Popescu NC, Reynolds SH. DLC-1 operates as a tumor suppressor
gene in human non-small cell lung carcinomas. Oncogene 2004, 23: 1405–1411
11 Fujiwara Y, Ohata H, Emi M, Okui K, Koyama K,
Tsuchiya E, Nakajima T, et al. A 3-Mb physical map of the chromosome
region 8p21.3-p22, including a 600-kb region commonly deleted in human
hepatocellular carcinoma, colorectal cancer, and non-small cell lung cancer.
Genes Chromosomes Cancer 1994, 10: 7–14
12 Patel U, Grundfest-Broniatowski S, Gupta M,
Banerjee S. Microsatellite instabilities at five chromosomes in primary breast
tumors. Oncogene 1994, 12: 3695–3700
13 Cunningham C, Dunlop MG, Wyllie AH, Bird CC.
Deletion mapping in colorectal cancer of a putative tumour suppressor gene in
8p22-p21.3. Oncogene 1993, 8: 1391–1396
14 Bova GS, Carter BS, Bussemakers MJ, Emi M, Fujiwara
Y, Kyprianou N, Jacobs SC, et al. Homozygous deletion and frequent
allelic loss of chromosome 8p22 loci in human prostate cancer. Cancer Res.
1993, 53: 3869–3873
15 Wu CL, Roz L, Sloan P, Read AP, Holland S,
Porter S, Scully C, et al. Deletion mapping defines three discrete areas
of allelic imbalance on chromosome arm 8p in oral and oropharyngeal squamous
cell carcinomas. Genes Chromosomes Cancer 1997, 20: 347–353
16 Wong CM, Lee JM, Ching YP, Jin DY, Ng IO.
Genetic and epigenetic alterations of DLC-1 gene in hepatocellular
carcinoma. Cancer Res 2003, 63: 7646–7651
17 Kim TY, Jong HS, Song SH, Dimtchev A, Jeong
SJ, Lee JW, Kim TY et al. Transcriptional silencing of the DLC-1
tumor suppressor gene by epigenetic mechanism in gastric cancer cells. Oncogene
2003, 22: 3943–3951
18 Pang JC, Chang Q, Chung YF, Teo JG, Poon WS,
Zhou LF, Kong X et al. Epigenetic inactivation of DLC-1 in
supratentorial primitive neuroectodermal tumor. Hum Pathol 2005, 36: 36–43
19 Sambrook J, Fritsch EF, Maniatis T. Molecular
Cloning. 2nd ed. New York: Cold Spring Harbor Laboratory Press 1989
20 Clark SJ, Harrison J, Paul CL, Frommer M. High
sensitivity mapping of methylated cytosines. Nucleic Acids Res 1994, 22: 2990–2997
21 Yuan BZ, Zhou X, Durkin ME, Zimonjic DB,
Gumundsdottir K, Eyfjord JE, Thorgeirsson SS et al. DLC-1 gene
inhibits human breast cancer cell growth and in vivo tumorigenicity.
Oncogene 2003, 22: 445–450
22 Zhou XL, Thorgeirsson SS, Popescu NC.
Restoration of DLC-1 gene expression induces apoptosis and inhibits both
cell growth and tumorigenicity in human hepatocellular carcinoma cells.
Oncogene 2004, 23: 1308–1313
23 Goodison S, Yuan J, Sloan D, Kim R, Li C,
Popescu NC, Urquidi V. The RhoGAP DLC-1 functions as a metastasis
suppressor in breast cancer cells. Cancer Res 2005, 65: 6042–6053
24 Dammann R, Strunnikova M, Schagdarsurengin U,
Rastetter M, Papritz M, Hattenhorst UE, Hofmann HS et al. CpG island
methylation and expression of tumour-associated genes in lung carcinoma. Eur J
Cancer 2005, 41: 1223–1236
25 Bais AJ, Gardner AE, McKenzie OL, Callen DF,
Sutherland GR, Kremmidiotis G. Aberrant CBFA2T3B gene promoter
methylation in breast tumors. Mol Cancer 2004, 3: 22–37
26 Kim DH, Kim JS, Ji YI, Shim YM, Kim H, Han J,
Park J. Hypermethylation of RASSF1A promoter is associated with the age
at starting smoking and a poor prognosis in primary non-small cell lung cancer.
Cancer Res 2003, 63: 3743–3746
27 Golub TR, Slonim DK, Tamayo P, Huard C,
Gaasenbeek M, Mesirov JP, Coller H et al. Molecular classification of
cancer: Class discovery and class prediction by gene expression monitoring.
Science 1999, 286: 531–537
28 Honorio S, Agathanggelou A, Schuermann M,
Pankow W, Viacava P, Maher ER, Latif F. Detection of RASSF1A aberrant
promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in
situ from breast cancer patients. Oncogene 2003, 22: 147–150
29 Feinberg AP. The epigenetics of cancer
etiology. Semin Cancer Biol 2004, 14: 427–432