Human GMDS gene fragment hypermethylation in chronic high level of arsenic exposure with and without arsenic induced cancer
© Chanda et al.; licensee Springer. 2013
Received: 19 June 2013
Accepted: 26 September 2013
Published: 24 October 2013
Arsenic, though a poor mutagen, is an accepted environmental carcinogen. Perturbation of DNA methylation pattern leading to aberrant gene expression has been hypothesized as the mechanism for arsenic induced carcinogenesis. We had earlier demonstrated the hypermethylation of promoter region of p53 and p16 genes in persons exposed to different doses of arsenic. Till now no genomic hot spot has been identified which is frequently hypermethylated or hypomethylated in persons chronically exposed to environmental arsenic. In the present work, we have identified one hypermethylated sequence by methyl-sensitive arbitrarily primed polymerase chain reaction in the peripheral blood leukocyte DNA of chronically arsenic exposed persons with and without arsenic induced skin cancer. The sequence is from GMDS gene responsible for fucose metabolism. Southern hybridization of the sequence to the amplification products of methyl sensitive restriction enzyme digested genome of persons exposed to different doses of arsenic indicated that methylation increased in a dose dependent manner.
According to (International agency for research on cancer1997) and National Research Council (NRC1999) arsenic is an important environmental toxicant and carcinogen. However, the mechanism of arsenic mediated carcinogenesis is not clear as arsenic is a poor mutagen (Rossman et al.1980; Jacobson and Moltanbano1985; Lee et al.1985) and does not induce significant point mutations. Biotransformation of arsenic, on the other hand, involves methylation of inorganic arsenic to organic monomethyl arsonic acid (MMA) and dimethyl arsinic acid (DMA), using the same methyl donor S-Adenosyl methionine (SAM) also involved in DNA methylation (Vahter1999). The interference of the DNA methylation pathway with arsenic detoxification pathway, as both the pathways require SAM, can lead to aberrant DNA methylation, resulting aberrant expression and/or silencing of genes Goering et al. (1999). Therefore, epigenetic alterations, particularly aberrant DNA methylation has been mooted as a possible mechanism of arsenic induced carcinogenesis (Ren et al.2010; Reichard and Puga2010).
Cytosine-5 methylation at the CpG islands in the regulatory sequence of a gene is one of the key mechanisms of gene inactivation. DNA methylation/demethylation seems to regulate a plethora of biological processes involving transcription, differentiation, development, DNA repair, recombination, and chromosome organization. Perturbation of DNA methylation has been correlated with many cases of cancer (Jones and Baylin2002). The hypothesis that arsenic perturbs DNA methylation has been tested successfully on tissue culture system (Mass and Wang1997), and later we demonstrated hypermethylation of the promoter region of p53 and p16 genes in DNA extracted from peripheral blood leucocytes of persons exposed to different doses of arsenic (Chanda et al.2006). A few highly exposed persons also showed p53 hypomethylation (Chanda et al.2006). Further arsenic induced genome wide hypermethylation has been demonstrated by us in DNA extracted from same population Majumder et al. (2010).
In this report we have further evaluated the hypothesis on a subsection of exposed population studied by isolating hypermethylated sequence from genomic DNA of arsenic exposed persons by methyl sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR). Differentially methylalated fragments have been identified and isolated from chronically arsenic exposed people. 7 persons of arsenic induced skin cancer out of 16 (all the cancer patients were recruited from previous study, Chanda et al.2006), have one common hypermethylated fragment of 565 bp (this was cloned and sequenced). The same sequence was also isolated from 3 chronically arsenic exposed persons out of 10 (all of these 10 subjects were recruited from previous study, Chanda et al.2006). The sequence is then analysed by bioinformatic tools (NCBI BLAST) to indicate that the fragment is actually situated in the human GDP mannose 4–6 dehydratase gene (GMDS gene). Southern hybridization of this fragment to amplified products from methyl sensitive restriction enzyme digested genomic DNA of persons exposed to arsenic in drinking water indicated that the sequence is indeed hypermethylated. The product of the identified gene is involved in fucose metabolism and it is reported that deletion of this gene results in cancer progression (Thompson et al.1992; Becker and Lowe2003;Yuan et al.2008). Though have been found here in small proportion this hypermethylated fragment may be act as a potential target (probe) for detecting aberrant methylation in chronic high level of arsenic exposure.
Materials and methods
Subjects of this study were the same set of our earlier study on arsenic induced DNA hypermethylation in p53 and p16 gene promoter region and are all residents of South & North 24 Parganas, West Bengal, India (Chanda et al.2006).
Criteria of diagnosis of arsenicosis and its severity are based on the parameters described earlier (GuhaMazumder et al.1998; GuhaMazumder2001; Chanda et al.2006). In this study only the subjects for p53 gene promoter hypermethylation group of the previous study had been chosen. Participants had been divided into the five groups A, B, C, D according to the concentration of arsenic in their drinking water, i.e. 0–50, 51–250, 251–500, 501–1000 μg/l respectively as earlier and group E with 500–1100 μg/l of arsenic suffering from arsenic induced skin cancer. As the concentration of arsenic in group A is within the permissible limit according to WHO and Medical Council of India, it was considered as the unexposed control group (NRC1999). Initially the number of participants in each group was 24, 12, 18, 15 and 16 in A, B, C, D and E group respectively (Chanda et al.2006). Among those, 11 subjects in group C, 10 subjects in group D and all the 16 subjects in group E were chosen for this study. All of the subjects chosen for MS-AP PCR have hypermethylated p53 promoter region compared to normal unexposed persons (Chanda et al.2006). All of the subjects studied for MS-AP-PCR were compared to normal unexposed subjects treated in similar way. In this study 12 of the normal unexposed subjects were recruited from group A.
Demographic data of study subjects taken from different arsenic exposure groups
Group A (0-50 μg/l) p53 methylation
Group C (250-500 μg/l) p53 methylation
Group D (501-1000 μg/l) p53 methylation
Group E (500-1100 μg/l) p53 methylation
N = 2; 0.08, 0.19
N = 2; 1.34, 1.62
N = 2; 1.50, 1.45
N = 3; 0.03, 0.23, 0.15
N = 3; 1.50, 2.78, 5.00
N = 4; 1.95, 2.04, 4.3 2.46
N = 3; 0.85, 1.09, 1.32
N = 1; 0.15
N = 1; 2.56
N = 3; 1.00, 1.62, 1.09
N = 3;0.18, 0.27, 0.27
N = 2; 1.56, 2.4
N = 2; 2.85, 4.46
N = 4; 1.42, 3.08, 2.09, 2.09
N = 3; 0.08, 0.09, 0.32
N = 2; 1.95, 4.66
N = 2 4.45, 2.7
N = 1; 2.55
N = 1; 2.15
N = 4; 2.20, 2.23, 1.62, 1.60
N = 1; 2.11
Avarage duration of exposure
Total number of samples
Written informed consent was obtained from all participants before drawing their blood. The name of the institute where human clinical studies were carried out is Institute of Post Graduate Medical Education and Research, Kolkata (IPGME&R), which is run by Govt. of West Bengal, a state government within the framework of Republic of India.
Molecular Biological and in silico experiments were carried out in University of Calcutta and Presidency University, Kolkata which are also run by Govt. of West Bengal. Ethical principles followed by the institute are guided by rules as formulated by Indian Council of Medical Research and these are in agreement with Helsinki declaration.
Determination of Arsenic concentration in urine and water
Level of arsenic in drinking water and urine was determined by atomic absorption spectrophotometer with hydride generation system (AAS) Atallah and Kalman (1991).
DNA isolation from blood
DNA was extracted from whole blood by conventional chloroform extraction method using 0.01% SDS and Proteinase K (0.1 mg/ml) (Miller et al.1998).
DNA isolation from tissue
DNA was extracted from cancer biopsy tissue samples by conventional phenol-chloroform (1: 1, v/v) extraction method and then by chloroform extraction followed by salting out using 0.01% SDS and Proteinase K (0.1 mg/ml) (Miller et al.1998).
p53 methylation status analysis
The p53 tumor suppressor gene methylation status was analyzed in each subject by the method described earlier (Chanda et al.2006).
Determination of clinical symptom score
Dermatological criteria and graduation of chronic arsenic toxicity for scoring sustem of skin manifestations
Moderate score = 2
Severe score = 3
Defuse Melanosis, Mild Spotty pigmentation, Leucomelanosis
Moderate Spotty pigmentation
Blotchy Pigmentation, Pigmentation of under surface of tongue, buccal mucosa
Mild Score = 1
Moderate score = 2
Severe score = 3
Slight thickening, or minute papules (<2 cm) in palm and soles
Multiple raised keratosis papules (2 to 5 cm) in palm & soles with diffuse thickening
Diffuse severe thickening, large discreet or confluent keratotic elevations (>5 cm), palm and soles (also dorsum of extremely and trunk)
Restriction enzyme digestion for arbitrarily primed PCR
Concentration and quality of isolated genomic DNA was determined UV–vis spectrophotometer (OD 260/280 >1.8). 300 ng of total genomic DNA isolated from persons, unexposed/exposed to arsenic through drinking water, was digested with 5 units of RsaI and 5units of HpaII restriction enzyme at 37°C overnight. HpaII is a methylation sensitive isoshizomer of MspI whose recognition sequence is CCGG. A sequence hypermethylated at this site would not be digested, whereas the unmethylated DNA would. The persons taken for MS-AP-PCR were from higher exposure groups of arsenic (251–500 μg/l, i.e. group C; 500–1000 μg/l, i.e. group D; and arsenic induced cancer group, E, with an exposure level of 500–1100 μg/l) and all have hypermethylated p53 promoter. Out of 18 in group C, 11 subjects were taken for MS-AP-PCR. All have hypermethylated p53 promoter region. In group D only 10 samples were chosen with hypermethylated p53 promoter having a median value of 2.63. In group E all the 16 samples were studied with p53 promoter hypermethylation with a median value of 1.62. The median value for p53 methylation in group A (unexposed control group) was 0.26 which is treated here as a basal value for normal unexposed persons (Chanda et al.2006). Demographic data and p53 methylation values for subjects included in this study are presented in Table 1.
Methyl sensitive arbitrarily primed PCR (MS-AP- PCR)
When RsaI + HpaII digested DNA was used as template in MS-AP-PCR using random primers that target CG-rich DNA sequences (Zhong and Mass2001), a series of amplified products were observed. Of these, a band present in PCR products of arsenic exposed DNA but absent in PCR products of similarly digested unexposed DNA represents the region of hypermethylation (Zhong and Mass2001). We used 3 different primers. Amongst these primers, OPN Hind12 (5’-AGCTTCTCCCTC-3’) gave one common hypermethylated fragment in arsenic induced cancer patients and in highly arsenic exposed persons when subjected to PCR amplification. The concentration of OPN Hind 12 in the PCR reaction mixture was 0.5μM. The PCR protocol was initial denaturation at 94°C for 5 minutes, 35 cycles at 94°C for 1 min, 40°C for 1 min, 72°C for 2 min, followed by 10 min at 72°C.
Isolation of candidate bands
Demographic data and p53 methylation status of subjects having GMDS gene hypermethylation
Conc. of arsenic in water μg/l
Duration of exposure yrs
Degree of pigmentation
Total urinary arsenic μg/l
p53 methylation value
++ + 3
++++ ++ 6
++++ ++ 6
++++ ++ 6
++++ ++ 6
DNA cloning in plasmid vector
The gel- recovered PCR product was re-amplified using the same PCR protocol with same primer. The amplified product was then purified by ethanol precipitation and cloned in E.coli XL1 blue strain using pTZ57R/T vector (TA cloning kit, Fermentas). The positive clones were identified by performing colony PCR with universal primers and sequenced.
Demographic data of subjects taken from different exposure group for southern hybridization
Conc. of arsenic in water (μg/l)
Duration of exposure yrs
Degree of pigmentation
Total urinary arsenic (μg/l)
p53 methylation value
Using the technique of MS-AP PCR, 1 common hypermethylated DNA fragment was identified from 10 different people with chronic high level of arsenic exposure with and with out cancer. Among 16 of the arsenic induced cancer patients studied (belonging to group E) 7 have the hypermethylated DNA fragment of 565 nucleotide base pair. Among 10 of group D subjects 3 have been identified to harbour this hypermethylated DNA fragment. Demographic data and p53 methylation status for these 10 subjects (with hypermethylated DNA) has been listed in Table 3. Interestingly, people from lower arsenic exposure (group C) did not have this hypermethylated gene fragment.
The fragment identified is a region of hypermethylation in comparison to normal unexposed persons. DNA sequence analysis revealed that the identified fragment has significant homology match (99%) to the sequence of human GMDS gene ( Accession no. NT_007592.15), Homo sapiens) (taken from GENEBANK database) after BLAST search. The sequence is situated in the intron between exon 1 and 2 of GMDS gene. (Genomic context: chromosome: 6; Maps: 6p24.1-25.3). It is the longest intronic sequence in GMDS gene (> 1,80,000 bp). This gene is involved in carbohydrate metabolism and generation of fucose. Fucose mediates initial contact between extravagating leucocytes and endothelial cells. Influence of fucose generating enzymes on leukocyte adhesion activity has been reported (Sullivan et al.1998; Eshel et al.2001).
Our identified fragment, OPN Aga 8, shows very low association with the DNA of <50 μg/l exposure group (Figure 1a). The degree of association increases gradually with the degree of arsenic exposure in higher exposure groups. The fragment isolated has a higher degree of association during hybridization with PCR product of person with arsenic induced cancer (Figure 1b).
GenBank accession numbers and size of GMD sequence of sampled taxa
Maximum composite likelihood estimate of the pattern of nucleotide substitution
Identified sequence OPN Aga 8
TCCCTCACTA CTCAAAGTTG ATGACTTCTT AAACCAAAAT GGTTGGTCAG AATCCAATCA AGAATATAAA GGCAACTGAA TAAATAAAAC CATAAAGTAA GTGTAAAATA CTAGTGTCCA GACATCTGAG ATGTATGTGG CTACTATGAA ACTTCCACAG CTGTACCGGC CGGGAGCTCA CGTGGTTCCC CAGGTTTAAC AGAACCCATT ACCAGTAAGA GTTTTATTTG CTTAATAAAT TACATTCTAA AGCACAATAG CCTAGGCTCA TAGCTGTAAA ATTGCCAAAT ATTGTCAATG ACCACTCTCT GGTCATAAAT AACAAAATAA TCTTGTGACT CATTGGATTT TTGATTCCCAAGGCGATTCT TTCTCGCCAT TACTCAAAAA TGTGAAAAAG TGCCTCTACGTGGCATTTTA TGGAGGATAT AAATTACTCA AAGGAGATGA CATAGGACAGATTTGTAGGC CGAGTAACAG GAACCAGCCA ACCAACTGTG TAAATTAAAGAACTAGTGAC AAAGAAGAGG GCTAGTGAAA GAATTCTGAA ATCCTAAGAA CAGAT
The mechanism by which arsenic contributes to the development of cancer is currently a subject of intense interest. Arsenic does not act as a point mutagen. However, metabolism of arsenic involves methylation of inorganic arsenate to dimethyl arsinic acid via alternating reduction of pentavalent arsenic to trivalent arsenic and addition of methyl group (Vahter1999; Donohue and Abernathy2001). The arsenic methyl transferase uses the same methyl donor SAM as DNA methyltransferase (Dnmt) and other methyltransferases. Interaction of arsenic methylation/detoxification pathway with DNA methylation pathway and consequent imbalance in DNA methylation has been envisaged. Increase of cytosine methyltransferase transcript after arsenic exposure has been reported (Zhong et al.2001), and this might explain the initial hypermethylation through excessive induction of the enzyme. On the other hand prolonged arsenic exposure may cause depletion of the SAM pool due to over consumption of the methyl groups by arsenic methyltransferase, and cause hypomethylation of DNA. Although we have failed to isolate any hypomethylated fragment from patients who have arsenic induced cancer or persons having chronic high level of exposure with systemic manifestations, yet, there was number of subjects with p53 promoter hypomethylation in our previous study (Chanda et al.2006). In fact decrease of tissue arsenic burden has been correlated with methionine intake in experiments with laboratory rats exposed to arsenic (Nandi et al.2005). Interestingly the fragment of GMDS gene isolated from subjects having high degree of arsenic exposure and relatively high degree of p53 methylation are from male subjects except one. We had not found any correlation between sex and p53 methylation status in our previous study (Chanda et al.2006). The hypermethylated gene fragment has been isolated from both smokers and nonsmokers although in the present study the number of smoker having GMDS gene intron hypermethylation is 8 out of 10. In our previous study we showed that there was no association between the p53 and p16 methylation status with individual’s smoking habit. In this study, due to small sample number, we could not carry out statistical evaluation for correlation between smoking habit and GMDS gene methylation. The fragment isolated in this study is from 9 male subjects and only one female subject out of 10 subjects. Increase in number of study subjects and consequent rise in the number of subjects having GMDS gene hypermethylation may provide definite information about the association (if any) of sex specificity of GMDS gene hypermethylation with arsenic exposure in human.
Such aberrant methylation of the genome leading to gene expression anomalies and have been mooted as possible mechanism of arsenic induced carcinogenesis. Cancer, which results from inappropriate expression of genes, may involve hypomethylation of protooncogene and/or hypermethylation of tumor suppressor genes that can alter the level of their expression and thereby promote cancer. In fact, there are recent observations that widespread methylation changes occur during tumor development (Jones and Baylin2002).
Overall, tumor cell DNA is hypomethylated compared to normal cell DNA and underexpression of Dnmt1 gene causes aggressive tumor induction in genetically engineered mice (Gaudet et al.2003). However, for some tumor suppressors like p16, p15 methylation is a common alternative to point mutation and in others like RASSF1A or H1C1, it is the only mechanism for tumor specific loss of function (Jones and Baylin2002). Silencing of genes like TIMP-3 through methylation has been associated with metastasis (Darnton et al.2005).
Methylation of DNA is maintained by a balance of the activity of DNA methyltransferase (Dnmt 1, Dnmt 3a and Dnmt 3b) and DNA demethylase (mbd2) activity. Inhibition of mbd2 by antisense expression results in inhibition of anchorage-independent growth of antisense transfected cancer cells or cells infected with an adenoviral vector expressing antisense mbd2 (Slack et al.2002). Expression of Dnmt mRNA is significantly high in gastric cancer in comparison to non-cancerous gastric mucosa (Fang et al.2004). Similarly level of mbd2 mRNA level is significantly lower in gastric cancer tissue than normal gastric mucosa (Fang et al.2004).
Previous works with human adenocarcinoma cell line in tissue culture showed that arsenic induces significant changes in methylation status in tumor suppressor gene p53 Mass and Wang (1997). Later, using arsenic exposed human kidney cell lines global hyper and hypomethylation has been demonstrated by the same group (Zhong et al.2001). DNA sequencing and SssI methylase assay were used for estimation of genomic CpG methylation level. Arsenic exposure of A 549 cells in culture resulted in a dose dependent increase in cytosine methylation in p53 gene and a small increase in global methylation Mass and Wang (1997). Later we have shown that arsenic induces genomic hypermethylation in chronically exposed persons Majumder et al. (2010). An increase in the rate of transcription of DNA methyltransferase gene in cells exposed to arsenite was detected by RT-PCR (Zhong et al.2001). Our group has demonstrated for the first time that there is dose dependent enhancement of methylation in the promoter region of p53 and p16 tumor suppressor genes of genomic DNA extracted from peripheral blood leucocytes of persons exposed to various doses of arsenic (Chanda et al.2006). However, both these genes are associated with cell cycling and repair, and the possibility exists that methylation perturbation observed is engineered through disturbances in cell cycle produced by arsenic, and is local, rather than a global effect of arsenic.
In the present work we have investigated that whether there is any probable common target for aberrant DNA methylation after arsenic exposure in exposed persons apart from p53 or p16 gene methylation. We have successfully identified one fragment of hypermethylated DNA from persons exposed chronically to arsenic and persons having arsenic cancer. The subjects have been chosen from our previous study population (Chanda et al.2006) having hypermethylated p53 promoter region with chronic high level of arsenic exposure with and without arsenic induced cancer. Therefore persons having GMDS gene intron hypermethylation also have p53 promoter hypermethylation. Thus this study reflects an association between the p53 promoter hypermethylation with GMDS gene intron hypermethylation in chronic high level of arsenic exposed people. The fragment was isolated from both peripheral blood leukocyte DNA and from cancer biopsy tissue of persons having arsenic induced cancer. The hypermethylated DNA fragment is from GMDS gene responsible for fucose metabolism. GMDS is the binding partner of tankyrase which is needed to be associated for the first step of fucose biosysnthesis (Bisht et al.2012). Oligosaccharides are involved in various aspects of life process including birth, differentiation, growth, inflammation, carcinogenesis, and cancer metastasis. Fucosylation is one of the most important oligosaccharide modifications in cancer. This type of glycomodification can be treated as a biomarker in cancer (Moriwaki et al.2009; Miyoshi et al.2012).
Fucosylated alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular haptoglobin have also been found in sera of patients with various carcinomas (Miyoshi et al.2012). Deletion mutation of the GMDS gene plays a pivotal role in fucosylation in human colon cancer. Loss of function mutation of this gene may lead to a virtually complete deficiency of cellular fucosylation, tumor progression and metastasis (Nakayama et al.2013) and transfection of the wild-type GMDS into HCT116 cells restored the cellular fucosylation. This type of GMDS mutation resulted in resistance to TRAIL-induced apoptosis followed by escape from immune surveillance (Moriwaki et al.2009; Haltiwanger2009; Moriwaki et al.2011) and thus promote carcinogenesis. Further, epigenetic regulation of fucosylation and TRAIL induced apoptosis in conjunction to cancer had been studied by same group (Moriwaki et al.2010). Although in the present study we have not shown any association with the level of fucose in patients with GMDS gene hypermethylation, but still GMDS gene fragment hypermethylation is associated with p53 hypermethylation with development of arsenic induced cancer (in group E) or severe skin manifestations (in group D) as a result of chronic high level of exposure. In the present study we have not work out the degree of correlation (if any,) between the GMDS intron hypermethylation and p53 promoter hypermethylation, (as quantitative analysis of GMDS gene hypermethylation has not been studied here), although this study signifies the association between p53 promoter hypermethylation and GMDS gene hypermethylation after chronic arsenic exposure.
The intronic fragment isolated in the present study showed hypermethylation in comparison to normal unexposed subjects. This epigenetic modification may be involve in transcriptional modification and may modify the ultimate cellular level of GMDS enzyme. As it is reported that aberrant methylation of introns or intergenic regions can regulate non coding RNA function to modify the degree of transcription of a gene and the exonal expression is dependent over the local methylation status rather than the promoter region (Cheung et al.2011). Consequences of intron methylation have also been studied by Hoivik et al. (2011) and Jowaed et al. (2010) in two separate studies where it was reported that intron methylation is associated with altered expression. Moreover, dense methylation surrounding transcription start site or near the first exon is tightly linked with gene silencing (Brenet et al.2011).
Till to date this is the first report of GMDS intron hypermethylation in chronic arsenic exposure with and without malignancy. Reports are also unavailable regarding association between p53, p16 gene hypermethylation and GMDS gene hypermethylation in human cancer as well as in arsenic induced cancer.
During the initial stage of the experiments we did observe some bands of hypomethylation, but we failed to clone them. It might be mentioned that in our previous investigations too, we observed far fewer hypomethylation cases. It is postulated that overexposure of arsenic and its biotransformation causes depletion of SAM, leading to hypomethylation of DNA. Hence extensive hypomethylation probably needs a very high exposure, which is achieved in artificial tissue culture systems, but rarely in real life situation. In the tissue culture experiments too, the study with cells exposed to arsenite for 2–4 weeks observed mostly hypermethylation and a few hypomethylation cases (Zhong et al.2001). Chronic exposure of 18 weeks at low dose, on the other hand produced extensive hypomethylation and transformation in rat hepatocyte cell line (Zhao et al.1997).
To sum up, this is the first report of GMDS gene fragment hypermethylation in the peripheral blood leukocyte DNA of persons exposed to arsenic. To ascertain this fragment of hypermethylation as a biomarker for arsenic induced cancer and chronic arsenic exposure researchers require repetition of such work in large sample group.
Help and advice of Dr. S Mukhopadhyay and Dr. S. Kundu at various stages of the work is acknowledged. DNGM Foundation provided partial financial support. No financial relationship exists between authors and the organization which have financially support the research.
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