Hypomethylation of the DNMT3L promoter in ocular surface squamous neoplasia.
The genetic predisposition to cancer has been well recognized, but increasing evidence points to the role of epigenetic mechanisms in the initiation and progression of cancer. A wide array of epigenetic changes, including genomewide hypomethylation, gene-specific hypermethylation, and aberrant histone modifications, are the hallmark of most cancers. (5-7) Both promoter methylation-mediated silencing of tumor suppressor genes and activation of oncogenes because of promoter hypomethylation have been well documented. (8-13) The DNA methyltransferase 3-like (DNMT3L) gene is a member of the DNA methyltransferase 3 (DNMT3) gene family (14) and regulates DNA methylation by interacting with the de novo methyltransferases DNA methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B). (15,16) DNMT3L is highly expressed in gametes and is a known factor responsible for the acquisition of imprints during gametogenesis. (17) In a previous study, we reported the loss of methylation at the DNMT3L promoter in cervical cancers. (18) Because the pathobiology of OSSN is similar to that of cervical squamous cell carcinoma, we hypothesized that OSSN may harbor similar epigenetic changes. Hence, our specific aim was to investigate the methylation status at the promoter of DNMT3L in squamous cell tumors of the ocular surface.
MATERIALS AND METHODS
The present pilot study was approved by the Institutional Review Board. Fresh tumor tissues (n = 6) were collected from patients with a clinical diagnosis of OSSN and stored in liquid nitrogen. After histologic confirmation of the diagnosis, the tissues were thawed for DNA extraction. Healthy conjunctival tissue specimens (n = 7) were collected from an ongoing project on characterization of limbal and conjunctival stem cells, which was also approved by the Institutional Review Board.
The homogenized tissue samples were suspended in DNA extraction buffer (100mM Tris--hydrogen chloride at pH 8.0, 50 mM ethylenediaminetetraacetic acid [EDTA], 1% sodium dodecyl sulfate, 200 mM sodium chloride, and 100 [micro]g proteinase K) and incubated overnight at 37[degrees]C. Genomic DNA was purified using the phenol-chloroform extraction method and ethanol precipitation.
Bisulfite Conversion and Polymerase Chain Reaction
DNA methylation analysis of the DNMT3L promoter was carried out using the sodium bisulfite-assisted genomic DNA conversion method described previously by Gokul et al (18) Briefly, 1 mg of genomic DNA was denatured and mixed with low-melting agarose (BMA, Rockland, Maine) and pipetted into cold, heavy mineral oil (Sigma-Aldrich, St Louis, Missouri) to form beads. These beads were incubated in a bisulfite modification solution first at 0[degrees]C for 30 minutes and then at 50[degrees]C for 3.5 hours. The beads were then incubated with 0.2 M sodium hydroxide (Sigma-Aldrich) and washed with 1X Tris-EDTA and sterile deionized water. The polymerase chain reaction (PCR) was performed using specific primers (hDNMT3LbisF: 5'-TTAGTTTTATTGAGTTTTTAATTTT-3' ;hDNMT3LbisR: 5' -TAATAAATTCAAAATTCCAATAAT-3' [MWG Biotech, Bangalore, India]) amplifying a 375-base pair region within the DNMT3L promoter. The amplification was performed in a thermal cycler (Eppendorf Master Cycler, Hamburg, Germany), with initial denaturation at 95[degrees]C for 5 minutes, followed by 35 cycles of denaturation at 94[degrees]C for 1 minute, annealing at 55[degrees]C for 1 minute, and extension at 72[degrees]C for 1 minute.
Cloning and Sequencing of PCR Products
The PCR products were ligated to a TA-based cloning vector (designed by inserting 2 Xcml sites in the p Blue Script SK+ [Stratagene] vector, which gives T [thymidine] and A [adenosine] overhang after digestion) and transformed into Escherichia coli (DH10B [Invitrogen, Carlsbad, California]) competent cells. Ampicillin-resistant colonies were screened for the presence of the insert by colony PCR. Plasmids were isolated from positive colonies and were sequenced using vector-specific primers. Multiple clones (range, 3-11 per sample) were sequenced to obtain a complete methylation profile.
The differences in DNA methylation levels at the DNMT3L promoter in OSSN and in healthy conjunctiva were analyzed using the t test for each CpG at all loci examined. The t test was done using 1-tailed distribution and taking into account unequal variance in the 2 data sets. The differences were considered to be statistically significant when the P values were less than .05. The efficacy of the bisulfite conversion was found to be 98.6% (SEM = 0.56%) for all cytosines converted to thymine in a non-CpG context.
We studied the methylation levels of 11 CpG dinucleotides in the DNMT3L promoter. The mean age of the 6 patients with OSSN included in the study was 50.2 years (range, 35-65 years); 4 patients were histologically diagnosed as having invasive tumors, and 2 had intraepithelial tumors. All 6 cases of OSSN showed hypomethylation at the DNMT3L promoter region as compared with the control samples, as shown in Figure 1.
Among the clones analyzed, most CpGs were methylated at an average rate of 60% in controls but at a rate of 39% in OSSN samples (obtained by calculating the percentage of methylation at each CpG dinucleotide for each sample and averaging across the control or tumor samples). The summary of the DNA methylation profiles for the controls and OSSN are presented in Figure 2. Of the tumor samples that we analyzed, OSSN4 showed a complete loss of DNA methylation at all the CpG dinucleotides except the 11th CpG (3 clones were screened for OSSN3; however, those 3 clones exhibited a uniform profile, except for CpG3 in clone 2). In the healthy conjunctiva tissues, the fourth CpG was found to be devoid of any methylation, whereas the 11th CpG was found to be methylated in all the clones (except in few clones of conjunctiva 3). The methylation profile of the 4th and 11th CpGs in OSSN cases resembled that of controls (except for CpG 11 in OSSN1, which was totally unmethylated). Hence, these CpG dinucleotides might not be informative with respect to methylation changes in OSSN.
The statistical significance of methylation differences at each CpG dinucleotide was determined by comparing healthy conjunctiva and OSSN samples. We found that the loss of methylation in OSSN cases was statistically significant (P < .05) at the third, sixth, and ninth CpGs (Table).
Because of the small number of samples available for each tumor grade, we could not get any statistically significant correlation with DNA methylation.
The variability observed in the methylation at individual CpGs, even between the different clones of the sample, could be attributed to 2 aspects of the technique. Because each clone represents a profile of the single cell, the heterogeneity between the cells could be due to difference in epigenetic states of each cell in a tissue. It is also possible that during sample collection, some healthy cells were collected along with the tumor cells, which could account for the variability in the methylation profile.
Different cell types are characterized by their unique gene expression patterns, which, in turn, are governed by a wide array of regulatory mechanisms. There are several epigenetic processes governing gene regulation. (19,20) Epigenetic modifications, especially DNA methylation, play a key role in that regulation. DNA methylation in the promoter region has been well known to bring about the silencing of the relevant gene. (21) Although the question remains as to whether these epigenetic changes are a cause or a consequence of cancer, many studies have supported the presence of such changes in various stages of cancer.22 Aberrant DNA methylation is a well-known epigenetic hallmark in almost all cancers, (23) including squamous cell carcinoma of cervix. (18) Alteration in DNA methyltransferases plays an important role in the development of cancer. (24) Given that the DNMT3L promoter has been shown to be hypomethylated in cervical cancers, we evaluated for methylation differences at this locus using a limited number of OSSN tumors.
Results from this pilot study indicate that varying degrees of hypomethylation can occur in the DNMT3L promoter region in OSSN. This loss of methylation could possibly lead to increased levels of gene expression at the DNMT3L locus in these tumors. Direct estimation of the level of expression of DNMT3L would be required to confirm this; however, that was not possible in this study because of limitations in the amount of tissue available. Increased levels of DNMT3L in cancer could lead to aberrant DNA methylation, and subsequent misregulation of a subset of genes, because of its stimulatory effect on DNMT3A and DNMT3B. (20) Although we found methylation differences at the third, sixth, and ninth CpGs to be statistically significant, the differences at the sixth CpG were more prominent. We speculate that this CpG could possibly be a suitable molecular marker, using a methylation-specific PCR strategy, which could serve as an adjunct screening method for OSSN. However, any use of this strategy as a molecular diagnosis tool for OSSN would need to be further validated on a larger sample.
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To our knowledge, this study is the first to have shown epigenetic changes in OSSN. Our data are corroborated by previous studies showing hypomethylation at the DNMT3L promoter in cervical cancer. As reported for cervical cancer, we could not determine any specific correlation between tumor grades in the OSSN cases examined studied and changes in DNA methylation because of the small numbers of tissues evaluated. In conclusion, we observed DNA hypomethylation at the DNMT3L promoter in OSSN. These observations suggest that epigenetic events play a role in the genesis of OSSN and raise the possibility that changes in methylation at this locus could be a potential biomarker for OSSN. Further studies to determine methylation and activity of the DNMT3L gene in a large number of cases are required to confirm these possibilities.
We thank the Hyderabad Eye Research Foundation and the Indian Council of Medical Research (ICMR; 5/4/6/13/02-NCDII) for funding this research. G.P.M. received his research fellowship from the ICMR project. G.G. is a senior research fellow of Council of Scientific and Industrial Research (CSIR).
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Guru Prasad Manderwad, MSc; Gopinathan Gokul, MSc; Chitra Kannabiran, PhD; Santosh G. Honavar, MD; Sanjeev Khosla, PhD; Geeta K. Vemuganti, MD, DNB, FNAMS
Accepted for publication November 24, 2009.
From the Ophthalmic Pathology Services (Mr Manderwad and Dr Vemuganti), the Kallam Anji Reddy Molecular Genetics Laboratory (Dr Kannabiran), and the Department of Ophthalmic Plastic Surgery, Orbit and Ocular Oncology (Dr Honavar), Kallam Anji Reddy Campus, L. V. Prasad Eye Institute, Hyderabad, India; and the Laboratory of Mammalian Genetics, Centre for DNA Fingerprinting and Diagnostics, Tuljaguda, Nampally, Hyderabad (Mr Gokul and Dr Khosla).
The authors have no other relevant financial interest in the products or companies described in this article.
Reprints: Geeta K. Vemuganti, MD, DNB, FNAMS, Ophthalmic Pathology Services, Hyderabad Eye Research Centre, Kallam Anji Reddy Campus, L. V. Prasad Eye Institute, L. V. Prasad Marg, Banjara Hills, Hyderabad 500 034, India (e-mail: email@example.com).
Methylation at Each CpG in the DNMT3L Promoter in Ocular Surface Squamous Neoplasia (OSSN) and Healthy Conjunctiva (a) Average Methylation Average CpG in Healthy Methylation P Dinucleotide Conjunctiva, % in OSSN, % Value 1 65.48 53.4 .24 2 57.2 40 .18 3 81.5 53.1 .047 4 5.2 8.36 .28 5 59.2 37.3 .10 6 87.2 38.4 .008 7 66.4 35.1 .07 8 66.4 40.2 .08 9 46.3 23.4 .04 10 43.9 30.3 .19 11 90 74.6 .20 (a) The significance of difference in methylation at each CpG dinucle- otide was calculated using the 1-tail distribution t test. The difference at the third, sixth, and ninth CpG was found to be statistically significant (P < .05).