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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.25 n.4 Temuco dic. 2007 


Int. J. Morphol, 25(4):817-824,2007.


Study of Methylation Pattern of de Novo DNA Methyltransferase Genes and its Correlation with DNA Methylation Pattern of RUNX3 in Individuals with Gastric Cancer from Northern Region of Brazil

Estudio del Estado de Metilación de Novo de Genes Metiltransferasas y su Correlación con el Patrón de Metilación de RUNX3 en Individuos con Cáncer Gástrico de la Región Norte del Brasil


*Eleonidas Moura Lima; **Mariana Ferreira Leal; *Fábio José Nascimento Motta; ****Paulo Pimentel de Assumpção; ****Maria Lucia Harada; **Marilia de Arruda Cardoso Smith; ****Rommel Rodríguez Burbano & *Cacilda Casartelli

* Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, Brazil.
** Departamento de Morfologia, Escola Paulista de Medicina, São Paulo, Brazil.
*** Servico de Cirurgia, Hospital João de Barros Barreto, Belém, Brazil.
**** Instituto de Ciências Biológicas, Universidade Federal do Para, Belém, Brazil.

Dirección para correspondencia

SUMMARY: Gastric cancer is the forth malignancy in frequency in the world. In the northern Brazil is the second neoplasia most frequent in males and the third most frequent in females. Genetic and epigenetic alterations are evolved on gastric carcinogenesis and DNA methylation is the epigenetic alteration better studied. We analyzed de novo DNA methyltransferases methylation pattern and its association with RUNX3 gene methylation pattern in Brazilian samples of intestinal-type and diffuse-type of gastric cancer. PCR methylation specific was used to evaluate DNA methylation pattern. Sixty-six samples were studied in this work. Only the gene RUNX3 presented altered methylation pattern, being methylated in 38.5% of gastric cancer intestinal-type samples and in 70% of gastric cancer diffuse-type samples and, by this reason, it should be evolved in the genesis of this neoplasia. There was a statistically significant difference among diffuse-type and intestinal-type samples (p=0.0418) and among normal and tumour tissues (p<0.0001) for RUNX3 gene but not to DNMT3A, DNMT3B e DNMT3 genes on CpG islands analyzed. Alteration of RUNX3 methylation pattern is not associated to de novo alteration of DNA methyltransferases methylation pattern on studied regionsTherefore, it becomes necessary a better comprehension of this phenomenon on gastric carcinogenesis.

KEY WORDS: Methylation; de novo DNA methyltransferases; RUNX3; Gastric cancer

RESUMEN: El cáncer gástrico es la cuarta patología más frecuente en el mundo. En el norte del Brasil, es la segunda neoplasia más frecuente en hombres y la tercera en mujeres. Alteraciones genéticas y epigenéticas relacionadas con la carcinogénesis gástrica y la metilación del DNA son las alteraciones epigenéticas mejor estudiadas. En este trabajo, analizamos el estado de novo de metilación de genes DNA metiltransferases y su asociación con el estado de metilación del gen RUNX3 en muestras de individuos brasileños con cáncer gástrico de los tipos intestinal y difuso. Fue usada la Reacción en Cadena de la Polimerasa (PCR), metilación específica, para analizar el estado de metilación del DNA. Fueron estudiados 66 tejidos tumorales. Solamente el gen RUNX3 presentó un estado de metilación alterado, estuvo metilado en 38,5% de las muestras de cáncer gástrico tipo intestinal y en 70% de muestras de cáncer gástrico tipo difuso, lo que sugiere que estaría relacionado con la génesis de esta neoplasia. Hubo una diferencia estadística significativa entre muestras de los tipos difuso e intestinal (p=0.0418) y entre tejidos normal y tumoral (p<0.0001)parael gen RUNX3. Esta asociación no fue encontrada para los genes DNMT3A, DNMT3B y DNMT3 en las islas CpG analizadas. Alteraciones del estado de metilación de RUNX3 no están asociadas con alteraciones de novo de genes DNA metiltransferases. De esta forma se hace necesaria una mejor comprensión de este fenómeno en la carcinogénesis gástrica.

PALABRAS CLAVE: Estado de metilación de novo; Genes; DNA metiltransferases; RUNX3; Cáncer gástrico.


Gastric cancer is the forth malignancy in frequency in the world and the second in mortality rate behind lung cancer, with approximately 650,000 deaths by year. Approximately 60% of all gastric cancer cases occur in developing countries. The areas with higher rates of incidence (>40/100,000) are Asian East, Andean Regions and South America (Stewart & Kleihues, 2003). In Brazil, the estimated number of new cases of gastric cancer for 2006 is 14,970 in males and 8,230 in females, being considered the second more frequent neopla-sia between males (11/100,000) and the third in females (6/ 100,000) of Brazilian Northern Region (INCA, 2005).

The increase of information about occurrence of genetic and epigenetic alteration in this neoplasia, sustain the existence of two routes for human gastric cancer: the mutation route, which evolves groups of genes related direct or indirectly with DNA repair mechanism; and the suppressor route, which joins groups of genes related to tumour suppression mechanism (Tamura, 2002; Tahara, 2004).

Epigenetic alterations, that are biochemical modifications related to modulation of genetic information, are listed below: histone modification (acetylation, phosphorylation, methylation and ubiquitylation) and DNA methylation (Li, 2002).

DNA methylation is the better-studied epigenetic alteration, where occurs the addition of a methyl radical to the base cytosine adjacent to guanine. This addition reaction is catalyzed by DNA methyltransferases, which use S-adenosil-methionine as a methyl donor [-CH3]. Its paper in cancer genesis would be related principally to genetic silencing (Baylin & Bestor, 2002). Literature corroborates the evolvement of this epigenetic alteration in carcinogenesis (Herman et al, 1996b; Esteller & Herman, 2002).

De novo DNA methyltransferases are enzymes evolved on the establishment of DNA methylation pattern during the earlier stages of embriogenesis because, differently ofDNMTl (DNA methyltransferase 1), they need nota hemi-methylated DNA to establish the existent human genome methylation pattern (Okano, Xie & Li, 1998; Okano et al, 1999; Siedlecki et al., 2003).

RUNX3\& one of the tree genes that belong to RUNX family, which probable function is tumor suppression in gastric cancer. Therefore, it would participate of the suppressor route of this neoplasia. This gene is located at lp36.1 chromosomal region, a frequently deleted region in many kinds of tumors (Coffman, 2003; Levanon et al, 2003).

The gene RUNX3 rarely suffers punctual mutation in gastric cancer, however its promoter region is frequently methylated. Its function as tumour suppressor is probably achieved byp21 gene activation, which has as gene product a cyclin-dependent kinase inhibitor (Chi et al, 2005).

Despite of the importance of de novo DNA methyltransferases in the establishment of human genome DNA methylation pattern, there is not a description of the methylation pattern of these genes and its correlation with epigenetic alterations in genes evolved with carcinogenesis, as RUNX3, and, specially, on gastric cancer genesis.

Comprehension of functional epigenetic pattern, specially DNA methylation found in regions rich in CpG dinucleotides, nominated CpG islands, can be understood as an interface between environmental conditions and its influence on genetic information modulation (Bird, 2002; Laird, 2003).

We analyzed the methylation pattern of DNMT3A, DNMT3B and DNMT3L genes and its association with RUNX3 methylation pattern, in Brazilian samples of gastric adenocarcinoma intestinal-type and Lauren diffuse-type (Lauren, 1965).


Sixty-six samples were studied in this work, being 20 samples from normal tissue, 26 samples of well-differentiated gastric adenocarcinoma and 20 samples of low-differentiated gastric adenocarcinoma, according to histopathological diagnosis based on Lauren's classification (Lauren). Samples were obtained at Para State Joáo de Barros Barreto University Hospital (HUJBB) after patients' clarified consent and Ethics Committee of HUJBB and Ribeiráo Preto Medical School Clinical Hospital approved the study. Approximately 1cm3 of fresh and micro dissected samples was used for DNA extraction, according to Rey et al. (1992).

Initially, 2 µg of DNA were diluted in 50µl de H20 distilled. Then, 5 µl of NaOH 3M were added to this solution, during 20 minutes at 42°C. In the next step 400 µl of Sodium bisulfite 3M prepared at procedure moment and 30 µl of hydroquinone 10mM were added. The mixture was incubated at 55°C, in a dark place by 16 hours. Then, DNA purification follows as recommended on purification kit (Wizard® DNA Clean-up System, Promega Corporation, Madison, WI).

MS-PCR technique (Methylation-specific PCR), developed by Herman et al, was used based on Shapiro et al, Hayatsu et al. and Wang et al. papers. Specific primers for CpG islands, located near or inside the promoter region of studied genes, were draw helped by Metn/'rtmerpiogram (Li & Dahiya, 2002) (Table I).

PCR were prepared in a final volume of 50µl containing 200µM of dNTPs, 2.0 mM of MgCl2, 50 ng of modified DNA, 200 pM of each primer and 0.5 U AmpliTaq GOLD (Applied Biosystems, Foster City, CA). PCR conditions were: one previous denaturation at 94°C for 2 minutes and 35, cycles, at 94°C, for 40 seconds, 50 - 60°C for 1 minute and 72°C for 40 seconds and a final extension at 72°C, for 5 minutes. MS-PCR products were visualized in poliacrylamide gel at 8% dyed with silver nitrate at 10%.

Direct product of PCR was used for sequencing reaction. On the first step of procedure, 5 µl of PCR product and 2 µl of ExoSAP-IT kit were used (USB Corporation, Cleveland, Ohio), as recommended by manufacturer. On the second step, a final volume of 10 µl were used to sequencing reaction, being 1 µl of mixture (PCR product), 5 µl of sequencing Kit DYEnamic ET Dye Terminator Cycle Sequencing Kit (GE Healthcare, Uppsala, Sweden). Sequencing of one methylated and one unmethylated sample of RÍ/AX3 gene, used as positive control for DNA modification technique by sodium bisulfite were also realized. For sequencing analysis, ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Perking Elmer, Alameda, CA) was used.

Statistical evaluation was performed using Fisher exact test found on TFPGA software (Miller, 1997).


MS-PCR results are presented on Fig. 1. Sequencing of a methylated and an unmethylated sample of R UNX3, used as positive controls for DNA modification by sodium bisulfite technique, presented methylated and unmethylated areas when compared to the unaltered sequence of DNA (Fig. 2).

Only RUNX3 presented an altered DNA methylation pattern, being methylated in 38.5% of gastric adenocarcinomas intestinal-type and in 70% of gastric adenocarcinomas diffuse-type. There was a statistically significant difference (p<0.05) among samples of diffuse and intestinal-types (p=0.0418) and among normal and tumour tissues (p<0.0001) only for RUNX3 (Table II). Considering the difference for this gene among the tissues, we can graphically group them in different ramifications of an epigenetic tree (Fig. 3).


In the human genome, approximately 50% of genes present high taxes of dinucleotides CG on their first exon or promoter region, nominated CpG island. However, in normal tissues most of these regions are "protected" of DNA methyltransferases action (Gardiner-Garden & Frommer, 1987; Antequera & Bird, 1993; Venter et al., 2001; Zhang et al., 2002) .

DNA methylation is a common process among tissues, being its pattern determined according to the stage of human development. Depending on the stage of cellular differentiation there are different enzymes related to its deftness on recognizing regions that should be methylated (Grace Goll & Bestor, 2005).

DNA methyltransferases can be separated in two groups: DNA methyltransferase 1 (DNMTl) and the family DNA methyltransferase 3 (DNMT3). DNMTl needs that DNA target be hemimethylated to obtain its greatest efficiency. This DNA methyltransferase is expressed in specific tissues confirming its expression in differentiated cells. Then, DNMTl is an enzyme responsible for maintain DNA methylation pattern, which at first, is established at the embryonic stage of human development (Gruenbaum et al., 1982; Bestor, 1988; Hermann, 2004).

DNMT3 family is composed by DNMT3A, DNMT3B and DNMT3L located, respectively, at 2p23, 20qll .2 and 21q22.3 chromosomal regions (Robertson et al, 1999; Xie et al, 1999; Hata et al., 2002). They are known as de novo DNA methyltransferases, because they could be evolved on the establishment of DNA methylation pattern of human genome, methylating the DNA free of methyl groups (Grace Goll & Bestor).

CpG islands are often "protected" of methylation process on normal tissues. Without this protection, CG rich regions once methylated are susceptible to mutation, a transition of C>T, because the 5-methylcitosine suffers spontaneous deamination and became a thymine. However, the mechanism that explains this protection should be elucidated (Gowher et al., 2005).

Despite of this protection, many genes evolved on cycle cell control, which present CpG islands, are inactivated by DNA methylation in gastric cancer. Then, neoplastic cells with these regions can be "unprotected" by DNA methylation (Kaneda et al., 2004).

Once that, CpG islands are free of methyl radical on normal tissue, have been suggested that de novo DNA methyltransferases could be evolved on DNA methylation process in cancer specially in gastric adenocarcinoma (Baylin et al., 1998; Rhee et al., 2002).

We analyzed the methylation pattern of de novo DNA methyltransferases, as in normal tissue as in tumour tissue, because genes that express these enzymes could be self-regulated, once that they present CpG islands too. Besides this analysis, methylation pattern of de novo DNA methyltransferases was related to alteration of DNA methylation pattern of RUNX3 gene (Waki et al., 2003; Kim et al., 2004).

Considering the analyzed regions of CpG islands, our results suggest that de novo DNA methyltransferases are not associated to alterations on RUNX3 methylation pattern in gastric adenocarcinoma, but we should consider that DNMT3B and DNMT3L genes present other CpG islands still unknown.

Once that DNA methylation pattern of CpG islands, as in normal tissue as in tumour, presented the same results for the genes DNMT3A, DNMT3B and DNMT3L, we suggest that these regions are not evolved on genie self-regulation process. Is probably that the alteration of RUNX3 methylation pattern in gastric cancer be associated to the loss of methylation pattern of DNMT3B on the unanalyzed region or could be associated to the loss of methylation pattern of DNMT1, because their expression levels are modified in many tumors (Xie et al.).

Some studies has show that DNA methyltransferases expression level are modified, as in gastric adenocarcinoma as in other tumors. DNTM3B is over expressed in colon cancer, as well as DNMT1. Being DNMT3B a de novo DNA methyltransferase, it could explain the methylation of CpG islands earlier "protected". Nevertheless, some studies suggest an association between DNMT3B and DNMT1 to promote an altered form of DNA methylation in many types of neoplasias (Xie et al.; Sato et al., 2002).

RUNX3 gene product is a transcription factor that binds to DNA and, probably, regulates the expression of kinase-depending inhibitors of cicline, being the protein p21 the most probably. Then, RUNX3 gene presents a tumour suppression function and then, it controls cellular grows.

The pathway that promotes the binding of RUNX3 gene product to target DNA seems to be activated by TGFb receptors. These receptors do signal transduction from plasmatic membrane to cellular nucleus by Smad complex and this complex draft other transcription factors, as protein p300, to reforcé RUNX3 gene product binding and the next function of this product (Lauren; Feng & Derynck, 2005).

Some studies showed that punctual mutations are rare on RUNX3 gene, as well as in other suppressor genes, as pS2 and PTEN, in gastric adenocarcinoma (Fujimoto et al., 2000; Lima et al., 2005). Our results demonstrate that RUNX3 is methylated as in intestinal-type as in diffuse-type of gastric adenocarcinoma, being statistically significative their association with gastric adenocarcinoma diffuse-type (p=0.043). Then, despite of being an epigenetic alteration also found in gastric adenocarcinoma intestinal-type, our results suggest that, for the genesis of gastric cancer, the modification of RUNX3 methylation pattern is an initial event on gastric adenocarcinoma diffuse-type, because 70% of our samples are methylated. On the other hand, on gastric adenocarcinoma intestinal-type, it occurs as a late event, once that this type presents intermediate lesion stages: benign, pre-cancerous and cancerous (Catalano et al., 2005).

Our results corroborate the hypothesis that RUNX3 has a modified DNA methylation pattern in gastric adenocarcinoma and, by this reason, should be evolved on the genesis of this neoplasia. Modification of its methylation pattern is not associated to modification of de novo DNA methyltransferases methylation pattern, located on the studied regions.

Understanding and characterization of epigenetic modifications, suddenly DNA methylation, is important on gastric cancer evolution, because this information could be useful to elaborate therapeutic interventions. By this reason, its necessary a better comprehension of this phenomenon, especially of the genes and pathways evolved.



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Correspondence to:

Dr. Rommel Rodriguez Burbano
Laboratorio de Citogenética Humana e Genética Toxicológica
Departamento de Biología
Centro de Ciências Biológicas
Universidade Federal do Pará
Campus Universitario do Guama
Av. Augusto Correa, 01
CEP 66075-900
Belém, Para



Phone:(091)88357972 Fax: (091) 32017601/

Received: 16-06-2007 Accepted: 08-10-2007

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). RRB had a PQ-2 fellowship (number 308256/2006-9) granted by CNPq. We wish to thank FAPESP, FAEPA, CAPES and FINEP (grants 0927-03).


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