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DNA methylation analysis of the tumor suppressor gene CDKN2B in Brazilian leukemia patients

Abstract

The aim of this work was to evaluate the methylation profile of the p15 (CDKN2B) gene in Brazilian patients with leukemia and to correlate the CDKN2B gene expression with the percentage of methylated CpG dinucleotides in its promoter region. Thirty-one samples from six patients with acute lymphocytic leukemia (ALL), four with chronic myeloid leukemia (CML), and 21 with acute myeloid leukemia (AML) were evaluated by MSP (Methylation-Specific PCR). The CDKN2B gene was found to be methylated in four (67%) of the six ALL samples and in 16 (76%) of the 21 AML samples, but in none of the four CML samples analyzed. We observed a correlation between the CDKN2B mRNA expression (RT-PCR) and the percentage of methylated CpG dinucleotides. Therefore, this study in Brazilian patients confirms that the CDKN2B gene is methylated in the majority of leukemia patients.

CpG island; DNA methylation; CDKN2B


HUMAN AND MEDICAL GENETICS

RESEARCH ARTICLE

DNA methylation analysis of the tumor suppressor gene CDKN2B in Brazilian leukemia patients

Patrícia Santos Pereira LimaI, II; Greice Andreoti MolffetaII; Amélia Góes de AraujoIII, IV; Marco Antônio ZagoIII, IV; Wilson Araújo da Silva Jr.II, IV

IDepartamento de Ciências Naturais, Universidade Estadual do Sudoeste da Bahia, Vitória da Conquista, BA, Brazil

IILaboratório de Genética Molecular e Bioinformática, Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

IIIDepartamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

IVCentro de Terapia Celular and Centro Regional de Hemoterapia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

Send correspondence to Send correspondence to: Patrícia Santos Pereira Lima Laboratório de Genética Molecular e Bioinformática, Fundação Hemocentro de Ribeirão Preto Rua Tenente Catão Roxo 2501 14051-140 Ribeirão Preto, SP, Brazil E-mail: psplima@rge.fmrp.usp.br

ABSTRACT

The aim of this work was to evaluate the methylation profile of the p15 (CDKN2B) gene in Brazilian patients with leukemia and to correlate the CDKN2B gene expression with the percentage of methylated CpG dinucleotides in its promoter region. Thirty-one samples from six patients with acute lymphocytic leukemia (ALL), four with chronic myeloid leukemia (CML), and 21 with acute myeloid leukemia (AML) were evaluated by MSP (Methylation-Specific PCR). The CDKN2B gene was found to be methylated in four (67%) of the six ALL samples and in 16 (76%) of the 21 AML samples, but in none of the four CML samples analyzed. We observed a correlation between the CDKN2B mRNA expression (RT-PCR) and the percentage of methylated CpG dinucleotides. Therefore, this study in Brazilian patients confirms that the CDKN2B gene is methylated in the majority of leukemia patients.

Key words: CpG island, DNA methylation, CDKN2B.

Introduction

DNA methylation is a covalent modification and results from the activity of a family of DNA methyltransferase (DNMT) enzymes which catalyze the transfer of a methyl group (CH3) from S-adenosylmethionine (SAM) to the cytosine residues at CpG dinucleotides (Strathdee and Brown, 2002). The distribution of CpG dinucleotides in the human genome is not uniform, but there are small stretches (0.5 kb to several kb) of CpG-rich DNA regions termed CpG islands (Galm et al., 2006). It has been estimated that the human genome contains about 29,000 CpG islands (Nephew and Huang, 2003). These CpG islands are usually located in the vicinity of genes, are often found near the promoters of widely expressed genes, and typically extend into the first exon (Jones, 2003). In contrast to CpG dinucleotides, which are dispersed throughout the genome, the cytosines within CpG islands, especially those associated with promoter regions, are normally unmethylated, allowing the expression of a gene. The exception to this unmethylated state of CpG islands involves the imprinted genes and X-chromosome inactivation, and this indicates the tight association of promoter DNA methylation with transcriptional silencing during normal mammalian development (Galm et al., 2006).

The methylation pattern of normal cells is kept through successive cellular divisions in adult tissues and the heritage of this information is called epigenetic inheritance (Laird, 2003). Therefore, epigenetics is the study of modifications in gene expression that are not caused by alterations of DNA sequence (Verma and Srivastava, 2002; Galm et al., 2006).

In tumorigenesis, the balance of the methylation state of normal cells is lost, and some of the possible alterations are: (I) transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation; and (II) histone deacetylation and global genomic hypomethylation. Hypomethylation contributes to carcinogenesis and is responsible for the chromosomal instability, reactivation of transposable elements, and loss of imprinting (Esteller and Herman, 2002). Therefore, the profile of hypermethylation promoters differs according to each cancer type, because for each tumor type specific genes are methylated. In addition to that, the epigenetic inactivation may affect all molecular mechanisms involved in cell immortalization and transformation. Last but not least, it seems that epigenetic changes are among the several early steps in carcinogenesis (Mukai and Sekiguchi, 2002).

According to the estimates of cancer incidence for 2008 by INCA (Instituto Nacional do Câncer – Brazilian National Cancer Institute), leukemias will afflict 5220 men and 4320 women. Hematologic neoplasias can jeopardize the lymphoid or myeloid lineages. Lymphoid neoplasias start from lymphoid lineage cells in different stages of maturation. Myeloid neoplasias result from a pluripotential progenitor cell mutation which maintain the capacity, albeit in an imperfect manner, of differentiation and maturation for each one of the myeloid lineages (Zago et al., 2001). Most acute leukemias appear to be the consequence of the collaboration between one class of mutations or gene rearrangements that confer a proliferative and/or survival advantage to hematopoietic progenitors and a second class of mutations that serve primarily to impair hematopoietic differentiation and subsequent apoptosis of cells (Kelly and Gilliland, 2002).

In hematological neoplasms, hypermethylation genes were identified in multiple fundamental pathways related to cancer, including cell cycle control (p15, p16, Rb1, p27, p73), DNA repair (O6MGMT), apoptosis inhibition (DAPK), tumoral metastasis (E-cadherin), and growth factors (ER, EphA3) (French et al., 2002). One of the most frequent methylated genes in leukemia, mainly in ALM, is CDKN2B (Herman et al., 1996; Issa et al., 1997; Wong et al., 2000; Toyota et al., 2001; Claus and Lubbert, 2003).

In this work, we determined the methylation profile of the CDKN2B gene in samples from Brazilian patients with acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and chronic myeloid leukemia (CML). We also evaluated the CDKN2B mRNA expression and the percentage of methylated CpG dinucleotides by sequencing of sodium bisulfite-treated DNA.

Material and Methods

Samples

Genomic DNA was extracted from bone marrow cells of six ALL patients, four CML patients, 21 AML patients and one sample of normal bone marrow, using a Super Quik Gene Kit (AGTC). The AML samples were subdivided based on French-American-British (FAB) into: M0 (1), M1 (2), M2 (10), M4 (2), M5 (4) and M6 (2). All patients signed a consent form approved by the Ethics Committee of the Institution.

Bisulfite DNA modification

Bisulfite DNA modification was accomplished in agreement with the technique described in Current Protocols In Human Genetics (Dracopoli et al., 1994). DNA (2 µg) was denatured with NaOH (2 M) for 10 min at 37 °C. Ten millimoles of hydroquinone and 3 M of sodium bisulfite at pH 5, both freshly prepared, were added and mixed, and then samples were incubated at 50 °C for 16 h. The modified DNA was purified using a Wizard DNA clean-up purification kit (Promega) according to the manufacturer's instructions and eluted into water. Modification was completed by treatment with NaOH 3 M for 5 min at room temperature, followed by ethanol precipitation. The DNA was resuspended in water and used immediately or stored at -80 °C.

Methylation-Specific PCR (MSP)

A modified DNA was used for two MSP reactions, both for the CDKN2B gene: one reaction specific for methylated DNA and other specific for unmethylated DNA. The primer sequences used were: CDKN2B-F(M) 5'-CGTTCG TATTTTGCGGTT-3'; CDKN2B-R(M) 5'-CGTACAATA ACCGAACGACCGA-3'; CDKN2B-F(U) 5' -TGTGATG TGTTTGTATTTTGTGGTT-3'; and CDKN2B-R(U) 5'-CCATACAATAACCAAACAACCAA-3' (Herman et al., 1996). For all reactions the amplification conditions were: bisulfite-modified DNA; 10X PCR buffer (Invitrogen); 50 mM MgCl2; 1.25 mM dNTP, and 300 ng/µL of each primer. Reactions were hot-started at 95 °C for 5 min before addition of 1.25 units of Taq polymerase (Invitrogen), followed by 35 cycles (30 s at 95 °C, 30 s at 61 °C for primer methylated or 60 °C for primer unmethylated, 30 s at 72 °C), and a final extension step of 4 min at 72 °C. Each PCR product was analyzed using 3% agarose gel, stained with ethidium bromide and directly visualized under UV illumination.

Sequencing of sodium bisulfite-treated DNA

Bisulfite-treated DNA was amplified by a nested-PCR protocol, using the following primers: CDKN2B-F1 5'-GGTTGAAGGAATAGAAATTT-3' and CDKN2B-R1 5'-ACACTCTTCCCTTCTTTCCC-3' for the first reaction; and CDKN2B-F2 5'-TTAGTTTTGGTTTTATTGG A-3' and CDKN2B-R2 5'-TCTCTCCTTCCTAAAAAAC C-3' for the second reaction. PCR was performed in a solution containing 10 X buffer (Biotools); 1.25 mM dNTP; 2.5 µM of each primer, and 2U of Taq DNA polymerase (Biotools). The PCR conditions were: 94 °C for 5 min and 55 °C for 2 min, followed by 35 cycles (72 °C for 1 min, 94 °C for 1 min, and 55 °C for 1 min) and 72 °C for 10 min for the first reaction. For the nested reaction the conditions were similar, only the annealing temperature being changed to 51 °C. The amplified products were sequenced directly in a MegaBace 1000 sequencer.

RNA isolation and reverse transcriptase PCR (RT-PCR)

Total RNA was isolated using TRIZOL (Invitrogen) and the cDNA was synthesized with the use of a High Capacity kit (Applied Biosystems). Amplification of CDKN2B mRNA was performed with specific primers: CDKN2B-RTF 5'-AACGGAGTCAACCGTTTCGG-3' and CDKN2B-RTR 5'-TGTGCGCAGGTACCCTGCA A-3' (Hoshino et al., 2002). The PCR conditions were as follows: 10 X buffer (Biotools); 1.25 mM dNTP; 20 pmol of each primer, and 2 U of Taq DNA polymerase (Biotools). PCR was initiated with one cycle at 95 °C for 1 min, followed by 30 cycles at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The internal control was the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The amplified products were analyzed on 1.5% agarose gel, stained with ethidium bromide and directly visualized under UV illumination.

Results

Methylation analysis

Different methylation patterns of the CDKN2B gene were detected among leukemia types analyzed by MSP. The gene was methylated in four (67%) of the six ALL samples and in 16 (76%) of the 21 AML samples, but in none of the four CML samples analyzed. Regarding AML subtypes, the frequency distribution of CDKN2B gene methylation was: M0 1/1, M1 2/2, M2 9/10, M4 0/2, M5 3/4, and M6 1/2. In 12 AML samples, amplification was achieved with both primers, specific for methylated DNA and for unmethylated DNA, and therefore these samples were classified as hemimethylated (Figure 1). For frequency estimates, these hemimethylated samples were considered as methylated.


Expression analysis

The expression of CDKN2B mRNA was evaluated in 19 samples with available RNA (six methylated, five unmethylated, and eight hemimethylated) and in one normal bone marrow sample. In the methylated samples, no CDKN2B mRNA expression was detected (Figure 2A). On the other hand, CDKN2B expression was detected in the unmethylated and in some hemimethylated samples (Figures 2B and C ). However, the level of expression was higher in the unmethylated than in the hemimethylated samples. The highest level of CDKN2B gene expression was detected in the normal bone marrow sample.



Sequencing analysis

The region analyzed (498 bp) after bisulfite treatment of the DNA is located approximately -234 to +264 bp from the transcription start site, comprising two CpG islands according to the MethPrimer program (Li and Dahiya, 2002). However, the CpG dinucleotides analyzed by sequencing contained only the region located -15 to +208 bp (223 bp) from the transcription start site that shows 26 CpG dinucleotides. The sequence amplified by MSP (147 bp), which shows 19 CpG dinucleotides, is within the sequenced region (Figure 3).


Thirteen out of the 31 samples analyzed by MSP were sequenced (one normal bone marrow, four methylated, four hemimethylated and four unmethylated). The methylation percentage for each sample was calculated as the number of methylated CpG dinucleotides in the total number of analyzed CpGs (Figure 4). The normal sample did not show any methylated CpG (data not shown). Seven out of eight samples, which were amplified with the primer set specific for the methylated DNA, presented a methylated CpG frequency of 46% to 100%. The exception was sample XIX (Figure 4), considered to be hemimethylated by MSP, which showed only 26% of methylated CpG dinucleotides. In the unmethylated samples, the percentage of methylated CpGs ranged from 15% to 46%.


Discussion

The CDKN2B gene encodes a 15-kDa protein that is a cyclin-dependent kinase inhibitor. The role of CDKN2B as a tumor suppressor in acute myelogenous leukemia (AML) was established in several previous studies (Herman et al., 1996; Issa et al., 1997; Wong et al., 2000; Toyota et al., 2001; Claus and Lubbert, 2003). Hypermethylation of CDKN2B CpG islands has been shown to occur in up to 80% of human AML, and this epigenetic state is associated with reduced expression (Markus et al., 2007).

In this study, analysis results of the methylated state of the CDKN2B gene, performed by MSP, were confirmed by direct sequencing and expression analysis. Our MSP results showed that CDKN2B was methylated in 76% of the AML samples. These results are in agreement with previous studies that found the CDKN2B gene to be methylated in more than 50% of the cases (Herman et al., 1996; Issa et al., 1997; Toyota et al., 2001; Claus and Lubbert, 2003).

Among patients with AML, Wong et al. (2000) reported CDKN2B methylation frequencies to be higher in subtypes M2, M3 or M4 than in M1, M5, M6 or M7. In nine of our 10 subtype M2 samples, the gene was methylated; for the other subtypes, the number of samples was too small to allow a correlation analysis.

In ALL, previous studies indicated different methylation frequencies for the CDKN2B gene, depending on the methodology used (Issa et al., 1997; Melki et al., 1999; Garcia-Manero et al., 2002; Melki and Clark, 2002; Chim et al., 2003). We found a higher frequency (67%) than that (40%) described by Chim and colleagues (2003), using the same methodology (MSP). This might be due to our smaller sample size (six samples) compared to theirs (25 samples). Even with a small number of LMC samples (four), our data showed the unmethylated state of the CDKN2B gene, which is in agreement with previous reports (Issa et al., 1997; Toyota et al., 2001).

A sample of genomic DNA usually consists of a large pool of molecules that may display methylation heterogeneity. This heterogeneity can be due to the fact that two alleles of any given genomic locus in a cell may differ in their methylation patterns, and that the DNA sample was derived from multiple cells with potentially different methylation patterns (Siegmund and Laird, 2002). This could explain the simultaneous amplification by MSP using primer sets for both methylated and unmethylated DNA. This methylation heterogeneity of samples can also be difficult to analyze by direct sequencing of sodium bisulfite-treated DNA, once it does not allow the methylation state of individual alleles to be determined. Hence, in some chromatograms the TCpG sequence was observed instead of TpG or CpG. We expected to find TpG in the case of an unmethylated dinucleotide and CpG in the case of a methylated dinucleotide. The term hemimethylated was also used to designate those CpG nucleotides for which the methylation state could not be defined by sequencing, as it occurred in CpG 04, 06 and 10 (Figure 5C).


In general, the amount of methylated CpG sites within a locus, as well as the number of methylated loci, increase in more advanced stages of cancer (Nephew and Huang, 2003). The consequence of this dynamic epigenetic gene silencing is that the degree of loss of protein production is not uniform throughout the tumor-cell population, unlike the one that is produced by the genetic changes (Jones and Baylin, 2002). We analyzed the expression of CDKN2B mRNA, which in general agreed both with the MSP results and with the percentage of methylated CpG dinucleotides, as determined by direct PCR sequencing. In the methylated samples that showed more than 80% of methylated CpG dinucleotides, no CDKN2B mRNA expression was observed by RT-PCR (Figures 2A and 4). In the unmethylated samples, a relationship between the expression level and the percentage of methylated CpG was observed, as in sample X (46% methylated CpG) and samples VIII and IX (15% and 17% methylated CpG, respectively) (Figures 2C and 4). The CDKN2B gene expression was very low when the gene was found to be hemimethylated by MSP (Figure 2B). CDKN2B mRNA expression differed between the normal bone marrow and the unmethylated samples (Figure 2C ). These unmethylated samples showed a low percentage of methylated CpG, which seems to correlate with the levels of mRNA transcription.

The percentage of methylated CpGs was similar (46%) in samples X and I (unmethylated and methylated, respectively). However, their level of gene expression and the number of hemimethylated CpGs (Figures 2D and 4) were different. There were 10 hemimethylated CpGs in sample I, while in sample X only 2 hemimethylated CpGs were identified. Hence, the methylation density of the region analyzed was higher in sample I than in sample X, and this might have caused the apparent difference in CDKN2B gene expression observed between these samples.

Another difference between the samples was the state of the methylated CpG 3 dinucleotide at the transcription factor Sp1 site. Sp1 is associated with the transcription of the CDKN2B gene and is a regulator of cell cycle progression (Pagliuca et al., 2000). Many factors are known to bind CpG-containing sequences, and some of them fail to bind when the CpG is methylated, therefore inhibiting transcription (Bird, 2002). CpG 3 and CpG 25 are part of two Sp1 sites (Figure 4), and in seven of the eight sequenced samples which were amplified by the specific primer set for methylated DNA at least one of these dinucleotide CpGs was methylated (Figure 4).

Methylation of specific targets may explain the observation that different hematopoietic malignancies harbor distinct methylation signatures (Rush and Plass, 2002). The cell type-specific pattern of hypermethylation suggests that the methylation of certain CpG islands may be used as disease marker and is also useful in the detection of minimal residual disease after chemotherapy. This pattern may therefore influence any adjuvant treatments (Melki and Clark, 2002) or be used as a marker for disease progression (Laird, 2003). Agrawal et al. (2007) showed increased CDKN2B methylation levels in the bone marrow of patients with acute leukemias in clinical remission and, according to these authors, the presence of aberrant DNA methylation in remission is a powerful indicator of a high risk of leukemia relapse.

In this first report on the methylation status of the CDKN2B gene in Brazilian leukemia patients, CDKN2B was found to be methylated, in agreement with data obtained in similar analyses performed in leukemia patients of other countries.

Acknowledgments

Support for this work was provided by the Brazilian agencies FAPESP, CNPq, Center for Cell-based Therapy and UESB. We also thank Marli H. Tavella, Cristiane F. Ayres, Adriana A. Marques and Anne Marie R. D. dos Santos for their excellent technical assistance.

Internet Resources

Received: August 23, 2007; Accepted: March 10, 2008.

Associate Editor: Emmanuel Dias Neto

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  • Send correspondence to:

    Patrícia Santos Pereira Lima
    Laboratório de Genética Molecular e Bioinformática, Fundação Hemocentro de Ribeirão Preto
    Rua Tenente Catão Roxo 2501
    14051-140 Ribeirão Preto, SP, Brazil
    E-mail:
  • Publication Dates

    • Publication in this collection
      18 Aug 2008
    • Date of issue
      2008

    History

    • Accepted
      10 Mar 2008
    • Received
      23 Aug 2007
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