J Korean Med Sci.  2008 Dec;23(6):1068-1089. 10.3346/jkms.2008.23.6.1068.

Chromosomal Losses are Associated with Hypomethylation of the Gene-Control Regions in the Stomach with a Low Number of Active Genes

• Jung YC
• Hong SJ
• Kim YH
• Kim SJ
• Kang SJ
• Choi SW
• Rhyu MG

• KMID: 1107535
• DOI: http://doi.org/10.3346/jkms.2008.23.6.1068

Abstract

Transitional-CpG methylation between unmethylated promoters and nearby methylated retroelements plays a role in the establishment of tissue-specific transcription. This study examined whether chromosomal losses reducing the active genes in cancers can change transitional-CpG methylation and the transcription activity in a cancer-type-dependent manner. The transitional-CpG sites at the CpG-island margins of nine genes and the non-island-CpG sites round the transcription start sites of six genes lacking CpG islands were examined by methylation-specific polymerase chain reaction (PCR) analysis. The number of active genes in normal and cancerous tissues of the stomach, colon, breast, and nasopharynx were analyzed using the public data in silico. The CpG-island margins and non-island CpG sites tended to be hypermethylated and hypomethylated in all cancer types, respectively. The CpG-island margins were hypermethylated and a low number of genes were active in the normal stomach compared with other normal tissues. In gastric cancers, the CpG-island margins and non-island-CpG sites were hypomethylated in association with high-level chromosomal losses, and the number of active genes increased. Colon, breast, and nasopharyngeal cancers showed no significant association between the chromosomal losses and methylation changes. These findings suggest that chromosomal losses in gastric cancers are associated with the hypomethylation of the gene-control regions and the increased number of active genes.

Keyword

Chromosomal Loss; Loss of Heterozygosity; CpG Methylation; Methylation-Specific PCR; Tissue Expression Profiles

MeSH Terms

Alu Elements/genetics
*Chromosome Deletion
CpG Islands/*genetics
*DNA Methylation
DNA, Neoplasm/chemistry/isolation & purification
Gene Expression Profiling
*Genes, Neoplasm
Humans
Long Interspersed Nucleotide Elements/genetics
Polymerase Chain Reaction
*Promoter Regions, Genetic
Stomach Neoplasms/*genetics

Figure

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  Fig. 1 Chromosomal losses detected in the gastric, colonic, mammary, and nasopharyngeal cancers (25 cases for each cancer type). (A) Individual chromosomal losses and (B) the number of chromosomal losses were evaluated by PCR-based analysis using 40 microsatellite markers on chromosomes 3p, 4p, 5q, 8p, 9p, 13q, 17p, and 18q.

• 

  Fig. 2 Methylation changes in the transitional-CpG sites of the 15 selected genes examined in four cancer types. The methylation status of the transitional-CpG sites was estimated using a semiquantitative methylation-specific PCR method. The methylation changes were scored based on the difference in the level of methylation between the normal and tumor tissues. The frequency of methylation changes in each transitional area is indicated as a percentage in 25 cancer cases. The level of chromosomal losses was evaluated by PCR-based loss-of-heterozygosity analysis. The cancer tissues were grouped into high-level chromosomal losses (H) four or more chromosomes and low-level chromosomal losses (L) involving less than four chromosomes.

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  Fig. 3 Comparison of the transitional-CpG methylation changes between the different cancer types (A) and between cancers with high-level (H) and low-level (L) chromosomal losses (B). The criteria for the level of chromosomal losses are described in the legend of Fig. 2. The frequency of the methylation changes in the six non-island CpG sites and nine CpG-island margins are indicated as a percentage in 25 cancer cases. p values were calculated for the differences in the frequency of methylation changes between four cancer types by a chi-square test.

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  Fig. 4 Methylation profiles of 15 transitional CpG sites examined in 11 tissue types. The estimation of CpG methylation obtained using semiquantitative methylation-specific PCR were divided into five levels (○, 0-20%; ⊘, 21-40%; ⊗, 41-60%; ⊛, 61-80%; ●, 81-100%). Differences in the mean level of methylation between the most highly methylated and least methylated tissue types are indicated by closed bars. Somatic tissues were classified into three germ-layer lineages, endoderm (EN), mesoderm (ME), and ectoderm (EC).

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  Fig. 5 Transcript populations of the embryonic stem cells, placenta, and somatic tissues in the SAGE libraries. (A) The number of the active genes and the number of tags expressed per active gene were calculated separately according to the presence or absence of CpG islands. The mean values of two to eight tissues are indicated for each tissue type. N, normal; C, cancer. All information about the SAGE data is listed in Supplementary Table 4. (B) Transcripts of the CpG-island-negative genes close to the L1 and Alu retroelements. The genes lacking the CpG islands were grouped according to the type of retroelements round the transcription start sites. The relative proportion of the gene-group transcripts in the total transcript population was calculated for each normal and cancer tissue. The transcript data of the nasopharynx was obtained from the EST libraries. All information regarding individual tissues is listed in Supplementary Table 4.

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