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LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability.

Estécio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, Jelinek J, Yang AS, Yan PS, Huang TH, Tajara EH, Issa JP - PLoS ONE (2007)

Bottom Line: Interestingly, some tumor samples in this group showed increase in LINE-1 methylation.Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples.We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa.

View Article: PubMed Central - PubMed

Affiliation: Department of Leukemia, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT

Background: Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer.

Methodology/principal findings: We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process.

Conclusion/significance: Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer.

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Related in: MedlinePlus

Methylated CpG Island Amplication (MCA)/CpG island microarray for repetitive DNA sequences. A) Relative abundance of hypermethylated and hypomethylated repeats for each CIMP/MSI group. A higher number of hypermethylated compared to hypomethylated repeats was observed for the CIMP+/MSI+group, and a gradual change in representation of hypermethylated and hypomethylated repeats was seen for the CIMP+/MSI-and CIMP-/MSI-groups, resulting in an overrepresentation of hypomethylated repeats in microsatellite stable groups. B) Validation of microarray results for LINE repeats. Note that CIMP/MSI groups with higher demethylation, as determined by bisulfite-pyrosequencing of LINE-1, presented also a higher number of hypomethylated LINE repeats by microarray analysis, as represented by a lower hyper/hypomethylation ratio. C) Unsupervised hierarchical clustering was applied to methylation data from a set of 770 repetitive DNA sequences across sixteen colorectal tumors paired with their normal appearing mucosa DNA. The colorectal tumors dendrogram is shown, and the sample ID for each case is included in the right. The terminal branches are color coded to represent the CIMP/MSI status of the tumor sample (red, CIMP+/MSI+; blue, CIMP+/MSI-; green, CIMP-/MSI-). Overall, samples of the same CIMP/MSI group clustered together, reinforcing the different methylation fate for repetitive DNA sequences methylation in each group. LINE, long interspersed nuclear elements; SINE, short interspersed nuclear elements, LTR, long terminal repeats; DNA repeats; Satellite repeats.
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pone-0000399-g003: Methylated CpG Island Amplication (MCA)/CpG island microarray for repetitive DNA sequences. A) Relative abundance of hypermethylated and hypomethylated repeats for each CIMP/MSI group. A higher number of hypermethylated compared to hypomethylated repeats was observed for the CIMP+/MSI+group, and a gradual change in representation of hypermethylated and hypomethylated repeats was seen for the CIMP+/MSI-and CIMP-/MSI-groups, resulting in an overrepresentation of hypomethylated repeats in microsatellite stable groups. B) Validation of microarray results for LINE repeats. Note that CIMP/MSI groups with higher demethylation, as determined by bisulfite-pyrosequencing of LINE-1, presented also a higher number of hypomethylated LINE repeats by microarray analysis, as represented by a lower hyper/hypomethylation ratio. C) Unsupervised hierarchical clustering was applied to methylation data from a set of 770 repetitive DNA sequences across sixteen colorectal tumors paired with their normal appearing mucosa DNA. The colorectal tumors dendrogram is shown, and the sample ID for each case is included in the right. The terminal branches are color coded to represent the CIMP/MSI status of the tumor sample (red, CIMP+/MSI+; blue, CIMP+/MSI-; green, CIMP-/MSI-). Overall, samples of the same CIMP/MSI group clustered together, reinforcing the different methylation fate for repetitive DNA sequences methylation in each group. LINE, long interspersed nuclear elements; SINE, short interspersed nuclear elements, LTR, long terminal repeats; DNA repeats; Satellite repeats.

Mentions: In addition to LINE-1 methylation analysis by bisulfite PCR and pyrosequencing, we also evaluated the methylation status of repetitive elements including LINE (long interspersed nuclear elements), SINE (short interspersed nuclear elements), LTR (long terminal repeats), DNA and satellite repeats, by coupling MCA (methylated CpG island amplification; 20) to a CpG island microarray containing a total of 770 spots representing repetitive DNA of different classes. The first analysis was performed by counting hypermethylated (log2ratio<1.0) and hypomethylated (log2ratio<−1.0) repeats separated according to their different classes (Figure 3A). For the CIMP+/MSI+samples, each one of the repeats classes except satellite repeats were found to be enriched for hypermethylation in tumor DNA compared to normal adjacent mucosa (hypermethylation/hypomethylation = 2.4-fold in average). The enrichment for hypermethylated sequences decreased sharply in CIMP+/MSI-and CIMP-/MSI-(0.88-and 0.71-fold, respectively). These findings suggest that there is a strong pressure for maintenance and/or de-novo methylation of repetitive elements in the MSI+group. Validation of our microarray method was done by comparing the results for LINE-1 to pyrosequencing data in the same colorectal samples. This analysis revealed that tumors with the lowest LINE-1 demethylation by pyrosequencing analysis showed the highest enrichment for hypermethylated LINE repeats (Figure 3B), with the inverse situation being observed for tumors with the highest LINE-1 demethylation. These results support that our microarray analysis is a suitable technique to access methylation changes in repetitive elements.


LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability.

Estécio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, Jelinek J, Yang AS, Yan PS, Huang TH, Tajara EH, Issa JP - PLoS ONE (2007)

Methylated CpG Island Amplication (MCA)/CpG island microarray for repetitive DNA sequences. A) Relative abundance of hypermethylated and hypomethylated repeats for each CIMP/MSI group. A higher number of hypermethylated compared to hypomethylated repeats was observed for the CIMP+/MSI+group, and a gradual change in representation of hypermethylated and hypomethylated repeats was seen for the CIMP+/MSI-and CIMP-/MSI-groups, resulting in an overrepresentation of hypomethylated repeats in microsatellite stable groups. B) Validation of microarray results for LINE repeats. Note that CIMP/MSI groups with higher demethylation, as determined by bisulfite-pyrosequencing of LINE-1, presented also a higher number of hypomethylated LINE repeats by microarray analysis, as represented by a lower hyper/hypomethylation ratio. C) Unsupervised hierarchical clustering was applied to methylation data from a set of 770 repetitive DNA sequences across sixteen colorectal tumors paired with their normal appearing mucosa DNA. The colorectal tumors dendrogram is shown, and the sample ID for each case is included in the right. The terminal branches are color coded to represent the CIMP/MSI status of the tumor sample (red, CIMP+/MSI+; blue, CIMP+/MSI-; green, CIMP-/MSI-). Overall, samples of the same CIMP/MSI group clustered together, reinforcing the different methylation fate for repetitive DNA sequences methylation in each group. LINE, long interspersed nuclear elements; SINE, short interspersed nuclear elements, LTR, long terminal repeats; DNA repeats; Satellite repeats.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1851990&req=5

pone-0000399-g003: Methylated CpG Island Amplication (MCA)/CpG island microarray for repetitive DNA sequences. A) Relative abundance of hypermethylated and hypomethylated repeats for each CIMP/MSI group. A higher number of hypermethylated compared to hypomethylated repeats was observed for the CIMP+/MSI+group, and a gradual change in representation of hypermethylated and hypomethylated repeats was seen for the CIMP+/MSI-and CIMP-/MSI-groups, resulting in an overrepresentation of hypomethylated repeats in microsatellite stable groups. B) Validation of microarray results for LINE repeats. Note that CIMP/MSI groups with higher demethylation, as determined by bisulfite-pyrosequencing of LINE-1, presented also a higher number of hypomethylated LINE repeats by microarray analysis, as represented by a lower hyper/hypomethylation ratio. C) Unsupervised hierarchical clustering was applied to methylation data from a set of 770 repetitive DNA sequences across sixteen colorectal tumors paired with their normal appearing mucosa DNA. The colorectal tumors dendrogram is shown, and the sample ID for each case is included in the right. The terminal branches are color coded to represent the CIMP/MSI status of the tumor sample (red, CIMP+/MSI+; blue, CIMP+/MSI-; green, CIMP-/MSI-). Overall, samples of the same CIMP/MSI group clustered together, reinforcing the different methylation fate for repetitive DNA sequences methylation in each group. LINE, long interspersed nuclear elements; SINE, short interspersed nuclear elements, LTR, long terminal repeats; DNA repeats; Satellite repeats.
Mentions: In addition to LINE-1 methylation analysis by bisulfite PCR and pyrosequencing, we also evaluated the methylation status of repetitive elements including LINE (long interspersed nuclear elements), SINE (short interspersed nuclear elements), LTR (long terminal repeats), DNA and satellite repeats, by coupling MCA (methylated CpG island amplification; 20) to a CpG island microarray containing a total of 770 spots representing repetitive DNA of different classes. The first analysis was performed by counting hypermethylated (log2ratio<1.0) and hypomethylated (log2ratio<−1.0) repeats separated according to their different classes (Figure 3A). For the CIMP+/MSI+samples, each one of the repeats classes except satellite repeats were found to be enriched for hypermethylation in tumor DNA compared to normal adjacent mucosa (hypermethylation/hypomethylation = 2.4-fold in average). The enrichment for hypermethylated sequences decreased sharply in CIMP+/MSI-and CIMP-/MSI-(0.88-and 0.71-fold, respectively). These findings suggest that there is a strong pressure for maintenance and/or de-novo methylation of repetitive elements in the MSI+group. Validation of our microarray method was done by comparing the results for LINE-1 to pyrosequencing data in the same colorectal samples. This analysis revealed that tumors with the lowest LINE-1 demethylation by pyrosequencing analysis showed the highest enrichment for hypermethylated LINE repeats (Figure 3B), with the inverse situation being observed for tumors with the highest LINE-1 demethylation. These results support that our microarray analysis is a suitable technique to access methylation changes in repetitive elements.

Bottom Line: Interestingly, some tumor samples in this group showed increase in LINE-1 methylation.Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples.We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa.

View Article: PubMed Central - PubMed

Affiliation: Department of Leukemia, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT

Background: Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer.

Methodology/principal findings: We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process.

Conclusion/significance: Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer.

Show MeSH
Related in: MedlinePlus