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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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Differential LINE-1 methylation among CIMP/MSI groups in primary colorectal carcinoma samples (CRCs). A) Colorectal tumor DNA and their normal appearing adjacent mucosa from sixty patients were evaluated for LINE-1 methylation. These tumors were previously evaluated for CpG island methylator phenotype (CIMP), using a panel of single-copy genes methylation analysis, and microsatellite instability (MSI) status, resulting in the identification of three CIMP/MSI groups. In normal appearing mucosa (top) little variation in LINE-1 methylation is observed between samples and CIMP/MSI groups (average methylation = 64.3%), while in tumor (bottom) several samples undergo high LINE-1 demethylation (25/60 tumor samples have methylation density bellow 55%), most notable in CIMP+/MSI-and CIMP-/MSI-groups. B) Relative LINE-1 demethylation in CRCs. Relative demethylation was calculated as the percent change of LINE-1 methylation in tumor compared to its normal appearing mucosa. Both CIMP+/MSI-and CIMP-/MSI-samples presented in average 16% demethylation for LINE-1, while no significant changes were observed for the CIMP+/MSI+samples. For the CIMP+group, 4–9% increase of methylation density for LINE-1 was observed for a small fraction of samples, most of them identified as CIMP+/MSI+samples.
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pone-0000399-g002: Differential LINE-1 methylation among CIMP/MSI groups in primary colorectal carcinoma samples (CRCs). A) Colorectal tumor DNA and their normal appearing adjacent mucosa from sixty patients were evaluated for LINE-1 methylation. These tumors were previously evaluated for CpG island methylator phenotype (CIMP), using a panel of single-copy genes methylation analysis, and microsatellite instability (MSI) status, resulting in the identification of three CIMP/MSI groups. In normal appearing mucosa (top) little variation in LINE-1 methylation is observed between samples and CIMP/MSI groups (average methylation = 64.3%), while in tumor (bottom) several samples undergo high LINE-1 demethylation (25/60 tumor samples have methylation density bellow 55%), most notable in CIMP+/MSI-and CIMP-/MSI-groups. B) Relative LINE-1 demethylation in CRCs. Relative demethylation was calculated as the percent change of LINE-1 methylation in tumor compared to its normal appearing mucosa. Both CIMP+/MSI-and CIMP-/MSI-samples presented in average 16% demethylation for LINE-1, while no significant changes were observed for the CIMP+/MSI+samples. For the CIMP+group, 4–9% increase of methylation density for LINE-1 was observed for a small fraction of samples, most of them identified as CIMP+/MSI+samples.

Mentions: The primary colorectal tumors presented a high variation in LINE-1 methylation among different samples (Figure 2A), and the stratification of these colorectal tumors and their normal adjacent tissue reveals a non-uniform variability in LINE-1 methylation. CRC with sporadic microsatellite instability (MSI), most of which are due to MLH1 promoter methylation, showed no difference in LINE-1 methylation between normal adjacent and cancer tissues (62.6%±1.1% versus 60.6%±1.7%, P = 0.33), with an average decrease in methylation of only 3.12%±2.3%. By contrast MSI-cases had a significant decrease in LINE-1 hypomethylation between normal adjacent and cancer tissues (64.6%±0.5% versus 53.8%±1.2%, P<0.0001). Apparently, LINE-1 hypomethylation was independent from CIMP status, since CIMP+/MSI-cases and CIMP-cases were equally hypomethylated (15.4%±2.7% versus 17.7%±2.7%, P = 0.56). This unequal distribution of relative demethylation by presence of microsatellite instability is represented in the Figure 2B, which illustrates the maintenance of LINE-1 methylation in CIMP+/MSI+tumors compared to normal appearing mucosa, while CIMP+/MSI-and CIMP-/MSI-undergo severe hypomethylation, with one case presenting an extreme relative demethylation (61.3%).


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)

Differential LINE-1 methylation among CIMP/MSI groups in primary colorectal carcinoma samples (CRCs). A) Colorectal tumor DNA and their normal appearing adjacent mucosa from sixty patients were evaluated for LINE-1 methylation. These tumors were previously evaluated for CpG island methylator phenotype (CIMP), using a panel of single-copy genes methylation analysis, and microsatellite instability (MSI) status, resulting in the identification of three CIMP/MSI groups. In normal appearing mucosa (top) little variation in LINE-1 methylation is observed between samples and CIMP/MSI groups (average methylation = 64.3%), while in tumor (bottom) several samples undergo high LINE-1 demethylation (25/60 tumor samples have methylation density bellow 55%), most notable in CIMP+/MSI-and CIMP-/MSI-groups. B) Relative LINE-1 demethylation in CRCs. Relative demethylation was calculated as the percent change of LINE-1 methylation in tumor compared to its normal appearing mucosa. Both CIMP+/MSI-and CIMP-/MSI-samples presented in average 16% demethylation for LINE-1, while no significant changes were observed for the CIMP+/MSI+samples. For the CIMP+group, 4–9% increase of methylation density for LINE-1 was observed for a small fraction of samples, most of them identified as CIMP+/MSI+samples.
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pone-0000399-g002: Differential LINE-1 methylation among CIMP/MSI groups in primary colorectal carcinoma samples (CRCs). A) Colorectal tumor DNA and their normal appearing adjacent mucosa from sixty patients were evaluated for LINE-1 methylation. These tumors were previously evaluated for CpG island methylator phenotype (CIMP), using a panel of single-copy genes methylation analysis, and microsatellite instability (MSI) status, resulting in the identification of three CIMP/MSI groups. In normal appearing mucosa (top) little variation in LINE-1 methylation is observed between samples and CIMP/MSI groups (average methylation = 64.3%), while in tumor (bottom) several samples undergo high LINE-1 demethylation (25/60 tumor samples have methylation density bellow 55%), most notable in CIMP+/MSI-and CIMP-/MSI-groups. B) Relative LINE-1 demethylation in CRCs. Relative demethylation was calculated as the percent change of LINE-1 methylation in tumor compared to its normal appearing mucosa. Both CIMP+/MSI-and CIMP-/MSI-samples presented in average 16% demethylation for LINE-1, while no significant changes were observed for the CIMP+/MSI+samples. For the CIMP+group, 4–9% increase of methylation density for LINE-1 was observed for a small fraction of samples, most of them identified as CIMP+/MSI+samples.
Mentions: The primary colorectal tumors presented a high variation in LINE-1 methylation among different samples (Figure 2A), and the stratification of these colorectal tumors and their normal adjacent tissue reveals a non-uniform variability in LINE-1 methylation. CRC with sporadic microsatellite instability (MSI), most of which are due to MLH1 promoter methylation, showed no difference in LINE-1 methylation between normal adjacent and cancer tissues (62.6%±1.1% versus 60.6%±1.7%, P = 0.33), with an average decrease in methylation of only 3.12%±2.3%. By contrast MSI-cases had a significant decrease in LINE-1 hypomethylation between normal adjacent and cancer tissues (64.6%±0.5% versus 53.8%±1.2%, P<0.0001). Apparently, LINE-1 hypomethylation was independent from CIMP status, since CIMP+/MSI-cases and CIMP-cases were equally hypomethylated (15.4%±2.7% versus 17.7%±2.7%, P = 0.56). This unequal distribution of relative demethylation by presence of microsatellite instability is represented in the Figure 2B, which illustrates the maintenance of LINE-1 methylation in CIMP+/MSI+tumors compared to normal appearing mucosa, while CIMP+/MSI-and CIMP-/MSI-undergo severe hypomethylation, with one case presenting an extreme relative demethylation (61.3%).

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