Limits...
CpG island methylation in a mouse model of lymphoma is driven by the genetic configuration of tumor cells.

Opavsky R, Wang SH, Trikha P, Raval A, Huang Y, Wu YZ, Rodriguez B, Keller B, Liyanarachchi S, Wei G, Davuluri RV, Weinstein M, Felsher D, Ostrowski M, Leone G, Plass C - PLoS Genet. (2007)

Bottom Line: Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer.The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression.This signature reflected gene transcription profiles and was detected only in advanced stages of disease.

View Article: PubMed Central - PubMed

Affiliation: Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, USA.

ABSTRACT
Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Global patterns of hypermethylation are tumor-type specific and nonrandom. The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression. Using restriction landmark genomic scanning on samples derived from tissue culture and in vivo models of T cell lymphomas, we found that MYC overexpression gave rise to a specific signature of CpG island hypermethylation. This signature reflected gene transcription profiles and was detected only in advanced stages of disease. The further inactivation of the Pten, p53, and E2f2 tumor suppressors in MYC-induced lymphomas resulted in distinct and diagnostic CpG island methylation signatures. Our data suggest that tumor-specific DNA methylation in lymphomas arises as a result of the selection of rare DNA methylation events during the course of tumor development. This selection appears to be driven by the genetic configuration of tumor cells, providing experimental evidence for a causal role of DNA hypermethylation in tumor progression and an explanation for the tremendous epigenetic heterogeneity observed in the evolution of human cancers. The ability to predict genome-wide epigenetic silencing based on relatively few genetic alterations will allow for a more complete classification of tumors and understanding of tumor cell biology.

Show MeSH

Related in: MedlinePlus

Analysis of Aberrant DNA Methylation in p53−, E2f2−, and Pten-Deleted Lymphomas(A) Graphical representation of the time-frame of tumor onset in EμSR-tTA;Teto-MYC (wild type), EμSR-tTA;Teto-MYC;p53−/− (p53−/−), EμSR-tTA;Teto-MYC;E2f2−/− (E2f2−/−), or EμSR-tTA;Teto-MYC;Teto-Cre;PtenLoxP/LoxP (Pten−/−) mice.(B) Examples of FACS profiles of normal thymocytes (normal) and tumor cells (tumors) derived from the different genetic backgrounds as indicated. The percentage of cells that are in each quadrant of the FACS profile is indicated.(C) Representative examples of RLGS profile sections of normal thymocytes derived from EμSR-tTA (normal p53+/+) and EμSR-tTA; p53−/− (normal p53−/−) mice and tumors derived from EμSR-tTA;Teto-MYC (tumor p53+/+) and EμSR-tTA;Teto-MYC; p53−/− (tumor p53−/−) mice. The position of DNA fragment 2D17 is indicated with arrows. Note the increase in the intensity of 2D17 in tumors deleted for the p53 tumor suppressor (compare p53+/+ and p53−/− tumor samples in the third and fourth panels).(D) Real time RT-PCR analysis of expression of 6D20 in normal thymic controls (white bars), EμSR-tTA;Teto-MYC tumors (black bars), or EμSR-tTA;Teto-MYC tumors deleted either for p53, E2f2, or Pten (grey bars) as indicated.(E) Summary of expression data from tumors derived in different genetic backgrounds obtained on analysis of eight RLGS fragments as indicated. Red boxes represent tumors with reduction in expression relative to normal thymic controls; yellow boxes indicate tumors with no change in expression. Repression thresholds for individual genes were chosen as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1994712&req=5

pgen-0030167-g004: Analysis of Aberrant DNA Methylation in p53−, E2f2−, and Pten-Deleted Lymphomas(A) Graphical representation of the time-frame of tumor onset in EμSR-tTA;Teto-MYC (wild type), EμSR-tTA;Teto-MYC;p53−/− (p53−/−), EμSR-tTA;Teto-MYC;E2f2−/− (E2f2−/−), or EμSR-tTA;Teto-MYC;Teto-Cre;PtenLoxP/LoxP (Pten−/−) mice.(B) Examples of FACS profiles of normal thymocytes (normal) and tumor cells (tumors) derived from the different genetic backgrounds as indicated. The percentage of cells that are in each quadrant of the FACS profile is indicated.(C) Representative examples of RLGS profile sections of normal thymocytes derived from EμSR-tTA (normal p53+/+) and EμSR-tTA; p53−/− (normal p53−/−) mice and tumors derived from EμSR-tTA;Teto-MYC (tumor p53+/+) and EμSR-tTA;Teto-MYC; p53−/− (tumor p53−/−) mice. The position of DNA fragment 2D17 is indicated with arrows. Note the increase in the intensity of 2D17 in tumors deleted for the p53 tumor suppressor (compare p53+/+ and p53−/− tumor samples in the third and fourth panels).(D) Real time RT-PCR analysis of expression of 6D20 in normal thymic controls (white bars), EμSR-tTA;Teto-MYC tumors (black bars), or EμSR-tTA;Teto-MYC tumors deleted either for p53, E2f2, or Pten (grey bars) as indicated.(E) Summary of expression data from tumors derived in different genetic backgrounds obtained on analysis of eight RLGS fragments as indicated. Red boxes represent tumors with reduction in expression relative to normal thymic controls; yellow boxes indicate tumors with no change in expression. Repression thresholds for individual genes were chosen as described in Materials and Methods.

Mentions: To test this prediction, we evaluated CpG island methylation in T cell lymphomas that developed in EμSR-tTA;Teto-MYC animals deleted for the p53, Pten, or E2f2 tumor suppressors. P53 and PTEN are the most frequently inactivated tumor suppressor genes in human cancer [29,30] and have been shown to have tumor suppressor function in T cells [31,32]. Recent studies in our laboratory indicate that E2f2 expression is significantly decreased in a number of human hematopoietic malignancies and its loss in mice accelerates T cell lymphomagenesis (G. L., unpublished data). Therefore, we interbred EμSR-tTA;Teto-MYC and p53−/− or E2f2−/− mice to generate cohorts of EμSR-tTA;Teto-MYC;p53−/− and EμSR-tTA;Teto-MYC;E2f2−/− animals. Because germ line inactivation of Pten results in embryonic lethality, EμSR-tTA;Teto-MYC mice were interbred with mice carrying the Teto-Cre transgene and a conditional allele of Pten (Teto-Cre;PtenLoxP). From these latter crosses we generated cohorts of EμSR-tTA;Teto-MYC;Teto-Cre;PtenLoxP/LoxP mice. Consistent with their tumor suppressor functions, loss of p53, Pten, or E2f2 significantly accelerated disease progression (p = 0.001; p = 0.005, and p = 0.0007, respectively; Figure 4A). In each case, FACS analysis confirmed that lymphomas in these mice were of T cell origin and consisted of either CD4 single- or CD4/CD8 double-positive cells (Figure 4B). We then analyzed the pattern of CpG island methylation in each tumor cohort by RLGS (Figures 4C and 5A). All four tumor groups had significant amounts of promoter hypermethylation; with an average of 1.8% of CpG islands hypermethylated in the EμSR-tTA; Teto-MYC cohort, 1.1 % in EμSR-tTA; Teto-MYC; p53−/− cohort (p = 0.06), 0.3% in EμSR-tTA; Teto-MYC; Teto-Cre;PtenLoxP/LoxP (p = 0.004), and 1.9% in EμSR-tTA; Teto-MYC; E2f2−/− (p = 0.885). Importantly, there was no detectable aberrant DNA methylation in control thymocytes isolated from age-matched EμSR-tTA;p53−/−, EμSR-tTA;Teto-Cre;PtenLoxP/LoxP, or EμSR-tTA;E2f2−/− mice (unpublished data). The aberrant DNA methylation detected in tumor samples was confirmed by 126 COBRA reactions performed on the same tumor samples that were used for RLGS analysis. This analysis revealed that in 74% of the cases evaluated, RLGS and COBRA assays yielded identical results (Figure S5 and unpublished data). In 22 % of cases COBRA detected aberrant DNA methylation events that were not identified by RLGS. This is not surprising, since COBRA is a more sensitive method to detect DNA methylation. In 4% of cases, COBRA assays failed to detect DNA methylation events that were detected by RLGS; this discrepancy likely reflects the different restriction sites analyzed by these methods.


CpG island methylation in a mouse model of lymphoma is driven by the genetic configuration of tumor cells.

Opavsky R, Wang SH, Trikha P, Raval A, Huang Y, Wu YZ, Rodriguez B, Keller B, Liyanarachchi S, Wei G, Davuluri RV, Weinstein M, Felsher D, Ostrowski M, Leone G, Plass C - PLoS Genet. (2007)

Analysis of Aberrant DNA Methylation in p53−, E2f2−, and Pten-Deleted Lymphomas(A) Graphical representation of the time-frame of tumor onset in EμSR-tTA;Teto-MYC (wild type), EμSR-tTA;Teto-MYC;p53−/− (p53−/−), EμSR-tTA;Teto-MYC;E2f2−/− (E2f2−/−), or EμSR-tTA;Teto-MYC;Teto-Cre;PtenLoxP/LoxP (Pten−/−) mice.(B) Examples of FACS profiles of normal thymocytes (normal) and tumor cells (tumors) derived from the different genetic backgrounds as indicated. The percentage of cells that are in each quadrant of the FACS profile is indicated.(C) Representative examples of RLGS profile sections of normal thymocytes derived from EμSR-tTA (normal p53+/+) and EμSR-tTA; p53−/− (normal p53−/−) mice and tumors derived from EμSR-tTA;Teto-MYC (tumor p53+/+) and EμSR-tTA;Teto-MYC; p53−/− (tumor p53−/−) mice. The position of DNA fragment 2D17 is indicated with arrows. Note the increase in the intensity of 2D17 in tumors deleted for the p53 tumor suppressor (compare p53+/+ and p53−/− tumor samples in the third and fourth panels).(D) Real time RT-PCR analysis of expression of 6D20 in normal thymic controls (white bars), EμSR-tTA;Teto-MYC tumors (black bars), or EμSR-tTA;Teto-MYC tumors deleted either for p53, E2f2, or Pten (grey bars) as indicated.(E) Summary of expression data from tumors derived in different genetic backgrounds obtained on analysis of eight RLGS fragments as indicated. Red boxes represent tumors with reduction in expression relative to normal thymic controls; yellow boxes indicate tumors with no change in expression. Repression thresholds for individual genes were chosen as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1994712&req=5

pgen-0030167-g004: Analysis of Aberrant DNA Methylation in p53−, E2f2−, and Pten-Deleted Lymphomas(A) Graphical representation of the time-frame of tumor onset in EμSR-tTA;Teto-MYC (wild type), EμSR-tTA;Teto-MYC;p53−/− (p53−/−), EμSR-tTA;Teto-MYC;E2f2−/− (E2f2−/−), or EμSR-tTA;Teto-MYC;Teto-Cre;PtenLoxP/LoxP (Pten−/−) mice.(B) Examples of FACS profiles of normal thymocytes (normal) and tumor cells (tumors) derived from the different genetic backgrounds as indicated. The percentage of cells that are in each quadrant of the FACS profile is indicated.(C) Representative examples of RLGS profile sections of normal thymocytes derived from EμSR-tTA (normal p53+/+) and EμSR-tTA; p53−/− (normal p53−/−) mice and tumors derived from EμSR-tTA;Teto-MYC (tumor p53+/+) and EμSR-tTA;Teto-MYC; p53−/− (tumor p53−/−) mice. The position of DNA fragment 2D17 is indicated with arrows. Note the increase in the intensity of 2D17 in tumors deleted for the p53 tumor suppressor (compare p53+/+ and p53−/− tumor samples in the third and fourth panels).(D) Real time RT-PCR analysis of expression of 6D20 in normal thymic controls (white bars), EμSR-tTA;Teto-MYC tumors (black bars), or EμSR-tTA;Teto-MYC tumors deleted either for p53, E2f2, or Pten (grey bars) as indicated.(E) Summary of expression data from tumors derived in different genetic backgrounds obtained on analysis of eight RLGS fragments as indicated. Red boxes represent tumors with reduction in expression relative to normal thymic controls; yellow boxes indicate tumors with no change in expression. Repression thresholds for individual genes were chosen as described in Materials and Methods.
Mentions: To test this prediction, we evaluated CpG island methylation in T cell lymphomas that developed in EμSR-tTA;Teto-MYC animals deleted for the p53, Pten, or E2f2 tumor suppressors. P53 and PTEN are the most frequently inactivated tumor suppressor genes in human cancer [29,30] and have been shown to have tumor suppressor function in T cells [31,32]. Recent studies in our laboratory indicate that E2f2 expression is significantly decreased in a number of human hematopoietic malignancies and its loss in mice accelerates T cell lymphomagenesis (G. L., unpublished data). Therefore, we interbred EμSR-tTA;Teto-MYC and p53−/− or E2f2−/− mice to generate cohorts of EμSR-tTA;Teto-MYC;p53−/− and EμSR-tTA;Teto-MYC;E2f2−/− animals. Because germ line inactivation of Pten results in embryonic lethality, EμSR-tTA;Teto-MYC mice were interbred with mice carrying the Teto-Cre transgene and a conditional allele of Pten (Teto-Cre;PtenLoxP). From these latter crosses we generated cohorts of EμSR-tTA;Teto-MYC;Teto-Cre;PtenLoxP/LoxP mice. Consistent with their tumor suppressor functions, loss of p53, Pten, or E2f2 significantly accelerated disease progression (p = 0.001; p = 0.005, and p = 0.0007, respectively; Figure 4A). In each case, FACS analysis confirmed that lymphomas in these mice were of T cell origin and consisted of either CD4 single- or CD4/CD8 double-positive cells (Figure 4B). We then analyzed the pattern of CpG island methylation in each tumor cohort by RLGS (Figures 4C and 5A). All four tumor groups had significant amounts of promoter hypermethylation; with an average of 1.8% of CpG islands hypermethylated in the EμSR-tTA; Teto-MYC cohort, 1.1 % in EμSR-tTA; Teto-MYC; p53−/− cohort (p = 0.06), 0.3% in EμSR-tTA; Teto-MYC; Teto-Cre;PtenLoxP/LoxP (p = 0.004), and 1.9% in EμSR-tTA; Teto-MYC; E2f2−/− (p = 0.885). Importantly, there was no detectable aberrant DNA methylation in control thymocytes isolated from age-matched EμSR-tTA;p53−/−, EμSR-tTA;Teto-Cre;PtenLoxP/LoxP, or EμSR-tTA;E2f2−/− mice (unpublished data). The aberrant DNA methylation detected in tumor samples was confirmed by 126 COBRA reactions performed on the same tumor samples that were used for RLGS analysis. This analysis revealed that in 74% of the cases evaluated, RLGS and COBRA assays yielded identical results (Figure S5 and unpublished data). In 22 % of cases COBRA detected aberrant DNA methylation events that were not identified by RLGS. This is not surprising, since COBRA is a more sensitive method to detect DNA methylation. In 4% of cases, COBRA assays failed to detect DNA methylation events that were detected by RLGS; this discrepancy likely reflects the different restriction sites analyzed by these methods.

Bottom Line: Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer.The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression.This signature reflected gene transcription profiles and was detected only in advanced stages of disease.

View Article: PubMed Central - PubMed

Affiliation: Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, USA.

ABSTRACT
Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Global patterns of hypermethylation are tumor-type specific and nonrandom. The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression. Using restriction landmark genomic scanning on samples derived from tissue culture and in vivo models of T cell lymphomas, we found that MYC overexpression gave rise to a specific signature of CpG island hypermethylation. This signature reflected gene transcription profiles and was detected only in advanced stages of disease. The further inactivation of the Pten, p53, and E2f2 tumor suppressors in MYC-induced lymphomas resulted in distinct and diagnostic CpG island methylation signatures. Our data suggest that tumor-specific DNA methylation in lymphomas arises as a result of the selection of rare DNA methylation events during the course of tumor development. This selection appears to be driven by the genetic configuration of tumor cells, providing experimental evidence for a causal role of DNA hypermethylation in tumor progression and an explanation for the tremendous epigenetic heterogeneity observed in the evolution of human cancers. The ability to predict genome-wide epigenetic silencing based on relatively few genetic alterations will allow for a more complete classification of tumors and understanding of tumor cell biology.

Show MeSH
Related in: MedlinePlus