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P. W. Chen, B. R. Ksander, H.-C. Li, J. `. Mellon, W. Yang; The Ocular Microenvironment Utilizes De Novo Methylation to Generate Tumor Escape Mutants by an Epigenetic Mechanism. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6140.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that tumor escape mutants develop in the immune privileged ocular microenvironment. Since the escape phenotype was permanent and developed within 7 days post anterior chamber injection, we hypothesized that the escape phenotype was established by epigenetic gene regulation.
Eye-derived P815 (P815e) tumor cells were established in DBA/2 mice injected in the anterior chamber with wild type P815 (P815wt) cells and harvested 10 days post injection. P815e cells were treated with the demethylating agent 5-Aza 2-Deoxycytidine (0.1uM, 5-Aza 2dC) for 72 hours and subcutaneously injected into the flank of immunized DBA/2 mice. De novo methyltransferase (DNMT) expression was identified by microarray analysis and confirmed by real-time PCR. DNA methylation analysis was performed using a global DNA methylation detection assay. Eye-derived P815 and P815wt cells were transfected with the PIVCIITA-luc methyltransferase-sensitive reporter gene. Luciferase activity was assessed after treatment of PIVCIITA-luc transfected cells with 50U of IFNg.
5-Aza 2dC treated P815e cells were rejected in immunized mice (P=0.0035) with 80% survival vs. 0% survival in immunized mice challenged with untreated P815e cells. Microarray analysis identified and real-time PCR confirmed upregulated expression of DNMT1, and DNMT3b 2.1-fold and 3-fold, respectively by P815e cells compared to DNMT expression by P815wt cells. Eye-derived P815 cells demonstrated a 24% increase in global DNA methylation compared to P815wt cells. PIVCIITA-luc transfected P815e cells demonstrated a 32-fold decrease in luciferase activity relative to the activity of PIVCIITA-luc transfected P815wt cells.
De novo methylation is responsible, in part, for the establishment of ocular tumor escape mutants suggesting that factors in the ocular microenvironment promote epigenetic gene regulation. Our results also imply that epigenetics may be a novel mechanism to support immune privilege.
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