Stage-Specific Alterations of DNA Methyltransferase Expression, DNA Hypermethylation, and DNA Hypomethylation during Prostate Cancer Progression in the Transgenic Adenocarcinoma of Mouse Prostate Model

Abstract

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system. (Mol Cancer Res 2008;6(8):1365 -74)

Opposing Roles of Dnmt1 in Early- and Late-Stage Murine Prostate Cancer

“…RNA samples were extracted and used for quantitative real-time reverse transcription-PCR (qRT-PCR) analysis as described previously (35). The following genes were analyzed: Dnmt1, Dnmt3a, Dnmt3b, and 18S rRNA.…”

“…Western blotting for Dnmt proteins was completed as described previously (35,37). Ponceau S total protein stain was used to confirm equivalent protein loading.…”

“…Quantification of 5-methyl-deoxycytidine (5mdC) levels was determined by liquid chromatography-electrospray ionization quadrapole mass spectrometry, as described previously (35,50). Bisulfite pyrosequencing of the murine B1 repetitive DNA element was done as described previously (35).…”

“…TRAMP mice are characterized by Dnmt mRNA and protein overexpression, altered DNA methylation, and altered gene expression during prostate cancer development (2,33,35,37). Of the three enzymatically active Dnmts, Dnmt1 shows the greatest level of overexpression in TRAMP, and this correlates with Rb inactivation, a key genetic event driving prostate cancer in the model (37).…”

“…Of the three enzymatically active Dnmts, Dnmt1 shows the greatest level of overexpression in TRAMP, and this correlates with Rb inactivation, a key genetic event driving prostate cancer in the model (37). Most critically, global DNA hypomethylation occurs during early and late disease stages, while DNA hypermethylation occurs primarily at late disease stages in TRAMP (35).…”