Figure 5.

LMP1 activates DNMT. (A) Western blot analysis of DNA methyltransferase 1 (DNMT1) in NPC076-LMP1 and NPC076-neo cells. Western blot analysis was performed as described in Fig. 1A. The protein level of DNMT1, PCNA, and LMP1 were detected by α-DNMT1 antibody 60B1220, α-PCNA antibody PC10, and α-LMP1 antibody S12, individually. Tubulin detected by α-tubulin antibody was used to normalize the amount of total protein in each preparation. (B) DNA methyltransferase activity assay in LMP1-expressing stable cell clones. DNA methyltransferase activity was measured in NPC076-LMP1 and MDCK-LMP1 or neo-control cells. (C) Kinetics of DNA methyltransferase activity in rAdLMP1-infected MCF-7 cells. Cells were infected with either rAdLMP1 or rAdLacZ at a multiplicity of infection of 100. Methyltransferase activity was determined as described in Materials and Methods. The data are an average of at least three independent experiments. The vertical bar represents standard deviation. Increase in C was determined by dividing the activity of cells infected with rAdLMP1 with that of cells infected with rAdLacZ. LMP1 and tubulin expression, shown at the bottom, were analyzed by Western blotting. (D) Methylation-specific PCR of E-cadherin promoter. MDA-MB-468 and MCF-7 cells were infected with rAdLMP1 or control virus, rAdLacZ, at multiplicity of infection of 100. Methylation-PCR analysis was carried out as described in Materials and Methods. U, unmethylated; M, methylated. The 116-bp PCR product represents the methylated state of island 1 within the E-cadherin promoter sequence, whereas the 97-bp product represents the unmethylated state. Uninfected cells were used as the unmethylated controls.