Immune Netw.  2012 Apr;12(2):58-65. 10.4110/in.2012.12.2.58.

Reactivation of Silenced WT1 Transgene by Hypomethylating Agents: Implications for in vitro Modeling of Chemoimmunotherapy

• Kwon YR
• Son MJ
• Kim HJ
• Kim YJ

• KMID: 2150735
• DOI: http://doi.org/10.4110/in.2012.12.2.58

Abstract

BACKGROUND
A cell line with transfected Wilms' tumor protein 1 (WT1) is has been used for the preclinical evaluation of novel treatment strategies of WT1 immunotherapy for leukemia due to the lack of appropriate murine leukemia cell line with endogenous WT1. However, silencing of the transgene occurs. Regarding the effects of hypomethylating agents (HMAs) on reactivation of silenced genes, HMAs are considered to be immune enhancers.
METHODS
We treated murine WT1- transfected C1498 (mWT1-C1498) with increasing doses of decitabine (DAC) and azacitidine (AZA) to analyze their effects on transgene reactivation.
RESULTS
DAC and AZA decreased the number of viable cells in a dose- or time-dependent manner. Quantification of WT1 mRNA level was analyzed by real-time polymerase chain reaction after mWT1-C1498 treated with increasing dose of HMA. DAC treatment for 48 h induced 1.4-, 14.6-, and 15.5-fold increment of WT1 mRNA level, compared to untreated sample, at 0.1, 1, and 10microM, respectively. Further increment of WT1 expression in the presence of 1 and 10microM DAC was evident at 72 h. AZA treatment also induced up-regulation of mRNA, but not to the same degree as with DAC treatment. The correlation between the incremental increases in WT1 mRNA by DAC was confirmed by Western blot and concomitant down-regulation of WT1 promoter methylation was revealed.
CONCLUSION
The in vitro data show that HMA can induce reactivation of WT1 transgene and that DAC is more effective, at least in mWT1-C1498 cells, which suggests that the combination of DAC and mWT1-C1498 can be used for the development of the experimental model of HMA-combined WT1 immunotherapy targeting leukemia.

Keyword

Wilms' tumor 1; Methylation; Transgene; Decitabine; Azacitidine

MeSH Terms

Azacitidine
Blotting, Western
Cell Line
Down-Regulation
Immunotherapy
Leukemia
Methylation
Models, Theoretical
Real-Time Polymerase Chain Reaction
RNA, Messenger
Transgenes
Up-Regulation
Wilms Tumor
Azacitidine
RNA, Messenger

Figure

• 

  Figure 1 Effects of HMA on cell viability. In all cases, cell viability was measured using MTS assay as described. (A) C1498 cells were cultured with increasing concentrations of HMA for 24 h, 48 h, and 72 h. mock-C1498 cells treated with 0.1~10µM DAC for 24 h up to 72 h. (B) mWT1-C1498 cells treated with 0.1~10µM DAC for 24 h up to 72 h. (C) mock-C1498 cells treated with 0.1~10µM AZA for 24 h up to 72 h. (D) mWT1-C1498 cells treated with 0.1~10µM AZA for 24 h up to 72 h. Samples were measured in triplicates.

• 

  Figure 2 (A) HMA mediates induction of WT1 in C1498 cells in a time- and dose-dependent manner by RT-PCR results. (B, C) Expression levels of WT1 mRNA were determined by real-time PCR in untreated and HMA treated mWT1-C1498 cells at 48 h and 72 h. Shown are the mean mRNA values normalized to GAPDH from at least three experiments. Error bars indicate the standard deviation. Data are expressed as mean±SD; *p<0.05, ***p<0.001.

• 

  Figure 3 DAC mediates induction of WT1 in mWT1-C1498 cells in a time-dependent manner. Expression of WT1 protein in mock-C1498 and mWT1-C1498 cells at 48 h and 72 h treated with 0, 0.1, 1, and 10µM DAC.

• 

  Figure 4 Dose-dependent hypomethylation induction by DAC in mWT1-C1498 cell lines. (A) A representative methylation analysis of four CpG-sites of the WT1 promoter using pyrosequencing. (B) Average percentage for each sample is displayed as bar graphs. Data are expressed as mean±SD; ***p<0.001.

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