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Reactivation of Silenced WT1 Transgene by Hypomethylating Agents - Implications for in vitro Modeling of
Yong-Rim Kwon1, Min-Jung Son, Hye-Jung Kim
1Laboratory of Hematological Disease and Transplant Immunology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.
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 10µM, respectively. Further increment of WT1 expression in the presence of 1 and 10µM 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.
Insights
Hypomethylating agents (HMAs) like decitabine (DAC) can reactivate silenced Wilms' tumor protein 1 (WT1) transgenes in leukemia models. DAC showed greater efficacy than azacitidine (AZA) in restoring WT1 expression for immunotherapy development.
Area of Science:
- Oncology
- Immunotherapy
- Molecular Biology
Background:
- A Wilms' tumor protein 1 (WT1)-transfected cell line is crucial for preclinical WT1 immunotherapy research in leukemia.
- Transgene silencing limits the utility of these models, necessitating strategies for reactivation.
- Hypomethylating agents (HMAs) are known immune enhancers with potential for gene reactivation.
Purpose of the Study:
- To investigate the effect of HMAs, specifically decitabine (DAC) and azacitidine (AZA), on WT1 transgene reactivation in a murine leukemia cell line.
- To evaluate the potential of HMAs in enhancing WT1 expression for combined immunotherapy strategies.
Main Methods:
- Murine WT1-transfected C1498 (mWT1-C1498) cells were treated with varying doses of DAC and AZA.
- WT1 mRNA levels were quantified using real-time polymerase chain reaction (RT-PCR).
- Protein expression and promoter methylation were analyzed via Western blot and methylation assays, respectively.
Main Results:
- Both DAC and AZA reduced viable cell counts in a dose- and time-dependent manner.
- DAC significantly increased WT1 mRNA levels in a dose-dependent manner, with notable increases at 48 and 72 hours.
- DAC demonstrated superior WT1 reactivation compared to AZA, correlating with decreased WT1 promoter methylation.
Conclusions:
- HMAs can effectively induce WT1 transgene reactivation in mWT1-C1498 cells.
- Decitabine (DAC) is more potent than azacitidine (AZA) in reactivating the WT1 transgene.
- The combination of DAC and mWT1-C1498 cells offers a promising experimental model for developing HMA-combined WT1 immunotherapy for leukemia.
