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Growth inhibition of cultured human Tenon's fibroblastic cells by targeting the E2F transcription factor
M Akimoto1, M Hangai, K Okazaki
1Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Abstract:
The transcription factor E2F regulates the expression of several genes concerned with cell growth. The ability to inhibit transcription by blocking E2F expression has great potential in the treatment of proliferative disorders. The effect of double-stranded phosphorothioate oligonucleotides containing E2F transcription factor cis element, a so called 'decoy' has examined on the growth of cultured human Tenon's fibroblastic cells. Human Tenon's fibroblastic cells were cultured and challenged by E2F decoy coated with the Hemagglutinating virus of Japan (HVJ) cationic liposomes (HVJ-CL). The outcome was evaluated using fluorescence microscopy, RT-PCR and growth assays. HVJ-CL facilitated the transfer of external oligonucleotides to cultured human Tenon's fibroblastic cells. The E2F decoy, transferred by HVJ-CL, inhibited simultaneously the expression of the mRNAs of several cell cycle related genes such as c-myc, cdc2, proliferative cell nuclear antigen, and dehydrofolate reductase. Entry into S phase was also reduced to 42.7% of the positive control by the E2F decoy. The total increase of DNA at four days was reduced to 59.7% of the positive control by 5 microM and 29.9% by 15 microM of E2F decoy. It is concluded that gene therapy using the E2F transcription factor offers a potential therapeutic modality for the treatment of proliferative disorders such as proliferative vitreoretinopathy and fibrosis following filtering surgery.
Insights
Gene therapy using E2F decoy oligonucleotides effectively inhibits cell proliferation. This approach shows promise for treating proliferative disorders by blocking key cell cycle gene expression.
Area of Science:
- Molecular Biology
- Gene Therapy
- Cell Biology
Background:
- The transcription factor E2F controls genes essential for cell growth.
- Inhibiting E2F expression offers a potential strategy for treating proliferative disorders.
- Decoy oligonucleotides targeting E2F cis elements can block gene transcription.
Purpose of the Study:
- To investigate the efficacy of E2F decoy oligonucleotides delivered via Hemagglutinating virus of Japan (HVJ) cationic liposomes (HVJ-CL) in inhibiting human Tenon's fibroblastic cell growth.
- To evaluate the impact of E2F decoy on cell cycle gene expression and DNA synthesis.
Main Methods:
- Culturing human Tenon's fibroblastic cells.
- Transfecting cells with E2F decoy oligonucleotides using HVJ-CL.
- Assessing oligonucleotide transfer, gene expression (RT-PCR), and cell proliferation (growth assays, DNA content analysis).
Main Results:
- HVJ-CL efficiently delivered E2F decoy oligonucleotides into cells.
- E2F decoy significantly inhibited the expression of cell cycle genes (c-myc, cdc2, PCNA, DHFR).
- Cellular S phase entry was reduced by 42.7%, and DNA increase was significantly reduced in a dose-dependent manner.
Conclusions:
- E2F decoy delivered by HVJ-CL effectively inhibits cell proliferation by targeting E2F.
- This gene therapy approach holds potential for treating proliferative disorders like proliferative vitreoretinopathy and post-surgical fibrosis.