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Updated: May 8, 2026
Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Post-translational regulation of gene expression using the ATF4 oxygen-dependent degradation domain for
Su Hee Cho1, Binna Oh, Hyun Ah Kim
1Department of Bioengineering, College of Engineering, Hanyang University , Seoul , Korea and.
Abstract:
Solid tumors have hypoxic regions in their cores, due to low blood supply levels. Therefore, hypoxia-specific gene regulation systems have been developed for tumor-specific gene therapy. In this study, the oxygen-dependent degradation (ODD) domain on activating transcription factor-4 (ATF4) was evaluated for post-translational regulation of proteins. The ATF4 ODD cDNA was amplified by RT-PCR, and a luciferase plasmid containing the ATF4 ODD domain, pSV-Luc-ATF4-ODD, was constructed. Luciferase expression was induced under hypoxia by the ATF4 ODD domain in transfection assays into N2A neuroblastoma cells, C6 glioblastoma cells, and U87 glioblastoma cells. In the transfection assay with pSV-Luc-ATF4-ODD, RT-PCR results showed that the mRNA level did not change under hypoxia. This suggests that the induction of luciferase under hypoxia was mediated by post-translational regulation. A plasmid expressing thymidine kinase from herpes simplex virus (HSV-tk), pSV-HSVtk-ATF4-ODD, was constructed with the ATF4 ODD cDNA. The transfection assay with pSV-TK-ATF4-ODD showed that the ATF4 ODD domain induced HSV-tk expression under hypoxia and facilitated the death of C6 cells in the presence of ganciclovir (GCV). Furthermore, pSV-HSVtk-ATF4-ODD induced caspase-3 activity in the hypoxic cells. In conclusion, the ATF4 ODD may be useful for hypoxia-specific gene therapy by post-translational regulation of gene expression.
Insights
The oxygen-dependent degradation domain of ATF4 can control gene expression in low-oxygen tumor environments. This finding supports its potential use in targeted gene therapy for solid tumors.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Solid tumors often contain hypoxic cores due to insufficient blood supply.
- Hypoxia-specific gene regulation is crucial for developing targeted tumor therapies.
Purpose of the Study:
- To investigate the potential of the oxygen-dependent degradation (ODD) domain of activating transcription factor-4 (ATF4) for post-translational gene regulation.
- To evaluate the ATF4 ODD domain's efficacy in hypoxia-specific gene therapy.
Main Methods:
- Amplification of ATF4 ODD cDNA via RT-PCR.
- Construction of luciferase and herpes simplex virus thymidine kinase (HSV-tk) reporter plasmids containing the ATF4 ODD domain.
- Transfection assays in N2A, C6, and U87 cell lines under normoxic and hypoxic conditions.
- Analysis of mRNA levels and protein expression, including caspase-3 activity.
Main Results:
- Luciferase expression was induced under hypoxia via the ATF4 ODD domain, independent of mRNA level changes, indicating post-translational regulation.
- The ATF4 ODD domain successfully induced HSV-tk expression under hypoxia in C6 glioblastoma cells.
- Hypoxic C6 cells transfected with the HSV-tk construct exhibited ganciclovir-induced death and increased caspase-3 activity.
Conclusions:
- The ATF4 ODD domain mediates gene expression through post-translational regulation in response to hypoxia.
- The ATF4 ODD domain shows promise as a tool for hypoxia-specific gene therapy in solid tumors.
- This system can be utilized to induce cell death in hypoxic tumor regions.
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