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Transfection with mdm2-antisense or wtp53 results in radiosensitization and an increased apoptosis of a soft tissue
1Institute of Pathology, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany.
Abstract:
Soft tissue sarcomas (STS) are mostly resistant after radiation treatment and are characterized by a rather low rate of apoptosis. The aim of this study was to test, in the p53 mutant STS cell line US8-93, the effect of a combined treatment with DNA transfection--either with mdm2 antisense oligodesoxynucleotides (mdm2-AS) or with a wild-type p53-plasmid (wtp53)--and the effects of irradiation on radiosensitivity. Mdm2-sense oligodesoxynucleotides (mdm2-SE) and a GFP-plasmid (GFP) were applied as controls. In order to evaluate the treatment radiation sensitization (clonogenic survival), apoptotic cell death and P53/MDM2-protein expression were determined. A moderately increased radiation sensitization was observed when comparing clonogenic survival after 2 Gy irradiation between cells transfected either with the control mdm2-SE (48%) or with mdm-2 AS (30%). At the same irradiation dose, clonogenic survival of wtp53-plasmid transfected cells (32%) was about 2-fold less than in the cells transfected with the control GFP-plasmid (61%). This enhancement factor of radiation sensitization was increased by about 3-fold at 4 Gy irradiation. Furthermore, an increase in apoptotic cells was already detectable by up to 7.7% (mdm2-AS) in comparison to 3.1% (mdm2-SE control) 72 hours after transfection. In parallel, the percentage of apoptotic cells could be further elevated after subsequent irradiation with 12 Gy by up to 15% (mdm2-AS) compared to 5.7% (mdm2-SE control). A striking result was obtained with the combined treatment of a wtp53 and 12 Gy irradiation which produced in 25% and 38.9% of apoptotic cells 48 hours and 72 hours after transfection, respectively. We can therefore conclude that the sensitivity of radiation therapy is enhanced by DNA transfection with wtp53 or mdm-2 AS ODNs for the correction of the p53-mdm2 balance in STS in vitro.
Insights
This study shows that DNA transfection with wild-type p53 (wtp53) or mdm2 antisense oligodeoxynucleotides (mdm2-AS) can enhance radiation sensitivity in soft tissue sarcomas (STS). These treatments correct the p53-mdm2 balance, improving radiation therapy outcomes in vitro.
Area of Science:
- Oncology
- Molecular Biology
- Radiotherapy Research
Background:
- Soft tissue sarcomas (STS) exhibit resistance to radiation therapy and low apoptosis rates.
- The p53-MDM2 pathway plays a critical role in regulating apoptosis and cellular response to DNA damage.
Purpose of the Study:
- To investigate the combined effects of DNA transfection and irradiation on radiosensitivity in a p53 mutant STS cell line.
- To evaluate the impact of restoring p53 function or modulating mdm2 expression on STS radiosensitivity and apoptosis.
Main Methods:
- Utilized p53 mutant STS cell line US8-93 for in vitro experiments.
- Employed DNA transfection with mdm2 antisense oligodeoxynucleotides (mdm2-AS) and wild-type p53-plasmid (wtp53).
- Assessed radiation sensitization via clonogenic survival, apoptotic cell death, and P53/MDM2 protein expression following irradiation at various doses (2 Gy, 4 Gy, 12 Gy).
Main Results:
- Transfection with mdm2-AS or wtp53 significantly enhanced radiation sensitization compared to control treatments (mdm2-SE, GFP-plasmid).
- wtp53 transfection approximately doubled radiosensitivity at 2 Gy and tripled it at 4 Gy.
- Combined wtp53 and 12 Gy irradiation markedly increased apoptotic cell percentages (25% at 48h, 38.9% at 72h).
- mdm2-AS also increased apoptosis, particularly when combined with 12 Gy irradiation (up to 15% vs. 5.7% in controls).
Conclusions:
- DNA transfection with wtp53 or mdm-2 AS ODNs enhances the sensitivity of radiation therapy in soft tissue sarcomas.
- Restoring the p53-mdm2 balance through these genetic modifications offers a promising strategy for improving STS treatment outcomes.
- These findings support the potential of targeted DNA-based therapies in combination with radiotherapy for managing radiation-resistant STS.
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