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A novel radiation-induced p53 mutation is not implicated in radiation resistance via a dominant-negative effect.

Sun Y, Myers CJ, Dicker AP, Lu B - PLoS ONE (2014)

Bottom Line: Limited sequencing was performed on the resistant cells created and compared to the parent cell line, leading to the identification of a novel mutation (del) at the end of the DNA binding domain of p53.These patterns held true after analysis of p53 overexpression in H460 cells; however, H1299 cells transfected with mutant p53 did not express p21, whereas those given WT p53 produced a significant amount, as expected.An MTS assay using H460 and H1299 cells transfected with WT or mutant p53 showed that the novel mutation did not improve cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Understanding the mutations that confer radiation resistance is crucial to developing mechanisms to subvert this resistance. Here we describe the creation of a radiation resistant cell line and characterization of a novel p53 mutation. Treatment with 20 Gy radiation was used to induce mutations in the H460 lung cancer cell line; radiation resistance was confirmed by clonogenic assay. Limited sequencing was performed on the resistant cells created and compared to the parent cell line, leading to the identification of a novel mutation (del) at the end of the DNA binding domain of p53. Levels of p53, phospho-p53, p21, total caspase 3 and cleaved caspase 3 in radiation resistant cells and the radiation susceptible (parent) line were compared, all of which were found to be similar. These patterns held true after analysis of p53 overexpression in H460 cells; however, H1299 cells transfected with mutant p53 did not express p21, whereas those given WT p53 produced a significant amount, as expected. A luciferase assay demonstrated the inability of mutant p53 to bind its consensus elements. An MTS assay using H460 and H1299 cells transfected with WT or mutant p53 showed that the novel mutation did not improve cell survival. In summary, functional characterization of a radiation-induced p53 mutation in the H460 lung cancer cell line does not implicate it in the development of radiation resistance.

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Related in: MedlinePlus

Novel mutation abrogates p53 binding.Cells were transfected with a plasmid containing either WT p53 or that containing the novel mutation. A. After transfection into p53 competent H460 cells, a luciferase assay shows WT p53 is capable of binding its consensus elements, whereas mutant p53 is not. B. When the same vectors were applied to naturally p53-deficient H1299 cells, this difference was much more pronounced. The sample transfected with the mutant p53 showed a small amount of luciferase activity, approximately 3× that of the vector, whereas the WT p53 induced 25× as much.
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pone-0087492-g006: Novel mutation abrogates p53 binding.Cells were transfected with a plasmid containing either WT p53 or that containing the novel mutation. A. After transfection into p53 competent H460 cells, a luciferase assay shows WT p53 is capable of binding its consensus elements, whereas mutant p53 is not. B. When the same vectors were applied to naturally p53-deficient H1299 cells, this difference was much more pronounced. The sample transfected with the mutant p53 showed a small amount of luciferase activity, approximately 3× that of the vector, whereas the WT p53 induced 25× as much.

Mentions: The development of luciferase reporter vectors has made analysis of the effects of mutation on the binding ability of various transcription factors quick and quantifiable. Examination of H460 cells transfected with WT p53 showed nearly twice the luciferase expression as the control, whereas cells transfected with the mutant p53 had slightly lower expression than the control (Fig. 6). When the same vectors were applied to naturally p53-deficient H1299 cells, the sample treated with the mutant p53 showed a small amount of luciferase activity, approximately 3× that of the vector, whereas the WT p53 induced 25× as much. These luciferase assays demonstrated the inability of mutant p53 to effectively bind its consensus elements, as evidenced by the failure to initiate downstream gene transcription.


A novel radiation-induced p53 mutation is not implicated in radiation resistance via a dominant-negative effect.

Sun Y, Myers CJ, Dicker AP, Lu B - PLoS ONE (2014)

Novel mutation abrogates p53 binding.Cells were transfected with a plasmid containing either WT p53 or that containing the novel mutation. A. After transfection into p53 competent H460 cells, a luciferase assay shows WT p53 is capable of binding its consensus elements, whereas mutant p53 is not. B. When the same vectors were applied to naturally p53-deficient H1299 cells, this difference was much more pronounced. The sample transfected with the mutant p53 showed a small amount of luciferase activity, approximately 3× that of the vector, whereas the WT p53 induced 25× as much.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928108&req=5

pone-0087492-g006: Novel mutation abrogates p53 binding.Cells were transfected with a plasmid containing either WT p53 or that containing the novel mutation. A. After transfection into p53 competent H460 cells, a luciferase assay shows WT p53 is capable of binding its consensus elements, whereas mutant p53 is not. B. When the same vectors were applied to naturally p53-deficient H1299 cells, this difference was much more pronounced. The sample transfected with the mutant p53 showed a small amount of luciferase activity, approximately 3× that of the vector, whereas the WT p53 induced 25× as much.
Mentions: The development of luciferase reporter vectors has made analysis of the effects of mutation on the binding ability of various transcription factors quick and quantifiable. Examination of H460 cells transfected with WT p53 showed nearly twice the luciferase expression as the control, whereas cells transfected with the mutant p53 had slightly lower expression than the control (Fig. 6). When the same vectors were applied to naturally p53-deficient H1299 cells, the sample treated with the mutant p53 showed a small amount of luciferase activity, approximately 3× that of the vector, whereas the WT p53 induced 25× as much. These luciferase assays demonstrated the inability of mutant p53 to effectively bind its consensus elements, as evidenced by the failure to initiate downstream gene transcription.

Bottom Line: Limited sequencing was performed on the resistant cells created and compared to the parent cell line, leading to the identification of a novel mutation (del) at the end of the DNA binding domain of p53.These patterns held true after analysis of p53 overexpression in H460 cells; however, H1299 cells transfected with mutant p53 did not express p21, whereas those given WT p53 produced a significant amount, as expected.An MTS assay using H460 and H1299 cells transfected with WT or mutant p53 showed that the novel mutation did not improve cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Understanding the mutations that confer radiation resistance is crucial to developing mechanisms to subvert this resistance. Here we describe the creation of a radiation resistant cell line and characterization of a novel p53 mutation. Treatment with 20 Gy radiation was used to induce mutations in the H460 lung cancer cell line; radiation resistance was confirmed by clonogenic assay. Limited sequencing was performed on the resistant cells created and compared to the parent cell line, leading to the identification of a novel mutation (del) at the end of the DNA binding domain of p53. Levels of p53, phospho-p53, p21, total caspase 3 and cleaved caspase 3 in radiation resistant cells and the radiation susceptible (parent) line were compared, all of which were found to be similar. These patterns held true after analysis of p53 overexpression in H460 cells; however, H1299 cells transfected with mutant p53 did not express p21, whereas those given WT p53 produced a significant amount, as expected. A luciferase assay demonstrated the inability of mutant p53 to bind its consensus elements. An MTS assay using H460 and H1299 cells transfected with WT or mutant p53 showed that the novel mutation did not improve cell survival. In summary, functional characterization of a radiation-induced p53 mutation in the H460 lung cancer cell line does not implicate it in the development of radiation resistance.

Show MeSH
Related in: MedlinePlus