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

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.

Figure 1. Identification of a novel radiation-induced p53 mutation that confers resistance to subsequent radiotherapy.

H460 cells (p53-competent) were exposed to sequentially higher doses of radiation, then incubated at 37°C for 8–10 days. One surviving (radiation-resistant) clone was selected and expanded to create a radiation-resistance H460 cell line (RR-H460). To confirm the effect of novel deletion on cell survival, a second radiation of 6 Gy was applied to cells from the parental and RR lines. A. Radiation resistance was confirmed by clonogenic assay after treatment with 0–6 Gy. A significant (p<0.007) difference in survival between the radiation resistant colony and the parent cell line was noted at doses of 4 or 6 Gy. SF = 0.2. B. Sequencing analysis revealed a novel deletion mutation at the end of the DNA binding domain of p53. Figure modified from the TP53 website (p53.free.fr).

Figure 2. Characterization of parent and RR cell line after treatment with 6 Gy radiation

A. Subsequent protein expression was analyzed by Western blot. The parent and derivative cell lines were found to have comparable levels of total caspase 3. Expression of p53, phosphorylated p53, p21, and caspase cleavage were all delayed in the radiation resistant cell line. The radiation resistant cell line clearly shows two forms of p53, mutant and WT. B. mRNA analysis of p21 expression indicates that, in the hours immediately after irradiation, transcription is initially higher in the RR cell line, but by 24-irradiation the levels are similar in the RR and parent cell lines.

Figure 3. Overexpression of the novel mutant p53 does not induce p21 expression in p53-deficient H1299 cells.

A. Overexpression of WT p53 or the deletion mutant in H460 cells resulted in higher expression of the truncated protein than the full length transcript; comparable levels of phosphorylated p53, p21, total caspase 3 and cleaved caspase 3 were observed. B. Overexpression of the same in H1299 cells resulted in similar protein expression patterns with the exception of p21, which was expressed as a result of transfection with WT p53 but not the deletion mutant. C. RT-PCR analysis confirms that similar levels of p21 are present in H460 cells transfected with the WT or mutated p53 and that H1299 cells transfected with WT p53 express much higher levels of p21 than those transfected with the mutant.

Figure 4. Deletion mutant fails to improve cell survival when expressed in conjunction with WT p53.

48%, whereas transfection with mutant p53 resulted in over 90% survival. In H460 cells, survival was over 90% regardless of p53 mutation status, showing definitively that the novel mutation did not improve cell survival unless present as the only available form of p53, and confirming its inability to act as a dominant negative mutation.

Figure 5. Overexpression of novel p53 mutation does not confer radiation resistance.

p53 variant does not affect radiation resistance in the presence of WT p53. Transfection with WT p53 results in slightly decreased survival after irradiation with 4: (SF = 0.2); WT: 1.05 (p = 0.074); DEL: 0.97 (p = 0.27).

Figure 6. 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.