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.
Highlights
Lung cancer is the number one cause of cancer-related deaths in the United States, and account for 2.4% of all deaths worldwide [1]
The H460 lung cancer cell line has intact p53; treatment with 20 Gy radiation produced a small number of radiation-resistant (RR) surviving cells, which were collected for study
Characterization of a novel p53 deletion mutant To determine the effect of our novel deletion on cell survival and the expression of p53 and downstream effector molecules responsible for cell cycle arrest and apoptosis, a second radiation of 6 Gy was applied to cells from the parental and RR cells
Summary
Lung cancer is the number one cause of cancer-related deaths in the United States, and account for 2.4% of all deaths worldwide [1]. The success of RT in lung cancer is greatly affected by a variety of factors including location, size, grade, extent of invasion, and individual tumor characteristics. Some of the cellular damage that causes cancer can induce resistance to treatments, making it a very real concern. Many labs have begun engineering cell lines expressing single mutations and using these to examine chemoand radioresistance. Some groups use molecular modeling to predict which mutations will have functional consequences
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