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(PP350) Differential response to irradiation of wild-type proliferating cell nuclear antigen (PCNA) promoter and mutant PCNA promoter from radiation sensitive wasted (wst) mice.
Petrikas, James*,1, Shivnani, Anand1, Cruz, Cecille2, Babbo, Angela2, Paunesku, Tatjana2, Woloschak, Gayle2, 1 Northwestern University, Chicago, IL, United States2 Northwestern University, Chicago, IL, United States
ABSTRACT- ABSTRACT BODY: Purpose/Objective: PCNA is a ring-like protein is involved in DNA replication and repair, serving as a scaffold for cellular machinery for DNA synthesis. PCNA is necessary for cell survival and interacts with many proteins, playing a key role in cellular commitment to proliferation, arrest and DNA repair. PCNA is a radiation inducible gene. PCNA transcription is regulated by p53, as p53 binding sites are present in the PCNA promoter. Ionizing radiation induces changes in PCNA distribution in cultured cells with enhanced levels in the nucleus; while PCNA over-expression occurs in cell lines with a capacity for adaptive survival responses to low-dose radiation. A radiosensitive mutant wasted (wst) mouse has mutations in its PCNA promoter and diminished expression in tissues. None of these wst-mouse specific mutations affect binding of p53 to the PCNA promoter. To determine if these mutations play a role in the radiation response of PCNA gene expression, we compared the mutant and wild-type promoter responses to irradiation. A difference in radiation response of these two promoters suggests that cis-elements mutated in the PCNA promoter of wst-mice play a role in the radiation response of the PCNA gene irrespective of p53 regulation. Materials/Methods: We cloned 2 kb segments of the PCNA promoters from a wst-mouse and its healthy littermate upstream of yellow fluorescent protein (YFP) as a reporter gene. These constructs were transiently transfected into a TF1 lymphoblast cell line (ATCC) that has wild-type p53 gene expression. Cells were grown in 5% CO2 in RPMI medium according to ATCC. Transient transfections were done using SuperFect reagent (Qiagen). X-ray irradiation was performed using an Elekta SLi multi-energy megavoltage (MV) medical linear accelerator. A dose of 3 Gy was delivered utilizing 4 MV photons over one minute. Flow cytometry was used to estimate YFP fluorescence. Results: We compared reporter gene expression controlled by wild type and mutant PCNA promoters in a p53 positive TF1 cell line grown in serum rich or serum depleted media. At 48 hours post-transfection, cells were irradiated at a dose of 3 Gy and, following 1 or 3 hour incubation, fixed. In unirradiated controls, in serum rich medium, the wild-type promoter is expressed more highly than the mutant promoter. The situation is reversed in serum depleted medium. One hour post-irradiation with 3 Gy, reporter gene expression in both types of media showed the same pattern: wild-type PCNA promoter controlled construct was more expressed. Three hours post-irradiation in serum rich medium, the reporter gene expression pattern was very similar to the situation in unirradiated control cells. In serum-depleted medium, wild-type promoter expression three hours post-irradiation was lower than in the control cells, while wst promoter controlled expression showed only a slight decrease. Conclusions: The data suggest that the wst-type PCNA promoter from radiosensitive mice does not respond naturally either to serum stimulation or irradiation, and while the trend of changes following irradiation is similar for both promoters, the wst promoter response to irradiation is always less pronounced. Therefore, the role of PCNA promoter mutation in the radiosensitivity of the wst-mouse may be in the initial abnormal expression of PCNA, followed by the less dynamic and less pronounced gene expression responses to irradiation.
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