Abstract
Tumor protein 53-induced nuclear protein-1 (TP53inp1) is expressed by activation via p53 and p73. The purpose of our study was to investigate the role of TP53inp1 in response of fibroblasts to ionizing radiation. γ-Ray radiation dose-dependently induces the expression of TP53inp1 in human immortalized fibroblast (F11hT) cells. Stable silencing of TP53inp1 was done via lentiviral transfection of shRNA in F11hT cells. After irradiation the clonogenic survival of TP53inp1 knockdown (F11hT-shTP) cells was compared to cells transfected with non-targeting (NT) shRNA. Radiation-induced senescence was measured by SA-β-Gal staining and autophagy was detected by Acridine Orange dye and microtubule-associated protein-1 light chain 3 (LC3B) immunostaining. The expression of TP53inp1, GDF-15, and CDKN1A and alterations in radiation induced mitochondrial DNA deletions were evaluated by qPCR. TP53inp1 was required for radiation (IR) induced maximal elevation of CDKN1A and GDF-15 expressions. Mitochondrial DNA deletions were increased and autophagy was deregulated following irradiation in the absence of TP53inp1. Finally, we showed that silencing of TP53inp1 enhances the radiation sensitivity of fibroblast cells. These data suggest functional roles for TP53inp1 in radiation-induced autophagy and survival. Taken together, we suppose that silencing of TP53inp1 leads radiation induced autophagy impairment and induces accumulation of damaged mitochondria in primary human fibroblasts.
Highlights
Ionizing radiation (IR) causes oxidative stress in DNA, proteins and lipids, but cells have a complex signal cascade to avoid ROS-induced damage and ensure their homeostasis
In normal human cells several ataxia telangiectasia mutated (ATM)/p53 associated genes such as TP53inp1, CDKN1A, and HDM2, as well as several tumor necrosis factor (TNF) receptor superfamily members were shown to be induced by IR [9,10]
Treatment with 2 Gy further increased the expression of TP53inp1 up to (2.613 ± 0.439, p = 0.025)
Summary
Ionizing radiation (IR) causes oxidative stress in DNA, proteins and lipids, but cells have a complex signal cascade to avoid ROS-induced damage and ensure their homeostasis. Many authors report that TP53inp has a role in the control of proliferation and apoptosis under stress condition and acts as a dual regulator of transcription and autophagy [11], but the precise role of TP53inp in the radiation induced cellular stress remains ambiguous. We show evidence of the dose-dependent transcription of TP53inp by IR. Until now, it is not yet known whether the level of TP53inp expression can affect the radiosensitivity of human fibroblasts and whether TP53inp can modify the effect of radiotherapy. We established a shRNA-mediated TP53inp silencing strategy to investigate the effect of TP53inp silencing on cell survival and sensitization to γ-radiation in human fibroblasts in vitro
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