Effect Of Nuclear Transposition Of PTEN On Radiation Damage Of Tumor Cells

BackgroundPTEN is a tumor suppressor gene that has been found mutations,deletions,and transcriptionalsilencing in many types of human malignancies.As a negative regulator of the classical PI3K/Akt cell growth signaling pathway,PTEN dephosphorylates phosphatidylinositol 3,4,5-triphosphate(PIP3)to phosphatidylinositol 4,5-diphosphate(PIP2)via its lipid phosphatase activity to inhibit cell survival,proliferation and angiogenesis.In addition,PTEN,independent of its phosphatase activity,can dephosphorylate proteins and peptide substrates required for tumor cell metabolism and inhibit tumor cell invasion.Because of the lack of a typical nuclear localization signal(NLS)on PTEN protein,it was initially thought that PTEN is fully localized in the cytoplasm.In recent years,a large number of studies have found that there are nuclear PTEN expression in human neurons,thyroid tissue,pancreatic cells,esophageal squamous cell carcinoma,vascular smooth muscle cells and intestinal mucosa,however,nuclear PTEN's specific biological function has been few reported.The nuclear translocation of proteins refers to the biological phenomenon that a large number of signaling proteins and molecules in eukaryotic cells move between the nucleus and cytoplasm through nuclear pore complexes.Dependent on the nuclear translocation,signal proteins and molecules can regulate the nuclear or cytoplasmic signaling pathways and other physiological or pathological processes more effectively and finely.UVB is part of the sunlight in the wavelength range of 280 nm-320 nm,a large number of publications show that excessive of UVB radiation can lead to the generation of reactive oxygen species(ROS),which induces DNA double-strand breaks and cell necrosis or apoptosis.While,whether the nuclear and cytoplasm distribution of PTEN will change in the UVB irradiation environment and what will happen for the biological behavior of a cell if it changes?To determine these issues can further reveal the nuclear and cytoplasmic functions of PTEN and may shed light on the treatment of clinical malignant tumors.PurposeThe purpose of this study was to investigate the subcellular localization of wild-type PTEN andits monoubiquitinated site mutant K13E-PTEN before and after DNA damage induced by UVB in U87cells,and their effect on the biological behavior of tumor cells.Hence,studying the phenomenon of subcellular localization of PTEN would contribute to further reveal the function of nuclear PTEN and the PTEN-related molecular mechanisms and also provide novel insights for clinical treatment of tumors.Method1.The cytotoxicity assay was conducted to detect the activity of U87 cells under different dosesof UVB irradiation after post-irradiation incubation.The toxicity of UVB on cells was explored to determine the suitable UV dose and time gradient for this project.2.U87 cells with overexpressed WT-PTEN and K13E-PTEN were constructed respectively.Thenucleus distributions of WT-PTEN and K13E-PTEN in U87 cells were detected by Western blot and fluorescence co-localization methods.Subsequently,U87 cells over-expressed WT-PTEN and K13E-PTEN were respectively irradiated with UVB radiation in a dose-gradient manner,and the effects of UVB on the nucleoplasm distribution of WT-PTEN and K13E-PTEN were compared.3.The different groups of U87 cells including control cells,overexpressed WT-PTEN andK13E-PTEN cells,and empty vector control cells were irradiated respectively with UV radiation in a dose-gradient manner.The viability of the different groups of cells was detected by MTT assay,and the Western blot was used to detect the expression of caspase-3/cleaved-caspase-3 in order to explore the effect of UVB on PTEN nuclear export/import and its function.Result1.The survive rate of cells was high after the UVB irradiation within the dose of 120mj/cm~2,indicating that the cells were resistant to a low dose of UVB;while when the UVB irradiation dose exceeds120mj/cm~2,with the increasing of UV irradiation,the survive rate of cells gradually decreased.Compared to the cells in the non-irradiation group,the proliferative activity of the irradiation group cells was significantly inhibited with the prolonged culture period.2.We found that PTEN was present both in cell nucleus and cytoplasm,and mainly localized inthe nucleus for the U87 cells overexpressing WT-PTEN.While in U87 cells over-expressed K13E-PTEN,PTEN expression was absent in the nucleus and conversely,it was located almost exclusively in the cytoplasm.However,after ultraviolet irradiation,the expression of K13E-PTEN and WT-PTEN in the nucleus was gradually enhanced.Fluorescent images showed that the expression level of WT-PTEN was higher than K13E-PTEN in nuclear.3.Compared with the control group,the survival rate of U87 cells overexpressingWT-PTEN/K13E-PTEN was lower and the apoptosis was more obvious under the same dose of UVB treatment.Compared with U87 cells over-expressing K13E-PTEN,the survival rate of U87 cells over-expressing WT-PTEN was significantly decreased under the same dose of UV treatment,but there was no significant difference in the degree of apoptosis.ConclusionOur studies showed that radiation can induce PTEN and its mutants into the nucleus,and itsapproach of nuclear import may be active transport or passive diffusion into the nucleus.After PTEN entering the nucleus,it synergizes with ultraviolet irradiation in causing the genomic damage and further promotes apoptosis.