engleski

The role of ΔNp73α in DNA damage response in normal and tumor human cells

ΔNp73α represents a potentially oncogenic isoform of p73 protein. Its overexpression has been detected in various human tumors often correlating with increased chemoresistance and worse prognosis. Exploring its tumorigenic properties upon ΔNp73α overexpression revealed cell-type and condition-dependent roles. Our aim is to investigate the effects of ΔNp73α overexpression on cycle progression of normal and tumor cells and expression of different cell cycle regulators after DNA damage. Normal human fibroblasts (NHF) and U2OS human osteosarcoma cell line were infected with retroviruses carrying ΔNp73α gene. Cells were treated with different doses of γ-irradiation (5 and 10 Gy) or topoisomerase II inhibitor ICRF-193, and harvested at different time points. The expression of proteins involved in cell cycle regulation and DNA damage response (p21, p27, p53, pRB, cA, cB1, Chk1, Chk2) was detected by Western blot. Distribution of cells in different cell cycle phases was measured by FACS analysis by determining the DNA content after propidium iodide staining. U2OS cells overexpressing ΔNp73α showed higher percentage of cells with 4N DNA content upon γ- irradiation compared to control cells, especially at longer time periods post-treatment (48 and 72h), and also increased number of cells with >4N DNA content (polyploidy). Significantly higher percentage of polyploid U2OS ΔNp73α overexpressing cells was also found after 48h of ICRF-193 treatment compared to control cells. The effect of ΔNp73α overexpression on cell-cycle distribution of NHF was weaker, possibly due to lower ectopic ΔNp73α levels compared to U2OS cells. Protein expression analysis of both cell types revealed various differences between controls and cells overexpressing ΔNp73α after both treatments. ΔNp73α isoform was shown to influence cell response to different genotoxic treatments in U2OS, and to a lesser extent in NHF cells. Effects obtained in response to ICRF-193 and γ-irradiation suggest its potential role in G2/M DNA damage checkpoint, representing interesting basis for more detailed analysis of signalling pathways involved.

ΔNp73; DNA damage; cell cycle control

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