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. 2010 Mar 29;107(16):7461–7466. doi: 10.1073/pnas.1002459107

Fig. 4.

Fig. 4.

p53 and GLS2 regulate DNA oxidation and ROS-mediated apoptosis. (A) HCT116 cells were untreated (control) or treated with daunorubicin (Dauno.; 100 nM) for 24 h and then fixed and stained using anti–8-OH-dG antibody and anti-p53 polyclonal antibody (FL) and visualized using Alexa Fluor–488– and -594–conjugated secondary antibodies. Nuclei were counterstained with DAPI and images were taken using a Keyence microscope. (B) HCT116 cells were treated as in A. Intensity of 8-OH-dG (Left) and p53 (Right) staining was quantified using Keyence software. The average of six random visual fields from two independent experiments is shown, with error bars representing SD. (C) HCT116 cells expressing WT p53 (p53+/+) were transfected with indicated RNAi for 24 h and then cells were either not treated (untreated) or treated with daunorubicin (100 nM) for another 24 h. DNA oxidation was detected as in A and quantified as in B. (D) U2OS cells were transfected with luciferase RNAi or GLS2 RNAi and then treated with daunorubicin (100 nM) as in C. DNA oxidation was detected and quantified as in C. (E) HCT116 (p53+/+) or (p53−/−) cells were transfected with luciferase RNAi (Luci) or GLS2 RNAi for 24 h and then were either not treated (control) or treated with daunorubicin (300 nM) for another 36 h. The amount of sub–G0/G1 cells was calculated using the Cell Quest program for FACS. Average of three independent experiments is shown, with error bars indicating SD. (F) Model for regulation of intracellular ROS levels by GLS2. Upon oxidative stress or DNA damage, p53 is stabilized and activated to induce several targets including antioxidant and prooxidant genes. One such target, GLS2, catalyzes the hydrolysis of glutamine to produce glutamate and NH4+ and functions as an antioxidant protein. In response to severe cellular stress or irreparable damage p53 transactivates prooxidant genes (PUMA, PIG3, Proline Oxidase), resulting in the elevation of intracellular ROS, and apoptosis. The balance between anti- and prooxidant genes and the differential regulation of p53 targets can determine the choice of cellular outcomes.