Roles Of P53and ROS In The Bystander Responses Induced By α-particle Irradiatied Hepatoma Cells

p53is a hot spot in the studies of tumor etiology and radiobiology, but it is still largely unknown in the influence of p53-reguated energy metabolism on radiation effects. Understanding of p53-regulated energy metabolism has significant roles in the investigations of tumor radiotherapy efficacy, radiation damage, carcinogenesis and even molecular epidemiology. Recently, it has been suggested that radiation induced bystander effect has important implications for the efficiency of radiotherapy but the underlying mechanism of cellular metabolism is widely unknown. The roles of SCO2(synthesis of cytochrome c oxidase2), a key effector for respiratory chain, and related signaling factor in α-particle induced bystander damage were investigated in a liver cell system.Radiation therapy has a very important role in the treatment of tumors. Our previous studies have shown that p53could regulate RIBE though a cytochrome c related pathway. To understand more detail roles of p53and its downstream signaling molecules in RIBE of hepatoma cells will be benefit to tumor therapy and provides better clinical guidance for radiation oncology treatment plan.Part I:Roles of p53and ROS in the bystander responses induced by irradiated hepatoma cells.Objective:To investigate the relationship among p53, ROS, and a-particle induced bystander effects within a liver cell system.Methods:Human hepatoma cells of HepG2with wild type p53(wtp53) and Hep3B (p53null) were irradiated with40cGy of a-particles, then were co-cultured with non-irradiated normal liver cells HL-7702for6h, then the incidence of micronucleus (MN) in the bystander HL-7702cells was analyzed. The expressions of total P53, phospho-P53(p-P53), SCO2and ROS in irradiated hepatoma cells were detected by western blot, PCR, enzyme activity and fluorescent probe DCFH-DA, respectively. In some experiments, the hepatoma cells were respectively treated p53inhibitor of PFT-a, SCO2inhibitor of NaHS, ROS scavenger of DMSO, p53siRNA, SCO2siRNA before irradiation.Results:Bystander damage of MN induction in HL-7702cells was observed when the HL-7702cells were co-culutred with40cGy oc-irradiated HepG2cells for6h, but no RIBE was observed when the HL-7702cells were co-cultured with a-irradiated Hep3B cells. The RIBE on HL-7702cells was abolished when the irradiated HepG2cells were pretreated with PFT-a, NaHS, DMSO or transfected with p53siRNA and SCO2siRNA. Meanwhile, p-P53protein, mRNA and the activity of SCO2, intracellular ROS were all increased in the irradiated HepG2cells but not in Hep3B cells and these expressions were eliminated by p53siRNA treatment. Moreover, irradiation-enhanced expressions of SCO2and ROS were also inhibited by SCO2siRNA in HepG2cells.Conclusions:Bystander effect can be induced by the irradiated HepG2cells with wild-type p53but notHep3B cells deficient of p53, and this p53-dependent RIBE has close relationship with irradiation enhanced SCO2and ROS. Threfore, a-particle induced bystander effect of hepatoma cells could be regulated through a p53-SCO2-ROS pathway. Part Ⅱ:Roles of p53in the RIBE of a multicellular systemObjective:To study the influence of p53on the RIBE within a cell co-culture system containing multi cell lines.Methods:Based on the above cell co-culture system with hepatoma cells and normal hepatic cells, adition suspension cells with different p53status were added into the co-culture system. These suspension cells included macrophage human U937cells (p53null), human lymphoblastoid cells HMy2.CIR (mutant p53) and human lymphoblastoid cells TK6(p53wild-type). In some experiments, TK6cells were treated PFT-a (an inhibitor of p53transcriptional functions) or PFT-μ,(an inhibitor of p53mitochondrial pathway), respectively. HepG2cells were irradiated with40cGy a-particles, then the bystander damage of MN induction in HL-7702cells were measured after co-culturing with the irradiated HepG2cells with or without addition of suspension cells in the co-culture system. ROS of irradiated HepG2cells were determined with flow cytometry.Results:Bystander MN in HL-7702cells was induced by the irradiated HepG2cells.When the suspension cells of U937or HMy were contained in the co-cultured system, the RIBE on HL-7702cells were significantly descreaed. But this RIBE was not influced when TK6cells were added into the co-culture system. Interestingly, when the TK6cells were treated with10μM PFT-μ. and then applied for multiply cell co-culture, the RIBE was not influenced, but when TK6cells were treated with20μM PFT-α, the MN induction in bystander HL-7702cells was reduced significantly. In addition, the ROS levels in the irradiated HepG2cells could be descreased by the suspension cells of U937, HMy, and TK6with PFT-α treatment, but it was not influenced by the suspension TK6cells with and without PTF-μ treatment.Conclusions:In the co-culture system containing multi-cell lines, as an middle mediator, the suspension cells with abnormal function of p53could influence the bystander responses induced by α-irradiated hepatoma cells by adjusting ROS generation in the irradiated cells.