The Role Of P53 In Radiation Induced Bystander Effect And Its Molecular Mechanism

Radiation induced bystander effect (RIBE) is the phenomenon that nonirradiated cells can respond to the signals released from nearby irradiated cells and then generate biological effects. The tumor suppressor p53 plays a crucial role in cellular response to DNA damage caused by ionizing radiation but it is still unclear whether p53 can modulate RIBE. In the present work, three different hepatoma cell lines, namely HepG2 (wild p53), PLC/PRF/5 (mutation p53), and Hep3B (p53 null) were irradiated with y-rays and then co-cultured with normal Chang liver cell (wild p53) in order to elucidate the mechanisms of RIBE. Results showed that only the irradiated HepG2 cells, rather than irradiated PLC/PRF/5 and Hep3B cells, could induce bystander effect of micronuclei (MN) formation in the neighboring Chang liver cells. When HepG2 cells were treated with 20?M pifithrin-a, an inhibitor of p53 function, or 5 ?M cyclosporin A (CsA), an inhibitor of cytochrome-c (cyt-c) release from mitochondria, the MN induction in bystander Chang liver cells was diminished. In fact, it was found that after irradiation cyt-c was released from mitochondria into the cytoplasm only in HepG2 cells in a p53-dependent manner but not in PLC/PRF/5 and Hep3B cells. Interestingly, when 50 ?M exogenous cyt-c was added into cell co-culture medium, RIBE was significantly triggered by the irradiated PLC/PRF/5 and Hep3B cells which previously failed to provoke a bystander effect. In addition, this exogenous cyt-c also partly recovered the RIBE induced by irradiated HepG2 cells under CsA treatment. Our results provide new evidence that the RIBE can be modulated by the p53 status of irradiated hepatoma cells and a p53-dependent release of cyt-c was involved in the RIBE. Meanwhile radiation-induced formation of intracellular nitric oxide (NO) was also reduced by CsA treatment in HepG2 cells. Both the depressed bystander effect and NO generation in the CsA-treated cells were reversed when 5 ?M cyt-c was in the presence of the medium during cell co-culture. But the cyt-c mediated overproductions of NO and bystander MN were abolished when the cells were pre-treated with s-methylisothiourea sulfate (SMT), an inhibitor of inducible nitric oxide synthase [1]. Moreover, intracellular reactive oxygen species (ROS) were also detected in the irradiated cells, but it was not varied after cyt-c treatment and hence was not a downstream signal of cyt-c. These data suggest that radiation induced cyt-c plays a profound role in regulating bystander response through iNOS triggered NO signal but not ROS.Recent evidence has demonstrated that, in addition to the therapeutic effect, irradiation could promote invasiveness of hepatocellular carcinoma cells. The irradiated cancer cells may also have impact on the invasion behavior of surrounding cells, which leads to the overall aggressive tumor progression. However, the roles and underlying mechanisms of p53 in the invasion of irradiated hepatoma cells and their nonirradiated bystanders have not been well investigated. In the present study, we found that the invasiveness of human hepatoma HepG2 cells could be increased by ?-ray irradiation. By pre-treating the cells with SU1498 (VEGFR2 inhibitor) and GM6001 (MMPs inhibitor), we demonstrated that this enhanced invasiveness was associated with the inter-activation between matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF) signaling, which was depressed by PFT-? (p53 inhibitor) or p53 siRNA. It was also found that the invasiveness of the co-cultured bystander cells was also elevated, which was properly related to the paracrine MMP-2 and VEGF through the irradiated cells in a p53 related pathway. Therefore, our data indicate that radiation induced upregulation of p53 is responsible for the promotion of VEGF-MMP-2 signaling pathway in both irradiated and bystander cells and hence is involved in the enhancement of hepatoma cell invasion.High LET radiation has several potential advantages over gamma-rays, one of which is that its effects may be independent of cellular p53 gene status. The purpose of this project is to investigate the novel bystander signals including mitochondrial malignancy and ROS generation by high LET-irradiated lymphocytes with different p53, namely TK6 (wild p53) and HMy2.CIR (HMy, mutation p53), and the following effects on bystander hepatocyte HL-7702, in order to to expound the role of p53 in LET-irradiation induced bystander effect. The results showed that after ?-ray irradiation, mitochondrial malignancy, P66 (Ser36) activation, and intracellular ROS were induced in TK6 cells but not in HMy cells, resulting in that a bystander effect on HL-7702 cells was only triggered by irradiated TK6 cells but not by irradiated HMy cells i.e., RIBE was in a p53 dependent manner with respect to ?-ray irradiation. But the heavy ion irradiation could induce a very remarkable mitochondrial malignancy, P66 activation and intracellular ROS generation in both TK6 and HMy cells and hence induced the bystander effect on HL-7702 cells. PFT-?, an inhibitor of p53, can only partly inhibit the ROS generation and bystander effect induced by 30 keV/?m carbon-irradiated TK6 cells but failed to suppress the bystander effect induced by either 70 keV/?m carbon or 180 keV/?m iron-irradiated TK6 cells. The mitochondrial inhibitors rotenone and oligomycin can inhibit heavy ion induced ROS generation in TK6 and HMy cells and the bystander effects on HL-7702 cells. It was clearly demonstrated that the bysander effect induced by low LET radiation is p53-dependent but by high LET irradiation is independent of p53 gene, which may be due to a p53-independent ROS generation through mitochondrial malignancy.Ionizing radiation can induce biological effects in non-irradiated cells, meanwhile, the bystander cells could also rescue the irradiated cells through a feedback signal stress. In order to elucidate the nature of this reciprocal bystander effect, we examined the interaction between a-irradiated human macrophages U937 cells and their bystander hepatocyte HL-7702 cells using a cell co-culture system. The results showed that after 6 h co-culture, mitochondria depolarization corresponding to apoptosis was significantly induced in HL-7702 cells, meanwhile the formation of micronuclei in the irradiated U937 cells was markedly decreased compared to that without cell co-culture treatment. This reciprocal effect was not observed when the cell membrane signaling was blocked by filipin that could inhibit cAMP transmitting from bystander cells to irradiated cells. After treatment of cells with exogenous cAMP, forskolin (an activator of cAMP) or KH-7 (an inhibitor of cAMP), we confirmed that the cAMP released from the bystander cells could protect the irradiated cells from radiation damage but the bystander cells itself became apoptotic due to cAMP deficient. In addition, knockdown p53 by RNA interference in bystander HL-7702 cells failed to evoke bystander effect that could be aggravated by cAMP inhibition. After co-culturing with irradiated U937 cells, p53 was activated in HL-7702 cells in response to the bystander stress. When p53 in HL-7702 cells was knocked down, there was no bystander response of mitochondrial depolarization was induced. Meanwhile, the forskolin and KH-7 failed to regulate the bystander effect in p53 siRNA HL-7702 cells. In conclusion, this study provided that cAMP could be released from bystander HL-7702 cells and compensated to a-irradiated U937 cells through a membrane signaling pathway and this cAMP communication plays a profound role in regulating the reciprocal effects.In conclusion, this thesis found that p53 is involved in low LET radiation induced bystander effect through p53-dependent mtROS. But high LET heavy ion could induce a bystander effect direct through regulation of mtROS independent of p53. Moreover, as a transcription factor, p53 could regulate the genes and proteins associated with cancer invasion and and play an important role in promoting invasioness in both irradiated and bystander cells. In addition, p53 function in bystander cells is also necessary for the RIBE.