Radiation-induced intercellular signaling mediated by cytochrome-c via a p53-dependent pathway in hepatoma cells

Abstract

The tumor suppressor p53 has a crucial role in cellular response to DNA damage caused by ionizing radiation, but it is still unclear whether p53 can modulate radiation-induced bystander effects (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 γ-rays and then co-cultured with normal Chang liver cell (wild p53) in order to elucidate the mechanisms of RIBE. Results showed that the radiosensitivity of HepG2 cells was higher than that of PLC/PRF/5 and Hep3B cells. Only irradiated HepG2 cells, rather than irradiated PLC/PRF/5 or Hep3B cells, could induce bystander effect of micronuclei (MN) formation in the neighboring Chang liver cells. When HepG2 cells were treated with 20 μM pifithrin-α, an inhibitor of p53 function, or 5 μM cyclosporin A (CsA), an inhibitor of cytochrome-c release from mitochondria, the MN induction in bystander Chang liver cells was diminished. In fact, it was found that after irradiation, cytochrome-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 μg/ml exogenous cytochrome-c was added into cell co-culture medium, RIBE was significantly triggered by irradiated PLC/PRF/5 and Hep3B cells, which previously failed to provoke a bystander effect. In addition, this exogenous cytochrome-c also partly recovered the RIBE induced by irradiated HepG2 cells even with CsA treatment. Our results provide new evidence that the RIBE can be modulated by the p53 status of irradiated hepatoma cells and that a p53-dependent release of cytochrome-c may be involved in the RIBE.

Figure 1

Dose responses of MN formation (a) and relative cell growth rate (b) in three human hepatoma cells (HepG2, PLC/PRF/5, Hep3B) irradiated with γ-rays. In some experiments, HepG2 and PLC/PRF/5 cells were treated with 20 μM PFT-α for 20 h before irradiation and during the subsequent cell culture period. Cell growth rate was measured on the fifth day after irradiation and was normalized to the sham-irradiated (0 Gy) cells.

Figure 2

MN formation in bystander Chang live cells that had been co-cultured with irradiated hepatoma cells. (a) Dose response of the yield of bystander MN in Chang live cells that were co-cultured with irradiated hepatoma cells for 12 h. **P<0.01, ***P<0.001 compared with the control without irradiation. (b) Time response of bystander MN in Chang live cells that were co-cultured with 3 Gy γ-irradiated hepatoma cells. *P<0.05, ***P<0.001 compared with the control without irradiation. Results correspond to the mean±s.e. of three independent experiments with three replicates in each case.

Figure 3

Effect of PFT-α and CsA on bystander MN induction in Chang live cells that had been co-cultured with 3 Gy γ-irradiated HepG2 cells for 12 h. ***P<0.001 compared with the control without irradiation or the indicated group with inhibitor treatment. Results correspond to the mean±s.e. of three independent experiments with three replicates in each case.

Figure 4

Western blot analysis of the expression of phospho-p53 (Ser15) and GAPDH in human hepatoma cells 12 h after 3 Gy γ-irradiation. In some experiments, HepG2 and PLC/PRF/5 cells were treated with 20 μm PFT-α 20 h before irradiation and during the subsequent cell culture period. (a) Immunoblots of phospho-p53 and GAPDH of the hepatoma cells under different conditions. (b) Relative level of phospho-p53 expression in the hepatoma cells. Values were normalized to GAPDH level in each sample, and then the ratio of each normalized value to its corresponding control (0 Gy) was calculated. ***P<0.001 compared with the control or PFT-α treated cells. All data were presented as the mean±s.e. for three experiments.

Figure 5

Release of cytochrome-c from three human hepatoma cells (HepG2, PLC/PRF/5, Hep3B) 12 h after 3 Gy γ-irradiation. In some experiments, HepG2 cells were treated with 20 μm PFT-α before and after irradiation or 1 h with 5 μM CsA before irradiation. (a) Cell image visualized by a fluorescence microscope. Chromatin was stained with 4′,6-diamidino-2-phenylindole and intracellular lipids were stained with Nile red. Immunolocalization of cytochrome-c (green), nuclear (blue) and cell outline morphology (red) were recorded and merged. The size bar in the photo is 20 μm. (b) The percentage of cytochrome-c-released cells in the population of HepG2 cells with and without PFT-α and CsA treatment, respectively. (c) The percentage of cytochrome-c released cells in the population of PLC/PRF/5 and Hep3B cells. *P<0.05 compared with the control without irradiation. ***P<0.001 to the control without irradiation or to the indicated group with inhibitor treatment. Results correspond to the mean±s.e. of three independent experiments with three replicates in each case. A full colour version of this figure is available at the Oncogene journal online.

Figure 6

Effect of exogenous cytochrome-c on bystander MN induction in the Chang live cells that had been co-cultured for 12 h with 3 Gy γ-irradiated PLC/PRF/5 cells (a), Hep3B cells (b) and HepG2 cells (c). **P<0.01, ***P<0.001 compared with the control without irradiation, *P<0.05, **P<0.01, ***P<0.001 compared with CsA-treated cells. Results correspond to the mean±s.e. of three independent experiments with three replicates in each case.