Relationship Between P53 And Bystander Effect Induced By The Radiated Hepatocellular Carcinoma Cells

Primary liver cancer is one of the most common malignant tumors in China. Along with the development of imaging technique and the 3-dimensional conformal radiotherapy treatment(3DCRT),radiotherapy has become a potential and important clinical option on the advanced stage of hepatoma.The curative effect of radiotherapy is relative to the radiosensitivity of tumor. Since hepatoma carcinoma cells(HCC) have different status of p53,including wild-type p53,mutant-type p53 and null-type p53,they always have different radiosensitivity.Recently,a new phenomenon so called as bystander effect has been reported.Due to this effect,the normal liver organ could be influenced by its neighbor of irradiated hepatoma cells,which may raise new problems of radiotherapy. Therefore,to investigate the relationships between p53 and radiosensitivity,between p53 and the bystander effects induced by irradiated HCC,may help us to evaluate curative effect of radiotherapy and provide theoretical evidence on how to preferably utilize the bystander effect to decrease the radiation side-effect and improve the efficiency of radiotherapy.Objectives1.Radiosensitivity of HCC with different types of p53For this purpose,HepG2(wtp53),PLC(mtp53) and Hep3B(null-p53) cells were irradiated and then cell proliferation and micronuclei incidence were measured.2.Role of p53 in bystander effect induced by irradiated HCCTo investigate the relationship between p53 and bystander effects induced by irradiated hepatoma cells,the expressions of p53 in targeted cells and bystander cells were detected.Method1.Radiosensitivity of liver cells Normal Chang liver cells,HepG2,PLC and Hep3B hepatoma cells were exposed toγ-ray of 0,1,3,5 and 7Gy,then the proliferation and the incidence of micronuclei (MN) of these cells were examined by cytometry and cytokinetic-blocking technique, respectively.For some experiments,HepG2 and PLC cells were pretreated with p53 inhibitor of PFT-αat the concentration of 20μmol/L.2.Relationship between p53 and the bystander effect induced by the irradiated hepatoma cellsHepG2,PLC and Hep3B cells were exposed toγ-rays at 0,1,3,5 and 7Gy,then co-cultured for 12 hours with Chang liver cells or identical tumor cells without irradiation,then the MN induction in the bystander cells was assayed.In the situation of 3Gy irradiation,the co-culture time was adjusted to 4h,12h and 24h so that the time response of bystander MN could be obtained.Furthermore,the induction of p53 phosphorylated protein(P-p53) of both irradiated cells and bystander cells was assayed by the method of western blotting when the target tumor cells were irradiated with 1,3 and 5Gy and further co-cultured with Chang liver cells for 0,1,2,4,8,12 or 24h.The expression of p53 mRNA in bystander cells was also measured by the method of real-time PCR when the target tumor cells were irradiated with 3Gyγ-rays. In some of above experiments,HepG2 and PLC cells were pretreated with 20μmol/L PFT-αbefore irradiation and then co-cultured with bystander cells where 0.1%DMSO was applied as the control of PFT-α,and the expressions of P53 and P21 in bystander cells were detected to evaluate the inhibiting effect of PFT-α.Results1.Effects of p53 on radiosensitivity of liver cellsIt was found that the radiosensitivity of hepatoma cells and Chang liver cells was relative to the status of p53.The results of cell proliferation test manifested that Hep3B cells of null-p53 had the highest radiosensitivity,HepG2(wtp53) and the PLC (mtp53) cells had the secondary radiosensitivity.For high dose irradiation,Chang liver cells had radiosensitivity similar to HepG2.The results of MN measurement indicated that HepG2 cell had the highest incidence of MN among those cell lines, and the followed MN incidence was Chang liver cells and PLC cells,but MN were rarely produced in the irradiated Hep3B cells with null-p53.In addition,PFT-αtreatment decreased greatly radiation induced cellular damage on HepG2 and PLC cells where MN induction in HepG2 cells was influenced much more.2.Relationship between p53 and bystander effect induced by irradiated hepatoma cellsIt was found that the bystander effect was relative to the status of p53 of irradiated hepatoma cells.Bystander MN could be observed in the cells that had been co-cultured with HepG2 cells(wtp53) but not that had been co-cultured with either PLC(mtp53) or Hep3B(null-p53).When the hepatoma cells were treated with PFT-αbefore irradiation,the bystander effect was diminished.Results from western blotting assay showed that the expressions of P-p53 in both irradiated hepatoma cells and bystander cells were relative to radiation dose and cell co-culture time.When the dose was 3Gy,the expression of P-p53 approached to a peak level.At this dose,the level of P-p53 in irradiated HepG2 cells increased to a peak value at 4h post-irradiation,and the P-p53 in the bystander cells had the highest level in the time range of 2-8 hrs of cell co-culture.The result of real-time PCR showed that p53 mRNA in bystander cells was not influenced by the cell co-culture treatment,indicating that the change of P53 is the consequence of post-transcriptional modification.Conclusions1.Radiosensitivity of hepatoma cells is relative to p53.Hep3B cells of null-p53 have the highest radiosensitivity,and the radiosensitivity of HepG2 with wtp53 is higher than PLC cells with mtp53.2.Bystander effect can be induced by the irradiated hepatoma cells with wtp53 but not by the hepatoma cells of null-p53 or mtp53.3.The expressions of P-p53 in both irradiated HepG2 cells and bystander Chang liver cells are relative to the irradiation dose and cell co-culture time.4.p53 regulates not only the radiosensitivity but also the bystander response.