ABSTRACT
Objective To investigate the radiobiological effects of radiation with different dose rates on human nasopharyngeal carcinoma cell line CNE-2 treated with or without a poly ADP-ribose polymerase (PARP) inhibitor,olaparib.Methods The concentration of olaparib used to treat cells equaled to the inhibition concentration IC10 of olaparib to CNE-2 cells.The CNE-2 cells were divided into acute radiotherapy (RT) group,fractionated radiotherapy (FRT) group,olaparib + RT group,and olaparib + FRT group.All groups were exposed to radiation of 0,1,2,3,5,7,and 10 Gy at a dose rate of 3 Gy/min.The delivery time for each dose point was 4 min in RT and 30 min in FRT.The colony forming assay was used to evaluate the survival of CNE-2 cells at each dose point.The multi-target,single-hit model was used to fit the cell survival curves and the parameters,D0,Dq,and SF2,were calculated.At dose points of 0.1,and 2 Gy,western blot was used to determine the expression of PARP-1 in the RT group and the FRT group and γH2AX in each group.Immunofluorescence was used to evaluate the γH2AX focus formation.A single factor analysis of variance was used to compare the 4 groups,and two two compared with SNK-q test.Results The IC10 value of olaparib to CNE-2 cells was 4.0 μmoL/L.At dose points of 1 and 2 Gy,the PARP-1 expression was significantly higher in the FRT group than in the RT group (P=0.029,0.022),while the γH2AX focus number was significantly smaller in the FRT group than in the other three groups (all P＜0.05);compared with the RT group,the D0,Dq,and SF2 values in the FRT group were increased by 11.67％,15.78％,and 23.61％,respectively;compared with the FRT group,the D0,Dq,and SF2 values in the Olaparib+ FRT group decreased by 11.19％,6.44％,and 13.26％,respectively;there were no significant differences in above indices between the RT group,the Olaparib+RT group,and the Olaparib+FRT group.Conclusions For the same radiation dose,fractionation reduces the relative dose rate and weakens the radiobiological effects.lowdose olaparib can compromise the single strand break repair induced by the decline of the relative dose rate in a fractionated irradiation mode,which promotes the formation of double-strand break and improves the radiobiological effects.
ABSTRACT
Objective To investigate the effects of radiation on the apoptosis rate and expression of 7 apoptosis-related genes in well-differentiated human nasopharyngeal carcinoma cell line CNE-1.Methods CNE-1 cells were cultured in vitro.The apoptosis rates of CNE-1 cells under 0-,2-,4-,6-,and 8-Gy radiation were measured by flow cytometry.The mRNA expression levels of Bcl-2,Bcl-xl,Bcl-w,Bax,Bak,Bad,and Bid were measured by RT-PCR.The Pearson test was used for analyzing the correlation of mRNA expression with apoptosis rate and radiation dose and the correlation between apoptosis rate and survival fraction.Results The early apoptosis rate of CNE-1 cells increased gradually as the radiation dose ranged from 0 to 6 Gy,but decreased when the radiation dose was 8 Gy; the late apoptosis rate of CNE-1 cells increased as the radiation dose ranged from 0 to 8 Gy.The mRNA expression of Bax was upregulated as CNE-1 cells were irradiated,and it was positively correlated with the early/late apoptosis rate and radiation dose (P =0.000 for all comparisons).The mRNA expression of Bcl-xl was downregulated,and it was negatively correlated with the early/late apoptosis rate (P =0.005 and 0.039) ; but it showed no correlation with radiation dose (P =0.369).The mRNA expression of Bcl-2 was upregulated and reached the peak level when the radiation dose was 4 Gy,and then it fell as the radiation dose increased.The mRNA expression of Bcl-w,Bcl-2,Bad,and Bid were not correlated with the early/late apoptosis rate (P =0.058 -0.894).There was no correlation between the apoptosis rate and survival fraction in CNE-1 cells (P =0.064).Conclusions Bax and Bcl-xl have some correlation with apoptosis rate in CNE-1 cells,but no correlation between the apoptosis rate and survival fraction was observed.
ABSTRACT
Objective To study the effect of E1A gene on the radiosensitivity of nasopharyngeal carcinoma (NPC) cells and its mechanism. Methods Ad-E1A gene was transfected into human NPC cells (CNE2), then the positive clones (CNE2-Ad-E1A) were identified by RT-PCR. CNE2 cells, CNE2 cells transfected with Ad-β-gal (CNE2-Ad-β-gal) and CNE2-Ad-E1A cells were irradiated with 0 Gy,2 Gy,4 Gy,6 Gy and 8 Gy respectively using 6 MV X-ray. Clone forming assays were carried out, cell survival curves were drawn and the sensitivity enhancing ratio (SER) was calculated. The redistributions of cell cy-cle were analyzed by flow cytometry. RT-PCR was used to detect the expression of wtp53. Results RT-PCR confirmed that E1A gene had been integTated into positively transfected cells and stably expressed. Cell survival curves showed that the SER of D0,Dq and SF_2 value was 1.37, 1.95 and 1.46 in CNE2-Ad-E1A cells. The D_0,D_q and SF_2 value was 1.57 Gy,1.82 Gy, 0.89 in CNE-2 cells and 1.53 Gy,1.78 Gy,0.82 in CNE2-Ad-β-gal cells, respectively. The G_2/M arrest was shown in CNE2-Ad-E1A cells. Moreover, the expression of wtp53 gene was markedly enhanced in Ad-E1A-CNE2 cells. Conclusions E1A gene can ef-fectively enhance the radiosensitivity of human NPC cells, which may be associated the enhancement of wt-p53 expression and G_2/M arrest.