Cdc25c在人肿瘤细胞低剂量辐射超敏感性发生机制中作用的研究
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摘要
【目的】研究Cdc25c在人肿瘤细胞低剂量辐射超敏感性发生机制中的作用。
【方法】流式细胞术分选计数后,再用克隆形成分析法分析人结肠癌细胞HT29和人宫颈癌细胞Siha在不同剂量辐射后的细胞存活情况,应用诱导修复模型对细胞存活分数进行拟合,验证是否存在HRS/IRR现象;流式细胞术检测人结肠癌细胞HT29和人宫颈癌细胞Siha在不同照射剂量点和时间点下的细胞周期分布情况;免疫印迹法检测人结肠癌细胞HT29和人宫颈癌细胞Siha接受不同剂量照射后磷酸化Cdc25c表达情况。
【结果】(1)人结肠癌HT29细胞存在HRS/IRR现象,人宫颈癌Siha细胞不存在HRS/IRR现象。(2)当辐射剂量<0.3 Gy时HT29细胞未发生G2/M期阻滞,Siha细胞出现了G2/M期的阻滞;当辐射剂量≥0.3 Gy时,两种细胞株均存在G2/M期的阻滞,且阻滞程度随照射剂量增加而增加。(3)在照射剂量<0.3 Gy时HT29细胞中磷酸化的Cdc25c不表达,而Siha细胞中可见磷酸化的Cdc25c表达;当照射剂量≥0.3 Gy时两种细胞株中均可见磷酸化的Cdc25c表达,表达活性与辐射剂量呈正相关。
【结论】人肿瘤细胞HRS/IRR现象的发生与G2/M期阻滞有关,检查点效应因子Cdc25c在G2/M期阻滞和人肿瘤细胞HRS/IRR的发生中具有重要作用。
Objective To study the effect of cdc25c and cell cycle checkpoint on HRS/IRR and the possible mechanism of HRS/IRR in tumor cells.
Methods Exponentially growing HT29 cells and Siha cells were irradiated with X-rays at different doses. Cell surviving fractions after irradiating with different radiation doses were measured by the clonogenic survival assay depend on (?)uorescence-activated cell sorter. Cell cycle distribution after irradiating was examined with flow cytometry. The level of Phospho-Cdc25c(216) in cells was detected by Western blot after irradiating.
Result HRS/IRR was observed in HT29 cells after low dose irradiating and not observed in Siha cells. The cell cycle distribution of HT29 cells had no change at dose below 0.3Gy. When the radiation dose was beyond 0.3Gy, G2/M phase arrest was observed in HT29 cells. Nevertheless, G2/M phase arrest was observed in Siha cells despite of radiation dose. At the dose less than 0.3Gy, the expression of Phospho- Cdc25C(ser216) was not observed in HT29 cells, but observed in Siha cells. When the dose is greater than 0.3Gy, Phospho-Cdc25c (ser216) was observed both in HT29 cells and in Siha cells.
Conclusions The activity of Cdc25c may play an important role in G2/M phase arrest and HRS/IRR.
【方法】流式细胞术分选计数后,再用克隆形成分析法分析人结肠癌细胞HT29和人宫颈癌细胞Siha在不同剂量辐射后的细胞存活情况,应用诱导修复模型对细胞存活分数进行拟合,验证是否存在HRS/IRR现象;流式细胞术检测人结肠癌细胞HT29和人宫颈癌细胞Siha在不同照射剂量点和时间点下的细胞周期分布情况;免疫印迹法检测人结肠癌细胞HT29和人宫颈癌细胞Siha接受不同剂量照射后磷酸化Cdc25c表达情况。
【结果】(1)人结肠癌HT29细胞存在HRS/IRR现象,人宫颈癌Siha细胞不存在HRS/IRR现象。(2)当辐射剂量<0.3 Gy时HT29细胞未发生G2/M期阻滞,Siha细胞出现了G2/M期的阻滞;当辐射剂量≥0.3 Gy时,两种细胞株均存在G2/M期的阻滞,且阻滞程度随照射剂量增加而增加。(3)在照射剂量<0.3 Gy时HT29细胞中磷酸化的Cdc25c不表达,而Siha细胞中可见磷酸化的Cdc25c表达;当照射剂量≥0.3 Gy时两种细胞株中均可见磷酸化的Cdc25c表达,表达活性与辐射剂量呈正相关。
【结论】人肿瘤细胞HRS/IRR现象的发生与G2/M期阻滞有关,检查点效应因子Cdc25c在G2/M期阻滞和人肿瘤细胞HRS/IRR的发生中具有重要作用。
Objective To study the effect of cdc25c and cell cycle checkpoint on HRS/IRR and the possible mechanism of HRS/IRR in tumor cells.
Methods Exponentially growing HT29 cells and Siha cells were irradiated with X-rays at different doses. Cell surviving fractions after irradiating with different radiation doses were measured by the clonogenic survival assay depend on (?)uorescence-activated cell sorter. Cell cycle distribution after irradiating was examined with flow cytometry. The level of Phospho-Cdc25c(216) in cells was detected by Western blot after irradiating.
Result HRS/IRR was observed in HT29 cells after low dose irradiating and not observed in Siha cells. The cell cycle distribution of HT29 cells had no change at dose below 0.3Gy. When the radiation dose was beyond 0.3Gy, G2/M phase arrest was observed in HT29 cells. Nevertheless, G2/M phase arrest was observed in Siha cells despite of radiation dose. At the dose less than 0.3Gy, the expression of Phospho- Cdc25C(ser216) was not observed in HT29 cells, but observed in Siha cells. When the dose is greater than 0.3Gy, Phospho-Cdc25c (ser216) was observed both in HT29 cells and in Siha cells.
Conclusions The activity of Cdc25c may play an important role in G2/M phase arrest and HRS/IRR.
引文
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3. Joiner MC, Marples B, Lambin P, et al. Low-dose hypersensitivity: current status and possible mechanisms. Int J Radiat Oncol Biol Phys, 2001;49:379-389.
4. Marples B, Wouters BG, Joiner MC. An association between the radiation- induced arrest of G2-phase cells and low-dose hyper-radiosensitivity: a plausible underlying mechanism? Radiat Res, 2003;160:38-45.
5. Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science, 1996; 274:1664-1672.
6. Kastan MB, Onyekwere O, Sidransky D, et al. Participation of p53 protein in the cellular response to DNA damage. Cancer Res, 1991;51:6304-6311.
7. Sinclair WK. Cyclic x-ray responses in mammalian cells in vitro. Radiat Res, 1968;33:620-643.
8. Xu B, Kim ST, Lim DS, et al. Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol, 2002;22:1049-1059.
9. Krempler A, Deckbar D, Jeggo PA, et al. An imperfect G2M checkpoint contributes to chromosome instability following irradiation of S and G2 phase cells. Cell Cycle, 2007;6:1682-1686.
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2. Short SC, Woodcock M, Marples B, et al. Effects of cell cycle phase on low-dose hyper-radiosensitivity. Int J Radiat Biol, 2003;79:99-105.
3. Joiner MC, Marples B, Lambin P, et al. Low-dose hypersensitivity: current status and possible mechanisms. Int J Radiat Oncol Biol Phys, 2001;49:379-389.
4. Jessus C, Ozon R. Function and regulation of cdc25 protein phosphate through mitosis and meiosis. Prog Cell Cycle Res, 1995;1:215-228.
5.陈俊妮,程晶,吴红革,等.细胞周期调控在HT29结肠癌细胞低剂量辐射超敏感性中作用的研究.中华放射肿瘤学杂志, 2009;18:204-205.
6. Joiner MC, Johns H. Renal damage in the mouse: the response to very small doses per fraction, 1988;114:385-398.
7. Skov KA. Radioresponsiveness at low doses: hyper-radiosensitivity and increased radioresistance in mammalian cells. Mutat Res, 1999;430:241-253.
8. Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis, 2002;23:687-696.
9. Niida H, Nakanishi M. DNA damage checkpoints in mammals. Mutagenesis, 2006;21:3-9.
10. Williams RS, Williams JS, Tainer JA. Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem Cell Biol, 2007;85:509-520.
11. Mitchell J, Smith GC, Curtin NJ. Poly(ADP-Ribose) polymerase-1 and DNA- dependent protein kinase have equivalent roles in double strand break repair following ionizing radiation. Int J Radiat Oncol Biol Phys, 2009;75: 1520-1527.
12. Lavin MF, Kozlov S. DNA damage-induced signalling in ataxia-telangiectasia and related syndromes. Radiother Oncol, 2007;83:231-237.
13. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases.Genes Dev, 2001;15:2177-2196.
14. Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science, 1996; 274:1664-1672.
15. Kastan MB, Onyekwere O, Sidransky D, et al. Participation of p53 protein in the cellular response to DNA damage. Cancer Res, 1991;51:6304-6311.
16. Sinclair WK. Cyclic x-ray responses in mammalian cells in vitro. Radiat Res, 1968;33:620-643.
17. Xu B, Kim ST, Lim DS, et al. Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol, 2002;22:1049-1059.
18. Krempler A, Deckbar D, Jeggo PA, et al. An imperfect G2M checkpoint contributes to chromosome instability following irradiation of S and G2 phase cells. Cell Cycle, 2007;6:1682-1686.
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