辐射损伤与细胞周期

辐射后的细胞可能发生恶性转化,可能死亡,另有一些细胞对辐射却产生适应性或抗性,最终长期存活下来。近年来研究发现,此差异与辐射对细胞周期的影响密切相关。辐射可阻断细胞周期活动及延长细胞周期,其中G1期、S期和G2/M期等细胞周期检查点(checkpoint)起决定作用,它们分别通过不同的信号途径对辐射所致的损伤进行调控,产生不同的辐射生物学效应。对该领域的深入研究不仅为进一步阐释辐射致癌提供一定的理论依据,而且为临床放疗增敏剂的研制提供新的思路。     

辐射损伤与细胞周期

辐射后的细胞可能发生恶性转化，可能死亡，另有一些细胞对辐射却产生适应性或抗性，最终长期存活下来。近年来研究发现，此差异与辐射对细胞周期的影响密切相关。辐射可阻断细胞周期活动及延长细胞周期，其中G1期、S期和G2/M期等细胞周期检查点(checkpoint)起决定作用，它们分别通过不同的信号途径对辐射所致的损伤进行调控，产生不同的辐射生物学效应。对该领域的深入研究不仅为进一步阐释辐射致癌提供一定的理论依据，而且为临床放疗增敏剂的研制提供新的思路。     

p16负向调控通路与辐射诱导的G1期阻滞

辐射诱导细胞发生G1期阻滞,其分子调控机制尚不十分清楚.近期文献报道,独立于p53基因之外的p16-Cyclins-CDKs(细胞周期素依赖性激酶)细胞周期负向调控通路在紫外线和电离辐射照后发生改变,提示此通路可能在辐射诱导的G1期阻滞中发挥至关重要的作用.     

p16负向调控通路与辐射诱导的G1期阻滞

辐射诱导细胞发生G1期阻滞，其分子调控机制尚不十分清楚。近期献报道，独立于p53基因之外的p16-Cyclins-CDKs(细胞周期素依赖性激酶)细胞周期负向调控通路在紫外线和电离辐射照后发生改变，提示此通路可能在辐射诱导的G1期阻滞中发挥至关重要的作用。     

细胞周期和细胞凋亡的改变在X线辐射损伤后皮肤愈合延迟中的作用

目的：探讨细胞周期紊乱和凋亡在X线辐射损伤后创伤愈合延迟中的作用。方法：选用局部软X线辐射损伤合并创伤大鼠模型，应用病理形态观察、流式细胞术等方法观察不同时相点创面组织的病理改变、细胞周期及细胞凋亡的变化。结果：组织病理观察表明，肉芽组织的结构不规则，胶原组织排列紊乱，胶原合成减少，创伤愈合延迟。在伤后3d--9d时间段内，辐射侧G0／G1期细胞增加，S期和G2／M期细胞减少，表明软X线局部照射后细胞发生了严重的G1期阻滞。在伤后13d--22d时间段内，辐射侧S期细胞逐渐增加，6d时达到峰值，大量的细胞停留在S期，S期显著延长。在3d--9d时间段内，辐射侧伤口组织细胞凋亡增加，6d时达到峰值，之后逐渐减少。在创伤愈合的整个过程中，辐射侧的细胞凋亡呈由低到高再到低的时相变化。对照例伤口组织细胞凋亡百分数在整个愈合过程中变化并不显著。结论：提示细胞周期紊乱和细胞凋亡增加可能是导致愈合延迟的重要机制。     

HIV-1 Tat 蛋白对细胞周期相关基因表达及辐射细胞周期阻滞的影响

目的：探讨HIV-1 Tat蛋白对细胞周期相关基因表达以及电离辐射诱发细胞周期阻滞的影响.方法：使用包含102个与DNA损伤修复和细胞周期相关的基因微阵列检测人横纹肌肉瘤细胞（TE671）及已转染tat基因的TE671细胞（TT2）基因表达谱的改变;使用流式细胞仪检测细胞周期变化;Western印迹检测蛋白表达变化.结果：在基因芯片的检测中发现,与DNA损伤修复及细胞周期调控相关的6个基因Cdc25C,KIF2C,Cdc20,DNA-PKcs,CTS1,Wee1在转染tat基因的细胞中表达下调;细胞周期检测发现TE671细胞和TT2细胞经4 Gy γ射线照射后表现出不同程度的G2/M期阻滞,但表达Tat的TT2细胞G2/M阻滞出现较TE671细胞晚,而在照射后48 h时TE671细胞G2/M期阻滞已恢复,但TT2细胞阻滞仍很显著.另外,TT2细胞S期阻滞时间延长.研究进一步发现细胞周期蛋白（cyclin）B1在TT2细胞中表达增强.结论：HIV-1Tat蛋白导致G2/M检验点功能紊乱,将影响细胞的辐射敏感性,本研究为了解AIDS合并肿瘤患者对放射治疗敏感性提供了重要实验数据.     

电离辐射对不同肿瘤细胞细胞周期的影响

目的 研究电离辐射对不同肿瘤细胞细胞周期的影响为肿瘤放疗及化疗提供科学依据。方法 处于细胞周期各时相的细胞百分数采用流式细胞术进行检测。结果 研究表明：电离辐射作用后,ＨｅｌａＳ３和Ｓ１８０细胞发生了Ｓ和Ｇ２期阻滞,而ＤＬ－４细胞则发生Ｇ１和Ｇ２期阻滞,Ｂ１６各时相细胞数无列出较高的辐射抗性。结论 电离辐射作用后,不同肿瘤细胞的辐射抗性、即辐射敏感性有较大差异。其细胞周期的变化规律亦不相同。     

三羟异黄酮对正常和辐射损伤小鼠骨髓造血细胞周期的影响

目的 三羟异黄酮（genistein,GEN）对正常和辐射损伤小鼠骨髓造血细胞（bone marrow hematopoietic cell,BMHC）的细胞周期、增殖能力及bc1-2基因表达的影响,以期阐明其防护放射性造血损伤的分子机制.方法 照射前24h,GEN以160 mg/kg体重剂量给予小鼠灌胃,流式细胞仪观察BMHCs细胞周期及增殖能力变化,RT-PCR及Western blot方法分析BMHCs bcl-2 mRNA和蛋白表达情况.结果 ①GEN可诱导正常小鼠BMHCs细胞周期一过性改变,即给药后1 d,BMHCs增殖抑制,大量细胞阻滞于G0/G1期;给药后2 d,GEN诱导BMHCs由G0/G1期向S期转换,S期细胞明显增多;4d后逐渐恢复正常.②照射前24 h给药,GEN可减少射线导致的BMHCs增殖抑制,使照后阻滞于G0/G1期细胞减少;S期和G2/M期细胞增多,细胞增殖能力较强.同时,GEN预处理组bcl-2 mRNA及蛋白的表达均较高.结论 改变BMHCs细胞周期、降低BMHCs的辐射敏感性、抑制BMHCs凋亡和提高残留BMHCs的增殖分化能力可能是GEN防护放射线造血损伤的分子机制之一.     

骨髓基质屏蔽白血病细胞抵抗药物诱导凋亡

目的:探索骨髓基质细胞抑制化疗药物诱导白血病细胞凋亡的可能机制.方法:体外分离培养骨髓基质细胞,与Jurkat细胞共培养.流式细胞仪检测DNR诱导Jurkat 细胞凋亡率和细胞周期分布.结果:共培养后骨髓基质细胞抑制DNR诱导的白血病细胞凋亡,而白血病骨髓基质保护效应强于正常骨髓基质细胞.共培养后Jurkat细胞出现G0/G1期阻滞.结论:G0/G1期阻滞是骨髓基质细胞保护白血病细胞的机制之一,但可能存在较细胞周期阻滞更复杂的机制.     

电离辐射诱导G2期阻滞的机制

电离辐射损伤后,细胞通过若干关卡来调控细胞周期的进程,使细胞有时间进行DNA修复,确保染色体组的完整性和遗传稳定性,减少突变的发生。不同的电离辐射使不同细胞产生G1、G2和S期等不同时相的变化,但电离辐射后G2期阻滞的现象十分普遍。近年来,对G2期阻滞机制的研究多集中在Chk1、Chk2和p53上。     

ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia.

PURPOSE: To provide an update on the product of the ATM gene mutated in the human genetic disorder ataxia-telangiectasia (A-T). SUMMARY: The product of the ATM gene mutated in the human genetic disorder A-T is a 350 kDa protein that plays a central role in the regulation of a number of cellular processes. It is a member of the phosphatidylinositol 3-kinase superfamily, but is more likely a protein kinase similar to another member of that family, i.e. DNA-dependent protein kinase (DNA-PK). A-T cells and fibroblasts derived from the atm -/- mouse are hypersensitive to ionizing radiation and defective in cell cycle checkpoint control. At present the nature of the lesion in damaged DNA recognized by ATM remains uncertain, but it is evident that a small number of residual strand breaks remain unrepaired in A-T cells, which may well account for the radiosensitivity. On the other hand, considerable progress has been achieved in delineating the role of ATM in cell cycle checkpoint control. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation. At the G1 /S interface ATM has been shown to play a central role in radiation-induced activation of the tumour suppressor gene product p53. ATM binds to p53 in a complex fashion and activates the molecule in response to breaks in DNA by phosphorylating it at serine 15 close to the N-terminus and by controlling other phosphorylation and dephosphorylation changes on the molecule. This in turn leads to the induction of p21/WAF1 and other p53 effector proteins before inhibition of cyclin-dependent kinase activity and G1 arrest. Emerging evidence supports a direct role for ATM at other cell cycle checkpoints. Other proteins interacting with ATM include c-Abl a protein tyrosine kinase, beta-adaptin an endosomal protein and p21 a downstream effector of p53. The significance of these interactions is currently being investigated. ATM also plays an important role in the regulation and surveillance of meiotic progression. The localization of ATM to both the nucleus and other subcellular organelles implicates this molecule in a myriad of cellular processes. CONCLUSION: ATM is involved in DNA damage recognition and cell cycle control in response to ionizing radiation damage. There is evidence that ATM may also have a more general signalling role.     

Review: ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia

Purpose: To provide an update on the product of the ATM gene mutated in the human genetic disorder ataxia-telangiectasia (A-T). Summary : The product of the ATM gene mutated in the human genetic disorder A-T is a 350kDa protein that plays a central role in the regulation of a number of cellular processes. It is a member of the phosphatidylinositol 3-kinase superfamily, but is more likely a protein kinase similar to another member of that family, i.e. DNA-dependent protein kinase (DNA-PK). A-T cells and fibroblasts derived from the atm /- mouse are hypersensitive to ionizing radiation and defective in cell cycle checkpoint control. At present the nature of the lesion in damaged DNA recognized by ATM remains uncertain, but it is evident that a small number of residual strand breaks remain unrepaired in A-T cells, which may well account for the radiosensitivity. On the other hand, considerable progress has been achieved in delineating the role of ATM in cell cycle checkpoint control. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation. At the G1/S interface ATM has been shown to play a central role in radiation-induced activation of the tumour suppressor gene product p53. ATM binds to p53 in a complex fashion and activates the molecule in response to breaks in DNA by phosphorylating it at serine 15 close to the N-terminus and by controlling other phosphorylation and dephosphorylation changes on the molecule. This in turn leads to the induction of p21/WAF1 and other p53 effector proteins before inhibition of cyclin-dependent kinase activity and G1 arrest. Emerging evidence supports a direct role for ATM at other cell cycle checkpoints. Other proteins interacting with ATM include c-Abl a protein tyrosine kinase, beta -adaptin an endosomal protein and p21 a downstream effector of p53. The significance of these interactions is currently being investigated. ATM also plays an important role in the regulation and surveillance of meiotic progression. The localization of ATM to both the nucleus and other subcellular organelles implicates this molecule in a myriad of cellular processes. Conclusion: ATM is involved in DNA damage recognition and cell cycle control in response to ionizing radiation damage. There is evidence that ATM may also have a more general signalling role.     

Lack of correlation between G1 arrest and radiation age-response in three synchronized human tumour cell lines

PURPOSE: To determine radiosensitivity as a function of cell age (the age-response) in three human tumour cell lines, and investigate the dependence of the age-response on G1 arrest and on cell-age heterogeneity in synchronized cell populations. MATERIALS AND METHODS: Variation in radiosensitivity throughout the cell cycle and G1 arrest was measured in mitotically selected populations of synchronized human tumour cells. In order to examine the effects of desynchronization and cell age heterogeneity on the measured age-response, a mathematical model was developed based on an existing kinetic model of the cell cycle. The model was used to describe the age-response for mitotically selected populations of cells, which was then compared with experimentally measured age responses. RESULTS: Three different human tumour cell lines had qualitatively similar age-responses, with periods of radiosensitivity in mitosis and in late G1 phase/early S phase, and periods of radioresistance in early/mid G1 phase and late S/G2 phase. Radiosensitivity appeared to increase in G1 phase before the onset of DNA synthesis. One of the cell lines displayed a prolonged G1 arrest after irradiation in G1 phase. Model results demonstrated that the measured age-responses were consistent with a simple model in which the cell cycle was divided into four regions. Radiosensitivity was assumed to be constant within each region, and changed abruptly at the borders between regions. CONCLUSIONS: Human tumour cell lines can exhibit qualitatively similar age-responses despite having markedly different G1 checkpoint responses. This suggests that modulation of the G1 arrest response may not prove to be a useful clinical strategy because it may not lead to significant cell age specific changes in radiosensitivity. The mathematical model of the radiation response of mitotically selected synchronized cells was a useful way to quantitatively describe cell age heterogeneity in these populations, and demonstrated the important impact of this heterogeneity on measured age-responses.     

Lack of correlation between G1 arrest and radiation age-response in three synchronized human tumour cell lines

Purpose: To determine radiosensitivity as a function of cell age (the age-response) in three human tumour cell lines, and investigate the dependence of the age-response on G1 arrest and on cell-age heterogeneity in synchronized cell populations. Materials and methods: Variation in radiosensitivity throughout the cell cycle and G1 arrest was measured in mitotically selected populations of synchronized human tumour cells. In order to examine the effects of desynchronization and cell age heterogeneity on the measured age-response, a mathematical model was developed based on an existing kinetic model of the cell cycle. The model was used to describe the age-response for mitotically selected populations of cells, which was then compared with experimentally measured age-responses. Results: Three different human tumour cell lines had qualitatively similar age-responses, with periods of radiosensitivity in mitosis and in late G1 phase/early S phase, and periods of radioresistance in early/mid G1 phase and late S/G2 phase. Radiosensitivity appeared to increase in G1 phase before the onset of DNA synthesis. One of the cell lines displayed a prolonged G1 arrest after irradiation in G1 phase. Model results demonstrated that the measured age-responses were consistent with a simple model in which the cell cycle was divided into four regions. Radiosensitivity was assumed to be constant within each region, and changed abruptly at the borders between regions. Conclusions: Human tumour cell lines can exhibit qualitatively similar age-responses despite having markedly different G1 checkpoint responses. This suggests that modulation of the G1 arrest response may not prove to be a useful clinical strategy because it may not lead to significant cell age specific changes in radiosensitivity. The mathematical model of the radiation response of mitotically selected synchronized cells was a useful way to quantitatively describe cell age heterogeneity in these populations, and demonstrated the important impact of this heterogeneity on measured age-responses.     

γ射线对培养的兔血管平滑肌细胞的抑制作用

目的 观察体外培养的兔动脉平滑肌细胞(SMCs）对7射线照射的剂量效应关系，探讨电离辐射抑制SMCs增殖的细胞生物学机理。方法 静止期SMCs分别给予γ射线2．5，5，10，20及30Gy一次性照射后继续培养4，24或72h。以^3H—TdR掺入法、流式细胞术分析、微核实验和电镜观察γ射线对SMCs的DNA合成、增殖周期和损伤的影响。结果 在2．5Gy以上剂量γ射线一次性照射时，SMCs增殖明显抑制，细胞G0／G1期阻滞，均呈剂量依赖性。2．5Gy以下剂量γ射线照射后24h左右可见细胞凋亡增加，2．5Gy以上剂量7射线照射可使SMCs发生坏死。结论 电离辐射能抑制SMCs增殖，使细胞产生G0／G1期阻滞，并呈剂量依赖关系。SMCs死亡方式与受照射剂量有关。     

Low-dose hyper-radiosensitivity in human hepatocellular HepG2 cells is associated with Cdc25C-mediated G2/M cell cycle checkpoint control

Purpose: Although the significance of cell cycle checkpoints in overcoming low-dose hyper-radiosensitivity (HRS) has been proposed, the underlying mechanism of HRS in human hepatocellular cells remains unclear. Therefore, the aim of this study was to characterize HRS inhuman hepatocellular HepG2 cells and to explore the molecular mechanism(s) mediating this response.

Materials and methods: HepG2 cells were exposed to various single doses of γ radiation (from 0?Gy to 4?Gy), and then were assayed at subsequent time-points. Survival curves were then generated using a linear-quadratic (LQ) equation and a modified induced repair model (MIRM). The percentage of cells in the G1, G2/M, and S phases of the cell cycle were also examined using propidium iodide (PI) staining and flow cytometry. Levels of total cell division cyclin 25C (Cdc25C) and phosphorylated Cdc25C were examined by Western blotting.

Results: Low-dose γ radiation (<0.3?Gy) induced HRS in HepG2 cells, while doses of 0.3, 0.5, and 2.0?Gy γ radiation significantly arrested HepG2 cells in the G2/M phase. While total Cdc25C levels remained unchanged after irradiation, levels of phosphorylated Cdc25C markedly increased 6, 16, and 24?h after treatment with 0.5 or 2.0?Gy radiation, and they peaked after 16?h. The latter observation is consistent with the G2/M arrest that was detected following irradiation.

Conclusions: These findings indicate that low-dose HRS in HepG2 cells may be associated with Cdc25C-mediated G2/M cell cycle checkpoint control.     

Molecular mechanism of cell death by radiation

p53 protein, a tumor suppressor protein, is accumulated and activated by ionizing radiation. It activates various downstream genes whose functions are involved in cell cycle arrest, apoptosis, and DNA repair. Although it was thought generally that G1 arrest by p53 activation after ionizing radiation was a transient phenomenon to facilitate DNA repair, we found that it is irreversible and permanent in both normal human cells and tumor cells. Because cells arrested irreversibly express various phenotypes, such as cell enlargement and expression of senescence associated-beta-gal, this is related to cellular senescence, but not to apoptosis. Therefore, we termed this phenomenon senescence-like growth arrest (SLGA). These results indicate that SLGA is the main form of cell death caused by ionizing radiation. SLGA can be utilized as an index of cancer therapy, because it is induced not only by radiation but also by anticancer drugs and is easy to examine by vital staining, thereby making the induction of SA-beta-gal an index.