基因组不稳定性与错配修复机制

辐射可以诱导细胞基因组发生不稳定性改变，表现为一系列的延迟突变表型。错配修复机制作为一种重要的复制后修复机制，在维持基因组稳定方面发挥着重要的作用。它是一种保守的修复机制，不仅可以通过异二聚体的形式直接参与修复过程，也可以通过cdc2磷酸化途径对细胞周期进行间接调控。因此，探讨基因组不稳定性与错配修复之间的相互关系，可能是我们深入了解电离辐射的损伤与修复机制的重要内容。     

基因组不稳定性与辐射致癌的分子机理

简要论述辐射诱发基因组不稳定性的形成及癌变的基本过程，影响基因组不稳定性及癌变的主要分子机制，如ＤＮＡ修复机制、细胞周期进程调控机制、抑癌基因及原癌基因突变与基因组不稳定性形成及癌变的关系。     

基因组不稳定性与辐射致瘤的分子机理

简要论述辐射诱发基因组不稳定性的形成及癌变的基因过程，影响基因组不稳定性及癌变的主要分子机制，如ＤＮＡ修复机制、细胞周期进程调控机制、抑癌基因及原癌基因突变且不稳定性形成及癌变的关系。     

散发性结直肠癌微卫生不稳定性与hMSH3和hMSH6基因突变的研究

目的：探讨散发性结肠癌中微卫星不稳定性及其与错配修复基因hMSH3和hMSH6的关系。方法：应用放射性同位素为基础的聚合酶链式反应（PCR）技术检测了48例散发性结直肠癌中4个位点（D2S1233、BAT－26、D17S261、D17S799）的微卫星不稳定性和错配修复基因hMSH3和hMSH6的突变。结果：（1）散发性结直肠癌的hMSH3、hMSH6基因突变率分别为：10．4％、25％；（2）D2S123、BAT－26、D17S261、D17S799 4个位点的不稳定性检出率分别为12．5％、18．8％、10．4％、8．3％；（3）hMSH3和hMSH6基因突变和微卫星不稳定性相关显著（P＜0．01）。结论：（1）错配修复基因突变引起微卫星不稳定性是散发性结肠癌发生的重要机制；（2）部分微卫星不稳定性是由错配修复基因hMSH3和hMSH6突变引起，其余的微卫星不稳定性可能涉及到其它错配修复基因；（3）可通过单独检测BAT－26微卫星位点的不稳定性来确定RER阳性，这在肿瘤的防治中将是一种更加简单，准确的分子手段而应用于临床。     

α粒子辐射与基因组不稳定性

α粒子照射后除了引起机体本身的可见的变化如细胞死亡、增殖、癌变,其引起的遗传损伤效应也日益受到人们的注意。越来越多的研究表明:辐射可引起基因组不稳定性的过程,使受照射细胞的应答反应传递到子代细胞中,并表现出一系列遗传学变化。基因组不稳定性的机制目前还不甚清楚,可能与旁效应、自由基、DNA修复缺陷、端粒功能失调以及基因大片段缺失等有关。     

X射线修复交叉互补基因功能的研究进展

对X射线修复交叉互补(XRCC)基因功能的研究极大地促进了对哺乳动物DNA损伤修复过程和遗传不稳定性致癌机制的理解。通过观察XRCC基因突变体的表型,可以对其功能进行鉴定。目前已鉴定的这一基因家族的多数成员均参与几种重要的DNA修复途径,包括碱基切除修复、同源重组修复和非同源末端重接。XRCC基因的鉴定及其在DNA损伤修复和维持遗传稳定性过程中发挥重要的作用。     

辐射诱导的基因组不稳定性

辐射诱导的基因组不稳定性的特点是受照细胞后代发生遗传变化的频率增加。基因组不稳定性的生物终点包括核型异常、基因突变和基因扩增及延迟的细胞增殖性死亡等。保持遗传信息稳定的一些关键基因的损伤对基因组不稳定性的发生和传递起重要作用,遗传外因子也会影响基因组不稳定性的发生。辐射诱导的基因组不稳定性对辐射致癌具有重要意义。     

X射线修复交叉互补基因功能的研究进展

对X射线修复交叉互补(XRCC)基因功能的研究极大地促进了对哺乳动物DNA损伤修复过程和遗传不稳定性致癌机制的理解。通过观察XRCC基因突变体的表型，可以对其功能进行鉴定。目前已鉴定的这一基因家族的多数成员均参与几种重要的DNA修复途径，包括碱基切除修复、同源重组修复和非同源末端重接。XRCC基因的鉴定及其在DNA损伤修复和维持遗传稳定性过程中发挥重要的作用。     

DNA损伤修复与肿瘤烷化剂化疗

DNA损伤修复能够使肿瘤细胞耐受化疗药物造成的DNA损伤而存活,因此,调节某种特殊的DNA修复路径可以增强化疗药物的疗效。另外,有些DNA修复路径在一些肿瘤细胞中是失活的。目前,以DNA修复蛋白O^6甲基鸟嘌呤-DNA甲基转移酶（MGMT）和错配修复蛋白作为调节靶标,提高化疗效果的联合用药策略正在进行各期临床实验。靶向DNA修复机制增强抗肿瘤化疗药物的疗效、克服肿瘤耐药正成为肿瘤个体化化疗研究的一个重要领域。     

α粒子辐射与基因组不稳定性

α粒子照射后除了引起机体本身的可见的变化如细胞死亡、增殖、癌变，其引起的遗传损伤效应也日益受到人们的注意。越来越多的研究表明：辐射可引起基因组不稳定性的过程．使受照射细胞的应答反应传递到子代细胞中，并表现出一系列遗传学变化。基因组不稳定性的机制目前还不甚清楚，可能与旁效应、自由基、DNA修复缺陷、端粒功能失调以及基因大片段缺失等有关。     

胃癌DNA错配修复基因hMSH2突变研究

采用ＰＣＲ、聚丙烯酰胺电泳、单链构象多态性分析及银染技术,检测３０例胃癌组织ＤＮＡ微卫星不稳定（ＭＳＩ）及ｈＭＳＨ２基因突变情况。探讨错配修复（ＭＭＲ）基因与胃癌的关系及发生的分子机制。结果显示３０例胃癌中有８例表现ＭＳＩ;有５例６次检测到ｈＭＳＨ２基因突变,其中生殖细胞突变２次,体细胞突变４次。本研究结果认为,胃癌组织ＤＮＡ中存在ＭＳＩ和ＭＲＲ基因突变。部分胃癌的发生机制可能为ＭＭＲ基因缺陷导致     

泛素连接酶CUL4A-DDB1复合体参与DNA损伤修复的研究进展

泛素化修饰在DNA损伤信号中发挥重要功能,包括细胞周期监控、DNA修复、细胞衰老和程序性死亡的调控。CUL4A-DDB1泛素连接酶通过DCAFs靶向调控特异性的底物,启动DNA切除修复机制对受损DNA进行修复。近期的研究表明CUL4A-DDB1泛素连接酶协助DNA修复因子与受损DNA的识别,来维持基因组的稳定性和正确性。     

Coming of age: 'Dysgenetics' - a theory connecting induction of persistent delayed genomic instability with disturbed cellular ageing

Introduction : In recent years a new phenomenon has manifested itself: delayed, persistent genomic instability. When cells are treated with carcinogens not only direct induction of chromosome aberrations and mutations takes place, but there is also an indirect induction: in the distant progeny of treated cells persistently enhanced levels of new chromosome aberrations and enhanced mutation rates are found. This persistent enhanced genomic instability is not due to the presence of lesions in the DNA induced by the treatment because the response can be transmitted to untreated cells. Apparently it is caused by a persistent dysfunctioning of the cell as a whole. Due to these findings a new model for multistep carcinogenesis emerges. According to this model the initiation of carcinogenesis is the induction of a state of persistent genomic instability that not only is responsible for enhanced mutation rates of oncogenes and tumor suppressor genes, but also predisposes to immortalization. This view could lead to a radical change in our views on carcinogenesis. Therefore understanding the mechanism is of utmost importance. Purpose : Up to now, the mechanism responsible for this persistent delayed genomic instability remains completely elusive and has only been described as 'unknown'. In this review the phenomenon is connected with a recent theory on cellular ageing and immortalization. Conclusion : Although highly speculative this review provides a framework for further experimental approaches that will contribute to our understanding of delayed genomic instability and possibly even to a better understanding of cellular ageing also.     

Coming of age: 'dysgenetics'--a theory connecting induction of persistent delayed genomic instability with disturbed cellular ageing

INTRODUCTION: In recent years a new phenomenon has manifested itself: delayed, persistent genomic instability. When cells are treated with carcinogens not only direct induction of chromosome aberrations and mutations takes place, but there is also an indirect induction: in the distant progeny of treated cells persistently enhanced levels of new chromosome aberrations and enhanced mutation rates are found. This persistent enhanced genomic instability is not due to the presence of lesions in the DNA induced by the treatment because the response can be transmitted to untreated cells. Apparently it is caused by a persistent dysfunctioning of the cell as a whole. Due to these findings a new model for multistep carcinogenesis emerges. According to this model the initiation of carcinogenesis is the induction of a state of persistent genomic instability that not only is responsible for enhanced mutation rates of oncogenes and tumor suppressor genes, but also predisposes to immortalization. This view could lead to a radical change in our views on carcinogenesis. Therefore understanding the mechanism is of utmost importance. PURPOSE: Up to now, the mechanism responsible for this persistent delayed genomic instability remains completely elusive and has only been described as 'unknown'. In this review the phenomenon is connected with a recent theory on cellular ageing and immortalization. CONCLUSION: Although highly speculative this review provides a framework for further experimental approaches that will contribute to our understanding of delayed genomic instability and possibly even to a better understanding of cellular ageing also.     

Role of RAD9-dependent cell-cycle checkpoints in the adaptive response to ionizing radiation in yeast, Saccharomyces cerevisiae

PURPOSE: To determine whether yeast cells (Saccharomyces cerevisiae) defective in damage-inducible cell-cycle arrest can invoke an adaptive response and become resistant to normally lethal doses of ionizing radiation. MATERIALS AND METHODS: Wild-type yeast cells, cells defective for DNA-damage-responsive G1 and G2 cell-cycle arrest (rad9delta), and cells defective for recombinational repair of DNA damage (rad50, 51, 52) were subjected to adapting treatments of heat or radiation and subsequently exposed to normally lethal doses of radiation. Survival, as measured by colony-forming ability, was compared with non-adapted, control cells. RESULTS: Wild-type and rad9delta cells became more resistant to potentially lethal doses of radiation after exposure to conditions that are known to elicit the adaptive response. Further, the relative magnitude of resistance developed by the normal, wild-type and rad9delta yeast cells was similar, with a dose modifying factor (at D1) for radiation-induced radiation resistance of 1.3 for both strains. Dose modifying factors (at D1) for heat-induced radiation resistance were 1.7 and 1.6 for wild-type and rad9delta cells, respectively. In contrast, none of the recombinational repair-defective cells exhibited radiation resistance after an adapting treatment. CONCLUSIONS: The ability of yeast cells to arrest in cell-cycle gap phases did not appear to contribute significantly to radiation resistance induced by radiation or heat. Instead, it is suggested that the adaptive response was due mainly to the existence and enhancement of cellular recombinational repair capacity, which was sufficient to repair any DNA damage without the requirement of a detectable cell-cycle delay.     

Role of RAD9-dependent cell-cycle checkpoints in the adaptive response to ionizing radiation in yeast,Saccharomyces cerevisiae

Purpose : To determine whether yeast cells (Saccharomyces cerevisiae) defective in damage-inducible cell-cycle arrest can invoke an adaptive response and become resistant to normally lethal doses of ionizing radiation. Materials and methods : Wild-type yeast cells, cells defective for DNA-damage-responsive G1 and G2 cell-cycle arrest (rad9 Δ) , and cells defective for recombinational repair of DNA damage (rad50, 51, 52) were subjected to adapting treatments of heat or radiation and subsequently exposed to normally lethal doses of radiation. Survival, as measured by colony-forming ability, was compared with non-adapted, control cells. Results : Wild-type and rad9 Δcells became more resistant to potentially lethal doses of radiation after exposure to conditions that are known to elicit the adaptive response. Further, the relative magnitude of resistance developed by the normal, wildtype and rad9 Δyeast cells was similar, with a dose modifying factor (at D 1) for radiation-induced radiation resistance of 1.3 for both strains. Dose modifying factors (at D 1) for heat-induced radiation resistance were 1.7 and 1.6 for wild-type and rad9 Δcells, respectively. In contrast, none of the recombinational repair-defective cells exhibited radiation resistance after an adapting treatment. Conclusions : The ability of yeast cells to arrest in cell-cycle gap phases did not appear to contribute significantly to radiation resistance induced by radiation or heat. Instead, it is suggested that the adaptive response was due mainly to the existence and enhancement of cellular recombinational repair capacity, which was sufficient to repair any DNA damage without the requirement of a detectable cell-cycle delay.     

Increased susceptibility to delayed genetic effects of low dose X-irradiation in DNA repair deficient cells

Abstract

Purpose: To examine whether the levels of micronuclei induction, as a marker for genomic instability in the progeny of X-irradiated cells, correlates with DNA repair function.

Materials and methods: Two repair deficient cell lines (X-ray repair cross-complementing 1 [XRCC1] deficient cell line [EM9] and X-ray repair cross complementing 5 [XRCC5; Ku80] deficient X-ray sensitive Chinese hamster ovary [CHO] cell line [xrs5]) were used in addition to wild-type CHO cells. These cells were irradiated with low doses of X-rays (up to 1 Gy). Seven days after irradiation, micronuclei formed in binucleated cells were counted. To assess the contribution of the bystander effect micronuclei induction was measured in progeny of non-irradiated cells co-cultured with cells that had been irradiated with 1Gy.

Results: The delayed induction of micronuclei in 1 Gy-irradiated cells was observed in normal CHO and EM9 but not in xrs5. In the clone analysis, progenies of xrs5 under bystander conditions showed significantly higher levels of micronuclei, while CHO and EM9 did not.

Conclusion: Genomic instability induced by X-irradiation is associated with DSB (double-strand break) repair, even at low doses. It is also suggested that bystander signals, which lead to genomic instability, may be enhanced when DSB repair is compromised.     

γ-H2AX分析及其在监测电离辐射所致DNA双链断裂中的应用前景

近几年的研究表明,组蛋白H2AX在DNA损伤修复、细胞周期检查点调控、基因组稳定的维持和肿瘤抑制中起着重要作用,而且在放射生物学中具有应用前景。磷酸化的组蛋白H2AX (y-H2AX)对DNA双链断裂快速敏感的反应使其成为DNA双链损伤的标志。y-H2AX分析也将在监测电离辐射尤其是低剂量电离辐射所致的DNA双链损伤中拥有广阔的应用前景。     

Investigation of G2-phase chromosomal radiosensitivity in hereditary non-polyposis colorectal cancer cells

Purpose : To investigate whether cells from hereditary nonpolyposis colorectal cancer (HNPCC) patients, a genetic condition characterized by constitutional mutations in DNA mismatch repair genes and associated with predisposition to colorectal carcinoma (CRC), could present a higher G2 chromosomal radiosensitivity. It is generally hypothesized that cancer predisposition in HNPCC is associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair but, to date, no data on G2 radiosensitivity have been reported for HNPCC. Materials and methods : Lymphoblastoid cell lines derived from six HNPCC patients heterozygous for MLH1, one HNPCC patient carrying a mutant MSH2 allele and three healthy controls were treated with 50 cGy of X-rays and sampled at various harvesting times, monitoring cell-cycle progression by 5-bromo-2-deoxyuridine (BrdUrd) incorporation in order to analyse chromosomal damage in the homogeneous G2 population. Results : There were no differences between lymphoblasts derived from patients in the frequency of G2 chromosomal aberrations induced by X-rays when compared with control cell lines. However, despite the absence of G2 radiosensitivity in HNPCC cells, lymphoblasts from patients heterozygous for MLH1 mutations showed a higher induction of chromatid exchanges. Conclusions : The observed possible incorrect rejoining of double-strand breaks in MLH1 heterozygotes would be an additional and important factor contributing to loss of heterozygosity in HNPCC patients.     

Investigation of G2-phase chromosomal radiosensitivity in hereditary non-polyposis colorectal cancer cells.

PURPOSE: To investigate whether cells from hereditary nonpolyposis colorectal cancer (HNPCC) patients, a genetic condition characterized by constitutional mutations in DNA mismatch repair genes and associated with predisposition to colorectal carcinoma (CRC), could present a higher G2 chromosomal radiosensitivity. It is generally hypothesized that cancer predisposition in HNPCC is associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair but, to date, no data on G2 radiosensitivity have been reported for HNPCC. MATERIALS AND METHODS: Lymphoblastoid cell lines derived from six HNPCC patients heterozygous for MLH1, one HNPCC patient carrying a mutant MSH2 allele and three healthy controls were treated with 50 cGy of X-rays and sampled at various harvesting times, monitoring cell-cycle progression by 5-bromo-2-deoxyuridine (BrdUrd) incorporation in order to analyse chromosomal damage in the homogeneous G2 population. RESULTS: There were no differences between lymphoblasts derived from patients in the frequency of G2 chromosomal aberrations induced by X-rays when compared with control cell lines. However, despite the absence of G2 radiosensitivity in HNPCC cells, lymphoblasts from patients heterozygous for MLH1 mutations showed a higher induction of chromatid exchanges. CONCLUSIONS: The observed possible incorrect rejoining of double-strand breaks in MLH1 heterozygotes would be an additional and important factor contributing to loss of heterozygosity in HNPCC patients.