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。     

X射线交叉互补修复基因多态性与肿瘤

DNA损伤是细胞最常见的损伤,DNA修复蛋白可以对DNA损伤进行修复,在维持生物体基因组的完整性和抑制肿瘤的发生中起着重要的作用。DNA损伤修复基因表达的正常与否与肿瘤的进展有关。该文对目前的研究热点——人类X射线交叉互补修复基因（XRCC）家族中XRCCl、XRCC2和Rad51基因多态性与肿瘤的关系进行了综述。     

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.     

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.     

Hyperthermic radiosensitization: mode of action and clinical relevance

PURPOSE: To provide an update on the recent knowledge about the molecular mechanisms of thermal radiosensitization and its possible relevance to thermoradiotherapy. SUMMARY: Hyperthermia is probably the most potent cellular radiosensitizer known to date. Heat interacts with radiation and potentiates the cellular action of radiation by interfering with the cells' capability to deal with radiation-induced DNA damage. For ionizing irradiation, heat inhibits the repair of all types of DNA damage. Genetic and biochemical data suggest that the main pathways for DNA double-strand break (DSB) rejoining, non-homologous end-joining and homologous recombination, are not the likely primary targets for heat-induced radiosensitization. Rather, heat is suggested to affect primarily the religation step of base excision repair. Subsequently additional DSB arise during the DNA repair process in irradiated and heated cells and these additional DSB are all repaired with slow kinetics, the repair of which is highly error prone. Both mis- and non-rejoined DSB lead to an elevated number of lethal chromosome aberrations, finally causing additional cell killing. Heat-induced inhibition of DNA repair is considered not to result from altered signalling or enzyme inactivation but rather from alterations in higher-order chromatin structure. Although, the detailed mechanisms are not yet known, a substantial body of indirect and correlative data suggests that heat-induced protein aggregation at the level of attachment of looped DNA to the nuclear matrix impairs the accessibility of the damaged DNA for the repair machinery or impairs the processivity of the repair machinery itself. CONCLUSION: Since recent phase III clinical trials have shown significant benefit of adding hyperthermia to radiotherapy regimens for a number of malignancies, it will become more important again to determine the molecular effects underlying this success. Such information could eventually also improve treatment quality in terms of patient selection, improved sequencing of the heat and radiation treatments, the number of heat treatments, and multimodality treatments (i.e. thermochemoradiotherapy).     

DNA double-strand break repair defects in syndromes associated with acute radiation response: at least two different assays to predict intrinsic radiosensitivity?

PURPOSE: Human diseases associated with acute radiation responses are rare genetic disorders with common clinical and biological features including radiosensitivity, genomic instability, chromosomal aberrations, and frequently immunodeficiency. To determine what molecular assays are predictive of cellular radiosensitivity whatever the genes mutations, the existence of a quantitative correlation between cellular radiosensitivity and unrepaired DNA double-strand breaks (DSB) repair defects was examined in a collection of 40 human fibroblasts representing 8 different syndromes. MATERIALS AND METHODS: A number of techniques such as pulsed-field gel electrophoresis, plasmid assay and immunofluorescence with antibodies against MRE11, MDC1, 53BP1 and phosphorylated forms of H2AX, DNA-PK were applied systematically. RESULTS AND CONCLUSIONS: Survival fraction at 2 Gy was found to be inversely proportional to the amount of unrepaired DSB, whatever the genes mutations and the assay applied. However, no single assay discriminates the full range of human radiosensitivity. Particularly, nuclear foci formed by the phosphorylation of H2AX do not predict well moderate radiosensitivities. Our findings suggest the existence of an ATM-dependent interplay between the activation of DNA-PK and MRE11. A classification of diseases according their cellular radiosensitivity, their molecular response to radiation and the functional assays permitting their evaluation is proposed.     

ADPRT抑制剂对人肿瘤细胞的放射增敏效应

目的：从细胞的克隆形成能力和细胞ＤＮＡ双链断裂及修复几方面探讨了ＡＤＰ－核糖基转移酶（ＡＤＰＲＴ）的特异性抑制剂３－氨基苯甲酰胺（３－ＡＢ）对人卵巢癌细胞株ＨＯＣ８的放射增敏效应。结果表明，３－ＡＢ能降低受照细胞的克隆形成能力；照射所诱发的初始ＤＮＡ双链断裂水平不受３－ＡＢ的影响，但细胞对双链断裂的修复能力受到抑制，表现为慢速修复水平下降，两方面的结果呈正相关。结论：通过脉冲电场凝胶电泳测定ＤＮＡ双链断裂及其修复水平，可以预测细胞的放射敏感性。     

The Bowman-Birk protease inhibitor enhances clonogenic cell survival of ionizing radiation-treated nucleotide excision repair-competent cells but not of xeroderma pigmentosum cells

PURPOSE: The radioprotective effect of the Bowman-Birk protease inhibitor (BBI) was previously shown to result from a TP53 dependent mechanism. Whether this effect involves specific DNA repair mechanisms is now tested. MATERIAL AND METHODS: Normal human fibroblasts were pre-treated with BBI before exposure to X-rays, UVB or to chemical agents (bleomycin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cisplatin). These agents were chosen because of their ability to induce different spectra of DNA damage. The radiometric agent bleomycin primarily induces double-strand breaks (dsb), which are repaired by recombination; MNNG results in alkylated bases which are repaired by base excision repair (BER); cisplatin results in DNA-crosslinks which are repaired mainly by nucleotide excision repair (NER); and finally UVB generates thymine dimers and thymine-cytosine-6-4 products which are also repaired by NER. Cell survival was analysed by colony formation assay and DNA dsb by constant field gel electrophoresis. The combined effect of BBI and X-rays was also tested for XP-fibroblasts, which are defective in NER. RESULTS: For normal human fibroblasts the radioprotective effect of BBI was clearly found by using a delayed plating procedure. The radioprotective effect was found to be unrelated to an altered induction or repair of radiation-induced DNA dsb. Pretreatment with BBI did not affect cell killing after exposure to bleomycin or MNNG, but resulted in a significant protection of cells exposed to cisplatin or UVB. These results indicate that pre-treatment with BBI did not alter recombination repair or BER, but was able to modify NER. The latter finding was supported by the observation made for XP-cells, where pretreatment with BBI failed to result in radioprotection after exposure to ionizing radiation. CONCLUSIONS: On the basis of these data it is proposed that the radioprotective effect of BBI is the result of an improved nucleotide excision repair mechanism.     

The Bowman-Birk protease inhibitor enhances clonogenic cell survival of ionizing radiation-treated nucleotide excision repair-competent cells but not of xeroderma pigmentosum cells

Purpose : The radioprotective effect of the Bowman-Birk protease inhibitor (BBI) was previously shown to result from a TP53 dependent mechanism. Whether this effect involves specific DNA repair mechansims is now tested. Material and methods : Normal human fibroblasts were pre-treated with BBI before exposure to X-rays, UVB or to chemical agents (bleomycin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cisplatin). These agents were chosen because of their ability to induce different spectra of DNA damage. The radiometric agent bleomycin primarily induces double-strand breaks (dsb), which are repaired by recombination; MNNG results in alkylated bases which are repaired by base excision repair (BER); cisplatin results in DNA-crosslinks which are repaired mainly by nucleotide excision repair (NER); and finally UVB generates thymine dimers and thymine-cytosine-6-4 products which are also repaired by NER. Cell survival was analysed by colony formation assay and DNA dsb by constant field gel electrophoresis. The combined effect of BBI and X-rays was also tested for XP-fibroblasts, which are defective in NER. Results : For normal human fibroblasts the radioprotective effect of BBI was clearly found by using a delayed plating procedure. The radioprotective effect was found to be unrelated to an altered induction or repair of radiation-induced DNA dsb. Pre-treatment with BBI did not affect cell killing after exposure to bleomycin or MNNG, but resulted in a significant protection of cells exposed to cisplatin or UVB. These results indicate that pre-treatment with BBI did not alter recombination repair or BER, but was able to modify NER. The latter finding was supported by the observation made for XP-cells, where pre-treatment with BBI failed to result in radioprotection after exposure to ionizing radiation. Conclusions : On the basis of these data it is proposed that the radioprotective effect of BBI is the result of an improved nucleotide excision repair mechanism.     

Dependence of adaptive response and its bystander transmission on the genetic background of tested cells

Abstract Purpose: Radiation-induced adaptive response (AR) is a phenomenon of increased radioresistance mediated by a low priming dose of ionizing radiation (IR) applied prior to a higher challenging dose. We have previously shown that in mouse-embryo fibroblasts (MEF) and human A549 cells, AR is associated with enhanced repair of DNA double-strand breaks (DSB) by the DNA-PK-dependent pathway of non-homologous end-joining (D-NHEJ). Importantly, AR was 'transmitted' to non-irradiated bystander cells through transfer of medium from cells that had received a priming dose of IR. Here, we examine the influence of the genetic background in these responses. Materials and methods: Two plasmid-based assays specifically designed to measure the efficiency of NHEJ and HRR (homologous recombination repair) were deployed. MEF and the primary human fibroblast cell lines HF12 and HF19 were exposed to 10 mGy to 5 Gy X-rays. Bystander effects were investigated using the medium-transfer technique. Results: In contrast to MEF, which induce robust AR to NHEJ, even as a bystander response, human fibroblasts fail to develop such phenomena. Conclusions: The development of AR is cell-type-specific. The same holds true for the development of AR as a bystander effect. Better understanding of the underlying mechanisms will help to understand the molecular basis of these differences in response.     

Excision Repair in u.v. (254 nm) Damaged Non-dividing Human Skin Fibroblasts: A Major Biological Role for DNA Polymerase Alpha

Summary

We have used the eukaryotic DNA polymerase α inhibitor, aphidicolin, and the polymerase β inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (< 3 J m^?2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 μg/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase β inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the β polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase α appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.     

The heritable path of human physical performance: from single polymorphisms to the “next generation”

Human physical performance is a complex multifactorial trait. Historically, environmental factors (e.g., diet, training) alone have been unable to explain the basis of all prominent phenotypes for physical performance. Therefore, there has been an interest in the study of the contribution of genetic factors to the development of these phenotypes. Support for a genetic component is found with studies that shown that monozygotic twins were more similar than were dizygotic twins for many physiological traits. The evolution of molecular techniques and the ability to scan the entire human genome enabled association of several genetic polymorphisms with performance. However, some biases related to the selection of cohorts and inadequate definition of the study variables have complicated the already difficult task of studying such a large and polymorphic genome, often resulting in inconsistent results about the influence of candidate genes. This review aims to provide a critical overview of heritable genetic aspects. Novel molecular technologies, such as next‐generation sequencing, are discussed and how they can contribute to improving understanding of the molecular basis for athletic performance. It is important to ensure that the large amount of data that can be generated using these tools will be used effectively by ensuring well‐designed studies.     

Molecular processes and radiosensitivity

Background

DNA double-strand breaks (DSB) are the most genotoxic lesions induced by ionizing radiation. At least 2 different pathways for DSB repair have been identified, homologous and non-homologous recombination.

Methods

Studies on X-ray-sensitive mutants have led to the identification of several genes involved in processing of DSB in bacteria, yeast and mammalian cells.

Results and Conclusion

In mammalian cells non-homologous recombination is the main pathway for DSB repair, while the role of homologous recombination in DSB repair awaits clarification. It is known that, in addition to DNA repair, other safeguards control the human, cellular response to ionizing radiation, such as cell cycle regulation and mechanisms involved in scavenging of free radicals produced by ionizing radiation.     

7Gyγ射线照射后4h小鼠骨髓差异表达基因的初步研究

目的 为探讨急性放射病骨髓损伤的分子机制,研究整体照射条件下,辐射前后骨髓基因表达的变化。方法 运用抑制消减杂交、cDNA阵列杂交及Northern杂交等方法,筛选C57BL/6J小鼠受7Gy60Co γ射线照射后4h骨髓组织差异表达基因。结果 筛选到一系列辐射后可能表达升高的基因,其功能涉及细胞周期调控、抗氧化、DNA损伤修复和造血免疫,实验确证CDKN1A及S100A8基因的差异表达。结论 辐射后差异表达基因的功能说明骨髓受辐射后即发生哺乳细胞普遍发生的辐射效应(如DNA损伤、细胞周期阻滞、过氧化反应),同时骨髓的造血免疫功能及神经内分泌调节发生改变。     

Ability to repair DNA double-strand breaks related to cancer susceptibility and radiosensitivity

Traditional radiobiology has aimed at elucidating the mechanism of radiosensitivity of cancer cells and normal cells. Because the mechanism of DNA double-strand break (DSB) repair, which is inherently important to radiosensitivity, was unknown, it has been difficult to obtain results applicable to clinical radiotherapy from traditional radiobiology research. Today, however, the molecular mechanism of DNA DSB repair has been elucidated because of the rapid advances in molecular biology. In DNA DSB repair, at least two major repair mechanisms, homologous recombination and nonhomologous end joining (NHEJ) have been reported. In the NHEJ pathway, DSBs are directly, or after processing of the DNA ends, rejoined at an appropriate chromosomal end. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA DSB repair by NHEJ. We have investigated how the ability of repair of DNA DSB influences cancer susceptibility and the radiosensitivity of tumors and normal tissues by focusing on the activity of DNA-PK. In the near future, research on DNA DSB repair mechanism will be able to be applied to research on carcinogenesis, prediction of radiosensitivity of tumors and normal cells, and sensitization of tumor cells.     

Enhanced excision repair activity in mammalian cells after ionizing radiation.

Monkey CV-1 cells which had received 5 Gy 12 h before harvesting lysates from their cell cultures contained approximately three times as much DNA excision repair enzyme activity as unirradiated cells. The activity was determined in crude cell lysates by the release of intermediate mobility DNA fragments and fragments with 3'-phosphoryl ends from 5'-32P-end labelled irradiated 95 bp alpha DNA. Different 3'-termini endow the fragments with differing mobilities, signifying steps in the processing of radiation damaged DNA. Similar results were obtained when Krebs II mouse tumour cells growing in mice as ascites received 5 Gy 12 h before harvest. The enzyme activities from CV-1 cells and from Krebs II cells were partially purified as 60-70 kDa proteins on Superose 12 or Ultrogel AcA-54 columns. Divalent cations were not required for enzyme activity. A 23 nucleotide long defined duplex oligodeoxynucleotide substrate containing a single 8-oxodG residue was also very actively cleaved by the partially purified cell enzymes. 8-oxoguanine is a major product of ionizing radiation's action on DNA and was recognized by the enzymes described here. The mechanism by which radiation increased excision repair activity of cellular enzymes is not understood.