Role of DNA double-strand break repair genes in cell proliferation under low dose-rate irradiation conditions

Radiation-induced DNA double-stand breaks (DSBs) lead to numerous biological effects. To elucidate the molecular mechanisms involved in cellular responses to low dose and low dose-rate radiation, it is informative to clarify the roles of DSB repair related genes. In higher vertebrate cells, there are at least two major DSB repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR). Here, it is shown that in chicken DT40 cells irradiated with gamma-rays at a low dose-rate (2.4 cGy/day), the growth delay in NHEJ-related KU70- and PRKDC (encoding DNA-PKcs)-defective cells were remarkably higher than in cells defective for the HR-related RAD51B and RAD54 genes. DNA-PKcs- defective human M059J cells also showed an obvious growth delay when compared to control M059K cells. RAD54(-/-)KU70(-/-) cells demonstrated their highest degree of growth delay after an X-irradiation with a high dose-rate of 0.9 Gy/min. However they showed a lower degree of growth delay than that seen in KU70(-/-) and PRKDC(-/-/-) cells exposed to low dose-rate irradiation. These findings indicate that cellular responses to low dose-rate radiation are remarkably different from those to high dose-rate radiation. The fact that both DT40 and mammalian NHEJ-defective cells were highly sensitive to low dose-rate radiation, provide a foundation for the concept that NHEJ-related factors may be useful as molecular markers to predict the sensitivity of humans to low dose-rate radiation.     

Misrepair of DNA double-strand breaks after exposure to heavy-ion beams causes a peak in the LET–RBE relationship with respect to cell killing in DT40 cells

To determine the radiobiological mechanisms underlying relative biological effectiveness (RBE) and the repair efficiencies of DNA double-strand breaks (DSBs) as a function of linear energy transfer (LET), we exposed cells of the chicken B-lymphocyte cell line DT40 and its DSB repair pathway-deficient derivatives to heavy-ion beams produced at the Heavy-Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (NIRS), Chiba, Japan. The relationship between LET and cell lethality was investigated in the DNA DSB repair gene knockouts Ku70^−/−, Rad54^−/−, and Ku70^−/−Rad54^−/−, and in the wild-type cells. We found that cell-cycle stage and activity of the DNA DSB repair pathways influence LET-mediated biological effects. An expected LET–RBE relationship was observed in the cells capable of DNA repair, but no peak was found in the RBE with respect to cell survival in the Ku70^−/−Rad54^−/− cells or in Ku70^−/− cells in the G1 and early S cell-cycle phases (when no sister chromatids were present and homologous recombination could not occur). These findings suggest that the peak in RBE is caused by deficient repair of the DNA DSBs.     

DNA-PK: the major target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via NHEJ pathway

The effect of wortmannin posttreatment was studied in cells derived from different species (hamster, mouse, chicken, and human) with normal and defective DNA-dependent protein kinase (DNA-PK) activity, cells with and without the ataxia telangiectasia (ATM) gene, and cells lacking other regulatory proteins involved in the DNA double-strand break (DSB) repair pathways. Clonogenic assays were used to obtain all results. Wortmannin radiosensitization was observed in Chinese hamster cells (V79-B310H , CHO-K1), mouse mammary carcinoma cells (SR-1), transformed human fibroblast (N2KYSV), chicken B lymphocyte wild-type cells (DT40), and chicken Rad54 knockout cells (Rad54-/-). However, mouse mammary carcinoma cells (SX9) with defects in the DNA-PK and chicken DNA-PK catalytic subunit (DNA-PKcs) knockout cells (DNA-PKcs-/-/-) failed to exhibit wortmannin radiosensitization. On the other hand, SCID mouse cells (SC3VA2) exposed to wortmannin exhibited significant increases in radiosensitivity, possibly because of some residual function of DNA-PKcs. Moreover, the transformed human cells derived from AT patients (AT2KYSV) and chicken ATM knockout cells (ATM-/-) showed pronounced wortmannin radiosensitization. These studies demonstrate confirm that the mechanism underlying wortmannin radiosensitization is the inhibition of DNA-PK, but not of ATM, thereby resulting in the inhibition of DSB repair via nonhomologous endjoining (NHEJ).     

Differences in sensitivity to DNA-damaging Agents between XRCC4- and Artemis-deficient human cells

Non-homologous end-joining (NHEJ) is the predominant pathway for the repair of DNA double-strand breaks (DSBs) in human cells. XRCC4 is indispensable to NHEJ and functions together with DNA ligase IV in the rejoining of broken DNA ends. Artemis is a nuclease required for trimming of some, but not all, types of broken DNA ends prior to rejoining by the DNA ligase IV/XRCC4 complex. To better understand the roles of these factors, we generated XRCC4- and Artemis-deficient cells from the human colon adenocarcinoma cell line HCT116 by gene targeting and examined their cellular responses to several DNA-damaging agents including X-rays. As anticipated, kinetic analyses of γ-H2AX foci and chromosomal aberrations after ionizing radiation (IR) demonstrated a serious incompetence of DSB repair in the XRCC4-deficient cells, and relatively moderate impairment in the Artemis-deficient cells. The XRCC4-deficient cells were highly sensitive to etoposide and 5-fluoro-2'-deoxyuridine as well as IR, and moderately sensitive to camptothecin, methyl methanesulfonate, cisplatin, mitomycin C, aphidicolin and hydroxyurea, compared to the parental HCT116 cells. The Artemis-deficient cells were not as sensitive as the XRCC4-deficient cells, except to cisplatin and mitomycin C. By contrast, the Artemis-deficient cells were significantly more resistant to hydroxyurea than the parental cells. These observations suggest that Artemis also functions in some DNA damage response pathways other than NHEJ in human cells.     

Repair of ionizing radiation induced DNA double strand breaks

DNA double-strand breaks (DSB) are created by ionizing radiation, an important environmental genotoxic agent. DSB are repaired by two mechanisms associated with recombination. In eukaryotic cells homologous recombination depends on genes belonging to the RAD52 epistatic group. Alternative pathway, DNA end-joining in non-homologous recombination involves DNA-dependent protein kinase (DNA-PK).     

Cellular response to pulsed low-dose rate irradiation in X-ray sensitive hamster mutant cell lines

The role of DNA repair mechanisms in the cellular response to low dose rate (LDR) irradiation was studied with the aim to gain insight in the process of sublethal damage (SLD) repair. Chinese hamster cell lines mutated in either DNA single strand break (ssb) repair or DNA double strand break (dsb) repair by non homologous end joining (NHEJ) and homologous recombination (HR), or showing an AT-like phenotype, were irradiated in plateau-phase either at high dose rate (HDR, 3.3 Gy/min) or at pulsed low dose rate (p-LDR, average 1 Gy/h). Cell survival after irradiation was assessed using the clonogenic assay. A change in sensitivity when the dose rate was decreased was observed for all parental cell lines and the DNA ssb repair mutant. No difference in cell survival after p-LDR versus. HDR irradiation was observed for the two NHEJ mutants, the AT-like mutant and the HR mutant. Based on these results we conclude that single strand break repair does not play a role in the dose rate effect. The AT like protein, functional NHEJ and XRCC3 are required for the dose rate effect.     

Non-homologous end-joining genes are not inactivated in human radiation-induced sarcomas with genomic instability

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.     

Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity

Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs^*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs^*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs^*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.     

Biotinylation of K12 in histone H4 decreases in response to DNA double-strand breaks in human JAr choriocarcinoma cells

We tested the hypothesis that biotinylation of K12 in histone H4 plays a role in the cellular response to double-strand breaks (DSB) of DNA in human cells. DSB were caused by treating choriocarcinoma JAr cells with etoposide. Biotinylation of K12 in histone H4 decreased by 50% as early as 10-20 min after initiation of treatment with etoposide. Biotinylation returned to initial levels 30-40 min after the addition of etoposide to the medium. Temporal patterns of K12-biotinylation were similar for human lymphoma cells. Phosphorylation of S14 of histone H2B and poly(ADP-ribosylation) of glutamate residues on histone H2A are known markers of DSB in DNA; these modifications increased 10-40 min after alterations in K12-biotinylation occurred. Decreased biotinylation of K12 of histone H4 was specific for DSB but was not detectable in response to single-strand breaks or the formation of thymine dimers. Biotin-deficient choriocarcinoma cells exhibited a 40% decrease in rates of survival in response to etoposide compared with biotin-sufficient controls. These studies suggest that the lack of biotinylation of K12 in histone H4 is an early signaling event in response to DSB.     

Mitotic cells can repair DNA double-strand breaks via a homology-directed pathway

The choice of repair pathways of DNA double-strand breaks (DSBs) is dependent upon the cell cycle phases. While homologous recombination repair (HRR) is active between the S and G2 phases, its involvement in mitotic DSB repair has not been examined in detail. In the present study, we developed a new reporter assay system to detect homology-directed repair (HDR), a major pathway used for HRR, in combination with an inducible DSB-generation system. As expected, the maximal HDR activity was observed in the late S phase, along with minimal activity in the G1 phase and at the G1/S boundary. Surprisingly, significant HDR activity was observed in M phase, and the repair efficiency was similar to that observed in late S phase. HDR was also confirmed in metaphase cells collected with continuous colcemid exposure. ChIP assays revealed the recruitment of RAD51 to the vicinity of DSBs in M phase. In addition, the ChIP assay for gamma-H2AX and phosphorylated DNA-PKcs indicated that a part of M-phase cells with DSBs could proceed into the next G1 phase. These results provide evidence showing that a portion of mitotic cell DSBs are undoubtedly repaired through action of the HDR repair pathway.     

32例三阴性乳腺癌患者易感基因胚系突变分析

目的:应用全外显子测序技术初步探讨三阴性乳腺癌(TNBC)患者易感基因突变情况。方法:收集本院就诊的32例TNBC患者,均经临床手术病理确诊。采集患者外周血提取基因组DNA进行全外显子组测序,通过生物信息学分析筛选与乳腺肿瘤相关的易感基因变异。结果:32例TNBC患者中14例检测到BRCA1/2罕见变异,明确致病性或可疑致病变异6例,突变携带频率为18.8%。其中BRCA1:c.5468-1_5474del和c.4749_4750del是较常见的突变;BRCA2:c.6027A>C为新的变异;BRCA2:c.3794G>T、c.7901T>A,BRCA1:c.4616T>C首次在中国人群中发现。除了BRCA1/2变异外,还检测到83个乳腺肿瘤易感基因变异,每个患者携带2.6个变异。2个以上患者携带的乳腺癌易感基因包括ALK、APC、CDH1、PTCH2、RB1CC1、RAD51D、RAD54L、TSC1等。结论:BRCA1/2是TNBC患者最重要的易感基因,其他与DNA损伤修复相关的基因突变可能与TNBC患者的表型有一定的相关性。     

Functional analysis of yeast homologue gene associated with human DNA helicase causative syndromes

Proteins having DNA helicase activity play very important roles in many processes involving DNA workings such as replication, repair, and recombination. In this decade, many DNA helicase genes have been cloned as the causative genes of human recessive heredity diseases. These are the causative genes for Xeroderma pigmentosum (XPB and XPD), Cockayne syndrome (CSB), diffuse collagen disease (Ku80), alpha-thalassmia (ATR-X), Bloom syndrome (BLM), Werner syndrome (WRN) and Rothmund-Thomson syndrome (RTS). The yeast homologue genes of these human DNA helicase genes exist. S. cerevisiae RAD25/SSL2, RAD3, RAD26, YKU80/HDF2 and RAD54 are the homologue for XPB/ERCC3, XPD/ERCC2, CSB/ERCC6, Ku80/XRCC5 and ATR-X/HX2, respectively. E coli. recQ gene and S. cerevisiae SGS1 are the homologue for all BLM, WRN and RTS. A search of whole genome of S. cerevisiae revealed that SGS1 is the sole homologue of recQ in S. cerevisiae. Thus it seems likely that SGS1 is a functional homologue of one or several human RecQ family genes. Many basic or essential functions are well conserved in the cells from lower eukaryotic to higher mammalian. The functional analysis in yeast could make an useful insight for the human homologue. To clarify the functions of S. cerevisiae Sgs1 and to get an insight into the functions of Blm, Wrn and Rts, in this study, we analyzed the phenotype of sgs1 disruptant and in detail the cause of the poor sporulation phenotype of sgs1 disruptants in relation to meiotic processes including meiotic recombination. The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate (MMS) and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants. The N-terminal 1-45 amino acid region and 698-1195 amino acid region of Sgs1, which including helicase domain and C-terminal RecQ conserved region with helicase activity, were required for complementation of MMS sensitivity and suppression of hyperrecombination of sgs1 disruptants in mitotic growth. The 126-400 and 596-1195 amino acid regions of Sgs1 were required for complementation of poor sporulation and of reduced meiotic functions. These regions required for the mitotic or meiotic functions of Sgs1 were well overlapped with the interaction regions of Top3 and Top2. Some of these results might explain the mechanism of the symptom of RecQ-related syndromes.     

Kinetic analysis of double-strand break rejoining reveals the DNA reparability of gamma-irradiated tobacco cultured cells

The rejoining efficiency of double-strand breaks (DSBs) was quantified by a DNA fragment-size analysis in tobacco protoplasts and CHO-K1 cells following gamma-ray irradiation in order to compare DNA reparability of higher plants with mammals. Results showed that the DSB rejoining efficiency of tobacco protoplasts is dependent on the temperature of post-irradiation cultivation and that it reaches a maximum at 27 degrees C, which represents the most suitable temperature for protoplast cultivation. The DSB rejoining kinetics of tobacco protoplasts were well represented by a biphasic-exponential equation: half of initial-induced DSBs were rejoined for 1 h and the others were almost rejoined within 4 h. We found that the DSB rejoining kinetics of tobacco protoplasts at 27 degrees C are the same as those of CHO-K1 cells at 37 degrees C. These findings indicate that the DSB rejoining efficiency of tobacco protoplasts and CHO-K1 cells are comparable at their respective cell cultivation temperatures, suggesting that DSB rejoining efficiency is little responsible for the higher radiation-tolerance of tobacco protoplasts.     

两种算法在非小细胞肺癌调强放疗中的剂量学比较

目的 比较两种算法在非小细胞肺癌（NSCLC）调强放疗（IMRT）中的剂量学差异,为非小细胞肺癌IMRT计划设计提供数据参考。方法 选择确诊为NSCLC的患者10例,分别制定采用Sliding Wnd（SLD）和Smart Sequence（SMT）算法的IMRT计划,比较两种算法在IMRT计划中的剂量分布、机器跳数、治疗时间以及二维剂量验证结果。结果 SLD算法的计划靶区D_2%、D_98%、D_mean、CI、HI值均优于SMT算法（P<0.05）,差异有统计学意义;SLD算法双肺的D_mean、V₂₀、V₃₀、V₄₀、V₅₀均优于SMT算法,差异有统计学意义;心脏、脊髓等其它参数相差不大,差异无统计学意义。SMT算法的机器跳数与SLD算法相比减少了48 MU,治疗时间减短了7.2 s,差异有统计学意义。SLD算法和SMT算法的IMRT计划二维剂量验证3%/3 mm的γ通过率分别为（99.2±0.5）%和（99.3±0.6）%,2%/2 mm的γ通过率分别为（95.1±1.5）%和（96.4±1.8）%（P>0.05）,差异无统计学意义。结论 两种算法的靶区剂量分布、危及器官受量以及二维剂量验证通过率均能满足临床要求,可应用于非小细胞肺癌IMRT计划设计。SLD算法的靶区剂量分布以及对双肺的保护均优于SMT算法,在NSCLC调强放疗中可优先考虑SLD算法。     

A pilot study of hypovitaminosis D in apparently healthy,veiled, Turkish women: severe vitamin D deficiency in 82%

A pilot study was performed in March 2001 in order to estimate the prevalence of hypovitaminosis D in veiled women in the Netherlands. In a group of 51 Turkish women aged 14-63 years, 42 (82%) were severely deficient (25-hydroxyvitamin D (25-(OH)D) < 20 nmol/l), and 4 (8%) were moderately deficient (25(OH)D: 20-30 nmol/l). About half of the deficient women complained of muscle pain, muscle weakness or fatigue. These results confirm the presence of a serious public health problem with regard to vitamin D amongst veiled women.     

No association between polymorphisms of the DNA repair gene<Emphasis Type="Italic">XRCC1</Emphasis> and cervical neoplasm risk

Objectives To investigate the association between genetic polymorphisms ofX-ray repair crosscomplementing group 1 (XRCC1) codons 194, 280, and 399 and cervical neoplasm susceptibility. Methods A community-based nested case-control study was conducted. The study population consisted of women living in Chiayi City, located in southwestern Taiwan, who had received pap smear screening between October, 1999, and December, 2000 (n=32,466). The potential cases were women having lesions greater than cervical intraepithelium neoplasm II (C1N2) reconfirmed by cervical biopsy. The potential controls (case: control=1∶2) were age matched (±2 yrs) and residency matched women who had had normal pap smears. In total, 100 cases (39 C1N2, 12 C1N3, 46 carcinoma in situ (CIS), and 3 invasive cancer) and 196 controls had the information on both questionnaire and data ofXRCC1 polymorphisms. Results The frequency ofArg/Arg, Arg/Gln, andGln/Gln in codon 399 among cases and controls was 54% (54/100), 38% (38/100), and 8% (8/100) and 58% (114/196), 37% (73/196), and 5% (9/196), respectively, which were not significantly different. No associations were also observed betweenXRCC1 codon 194 and 280 genotypes and cervical neoplasm. While dichotomized by age (<40 vs. ≥40 yrs), smoking status (active and passive smokers vs. non-smokers), and disease status (C1N2 and C1N3 vs. CIS and invasive cancer), the results remained insignificant. Conclusions The present findings suggest thatXRRC1 codon 194, 280 and 399 genotypes may not influence cervical neoplasm risk in the Taiwanese population.     

Identification of a new G-to-A transition mutation at nucleotide position 129 of the Xrcc4 gene in ionizing radiation-hypersensitive mutant LX830 cells

The mouse lymphoma cell line LX830 is an X-ray-hypersensitive mutant. Complementation tests between LX830 cells and radiation-sensitive mutants of M10 (Xrcc4 deficient cells) or SX10 (DNA ligase IV deficient cells) cells showed that M10 cells did not complement LX830 cells, but SX10 cells did, suggesting that LX830 cells would belong to the X-ray-cross complementation group (XRCC4). A sequence analysis of Xrcc4 cDNA in LX830 cells disclosed a transition of G to A at nucleotide position 129, which resulted in a change of tryptophan (43) to a termination codon. Transfection of the mouse Xrcc4 cDNA rescued the X-ray sensitivity of the mutant cells. LX830 is an Xrcc4-deficient cell line bearing a termination codon in exon 2 of the Xrcc4 gene and no wild-type Xrcc4 gene.     

双链断裂修复蛋白hKu70缺陷细胞株的建立及其生物学特性

目的 建立并鉴定DNA双链断裂(DSB)修复蛋白hKu70缺陷细胞株,并观察该缺陷细胞的某些生物学效应,用于AKu70基因功能及职业有害因素对DNA双链断裂修复影响的研究。方法 用构建的AKu70基因反义RNA绿色荧光蛋白真核表达载体(pEGFP—CI—K)转染人胚肺成纤维细胞(HLF),用蛋白兔疫印迹法鉴定转染细胞中AKu70基因的表达水平。同时观察转染细胞生长形态,绘制生长曲线,软琼脂培养法鉴定恶性程度。结果 pEGFP—CI—K载体在转染细胞内可较稳定表达,hKu70蛋白缺陷细胞株AKu70基因的蛋白表达水平下降了42％,转染后hKu70蛋白缺陷细胞生长形态、生长速度无明显变化,软琼脂培养未见细胞集落。结论 成功建立和鉴定了hKu70蛋白缺陷细胞株,该缺陷不足以单独引起可观察的某些生物学效应。