衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

ADP-核糖基转移酶的抑制对γ线所致肿瘤细胞DNA损伤的加强作用

本文探讨了ADP—核糖基转移酶(ADPRT)的活性、DNA单链断裂(SSB)重接和细胞潜在致死质损伤修复(PLDR)三者的关系。证明了ADPRT的特异性抑制剂3—氨基苯甲酰胺(3AB)能阻抑γ线所致的小鼠腹水瘤细胞DNA SSB的重接和PLDR。为增强放射治疗的效果提供了可能的新途径。     

哺乳动物细胞DNA辐射损伤与修复的研究 Ⅰ.γ-线引起人外周血转化淋巴细胞DNA链的断裂与重接

~(60)Coγ-线照射人外周血转化淋巴细胞后,即刻经碱性蔗糖梯度(5～20%〕超离心沉降分析,结果表明随照射剂量(1～6Krad)增大,DNA单链断裂亦增加。3Krad照射细胞经与自体血清37℃保温在2小时以内,DNA断裂链重接分子随保温时间的延长而逐渐增加,然未见完全修复。若继续保温至8小时后,DNA重接分子又发生断裂,至24小时后已重接好的DNA沉降峰几乎消失,并在离心管口出现DNA降解分子,同时细胞活力降低,较保温前下降28%。可以设想DNA重接分子的再断裂是一种不可逆的生化变化,且与细胞死亡有关。     

哺乳动物细胞DNA辐射损伤与修复的研究——Ⅰ.γ-线引起人外周血转化淋巴细胞DNA链的断裂与重接

~(60)Coγ-线照射人外周血转化淋巴细胞后,即刻经碱性蔗糖梯度(5～20%)超离心沉降分析,结果表明随照射剂量(1～6Krad)增大,DNA单链断裂亦增加。3Krad照射细胞经与自体血清37℃保温在2小时以内,DNA断裂链重接分子随保温时间的延长而逐渐增加,然未见完全修复。若继续保温至8小时后,DNA重接分子又发生断裂,至24小时后已重接好的DNA沉降峰几乎消失,并在离心管口出现DNA降解分子,同时细胞活力降低,较保温前下降28%。可以设想DNA重接分子的再断裂是一种不可逆的生化变化,且与细胞死亡有关。     

羟磷灰石离心法及其在细胞DNA辐射损伤和修复研究中的应用

Ahnstrm等在1973年首先用羟磷灰石(以下简称HA)层析法检测DNA单链断裂。该法较经典的硷性蔗糖梯度离心法操作简便,灵敏度高。我们根据本室条件,按照Kanter等的方法稍加改进,用国产HA建立了HA离心分离检测DNA单链断裂及其重接的技术,并用该法观察了小鼠白血病L_(7712)细胞DNA单链断裂及其重接。材料与方法(一)细胞取郑升等建立的小鼠腹水型淋巴性白血病细胞(L_(7712)),由615纯种小鼠     

高温对人淋巴细胞DNA单链断裂及其修复的影响

高温作为辐射增敏技术与辐射相结合,用于临床治疗肿瘤已取得显著的疗效。高温能引起淋巴细胞DNA单链断裂,高温对辐射引起的淋巴细胞DNA单链断裂有协同作用,并且这种协同作用与加热和辐照的顺序有关。高温还能极大地抑制受照淋巴细胞DNA单链断裂的重接修复。     

用脉冲电场凝胶电泳检测电离辐射所致哺乳动物细胞DNA双链断裂

 本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。     

竹红菌甲素光敏作用所致的HeLa细胞DNA单链断裂重接的研究

本文采用羟基磷灰石离心法对竹红菌甲素光敏作用所致的HeLa细胞DNA的单链断裂进行荧光定量测定。以单链DNA的含量变化衡量DNA链断裂程度及其修复活性,并与γ射线的作用进行了比较。细胞经γ射线和甲素的光敏作用后,DNA产生单链断裂,当细胞存活率为0.1时,前者是6.0×10~(-10)断裂/道尔顿,后者是2.3×10~(-10)断裂/道尔顿,两者之比为2.8。细胞本身具有修复能力,当最初的单链断裂频率相等时,γ射线处理的细胞其DNA单链断裂重接能力高于光敏作用的细胞。后者的修复能力能被羟基脲、放线菌素D及丁酸钠等所抑制。细胞修复能力的差别指出两种作用产生的单链断裂的化学性质有重要差别。     

DNA氧化性损伤与端粒缩短

末端复制问题（the end replication problem）不能完全解释端粒在某些细胞分裂过程中迅速缩短的现象.40％的高压氧下细胞传代次数降低,端粒缩短速率增大,细胞出现衰老特征,端粒DNA上单链断裂积累.推测端粒缩短的主要原因在于衰老过程中或氧胁迫下端粒DNA单链断裂增多,使端粒末端单链片段在DNA复制时丢失.端粒酶和活性氧对端粒长度的正负调控作用的准确机制还有待于更深入的研究.     

PNKP在DNA损伤修复中的作用

多聚核苷酸激酶/磷酸酶(polynucleotide kinase/phosphatase,PNKP)是一种DNA末端修复酶,同时具有激酶和磷酸酶活性,在DNA单链断裂修复途径、碱基切除修复途径以及DNA双链断裂修复中的非同源末端连接途径中发挥着至关重要的作用。近年来,由于一种与PNKP相关的常染色体隐性遗传病——MCSZ综合征的发现,使得人们对PNKP的关注度进一步增加。笔者从与PNKP相互作用的X射线交叉互补修复基因1(X-ray repair cross-complementing group 1,XRCC1)、X射线交叉互补修复基因4(X-ray repair cross-complementing group 4,XRCC4)和毛细血管扩张性共济失调突变基因(ataxia-telangiectasia mutated,ATM)入手,对PNKP在DNA损伤修复中的作用进行概述。     

亚硒酸钠诱发的晶状体上皮细胞DNA损伤及修复

观察了亚硒酸钠（Ｎａ２ＳｅＯ３）在体外作用于大鼠晶状体上皮细胞（ＲＬＥｃｅｌｌｓ）而造成的ＤＮＡ单链断裂（ｓｉｎｇｌｅｓｔｒａｎｄｂｒｅａｋｓ,ＳＳＢ）,并对其ＤＮＡ损伤、修复动力学做了初步研究．发现ＳＳＢ严重程度与亚硒酸钠的浓度呈线性相关,其ＳＳＢ重接修复约在３０～６０ｍｉｎ内完成．还作了有关非程序ＤＮＡ合成（ＵＤＳ）的检测,发现与ＳＳＢ相比,ＵＤＳ发生迟且持续时间更长,提示Ｎａ２ＳｅＯ３可能在体外对大鼠晶状体上皮细胞除造成ＳＳＢ以外,还可能造成其它种类的ＤＮＡ损伤．     

Heavy ion-induced plasmid DNA damage in aerated or deaerated conditions

Using the plasmid relaxation assay, the induction of single strand breaks (SSB) and base damages was investigated in air-dried plasmid DNA irradiated under air or under vacuum, with two high LET particles. We first observed that an irradiation with 12C5+ ion produced less of both damages when performed in a vacuum rather than in the presence of air. This could be due to the presence of O2 which increases the primary radicalar effects in the latter case. Another explanation is a difference in the degree of hydration of the DNA molecules. Indeed, under vacuum only the water molecules tightly bound to DNA will persist. In contrast, in the presence of air, the outer hydration shell enhances the amount of hydroxyl radicals available for the radiolytic attack. However, no difference in the SSB induction was observed when DNA was irradiated with 36S16+ ion in the presence of air or under vacuum. This is likely due to the LET effect which partly cancels the production of radicals by recombination and increases the formation of superoxide anions in the track. Similarly, the lower induction of damage by 36S16+ irradiation in comparison with the 12C5+ ion is a consequence of the higher ionizing density for 36S16+ than for 12C5+ ions. Meanwhile, for both ions, base damages are not detected when DNA is irradiated under vacuum, whereas they are as frequent as SSB when irradiation is performed in the presence of air. Altogether, these observations support the idea that SSB and base damage are not formed by the same mechanism.     

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).

Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.

Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.

DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.     

DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.     

Rufloxacin induced photosensitization in bio-models of increasing complexity.

Rufloxacin belongs to the class of fluoroquinolones that act mainly as specific inhibitors of bacterial Topoisomerase II. These drugs are widely known to be involved in various diseases ranging from cutaneous reactions to aging. The type II photosensitizing activity of Rufloxacin has been already demonstrated on calf thymus DNA and free nucleosides. The aim of this study is to examine in control untreated and UVA irradiated human fibroblasts the modifications on DNA status induced by Rufloxacin added in the culture medium. This allows to investigate the photosensitizing activity of Rufloxacin in a more complex cell model. Fibroblasts, either in the presence or in the absence of Rufloxacin, were exposed to UVA irradiation for different times. An experimental protocol was followed in order to evaluate the amount of single-strand breaks (SSB) and double-strand breaks (DSB) DNA fragmentation by comet assay, and plasmid photocleavage. The presence of oxidized bases was also evaluated using the 8-OH-dGuo test. The comet assay test was also employed to assess cellular repair capacity. The intracellular drug concentration was verified by HPLC-MS. The results confirming the role of Rufloxacin as photosensitizer were: (i) a time-dependent increase in DNA fragmentation when fibroblasts were irradiated in the presence of Rufloxacin; (ii) the efficiency of the cellular repair machinery to be exhaustive after 2 h (whereas no correlation between irradiation time and DNA damage repair was observed with a higher level of DNA fragmentation after shorter irradiation times); (iii) the increased number of cells exhibiting high DNA fragmentation, seen as comets with long tails, was not accompanied by a similar large extent of oxidised DNA base formation, as measured by 8-OH-dGuo analysis; (iv) the double helix SSB, formed in plasmid photosensitization, agreed with the comet assay results, pointing out a good correlation among the cell system and the simpler models used.     

Damage clusters after gamma irradiation of a nanoparticulate plasmid DNA peptide condensate

We have gamma-irradiated plasmid DNA in aqueous solution in the presence of submillimolar concentrations of the ligand tetra-arginine. Depending upon the ionic strength, under these conditions, the plasmid can adopt a highly compacted and aggregated form which attenuates by some two orders of magnitude the yield of damage produced by the indirect effect. The yields of DNA single- and double-strand breaks (SSB and DSB) which result are closely comparable with those produced in living cells. The radical lifetimes, diffusion distances, and track structure are expected to be similarly well reproduced. After irradiation, the aggregation was reversed by adjusting the ionic conditions. The approximate spatial distribution of the resulting DNA damage was then assayed by comparing the increases in the SSB and DSB yields produced by a subsequent incubation with limiting concentrations of the eukaryotic base excision repair enzymes formamidopyrimidine-DNA N-glycosylase (the FPG protein) and endonuclease III. Smaller increases in DSB yields were observed in the plasmid target that was irradiated in the condensed form. By modeling the spatial distribution of DNA damage, this result can be interpreted in terms of a greater extent of damage clustering.     

Experimental aspects of in vitro and in vivo photochemotherapy

The selectivity of in vitro photodynamic reactions and the in vivo effects induced by PRT, whether the irradiation is applied interstitially or externally, still remains unclear. In vitro studies were performed using leukemic cell lines and syngeneic normal hemopoietic progenitors. For these, cells incubated with hematoporphyrin derivative (HPD) and non-incubated cells were irradiated with an argon laser. Data were obtained as the count of cell colonies found after a 7-day incubation period on semi-solid collagen gel medium. In vivo studies employed the HT 29 tumor model grafted into nude mice. Both animals injected with HPD and non-infected controls were irradiated with a dye laser pumped by an argon laser (Coherent) using a 400 micron optic fiber located either at a distance of 65 mm from the skin or inserted into the tumor. The temperature increase occurring during PRT was measured using non-absorbing thermocouples. In vitro, after HPD treatment and argon irradiation leukemic cells showed a greater phototoxicity (greater than 2 log10) than did the normal cells (0.25 log10). In vivo, when the heat rise is very similar (less than 4 degrees C) in both the tissues irradiated externally and those irradiated interstitially after HPD injection, histological examination of these did not reveal any quantitative differences (90% of tumor mass). These results are discussed.     

Exposure of mammalian cells to 60-Hz magnetic or electric fields: analysis of DNA repair of induced, single-strand breaks

DNA damage was induced in isolated human peripheral lymphocytes by exposure at 5 Gy to 60Co radiation. Cells were permitted to repair the DNA damage while exposed to 60-Hz fields or while sham-exposed. Exposed cells were subjected to magnetic (B) or electric (E) fields, alone or in combination, throughout their allotted repair time. Repair was stopped at specific times, and the cells were immediately lysed and then analyzed for the presence of DNA single-strand breaks (SSB) by the alkaline-elution technique. Fifty to 75 percent of the induced SSB were repaired 20 min after exposure, and most of the remaining damage was repaired after 180 min. Cells were exposed to a 60-Hz ac B field of 1 mT; an E field of 1 or 20 V/m; or combined E and B fields of 0.2 V/m and 0.05 mT, 6 V/m and 0.6 mT, or 20 V/m and 1 mT. None of the exposures was observed to affect significantly the repair of DNA SSB.     

The binding of SSB-proteins with DNA in chromatin of Ehrlich ascites carcinoma cells]

Using UV-induced cross-linking between proteins and DNA, the contacts between single-stranded DNA-binding proteins (SSB proteins) and chromatin DNA have been demonstrated. Ehrlich ascites tumour DNA was labeled in vivo by inoculation of tumour-bearing mice with 3H-thymidine. The cells were irradiated with the UV light dose of 3000 J/m2, destroyed in a Triton X-100-containing hypotonic medium, and separated by centrifugation into the extrachromatin fraction and chromatin. Chromatin DNA was digested with DNAase 1, and the chromatin proteins were extracted with 2 M NaCl-polyethyleneglycol. SSB proteins from the extrachromatin fraction and chromatin were purified. Only SSB proteins from UV-irradiated cell chromatin appeared to possess a high specific radioactivity which exceeded 7.5-fold that of non-irradiated cells. There were no differences between chromatin SSB proteins in control and irradiated cells as could be evidenced from SDS electrophoresis data. It is assumed that in irradiated cells SSB proteins of DNA-digested chromatin are covalently cross-linked with DNA fragments.     

Glucose oxidase-produced H2O2 induces Ca2+-dependent DNA damage in human peripheral blood lymphocytes

DNA of lymphocytes from human peripheral blood was analyzed by using the single cell gel electrophoresis technique (comet assay). The cells were used either as received from the donors or after treatment with various concentrations of the H2O2-generating enzyme glucose oxidase, in order to achieve a continuous flow of H2O2. The formation of single strand breaks (SSB) was dose-related but the time course of the induction of SSB by relatively low concentrations of glucose oxidase was of a biphasic mode with a fast increase 2 to 5 min after the addition of glucose oxidase followed by a gradual decrease toward the original base level during the next 35 to 60 min. This response of the cells appears to be based on the activation of already existing defense system(s) because it was shown that H2O2 is continuously released during the reaction time and the inhibition of protein synthesis does not affect the observed pattern. Supplementation of the growth medium with various antioxidants resulted in substantial protection only when the agents were taken up by the cells. The presence of the intracellular calcium chelator BAPTA protected the cells from H2O2-induced DNA damage in a dose-dependent manner. Only at the higher rate of H2O2-generation considerable DNA damage was observed in the presence of BAPTA.These results suggest that H2O2, at low concentrations induces DNA damage through intracellular Ca2+ -mediated processes, which lead to DNA strand breaks possibly by endonuclease activation.