Hyperactivation of DNA-PK by Double-Strand Break Mimicking Molecules Disorganizes DNA Damage Response

Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an “all-or-none” pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a “false” DNA damage signaling that disorganizes the DNA repair system.     

Delivery of siRNA Using CXCR4-targeted Nanoparticles Modulates Tumor Microenvironment and Achieves a Potent Antitumor Response in Liver Cancer

Antiangiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). However, the only clinically approved systemic antiangiogenic agent for advanced HCC is sorafenib, which exerts considerable toxicity. Moreover, acquired resistance to antiangiogenic therapy often develops and restricts the therapeutic efficacy of this treatment. Hence, in this study, we develop a CXCR4-targeted lipid-based nanoparticle (NP) formulation to specifically deliver vascular endothelial growth factor (VEGF) siRNA as an antiangiogenic substance into HCC. AMD3100, a CXCR4 antagonist, is added into NPs to serve as both a targeting moiety and a sensitizer to antiangiogenic therapy. We demonstrate that AMD-modified NPs (AMD-NPs) can efficiently deliver VEGF siRNAs into HCC and downregulate VEGF expression in vitro and in vivo. Despite the upregulation of the SDF1α/CXCR4 axis upon the induction of hypoxia after antiangiogenic therapy, CXCR4 inhibition by AMD-NPs in combination with either conventional sorafenib treatment or VEGF siRNA prevents the infiltration of tumor-associated macrophages. These dual treatments also induce synergistic antiangiogenic effects and suppress local and distant tumor growth in HCC. In conclusion, the tumor-targeted multifunctional AMD-NPs that co-deliver VEGF siRNA and AMD3100 provide an effective approach for overcoming tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC.     

Sex Modulates Intestinal Transformation by the Tumor‐Suppressor GCC

Background and Aims: Ovarian hormones oppose colorectal cancer, although mechanisms remain undefined. Similarly, the most commonly lost gene products in intestinal neoplasia include guanylin and uroguanylin, paracrine hormones for guanylyl cyclase C (GCC), which recently emerged as a tumor suppressor. However, the molecular intersection between intestinal paracrine and systemic sex hormones opposing intestinal neoplasia has not been explored. Methods: Intestinal tumorigenesis was quantified in wild type (Gcc^+/+) and GCC‐deficient (Gcc^−/−) mice carrying mutations in adenomatous polyposis coli (Apc) (Apc^Min/+) or exposed to the carcinogen azoxymethane (AOM). Proliferation of epithelial cells was examined employing cell cycle markers. Results: Deletion of Gcc increased tumor multiplicity and growth in colons and small intestines, respectively, of Apc^{Min /+} mice. While changes in multiplicity and growth increased tumor burden, females exhibited approximately 60% (p= 0.040) of the burden in males. Similarly, female Gcc^−/− mice treated with AOM exhibited approximately 40% (p= 0.048) of the burden in males. Moreover, Gcc deletion promoted epithelial cell proliferation, quantified by increases in β‐catenin, cMyc, cyclin D1, and phosphorylated retinoblastoma protein (pRb), in males but not females. Conclusion: There is a previously unappreciated interaction between sex and GCC signaling restricting crypt cell proliferation. Thus, the invariable loss of guanylin and uroguanylin resulting in tumorigenesis is mitigated in females by hormonal components of the ovarian axis. In the context of the universal overexpression of GCC by tumors, these observations highlight the combination of GCC paracrine and ovarian hormones for targeted prevention and therapy of colorectal cancer.     

单细胞电泳法检测肿瘤患者化疗后淋巴细胞DNA损伤

应用单细胞电泳 (SCGE)检测肿瘤患者环磷酰胺化疗后外周血T淋巴细胞DNA损伤。在标准SCGE条件下应用彗星图象分析系统定量分析人T淋巴细胞DNA的损伤程度。彗星图象分析研究结果显示 ,肿瘤患者外周血T淋巴细胞DNA损伤明显强于正常对照组 ,肿瘤患者外周血T淋巴细胞拖尾动量为 10 42± 1 98,正常人群为 1 2 6± 0 77;两者差异极显著 (P <0 .0 1)。肿瘤患者化疗后T淋巴细胞的拖尾长度为 3 3 69± 7 56μm ,DNA损伤的百分比为 3 1 5± 5 46% ;正常人群T淋巴细胞的拖尾长度和DNA损伤的百分比分别为 16 2± 1 5μm和7 46± 1 15% ;两组数据相比差异极显著 (P <0 .0 1)。结论 :单细胞电泳可快速灵敏地检测细胞DNA损伤 ,能用于肿瘤化疗患者的流行病学观察。对于指导临床用药和预后也有指导意义     

Inhibition of Breast Tumor Progression by Systemic Delivery of the Maspin Gene in a Syngeneic Tumor Model

Maspin has been shown to possess tumor-suppressing activity against breast tumor growth and metastasis. To test the therapeutic value of the maspin gene (SERPINB5) in breast cancer, we established a syngeneic breast tumor metastasis model. This model involved the implantation of mammary tumor cells orthotopically to mammary gland and allowed tumors to grow within the gland and become metastatic to other organs. The mammary tumor cells were initially isolated from MMTV-polyoma virus middle T transgenic mice and were selected in vitro for high invasiveness. Here, we demonstrate that the mammary tumor cells were highly invasive and metastatic. Overall, 100% of tumor-transplanted mice developed lung metastasis. Using nonviral liposome as a carrier, we delivered SERPINB5 to mice bearing mammary tumors. Our data showed that both primary tumor growth and metastasis were significantly inhibited in this syngeneic metastasis model. Such inhibition is mediated by SERPINB5 transgene through increased apoptosis in SERPINB5-treated tumors. Thus, SERPINB5 can be used in gene therapy against breast tumor growth and metastasis.     

Absence of Systemic Immune Response to Adenovectors After Intraocular Administration to Children With Retinoblastoma

The ocular environment has been shown to induce tolerance to locally administered antigens. We therefore investigated whether there was a systemic immune response against adenoviral vectors injected into the vitreous of retinoblastoma patients enrolled in a phase 1 clinical trial of adenoviral-mediated thymidine kinase gene transfer. Sections of enucleated eyes were immunostained with antibodies against inflammatory cells. A trend toward increasing numbers of plasma cells, T cells, macrophages, and antigen-presenting cells was observed in the injected subjects'' eyes, but systemically, there was no significant increase in the number of adenovirus-specific cytotoxic T lymphocytes (CTLs) or in adenovirus neutralizing antibodies. Therefore, in contrast to studies showing significant immunogenicity of Ad-RSVtk following injection into extraocular tumors, injection into the eye produces only a mild local inflammatory response without evidence of systemic cellular or humoral immune responses to adenovirus.     

Tai Chi Improves Oxidative Stress Response and DNA Damage/Repair in Young Sedentary Females

[Purpose] This study was to examine the effects of 12 weeks of Tai Chi (TC) exercise on antioxidant capacity, and DNA damage/repair in young females who did not perform regular physical exercise. [Subjects and Methods] Ten female students from a Chinese university voluntarily participated in this program. All of them practiced the 24-form simplified Tai Chi, 5 times weekly, for 12 weeks. Plasma levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), glutathione (GSH), hydroxyl radical inhibiting capacity (OH·-IC), 8-hydroxy-2’-deoxyguanosine (8-OHdG), and 8-oxoguanine DNA glycosylase (OGG1) were measured at 0, 8, and 12 weeks. Heart rate (HR) was monitored during the last set of the training session at 4, 8, and 12 weeks. [Results] Plasma SOD and OH·-IC levels were increased at 8 and 12 weeks compared to the baseline (0 weeks). Gpx and GSH levels did not change significantly throughout the study period. The plasma MDA level was decreased significantly at 8 weeks but not at 12 weeks compared to the baseline value. While the plasma 8-OHdG level did not change throughout the study period, the plasma OGG1 level was significantly increased at 8 and 12 weeks compared to the baseline value. [Conclusion] TC practice for 12 weeks efficiently improved the oxidative stress response in young females who did not perform regular physical exercise. The TC exercise also increased the DNA repairing capacity.Key words: Tai Chi, Oxidative stress, DNA damage     

泛素连接酶 Fbw7 的调控异常及在肿瘤中的作用

泛素蛋白酶系统对于维持细胞正常生理功能具有重要作用,Fbw7 是 E3 泛素连接酶的底物结合单位,参与泛素化降解与细胞增殖、分化、凋亡有关的重要分子,其调控异常在肿瘤细胞中极为常见。Fbw7 调控的底物包括一系列促癌分子和癌症相关转录因子,被认为是重要的抑癌分子。比如 Fbw7 可以通过调控 Cyclin E、c-Myc、Aurora A 减少因细胞周期异常而造成的染色体不稳,通过调控 p63、Mcl1 来影响细胞损伤修复并增加细胞凋亡,通过调控 TGFβ、mTOR 抑制肿瘤转移,再者可以通过对 Notch 和 Bcl2 家族分子的调控增加肿瘤细胞对化疗的敏感性。因此稳定 Fbw7 的表达可以抑制肿瘤表型的产生和发展,本文就 Fbw7 结构功能、突变机制,调控通路及其在肿瘤发生发展中的作用进行综述。     

Therapeutic Silencing of miR-214 Inhibits Tumor Progression in Multiple Mouse Models

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Role of Attachment in Response to Pet Loss

This study examined the impact of attachment on grief severity following the death of a pet. Seventy-one participants who had lost a dog or cat within the past year completed a set of measures that included an attachment measure assessing individual differences in attachment anxiety and avoidance, strength of the past attachment to the pet, the continuing bond with the deceased pet, social support, and complicated grief symptoms. Attachment anxiety and strength of the past attachment to the pet were each uniquely predictive of more severe grief. Furthermore, the continuing bond to the deceased pet partially mediated the impact of strength of the past attachment to the pet on grief severity. No significant mediators of the effect of attachment anxiety on grief were found, however. The results highlight the importance of distinguishing strength of attachment from attachment security in examining the effect of attachment on response to pet loss.     

Genome-Scale Approaches to Investigate Oxidative DNA Damage

In the trend of biological science after the completion of the human genome project, appreciation of an organism as a system rather than the sum of many molecular functions is necessary. On the investigation of DNA damage and repair, therefore, the orientation toward systematic and comprehensive genome-scale approaches is rapidly growing. The immunoprecipitation-based technique combined with high-density microarrays is one of the promising methods to provide access to such novel research strategies. We propose this sort of research area as oxygenomics.     

Peroxidase-Thiocyanate-Peroxide Antibacterial System Does Not Damage DNA

The hypothiocyanite ion (OSCN^-) is a normal component of human saliva. It is a highly reactive oxidizing agent, and at concentrations above the values normally found in human saliva, it inhibits the growth and metabolism of oral bacteria. This finding has led to the suggestion that antibacterial properties of human saliva might be enhanced in vivo by appropriate supplements which elevate OSCN^- concentrations. Since DNA is sensitive to oxidizing agents (hydrogen peroxide attacks nucleosides), high concentrations of OSCN^- in human saliva might damage DNA and produce deleterious effects on the oral mucosa. In the present study, the effect of high OSCN^- concentrations on several mutagen-sensitive Salmonella typhimurium strains was determined. These strains are used to detect base-pair substitutions and frameshift mutations. We also studied the effects of OSCN^- on a Saccharomyces cerevisiae (yeast) strain commonly employed as a test cell for evaluating the potential of a compound to produce gene conversion, mitotic crossing-over, or reverse mutation. By recording the UV spectra of mixtures of calf thymus DNA and OSCN^-, we explored the possible in vitro reactions of this oxidizing agent with eucaryotic genetic material. Our results show that, at concentrations above 10 μM, OSCN^- is toxic for the tested Salmonella typhimurium strains. The mutant strains with defects in cell wall lipopolysaccharides are killed more readily by OSCN^- than is the strain lacking these defects. However, OSCN^- was not mutagenic for any of the tested strains. Saccharomyces cerevisiae was not affected by OSCN^- even at concentrations above 800 μM. Calf thymus DNA was not oxidized by OSCN^-. We conclude that the elevated concentrations of OSCN^- required to produce antibacterial effects in the human mouth pose no threat to the genetic material of host tissues.     

自发性疼痛降低腹侧海马CA1到前额叶边缘下皮层通路功能连接性并且调控大鼠慢性炎症痛的发展

疼痛会引起自发性脑区活动的波动性改变和相关脑区环路的持续神经可塑性变化。有研究提示海马与内侧前额叶皮层(m PFC)脑区活动与自发性疼痛和慢性疼痛相关,但该观点缺乏直接的证据。本研究结合长时程在体电生理记录与行为学实验,发现持续性自发性疼痛会显著破坏炎症痛模型大鼠中腹侧海马CA1-边缘下皮层(v CA1-IL)的功能连接水平以及海马对IL神经元活动的调节程度。采用化学遗传和光遗传学调控技术特异性激活v CA1-IL通路可减轻自发性疼痛。对v CA1-IL通路进行中脑源性神经营养因子(BDNF)的特异性过表达能够活化通路功能,缓解自发性疼痛,加速炎症性疼痛的全面恢复。本研究确定了一种与自发性疼痛高度相关的神经通路。     

Timed Somatic Deletion of p53 in Mice Reveals Age-Associated Differences in Tumor Progression

Inactivating mutations in the p53 tumor suppressor gene occur often in the progression of human cancers. p53 inhibits the outgrowth of nascent cancer cells through anti-proliferative actions (including induction of apoptosis or senescence). To test p53 tumor suppressor functions in a novel experimental context, we somatically deleted both p53 alleles in multiple tissues of mice at various ages. Mice homozygously deleted for p53 at 3 months of age showed a longer tumor latency compared to mice deleted for p53 at 6 and 12 months of age. These results are consistent with a model in which tissues accumulate oncogenically activated cells with age and these are held in check by wildtype p53. We also deleted p53 before, concurrent with, and after treatment of mice with ionizing radiation (IR). The absence or presence of p53 during IR treatment had no effect on radiation-induced lymphoma latency, confirming that the immediate p53 damage response was irrelevant for cancer prevention. Even the presence of wildtype p53 for up to four weeks post-IR provided no protection against early lymphoma incidence, indicating that long term maintenance of functional p53 is critical for preventing the emergence of a cancer. These experiments indicate that sustained p53 anti-oncogenic function acts as a final or near final line of defense preventing progression of oncogenically activated cells to malignant tumors.     

视网膜母细胞瘤组织中赖氨酸羟化酶2表达及其对瘤细胞迁移和侵袭的影响

目的　探讨前胶原赖氨酸 -2-酮戊二酸 -5-双加氧酶 2（procollagen-lysine 2-oxoglutarate 5-dioxygenase 2, PLOD2）在视网膜母细胞瘤中的表达,以及下调该基因对细胞迁移和侵袭的影响。方法　选取 2011年 3月～ 2019年 3月在湖北省大冶爱尔眼科医院和中国人民解放军广州军区武汉总医院行眼球摘除手术治疗的视网膜母细胞瘤患儿 73例,同期,留取正常视网膜组织 32例作为对照组,免疫组织化学法检测视网膜母细胞瘤和对照组组织中 PLOD2蛋白表达,培养 Y79细胞并分为 PLOD2干扰组（ I组）、阴性对照组（ NC组）和空白组（ B组）,分别转染 PLOD2干扰序列、阴性对照序列及不作任何处理, RT-qPCR检测 PLOD2 mRNA表达, Transwell法检测细胞迁移和侵袭能力, Western blot法检测 PLOD2, E-钙黏蛋白（ E-cadherin,E-cad）和 N-钙黏蛋白（ N-cadherin,N-cad）表达。结果　与对照组相比,在视网膜母细胞瘤组织中 PLOD2蛋白阳性表达率升高（ 75.34% vs 25.00%）,差异有统计学意义（ χ2=23.493,P＜ 0.001）;与国际眼内视网膜母细胞瘤分类（ intraocular international retinoblastoma classife,IIRC）D期［18(60%)］、未发生脉络膜浸润［ 31(65.96%)］和未发生视神经浸润［22(62.86%)］比较, IIRC分期 E期［37(86.05%)］、发生脉络膜浸润［24(92.31%)］和发生视神经浸润［ 33(86.84%)］的视网膜母细胞瘤患者组织中 PLOD2蛋白阳性表达率明显升高,差异均有统计学意义（χ2=6.453,6.256,5.642,均 P＜ 0.05）;与 NC组和 B组比较,在 I组细胞中 PLOD2 mRNA相对表达量明显降低（ 0.27±0.09 vs 1.01±0.08,1.00±0.05）,差异有统计学意义（ F=183.919,P＜ 0.001）;与 NC组和 B组比较,下调 PLOD2表达后细胞迁移能力和侵袭能力均显著降低（ F=10.163,24.094,均 P＜ 0.05）;与 NC组和 B组比较,下调 PLOD2表达后 N-cad蛋白相对表达量明显降低,而 E-cad蛋白相对表达量明显升高,差异均有统计学意义（ F=62.122, 36.641, 均 P＜ 0.001）。结论　视网膜母细胞瘤组织 PLOD2蛋白呈高表达,下调 PLOD2可抑制 EMT而降低 Y79细胞的迁移和侵袭力。     

Terminal Differentiation of Cardiac and Skeletal Myocytes Induces Permissivity to AAV Transduction by Relieving Inhibition Imposed by DNA Damage Response Proteins

Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain, and retina. The reason for their exquisite tropism for these cells has long remained elusive. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DNA damage response (DDR) and that this markedly induces permissivity to AAV transduction. We observed that expression of members of the MRN complex (Mre11, Rad50, Nbs1), which bind the incoming AAV genomes, faded in cardiomyocytes at ~2 weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincided with increased AAV permissivity. Treatment of proliferating cells with short-interfering RNAs (siRNAs) against the MRN proteins, or with microRNA-24, which is normally upregulated upon terminal differentiation and negatively controls the Nbs1 levels, significantly increased permissivity to AAV transduction. Consistently, delivery of these small RNAs to the juvenile liver concomitant with AAV markedly improved in vivo hepatocyte transduction. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction.