Validation of 64Cu-ATSM damaging DNA via high-LET Auger electron emission

Radioactive copper (II) (diacetyl-bis N4-methylthiosemicarbazone) (Cu-ATSM) isotopes were originally developed for the imaging of hypoxia in tumors. Because the decay of a ⁶⁴Cu atom is emitting not only positrons but also Auger electrons, this radionuclide has great potential as a theranostic agent. However, the success of ⁶⁴Cu-ATSM internal radiation therapy would depend on the contribution of Auger electrons to tumor cell killing. Therefore, we designed a cell culture system to define the contributions to cell death from Auger electrons to support or refute our hypothesis that the majority of cell death from ⁶⁴Cu-ATSM is a result of high-LET Auger electrons and not positrons or other low-LET radiation. Chinese hamster ovary (CHO) wild type and DNA repair–deficient xrs5 cells were exposed to ⁶⁴Cu-ATSM during hypoxic conditions. Surviving fractions were compared with those surviving gamma-radiation, low-LET hadron radiation, and high-LET heavy ion exposure. The ratio of the D₁₀ values (doses required to achieve 10% cell survival) between CHO wild type and xrs5 cells suggested that ⁶⁴Cu-ATSM toxicity is similar to that of high-LET Carbon ion radiation (70 keV/μm). γH2AX foci assays confirmed DNA double-strand breaks and cluster damage by high-LET Auger electrons from ⁶⁴Cu decay, and complex types of chromosomal aberrations typical of high-LET radiation were observed after ⁶⁴Cu-ATSM exposure. The majority of cell death was caused by high-LET radiation. This work provides strong evidence that ⁶⁴Cu-ATSM damages DNA via high-LET Auger electrons, supporting further study and consideration of ⁶⁴Cu-ATSM as a cancer treatment modality for hypoxic tumors.     

Interstitial telomeric sequences are not preferentially involved in the chromosome damage induced by the methylating compound streptozotocin in chinese hamster cells

The effect of the methylating compound streptozotocin (STZ) on interstitial telomeric sequences (ITSs) was investigated in Chinese hamster ovary (CHO) cells by using peptide nucleic acid‐fluorescence in situ hybridization with a pantelomeric probe. Cells were exposed to increasing concentrations of STZ, and chromosomal aberrations were analyzed at the first mitosis after treatment. The frequency of chromosomal aberrations directly involving ITSs increased in STZ‐treated cells by a factor of 2.6 (2 mM) and 3.6 (4 mM) when compared with the frequency of these aberrations in control cells (P < 0.05). However, no significant differences were found between control and exposed cells in the percentage of aberrations directly involving ITSs, demonstrating that these repeat regions were not preferentially involved in the chromosome damage induced by STZ. In addition, STZ did not alter telomerase activity, suggesting that this enzyme may not be involved in the induction of chromosomal aberrations by this compound. Environ. Mol. Mutagen., 2013. © 2012 Wiley Periodicals, Inc.     

旋毛虫DNA疫苗在中国仓鼠卵巢细胞中的表达

目的　观察编码旋毛虫相对分子质量(Mr)31000抗原的DNA疫苗(重组真核表达质粒pcDNA3 TspE1)在中国仓鼠卵巢(CHO)细胞中的体外表达 ,并分析其表达产物的抗原性。　方法　通过用阳离子脂质体Lipofectamine2000将重组质粒pcDNA3 TspE1转染CHO细胞,G418筛选阳性克隆,用逆转录聚合酶链反应(RT PCR)、间接荧光抗体试验(IFAT)、十二烷基硫酸钠 聚丙烯酰胺凝胶电泳(SDSPAGE)和蛋白质印迹法(Westernblotting)对表达产物进行鉴定。　结果　RT PCR结果显示,pcDNA3 TspE1转染的CHO细胞在876bp处有一条带,而用空质粒pcDNA3转染的CHO细胞未出现条带,表明pcDNA3 TspE1转染细胞中有TspE1基因转录。IFAT结果显示,pcDNA3 TspE1转染的CHO细胞与重组融合蛋白免疫小鼠血清反应呈现亮绿色荧光 ,而pcDNA3转染的CHO细胞及未转染细胞呈现橘黄色。Westernblotting显示在pcDNA3 TspE1转染的CHO细胞培养液中存在有一Mr约31000的蛋白带 ,且该条带能被重组融合蛋白免疫小鼠血清、旋毛虫肌幼虫可溶性抗原免疫兔血清、感染旋毛虫的小鼠及旋毛虫病患者血清识别。　结论　重组质粒pcDNA3 TspE1可转染CHO细胞,旋毛虫TspE1基因可在转染的CHO细胞中表达,表达蛋白能分泌到细胞培养上清中且具有旋毛虫     

A tamoxifen derivative,N,N-diethyl-2-[4-(phenylmethyl) phenoxy] ethanamine,selectively targets P-glycoprotein-positive multidrug resistant Chinese hamster cells

DPPE, a tamoxifen derivative with antihistamine activity, was previously shown to potentiate the toxicity of chemotherapeutic drugs. Recently, a Phase III clinical study using doxorubicin with DPPE demonstrated significant increase in the overall survival of breast cancer patients. In this study we examined the effects of DPPE alone on the growth of drug sensitive and P-gp positive CHO cell line. Our results demonstrate DPPE is selectively toxic to P-gp positive cells and the sensitivity to DPPE alone correlated with the levels of P-gp expression. Moreover, in MDR cells, DPPE-induced apoptosis was significantly reduced with Bcl2 overexpression and in the presence of P-gp ATPase inhibitor, PSC833. Furthermore, knockdown of P-gp expression in MDR cells with P-gp-siRNA reversed DPPE sensitivity and increased their sensitivity to doxorubicin and taxol but not to cisplatin. The addition of DPPE to membrane fractions led to dose-dependent increase in P-gp ATPase that was inhibited with PSC833. Moreover, incubation of P-gp positive cells with DPPE led to a significant increase in superoxide levels and a drop in cellular ATP and GSH pools that were reversible with inhibitors of P-gp ATPase. The combined presence of DPPE and the mitochondria electron transport complex III inhibitor, antimycin A, synergized in their effects on the growth of MDR cells but had no effect on the growth of parental drug sensitive cells. Collectively, the results of this study provide a possible mechanism that may be relevant to the clinical results of DPPE in breast cancer trial and demonstrates DPPE as P-gp collateral sensitivity drug.     

Isolation,synthesis and characterization of ω-TRTX-Cc1a,a novel tarantula venom peptide that selectively targets L-type CaV channels

Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (Ca_V) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of Ca_V2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba²⁺ currents (I_Ba) mediated by L-type (Ca_V1.2 and Ca_V1.3) Ca_V channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC₅₀) values of 825 nM and 2.24 μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited I_Ba mediated by high voltage-activated Ca_V channels but did not affect low voltage-activated T-type Ca_V channels. Cc1a exhibited weak activity at Na_V1.5 and Na_V1.7 voltage-gated sodium (Na_V) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1–E5) or C-terminus (ΔW35–V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type Ca_V channels.     

鼠源抗过敏融合蛋白的瞬时表达及纯化

采用流加和降温培养工艺,实现在5 L搅拌式生物反应器规模的瞬时基因表达。首先比较了两种表达载体在CHOS细胞中的瞬时基因表达,发现pIDmAAFP载体介导的瞬时转染表达可以获得mAAFP的高水平表达。采用pIDmAAFP载体在1.3 L搅拌式生物反应器中进行瞬时基因表达,与分批培养相比,通过流加和降温培养工艺对转染过程进行控制,培养时间延长1倍,蛋白表达浓度提高8倍。在5 L搅拌式生物反应器中对上述工艺进行成功放大,mAAFP质量浓度达到25 mg/L。之后,利用rProtein A Sepharose Fast Flow介质纯化mAAFP,并通过SDSPAGE、Western blot验证其纯度,为下一步利用小鼠模型研究蛋白功效和作用机理做好准备。     

Prestin蛋白的稳定高表达卵巢细胞株的建立

目的:建立体外稳定高表达耳蜗外毛细胞马达蛋白(prestin)的多克隆中国仓鼠卵巢细胞株(CHO)。方法:采用prestin质粒转染CHO细胞进行表达,高浓度博来霉素400~800mg/L筛选出多克隆细胞株,传代培养,同时长时间维持低浓度抗生素(200~400mg/L)。收集所筛选的多克隆细胞株,提取总RNA,采用针对prestin蛋白不同片段设计的3对引物,进行逆转录-聚合酶链反应(RT-PCR),对扩增产物进行凝胶电泳分析。采用免疫荧光染色方法鉴定所筛选多克隆细胞株的prestin蛋白表达。结果:采用3对不同的prestin引物的RT-PCR结果均为阳性,而对照组则为阴性;利用prestin蛋白的特异性抗体,对所筛选的多克隆细胞株进行免疫荧光染色鉴定结果显示阳性率>90%。结论:成功获得稳定高表达prestin蛋白的多克隆CHO细胞株,转染表达率高。     

十溴联苯醚BDE-209对人卵巢癌细胞及仓鼠卵巢上皮细胞生长的影响

目的:探讨十溴联苯醚(BDE-209)对人卵巢癌细胞株OVCAR-3及中国仓鼠卵巢上皮细胞株CHO细胞增殖和细胞周期的影响。方法:采用MTT检测法、免疫荧光化学检测法以及流式细胞仪等方法观察不同浓度BDE-209(0～100nmol/L)作用于OVCAR-3和CHO细胞0～72h,其细胞形态学变化、OD值改变、K i67表达变化及细胞周期的变化。结果:(1)0～100nmol/L BDE-209作用于细胞72h,倒置显微镜下观察随着浓度增高细胞增殖明显,细胞间隙变小甚至消失,细胞密集排布,细胞形态变小;(2)MTT法检测发现BDE-209促进两种细胞增殖,其增殖率呈时间依赖性和浓度依赖性;(3)0～100nmol/L BDE-209作用72h后,K i67阳性细胞数量增多,表达强度增强,呈现浓度依赖性;(4)流式细胞术检测发现BDE-209改变了细胞周期分布,OVCAR-3细胞G2/M期及CHO细胞S期细胞比例明显增高,且增殖指数(PI)随药物浓度增加而升高。结论:BDE-209明显促进OVCAR-3细胞和CHO细胞体外增殖,并使G2/M期或S期细胞比例增加。     

Helicobacter pylori binds von Willebrand factor and interacts with GPIb to induce platelet aggregation

BACKGROUND & AIMS: Clinical studies have suggested an association between cardiovascular disease and infection with Helicobacter pylori. We examined the effect of H. pylori on platelets and the mechanism of the interaction. METHODS: Three of 5 strains of H. pylori induced platelet aggregation with a lag time of 5 +/- 2 minutes that was independent of the toxigenic genes cagA and vacA. Aggregation was inhibited completely by aspirin and a glycoprotein (GP) IIb/IIIa antagonist. Aggregation also was inhibited by monoclonal antibodies that prevented the von Willebrand factor (vWF) interaction with GPIb. vWF-coated H. pylori bound to cells transfected with GPIbalpha but not to mock transfected cells and this was inhibited by an antibody to GPIb. RESULTS: The interaction with platelets appeared to be mediated by vWF because platelet aggregation was blocked by an antibody to vWF. Moreover, a strain of H. pylori that induced platelet aggregation bound vWF to a greater extent than a nonaggregating strain. Aggregation also required IgG and could be inhibited by an antibody to the platelet IgG receptor (FcgammaRIIA). CONCLUSIONS: Some strains of H. pylori induce platelet activation mediated by H. pylori-bound vWF interacting with GPIb, and supported by IgG. These platelet-H. pylori interactions may contribute to the pathogenesis of H. pylori-associated peptic ulcer disease and to the association between H. pylori infection and cardiovascular disease, whereas local platelet effects may contribute to the pathogenesis of H. pylori-associated peptic ulcer disease.