Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma

Background

There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.

Methods and Findings

Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).

Conclusions

Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.Trial registration: http://www.ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT00047073","term_id":"NCT00047073"}}NCT00047073).     

鸡氨肽酶H在大肠杆菌中的表达、纯化与部分酶学性质分析

氨肽酶H(Aminopeptidase H, APH))是生物组织内一种常见的氨基内肽酶, 但因为天然材料中含量很低, 基本无法深入研究其催化机理、功能与结构的关系及其在生物体内的确切功能。从鸡肝组织克隆了APH的全基因序列, 并把该序列亚克隆到载体pET22b(+)上, 然后转化大肠杆菌Rosetta(DE3), 构建了APH的表达菌株。该菌株经IPTG诱导, 在SDS-PAGE上明显出现一条与天然APH理论分子量一致的新增蛋白带, 该条带的浓度随着表达时间的延长逐渐加深; 6 h基本达到平衡, 此时重组蛋白占总蛋白的16.7%, 表达水平高达94.7 mg/L。对表达产物进行了活性检测、纯化和酶学性质分析, 发现重组蛋白在亚基构成, 热稳定性, 最适pH等方面与天然APH基本相同, 据此可以确认表达产物确实是APH, 发酵液总活力达到1636 u/L。这些结果为APH的催化机理及其在生物体内的功能的阐明奠定了重要的物质基础。     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

NaCl胁迫对唐古特白刺愈伤组织的生理效应

以唐古特白刺(Nitraria tangutorum Bobr.)愈伤组织为材料,研究低(75 mmol/L)、中(150 mmol/L)、高(300 mmol/L)浓度NaCl处理下其膜脂过氧化、抗氧化酶活性及渗透性调节物含量的变化,试图从细胞水平揭示唐古特白刺适应盐环境的生理机制。结果显示:(1)唐古特白刺愈伤组织中MDA含量在低浓度NaCl处理下与对照无显著性差异,而在中高浓度下显著升高。(2)白刺愈伤组织超氧化物歧化酶(SOD)活性在各浓度NaCl胁迫下均比对照显著升高,且此效应无浓度依赖性;其过氧化氢酶(CAT)活性随胁迫浓度的增加呈先升高后降低的变化趋势,在中低浓度下比对照显著增加,高浓度下显著降低为对照的64%;其过氧化物酶(POD)活性在低浓度下无显著性变化,在中高浓度显著下降为对照的55%和29%。(3)随NaCl胁迫浓度的增加,白刺愈伤组织中脯氨酸、可溶性糖和可溶性蛋白含量均呈先升高后降低的变化趋势,且除高浓度下可溶性蛋白含量约降为对照的87%外,其余NaCl胁迫处理的3种渗透调节物含量均高于对照。研究发现,白刺愈伤组织在低浓度的盐胁迫下具有较强的抗氧化酶活性和渗透性调节作用,因而表现出较强的抗氧化作用,但高浓度NaCl胁迫对白刺愈伤组织造成了显著的氧化损害。     

梭曼对大鼠应激性体温过高的作用及中枢和外周胆碱能受体阻断剂对其效应的影响

目的：观察梭曼对大鼠应激性体温过高的抑制作用以及中枢和外周胆碱能受体阻断剂对其效应的影响。方法：用无线遥测技术测量大鼠的体温,观察皮下注射梭曼、东莨菪碱、甲基东莨菪碱和吡啶斯的明对开放环境中大鼠应激性体温过高的影响。用分光光度技术测定血浆中胆碱酯酶活性。结果：①对照组大鼠在开放实验箱中体温升高达0.96℃,而注射梭曼动物体温只升高了0.55℃。中枢性胆碱能受体阻断剂东莨菪碱几乎完全阻断梭曼对应激体温过高的抑制作用,而外周胆碱能受体阻断剂甲基东莨菪碱则能明显增强梭曼对应激性体温过高的抑制作用。②外周抗胆碱酯酶剂吡啶斯的明能使血浆胆碱酯酶的活性降低至52%,同时明显提高开放环境中大鼠应激性体温过高的反应。甲基东莨菪碱几乎可以阻断吡啶斯的明对应激体温过高反应的影响。结论：神经毒剂梭曼可改变大鼠在开放环境中应激性体温过高的反应能力,其作用主要是通过中枢毒蕈碱型胆碱能通路所致。此外,外周胆碱能神经参与大鼠开放应激性体温过高的形成过程。     

Modulation of kinase-inhibitor interactions by auxiliary protein binding: crystallography studies on Aurora A interactions with VX-680 and with TPX2

VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 A resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a pi-pi interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggest that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.     

Prevalence of asthma among Chinese adolescents living in Canada and in China

Background

Studies of the prevalence of asthma among migrating populations may help in identifying environmental risk factors.

Methods

We analyzed data from Vancouver, Canada, and from Guangzhou, Beijing and Hong Kong, China, collected during phase 3 of the International Study of Asthma and Allergies in Childhood. We subdivided the Vancouver adolescents according to whether they were Chinese immigrants to Canada, Canadian-born Chinese or Canadian-born non-Chinese. We compared the prevalence of asthma and wheezing among Chinese adolescents born in Canada, Chinese adolescents who had immigrated to Canada and Chinese adolescents living in China.

Results

Of 7794 Chinese adolescents who met the inclusion criteria, 3058 were from Guangzhou, 2824 were from Beijing, and 1912 were from Hong Kong. Of 2235 adolescents in Vancouver, Canada, 475 were Chinese immigrants, 617 were Canadian-born Chinese, and 1143 were Canadian-born non-Chinese. The prevalence of current wheezing among boys ranged from 5.9% in Guangzhou to 11.2% in Canadian-born Chinese adolescents. For girls, the range was 4.3% in Guangzhou to 9.8% in Canadian-born Chinese adolescents. The prevalence of ever having had asthma ranged from 6.6% to 16.6% for boys and from 2.9% to 15.0% for girls. Prevalence gradients persisted after adjustment for other environmental variables (odds ratios for ever having had asthma among Canadian-born Chinese compared with native Chinese in Guangzhou: 2.72 [95% confidence interval 1.75–4.23] for boys and 5.50 [95% confidence interval 3.21–9.44] for girls; p < 0.001 for both). Among Chinese adolescents living in Vancouver, the prevalence of ever wheezing increased with duration of residence, from 14.5% among those living in Canada for less than 7 years to 20.9% among those living their entire life in Canada. The same pattern was observed for the prevalence of ever having had asthma, from 7.7% to 15.9%.

Interpretation

Asthma symptoms in Chinese adolescents were lowest among residents of mainland China, were greater for those in Hong Kong and those who had immigrated to Canada, and were highest among those born in Canada. These findings suggest that environmental factors and duration of exposure influence asthma prevalence.The prevalence of asthma symptoms exhibits large geographic variations, even among genetically similar groups,^1,2 which suggests that differences may reflect variation in environmental factors. Epidemiologic studies have demonstrated associations between asthma and exposure to household allergens,³ pets,⁴ environmental tobacco smoke⁵ and environmental pollution,⁶ as well as sex,⁷ obesity,⁸ number of siblings and birth order,⁹ and maternal education.¹⁰ Increasing “westernization” of environmental factors (such as changes in maternal diet, smaller family size, fewer infections during infancy, increased use of antibiotics and vaccination, less exposure to rural environments and improved sanitation) has been associated with an increased risk of childhood asthma.¹¹ Conversely, the hygiene hypothesis proposed by Strachan in 1989 suggested that infections and contact with older siblings may reduce the risk of allergic diseases.¹²Migration studies examining children of the same ethnic background living in different environments for part or all of their lives may help to identify factors relevant to the development of diseases and may explain some of the observed geographic variations in prevalence. In the International Study of Asthma and Allergies in Childhood, prevalence rates for asthma in Canada were among the highest in the world, whereas those in China were among the lowest.² This difference could reflect genetic or environmental factors. China has been and continues to be a major source of international migration.^13,14 Of immigrants in Vancouver, Canada, who landed between 1985 and 2001, half were born in East Asia, mainly Hong Kong and mainland China.¹⁵ Few studies on the prevalence of asthma among immigrants have been undertaken in Canada,¹⁶ and data for Chinese people living in Canada are not available.We hypothesized that the prevalence of asthma would be highest among Canadian-born Chinese adolescents, lower among Chinese adolescents who had immigrated to Canada and lowest among Chinese adolescents living in China. We further hypothesized that, among Chinese immigrants to Canada, prevalence rates of asthma would relate to duration of residence in Canada.     

Dynamic property is a key determinant for protein-protein interactions

Dynamic property is highly correlated with the biological functions of macromolecules, such as the activity and specificity of enzymes and the allosteric regulation in the signal transduction process. Applications of the dynamic property to protein function researches have been discussed and encouraging progresses have been achieved, for example, in enzyme activity and protein-protein docking studies. However, how the global dynamic property contributes to protein-protein interaction was still unclear. We have studied the dynamic property in protein-protein interactions based on Gaussian Network Model and applied it to classify biological and nonbiological protein-protein complexes in crystal structures. The global motion correlation between residues from the two protomers was found to be remarkably different for biological and nonbiological complexes. This correlation has been used to discriminate biological and nonbiological complexes in crystal and gave a classification rate of 86.9% in the cross-validation test. The innovation of this feature is that it is a global dynamic property which does not rely directly on the interfacial properties of the complex. In addition, the correlation of the global motions was found to be weakly correlated with the dissociation rate constant of protein complexes. We suggest that the dynamic property is a key determinant for protein-protein interaction, which can be used to discriminate native and crystal complexes and potentially be applied in protein-protein dynamic rate constants estimations.     

Mao Zedong's fight against schistosomiasis

In 1956, Mao Zedong began a mass campaign against schistosomiasis in the People's Republic of China. The campaign, which integrated mass mobilization, science, agricultural production, local construction projects, and prophylactic measures, was fueled primarily by a determination to accelerate China's agricultural development. The initial success of this campaign encouraged Mao to embark on the next stage of socialism, the Great Leap Forward. As attention was diverted away from schistosomiasis, however, the disease has again become a major burden to the health of the country.     

Replication of nonautonomous retroelements in soybean appears to be both recent and common

Retrotransposons and their remnants often constitute more than 50% of higher plant genomes. Although extensively studied in monocot crops such as maize (Zea mays) and rice (Oryza sativa), the impact of retrotransposons on dicot crop genomes is not well documented. Here, we present an analysis of retrotransposons in soybean (Glycine max). Analysis of approximately 3.7 megabases (Mb) of genomic sequence, including 0.87 Mb of pericentromeric sequence, uncovered 45 intact long terminal repeat (LTR)-retrotransposons. The ratio of intact elements to solo LTRs was 8:1, one of the highest reported to date in plants, suggesting that removal of retrotransposons by homologous recombination between LTRs is occurring more slowly in soybean than in previously characterized plant species. Analysis of paired LTR sequences uncovered a low frequency of deletions relative to base substitutions, indicating that removal of retrotransposon sequences by illegitimate recombination is also operating more slowly. Significantly, we identified three subfamilies of nonautonomous elements that have replicated in the recent past, suggesting that retrotransposition can be catalyzed in trans by autonomous elements elsewhere in the genome. Analysis of 1.6 Mb of sequence from Glycine tomentella, a wild perennial relative of soybean, uncovered 23 intact retroelements, two of which had accumulated no mutations in their LTRs, indicating very recent insertion. A similar pattern was found in 0.94 Mb of sequence from Phaseolus vulgaris (common bean). Thus, autonomous and nonautonomous retrotransposons appear to be both abundant and active in Glycine and Phaseolus. The impact of nonautonomous retrotransposon replication on genome size appears to be much greater than previously appreciated.