检测AKT1基因E17K突变的高分辨率熔解曲线法（HRM）的建立及应用

建立并优化HRM方法检测AKT1基因的E17K(49G>A)突变,并在85例非小细胞肺癌中应用该法检测AKT1基因的E17K突变.设计、合成针对AKT1基因的E17K突变的引物、探针,优化不对称PCR条件及高分辨率熔解曲线(HRM)基因突变分析方法.对85例非小细胞肺癌标本提取的基因组DNA,应用HRM基因突变分析方法和直接测序的方法检测了AKT1基因的E17K突变情况.经优化后,设计的引物在不对称PCR反应条件下可扩增出良好的条带.检测探针与野生型模板的扩增产物杂交后,其解链温度要高于与突变型产物杂交的解链温度.野生型和突变型的质粒模板所产生的熔解曲线可见显著差异,相应的Tm值相差约 3.5℃.经HRM基因突变分析方法和直接测序法检测,在85例非小细胞肺癌标本检测到1例AKT1基因的E17K位点为杂合突变型,其余均为野生型.本研究建立的对AKT1 E47K突变检测的HRM方法是一种灵敏、准确、快速的方法.另外,本研究仅在非小细胞肺癌患者中发现频率极低的AKT1 E47K突变.     

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.     

Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.     

Tumour maintenance is mediated by eNOS

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.     

桂枝茯苓汤含药血清对子宫内膜癌细胞的影响

为探究桂枝茯苓汤含药血清是否通过调控3-磷酸肌醇激酶(PI3K)、蛋白激酶B(AKT),参与调控子宫内膜癌细胞增殖、上皮-间质转化(EMT)与凋亡,将72只SD大鼠分为健康组、桂枝茯苓汤高剂量组、桂枝茯苓汤中剂量组、桂枝茯苓汤低剂量组和奥沙利铂组,对各组大鼠灌胃相应剂量药物,从大鼠的腹主动脉取血制备含药血清,培养子宫内膜癌HEC-B细胞,分为对照组、含药血清高剂量组、含药血清中剂量组、含药血清低剂量组、奥沙利铂血清组、含药血清高剂量+通路激活剂组,向各组细胞培养基中加入相应血清及试剂.采用四甲基偶氮唑蓝(MTT)法检测各组细胞增殖情况,流式细胞术检测各组细胞凋亡情况,Western Blot法检测EMT相关蛋白E-钙黏蛋白(E-cadherin)、细胞波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)表达水平及PI3K/AKT通路相关蛋白磷酸化水平.结果表明：与对照组相比,含药血清高剂量组、含药血清中剂量组、含药血清低剂量组的细胞生存率、Vimentin和N-cadherin蛋白表达量、PI3K和AKT磷酸化程度均显著降低(P<0.05),细胞凋亡率、E-cadh...     

Oncogenically active MYD88 mutations in human lymphoma

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.     

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.     

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ～600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.     

黄芪丹参配伍提取物经PI3K-AKT信号通路对心肌梗塞大鼠的影响

观察黄芪丹参配伍提取物对心肌梗塞(myocardial infarction,MI)模型大鼠心肌中磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase,PI3-K)-蛋白质丝氨酸苏氨酸激酶(v-akt murine thymoma viral oncogene homolog,AKT)信号通路的影响。8周龄SD雄性大鼠采用“冠状动脉左前降支结扎法”复制成功后,随机分为假手术对照组、模型组、黄芪丹参配伍低、中、高剂量组(10、20、40mg.kg^-1.d^-1)。对应灌胃给药4周后采用ELISA法、RT-qPCR法和Wstern blot法观察经PI3K-AKT信号通路对MI的干预作用。结果表明：黄芪丹参配伍提取物随着浓度增加可明显提高PI3K、AKT表达(P0.05,P0.01)。可见黄芪丹参配伍提取物能增强PI3K-AKT信号通路,对保护心肌细胞起到积极作用。     

Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa.

Hepatocellular carcinoma (HCC) is a prevalent cancer in sub-Saharan Africa and eastern Asia. Hepatitis B virus and aflatoxins are risk factors for HCC, but the molecular mechanism of human hepatocellular carcinogenesis is largely unknown. Abnormalities in the structure and expression of the tumour-suppressor gene p53 are frequent in HCC cell lines, and allelic losses from chromosome 17p have been found in HCCs from China and Japan. Here we report on allelic deletions from chromosome 17p and mutations of the p53 gene found in 50% of primary HCCs from southern Africa. Four of five mutations detected were G----T substitutions, with clustering at codon 249. This mutation specificity could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1 (ref. 7), a food contaminant in Africa, which is both a mutagen that induces G to T substitution and a liver-specific carcinogen.     

候选药物TW918对K-Ras突变型人肺腺癌A549细胞的体外抑制作用

探讨候选药物TW918对K-Ras突变型人肺腺癌A549细胞的体外抑制作用.以MTT法检测TW918对A549细胞的增殖抑制作用;流式细胞术检测A549细胞周期和凋亡的变化;Western blotting 检测相关蛋白的表达.结果表明:候选药物TW918可以以时间和剂量依赖性地抑制A549细胞的增殖,且作用72 h时抑制效果比吉非替尼高9.93倍;引起细胞周期阻滞于G1期,并诱导细胞发生凋亡;降低A549细胞中p-EGFR的表达,并抑制其下游PI3K/AKT/mTOR信号通路的激活.TW918对K-Ra     

Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome

Tumour suppressor genes, whose usual function seems to be controlling normal cell proliferation, have been implicated in many inherited and sporadic forms of malignancies Much evidence supports the concept of tumour formation by loss-of-function mutations in suppressor genes, as predicted by the two-hit model of Knudson and DeMars. The suppressor gene, p53, is affected in such a manner by numerous mutations, which occur in a variety of human tumours. These mutations usually represent the loss of one allele and the substitution of a single base in the other. We have now analysed the p53 gene in a family affected by Li-Fraumeni syndrome, a rare autosomal dominant syndrome characterized by the occurrence of diverse mesenchymal and epithelial neoplasms at multiple sites. In some instances the neoplasms seem to be related to exposure to carcinogens, including ionizing radiation. The Li-Fraumeni family that we studied had noncancerous skin fibroblasts (NSF) with an unusual radiation-resistant phenotype. DNA derived from the NSF cells of four family members, spanning two generations, had the same point mutation in codon 245 (GGC----GAC) of the p53 gene. This mutation leads to substitution of aspartic acid for glycine in one of the regions identified as a frequent target of point mutations in p53. The NSF cell lines with the mutation also retained the normal p53 allele. This inherited p53 mutation may predispose the members of this family to increased susceptibility to cancer.     

大肠杆菌ATCase抗反馈抑制突变体的构建及其对胞苷积累的影响

为解决胞苷生物合成途径中天冬氨酸氨甲酰转移酶受胞苷三磷酸反馈抑制调节的问题,通过对其碱基序列和蛋白质结构分析,利用基因定点突变的方法构建了大肠杆菌的ATCase突变酶,得到三个突变体:M1(H20L)、M2(K60E)、M3(K94E),并在E.coli DH5α中对融合蛋白进行了表达.酶活测定表明,M1、M2、M3的ATCase酶相对活性都比野生型M0的高,分别为野生型M0的1.10、1.22和1.37倍,且比活力都有不同程度提高.与含野生型pyrBI基因的M0相比,含突变型基因的M1、M2和M3均对15,mmol/L的CTP具有强的抗反馈抑制作用,且M1、M2和M3的抗CTP反馈抑制作用分别是M0的5.4、6.0和8.5倍.最后将各突变质粒转入到E.coli Cyt10(Δcdd)中进行发酵培养,结果表明,与未含突变基因菌株相比,各含突变基因菌株的胞苷积累量均有不同程度的提高,说明ATCase定点突变使胞苷的合成积累途径得到了不同程度的强化.     

树干毕赤氏酵母木糖还原酶Lysine270突变的理性设计

木糖代谢过程中木糖还原酶（XR）和木糖醇脱氢酶（XDH）的氧化还原不平衡是利用纤维素生成酒精的关键问题之一.前期研究发现Lysine270是形成树干毕赤氏酵母木糖还原酶（P. stipitis XR）与NAD(P)结合口袋的关键氨基酸之一.为研究Lysine270对P. stipitis XR辅酶偏好性的影响,本研究将Lysine270用其它19种氨基酸替代,构建19种不同的XR突变子,利用同源建模和分子对接的方法评价不同突变子与NAD(P)之间的相互作用,并从中选择两个突变子K270R和K270N进行试验验证.树干毕赤氏酵母木糖还原酶K270R、K270N突变基因及野生基因WT-XYL1分别在大肠杆菌中进行了表达,表达后的蛋白经His-Tag纯化柱纯化后再进行辅酶偏好性及酶学性质研究.结果发现,Lysine270突变直接影响XR与NAD(P)的Km值,通过理性选择得到的K270N突变子的辅酶依赖性由NADPH完全逆转为NADH.     

BRAF mutation predicts sensitivity to MEK inhibition

The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members. Using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumour growth in BRAF mutant xenografts, whereas RAS mutant tumours were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumours, and offer a rational therapeutic strategy for this genetically defined tumour subtype.