CN1965076B - 由wrn介导端粒引发的细胞信号传导的调节 - Google Patents
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Abstract
本发明描述了WRN调节剂的用途。WRN激活剂可以用来诱导生长抑制、细胞凋亡或增殖性衰老，而WRN抑制剂可以用来减少生长抑制、细胞凋亡或增殖性衰老。本发明还描述了鉴别WRN调节剂的方法。
Description
发明背景
技术领域
本发明涉及信号传导途径的调节。更具体地说，本发明涉及端粒引发的衰老、凋亡、皮肤晒黑(tanning)及其他DNA损伤应答的调节。
相关技术描述
随着人口老龄化，发达世界中的人类癌症患病率随之增高。尽管进行了广泛的研究，近年来对于一些就诊时的癌症类型和疾病期，发病率和死亡率仍然没有得到显著改善。在癌症进展期间，肿瘤细胞越来越不受负调控的控制，包括对于衰老和凋亡的抗性，而衰老和凋亡是如何调节正常细胞与它们的组织特异性环境的相互作用的重要方面。
细胞衰老已经被认为是抗癌的一种重要防御手段。广泛的证据暗示在衰老过程中端粒渐进缩短(由不能复制染色体3′末端引起)或其它形式的端粒机能障碍。在生殖系细胞和大部分癌细胞中，永生化与由端粒酶作用产生的保持端粒长度相关，该端粒酶复合物能够把TTAGGG重复片段添加到染色体的3′末端。端粒，即TTAGGG的串联重复序列，其末端是具有大约150-300碱基的3′单链突出端的环状结构，其隐藏在近端端粒双链DNA内并由端粒重复序列结合因子(telomeric repeat binding factor，TRF)尤其是TRF2所稳定。TRF2的显性-阴性形式(TRF2DN)的异位表达破坏端粒环状结构，暴露3′突出端，引起DNA损伤应答，在原代成纤维细胞、纤维肉瘤细胞和其它几种恶性细胞类型中引起衰老。
衰老还可能被广泛的DNA损伤或某些癌基因的过表达所剧烈加速。能酶促维持或构建端粒长度的端粒酶反转录酶催化亚基(telomerase reversetranscriptase catalytic subunit，TERT)的异位表达能绕过衰老过程随后导致一些人类细胞类型的永生化，强烈提示复制型衰老的端粒依赖机制。此外，尽管恶性细胞经常具有比正常衰老细胞更短的端粒，但通常恶性细胞表达TERT和/或含有允许细胞绕过衰老应答并无限增殖的突变。然而，一些肿瘤细胞应答于多种抗癌剂而经历衰老，表明获得永生化并不必然意味着丧失对于DNA损伤的基本细胞应答。
人类细胞的衰老主要取决于p53和pRb途径。肿瘤抑制因子p53在细胞应激应答机制中扮演一个重要角色，它能把多种不同刺激例如DNA损伤、转录或复制的失控、癌基因转化和一些化疗药物引起的微管异常转化为细胞生长抑制或凋亡。当被激活时，p53引起细胞生长抑制或程序性自杀性细胞死亡(凋亡)，其依次作为基因组稳定性的重要控制机制。特别地，p53通过从细胞群体中除去遗传损伤细胞来控制基因组稳定性，因而它的一个主要功能就是防止肿瘤形成。
完整的肿瘤抑制因子pRb途径也有助于防止肿瘤发生。在不含有野生型p53的pRb-/-肿瘤细胞中，导入pRb能诱发衰老。虽然宫颈癌细胞常常保留有野生型p53和pRb基因，但是HPV E6和E7蛋白分别干扰了p53和pRb途径。在宫颈癌细胞系中病毒E2蛋白的异位表达能抑制HPV E6和E7的基因转录并诱导迅速显著的衰老应答，再次证实了p53和pRb在癌细胞衰老中的重要作用。
仅仅抑制p53或pRb途径是不足以让成纤维细胞绕过复制型衰老的。实际上，不论转染了SV 40 T抗原还是转导了腺病毒E1A+E1B或者HPVE6+E7组合的人类成纤维细胞，都能同时抑制p53和pRb途径，具有延长的生存期并能逃避复制型衰老。
DNA双链断裂对哺乳动物细胞是极端的细胞毒性。在真核生物中高度保守的MRN复合物参与双链断裂的修复。MRN复合物形成后能立即粘附于双链断裂位点。在细胞周期中的S期，MRN复合物也迁移到端粒处与端粒重复序列结合因子(TRF)结合。
MRN复合物由Mre11、Rad50和NBS(p95)组成。作为Mre11/p95/Rad50复合物的一部分，Mre11与细胞周期S期的端粒结合。Mre11是一种偏好DNA链3′末端的核酸外切酶。人们认为Mre11的活性依赖于与Rad50的相互作用，Rad50是一种ATP酶。Nbs1被认为参与MRN复合物的核定位以及其在双链断裂位点处的装配。
已知在维尔纳氏综合征(Werner’s Syndrome)中的突变蛋白，WRN蛋白，与MRN复合物相互作用(Cheng et al.，2004，vol.2004)。维尔纳氏综合征是一种常染色体隐性疾病，其特征为过早老化、增大的恶性度和基因组不稳定性。WRN是一种既含有解旋酶又含有3′到5′核酸外切酶结构域的核蛋白(Oshima，J.，2002，Bioessays 22，894-901)。迄今为止，在维尔纳氏综合征中已鉴定出的全部突变都是WRN截短，其能从蛋白的COOH末端除去核定位信号(Oshima，J.，2002)。因此，人们认为在维尔纳氏综合征中的WRN突变通过阻止蛋白到达它的细胞核作用位点而产生无功能的表型。来自维尔纳氏综合征病人的细胞无论是在基线水平还是在DNA损伤之后均显示出缺失和易位水平提高，提示WRN蛋白参与DNA修复、复制和重组(Opresko et al.，2003，Carcinogenesis 24，791-802)。比起与其年龄匹配相当的对照，维尔纳氏综合征细胞也过早地衰老(Martin et al.，1970，Lab Invest23，86-92)，还表明其加速了端粒缩短(Schulz et al.，1996，Hum Genet 97，750-4)。
除了与MRN复合物相互作用之外，还知道WRN与其它参与DNA损伤应答和DNA修复/复制的蛋白相互作用，如DNA-PK/Ku(Karmakar et al.，2002，Nucleic Acids Res 30，3583-91)、p53(Brosh et al.，2001，J Biol Chem 276，35093-102)和在早衰老化综合症(premature aging syndrome)、布卢姆综合征(Bloom’s Syndrome)中突变的解旋酶、BLM(von Kobbe et al.，2002，J BiolChem 277，22035-44)。此外，WRN与端粒重复序列结合因子2(TRF2)相互作用，该相互作用改变了对促进3′到5′消化端粒DNA的WRN核酸外切酶活性的特异性(Machwe et al.，2004，Oncogene 23，149-56；Opresko et al.，2002，J Biol Chem 277，41110-9)。这些数据共同表明了WRN在DNA新陈代谢和端粒保持中的关键作用。然而WRN在这些途径中的明确作用尚未理解。
癌症典型用高毒性疗法进行治疗，例如化疗和放疗，这些疗法对全部增殖细胞不论其正常还是恶性都造成同样的损伤。这些治疗的副作用包括了对淋巴系统、造血系统和肠上皮的严重破坏以及毛发脱落。一直寻求更安全和更有效的癌症疗法，尤其是那些通过相比于正常增殖细胞能优先靶向于恶性细胞从而避免某些或全部这种副作用的替代疗法。
发明概述
本发明涉及一种基于细胞的筛选WRN调节剂的方法，其包含在调节剂被细胞摄取的条件下把候选调节剂与表达WRN的细胞接触，以及测定与DNA损伤应答途径激活相关的细胞性质，该性质包括但不限于细胞增殖、细胞生存力、细胞形态学、SA-β-Gal活性及p53或p95的磷酸化、ATM的磷酸化、H2AX的磷酸化、S期停滞诱导或凋亡的诱导。调节剂可以通过与对照比较改变的性质而鉴定。候选调节剂可以是特异结合WRN的物质。WRN可指具有核酸外切酶活性的WRN的片段、同系物、类似物或变体。
本发明还涉及一种筛选特异结合于WRN的物质的体外方法，其包含把候选物与WRN接触，并测定候选物是否特异结合于WRN。WRN可以附着于一种固相支持物上。或者，候选物可以附着于一固相支持物。
本发明还涉及一种筛选WRN调节剂的体外方法，其包含在体外在有WRN核酸底物存在的情况下把候选调节剂与WRN接触，并测定底物的水解。调节剂可以通过与对照比较底物核酸的水解变化而鉴定。核酸底物可以是与(TTAGGG)n有至少33％核苷酸序列相同性的寡核苷酸，其中n＝1-20。或者，核酸底物可以是与(TTAGGG)n有至少50％核苷酸序列相同性的寡核苷酸，其中n＝1-20。底物核酸的水解可以通过UV吸收、标记寡核苷酸凝胶分析、或回收非可沉淀的核苷酸碱基(recovery ofnon-precipitatable nucleotide bases)进行测定。
本发明还涉及一种基于细胞筛选DNA损伤途径调节剂的方法，其包含在调节剂被细胞摄取的条件下并有寡核苷酸存在的情况下把候选调节剂与表达WRN的细胞接触，以及测定与DNA损伤应答途径激活相关的细胞性质，该性质包括但不限于细胞增殖、细胞生存力、细胞形态学、衰老相关β-半乳糖苷酶(SA-β-Gal)活性以及p53或p95的磷酸化；ATM的磷酸化、H2AX的磷酸化、S期停滞的诱导或凋亡的诱导。调节剂可以通过与对照比较性质的改变而鉴定。寡核苷酸可与(TTAGGG)n有至少33％核苷酸序列相同性，其中n＝1-20。寡核苷酸可与(TTAGGG)n有至少50％核苷酸序列相同性，其中n＝1-20。WRN可以WRN的片段、同系物、类似物或变体的形式表达，且其具有核酸外切酶活性。
如上所述用于基于细胞的筛选方法中的细胞可以是癌细胞。所述用于所述基于细胞的筛选方法中的细胞可以通过端粒酶反转录酶或ALT途径保持端粒。在上述体外及基于细胞的筛选方法中所述的候选调节剂和物质可以是碳水化合物、单糖、寡糖、多糖、氨基酸、肽、寡肽、多肽、蛋白质、核苷、核苷酸、寡核苷酸、多核苷酸、脂质、类维生素A、类固醇、糖肽、糖蛋白、蛋白聚糖、或有机小分子。
本发明还涉及包含WRN激活剂的组合物的用途。激活剂可以用来治疗癌症、诱导凋亡、诱导细胞衰老、抑制促进皮肤晒黑、促进细胞分化或促进免疫抑制。激活剂可以是WRN的寡核苷酸激活剂，其可与(TTAGGG)n有至少33％核苷酸序列相同性，其中n＝1-20。激活剂可以是WRN的寡核苷酸激活剂，其可与(TTAGGG)n有至少50％核苷酸序列相同性，其中n＝1-20。大约一个到大约十个最前端的3′-核苷酸键可被3′-5′核酸酶水解。
本发明还涉及包含WRN抑制剂的组合物的用途。该抑制剂可以用于抑制凋亡、抑制细胞衰老、促进生长、抑制皮肤晒黑、抑制细胞分化、或减少癌症治疗副作用。该组合物可以与化疗或电离辐射组合给与。该抑制剂可以是WRN的寡核苷酸抑制剂，其可与(TTAGGG)n有至少33％核苷酸序列相同性，其中n＝1-20。该抑制剂可以是WRN的寡核苷酸抑制剂，其可与(TTAGGG)n有至少50％核苷酸序列相同性，其中n＝1-20。大约0个到大约10个最前端的3′-核苷酸键可被3′-5′核酸酶水解。
本发明还涉及一种包含寡核苷酸的组合物，该寡核苷酸与(TTAGGG)n有至少33％核苷酸序列相同性以及有至少一个不可水解的核苷酸间键，其中n＝1-20。大约一个到大约十个最前端的3′-核苷酸键可被3′-5′核酸酶如WRN水解。所述寡核苷酸可以与TTAGGG有至少33％的核苷酸序列相同性。所述寡核苷酸也可以与TTAGGG有至少50％的核苷酸序列相同性。所述寡核苷酸还可以具有序列GTTAGGGTTAG。所述不可水解的键可以是硫代磷酸酯。所述寡核苷酸可以是PNA。
附图说明
图1A-1H示出碘化丙锭染色的Jurkat细胞(永生化T淋巴细胞)经稀释剂处理(图1A和1E)；40μM 11mer-1 pGTTAGGGTTAG(SEQ ID NO：2)处理(图1B和1F)；40μM 11mer-2 pCTAACCCTAAC(SEQ ID NO：3)处理(图1C和11G)；40μM 11mer-3 pGATCGATCGAT(SEQ ID NO：4)处理(图1D和1H)的FACS分析。Jurkat细胞在分析前用上述试剂处理48小时(图1A-1D)或72小时(图1E-1H)。
图2A-2F为细胞添加以下物质后荧光激活细胞分选结果图：图2A，稀释剂；图2B，0.4μM 11mer-1；图2C，0.4μM 11mer-1-S；图2D，稀释剂；图2E，40μM 11mer-1；图2F，40μM 11mer-1-S。
图3A-3G为细胞添加以下物质后荧光激活细胞分选结果图：图3A，稀释剂；图3B，10μM 11mer-1；图3C，10μM 11mer-1及1μM 11mer-1-S；图3D，10μM 11mer-1及5μM 11mer-1-S；图3E，10μM 11mer-1及10μM 11mer-1-S；图3F，20μM 11mer-1-S；图3G，10μM 11mer-1-S。
图4所示柱形图为用稀释剂、pTpT或pTspT处理后的细胞黑色素含量(pg/细胞)。
图5所示柱形图为经稀释剂、11mer-1或11mer-1-S处理后的细胞黑色素含量(pg/细胞)。
图6所示柱形图为经如下处理后的细胞黑色素含量(pg/细胞)：空白处理(无照射，无寡核苷酸)，或紫外线(UV)照射，或无照射但给予pTspT，或UV照射并给予pTspT。
图7所示为核苷酸序列SEQ ID NO：2的寡核苷酸图，其由硫代磷酸酯键合成。
图8所示柱形图为在正常新生人成纤维细胞培养时图7所述的硫代磷酸寡核苷酸1、2、3及4在导致衰老中的作用的检测结果，由β-半乳糖苷酶活性阳性染色细胞指示。寡核苷酸″11-1″表示用完全使用硫代磷酸酯键合成的SEQ ID NO：2处理的成纤维细胞培养物。″Dil″表示经不含寡核苷酸的稀释剂处理的成纤维细胞培养物。
图9示出T-oligo诱导p53及H2AX磷酸化的能力由于WRN敲低(knockdown)而降低。在T-oligo处理当天，WRN蛋白水平设定为在“0Hrs”。加入T-oligo(40μM，T)或稀释剂(D)，24小时或48小时后收集细胞进行Western分析。对照成纤维细胞是空白(IR，-)或经10Gy IR(IR，+)照射并且1小时后收集。western印迹用识别WRN、p53磷酸丝氨酸15或磷酸H2AX的抗体探查。
图10示出T-oligo可导致ATM相关的DNA依赖蛋白激酶(DNA-PK)催化亚基(DNA-PKCS)的磷酸化。ATM与DNA-PKCS的位置已标示出。经空白处理与经照射处理(10Gy IR)的成纤维细胞作为对照。
图11示出响应于T-oligo处理的p53的丝氨酸37被磷酸化。
图12示出WRN蛋白″感知″暴露的端粒突出端及T-oligo、导致激活ATM与DNA-PK并导致与p53以及其它底物的磷酸化的可能机制。
发明详述
本发明基于这样一个发现：WRN参与DNA损伤样信号传导，该信号传导是由端粒3′突出序列的水解引发的。端粒序列的水解引发对于应答DNA损伤的保护性细胞应答中起重要作用的信号级联，DNA损伤包括但不限于包括细胞衰老、皮肤晒黑及凋亡。如本申请及以其全文并入参考的共同未决国际专利申请No.PCT/US2004/000819所述，3’末端核酸酶消化被阻断的T-oligo丧失了激发DNA损伤应答的能力。鉴于WRN在参与DNA复制和修复及端粒维持中作为3′-5′核酸酶，所以WRN可以成为T-oligo的核″传感器″。
不局限在理论上，我们相信如UV照射、DNA氧化损伤或DNA加合物致癌的形成或年龄相关端粒缩短的DNA损伤都将降低端粒环的稳定性，使包含TTAGGG重复序列的3′突出序列暴露出来。随后，端粒相关蛋白将以序列依赖方式附着在突出端，单独或以复合体一部分的形式作为WRN的“锚”。接着，WRN开始由3′端水解端粒突出序列，引起进一步的信号级联放大，最终导致细胞周期停滞、基因诱导、凋亡与/或衰老的生物学终点。此处所列数据提示不论单独还是作为复合体如Mre11/Rad50/p95(Nbs1)复合物的一部分，WRN蛋白可以“感知”并降解T-oligo，使ATM与DNA-PK激酶活化，随后导致下游底物的磷酸化(图12)。
根据WRN在所述的信号传导途径中的作用，预期WRN激活剂可以激活DNA损伤应答途径，而不论是否存在DNA损伤或端粒环破坏。类似地，预期即使在存在DNA损伤或端粒环破坏的情况下，WRN抑制剂也可以抑制信号转导途径。
在揭示和描述本产品、组合物和方法之前，应理解本文所用术语仅仅为了表述具体的实施方案，而非意图限制。必须注意的是，除非本文另有明确说明，说明书及其权利要求书所用的单数形式″一″、″所述″同时也包括了复数情况。
本申请通篇引用了专利及公开文献，这些出版物的公开内容以其全文并入本申请作为参考，是为了更加充分的描述本发明所属技术领域的状况。
1.定义
如本文所用，术语″激活剂″是指任何可激活蛋白或使蛋白活性提高的物质。
如本文所用，术语″给予″用来描述调节剂的量时，是指该药物的单一或多次剂量。
如本文所用，术语″类似物″在用于肽或多肽时，指一种肽或多肽，其与常规氨基酸组相比包含一或多个非标准氨基酸或其他结构变化，并优选保持了至少一种生物学活性；当用于寡核苷酸时，指一种寡核苷酸，其包含一或多个除核苷酸间的磷酸二酯键外的核苷酸间键，并优选保持了至少一种生物学活性。
如本文所用，术语″抗体″指IgG、IgM、IgA、IgD或IgE类抗体或其片段或衍生物，包括Fab、F(ab′)2、Fd和单链抗体，双抗体(diabody)、双特异性抗体、双功能抗体及其衍生物。所述抗体可为单克隆抗体、多克隆抗体、亲和性纯化抗体或其混合物，其对所需表位或衍生于其的序列显示出充分的结合特异性。所述抗体亦可为嵌合抗体。所述抗体可为本领域公知的一或多个化学物、肽或多肽部分附着所衍生化。所述抗体可与化学部分缀合。
如本文所用，术语″凋亡″指细胞的一种死亡形式，包括但不限于细胞体积进行性萎缩并保持胞质细胞器的完整性；光镜或电镜下可见的染色质固缩(即核固缩)；和/或DNA断裂为可通过离心沉降法检测到的核小体大小的片段。当细胞失去膜完整性时(例如质膜出泡)并伴有吞噬细胞对完整细胞碎片(凋亡小体)的吞食时，细胞死亡。
如本文所用，术语″生物学活性″在用于肽或多肽的类似物、衍生物、片段、同源物或变体时，指所述类似物、衍生物、片段、同源物或变体保留了所述肽或多肽的至少一种活性，包括但不限于被特异性抗体结合的能力；在用于WRN时，包括但不限于解旋酶活性、3′-5′核酸外切酶活性及与MRN复合物相互作用的能力；在用于寡核苷酸的类似物、衍生物、片段、同源物或变体时，指所述类似物、衍生物、片段、同源物或变体如Maniatis et al.，在Molecular Cloning(A Laboratory Manual)，Cold Spring Harbor Laboratory，1982(其内容引入本申请作为参考)所述在严格杂交条件下与所述寡核苷酸进行杂交。
如本文所用，术语″癌症治疗″指本领域公知的任何治疗癌症的方法，包括但不限于化疗与放射疗法。
如本文所用，术语″组合″用来描述给予一种调节剂及另外的治疗时，指所述调节剂可在另外的治疗之前、之后或同时或其组合时给予。
如本文所用，术语″衍生物″在文中用于肽或多肽时，指其存在除一级结构(氨基酸与氨基酸类似物)外的差别并优选保持了至少一种生物学活性的肽或多肽；在文中用于寡核苷酸时，指其存在除核苷酸序列外的差别并优选保持了至少一种生物学活性的寡核苷酸。值得注释的是，肽或多肽的衍生物可通过糖基化，即一种翻译后修饰来区别。例如，由于在异源系统中表达，肽或多肽可能具有糖基化形式。如果保留了至少一种生物学活性，那么这些肽或多肽是本发明的衍生物。其他衍生物包括但不限于具有共价修饰的N-或C-端的融合肽或融合多肽、PEG化的肽或多肽、与脂质部分结合的肽或多肽、烷基化的肽或多肽、通过氨基酸侧链功能基团与其他肽、多肽或化学物连接的肽或多肽以及本领域所理解的其它修饰形式。
如本文所用，术语″片段″在文中用于肽或多肽时，指任何肽或多肽片段，优选约5-300个氨基酸长，更优选约8-50个氨基酸长，并优选保持了至少一种生物学活性；在文中用于寡核苷酸时，指任何寡核苷酸片段，优选是约2-250个核苷酸，更优选约2-20个核苷酸长，并优选保持了至少一种生物学活性。典型的肽或多肽片段的例子为8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个氨基酸长。典型的寡核苷酸片段的例子为2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸长。
如本文所用，术语″同源物″在文中用于肽或多肽时，指具有共同进化祖先或具有至少50％的同源性并优选保持了至少一种生物学活性的肽或多肽；在文中用于寡核苷酸时，指具有共同进化祖先或具有至少50％的同源性并优选保持了至少一种生物学活性的寡核苷酸。
如本文所用，术语″抑制″在用于蛋白活性时指防止、遏制、阻止或消除酶活性。
如本文所用，术语″治疗″在用于保护哺乳动物防止发生疾病时指防止、遏制、阻止或消除所述疾病。防止所述疾病包括在该疾病发生前给予哺乳动物本发明的组合物。遏制所述疾病包括在该疾病发生之后但在其临床症状之前给予哺乳动物本发明的组合物。阻止所述疾病包括在该疾病的临床症状发生后给予哺乳动物本发明的组合物使所述疾病减轻或防止恶化。消除所述疾病包括在该疾病的临床症状发生后给予哺乳动物本发明的组合物使该哺乳动物不再患有所述疾病。
如本文所用，术语″变体″在文中用于肽或多肽时，指由于氨基酸的插入、缺失或保守置换造成氨基酸序列差异并优选保持了至少一种生物学活性的肽或多肽；在文中用于寡核苷酸时，指由于核苷酸的插入、缺失或置换造成核苷酸序列差异并优选保持了至少一种生物学活性的寡核苷酸。
2.调节剂
a.WRN调节剂
本发明涉及WRN活性调节剂。该调节剂可以诱导或增强WRN活性。该调节剂也可以抑制或降低WRN活性。该调节剂可以是人工合成的化合物或是自然存在的化合物。该调节剂可以是低分子量化合物、寡核苷酸、多肽或肽、或其片段、类似物、同源物、变体或衍生物。
寡核苷酸调节剂可以是与(TTAGGG)n具有至少大约33％到100％或至少50％到大约100％核苷酸序列相同性的寡核苷酸，n为大约1到20。如本文所用，″(TTAGGG)n″在文中用于对比核酸序列时，作为参考核酸。序列相同性通过将寡核苷酸与参考核酸进行比对并用(a)比对相同的核苷酸数目除以(b)总的寡核苷酸碱基对数目来计算。例如，11bp的寡核苷酸序列GTTAGGGTTAG与(TTAGGG)2具有＞91％的序列相同性。
所述寡核苷酸可以是包括但不限于单链、双链或其组合的形式。所述寡核苷酸优选包含大约2至大约2000个核苷酸、更优选为大约2至大约200个核苷酸单链3′末端。所述寡核苷酸也可以是EST。可特别预期的还可以是所述寡核苷酸的类似物、衍生物、片段、同源物或变体。
如实施例所示，本发明中的某些寡核苷酸在细胞中抑制细胞增殖及诱导凋亡，而本发明的其它寡核苷酸抑制生长停滞及抑制凋亡。寡核苷酸活性的不同依赖于3′可水解核苷酸间键的数目。改变3′可水解核苷酸间键的数目，则该寡核苷酸的作用也改变。
不局限在理论上，我们认为寡核苷酸被WRN识别并作为3′外切酶WRN的底物。其推论是，包含3′不可水解核苷酸间键的底物寡核苷酸作为WRN的拮抗剂或抑制剂。决定WRN活性水平的其它因素包括但不限于3′可水解核苷酸间键的总浓度、碱基序列和G含量。
如果(i)一个核苷酸间键是在生理条件下可被WRN水解的磷酸二酯键或其类似物，及(ii)所有位于其3′方向的核苷酸间键也都是可水解的，则根据本发明，认为该核苷酸间键是可水解的。如果核苷酸间键在生理条件下不被WRN水解，则根据本发明，认为其是不可水解的，无论3′端可水解的核苷酸间键的数目为何。不可水解的核苷酸间键的代表性例子包括但不限于硫代磷酸酯键和肽核酸键(PNA)。
在本发明的一个实施方案中，所述寡核苷酸包含可水解核苷酸间键。该寡核苷酸可以包含约1至约200个可水解核苷酸间键。所述寡核苷酸也包含不可水解核苷酸间键。该寡核苷酸可包含约0至约199个不可水解核苷酸间键。
在另一实施方案中，寡核苷酸包含不可水解键。该寡核苷酸可包含约1至约200个不可水解核苷酸间键。寡核苷酸也可以包含可水解核苷酸间键。该寡核苷酸包含约0-5个可水解核苷酸间键。优选的寡核苷酸是本申请所述和如并入本文参考的2002年4月12日提交的共同未决美国专利申请No.10/122,630所述的T-oligo。
b.DNA损伤途径调节剂
本发明还涉及DNA损伤途径的调节剂。该调节剂可以诱导DNA损伤途径。该调节剂也可以抑制DNA损伤途径。所述调节剂可以是人工合成化合物或天然存在的化合物。该调节剂可以是低分子量化合物、多肽或肽、或其片段、类似物、同源物、变体或衍生物。
3.组合物
本发明还涉及包含上述调节剂的组合物。该组合物可以包含WRN激活剂。该组合物也可以包含WRN抑制剂。该组合物还可以包含超过一种的本发明的调节剂。该组合物亦可以包含一或多种调节剂以及其它治疗剂。
在本发明的一个实施方案中，该组合物包含了本发明的寡核苷酸。该寡核苷酸可以包含可水解核苷酸间键或不可水解核苷酸间键或其组合。在一个优选的实施方案中，该寡核苷酸是WRN激活剂。在另一个优选实施方案中，寡核苷酸是WRN抑制剂。如上所述，基于可水解核苷酸间键的总浓度，可以调节寡核苷酸的活性以诱导或者抑制WRN。
a.制剂
本发明的组合物可按常规方式制成片剂或锭剂。例如，口服片剂和胶囊可含有常规赋形剂，包括但不限于粘合剂、填充剂、润滑剂、崩解剂和增湿剂。粘合剂包括但不限于糖浆、阿拉伯树胶、明胶、山梨醇、黄蓍胶、淀粉胶和聚乙烯吡咯烷酮。填充剂包括但不限于乳糖、蔗糖、微晶纤维素、玉米淀粉、磷酸钙和山梨醇。润滑剂包括但不限于硬脂酸镁盐、硬脂酸、滑石、聚乙二醇和二氧化硅。崩解剂包括但不限于马铃薯淀粉和淀粉乙醇酸钠。增湿剂包括但不限于月桂硫酸酯钠。片剂可按本领域熟知方法包衣。
本发明的组合物亦可为液体制剂，包括但不限于含水或含油悬液、溶剂、乳状液、糖浆和酏剂。该组合物也可制成干产物，使用前用水或其他合适的载体建立(constitution)。这些液体制剂可含有添加剂，包括但不限于悬浮剂、乳化剂、非水载体及防腐剂。悬浮剂包括但不限于山梨醇糖浆、甲基纤维素、葡萄糖/蔗糖糖浆、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶和氢化食用脂肪。乳化剂包括但不限于卵磷脂、脱水山梨糖醇单油酸酯和阿拉伯树胶。非水载体包括但不限于食用油类、杏仁油、分馏椰子油、油酯、丙二醇和乙醇。防腐剂包括但不限于对羟基苯甲酸甲酯或对羟基苯甲酸丙酯和山梨酸。
本发明的组合物亦可制为栓剂，其可以含有栓剂基质，包括但不限于可可脂或甘油酯。本发明的组合物亦可制为吸入剂，其可以为包括但不限于以干粉给予的溶液、悬液或乳浊液的形式或以使用喷射剂(如二氯二氟甲烷或三氯氟甲烷)的气雾剂的形式。本发明的组合物亦可制成包含水或非水载体的经皮制剂，包括但不限于乳膏、软膏、洗液、浆糊(paste)、药膏(medicated plaster)、药膏(patch)或膜。
本发明的组合物亦可制为胃肠外给予，包括但不限于通过注射或连续输液。注射制剂可为含油或含水载体的悬液、溶液或乳状液，并可含配制剂包括但不限于悬浮剂、稳定剂和分散剂。该组合物亦可以干粉形式提供，用合适的载体包括但不限于无菌、无热源水重建(reconstitution)。
本发明的组合物还可制为贮存制品(depot preparation)，其可通过植入或肌内注射给予。该组合物可以用合适的聚合物或疏水材料(例如，可接受油的乳浊液)、离子交换树脂或略溶性衍生物(例如略溶性盐)来配制。
本发明的组合物还可制为脂质体制品。脂质体制品可含有可以穿透感兴趣细胞或角质层并能够与细胞膜融合的脂质体，最终将脂质体内涵物传送入细胞。例如，可使用以下专利所描述的脂质体：Yarosh的美国专利5,077,211、Redziniak et al.的美国专利4,621,023或Redziniak et al.的美国专利4,508,703。用于靶向皮肤病的本发明组合物可以在哺乳动物皮肤暴露于紫外线或引起氧化损害的因素之前、之中或之后给予。其他合适的制剂还应用niosome。Niosome是与脂质体类似的脂质载体，其具有主要由非离子脂质组成的膜，其一些形式可以有效的携带化合物穿过角质层。
4.治疗方法
a.WRN激活剂
本发明诱导或增强WRN活性的调节剂可单独或与其他治疗方式组合用于治疗与生长停滞、凋亡或增殖性衰老的机制丧失相关的疾病。这些病症的代表性例子包括但不限于过度增生性疾病，如癌症和超过正常范围的细胞的良性生长，例如在牛皮癣、成纤维细胞肥厚性瘢痕与瘢痕疙瘩中的角质细胞的良性生长；或在某种自身免疫紊乱中的某些淋巴细胞亚群的良性生长。这些方法治疗的癌症具有多样化的表现形式且产生自机体的不同细胞类型及器官中，例如宫颈癌、淋巴瘤、骨肉瘤、黑色素瘤及其他皮肤癌和白血病。所述疗法指向的癌细胞的类型包括乳腺、肺、肝、前列腺、胰、卵巢、膀胱、子宫、结肠、脑、食道、胃和甲状腺。所述调节剂也可用来抑制促进皮肤晒黑、促进细胞分化和免疫抑制。
在本发明的一个实施方案中，包含可水解核苷酸间键的本发明寡核苷酸用于通过给予有需要这种治疗的患者所述寡核苷酸来治疗与生长停滞、凋亡或增殖性衰老的机制丧失相关的疾病。该寡核苷酸也可包含不可水解核苷酸间键。如上所述，寡核苷酸的活性可以基于可水解核苷酸间键的总浓度来调节以诱导生长停滞或凋亡。该寡核苷酸可与本发明中的调节剂或其他治疗方式组合给予。
在一个优选实施方案中，寡核苷酸用来治疗癌症，所述癌症选自宫颈癌、淋巴瘤、骨肉瘤、黑色素瘤、皮肤癌、白血病、乳腺癌、肺癌、肝癌、前列腺癌、胰腺癌、卵巢癌、膀胱癌、子宫癌、结肠癌、脑癌、食道癌、胃癌和甲状腺癌。
T-oligo在小鼠模型中可如UV照射一样有效地阻断过敏性接触性超敏反应的诱导与引发，其通过上调已知免疫抑制介质TNF-α与IL10实现。因此，局部或全身性的WRN激活剂可以代替类固醇治疗，例如牛皮癣、湿疹的淋巴细胞介导的皮肤疾病以及淋巴细胞介导的系统性疾病如类风湿性关节炎、多发性硬化、红斑狼疮和许多其他的疾病的治疗。
b.WRN抑制剂
本发明的可抑制或降低WRN活性的调节剂可单独或与其他疗法组合用来治疗与生长停滞、凋亡或增殖性衰老相关的疾病。这种疾病的代表性例子包括但不限于暴露于UV照射和如化疗和放疗的癌症治疗对正常组织的副作用或促进抑制暴露于日光引起的正常皮肤晒黑的应答。该调节剂也可以用来抑制细胞分化。
在另一个实施方案中，含不可水解核苷酸间键的本发明寡核苷酸用于通过给予有需要这种治疗的患者所述寡核苷酸来治疗与生长停滞或凋亡相关的疾病。该寡核苷酸也可包含可水解核苷酸间键。如上所述，寡核苷酸的活性可以基于可水解核苷酸间键的总浓度来调节以抑制生长停滞或抑制凋亡。该寡核苷酸可与本发明调节剂或其他治疗方式组合给予。
在一个优选的实施方案中，该寡核苷酸被用来治疗的疾病选自UV辐射暴露和如化疗和放疗癌症治疗方法的副作用。
c.给予
本发明的组合物可以任何方式给予，包括但不限于口服、胃肠外、舌下、经皮、直肠、跨粘膜、局部、吸入、口腔给予或其组合。胃肠外给予包括但不限于静脉内、动脉内、腹膜内、皮下、肌内、鞘内及关节内给予。
d.剂量
用于治疗所需的组合物的治疗有效量根据要治疗疾病的性质、所需活性的时间长短以及病人的年龄和状况变化，并最后由主治医师作出决定。通常，用于成人治疗的典型剂量范围是0.001mg/kg到大约200mg/kg/天。剂量可以是约1μg/kg到约100μg/kg/天。所需剂量应方便单剂量给予或以合适的间隔多剂量给予，例如每天二次、三次、四次或更多次亚剂量。通常多剂量给药是需要或必需的。
调节剂的剂量可是任何剂量，包括但是不限于约1μg/kg、25μg/kg、50μg/kg、75μg/kg、100μg/kg、125μg/kg、150μg/kg、175μg/kg、200μg/kg、225μg/kg、250μg/kg、275μg/kg、300μg/kg、325μg/kg、350μg/kg、375μg/kg、400μg/kg、425μg/kg、450μg/kg、475μg/kg、500μg/kg、525μg/kg、550μg/kg、575μg/kg、600μg/kg、625μg/kg、650μg/kg、675μg/kg、700μg/kg、725μg/kg、750μg/kg、775μg/kg、800μg/kg、825μg/kg、850μg/kg、875μg/kg、900μg/kg、925μg/kg、950μg/kg、975μg/kg或1mg/kg。
5.筛选方法
本发明还涉及鉴定WRN活性调节剂的筛选方法。此外，本发明涉及鉴定DNA损伤途径调节剂的筛选方法。筛选方法可以多种方式进行，包括但不限于体外检测、基于细胞的检测、遗传学检测和体内检测。
WRN调节剂可通过筛选与WRN特异性结合的底物而鉴定。本领域技术人员可通过使用许多标准技术体外鉴定特异结合底物，所述技术包括但不限于免疫沉淀和亲和层析。本领域技术人员也可通过使用许多标准技术应用遗传筛选来鉴定特异结合底物，所述技术包括但不限于酵母双杂交与噬菌体展示。特异结合底物还可通过使用高通量筛选方法包括但不限于将WRN附着到固体基质如芯片上(例如玻璃、塑料或硅)来进行鉴定。
WRN调节剂也可通过体外筛选调节WRN活性的底物来鉴定。通过将WRN与疑似调节剂接触、确定疑似调节剂是否可以改变WRN的活性从而鉴定调节剂。WRN的活性可通过测定WRN的核酸底物的水解而测定。测定核酸底物水解的方法包括但不限于测量UV吸收以及优选标记的寡核苷酸的凝胶分析或非沉淀核苷酸碱基的回收。
WRN调节剂可通过筛选在基于细胞的分析中调节WRN活性的底物而鉴定。类似地，DNA损伤途径的调节剂也可得到鉴定。使疑似调节剂与细胞接触并测定疑似调节剂是否可以改变凋亡、衰老、或p53、p95、ATM、H2AX的磷酸化、S期停滞的诱导或凋亡诱导的水平从而鉴定调节剂。如上所述，候选调节剂可以是特异性结合WRN的底物。测定凋亡调节的方法包括但不限于在FACS分析中测定sub-G0/G1峰的大小、TUNEL分析、DNAladder分析、膜联蛋白分析或ELISA分析。衰老的调节可以通过检测衰老相关的β-半乳糖苷酶活性或细胞产量增加的失败或磷酸化pRb的失败或促有丝分裂刺激后的3H-胸腺嘧啶掺入的失败来确定。p53活性的调节可以通过凝胶迁移分析测定p53丝氨酸15或丝氨酸37的磷酸化来进行检测，可通过p53启动子驱动的CAT或荧光素酶构建体的读取(read-out)进行测定，或通过p53调节的基因产物如p21的诱导进行测定。p95活性的调节可以通过在western印迹分析中p95条带的迁移从而检测p95的丝氨酸343的磷酸化或由FACS分析检测S期停滞来进行测定。WRN调节剂还可通过筛选在体内调节肿瘤发生的底物来鉴定。
任何细胞都可用于基于细胞的分析。优选地，本发明中使用的细胞包括哺乳动物细胞，更优选的是人类细胞与非人灵长类细胞。合适细胞的代表性例子包括但不限于原代(正常)人类皮肤成纤维细胞、表皮角质细胞、黑色素细胞和相应的永生化或转化细胞系；以及原代、永生化的或转化的鼠细胞系。蛋白磷酸化的量可通过使用本领域技术标准来测定，包括但不限于比色法、发光法、荧光测定法和western印迹。
将疑似调节剂通过如混合的方式加入细胞中的条件是这样的条件，其中在基本上没有其他可干扰凋亡或信号传导的调节化合物存在的情况下，该细胞会发生凋亡或信号转导。有效的条件包括但不限于允许细胞生长的合适的培养基、温度、pH和氧条件。合适的培养基典型是固体或液体培养基，其包含生长因子及可同化的碳、氮和磷源，以及合适的盐、矿物质、金属及其他营养物如维生素，还包括细胞可以在其中进行培养以展示凋亡或信号传导的有效的培养基。例如，对于哺乳动物细胞来说，所述培养基可为含10％胎牛血清的Dulbecco modified Eagle′s培养基。
细胞可在多种容器中培养，所述容器包括但不限于组织培养瓶、试管、微孔板和培养板(petri plate)。培养应在具有对细胞适宜的温度、pH和二氧化碳含量的条件下进行。这些培养条件也是本领域常规技术。
将疑似调节剂加入细胞的方法包括电穿孔、显微注射、细胞表达(即使用一个表达系统，包括裸核酸分子、重组病毒、逆转录病毒表达载体和腺病毒表达)、在培养基中加入试剂、使用金属配对试剂以及使用用于细胞通化的表面活性剂。
候选调节剂可以是天然分子，如碳水化合物、单糖、寡糖、多糖、氨基酸、肽、寡肽、多肽、蛋白质、核苷、核苷酸、寡核苷酸、多核苷酸，包括DNA及DNA片段、RNA及RNA片段等、脂质、类视黄醇(retinoid)、类固醇、糖肽、糖蛋白、蛋白聚糖等；或天然分子的类似物及衍生物，如肽模拟物(peptidomimetics)等；以及非天然分子，如″小分子″有机化合物。术语″小分子有机化合物″通常指分子量小于大约1000、优选小于大约500的有机化合物。
候选调节剂可能存在于文库(即化合物的集合)中，该文库可以任何方法制备或获得，包括但不限于组合化学技术、发酵方法、植物与细胞提取等。制备组合文库的方法为本领域所熟知。参见例如E.R.Felder，Chimia 1994，48，512-541；Gallop et al.，J.Med.Chem.1994，37，1233-1251；R.A.Houghten，Trends Genet.1993，9，235-239；Houghten et al.，Nature 1991，354，84-86；Lamet al.，Nature 1991，354，82-84；Carell et al.，Chem.Biol.1995，3，171-183；Madden et al.，Perspectives in Drug Discovery and Design 2，269-282；Cwirlaet al.，Biochemistry 1990，87，6378-6382；Brenner et al.，Proc.Natl.Acad.Sci.USA 1992，89，5381-5383；Gordon et al.，J.Med.Chem.1994，37，1385-1401；Lebl et al.，Biopolymers 1995，37177-198及本文引用的参考文献。
以下非限制性实施例举例说明了本发明的多个方面。
实施例
实施例1
寡核苷酸可诱导凋亡
将与端粒突出端重复序列(TTAGGG；SEQID NO：1)同源的寡核苷酸、序列(11mer-1：pGTTAGGGTTAG；SEQ ID NO：2)、与这个序列互补(11mer-2：pCTAACCCTAAC；SEQID NO：3)以及端粒序列不相关的(11mer-3：pGATCGATCGAT；SEQ ID NO：4)加入Jurkat细胞培养物中，Jurkat细胞是一种人类T细胞系，据报导其可由于应答端粒破坏而发生凋亡。在48小时内，用40μM的SEQ ID NO：2处理的细胞有50％积聚在S期，而对照细胞相比只有25-30％(p＜0.0003，非配对t-检验；见图1A-1D)；72小时，通过sub-G0/G1 DNA含量确定13％的这些细胞凋亡，对照组相比为2-3％(p＜0.007，非配对t-检验；见图1E-1H)。96小时，11mer-1处理的细胞有20±3％凋亡，对照组为3-5％(p＜0.0001，非配对t-检验)。为排除11mer-1所独有的效应是由于其被优先摄取所引起的，使用3′端以亚磷酰胺荧光素标记的寡核苷酸处理Jurkat细胞，然后进行共聚焦显微镜与FACS分析。细胞的荧光强度在所有处理后4小时和24小时相同。Western分析显示在加入11mer-1 24小时后p53增加以及转录因子E2F1水平伴随增加，但在11mer-2或11mer-3中未见，已知E2F1在诱导凋亡过程中与p53协同作用并可以p53依赖方式诱导人类成纤维细胞衰老表型以及调节S期关卡(checkpoint)。
实施例2
端粒突出端同源物11mer-1的硫代磷酸物不诱导凋亡
将Jurkat人类T细胞培养物用稀释剂、11mer-1(SEQ ID NO：1)或硫代磷酸11mer-1(11mer-1-S)处理96小时，然后收集进行FACS分析。检测两种寡核苷酸浓度，0.4μM(图2A-2C)与40μM(图2D-2F)。在0.4μM，两种寡核苷酸都不能影响预期的Jurkat细胞的对数生长细胞周期图。在40μM，sub-G0/G1峰显示11-mer-1诱导了广泛的凋亡，而11mer-1-S没有作用。
实施例3
11mer-1的硫代磷酸物阻断了磷酸骨架11mer-1诱导的S期停滞
仅用11mer-1(SEQ ID NO：2)或在11mer-1-S浓度提高的情况下用11mer-1处理角质细胞系(SSC12F，100,000细胞/38cm2)培养物48小时。如前述实施例1所示，通过FACS(Becton-Dickinson FacScan)证明11mer-1诱导了S期停滞。43％的细胞处于S期，在经稀释剂处理的对照细胞中为26％。然而，在提高浓度的硫代磷酸11mer-1加入这些培养物中时，几乎没有细胞被停滞(图3A-3G)。当11mer-1∶11mer-1-S的比率为2∶1时可见这种停滞被完全抑制。11mer-1-S自身不诱导S期停滞。
实施例4
硫代磷酸形式的端粒寡核苷酸减弱了组成性和UV诱导的色素沉积且不刺激黑素原生成
用100μM的pTpT或硫代磷酸pTpT(pTspT)(图4)或40μM的11mer-1或硫代磷酸11mer-1(11mer-1-S)(图5)处理S91小鼠黑色素瘤细胞(100,000细胞/38cm2)培养物6天，然后收集，计数，并检测黑色素含量。pTpT和11mer-1(分别见图4和图5)刺激了这些细胞中的黑素原生成，而pTspT和11mer-1-S则没有(分别见图4和图5)。此外，pTspT(图4)和11mer-1-S(图5)都减少了这些细胞中的组成性色素沉积，提示在端粒修复/复制时这个序列的慢性暴露可提供持续的、低水平的黑素原生成信号而且这个信号被pTspT与11mer-1-S阻断。
实施例5
硫代磷酸pTspT抑制UV诱导的黑素原生成
对双份S91细胞培养物(100,000细胞/39cm2)进行假照射或以来自1kW氙弧太阳模拟器(XMN 1000-21，Optical Radiation，Azuza，CA)的5mJ/cm2太阳模拟光进行照射，使用研究性辐射计(型号IL1700A，InternationalLight，Newburyport，MA)将该模拟器设定在285-5nm处。两个空白照射板随后补加100μM的pTspT，两个照射培养物也用pTspT类似处理。一周后，收集细胞，计数，使用1N NaOH溶解细胞沉淀并在475nm处测量光密度来分析黑色素含量。UV照射使这些细胞中的黑色素含量加倍。然而，加入pTspT可以阻断这种应答(图6)。另外，pTspT在空白照射培养物中减少了组成性色素沉积，与图4及图5所示数据类似。
实施例6
水解T-oligo对于活性是必需的
合成基于SEQ ID NO：2的寡核苷酸。寡核苷酸1完全以硫代磷酸骨架合成。寡核苷酸2在每个末端都具有两个硫代磷酸酯键，其他中间部分的键均为磷酸二酯键。寡核苷酸3在5′末端具有二个硫代磷酸酯键(5′端阻断)，其余键均为磷酸二酯键。寡核苷酸4在3味端具有二个硫代磷酸酯键(3′端阻断)，其余键均为磷酸二酯键。参见图7。
将这些寡核苷酸加入正常的新生成纤维细胞培养物中。48小时后收集细胞并通过western印迹分析p53丝氨酸15磷酸化以及p95/Nbsl磷酸化。其它培养物在寡核苷酸存在的情况下继续培养一周，然后对衰老相关β-半乳糖苷酶活性(SA-β-Gal)进行细胞染色，β-半乳糖苷酶阳性细胞被记分，并计算占细胞总数的百分比(图8)。
具有核酸酶可接近3′末端的寡核苷酸在刺激″早期″应答如p53和p95/Nbsl磷酸化时是最有效的。而具有核酸酶可接近5′末端的寡核苷酸也能够在一周后诱导衰老表型，但是在48小时不引起磷酸化反应，提示3′-5′核酸酶的敏感性对于诱导衰老的活性是优选的。
实施例7
下调WRN蛋白水平阻断T-oligo应答
用50ρmol的WRN siRNA或50ρmol针对对照绿色荧光蛋白(GFP)的siRNA处理正常新生成纤维细胞连续两天。第二次siRNA处理后一天，收集代表性的培养物做wester印迹分析以评估WRN siRNA去除所述蛋白的效力。同时，双份WRN siRNA处理和GFP siRNA处理的培养物给予40μM T-oligo(11mer-1；SEQ ID NO：2)或等量的稀释剂。在加入T-oligo或稀释剂后1或2天收集所有条件的细胞并通过western印迹分析检测p53丝氨酸15的磷酸化及组蛋白H2AX的磷酸化，这两种现象均能很好的记录DNA损伤应答(Lambert et al.，1998，J Biol Chem 273，33048-53；Burma et al.，2001，J Biol Chem 276，42462-7)。作为对照，包括来自空白照射细胞和用10Gy电离辐射(IR)照射的细胞的细胞裂解物。印迹用特异于WRN、p53磷酸丝氨酸15和磷酸H2AX的抗体探查。图9所示数据表明WRN的“敲低”显著降低了第0天(加入T-oligo当天)的WRN水平以及T-oligo诱导p53和H2AX磷酸化的能力。
实施例8
DNA-PK介导T-oligo作用
用40μM的T-oligo或等体积的稀释剂处理正常的新生儿成纤维细胞4、6、8、16、24或48小时，然后收集并用抗ATM磷酸丝氨酸1981的抗体通过western印迹分析。图10显示T-oligo处理在成纤维细胞中导致表观分子量为450kDa的蛋白质磷酸化，该蛋白质迁移至ATM(370kDa)的分子标准的上方。在后续实验中，此磷酸化蛋白质示出与ATM相关的DNA依赖性蛋白激酶(DNA-PKcs)的催化亚基共迁移，有理由预期其结合抗高度同源的ATM蛋白的抗体。
正如T-oligo处理后的情形(图10)所示，由于DNA-PKcs在激活时自身磷酸化(Ding et al.，2003，Mol Cell Biol 23，5836-48)并且通过异源二聚体Ku蛋白(DNA-PK复合物的一部分)与WRN相关，所以分析成纤维细胞的p53丝氨酸37的磷酸化，该位点显示被DNA-PK磷酸化(Lees et al.，1992，Mol Cell Biol 12，5041-9)。新生儿成纤维细胞如上述处理并类似地用抗丝氨酸37磷酸化的p53抗体进行分析。Western分析显示p53丝氨酸37在应答T-oligo处理时被磷酸化(图11)。
Claims (3)
1.一种鉴定WRN调节剂的方法，包括：
(a)提供一种表达WRN的细胞；
(b)在候选调节剂被该细胞摄取的条件下将候选调节剂与所述细胞接触；
(c)进一步将步骤(b)的所述细胞和步骤(a)的对照细胞和寡核苷酸GTTAGGGTTAG接触，及
(d)测量步骤(c)的所述细胞中的p53磷酸化，
其中与仅和所述寡核苷酸接触的所述对照细胞相比，所述和候选调节剂以及所述寡核苷酸接触的细胞中的p53磷酸化的变化指示所述候选调节剂是WRN调节剂。
2.根据权利要求1的方法，其中所述细胞是癌细胞。
3.根据权利要求2的方法，其中所述的候选调节剂选自碳水化合物、氨基酸、肽、蛋白质、核苷、核苷酸、寡核苷酸、多核苷酸、脂质、类维生素A、类固醇、糖肽、糖蛋白和蛋白聚糖。
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| EP (2) | EP2434009A1 (zh) |
| JP (1) | JP2007537758A (zh) |
| KR (1) | KR20070030219A (zh) |
| CN (2) | CN101869575A (zh) |
| AU (1) | AU2005245932A1 (zh) |
| BR (1) | BRPI0511142A (zh) |
| CA (1) | CA2566859C (zh) |
| HK (1) | HK1104062A1 (zh) |
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| US20100086941A1 (en) * | 2008-10-01 | 2010-04-08 | Adami Guy R | Methods for determining aged based accumulation of senescent cells using senescence specific DNA damage markers |
| US20110052676A1 (en) * | 2009-09-01 | 2011-03-03 | James Vincent Gruber | Composition For Delaying Cellular Senescence |
| RU2550267C2 (ru) * | 2012-12-12 | 2015-05-10 | Елена Андреевна Чирясова | Способ воздействия на пролиферативный статус клеток с помощью специфических нуклеотидных последовательностей g-цепи теломерной днк человека |
| US11530413B2 (en) | 2017-07-21 | 2022-12-20 | Novartis Ag | Compositions and methods to treat cancer |
| US20210371855A1 (en) * | 2018-06-15 | 2021-12-02 | Ideaya Biosciences, Inc. | Methods of inhibiting proliferative cells |
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| FR2521565B1 (fr) | 1982-02-17 | 1985-07-05 | Dior Sa Parfums Christian | Melange pulverulent de constituants lipidiques et de constituants hydrophobes, procede pour le preparer, phases lamellaires lipidiques hydratees et procede de fabrication, compositions pharmaceutiques ou cosmetiques comportant des phases lamellaires lipidiques hydratees |
| FR2534487B1 (fr) * | 1982-10-15 | 1988-06-10 | Dior Christian Parfums | Procede d'homogeneisation de dispersions de phases lamellaires lipidiques hydratees, et suspensions obtenues par ce procede |
| US5077211A (en) * | 1988-07-06 | 1991-12-31 | Applied Genetics, Inc. | Purification and administration of dna repair enzymes |
| WO2001074342A2 (en) * | 2000-03-31 | 2001-10-11 | Trustees Of Boston University | Use of locally applied dna fragments |
| US6090620A (en) * | 1995-12-29 | 2000-07-18 | University Of Washington | Genes and gene products related to Werner's syndrome |
| US6228583B1 (en) * | 1997-08-04 | 2001-05-08 | Massachusetts Institute Of Technology | Assays for compounds which extend life span |
| SE0200929D0 (sv) | 2002-03-26 | 2002-03-26 | Alstom Switzerland Ltd | An apparatus and method for suspending a stator core of an electric generator |
| AU2004233188A1 (en) | 2003-04-11 | 2004-11-04 | Trustees Of Boston University | Modulation of telomere-initiated cell signaling |
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Non-Patent Citations (2)
| Title |
|---|
| P. Pichierri 等人.Werner＇s syndrome lymphoblastoid cellsarehypersensitivetotopoisomerase II inhibitors in the G2 phaseofthe cell cycle..Mutation Research459 2.2000,459(2),正文127页2.1-130页3.1节. |
| P. Pichierri 等人.Werner＇s syndrome lymphoblastoid cellsarehypersensitivetotopoisomerase II inhibitors in the G2 phaseofthe cell cycle..Mutation Research459 2.2000,459(2),正文127页2.1-130页3.1节. * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2005113764A3 (en) | 2006-03-30 |
| CN1965076A (zh) | 2007-05-16 |
| TR200606490A2 (tr) | 2008-05-21 |
| KR20070030219A (ko) | 2007-03-15 |
| EP2434009A1 (en) | 2012-03-28 |
| US20080221052A1 (en) | 2008-09-11 |
| AU2005245932A1 (en) | 2005-12-01 |
| BRPI0511142A (pt) | 2007-12-04 |
| RU2006144817A (ru) | 2008-06-27 |
| CA2566859A1 (en) | 2005-12-01 |
| NZ551323A (en) | 2010-03-26 |
| EP1761629A2 (en) | 2007-03-14 |
| MXPA06013263A (es) | 2007-08-14 |
| JP2007537758A (ja) | 2007-12-27 |
| NO20065782L (no) | 2006-12-18 |
| WO2005113764A2 (en) | 2005-12-01 |
| IL179199A0 (en) | 2007-03-08 |
| HK1104062A1 (en) | 2008-01-04 |
| CA2566859C (en) | 2011-07-19 |
| TR200606490T1 (tr) | 2007-01-22 |
| IL179199A (en) | 2010-12-30 |
| CN101869575A (zh) | 2010-10-27 |
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