CN102137927B - 鉴定脆性组氨酸三联体(Fhit)相互作用的方法及其用途 - Google Patents
鉴定脆性组氨酸三联体(Fhit)相互作用的方法及其用途 Download PDFInfo
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Abstract
本文提供使用Fhit基因诊断、预后和治疗癌症相关疾患的方法和组合物。
Description
对相关申请的交叉引用和关于资助研究的声明
本发明要求2007年10月26日提交的临时专利申请序列第60/000,480号的权益。本发明是在NCI资助号CA77738和CA78890的政府支持下进行的。政府在本发明中享有某些权利。
发明背景
FHIT基因包括染色体3p14.2的最具活性的常见的脆性位点(1,2)。由于等位基因丢失、基因组重排、启动子超甲基化或其组合,Fhit表达在大多数类型的人肿瘤的大部分中是丢失的或降低的(3,4)。Fhit敲除小鼠显示对癌症发展的增加的易感性(5,6),并且FHIT基因治疗在暴露于致癌原的Fhit-缺陷小鼠中预防肿瘤(7,8)。癌细胞中通过稳定转染的Fhit恢复在体外几乎没有作用,除非将细胞暴露于应激,包括体内裸小鼠环境应激(9);病毒介导的Fhit恢复,一种同时提供应激和Fhit表达的过程,在体内遏制肿瘤发生并在体外触发许多类型的恶性细胞的凋亡(10-13),所述恶性细胞包括肺癌细胞。
在肺增生病变中,DNA损伤关卡基因(checkpoint gene)已经被激活,导致关卡蛋白中突变的选择和肿瘤进展(14、15)。FHIT内FRA 3B处的DNA改变的证据伴随增生和关卡激活。在其他抑制基因的表达发生病理变化或改变之前,FHIT等位基因的丢失发生于吸烟者的正常外观的支气管上皮细胞中(16-18)。
Fhit表达受到暴露于DNA损伤剂的下调(19),并且Fhit在对此类剂的应答中起作用(20,21),而Fhit-缺陷细胞逃避凋亡并积累突变。
虽然Fhit表达在几种实验模型中通过涉及外在和内在凋亡途径的半胱天冬酶(caspase)依赖性机制触发凋亡,但是关于此过程的早期事件以及Fhit丢失如何参与肿瘤启动仍知之甚少。
因此,存在对在需要其的受治疗者中改变FHIT表达的方法的需要。还存在对可用于在需要其的受治疗者中改变FHIT表达的组合物的需要。
发明概述
在广泛的方面,提供了鉴定直接与Fhit相互作用以影响终点为凋亡的下游信号途径的蛋白的方法。在一个实施方案中,在用AdFHIT-His6病毒感染肺癌细胞之后,细胞内的蛋白被化学交联。鉴定并表征了与Fhit连接的蛋白和受它们影响的途径。
在另一广泛的方面,本文提供了诊断受治疗者是否具有癌症相关疾患或处于发展所述疾患的风险的方法,该方法包括测量受治疗者的测试样品中至少脆性组氨酸三联体(Fhit)基因的水平,其中相对于对照样品中对应的Fhit基因产物的水平,测试样品中所述Fhit基因产物的水平的改变指示受治疗者具有癌症相关疾患或处于发展所述疾患的风险。
当根据附图理解下列优选实施方案详述时,本发明的各种目标和优点将对本领域技术人员是明显的。
附图简述
本专利或申请文件含有至少一幅彩色附图。含有彩色附图的本专利或专利申请公布的拷贝将在提出请求并支付必要的费用后由美国专利商标局提供。
图1A-1H-Fhit蛋白在胞质溶胶和线粒体中的亚细胞定位。
图1A,以抗Fhit血清对用PonA处理48h的H1299细胞(D1)进行免疫荧光显微术;使用异硫氰酸荧光素(绿色)-轭合的抗兔免疫球蛋白(IgG)检测Fhit染色;鉴定线粒体的Mito-Tracker Red染色显示与Fhit的部分共定位。第四个图(右下方)中的黄色显示共定位点。
图1B,以penta-His抗体进行的A549AdFHIT(左)或AdFHIT-His6-感染的细胞(右)的免疫电子显微术显示Fhit的线粒体定位(右);以AdFHIT感染的A549细胞充当对照并显示仅有少量分散的粒状物(左图)。
图1C,使用抗Fhit对AdFHIT-感染的A549亚细胞级分的免疫印迹分析表明胞质溶胶、膜、细胞骨架和线粒体中的Fhit蛋白分布。
图1D,对用碳酸钠(图1E)和浓度增加的毛地黄皂苷(图1F)处理后AdFHIT-His6感染的A549细胞的线粒体蛋白的免疫印迹分析表明,Fhit主要分布于线粒体基质中;用Fhit和CoxIV抗血清探测滤膜;图1F中的泳道代表用0%、0.10%、0.15%和0.20%毛地黄皂苷处理后的上清液。
图1G,使用抗Fhit对MKN74/E4和MKN74/A116细胞(稳定表达外源Fhit),和图1H,HCT116(内源Fhit阳性克隆癌细胞系)的亚细胞级分的免疫印迹分析证实了Fhit的线粒体定位;GAPDH和CoxIV抗血清充当对照。
图2A-F-外源和内源Fhit与内源Hsp60、Hsp10和Fdxr蛋白形成复合体。将用重组Fhit-His6蛋白分离的蛋白复合体在聚丙烯酰胺凝胶上分离,并用针对Hsp60(图2A)、Hsp10(图2B)和Fdxr(图2C)的抗血清探测;在后一图中,其于线粒体分离后制备,显示Fhit在线粒体中以时间依赖性方式募集Fdxr。以有或无DSP下用AdFHIT-His6感染A549细胞后分离的蛋白对滤膜进行上样。
图2D,用AdFHIT感染A549细胞后以抗Hsp60进行的免疫共沉淀;以Hsp60、Fhit和Hsp10抗血清探测滤膜。
图2E,用V5标签的FDXR基因和FHIT质粒共转染A549细胞;用抗V5进行免疫沉淀并用Fdxr和Fhit抗血清进行检测。
图2F,来自DSP处理的Fhit阳性HCT116细胞的内源相互作用物蛋白(Fdxr和Hsp10)的免疫沉淀和免疫印迹检测。以针对每种靶蛋白的抗血清探测滤膜。内源Fhit与Hs p10和Fdxr共沉淀。
图3A-D-Hsp60/Hsp10的敲低降低了线粒体中的Fhit水平。
图3A,使用H6抗体对AdFHIT-His6感染的A549细胞的亚细胞级分(胞质溶胶和线粒体)进行的镍-H6下拉(pull down)实验;将溶胞产物与镍珠孵育以分离DSP交联的Fhit-His6蛋白复合体并上样至4-20%聚丙烯酰胺凝胶。感染后24h,Hsp60-Fhit复合体存在于两个区室中;感染后48h,复合体在两个区室中同样是可检测的,并且Fhit复合体蛋白的增加显示与AdFHIT-His6感染后48h Fhit蛋白的增加相关,线粒体中有轻微增加(对输入样品进行光密度法分析)。
图3B,对Hsp60/Hsp10沉默72h后Fhit阳性D1细胞中的Hsp60、Hsp10、Fhit和GAPDH的免疫印迹分析,其显示CHX追踪(30μg/ml)1-12h后的Fhit、Hsp60和Hsp10水平。
图3C,用Hsp60和Hsp10 siRNA转染后72h和m.o.i.1的AdFHIT感染后24h,使用Hsp60、Hsp10、Fhit、GAPDH和CoxIV抗血清,对A549细胞的胞质溶胶/线粒体蛋白级分的免疫印迹分析。Hsp60/10沉默未显示影响Fhit胞质溶胶水平,但与线粒体级分中Fhit的下降有关。零乱(Scrambled)(Scr)siRNA用作对照。
图3D,“内源”Fhit复合体蛋白的亚细胞分级和免疫沉淀。将有和无过氧化物处理的PonA-诱导的D1和E1细胞分级为胞质溶胶和线粒体,并评估诱导后48h亚细胞级分中Fhit和相互作用物(左侧)的存在;每个泳道上样25μg蛋白。内源Hsp60共沉淀Fhit和Fdxr。
图4A-F-Fhit表达诱导用过氧化物处理细胞后胞内ROS产生。
图4A,在有和无5小时H2O2处理下,用FHIT质粒转染后48h的A549细胞中ROS评估的荧光激活细胞分选仪(FACS)分析。空载体转染的细胞充当对照。根据氢化乙啡啶(hydroethidine)被O2氧化产生的乙啡啶的荧光确定胞内超氧化物。M2指ROS阳性细胞的分数。
图4B,通过D1和E1细胞中氢化乙啡啶氧化产生的荧光进行ROS评估的FACS分析;PonA处理后48h,将细胞用0.5和1.0mM H2O2处理5h,并测量氧化应激;%阳性指指示ROS的荧光细胞的分数。重复这些实验三次,得到了相似的结果。
图4C,应激条件下H1299Fhit表达细胞(D1)中增加的绿色荧光DCF信号。Fhit诱导后48h和H2O2处理E1和D1细胞5小时后,将细胞与2′,7′-二氯二氢荧光素二乙酸酯孵育,2′,7′-二氯二氢荧光素二乙酸酯是在ROS存在下可被氧化为高度绿色荧光染料DCF的ROS指示物(放大率x40)。
图4D,对E1和D1细胞进行MTS细胞存活测定。将细胞用PonA处理48h,然后用浓度增加的H2O2(0.125、0.25和0.5mM)处理4h。在H2O2处理后24h进行分析。柱报告四个实验的平均值±S.E。每个点测量四个重复,并计算标准偏差;认为p<0.05是显著的。
图4E,氧化应激处理后48h,D1和E1细胞周期动力学的FACS分析。将细胞用PonA处理48h,然后用浓度增加的H2O2(0.25和0.5mM)处理4h。在H2O2处理后48h进行分析。所有实验以三个重复进行两次。
图4F,5mM PonA刺激和所示浓度下的5小时H2O2处理后H1299/D1和H1299/E1细胞的集落形成测定。
图5A-H.-Fhit病毒转导触发的凋亡可通过其与Fdxr的相互作用介导。
图5A,使用针对Fdxr、Fhit和GAPDH的抗血清的免疫印迹分析。蛋白是在用PonA处理后48h从E1(对照)和D1细胞提取的。
图5B,用25μM蛋白酶体抑制剂MG132进行4小时处理后,D1和E1细胞中Fdxr表达的免疫印迹分析。GAPDH检测显示相等的蛋白上样。
图5C,D1细胞和E1细胞中Fdxr、Fhit和GAPDH的免疫印迹分析显示CHX追踪(30μg/ml)4-12h后的Fdxr水平,所述D1细胞表达Fhit。基于GAPDH水平的光密度法显示Fhit存在下增强的Fdxr稳定性。
图5D,用AdFHIT m.o.i.50和100感染后FDXR+/+/+和FDXR+/-/-细胞周期动力学的FACS分析。实验在感染后48h进行,并重复三次,得到相似结果。Ad GFP-感染细胞的谱图与非感染的细胞的谱图(未显示)相似。
图5E,显示用AdFHIT m.o.i.50和100感染FDXR+/+/+和FDXR+/-/-后Fdxr、Fhit和GAPDH的表达的免疫印迹分析。蛋白质在感染后48h提取。
图5F,AdFHIT m.o.i.50后48h的FDXR表达的实时RT-PCR分析。将PCR产物对GADPH和肌动蛋白表达进行标准化,并且每个点重复四次;对照和Fhit阳性样品之间的差异不显著。
图5G,半胱天冬酶3和Parp1激活。使用Fhit、半胱天冬酶3、Parp1抗血清,对m.o.i.50的AdFHIT和Ad GFP感染后48、72和96h的HCT116 FDXR+/+/+细胞的总细胞溶胞产物的免疫印迹分析。GAPDH和CoxIV充当内部蛋白标志物。
图5H,使用Fhit和细胞色素c抗血清,对m.o.i.50的AdFHIT和Ad GFP感染后48、72和96h的HCT116 FDXR+/+/+细胞的胞质溶胶/线粒体级分的免疫印迹分析。GAPDH和β-肌动蛋白充当内部蛋白标志物。
图6A-E-Fhit增强癌细胞对紫杉醇和顺铂的敏感性。
对E1和D1细胞进行MTS测定。将细胞用PonA处理48h,然后用紫杉醇(50-500ng/ml)(图6A)或顺铂(0.05-0.2mM)(图6B)处理24或48h。条报告四个实验的平均值±S.E。每个点测量四个重复并计算标准偏差;图6A和图6B中方括号旁边的星号指示D1和E1细胞对药物应答的统计学显著的差异,p<0.05。
图6C和图6D,图显示E1和D1细胞的流式细胞术分析的代表性结果。将细胞用PonA处理48h,然后用紫杉醇(50-500ng/ml)(图6C)或顺铂(0.05-0.2mM)(图6D)处理。每个数据点在24、48和72h测量三个重复(显示了48h的数据)。
图6E,半胱天冬酶3和Parp1切割:使用Fhit、半胱天冬酶3和Parp1抗血清,对用紫杉醇(50和100ng/ml)或顺铂(0.05和0.1mM)处理48h后PonA诱导的D1细胞的总细胞溶胞产物的免疫印迹分析。GAPDH充当上样对照。
图7.表1通过质谱法分离的候选Fhit蛋白配偶体。在A549AdFHIT-H6感染的细胞样品中选择性捕获的蛋白质。列出了鉴定的肽的氨基酸序列、Mascot评分和蛋白质序列覆盖率。
图8A-8C.Ad-His6生物活性与AdFHIT相当。
图8A,用Ad-His6、MOI 20感染的A549细胞的蛋白质印迹分析。通过抗pentaHis和抗Fhit血清检测Fhit-His蛋白。Ad FHIT和Ad-His6二者均携带通过FHIT下游的内部核糖体进入序列受到CMV5启动子调控的GFP cDNA。使用γ-微管蛋白对样品上样进行标准化。
图8B,用Ad-His 6、MOI 15感染后96hr的A549细胞的流式细胞术分析。上图指示感染细胞的subG1 DNA含量(实验重复三次;subG1级分的平均值:Ad FHIT为22%+/-4.3,Ad-His6为29%+/-5;差异不是统计学显著的;下图显示具有指示凋亡的成熟半胱天冬酶-3的细胞的百分比。用Ad-His6感染的A549细胞中细胞死亡的程度与在用Ad FHIT感染后获得的结果相当。
图8C,Fhit-His6的体内交联。His6下拉和交联逆转条件后的细胞溶胞产物通过4-20%梯度SDS-PAGE分离的凝胶的银染色。内部阴性对照包括Ad FHIT感染的细胞(交联的,CL)和Ad-His6感染的细胞(未交联的,NT)的His6下拉。
图9A-9F.通过纳米孔(nanobore)LC-MS/MS鉴定的候选Fhit蛋白配偶体的初步验证。显示了AdFHIT-His6和对照样品的选择离子色谱图(Selected ion chromatograms,SIC)。六个SIC对报告了下列六个m/z值的离子电流:1)672.8(保留时间为30min的峰鉴定为属于Hsp60的胰蛋白酶肽TVIIEQSWGSPK[SEQ ID NO:5]),2)685.4(保留时间为32min的峰鉴定为属于Aldh2的胰蛋白酶肽LGPALATGNVVVMK[SEQ ID NO:22]),3)617.3(保留时间为39min的峰鉴定为属于Mdh的胰蛋白酶肽IFGVTTLDIVR[SEQ ID NO:10]),4)658.4(保留时间为26min的峰鉴定为属于Hsp10的胰蛋白酶肽VLQATVVAVGSGSK[SEQ ID NO:19]),5)551.7(保留时间为28min的峰鉴定为属于Etfb的胰蛋白酶肽EIDGGLETLR[SEQ ID NO:15]),6)598.3(保留时间为23min的峰鉴定为属于Fdxr的胰蛋白酶肽FGVAPDHPEVK[SEQ ID NO:23])。用红色箭头指示的感兴趣的肽专一地存在于Ad-His6样品中。
图10.表2,Fhit在MKN74胃癌细胞中诱导ROS产生。ROS评估使用携带p53突变等位基因并表达外源Fhit的人胃癌细胞系MKN74A116进行;Fhit阴性MKN74E4细胞用作对照。为了诱导ROS产生,我们用0.5、1.0和2.0mM H2O2处理MKN74细胞5hr。结果表明与对照相比,表达外源Fhit的细胞中有显著更高的ROS产生率;2mM H2O2处理后,在Fhit-表达细胞中观察到毒性。数字报告四个实验的平均值±S.E.。
优选实施方案详述
应理解,上面的一般描述和下面的详细描述仅是示例性的和解释性的,并不意图限制本文的教导的范围。在本申请中,除非另外指明,否则使用单数时包括复数。
在权利要求和/或说明书中,当与术语“包含(comprising)”联合使用时,词“一种(a)”或“一种(an)”的使用可意指“一种(one)”,但其也与“一种或多种”、“至少一种”和“一种或多于一种”一致。
另外,“包含(comprise)”、“含有(contain)”和“包括(include)”或这些根词的修饰形式,例如但不限于“包含(comprises)”、“含有(contained)”和“包括(including)”,并不意图是限制性的。术语“和/或”意指前后的术语可以是一起的或单独的。出于例证目的,但不是作为限制,“X和/或Y”可意指“X”或“Y”或“X和Y”。
如本文所用的术语“其组合”指在该术语之前所列的条目的所有排列和组合。例如,“A、B、C或其组合”意图包括以下的至少一种:A、B、C、AB、AC、BC或ABC,以及,如果在具体环境中顺序是重要的,还包括BA、CA、CB、ACB、CBA、BCA、BAC或CAB。
如本文可交换使用的,“基因产物”、“DNA”和“基因”在本文可交换使用。
本文可使用以下缩写词:GAPDH,甘油醛-3-磷酸脱氢酶;DSP,二硫代二(琥珀酰亚胺丙酸酯);LC-MS/MS,液相色谱串联质谱;Fdxr,铁氧还蛋白还原酶;PonA,松甾酮A;m.o.i.,感染复数;ROS,活性氧簇(reactive oxygen species);FU,5-氟尿嘧啶;DCFH-DA,二氯荧光素二乙酸酯;DCF,2′,7′-二氯荧光素;CHX,环己酰亚胺;siRNA,小干扰RNA;RT,反转录酶;MTS,3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑。
本文使用的各部分的标题仅是为了组织目的,而不应以任何方式视为限制所描述的主题。本申请中引用的所有文献和相似材料,包括专利、专利申请、文章、专著、论文和互联网网页,出于任何目的而通过引用明确地整体并入。如果所并入的文献和相似材料中的一种或多种以与本申请中的术语定义矛盾的方式定义或使用了该术语,则以本申请为准。
Fhit蛋白在大多数癌症中是丢失的,其恢复抑制肿瘤发生,并且病毒介导的FHIT基因治疗在临窗前模型中诱导凋亡并抑制肿瘤。使用蛋白质交联和蛋白质组学方法表征参与触发Fhit-介导的凋亡的Fhit蛋白复合体。该复合体包括介导Fhit稳定性并可影响向线粒体的输入的Hsp60和Hsp10,在线粒体中,该复合体与铁氧还蛋白还原酶相互作用,铁氧还蛋白还原酶负责经由铁氧还蛋白将电子从NADPH转移给细胞色素P450。病毒介导的Fhit恢复增加胞内活性氧簇的产生,然后是氧化应激条件下肺癌细胞凋亡的增加;相反,Fhit-阴性细胞逃避了凋亡,其携带可促成增加的突变率的严重氧化型DNA损伤。Fhit相互作用蛋白的表征鉴定了Fhit-介导的凋亡途径的直接效应物,由于Fhit的丢失,该途径在大多数癌症中是丢失的。
对Fhit-相互作用蛋白的更早检索指出了几种候选蛋白,其中没有一个可通过免疫共沉淀实验被我们确认为相互作用物,这些候选蛋白包括Ubc9、α-微管蛋白和Mdm2(35-37)。为了重新着手于Fhit蛋白相互作用物的问题,使用了如下:用于交联后Fhit复合体纯化的腺病毒转导的Fhit-His6,并鉴定了Fhit-连接蛋白、Hsp60、Hsp10和Fdxr;这些蛋白的亚细胞定位暗示线粒体可能是Fhit活性的焦点。Hsp“应激蛋白”作为分子伴侣蛋白执行诸如蛋白易位、折叠和装配的功能(38)。AdFHIT感染后Fhit与Hsp60/Hsp10相互作用的发现暗示,在激活凋亡途径之前,Hsp复合体对Fhit稳定性以及可能地对于它的正确折叠以输入到线粒体,可能是重要的,我们通过敲低AdFHIT-感染的肺癌细胞中的Hsp60、Hsp10或二者表达研究了这一暗示;CHX追踪后评估了表达可诱导的Fhit的肺癌细胞系H1299D1细胞中的Fhit稳定性。敲低Hsp60和Hsp10二者后,分离的线粒体中的Fhit蛋白降低,强化了Fhit-Hsp60/10相互作用参与Fhit稳定性和/或正确折叠以输入到线粒体的提议。
HCT116结肠癌细胞中FDXR基因的靶向破坏显示其是存活必需的;基因拷贝数的降低造成对5-氟尿嘧啶-诱导的凋亡的敏感性的降低(29),而FDXR是p53家族的靶基因(30)。Fdxr的过表达使结肠癌细胞对H2O2、5-氟尿嘧啶和阿霉素-诱导的细胞死亡敏感,表明Fdxr通过在线粒体中产生氧化应激促成p53-介导的凋亡。因此,激活的p53部分是通过ROS诱导响应于细胞应激的凋亡,并且p53同时增加FDXR基因的转录,这通过增加ROS-诱导的凋亡依次增强了p53功能(29,30)。
现在本文显示了Fhit在线粒体级分中的存在;当Fhit被过表达或Fhit-表达细胞受到应激时,Fhit可保护Fdxr不受蛋白体降解,导致Fdxr蛋白水平的增加,这与ROS的产生有关并跟随有凋亡。Fhit不影响FDXR转录水平但可影响蛋白的稳定性。在缺乏Fhit和p53二者的H1299细胞中,Fdxr过表达增加对ROS-诱导的细胞死亡的敏感性,表达可诱导的Fhit或p53的H1299细胞对ROS-诱导的细胞死亡敏感;缺乏Fhit、p53或二者的癌细胞将缺乏增加Fdxr表达的方式,并将对氧化损伤更不敏感,并将存活。
Fhit在凋亡中通过与Fdxr相互作用的线粒体功能的发现,现在延伸了在大多数癌症中顺序丢失的并参与对DNA损伤的应答的重要肿瘤抑制物Fhit和p53的功能平行物,并阐明它们的差异,p53充当转录水平的Fdxr调控物,而Fhit充当转录后Fdxr调控物。对Fhit抑制途径的直接下游效应物的描绘将导致对可影响激活Fhit途径的预防和治疗策略的Fhit功能的机制研究。
ROS产生对Fhit-介导的凋亡至关重要的发现强调了Fhit丢失作为阴性预后因子在不同临床环境中的重要性;例如,评估肿瘤发生前或肿瘤病症中的Fhit状态可预测对抗氧化剂治疗的应答。
为了鉴定与Fhit相互作用以实现下游凋亡途径的蛋白,本文中本发明人在肺癌细胞中于病毒介导的Fhit过表达后交联了细胞内的蛋白,并表征了与Fhit相关的蛋白和它们所影响的途径。
结果
Fhit蛋白复合体的分离-为了鉴定Fhit-相互作用蛋白,我们产生了携带在其3′端用编码His6表位标签的序列修饰的FHITcDNA的腺病毒(AdFHIT-His6)。A549细胞中表达的该标签的Fhit蛋白的生物活性与野生型Fhit活性相当(图8)。
对Fhit-诱导的凋亡易感的A549肺癌衍生细胞(10)用AdFHIT或AdFHIT-His6进行感染,并用DSP处理,DSP是横穿膜并于体内固定复合体中的蛋白的交联剂。裂解细胞,并用对His6表位标签亲和的镍珠分离蛋白。用二硫苏糖醇处理纯化蛋白以切割DSP并使复合体解离,并通过胰蛋白酶消化;通过LC-MS/MS鉴定蛋白成分(图7-表1和图9)。
鉴定了六种蛋白,所有蛋白均具有线粒体定位:Hsp60和Hsp10、铁氧还蛋白还原酶(Fdxr)、苹果酸脱氢酶、电子传递黄素蛋白和线粒体醛脱氢酶2;Hsp60和Hsp10还分布于胞质溶胶(23)。
Fhit亚细胞定位-因为候选Fhit相互作用物是线粒体蛋白,所以本发明人在此确定了缺乏线粒体定位信号的Fhit是否定位于这些细胞器。用诱导子PonA处理携带可诱导的FHIT cDNA的Fhit阴性H1299肺癌细胞(D1细胞)48h,并使用抗Fhit血清和线粒体标志物MitoTracker Red 580评估Fhit亚细胞定位的间接免疫荧光检测;Fhit荧光信号(绿色染色,图1A)是细胞质定位的并与MitoTracker Red染料部分共定位(黄色染色,图1A,右下),表明外源Fhit定位于线粒体和胞质溶胶。抗Fhit特异性得到Fhit阴性H1299克隆E1细胞中缺乏绿色荧光的证实(未显示)。为了证实线粒体定位,通过抗pentaHis染色于48h后用免疫电子显微术检查以m.o.i.20用AdFHIT-His6或AdFHIT感染的A549细胞;FhitHis6-转导的细胞展示线粒体中有显著数量的金粒(图1B,右图),而AdFHIT-转导的细胞显示稀疏的反应性(图1B,左图)。
为了评估Fhit的亚线粒体定位,从如上所述的用AdFHITm.o.i.1感染的A549细胞纯化线粒体。碳酸钠方法是非破坏性方法,其允许在诱导展开膜层产生后有效释放胞内膜的可溶蛋白和外周膜蛋白到上清液中;另外,它允许回收膜的整合蛋白(沉淀)(24)。
图1E显示Fhit是可溶级分中唯一可检测的。为了进一步限定Fhit的亚线粒体定位,用0.10%和0.15%毛地黄皂苷处理线粒体以选择性破坏线粒体外膜,释放膜间腔和基质中含有的蛋白;如图1F所示,外膜和内膜的逐渐破坏释放增加量的Fhit蛋白,暗示Fhit主要分布于内膜的腔侧或线粒体基质中。线粒体定位在稳定表达外源Fhit的胃癌衍生的MKN74A116细胞(9)和表达内源Fhit的HCT116结肠癌细胞中得到证实(图1G和图1H)。
Fhit与Hsp60、Hsp10和Fdxr相互作用-在候选相互作用物蛋白中,发明人集中于为可能的伴侣蛋白的Hsp60和Hsp10,以及被p53反式激活并参与对治疗药物的应答的线粒体呼吸链蛋白Fdxr(25)。为了证实相互作用,在有或无DSP下,以m.o.i.20用AdFHIT或AdFHIT-His6感染A549细胞。通过His6标签纯化Fhit复合体,并用针对Hsp60、Hsp10和Fdxr的抗血清检测共纯化的蛋白;Hsp60和Fdxr仅在暴露于DSP的细胞的溶胞产物中检测到(图2A和图2C),而Hsp10在无交联下也是可检测的(图2B)。
感染后的时间过程实验显示Fhit对Fdxr的募集(图2C);另外,内源Hsp60在DSP不存在下免疫共沉淀Fhit和Hsp10(图2D)。
为了验证相互作用的特异性,我们产生了具有3′V5表位标签的FDXR cDNA表达质粒。用FDXR-V5和FHIT质粒共转染A549细胞,并用单克隆抗V5沉淀蛋白;共沉淀的Fhit仅在DSP交联后可检测(图2E)。
为了确定这些蛋白是否还与内源Fhit相互作用,发明人从DSP-处理的Fhit阳性HCT116细胞中免疫沉淀了每种内源候选相互作用物蛋白并寻找内源Fhit的共沉淀(图2F)。
内源Fhit与Hsp10和Fdxr共沉淀,证实了内源Fhit在线粒体中的存在以及在不存在应激下其与内源伴侣蛋白和呼吸链蛋白的相互作用。
Hsp60/10相互作用影响Fhit稳定性和/或线粒体输入-Hsp60和Hsp10是在复合体中发现的分子伴侣蛋白(26)并可对于蛋白的折叠和输入到线粒体是重要的。本发明人现在在此认为Hsp60/10复合体负责Fhit的正确折叠和线粒体寻址。
为了研究这些相互作用的定位,用AdFHIT-His6m.o.i.5感染A549细胞,并从交联后的胞质溶胶和线粒体级分中收集蛋白溶胞产物。通过Fhit-H6-镍下拉分离复合体,在聚丙烯酰胺凝胶上分离,并用Hsp60和Fhit抗血清探测滤膜。在感染后24和48h,在胞质溶胶和线粒体中观察到与Hsp60的相互作用(图3A),与这些时间的Fhit表达增加相称(输入),如图3A所示。
为了确定Fhit-Hsp60/10相互作用对于Fhit蛋白的稳定性是否重要,用Hsp60和Hsp10 siRNA转染具有可诱导的Fhit表达的H1299(D1细胞),并在转染后72h,在1、6和12h进行CHX追踪,评估Fhit蛋白表达并将其与用零乱序列转染的细胞进行比较。
如图3B所示,在Hsp60和Hsp10沉默后CHX的6和12h,存在Fhit表达的强烈降低(在1和12h,分别为从1至0.4)。接下来,将Hsp60和Hsp10 siRNA分别或组合地转染入A549细胞;24h后,用AdFHIT m.o.i.1感染细胞,并在24h后分级胞质溶胶和线粒体。沉默两种Hsp后,与对照相比,胞质溶胶中的Fhit水平未受影响但线粒体中的降低(图3B),显示Hsp60/10复合体可介导病毒转导的Fhit稳定和线粒体定位。如果Hsp60和Hsp10参与Fhit病毒转导后的Fhit稳定性,则以下也是真实的:具有较少Fhit的细胞区室将受到Fhit稳定性降低的影响。本发明人在此还检查了表达可诱导的Fhit的H1299D1细胞中的Fhit复合体,使用Fhit阴性E1细胞作为对照;D1细胞中Fhit诱导后48h(图3C,左图),有和无H2O2下,在D1和E1细胞的胞质溶胶和线粒体中的Fhit复合体蛋白的分布是相似的。
有或无H2O2下,PonA诱导后48h,从这些细胞的总细胞溶胞产物中免疫沉淀Hsp60,并共沉淀Fhit和Fdxr(图3C,右图)。D1细胞中Fhit表达的诱导并没有引起体外生物学变化;因此Fhit复合体并不是作为凋亡的结果形成的。有和无应激条件下,在PonA-诱导的Fhit表达后在D1细胞中进行时间过程实验,以确定是否存在Fhit蛋白相互作用物的生物学变化。共同发明人未检测Fhit表达后的定位变化。
Fhit诱导活性氧簇(ROS)的产生-Fdxr是54-kDa黄素蛋白,它定位于线粒体内膜的基质侧,并负责经由单电子穿梭铁氧还蛋白-细胞色素P450将电子从NADPH传递至底物(27)。在底物限制条件下,电子从此穿梭系统中泄漏并产生ROS(28)。Fdxr通过ROS产生介导结直肠癌细胞中的p53-依赖性、5-氟尿嘧啶-诱导的凋亡(29,30),ROS是有效的胞内氧化剂和凋亡的调节物(31)。
本发明人随后在此研究确定了ROS产生是否可参与Fhit-介导的凋亡。Fdxr的过表达增加了肿瘤细胞对H2O2处理通过ROS产生引起的凋亡的敏感性(29,30)。发明人检查了在用FHIT表达质粒瞬时转染后,有和无H2O2处理下A549细胞中的ROS产生。通过测量乙啡啶荧光评估胞内超氧化物,乙啡啶荧光是氢化乙啡啶被超氧化物氧化的结果。测量用浓度增加的H2O2刺激后5h的胞内超氧化物。FHIT-转染的细胞中的ROS产生是约3倍高(在0.5mM H2O2下,为16.7%对比5.4%。而在1.0mM H2O2下,为18.8%对比7.7%)。2mM H2O2对Fhit-表达细胞是毒性的,但对不表达的细胞则是无毒的(图4A)。
使用分别携带PonA-可诱导的FHIT和空载体表达质粒的p53和Fhit阴性的肺癌衍生H1299D1和E1克隆进行了相似的实验;用5μMPonA处理细胞,并在48h时用0.5和1.0mM H2O2处理;在Fhit-阳性D1细胞中的%ROS-阳性细胞比在E1对照细胞中的高(分别为在0.5mM H2O2下的20%对比3.5%和在1.0mM H2O2下的78%对比25%)(图4B)。
这些结果与使用人胃癌衍生细胞MKN74A116的实验相似(图10),MKN74A116表达突变的p53(32)并稳定表达外源Fhit(9)。
为了进一步研究氧化应激过程中Fhit重建后的ROS产生,使用DCFH-DA测量Fhit-过表达细胞的氧化还原态。过氧化物酶、细胞色素c和Fe2+可在H2O2存在下将DCFH-DA氧化为荧光2′,7′-二氯荧光素(DCF);因此,DCF指示H2O2水平和过氧化物活性。在应激条件下,与E1细胞相比,在D1细胞中检测到增加的DCF荧光(图4C)。
还通过H2O2处理后24h的MTS细胞毒性测定评估了H2O2处理后Fhit-表达细胞中细胞活力的下降。H2O2处理在E1和D1两种细胞中均导致细胞活力下降或生长停止,但是这一表型在D1细胞中更加显著(图4D)。
为了确定有或无Fhit下的H2O2处理是否可影响细胞活力或细胞周期动力学,我们进行了流式细胞术(图4E);当Fhit存在时,在应激条件下,存在0.25和0.5mM H2O2处理后48h G2/M停止的一致增加,分别为45.5%和49.5%,相比之下,在相同条件下,E1细胞中为27.5%和29%。
为了评估G2/M停止是否可影响细胞的长期活力,进行了集落测定(图4F)。在暴露于0.25mM或更高浓度的H2O2之后,未检测到Fhit-表达细胞的集落。
Fhit诱导的ROS产生是Fdxr依赖性的-为了评估Fdxr在Fhit-介导的ROS产生中的作用,发明人检查了Fhit诱导后D1细胞中的Fdxr水平,并观察到与E1细胞相比其表达增加了2.4倍(图5A),这一增加不是因为转录的增加,如实时RT-PCR所确定的(图5F)。
接下来,发明人测量了在有或无Fhit表达下,在蛋白酶体降解抑制剂MG132存在下的Fdxr水平;MG132处理后4h,与E1细胞相比,D1细胞中观察到Fdxr蛋白的显著增加(图5B),显示Fhit保护Fdxr免受蛋白酶体降解。
通过4-12小时CHX追踪评估了在Fhit蛋白存在或不存在下的Fdxr降解率(图5C);与D1细胞相比,Fhit阴性E1细胞中的Fdxr降解率更高(从1降至0.3),而D1细胞中没有明显的下降。因此,本发明人在此现在认为Fhit通过保护Fdxr蛋白免受蛋白酶体降解来防止它失去稳定性。
使用表达内源野生型p53和Fhit并携带三个FDXR等位基因(FDXR+/+/+)的HCT116结肠癌细胞和两个等位基因被敲除的HCT116FDXR+/-/-细胞(28),来确定AdFHIT诱导的凋亡是否受Fdxr表达水平的影响;FDXR无效状态与活力不相容(29)。
用AdFHIT m.o.i.50或100感染这些细胞并评估感染后48和72h的凋亡。野生型HCT116细胞(FDXR+/+/+)以剂量依赖方式易感外源Fhit介导的凋亡,因为在m.o.i.50和100下的sub-G1细胞分数分别为12.1%和18.8%;FDXR+/-/-细胞在48和72h对Fhit诱导的细胞死亡是不应的(数据未显示),在m.o.i.50和100下的sub-G1群体为4.7%和4.3%(图5D)。
Fhit过表达导致FDXR+/+/+和FDXR+/-/-两种细胞中增加的Fdxr蛋白水平(图5E),并且FDXR+/-/-细胞在感染后72h经受Fhit-介导的凋亡。
Fhit介导的Fdxr表达的增加不是在转录水平,如通过实时RT-PCR所确定的(图5F),并因此与p53的转录激活无关。
为了更好地确定AdFHIT感染后在FDXR+/+/+细胞中检测的sub-G1峰是否与凋亡诱导相关,在半胱天冬酶3和Parp1切割的48、72和96h进行了时间过程实验,并与Ad GFP感染的细胞进行了比较(图5G)。
在病毒介导的Fhit过表达后48、72和96h,观察到了半胱天冬酶3切割和相关的Parp1切割。在用AdFHIT m.o.i.100感染HCT116细胞之后,评估了细胞色素c从线粒体释放到胞质溶胶的时间过程(图5H);与GFP感染的细胞相比,在HCT116 FDXR+/+/+细胞中观察到进行性细胞色素c释放,指示Fhit过表达的HCT116 FDXR+/+/+细胞中凋亡级联的开始。
Fhit增强化疗剂的ROS相关的效应-胞内ROS的产生是用紫杉醇处理诱导的肺癌细胞凋亡中的早期事件(33)。发明人对有或无诱导的Fhit表达的H1299D1和E1细胞测试了紫杉醇。在诱导Fhit表达之后,D1细胞比E1细胞对紫杉醇更加敏感(图6A),如通过MTS细胞活力测试所测量的。顺铂诱导Fdxr表达,而顺铂诱导的凋亡途径与ROS产生有关(34)。
Fhit表达的D1细胞比E1细胞对顺铂更加敏感,如24和48h的MTS测定所测量的(图6B)。
为了检查药物处理后的细胞活力,我们进行了流式细胞术分析(图6,图6C和图6D);用增加的紫杉醇浓度(50-500ng/ml)处理的PonA-诱导的D1和E1细胞显示增加的48h sub-G1群体:对于D1细胞分别为9.6%、36%和40%,相比之下E1细胞的为4%、16.7%和30%(图6C)。
相似地,增加的顺铂浓度(0.05-0.2mM)导致增加的48h sub-G1群体:D1细胞中的分别为5%、16.2%和30%,相比之下E1细胞的为2.3%、7%和14.6%(图6C)。
在24和72h(数据未显示),也检测到与E1细胞相比D1中增加的sub-G1群体。为了确定D1细胞的sub-G1级分是否代表凋亡细胞,制备了48h的药物处理细胞的溶胞产物,并进行了半胱天冬酶3和Parp1切割的免疫印迹分析(图6D)。
与未处理的细胞(对照)相比,观察到紫杉醇(50和100ng/ml)和顺铂(0.05和0.1mM)处理后激活的半胱天冬酶3和相关的Parp1切割。本发明人在此现在认为Fhit表达通过与Fdxr一起参与ROS产生增加了对氧化损伤的敏感性。
实施例I
材料和方法
细胞、载体和抗血清-A549、H1299、MKN74-E4和A116和HCT116细胞在加10%胎牛血清和青霉素/链霉素(Sigma)的RPMI 1640培养基上维持。用于制备重组腺病毒的HEK293细胞(Microbix)在加10%胎牛血清和青霉素/链霉素的Dulbecco改良的Eagle培养基中培养。AdFHIT-His6病毒的制备如本文的实施例II中所述。[His6-SEQ IDNO:32][penta-His-SEQ ID NO:33]。
从人脑部cDNA(Clontech)中克隆了全长FDXR,将其亚克隆到pcDNA3.1/V5-HisTOPO TA载体(Invitrogen)并测序;详情如在下文所附方法所述。使用LipofectamineTM(Invitrogen)根据生产商的指导转染细胞。
蛋白质印迹分析-如(13)所述使用以下进行免疫印迹分析:单克隆抗pentaHis(Qiagen);兔多克隆抗Fhit(Zymed LaboratoriesInc.);针对GFP、Hsp60、Hsp10和细胞色素c的兔多克隆抗血清(SantaCruz Biotechnology);兔多克隆抗Fdxr(Abcam);单克隆抗CoxIV(Molecular Probes);抗V5(Sigma);抗Parp1(Santa CruzBiotechnology);和抗半胱天冬酶3(Cell Signaling)。蛋白水平相对于β-肌动蛋白或/和GAPDH3水平进行标准化,用适当的抗血清(Santa Cruz Biotechnology)进行检测。
质谱研究-将蛋白沉淀溶解,并通过胰蛋白酶消化,如本文所述。注射肽混合物用于LC-MS/MS分析。通过数据库搜索鉴定蛋白后,进行LC-MS/MS数据检查以评估属于用AdFHIT-His 6感染的细胞的样品中的候选配偶体蛋白的质量峰的唯一存在。
蛋白质相互作用分析-在15mM Tris-Cl、pH 7.5、120mM NaCl、25mM KCl、2mM EGTA、0.1mM二硫苏糖醇、0.5%Triton X-100、10mg/ml亮肽素、0.5mM苯甲磺酰氯中提取蛋白。在有或无二硫代二(琥珀酰亚胺丙酸酯)(DSP)下,通过如下进行免疫共沉淀实验:将1mg总蛋白与轭合琼脂糖凝胶(Sepharose)的Hsp60、Hsp10、Fdxr、penta-His和V5抗血清在4℃下孵育2h;清洗后,将珠在1x SDS样品缓冲液中煮沸并在4-20%聚丙烯酰胺凝胶(Bio-Rad)上分离蛋白,转移至聚(偏二氟乙烯)滤膜(poly(vinylidene difluoride)filter)(Millipore),并用特异性抗血清探测。
Fhit蛋白的亚细胞定位-通过使用抗Fhit血清的间接免疫荧光检测在松甾酮A(PonA)-诱导的、Fhit-表达H1299D1细胞中对Fhit进行亚定位,并通过使用抗pentaHis在免疫电子显微图中检测A549AdFHIT-His6-感染的细胞中的FhitHis6对Fhit进行亚定位。在分级研究中,使用线粒体/胞质溶胶分级试剂盒分离线粒体,并使用FractionPREPTM细胞分级系统从胞质溶胶、膜、核和细胞骨架中提取蛋白(Biovision Research Products)。对于根据Dahéron等人的方法(22)的亚线粒体定位,将线粒体重悬于在冰上的0.1M碳酸钠、pH11.5保持30min,且定期涡旋,并如本文所述进行分级。
流式细胞术-以m.o.i.50和100用AdFHIT或Ad GFP感染HCT116FDXR+/+/+和FDXR+/-/-细胞,并在感染后48h进行评估。用0.25和0.5mMH2O2或用化疗药物处理PonA-诱导的H1299D1和E1细胞并孵育不同的时间,如文本和图中所示。对于两种实验,收集细胞,用磷酸缓冲盐水清洗并重悬于冷70%乙醇。对于分析,将细胞离心沉淀,在磷酸缓冲盐水中清洗,并在室温重悬于0.1mg/ml碘化丙啶/Triton X-100染色溶液(0.1%Triton X-100、0.2mg/ml无DNA酶的RNA酶A)保持30,并通过流式细胞术分析。
胞内活性氧簇(ROS)的评估-通过氢化乙啡啶(二氢乙啡啶-HE;Molecular Probes)氧化造成的乙啡啶荧光测量胞内超氧化物。在37℃下,用0.5、1.0、2.0和4.0mM H2O2处理稳定表达Fhit的MNK74细胞、瞬时表达Fhit的A549细胞和可诱导地表达Fhit的H1299细胞;4h后,向细胞中添加氢化乙啡啶(10μM)并在37℃下孵育15min。通过流式细胞术测量荧光。二氯荧光素二乙酸酯(DCFH-DA)(Molecular Probes)用于表达诱导的Fhit的D1细胞,用H2O2(0.1至1.0mM)施加应激,用10μM DCFH-DA处理,在37℃下孵育1h。通过流式细胞术在FAC-Scan流式细胞仪上和通过荧光显微术测量DCF荧光。
Hsp60和Hsp10沉默-通过Lipofectamine 2000试剂(Invitrogen)和6μg Hsp60和/或Hsp10 siRNA(Dharmacon目录号分别为NM_002156[GenBank]和NM_002157[GenBank])转染8x105/孔(6孔板)的A549肺癌细胞;48h后,以m.o.i.1用AdFHIT感染细胞,并在24h后收集用于胞质溶胶/线粒体蛋白分级。通过SDS-PAGE和免疫印迹分析蛋白;用Hsp60、Hsp10和Fhit抗血清探测滤膜。蛋白上样用GAPDH和CoxIV进行标准化。1x106H1299D1和E1肺癌细胞如上所述地进行转染,并在转染后24h,对细胞进行PonA-诱导;诱导后48h,进行1、4、6和12h环己酰亚胺(CHX)(10μg/ml)追踪,并如本文所述地分析蛋白溶胞产物。
实对RT-PCR-使用TRIzol试剂(Invitrogen)分离的总RNA在DNA酶处理(Ambion)后直接通过使用SuperScript First-Strand(Invitrogen)的反转录加工为cDNA。使用Power SYBR Green PCRMaster Mix(Applied Biosystems)通过qPCR扩增靶序列。FDXR引物为:正向,3′-TCGACCCAAGCGTGCCCTTTG-5′[SEQ ID No.24];反向,3′-GTGGCCCAGGAGGCGCAGCATC-5′[SEQ ID No.25]。使用肌动蛋白和GADPH基因将样品标准化。
实施例III
重组腺病毒的产生-携带野生型FHIT cDNA的重组腺病毒(AdFHIT)按照之前的描述制备(Ishii等人,2001 Cancer Res 61:1578-1584)。His标签的FHIT cDNA通过使用下列寡核苷酸的PCR产生:5′-ACgTggATCCCTgTgAggACATgTCgTTCAgATTTggC-3′(正向)[SEQ IDNO:26]和5′-TTgTggATCCTTATCAgTgATggTgATggTgATgCgATCCTCTCTgAAAgTAgCCCgCAg-3′[SHQ ID NO:27]。这些引物设计为具有BamHI限制位点以亚克隆至转移载体pAdenoVator-CMV5-IRES-GFP。Ad-His 6是使用AdenoVatorTM试剂盒(Qbiogene,Carlsbad,CA)按照生产商的方法产生的。用作对照的Ad GFP购自Qbiogene(Carlsbad,CA)。
携带FDXR cDNA的重组表达载体的产生-使用以下引物从人脑部cDNA(Clontech,Palo Alto,CA)扩增野生型铁氧还蛋白还原酶全长:5’-CTgTTCCCAgCCATggCTTCgCgCTg-3’(正向)[SEQ ID NO:28]和5’-TCAgTggCCCAggAggCgCAgCATC-3’[SEQ ID NO:29]。将扩增产物亚克隆至pcDNA 3.1/V5-His TOPO TA载体(Invitrogen,Carlsbad,CA)。测序排除了扩增产物中的突变。
对于V5标签的铁氧还蛋白还原酶cDNA的制备,使用排除反向引物中的FDXR生理终止密码子的相同引物序列进行PCR扩增。也就是说,野生型和V5标签的铁氧还蛋白还原酶(FDXR)cDNA二者均是通过使用人铁氧还蛋白还原酶基因(GenBank登录号NM_024417)的野生型编码序列作为模板制备的。该铁氧还蛋白还原酶编码序列是从人脑部cDNA(Clontech)扩增的。
用于产生V5标签的FDXR cDNA的引物为:正向:5’-CTgTTCCCAgCCATggCTTCgCgCTg-3’[SHQ ID NO:30];和反向:5’-gTggCCCAggAggCgCAgCATC-3’[SEQ ID NO:31]。应注意,除了用于产生V5标签的cDNA的反向引物之外,寡核苷酸序列是相同的,在所述反向引物中,FDXR的生理终止密码子被省略以符合读框地将FDXR编码序列与V5标签融合。
将扩增产物亚克隆至pcDNA3.1/V5-HisTOPO TA载体(Invitrogen,Carlsbad,CA)。测序排除扩增产物中的突变。
在某些实施方案中,能够分离Fhit-His6的腺病毒包括携带FHIT-His6 cDNA的腺病毒。FHIT-His6 cDNA可通过使用人FHIT基因(GenBank登录号NM_002012)的野生型编码序列作为模板来制备。为了在最终Fhit产物的C-末端引入多聚组氨酸标签,野生型FHIT编码序列的PCR扩增使用设计为去除生理终止密码子并向内源FHIT序列添加后接人工终止密码子的编码六个组氨酸的18bp片段的反向引物进行。另外,正向和反向两种引物均携带BamHI限制位点以易于亚克隆。用于此扩增的寡核苷酸序列是如下序列:正向:5′-ACgTggATCCCTgTgAggACATgTCgTTCAgATTTggC-3′[SEQ ID NO:26];和反向:5’-TTgTggATCCTTATCAgTgATggTgATggTgATgCgATCCTCTCTgAAAgTAgACCCgCAg-3’[SEQID NO:27]。对PCR扩增产物进行测序以在克隆到Ad5重组基因组(AdenoVatorTM,一种由Qbiogene购买的载体)之前排除随机突变。
在某些实施方案中,分离外源过表达Fhit-His6的方法包括使用携带FHIT-His6 cDNA的腺病毒,其中通过His标签分离Fhit-His6。Fhit-His6代表通过Ad FHIT-His6载体被驱动在哺乳动物细胞中表达的重组蛋白。His6表位允许通过利用Ni-NTA系统回收重组Fhit-His6蛋白以及与所述重组蛋白本身相互作用的蛋白复合体。该系统是从Qiagen可商业获得的。简要而言,用Ad FHIT-His6感染人A549癌细胞;感染后48小时,使用购自Pierce的交联剂二硫代二(琥珀酰亚胺丙酸酯)[DSP]进行胞内蛋白复合体的光交联以稳定活细胞内的蛋白复合体。在蛋白提取缓冲液中破裂细胞,并使用Ni-NTA磁珠技术分离Fhit-His6蛋白复合体,该技术是通过利用His6标签对此类珠的强亲和力。然后通过质谱研究分离的Fhit-His6蛋白复合体以鉴定该复合体中存在的所有蛋白。
在某些实施方案中,携带脆性组氨酸三联体(Fhit)FHIT cDNA的重组腺病毒可被修饰为在FHIT cDNA的3′带有编码六组氨酸表位标签的序列(AdFHIT-His6)。
在某些实施方案中,在至少一个细胞中介导凋亡过程的方法包括使所述细胞暴露于足以在所述细胞中介导凋亡过程的量的脆性组氨酸三联体(Fhit)基因产物。
在某些实施方案中,在细胞中诱导凋亡过程的方法包括使所述细胞暴露于足以在所述细胞中引起活性氧簇(ROS)产生的量的脆性组氨酸三联体(Fhit)基因产物。
在某些实施方案中,在至少一个细胞中介导凋亡过程的方法包括:使所述细胞暴露于足量的脆性组氨酸三联体(Fhit)基因产物以允许Fhit进入细胞的线粒体并与细胞中的Fdxr蛋白相互作用,从而引起与ROS产生相关的Fdxr蛋白水平增加,并引起细胞中凋亡过程的变化。
应注意,在先前的研究中,已广泛证明Fhit阴性癌细胞中的Fhit蛋白过表达能够触发程序性细胞死亡(或凋亡)。在本发明中,发明人提供了关于Fhit蛋白在凋亡过程中的作用的基本原理。事实上,用Ad FHIT(以感染复数50或MOI50,即每个细胞50个病毒粒子)对癌细胞进行的FHIT基因治疗负责Fhit过表达;然后,新合成的重组蛋白被其相互作用物Hsp60/Hsp10从胞质溶胶带到线粒体,在线粒体中,Fhit与属于呼吸链的蛋白FDXR(铁氧还蛋白还原酶)相互作用。此相互作用导致线粒体产生ROS(活性氧簇)。ROS代表启动凋亡过程的内在(或线粒体)途径的早期步骤;事实上,它们诱导线粒体膜的损伤,该损伤依次释放细胞色素c到胞质溶胶。该步骤对于凋亡的执行是至关重要的,因为细胞色素c与其他胞质溶胶分子(即Apaf-1和半胱天冬酶3酶原(pro-caspase 3))促成凋亡体的产生,凋亡体是能够驱动细胞以不可逆方式凋亡的多蛋白复合体。
研究凋亡常用的方法在于通过流式细胞术分析(BectonDickinson)检测成熟半胱天冬酶-3(初期凋亡的指示物)[有关参考文献,参见Trapasso等人,2003,PNAS,100,1592-1597]。
在某些实施方案中,制备V5标签的铁氧还蛋白还原酶cDNA的方法包括用以下引物序列进行PCR扩增:5′-CTgTTCCCAgCCATggCTTCgCgCTg-3′(正向)[SEQ ID NO:28]和5′-TCAgTggCCCAggAggCgCAgCATC-3′[SEQ ID NO:29],并将扩增产物亚克隆至pcDNA3.1/V5-HisTOPO TA载体。
野生型和V5标签的铁氧还蛋白还原酶(FDXR)cDNA二者均是通过使用人铁氧还蛋白还原酶基因(GenBank登录号NM_024417)的野生型编码序列作为模板制备的。该铁氧还蛋白还原酶编码序列是从人脑部cDNA(Clontech)扩增的。也就是说,用于扩增野生型FDXR cDNA的引物为:正向:5’-CTgTTCCCAgCCATggCTTCgCgCTg-3’[SEQ ID NO:28];反向:5’-TCAgTggCCCAggAggCgCAgCATC-3’[SEQ ID NO:29]。
用于产生V5标签的FDXR cDNA的引物为:正向:5’-CTgTTCCCAgCCATggCTTCgCgCTg-3’[SEQ ID NO:30],和反向:5’-gTggCCCAggAggCgCAgCATC-3’[SEQ ID NO:311。应注意,除了用于产生V5标签的cDNA的反向引物之外,寡核苷酸序列是相同的,在所述反向引物中,FDXR的生理终止密码子被省略以符合读框地将FDXR编码序列与V5标签融合。
最后,将两种产物亚克隆至pcDNA3.1/V5-HisTOPO TA表达载体(购自Invitrogen),然后测序以评估两种产物不合随机突变。
在某些实施方案中,用于产生重组腺病毒的方法包括制备携带野生型FHIT cDNA的重组腺病毒(AdFHIT);和使用以下寡核苷酸进行的PCR产生His标签的FHIT cDNA:5′-ACgTggATCCCTgTgAggACATgTCgTTCAgATTTggC-3′(正向)[SEQ ID NO:26],和5′-TTgTggATCCTTATCAgTgATggTgATggTgATgCgATCCTCTCTgAAAgTAgACCCgCAg-3′[SEQ ID NO:27]。FHIT-His6 cDNA的制备如本文所述。重组腺病毒载体Ad FHIT-His6根据生产商的建议制备(Qbiogene)。简要而言,用BamHI消化扩增的FHIT-His6 PCR片段,并亚克隆至BamHI线性化的转移载体pAdenoVator-CMV5-IRES-GFP。将pAdenoVator-CMV5-IRES-GFP/FHIT-His6与E1/E3缺失的Ad5主链病毒DNA共转染到293细胞。通过使用特异性penta-His抗体(Qiagen)的蛋白质印迹筛选病毒噬斑中Fhit-His6蛋白的存在。噬斑纯化一个阳性克隆并在293细胞中扩增。冷冻/解冻循环之后,在两个连续的氯化铯梯度上纯化上清液中的腺病毒。通过TCID50法滴定重组腺病毒并分成等份。病毒贮液在-80℃储存。最后,携带野生型FHIT cDNA的重组腺病毒(Ad FHIT)是Trapasso等人(2003,PNAS,100,1592-1597)以前制备的。
线粒体定位研究-使用共聚焦显微术通过免疫荧光评估Fhit蛋白分布;具有可诱导的FHIT cDNA的H1299 D1细胞和具有空载体的E1细胞用PonA处理48hr,并用Mitotracker Red 580(M-22425,Molecular Probes,Eugene,OR)以500nM的工作浓度在生长条件下对活细胞染色40min。固定细胞,并通过在冰冷的丙酮中孵育5min然后在PBS中清洗使细胞可渗透。使细胞与5%BSA孵育1hr以阻断非特异的相互作用然后与工作浓度为1.6μg/ml的Fhit抗血清(Zymed,S.San Francisco,CA)孵育过夜,用PBS清洗,并用AlexaFluor 488驴抗兔IgG(Molecular Probes)孵育。将载玻片封闭在封闭介质中以获得DAPI(Vector,Burlingane,CA)的荧光并显现。对于Fhit的免疫电子显微术定位,用AdHis6或AdFHIT MOI 5感染的A549细胞在于PBS pH7.2的4%多聚甲醛中在4℃下固定30min,用PBS清洗3次,并使用在于PBS中的0.05%氢化硼钠中孵育30min还原剩余的游离醛基。PBS清洗之后,用于PBS中的50mM甘氨酸封闭样品30min,在PBS中清洗两次,并在25%和50%乙醇中各脱水15min,然后换3次70%乙醇,每次15min。然后用2∶1的70%乙醇+硬质LR White树脂(Electron Microscopy Sciences,Hatfield,PA)渗透样品1hr,1∶2的70%乙醇+LR White渗透1hr,100%LR White渗透1hr和100%LRW在4℃下过夜。第二天细胞再接受多于2次100%LR White更换,并在58℃下于明胶胶囊中聚合20-24hr。使用Reichert UCTUltramicrotome和钻石刀切出900nm薄切片并置于镍网上。使镍网以切片侧向下浮于PBS液滴5min,在室温(RT)下浮于在PBS中的5%山羊血清1hr,然后在潮湿室中在4℃下浮于含有0.1%BSA和0.05%Tween-20(BSA/Tw)的PBS稀释的20mg/ml的Penta-His小鼠单克隆抗体(Qiagen,Valencia,CA)或单独的BSA/Tw过夜。第二天,用PBS洗涤网6次,每次5分钟,然后与1∶10稀释于BSA/Tw的山羊抗小鼠10nm胶体金轭合物(Ted Pella,Redding,CA)在RT下孵育2hr。用PBS洗涤网6次,每次5分钟,用DI H2O润洗,然后用2.5%乙酸双氧铀水溶液后染色3min。在配备有US1000 Gatan 2K数码相机的Tecnai 12电子显微镜上收集图像。
样品消化和LC-MS/MS分析-将用Ni-NTA珠分离的蛋白用冷丙酮沉淀,并重悬于用0.1M Tris-HCl缓冲至pH 8的6M尿素。通过加入DTT(终浓度10mM,在37℃下孵育1hr)和碘乙酰胺(终浓度25mM,在37℃下孵育1hr)分别进行蛋白还原和烷基化。用DTT(另外的5mM)中和过量的碘乙酰胺后,通过用1mM CaCl2稀释将尿素浓度降低至1.5M。使用50ng TPCK处理的胰蛋白酶(Sigma)进行过夜消化。总消化溶液的体积为100μl。
使用来自Dionex(Sunnyvale,CA)的Ultimate nano LC系统进行色谱。在将RP阱转换为与75μmX150mm Pepmap C18 nano LC柱联机之前,将消化混合物(30μl)直接注射于Pepmap C18RP筒(0.3mmID X 5mm长)并用H2O/三氟乙酸(TFA)/乙腈97.9∶0.1∶2(v/v/v)洗涤10分钟。使用从5%B至50%B的45-min线性梯度以300nl/min实现肽的梯度洗脱。流动相A为H2O/乙腈/甲酸(FA)/TFA97.9∶2∶0.08∶0.02(v/v/v/v);流动相B为H2O/乙腈/FA/TFA4.9∶95∶0.08∶0.02(v/v/v/v)。
MS检测在以正离子模式操作的Applied Biosystems(Framingham,MA)QSTAR XL hybrid LC-MS/MS上进行,纳米电喷雾电位为1800V、气帘气为15单位,CAD气为3单位。信息依赖性采集(IDA)通过在持续2秒钟的从400至1200m/z的完全TOF-MS扫描之后选择两个丰度最高的峰进行MS/MS分析来实行。两个MS/MS分析都在增强模式(2秒/扫描)下进行。
LC-MS/MS数据分析-通过询问Mascot搜索引擎(www.matrixscience.com)(2006年6月登录)上的Swiss Prot数据库搜索MS/MS谱。使用以下搜索参数。MS耐受性:50ppm;MS/MS耐受性:1Da;甲硫氨酸氧化(可变修饰);半胱氨酸脲甲基化(固定修饰);酶:胰蛋白酶;最大未切割数:1。
比较了分别从用Ad FHIT-His 6和对照感染的A549获得的蛋白列表,并保留专一地存在于A549-Ad FHIT-His6列表中的蛋白用于进一步的验证。作为第一验证方法,使用选择离子色谱(SIC)展示模式检查LC-MS/MS原始数据。通过SIC比较,可评估鉴定为属于检查的六种候选蛋白的感兴趣的肽在Ad FHIT-His6样品中的专一存在。此验证步骤已经为六种候选蛋白的特异性捕获提供了相当强的证据。另外,通过生化和功能测定进一步验证了这些发现。
根据专利法规的规定,在本发明的优选实施方案中解释和例证了本发明的原理和操作模式。然而,必须理解,本发明可以以不同于所具体解释和例证的方式实施而不悖离其精神或范围。
参考文献
上文讨论的参考文献和以下参考文献,在它们对本文所述内容提供示例性方法或其它补充细节的程度上,通过引用具体地并入本文。
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Claims (2)
1.一种能够分离Fhit-His6的腺病毒在制备药物中的用途,所述药物用于诱导活性氧簇的产生,其中该腺病毒携带FHIT-His6cDNA。
2.FHIT在制备药物中的用途,所述药物用于诱导活性氧簇的产生。
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WO2009055773A3 (en) | 2009-06-18 |
EP2201112A2 (en) | 2010-06-30 |
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CN102137927A (zh) | 2011-07-27 |
US20150010647A1 (en) | 2015-01-08 |
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AU2008316577A1 (en) | 2009-04-30 |
US20120010092A1 (en) | 2012-01-12 |
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