Antibodies elicited against cis-diamminedichloroplatinum(II)-modified DNA are specific for cis-diamminedichloroplatinum(II)-DNA adducts formed in vivo and in vitro.
Antibodies elicited against cis-diamminedichloroplatinum(II)-modified DNA are specific for cis-diamminedichloroplatinum(II)-DNA adducts formed in vivo and in vitro.
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针对顺式二氯二氨铂 (II) 修饰的 DNA 引发的抗体对体内和体外形成的顺式二氯二氨铂 (II)-DNA 加合物具有特异性。
DOI：
10.1073/pnas.79.21.6443
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发表时间：
1982
影响因子：
11.1
通讯作者：
Yuspa,SH
中科院分区：
文献类型：
--
作者：
Poirier,MC;Lippard,SJ;Zwelling,LA;Ushay,HM;Kerrigan,D;Thill,CC;Santella,RM;Grunberger,D;Yuspa,SH
用抗肿瘤药物顺-二氨二氯铂（II）（cis-DDP）修饰至4.4%（铂药物/核苷酸比= 0.044）的小牛胸腺DNA诱导的兔抗血清含有对铂修饰的DNA免疫原以及对在培养的小鼠白血病L1210细胞和暴露于cis-DDP的荷瘤小鼠腹水中的L1210细胞中形成的Pt-DNA加合物具有特异性的抗体。铂修饰的DNA静电复合甲基化牛血清白蛋白和注射到兔子。早期bleeding的衍生的抗血清被用来建立竞争性酶联免疫吸附试验（ELISA），证明特异性的铂修饰的DNA，但不为DNA或铂药物单独。在ELISA中，如通过原子吸收光谱法测定的，在0.5 nM Pt（在DNA上）的浓度下发生50%抑制。该值对应于10（7）个核苷酸中一种加合物的可检测性下限，每个微量滴定孔加入50微克样品DNA。通过ELISA发现，从暴露于增加剂量的Pt药物的培养的小鼠L1210细胞中分离的DNA每微克DNA含有0.2至10.0 fmol的Pt加合物。这些水平保持稳定长达4小时后，1小时的药物治疗，在此期间DNA链间交联的发展。因此，抗血清似乎对DNA链间交联不具有特异性。L1210细胞的DNA暴露于trans-diamminedichloropatinum（II）和L-苯丙氨酸氮芥在ELISA中没有识别。注射顺式-DDP后5小时，从携带L1210肿瘤的小鼠腹水细胞中制备的DNA发现每微克DNA含有约2fmol Pt。这项工作确立了体外制备的顺式-DDP-DNA加合物与体内Pt药物与其生物靶点DNA的结合有关，并为研究Pt抗癌药物的作用机制开辟了新的途径。
Rabbit antiserum elicited against calf thymus DNA modified to 4.4% (Pt drug/nucleotide ratio = 0.044) with the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) contains antibodies specific for the Pt-modified DNA immunogen as well as for Pt-DNA adducts formed in both cultured mouse leukemia L1210 cells and in L1210 cells from the ascites fluid of tumor-bearing mice exposed to cis-DDP. Pt-modified DNA was electrostatically complexed to methylated bovine serum albumin and injected into rabbits. Early bleedings of the derived antiserum were used to establish a competitive enzyme-linked immunosorbent assay (ELISA), which demonstrated specificity for the Pt-modified DNA but not for DNA or the Pt drug alone. In the ELISA, 50% inhibition occurred at a concentration of 0.5 nM Pt (on DNA) as determined by atomic absorption spectroscopy. This value corresponds to a lower limit of detectability of one adduct in 10(7) nucleotides, with 50 micrograms of sample DNA added per microtiter well. DNA isolated from cultured mouse L1210 cells exposed to increasing doses of the Pt drug was found by ELISA to contain from 0.2 to 10.0 fmol of Pt adduct per microgram of DNA. These levels remained stable for up to 4 hr after a 1-hr drug treatment, during which time DNA interstrand crosslinks developed. Thus, the antiserum appears not to be specific for DNA interstrand crosslinks. DNAs from L1210 cells exposed to trans-diamminedichloroplatinum(II) and L-phenylalanine mustard were not recognized in the ELISA. DNA prepared from the ascites cells of mice bearing the L1210 tumor 5 hr after injection of cis-DDP was found to contain about 2 fmol of Pt per microgram of DNA. This work establishes that cis-DDP-DNA adducts prepared in vitro are relevant to the in vivo binding of the Pt drug to its biological target, DNA, and opens new avenues for studying the mechanism of action of the Pt anticancer drugs.