Monjardet-Bas Véronique, Chottard Jean-Claude, Kozelka Jirí
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université René Descartes, UMR 8601 CNRS, 45 rue des Saints-Pères, 75270 Paris, France.
Chemistry. 2002 Mar 1;8(5):1144-50. doi: 10.1002/1521-3765(20020301)8:5<1144::aid-chem1144>3.0.co;2-k.
The antitumor drug cisplatin forms two kinds of guanine-guanine cross-links with DNA: intrastrand, occurring mainly at GG sites, and interstrand, formed at GC sites. The former are generally more abundant than the latter, at least in experiments with linear duplex DNA. The formation of interstrand cross-links requires partial disruption of the Watson-Crick base pairing, and one could therefore expect the cross-linking reaction to be rather slow. In contrast with this expectation, kinetic measurements reported here indicate that interstrand cross-linking is as fast as intrastrand, or even faster. We have investigated the reactions between two hairpin-stabilized DNA duplexes, containing either a d(TGCA)(2) sequence (duplex TGCA) or a d(G(1)G(2)CA)-d(TG(3)CC) sequence (duplex GGCA), and the diaqua form of cisplatin, cis-Pt(NH(3))(2)(H(2)O)(2), in an unbuffered solution kept at pH 4.5 +/- 0.1 and 20 degrees C. Using HPLC as the analytical method, we have determined the platination (first step) and chelation (second step) rate constants for these reaction systems. Duplex TGCA, in which the two guanines are quasi-equivalent, is found to be platinated very slowly (k=0.5 +/- 0.1M(-1)s(-1)) and to form the final interstrand cross-link very rapidly (k=13 +/- 3 x 10(-3) s(-11)). For GGCA, we find that G(1) is platinated rapidly (k=32 +/- 5M(-1)s(-1)) to form a long-lived monoadduct, which is only slowly chelated (k=0.039 +/- 0.001 x 10(-3) s(-1)) by G(2) (intrastrand), while G(2) is platinated one order of magnitude more slowly than G(1) (k=2.0 +/- 0.5M(-1)s(-1)) and chelated fairly rapidly both by G(1) (intrastrand: k=0.4 +/-0.1 x 10(-3) s(-1)) and G(3) (interstrand: k=0.2 +/- 0.1 x 10(-3) s(-1)); finally, G(3) is platinated at about the same rate as G(2) (k=2.4 +/- 0.5M(-1)s(-1)) and chelated very rapidly by G(2) (interstrand: k=10 +/- 4 x 10(-3) s(-1)). These results suggest that the low occurrence of interstrand cross-links in cisplatinated DNA is due to an extremely slow initial platination of guanines involved in d(GC)(2) sequences, rather than to a slow cross-linking reaction.
抗肿瘤药物顺铂与DNA形成两种鸟嘌呤 - 鸟嘌呤交联：链内交联，主要发生在GG位点；链间交联，形成于GC位点。前者通常比后者更丰富，至少在对线性双链DNA的实验中如此。链间交联的形成需要部分破坏沃森 - 克里克碱基对，因此可以预期交联反应相当缓慢。与这种预期相反，此处报道的动力学测量表明链间交联与链内交联一样快，甚至更快。我们研究了两个发夹稳定的DNA双链体之间的反应，其中一个含有d(TGCA)(2)序列（双链体TGCA），另一个含有d(G(1)G(2)CA)-d(TG(3)CC)序列（双链体GGCA），以及顺铂的二水合物形式顺 - Pt(NH(3))(2)(H(2)O)(2)，反应在pH值为4.5±0.1且温度为20℃的无缓冲溶液中进行。使用高效液相色谱法作为分析方法，我们测定了这些反应体系的铂化（第一步）和螯合（第二步）速率常数。发现双链体TGCA中两个鸟嘌呤几乎等效，其铂化非常缓慢（k = 0.5±0.1M(-1)s(-1)），而形成最终链间交联非常迅速（k = 13±3×10(-3)s(-11)）。对于双链体GGCA，我们发现G(1)迅速铂化（k = 32±5M(-1)s(-1)）形成一个长寿命的单加合物，该单加合物仅被G(2)（链内）缓慢螯合（k = 0.039±0.001×10(-3)s(-1)），而G(2)的铂化比G(1)慢一个数量级（k = 2.0±0.5M(-1)s(-1)），并且被G(1)（链内：k = 0.4±0.1×10(-3)s(-1)）和G(3)（链间：k = 0.2±0.1×10(-3)s(-1)）相当迅速地螯合；最后，G(3)的铂化速率与G(2)大致相同（k = 2.4±0.5M(-1)s(-1)），并被G(2)（链间：k = 10±4×10(-3)s(-1)）非常迅速地螯合。这些结果表明，顺铂化DNA中链间交联发生率低是由于参与d(GC)(2)序列的鸟嘌呤初始铂化极其缓慢，而不是交联反应缓慢。
