Beckerbauer L, Tepe J J, Cullison J, Reeves R, Williams R M
School of Molecular Biosciences, Biochemistry/Biophysics, Washington State University, Pullman, WA 99164-4660, USA.
Chem Biol. 2000 Oct;7(10):805-12. doi: 10.1016/s1074-5521(00)00028-4.
Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity.
Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent.
We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.
高迁移率族（HMG）I/Y染色质癌蛋白家族的过表达被认为是肿瘤细胞转化和几种人类癌症转移潜能增加的临床诊断标志物。因此，这些小沟DNA结合癌蛋白是抗肿瘤化疗的一个有吸引力的靶点。FR900482代表一类新型抗肿瘤药物，它与DNA小沟结合，与结构相关的丝裂霉素C类抗肿瘤药物相比，宿主毒性大大降低。我们报告了FR900482在体内使DNA与HMG I/Y发生共价交联，这是与小沟结合癌蛋白的共价DNA-药物-蛋白质交联的首个例子，也是这些化合物发挥抗肿瘤活性的潜在新机制。
使用改良的染色质免疫沉淀方法，对在体内被FR900482与HMG I/Y蛋白共价交联的DNA片段进行聚合酶链反应扩增、分离和表征。对照细胞的核样本中没有DNA片段，而用FR900482处理的细胞的核样本中含有在体内被药物与小沟结合的HMG I/Y蛋白交联的DNA片段。额外的对照实验表明，该药物在体内也能交联其他非致癌性小沟结合蛋白（HMG-1和HMG-2），但不能交联大沟结合蛋白（Elf-1和NFκB）。我们的结果首次证明FR900482在体内能交联多种小沟结合蛋白，并表明HMG I/Y癌蛋白的交联可能参与了作为化疗药物的作用方式。
我们已经表明，本研究中以FR900482为代表的FR类抗肿瘤抗生素能够在体内使HMG I/Y癌蛋白与DNA产生共价交联。这类化合物在DNA小沟中交联HMG I/Y蛋白的能力代表了在活细胞中药物诱导的特定癌症相关蛋白与DNA交联的首次证明。我们还证明了FR900482在体内能交联其他小沟结合蛋白（本研究中的HMG-1和HMG-2）；然而，由于HMG I/Y是目前已知的唯一小沟结合癌蛋白，这些非组蛋白染色质蛋白有可能是这类药物重要的体内靶点。由于其大大降低的宿主毒性和优异的DNA链间交联功效，这些化合物已被评估为丝裂霉素C极具吸引力的临床替代物。FR900482在体内使HMG I/Y癌蛋白与核DNA交联的能力可能显著阐明了这类抗癌药物的抗肿瘤功效。
