DNA Repair and Cell Cycle Checkpoints as Targets for Ant

Et743(NSC 648766)是一种正在进行II/III期临床试验的新型抗癌药物。这种药物之所以引人注目，是因为它的临床活性和独特的作用机制。在肉瘤以及卵巢癌和乳腺癌中也观察到了反应，众所周知，肉瘤对其他已知的治疗方法具有耐药性。Et743与其他临床使用的抗癌药物不同，因为它在DNA小沟中的特定鸟嘌呤处形成共价加合物，而且它选择性地阻止受富含鸟嘌呤序列的转录因子(如NF-Y、Sp1和SXR转录因子)调控的基因的转录。为了进一步阐明Et743的作用机制，我们从NCI抗癌细胞筛选的人结肠癌细胞系(HCT116/ER5和SW620/ER0.5)中获得了两个耐Et743的细胞系。两种细胞系均缺乏DNA核苷酸切除修复(NER)基因XPG，XPG基因的转染可恢复HCT116/ER5细胞对Et743的敏感性。我们还发现，缺失NER基因XPG、XPA、XPD或XPF的着色性干皮病细胞对Et743具有抗性。通过与野生型基因互补来恢复敏感性。此外，对缺乏XPC或Cockayne综合征基因(CsA和CSB)的细胞的研究表明，药物敏感性具体依赖于NER的转录偶联途径(TC-NER)，而不是全球基因组途径(GG-NER)。这些发现导致我们提出Et743与转录偶联NER(TC-NER)机制相互作用，诱导致命性DNA链断裂。Et743定义了一类新的抗癌药物，其中增强的抗增殖活性与增强的细胞DNA修复能力平行。这些发现引导我们研究顺铂对NER的依赖。我们发现，缺陷的TC-NER使细胞对顺铂敏感，而缺陷的全球基因组修复(GG-NER)不影响顺铂的反应。Et743和顺铂相对于TC-NER的活性之间的互补性表明Et743用于顺铂耐药肿瘤，反之亦然。已经提出了一项临床方案，用于Et743对顺铂耐药的卵巢癌的I期临床试验(与NCI CCR病理分会Elise Kohn博士合作)。计划进行进一步的分子研究，以确定Et743诱导的DNA单链断裂对转录和链特异性的依赖性。我们还通过使用NER缺失、XPD和XPD互补的细胞的微阵列分析来研究TC-NER依赖的转录抑制。 另一个与DNA修复相关的发现是APE-1(APEX)在细胞凋亡DNA片段化中的作用。我们之前曾报道在人类白血病HL60细胞中存在一种未知的凋亡核酸酶(AN34)。经过纯化和测序，我们发现AN34与人无嘌呤核酸内切酶(APE-1=APEX=ref-1)相对应。这一鉴定已被免疫印迹和免疫阻断实验所证实。生化检测表明，caspase3在细胞凋亡过程中可激活APEX的内切核酸酶和3‘外切核酸酶。 由于大多数癌症的细胞周期检查点通路(P53、pRb)和细胞周期机制(细胞周期蛋白、细胞周期蛋白依赖的激酶抑制物，如p16)发生改变，我们正在探索细胞周期检查点的抑制剂作为新的抗癌药物。7-羟基金孢菌素(UCN-01)是一种正在进行II/III期临床试验的新型抗癌药物。我们发现UCN-01与DNA损伤剂，如拓扑异构酶抑制剂和作用于细胞周期S期的药物具有协同作用。这种协同作用与S阶段检查点的取消有关，该检查点由两个蛋白激酶Chk1和Chk2控制。我们发现UCN-01同时抑制Chk1和Chk2，我们正在研究Chk2在癌细胞细胞周期检查点反应中的作用。我们已经将Chk2表达为重组蛋白，并正在进行使用高通量筛选发现Chk2抑制剂的初步实验(与Shoemaker博士和Scudiero，DTP，NCI合作)。

Ecteinascidin 743 (Et743) (NSC 648766) is a novel anticancer agent in Phase II/III clinical trials. This drug is remarkable because of its clinical activity and its unique mechanism of action. Responses have been observed in sarcomas, which are notoriously resistant to other known treatments, as well as in ovarian and breast cancer. Et743 differs from other clinically used anticancer agents because it forms covalent adducts at specific guanines in the DNA minor groove and because it selectively block transcription of genes regulated by transcription factors that bind to guanine-rich sequences (such as NF-Y, Sp1, and SXR transcription factors). To further elucidate the mechanism of action of Et743, we have generated two Et743-resistant cell lines from human colon carcinoma cell lines from the NCI Anticancer Cell Screen (HCT116/ER5 and SW620/ER0.5). Both cell lines were found to be deficient for the DNA nucleotide excision repair (NER) gene, XPG, and transfection of XPG cDNA restored the Et743 sensitivity in the HCT116/ER5 cells. We also found that Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD, or XPF were resistant to Et743. Sensitivity was restored by complementation with wild-type genes. Moreover, studies with cells deficient in XPC or in the Cockayne Syndrome genes (CSA and CSB) indicated that the drug sensitivity is dependent specifically on the transcription-coupled pathway of NER (TC-NER) rather than on the global genome pathway (GG-NER). These findings led us to propose that Et743 interacts with the transcription-coupled NER (TC-NER) machinery to induce lethal DNA strand breaks. Et743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability. These findings led us to study the NER-dependence for cisplatin. We found that defective TC-NER sensitizes cells to cisplatin, whereas defective global genome repair (GG-NER) did not affect cisplatin response. The complementary between the activities of Et743 and cisplatin with respect to TC-NER suggests the use of Et743 in cisplatin-resistant tumors and vice-versa. A clinical protocol has been proposed for a Phase I clinical trial of Et743 in ovarian cancers resistant to cisplatin (Collaboration with Dr. Elise Kohn, Pathology Branch, CCR, NCI). Further molecular studies are planned to determine the transcription- and the strand-specific-dependence of the DNA single-strand breaks induced by Et743. We are also looking at TC-NER-dependent transcription inhibition by microarray analyses using NER-deficient, XPD, and XPD-complemented cells. Another finding related to DNA repair is the implication of APE-1 (APEX) in apoptotic DNA fragmentation. We had previously reported the presence of an unidentified apoptotic nuclease (AN34) in human leukemia HL60 cells. After purification and peptide sequencing, we found that AN34 corresponds to human apurinic endonuclease (APE-1 = APEX = REF-1). This identification has been confirmed by immunoblotting and immunoblocking experiments. Biochemical assays demonstrated that caspase 3 can activate the endo- and 3'-exo-nuclease of APEX during apoptosis. Because most cancers have alterations in the cell cycle checkpoint pathways (p53, pRb) and cell cycle machinery (cyclins, cyclin-dependent kinase inhibitors - such as p16), we are exploring inhibitors of cell cycle checkpoints as novel anticancer agents. 7-hydroxystaurosporine (UCN-01) is a novel anticancer agent in phase II/III clinical trials. We found that UCN-01 is synergistic with DNA damaging agents such as topoisomerase inhibitors and drugs that act during the S-phase of the cell cycle. This synergism has been related to an abrogation of the S-phase checkpoint, which is controlled by 2 protein kinases, Chk1 and Chk2. We found that UCN-01 inhibits both Chk1 and Chk2, and we are investigating the role of Chk2 in cell cycle checkpoint response in cancer cells. We have expressed Chk2 as a recombinant protein and preliminary experiments are ongoing to discover Chk2 inhibitors using a high throughput screen (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI).