DNA Repair and Cell Cycle Checkpoints as Targets for Ant

DNA 修复和细胞周期检查点作为 Ant 的目标

• 批准号: 6761648
• 负责人: YVES POMMIER
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6761648
• 关键词: DNA damage; DNA repair; DNA topoisomerases; antineoplastics; apoptosis; cell cycle; cytotoxicity; drug interactions; enzyme inhibitors; enzyme mechanism; neoplasm /cancer pharmacology; tissue /cell culture

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).

Ecteinascidin 743 (Et743) （NSC 648766）是一种处于II/III期临床试验的新型抗癌药物。该药因其临床活性和独特的作用机制而引人注目。在众所周知对其他已知治疗方法具有耐药性的肉瘤以及卵巢癌和乳腺癌中已经观察到这种反应。Et743不同于其他临床使用的抗癌药物，因为它在DNA次要凹槽的特定鸟嘌呤上形成共价加合物，因为它选择性地阻断与富含鸟嘌呤序列结合的转录因子（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Et743耐药细胞系。两种细胞系均缺失DNA核苷酸切除修复（NER）基因XPG，转染XPG cDNA可恢复HCT116/ER5细胞对Et743的敏感性。我们还发现缺乏NER基因XPG、XPA、XPD或XPF的着色性干皮细胞对Et743具有抗性。通过与野生型基因的互补恢复敏感性。此外，对XPC或Cockayne综合征基因（CSA和CSB）缺失的细胞的研究表明，药物敏感性特异性依赖于NER的转录偶联途径（TC-NER），而不是全球基因组途径（GG-NER）。这些发现使我们提出Et743与转录偶联NER （TC-NER）机制相互作用诱导致命的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修复能力并行。这些发现促使我们研究顺铂的ner依赖性。我们发现有缺陷的TC-NER使细胞对顺铂敏感，而有缺陷的全局基因组修复（GG-NER）不影响顺铂反应。Et743和顺铂在TC-NER方面的互补性表明，Et743可用于顺铂耐药肿瘤，反之亦然。Et743治疗顺铂耐药卵巢癌的I期临床试验已提出临床方案（与NCI CCR病理学分部Elise Kohn博士合作）。进一步的分子研究计划确定Et743诱导的DNA单链断裂的转录特异性和链特异性依赖。我们还通过使用ner缺陷、XPD和XPD补充细胞的微阵列分析来研究tc - ner依赖性转录抑制。