Regulation and Function of the p14ARF/topoisomerase I Complex in Cancer

p14ARF/拓扑异构酶 I 复合物在癌症中的调节和功能

• 批准号: 7653663
• 负责人: RUTH A GJERSET
• 金额: $ 37.35万
• 依托单位: TORREY PINES INST FOR MOLECULAR STUDIES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-10 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7653663
• 关键词: Address; Affect; Antibodies; Antineoplastic Agents; Binding; Biochemical; Biological; Biological Assay; Biological Process; C-terminal; CDKN2A gene; Camptothecin; Cancer cell line; Cell Culture Techniques; Cell Line; Cell Nucleolus; Chromatin; Clinical; Complex; DNA; DNA Binding; Defect; Diagnostic; Down-Regulation; Drug Delivery Systems; Enzymes; Freezing; Frequencies; Goals; Human; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Lead; Malignant Neoplasms; Maps; Mediating; Modeling; Molecular; Molecular Biology Techniques; Mutation; Nonhomologous DNA End Joining; Nude Mice; Pathway interactions; Pensions; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Play; Protein Kinase C; Proteins; Regulation; Resistance; Role; Serine; Small Interfering RNA; Specimen; Surrogate Markers; TP53 gene; Testing; Therapeutic; Tissues; Treatment Failure; Tumor Suppressor Proteins; Type I DNA Topoisomerases; base; cancer cell; cancer therapy; casein kinase II; chemotherapeutic agent; design; human TOP1 protein; improved; in vitro Assay; in vivo; insight; irinotecan; neoplastic; neoplastic cell; novel; overexpression; public health relevance; response; synthetic construct; tumor

DESCRIPTION (provided by applicant): The long term goal of this study is to develop improved ways to suppress cancer through modulation of topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, and to further elucidate how the p14ARF/topoisomerase I complex contributes to the mechanism of cancer and to the therapy response. p14ARF, which plays a well-established role in the p53 pathway, has recently been found to engage in a novel interaction with topoisomerase I. The interaction requires topoisomerase I serine phosphorylation, results in activation of topoisomerase I activity, and leads to enhanced cellular sensitivity to a potent class of camptothecin-related chemotherapeutic agents that target topoisomerase I. Cancer-associated abnormalities that reduce phosphorylation of topoisomerase I, also abolish complex formation with p14ARF and correlate with therapy resistance in cell culture. Though highly relevant to the mechanism and treatment of cancer, the regulation of this complex remains poorly understood. A better molecular understanding of how the interaction is regulated and how it leads to activation of topo I and therapy sensitization will therefore further elucidate the roles of both proteins in cancer, and could lead to improved diagnostic strategies for identifying resistant tumors as well as improved therapeutic strategies for reversing resistance, a major cause of treatment failure. The Specific Aims of this project are to (1) Determine the molecular features of the p14ARF/topoisomerase I interaction, (2) Define the molecular mechanism of p14ARF-mediated activation of topoisomerase I, (3) Determine how frequently abnormalities in p14ARF/topoisomerase I complex formation occur in cancer and whether they correlate statistically with clinical attributes of tumors, and (4) Determine the therapeutic potential of p14ARF-mediated therapy sensitization. The study will employ molecular biology techniques to identify binding domains on topoisomerase I in Aim I, and biochemical assays with defined synthetic DNA substrates and purified p14ARF and topo I proteins to address mechanistic issues in Aim II. For Aim III, the study will analyze specimens of normal and neoplastic tissue by immunoprecipitation/western analysis and immunofluorescence. Aim IV will employ cell culture-based viability assays and human tumor xenograph models in nude mice. Together this combination of approaches is designed to clarify the biological function of the p14ARF/topoisomerase I complex and determine its therapeutic and diagnostic potential. PUBLIC HEALTH RELEVANCE: The present study will develop a rationale for improved therapeutic approaches to cancer through modulation of the essential cellular enzyme, topoisomerase I, a target for a highly potent class of chemotherapeutic drugs. A novel cellular complex between topoisomerase I and the p14ARF tumor suppressor, a central player in cancer, sensitizes cancer cells to topoisomerase I-targeted drugs, and cancer associated abnormalities in complex formation correlate with therapy resistance, a major obstacle to successful treatment of cancer. By elucidating the regulation and function of this complex in cancer, this study will provide further insight into the roles of these two proteins in cancer, and establish a basis for exploiting the complex therapeutically and diagnostically.

描述（由申请人提供）：这项研究的长期目标是通过调节拓扑素酶I，这是类似摄影剂的化学治疗药物的重要目标，开发改进的方法来抑制癌症，并进一步阐明P14ARF/拓扑异构酶I如何为癌症和治疗反应有助于癌症。 p14ARF, which plays a well-established role in the p53 pathway, has recently been found to engage in a novel interaction with topoisomerase I. The interaction requires topoisomerase I serine phosphorylation, results in activation of topoisomerase I activity, and leads to enhanced cellular sensitivity to a potent class of camptothecin-related chemotherapeutic agents that target topoisomerase I.降低拓扑异构酶I的磷酸化的癌症相关异常，也取消了与P14arf的复合物形成，并与细胞培养中的治疗耐药性相关。尽管与癌症的机制和治疗高度相关，但对这种复合物的调节仍然很少理解。因此，更好地理解了对相互作用的调节方式，以及它如何导致TOPO I和治疗敏化的激活将进一步阐明两种蛋白质在癌症中的作用，并可能导致改善诊断抗性肿瘤以及改善治疗策略的诊断策略，以改善抗药性，抗药性，治疗失败。 The Specific Aims of this project are to (1) Determine the molecular features of the p14ARF/topoisomerase I interaction, (2) Define the molecular mechanism of p14ARF-mediated activation of topoisomerase I, (3) Determine how frequently abnormalities in p14ARF/topoisomerase I complex formation occur in cancer and whether they correlate statistically with clinical attributes of tumors, and （4）确定P14ARF介导的治疗敏化的治疗潜力。该研究将采用分子生物学技术来鉴定AIM I中的拓扑异构酶I上的结合结构域，以及具有定义的合成DNA底物和纯化的P14ARF和TOPO I蛋白质的生化测定，以解决AIM II中的机械问题。对于AIM III，该研究将通过免疫沉淀/西方分析和免疫荧光分析正常和肿瘤组织的标本。 AIM IV将在裸鼠中采用基于细胞培养的生存力测定和人类肿瘤Xenographing模型。这种方法的组合旨在阐明p14arf/拓扑异构酶I复合物的生物学功能，并确定其治疗和诊断潜力。公共卫生相关性：本研究将通过调节基本细胞酶，拓扑异构酶I的调节，为改善癌症的治疗方法发展一个理由，这是高度有效的化学治疗药物类别的靶标。癌酶I和P14ARF肿瘤抑制剂（癌症的中心参与者）之间的一种新型细胞复合物使癌细胞对拓扑异构酶I靶向的药物以及癌症相关的复杂形成异常相关，这与治疗耐药性相关，是成功治疗癌症的主要障碍。通过阐明该复合物在癌症中的调节和功能，这项研究将进一步了解这两种蛋白质在癌症中的作用，并为通过治疗和诊断而利用该复合物的基础。