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Mechanisms of Cytotoxicity and Radiosensitization for Antimetabolites

抗代谢物的细胞毒性和放射增敏机制

基本信息

批准号：
8479128
负责人：
DONNA S. SHEWACH
金额：
$ 24.29万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-08 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8479128
关键词：
A549 Abbreviations Adverse effects Animals Antimetabolites Antineoplastic Agents Biological Assay Cell Cycle Progression Cell Line Cells Chinese Hamster Ovary Cell Clinic Comet Assay Cytarabine DNA DNA Damage DNA Double Strand Break DNA Mismatch Repair Protein MLH1 DNA Repair DNA Synthesis Inhibition DNA biosynthesis Data Deoxyribonucleosides Diphosphates Dose Drug Targeting Enzymes Failure Floxuridine Genetic Recombination Goals Grant HCT116 Cells HT29 Cells High Pressure Liquid Chromatography Human In Vitro Lesion MCF7 cell MLH1 gene MSH2 gene Mediating Metabolic Methotrexate Mismatch Repair Modeling Mus Normal tissue morphology Nucleotides Pathway interactions Patients Pemetrexed Pharmaceutical Preparations Proteins Protocols documentation Radiation therapy Radiation-Sensitizing Agents Radiosensitization Ribonucleotide Reductase Ribonucleotide Reductase Inhibitor Ribonucleotide Reductase Subunit Role SW620 Schedule Solid Neoplasm Subfamily lentivirinae Testing Thymidylate Synthase Thymidylate Synthase Inhibitor Toxic effect Translations analog anticancer activity base cancer cell cancer therapy cell killing cytotoxic cytotoxicity gemcitabine homologous recombination hydroxyurea improved in vivo irradiation neoplastic cell novel novel strategies prevent public health relevance recombinational repair repaired response ribonucleotide reductase M2 small hairpin RNA tripolyphosphate tumor tumor xenograft

项目摘要

DESCRIPTION (provided by applicant): Gemcitabine is an antimetabolite with broad solid tumor activity in patients. In addition, it is a potent radiation sensitizer. During the previous grant period, we investigated the metabolic and repair pathways important for cytotoxicity and radiosensitization with gemcitabine. Mechanistic studies demonstrated that inhibition of ribonucleotide reductase, mediated by the diphosphate of gemcitabine, was responsible primarily for inhibition of DNA synthesis, whereas incorporation of the analog into DNA contributed more to cytotoxicity. In addition, ribonucleotide reductase-mediated decrease in deoxynucleotides correlated strongly with radiosensitization. We tested the hypothesis that the imbalance in deoxynucleotides led to misincorporation of nucleotides into DNA which, if not repaired prior to irradiation, resulted in radiosensitization. Results demonstrated that gemcitabine produced mismatches in DNA, which occurred only at radiosensitizing concentrations and persisted only after irradiation. Radiosensitization with gemcitabine was enhanced by mismatch repair deficiency, which increased mismatches in DNA, and suppression of the p53-inducible ribonucleotide reductase subunit p53R2, which prolonged the deoxynucleotide pool imbalances. In contrast, mismatch repair deficiency decreased cytotoxicity with gemcitabine. We now propose that these mechanisms can be generalized to other antimetabolites that produce alterations in deoxynucleotide pools and function as radiosensitizers, such as hydroxyurea (ribonucleotide reductase inhibitor), and fluorodeoxyuridine, methotrexate and pemetrexed (thymidylate synthase inhibitors) to produce the first unifying hypothesis for antimetabolite radiosensitization. We will also evaluate cytotoxicity and radiosensitization of shRNA-mediated suppression of the enzymes targeted by these antimetabolites, and compare the in vivo antitumor efficacy of shRNA enzyme suppression with antimetabolites alone or with ionizing radiation. New data indicate that late- occurring DNA double strand breaks which are unable to be repaired by homologous recombination are responsible for radiosensitization with gemcitabine. The DNA damage and repair pathways, which may include ATM, ATR and homologous recombination, that produce radiosensitization will be evaluated. Preliminary results indicate that suppression of either thymidylate synthase or the R2 subunit of ribonucleotide reductase with shRNA is at least as effective as antimetabolites in producing radiosensitization. Mechanistic studies will aid in developing a general hypothesis for cytotoxicity and radiosensitization with the antimetabolites and shRNAs. In vivo animal studies will explore dosing antimetabolites or shRNA for radiosensitization based on the amount of drug or shRNA that will decrease dNTPs and produce misincorporated deoxynucleotides in tumor cell DNA. We further hypothesize that suppression of R2 in vivo will produce superior radiosensitization with lower normal tissue toxicity. These mechanism-based studies have high potential for translation to clinic to improve radiosensitization protocols for patients.

描述（由申请方提供）：吉西他滨是一种抗代谢药，在患者中具有广泛的实体瘤活性。此外，它还是一种有效的辐射敏化剂。在上一个资助期间，我们研究了代谢和修复途径的细胞毒性和放射增敏与吉西他滨的重要性。机制研究表明，抑制核糖核苷酸还原酶，介导的吉西他滨的二磷酸，是主要负责抑制DNA合成，而类似物掺入DNA的细胞毒性贡献更多。此外，核糖核苷酸还原酶介导的脱氧核苷酸减少与放射增敏密切相关。我们测试的假设，即在脱氧核苷酸的不平衡导致错误的核苷酸掺入DNA，如果不修复前照射，导致放射增敏。结果表明，吉西他滨在DNA中产生错配，仅在放射增敏浓度下发生，并且仅在照射后持续。错配修复缺陷增加DNA错配，抑制p53诱导型核糖核苷酸还原酶亚基p53 R2延长脱氧核苷酸池失衡，从而增强吉西他滨的放射增敏作用。相反，错配修复缺陷降低了吉西他滨的细胞毒性。我们现在提出，这些机制可以推广到其他抗代谢物，产生脱氧核苷酸池的变化，并作为放射增敏剂，如羟基脲（核糖核苷酸还原酶抑制剂），氟脱氧尿苷，甲氨蝶呤和培美曲塞（胸苷酸合成酶抑制剂），以产生抗代谢物放射增敏的第一个统一的假设。我们还将评估这些抗代谢物靶向的酶的shRNA介导的抑制的细胞毒性和放射增敏性，并比较shRNA酶抑制与单独的抗代谢物或电离辐射的体内抗肿瘤功效。新的数据表明，不能通过同源重组修复的晚期发生的DNA双链断裂是吉西他滨放射增敏的原因。将评价产生放射增敏作用的DNA损伤和修复途径，可能包括ATM、ATR和同源重组。初步结果表明，抑制胸苷酸合成酶或核糖核苷酸还原酶的R2亚基与shRNA至少是一样有效的抗代谢物在产生放射增敏。机制研究将有助于开发抗代谢物和shRNA的细胞毒性和放射增敏的一般假设。体内动物研究将基于药物或shRNA的量探索用于放射增敏的抗代谢物或shRNA的剂量，所述药物或shRNA的量将减少dNTP并在肿瘤细胞DNA中产生错误掺入的脱氧核苷酸。我们进一步假设，抑制R2在体内将产生上级放射增敏与较低的正常组织毒性。这些基于机制的研究具有很高的潜力，可转化为临床，以改善患者的放射增敏方案。