THYMIDYLATE SYNTHASE IN HEAD AND NECK CANCER

胸苷酸合酶在头颈癌中的作用

• 批准号: 2108872
• 负责人: JOHN J MCGUIRE
• 金额: $ 14.02万
• 依托单位: ROSWELL PARK CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-21 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2108872
• 关键词: DNA replication; acid aminoacid ligase; antineoplastics; athymic mouse; cytotoxicity; disease /disorder model; drug administration rate /duration; drug hypersensitivity; drug resistance; drug screening /evaluation; enzyme inhibitors; flow cytometry; folate; gastrointestinal epithelium; head /neck neoplasm; intracellular transport; model design /development; neoplasm /cancer pharmacology; phenotype; squamous cell carcinoma; thymidylate synthase; tissue /cell culture

The goal of this research is to improve selective chemotherapy for head and neck (HN) squamous cell carcinoma (SCC) by targeting the folate- dependent enzyme thymidylate synthase (TS). Current antifolate therapy (methotrexate; MTX) is used in HNSCC, but resistance often occurs to chemotherapy regimens in which this dihydrofolate reductase (DHFR) inhibitor is employed thus limiting both response rate and long-term survivors. However, since folate metabolism is vulnerable in HNSCC, antifolates unrelated to MTX may be therapeutically superior. The folate- dependent enzyme TS, because of its role as the only de novo source of thymidine nucleotides for DNA synthesis, is a key target for new antifolates. We propose to explore the cellular/biochemical pharmacology of three new TS-directed inhibitors in in vitro human HNSCC models. These data will be used to optimally design therapeutic studies in mice proposed in Project 2 of this Interactive RO1 (Dr. Y. Rustum) and to define crucial determinants of response to these drugs that should be monitored in vivo (Project 2). This goal will be addressed through these Specific Aims: 1. Characterize human HNSCC cell lines to develop model systems for studying TS inhibitors. FaDu, from a human pharynx SCC, and A253, from a human epidermoid carcinoma of the neck, cell lines will be used since they represent intrinsic sensitivity (FaDu) and resistance (A253) to MTX in brief exposures, such as occur clinically. Another factor in their choice is that A253 and FaDu produce well-differentiated and less differentiated squamous cell carcinomas, respectively, in nude mice and thus can be used in in vivo studies (Project 2). Growth characteristics, cloning efficiency, and effects of supplements will be assessed during culture as monolayers and as multicell spheroids (MS). 2. Define the cellular and biochemical pharmacology in human HNSCC models and normal murine intestine of D1694, AG331, and 1843U89 (three folate- related TS inhibitors) as single agents. Concentration/schedule dependence of monolayer HNSCC cell growth inhibition by the drugs as single agents will be fully defined; clonogenic survival will be measured as appropriate. The mechanism of action of the drugs will be studied using isolated enzymes (TS, folylpolyglutamate synthetase, and DHFR), metabolite protection, and measurement of whole cell transport/metabolism and folate pools to identify crucial determinants of response. Other parameters would be investigated as dictated by results. Analogous studies in HNSCC MS, would be undertaken. Results from monolayer and MS studies will be correlated with in vivo results (Project 2). Specific comparative studies using normal mouse intestinal epithelium will be used to explore selective toxicity of these drugs. Useful combinations of TS inhibitors and selected agents would be studied in a renewal. 3. Select sublines of the HNSCC models with acquired resistance to each TS inhibitor and characterize the resistance phenotype. Sublines of HNSCC models resistant to the drugs will be selected in monolayers using both continuous and intermittent drug exposure, since the exposure schedule may influence resistance frequency and/or resistance phenotype. Biochemical/cellular study of resistant sublines will be performed as for parental cells. Studies of resistance in the HNSCC MS model would be initiated following a similar protocol. Resistance in both in vitro models will be correlated with resistance in vivo (Project 2).

本研究的目的是提高选择性化疗的头部 和颈部（HN）鳞状细胞癌（SCC），通过靶向叶酸- 依赖性酶胸苷酸合成酶（TS）。 当前抗叶酸治疗 （甲氨蝶呤; MTX）用于HNSCC，但经常发生耐药性， 二氢叶酸还原酶（DHFR） 因此限制了反应速率和长期 幸存者然而，由于叶酸代谢在HNSCC中是脆弱的， 与MTX无关的抗叶酸剂可能在治疗上更上级。 叶酸- 依赖酶TS，因为它作为唯一的从头来源的作用， 用于DNA合成的胸苷核苷酸，是新的 抗叶酸剂我们建议探索细胞/生化药理学 在体外人HNSCC模型中的三种新的TS定向抑制剂。这些 数据将用于最佳设计小鼠治疗研究， 在本交互式RO 1的项目2中（Y. Rustum）并定义关键 应在体内监测对这些药物的反应的决定因素 （项目2）。这一目标将通过以下具体目标实现： 1.表征人HNSCC细胞系以开发用于以下的模型系统： 研究TS抑制剂FaDu，来自人咽部SCC，和A253，来自人咽部SCC。 人颈部表皮样癌，将使用细胞系，因为它们 代表MTX的固有敏感性（FaDu）和耐药性（A253）， 短暂暴露，如临床上发生的情况。他们选择的另一个因素是 A253和FaDu产生分化良好和分化较低细胞 鳞状细胞癌，因此可以使用 体内研究（项目2）。 生长特征、克隆 在培养过程中，将评估补充剂的效率和效果， 单层和多细胞球状体（MS）。 2.定义人类HNSCC模型中的细胞和生化药理学 和D1694、AG 331和1843 U89（三种叶酸- 相关TS抑制剂）作为单一药剂。浓度/时间表依赖性 单层HNSCC细胞生长抑制的药物作为单一药物 将被完全定义;克隆生存率将被测量为 适当将使用以下方法研究药物的作用机制 分离的酶（TS、叶酰聚谷氨酸合成酶和DHFR）、代谢物 保护和测量全细胞转运/代谢和叶酸 确定应对措施的关键决定因素。其他参数将 根据结果进行调查。HNSCC MS中的类似研究， 将进行。单层和MS研究的结果将 与体内结果相关（项目2）。具体比较研究 使用正常小鼠肠上皮将用于探索选择性的 这些药物的毒性。TS抑制剂和选择的组合的有用组合 代理商将在更新中进行研究。 3.选择对每种TS具有获得性抗性的HNSCC模型的亚系 抑制剂并表征耐药表型。HNSCC亚系 对药物有抗性的模型将在单层中使用两种方法来选择， 连续和间歇性药物暴露，因为暴露时间表可能 影响抗性频率和/或抗性表型。 将对耐药亚系进行生化/细胞研究， 亲代细胞HNSCC MS模型中的抗性研究将是 开始遵循类似的协议。 两种体外耐药性 模型将与体内耐药性相关（项目2）。