BIOCHEMICAL STUDIES OF ACQUIRED CHEMOTHERAPY RESISTANCE

此应用程序的总体目标是在临床上识别 伊立替康产生耐药的相关机制 局部晚期非小细胞肺癌患者的基础化疗 肺癌(NSCLC)。伊立替康是一种喜树碱的衍生物， 在非小细胞肺癌中的显著活动。当前对当地的管理 先进的非小细胞肺癌在诊断时结合了手术分期， 化疗，然后手术切除肿瘤 肿瘤，从而提供了一个独特的机会来获得足够的 术前化疗前后的病理资料分析 为进行建议的实验室研究。喜树碱 稳定拓扑异构酶I：DNA共价复合体的研究 单链蛋白连锁DNA断裂(PLDB)。PLDB可以 随后被转换为dsDNA断裂，这些断裂可能 致命的。细胞对喜树碱的耐药性可能与步骤有关 在公共分贝形成之前发生的，例如减少 TOPO I，或在PLDB形成后发生的步骤的结果， 例如增强修复DNA损伤的能力。为了 描述体内对喜树碱的耐药性是如何产生的， 三个周期前、后采集的非小细胞肺癌肿瘤组织 伊立替康联合顺铂的手术化疗 被分析。从肿瘤制备的单细胞悬浮液中 喜树碱体外孵育对dsDNA损伤和pLdB的影响 将对队形进行分析。药敏比较会 确定接受有限的非治愈性化疗是否 引起耐药性，以及耐药性是在之前还是之后产生的 公共分贝的形成。细胞内的浓度、活性、 分布和拓扑异构酶I对伊立替康的亲和力将为 其特征是确定这些特定生物化学中的哪一种 基因改变导致体内对伊立替康产生耐药性。其他内容 研究将根据是否对DNA进行分析来指导 病变提示在治疗过程中获得的耐药性发生。 在形成PLDB之前和之后。在完成以下工作后 这是第一次临床试验，第二次研究将利用 伊立替康联合紫杉醇。生化研究 分析耐药的临床相关机制 喜树碱将被重复使用。表型的比较 在肿瘤中观察到的变化将提供对 伊立替康的耐药机制 与不同类别的化疗药物联合使用。在……里面 此外，由于多药耐药(MDR)可能与 紫杉醇的活性，我们将评估这些肿瘤的 多药耐药在术前暴露前后的表达 化疗。这些药物外排相关的过度表达 蛋白质将通过免疫细胞化学和功能测定 用99TcSESTAMIDI活体扫描。如果表达的是 非小细胞肺癌患者术前化疗后多药耐药增加 含有紫杉醇，然后选择肿瘤内的克隆 这些MDR的表达可能与最终治疗失败有关。 这将支持调制器正在进行的额外试验 包括在最初的治疗中以根除这些克隆人。这些 生化方法应该能更好地理解 非小细胞肺癌获得性耐药机制的研究 并导致治疗这种疾病的新策略。

The overall aim of this application is to identify the clinically relevant mechanisms of resistance which develop to irinotecan- based chemotherapy in patients with locally advanced non small cell lung cancer (NSCLC). Irinotecan is a camptothecin derivative with significant activity in NSCLC. Current management of locally advanced NSCLC incorporates surgical staging at diagnosis, treatment with chemotherapy, and then surgical resection of the tumor, thus providing a unique opportunity to obtain sufficient pathologic material before and after pre-operative chemotherapy in order to conduct the laboratory studies proposed. Camptothecins stabilize the topoisomerase (topo) I: DNA covalent complex in a single strand protein linked DNA break (PLDB). PLDBs can subsequently be converted to dsDNA breaks which are potentially lethal. Cellular resistance to camptothecin may be related to steps occurring before the formation of the PLDB, such as reduction in topo I, or result from steps occurring after formation of the PLDB, such as an enhanced capacity to repair DNA damage. In order to characterize how resistance to camptothecin develops in vivo, NSCLC tumor tissue obtained before and after three cycles of pre- operative chemotherapy consisting of irinotecan and cisplatin will be analyzed. In single cell suspensions prepared from tumor and incubated in camptothecin in vitro, dsDNA damage and PLDB formation will be analyzed. Comparison of drug sensitivity will determine whether limited exposure to non-curative chemotherapy induces resistance and whether resistance develops before or after formation of the PLDB. The concentration, activity, intracellular distribution, and topoisomerase I affinity for irinotecan will be characterized to determine which of these specific biochemical alterations contribute to resistance to irinotecanin vivo. Additional studies will be directed according to whether analysis of DNA lesions suggest that resistance acquired during treatment occurs before of after formation of the PLDB. Following completion of this first clinical trial, a second study will be performed utilizing the combination of irinotecan plus paclitaxel. Biochemical studies to analyze clinically relevant mechanisms of resistance to camptothecins will be repeated. Comparison of the phenotype changes observed in the tumor will provide insight into the mechanisms of resistance which are invoked toirinotecan when it is combined with different classes of chemotherapeutic agents. In addition, since multidrug resistance (MDR) may be relevant to the activity of paclitaxel, we will evaluate these tumors for the expression of MDR before and after exposure to pre-operative chemotherapy. The overexpression of these drug efflux associated proteins will be measured by immunocytochemistry and functionally by in-vivo scanning with 99Tc-sestamidi. If the expression of MDR is increased in NSCLC following pre-operative chemotherapy containing paclitaxel, then selection of clones within the tumor which express MDR may be relevant to ultimate treatment failure. This would support additional trials in which modulators are included in the initial treatment to eradicate these clones. These biochemical approaches should result in a better understanding of the mechanisms of acquired resistance to chemotherapy in NSCLC and lead to new strategies to treat this disease.