Cellular Pharmacology in Cancer Chemotherapy

涉及核苷类似物代谢的耐药机制在原发肿瘤样本中并不普遍。由于将类似物掺入DNA通常对其细胞毒性至关重要，因此临床相关的耐药机制可能涉及将伪造核苷酸掺入DNA后的信号传导过程。在体外或治疗过程中，白血病细胞暴露于核苷类似物的细胞抑制剂浓度下，终止DNA合成并阻止细胞周期在S期的进展。在移除药物后，一部分细胞恢复进展并重新繁殖疾病。因此，细胞有能力延缓细胞周期进程，阻止DNA合成，从而限制类似物的掺入，这可能是一种保护细胞潜在毒性的防御机制。我们假设，一旦分子机制感知到核苷酸类似物与DNA的结合，信号被激活以阻止DNA复制，限制类似物分子进一步与DNA的结合，并允许修复发生。一旦类似物被清除，抑制信号被移除，细胞继续循环。这将构成一种正常的细胞防御反应，可能表现为实验和临床耐药性。作为一个推论，我们假设细胞周期调节过程的扰动可能产生导致细胞死亡的相互冲突的信号，从而绕过细胞防御机制。该项目旨在建立和验证人体实体肿瘤细胞对治疗性核苷类似物的S期特异性细胞周期阻滞模型，并研究失调的后果。下面的问题将评估这些假设。首先，对核苷类似物反应的S相阻滞的分子决定因素是什么？我们将开发核苷类似物诱导的S期阻滞细胞系模型。这些将用于比较S期阻滞细胞和S期富集群体中与细胞周期蛋白A相关的Cdk2激酶活性的变化，细胞周期蛋白A是驱动细胞进入和通过S期的复合物。Cdk2的抑制性磷酸化在包括Cdc25A、Chkl和Chk2的调控层次中的作用将被定义，我们将评估周期蛋白依赖性激酶抑制剂（如p21）在S期阻滞细胞中Cdk2激酶活性方面的作用。其次，干预对核苷类似物阻滞细胞存活调节的生物学作用和分子后果是什么？我们将使用激酶抑制剂，如UCN-01，在单独使用时不影响生长的浓度下，失调S期阻滞机制。调控机制将评估活性的特定改变，这些改变可能与细胞周期进展或凋亡过程的开始有关。第三，S期阻滞失调对细胞活力的影响是什么？我们将通过激活凋亡反应的信号，如应激反应级联、BAX上调和前caspase-9激活，或通过破坏包括PI 3-激酶或NFkappaB在内的存活途径的刺激，寻找与S期阻滞功能障碍的因果关系的证据。我们将研究可能启动S相阻滞和对其失调的反应的传感器。这些问题的答案应该产生对核苷类似物潜在耐药机制的分子药理学的新认识。

Resistance mechanisms that involve the metabolism of nucleoside analogues are not prevalent in primary tumor samples. Because incorporation of the analogues into DNA is generally essential to their cytotoxicity, clinically relevant resistant mechanisms are likely to involve signaling processes subsequent to the incorporation of the fraudulent nucleotides into DNA. Exposure of leukemia cells, either in vitro or during therapy, to cytostatic concentrations of nucleoside analogues, terminates DNA synthesis and arrests cell cycle progression in S phase. Subsequent to the removal of the drug a portion of the cells resume progression and re-population of the disease. Thus, the ability of cells to enact a delay in cell cycle progression that stops DNA synthesis, thereby limiting the incorporation of analogue, may be a defense mechanism that spares cells potential toxicity. We hypothesize that once molecular mechanisms sense the incorporation of a nucleotide analogue into DNA, signals are activated to block DNA replication, limiting further incorporation of analogue molecules into DNA and allowing repair to occur. Once the analogue is cleared, the inhibitory signal is removed and cells continue cycling. This would constitute a normal cellular defense response that may manifest itself as experimental and clinical resistance. As a collolary, we postulate that perturbation of the cell cycle regulatory processes may generate conflicting signals that result in cell death, thereby circumventing the cellular defense mechanism. This project seeks to establish and validate a model for S phase- specific cell cycle arrest by human solid tumor cells in response to therapeutic nucleoside analogues, and to investigate the consequences of dysregulation. The following questions will evaluate these hypotheses. First, what are the molecular determinants of the S phase arrest in response to nucleoside analogues? We will develop cell line models for nucleoside analogue-induced S phase arrest. These will be used to compare in S phase arrested cells and S phase enriched populations the alterations of the kinase activity of Cdk2 associated with cyclin A, the complex that drive cells into and through S phase. The role of inhibitory phosphorylations of Cdk2 in the regulatory hierarchy involving Cdc25A, Chkl and Chk2 will be defined, and we will evaluate the role of cyclin dependent kinase inhibitors such as p21 with respect to the kinase activity of Cdk2 in S phase arrested cells. Second, what are the biological actions and molecular consequences of intervention in the regulation of survival in nucleoside analogue-arrested cells? We will dysregulate the S phase arresting mechanism using kinase inhibitors such as UCN-01 at concentrations that do not affect growth when used alone. The regulatory mechanisms will be evaluated for specific alterations in activity that may be associated with either cell cycle progression or with initiation of apoptotic processes. Third, what are the consequences of S phase arrest dysregulation on cell viability? Evidence for a cause and effect relationship with S phase arrest dysfunction will be sought with signals that activate apoptotic responses such as stress response cascades, up-regulation of BAX, and pro- caspase-9 activation, or with stimuli that abrogate survival pathways involving PI 3-kinase or NFkappaB. We will investigate the sensors that may initiate the S phase arrest and responses to it dysregulation. Answers to these questions should generate a new understanding of the molecular pharmacology of potential resistance mechanisms to nucleoside analogues.