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通常对它们的细胞毒性是必不可少的，临床上相关的耐药机制可能涉及将欺诈性核苷酸掺入DNA后的信号传递过程。无论是在体外还是在治疗过程中，白血病细胞暴露于抑制浓度的核苷类似物，终止DNA合成，并阻止细胞周期进展在S阶段。在药物去除后，部分细胞恢复疾病的进展和重新繁殖。因此，细胞能够延缓细胞周期进程，停止DNA合成，从而限制类似物的掺入，这可能是避免细胞潜在毒性的一种防御机制。我们假设，一旦分子机制检测到核苷酸类似物被掺入DNA，信号就会被激活以阻止DNA复制，限制类似分子进一步掺入DNA并允许修复发生。一旦类似物被清除，抑制信号就会被移除，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。作为推论，我们假设细胞周期调控过程的扰动可能会产生相互矛盾的信号，导致细胞死亡，从而绕过细胞防御机制。本项目旨在建立并验证治疗性核苷类似物引起的人实体瘤细胞S时相特异性细胞周期停滞的模型，并研究调控失调的后果。以下问题将评估这些假设。首先，核苷类似物引起S停滞的分子决定因素是什么？我们将建立核苷类似物诱导S期阻滞的细胞系模型。这将被用来比较在S期阻滞细胞和S期富集细胞中与细胞周期蛋白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期的复合体。我们将确定CDK2的抑制磷酸化在涉及CDc25A、ChK1和ChK2的调控体系中的作用，并将评估细胞周期蛋白依赖的激酶抑制剂如p21在S期阻滞细胞中对CDK2的激酶活性的作用。第二，干预核苷类似物抑制细胞存活调控的生物学作用和分子后果是什么？我们将使用单独使用时不影响生长的浓度的激酶抑制剂，如UCN-01，来扰乱S期的阻止机制。将对调节机制进行评估，以确定可能与细胞周期进展或启动凋亡过程相关的活动的特定变化。第三，S时相阻滞失调对细胞活力有何影响？将通过激活细胞凋亡反应的信号，如应激反应级联反应、bax上调和caspase-9原激活，或通过阻断PI 3-激酶或NFkappaB等生存通路的刺激，寻找与S期停滞功能障碍存在因果关系的证据。我们将研究可能启动S时相停止的传感器以及对其异常调节的反应。对这些问题的回答应该会对核苷类似物潜在耐药机制的分子药理学产生新的理解。

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.