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.
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