Although several mechanisms of resistance to nucleoside analogs that focus on their cellular metabolism have ben generated in cells in vitro, none of these appear to be prevalent in the leukemia cells of patients whose disease is resistant to therapy. Because incorporation of the analogs into DNA is essential to their cytotoxicity, clinically relevant resistant mechanisms are likely to involve signaling processes subsequent to the incorporation of the fraudulent nucleosides into DNA. When leukemia cells, either in vitro or in the marrow, are exposed to cytostatic concentrations of nucleoside analogs, they terminate DNA synthesis and arrest cell cycle progression in S phase. Subsequent to the removal of the drug cells resume progression and population growth. Thus, the ability of cells to enact delay in cell cycle progression and population growth. Thus, the ability of cells to enact a delay in cell cycle progression that stops DNA synthesis, thereby limiting the incorporation of analog, may be a defense mechanism that spares cells potential toxicity We hypothesize that once cells perceive the incorporation of a nucleoside analog into DNA, signals are issued to block DNA replication, limiting further incorporation of analog molecules into DNA and allowing repair to occur. Once the analog is cleared, the inhibitory signal is removed and cells continue cycling. This would constitute a normal defense response which may manifest itself as experimental and clinical resistance. Perturbation of the cell cycle regulatory processes may generate conflicting signals that result in cell death, thereby circumventing the cellular defense mechanism. Furthermore, we postulate that cells that are compromised in cell cycle progression may be sensitized to agents that inhibit survival pathways or activate apoptotic cascades. This project seeks to establish and validate a model for S phase-specific cell cycle arrest in response to therapeutic nucleosides analogs, and are the molecular determinants of the S phase arrest in response to nucleoside analogs? Using leukemia cell lines, develop a model for nucleoside analog-induced S phase arrest. Compare in S phase arrested cells and S phase enriched populations the kinase activity of Cdk2 associated with either cyclin E or cyclin A, the complexes that drive cells through S phase. Evaluate the role of inhibitory phosphorylations of Cdk2, and of association of cyclin-dependent kinases with the cyclin dependent kinase inhibitors, 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 analog arrested cells? Intervene with survival or apoptotic mechanisms in analog arrested cells using kinase inhibitors, e.g., UCN-01, flavopiridol, at concentrations that do not affect growth when used alone. Evaluate whether the S phase arrest has been abrogated allowing cells to progress in the cell cycle, or if cells begin the apoptotic process directly. Quantitate the actions of kinase inhibitors on the molecular regulators of the S phase arrest, (Cdk2/cyclin A kinase activity, phosphorylation status, and association with P21). Evaluate alterations in the molecular regulation of the PI-3-Kinase-Akt-Bad survival pathway as a means of mediating cell death. Third, can primary AML cells serve as in vitro models of cellular and molecular determinants of nucleoside analog- induced S phase arrest and interventions? Evaluate the ability of cytostatic levels of S phase specific nucleosides analogs to arrest cell cycle progression of primary AML cells in vitro. Quantitate the ability of non-toxic concentrations of kinase inhibitors to kill S arrested cells. Seek a molecular basis for the actions of the combinations. Compare the responses among individuals as indicators of sensitivity and resistance. Finally, can the cellular and molecular determinants of nucleoside analog- induced S phase arrest and interventions be modeled with primary AML cells during a clinical trial? Conduct clinical trials that will permit validation of the working hypotheses in primary leukemia cells isolated from patients with relapsed AML during therapy with ara-C and UCN-01.
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