The long term objectives of this proposal are to identify the molecular mechanisms by which cytokine growth factors mediate release from DNA damage-induced checkpoints in cell cycle progression. Cell cycle arrest following DNA damage provides a period during which repair mechanisms can restore DNA integrity prior to replication. Failure of DNA-damaged cells to enter a growth arrest allow the accumulation of new mutations which contribute to tumorigenic development. Thus, the potential for cytokine growth factors, or elements of signaling pathways activated by cytokines, to contribute to the prevention of appropriate responses to DNA damage adds yet another dimension to their possible involvement in the development of cancer cells from normal cells. Previously, it was demonstrated that release from DNA damage-induced cell cycle arrest in hematopoietic cells correlated with an activity associated with a C- terminal domain of the erythropoietin receptor.
The specific aims of this proposal are to 1) map cytokine receptor domains required for checkpoint release, 2) determine the requirement for specific signaling pathways in cytokine-induced checkpoint release, 3) determine if known mediators of checkpoint arrest are targets of cytokine signaling pathways mediating the release from DNA damage-induced arrest. To accomplish these specific aims, the ability of cytokines, such as erythropoietin and interleukin-3, to release a UV-irradiation-induced G/1 cell cycle arrest will be assessed using mutants of the cytoplasmic domains of their receptors. The ability of mutant receptors to release the G/1 checkpoint will then be correlated with the activation of specific signaling pathways. Likewise, the role that specific signaling pathways play in release of the G/1 checkpoint will be assessed by analyzing the effects of dominant-negative and dominant-active signaling components or inhibitory drugs. Finally, we will directly assay the status of G/1 cell cycle regulators, including cyclin D-dependent kinases and their inhibitors, and correlate their activities with the UV-induction and cytokine release of the G/1 checkpoint.
