Eukaryotic cells have evolved an elaborate network of genome surveillance and repair proteins to insure that DNA replication will occur in an accurate and timely fashion. This surveillance pathway is termed the S phase replication checkpoint. Defects in this 'caretaker'machinery lead to genetic instability, a hallmark feature of human cancers. The replication checkpoint monitors the progress of replication forks, and when fork stalls, transmits signals that delay S-phase progression, and maintains the stability of stalled forks so that DNA replication can resume after the initial error is corrected. Two key components of the replication checkpoint are the apical protein kinase, ATR, and its downstream target kinase, Chk1. Loss of ATR or Chk1 function is lethal, even in the absence of extrinsic genotoxic stress, underscoring the importance of the replication checkpoint in the maintenance of cell viability. In preliminary work, we tested the hypothesis that certain anti-tumor agents, such as the topoisomerase 1 (Top I) poisons (e.g., camptothecin (CPT)) selectively kill cancer cells through the induction of protracted, high-intensity replication stress. Our studies unexpectedly revealed that treatment with CPT or other replication stress inducers (e.g., deep hypoxia or methylmethane sulfonate) triggers the ubiquitin-dependent degradation of Chk1 in both normal and transformed human cells. The degradation of Chk1 was dependent on the Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, and the consequences of severe Chk1 destruction were replication fork collapse and ultimate cell death. Remarkably, defects in the Chk1 degradation pathway confer resistance to the cytotoxic effect of CPT - a major problem with this class of drugs in the clinical arena. Thus, this novel layer of Chk1 regulation has important implications for our understanding of replication checkpoint signaling, as well as mechanisms of anticancer resistance in cancer patients. I now propose to elucidate the underlying mechanisms and biological significance of the stress-induced Chk1 destruction through pursuit of the following specific aims: In the mentored-phase, (1) To identify the F-box proteins of the E3 ligase complexes responsible for Chk1 destruction;In the independent phase, (2) To identify the putative 'Degron'region and the lysine residues targeted for ubiquitin modification in Chk1;(3) To investigate the roles of de-phosphorylation in Chk1 degradation;(4) To characterize the molecular mechanisms underlying the defect in Chk1 degradation in CPT-resistant cancer cells. Relevance: The results of these studies will not only advance our understanding of the genotoxic stress response machinery in human cells, but also provide novel insights into the causes of genetic instability and anticancer drug resistance in cancer cells, and these lines of research have direct implications for the development of novel therapeutic agents targeted against tumors.
The results of these studies will not only advance our understanding of the genotoxic stress response machinery in human cells, but also provide novel insights into the causes of genetic instability and anticancer drug resistance in cancer cells, and these lines of researches have direct implications for the development of novel therapeutic agents targeted against tumors.
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