Quantitative proteomics reveals a "poised quiescence" cellular state after triggering the DNA replication origin activation checkpoint.
An origin activation checkpoint has recently been discovered in the G1 phase of the mitotic cell cycle, which can be triggered by loss of DNA replication initiation factors such as the Cdc7 kinase. Insufficient levels of Cdc7 activate cell cycle arrest in normal cells, whereas cancer cells appear to lack this checkpoint response, do not arrest, and proceed with an abortive S phase, leading to cell death. The differential response between normal and tumor cells at this checkpoint has led to widespread interest in the development of pharmacological Cdc7 inhibitors as novel anticancer agents. We have used RNAi against Cdc7 in combination with SILAC-based high resolution MS proteomics to investigate the cellular mechanisms underlying the maintenance of the origin activation checkpoint in normal human diploid fibroblasts. Bioinformatics analysis identified clear changes in wide-ranging biological processes including altered cellular energetic flux, moderate stress response, reduced proliferative capacity, and a spatially distributed response across the mitochondria, lysosomes, and the cell surface. These results provide a quantitative overview of the processes involved in maintenance of the arrested state, show that this phenotype involves active rather than passive cellular adaptation, and highlight a diverse set of proteins responsible for cell cycle arrest and ultimately for promotion of cellular survival. We propose that the Cdc7-depleted proteome maintains cellular arrest by initiating a dynamic quiescence-like response and that the complexities of this phenotype will have important implications for the continued development of promising Cdc7-targeted cancer therapies.
Mulvey
Claire
C
Centre for Molecular Medicine, Rayne Institute, Division of Medicine, University College London, London, U.K.
Tudzarova
Slavica
S
Crawford
Mark
M
Williams
Gareth H
GH
Stoeber
Kai
K
Godovac-Zimmermann
Jasminka
J
D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004261:DNA Replication / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D018741:Replication Origin / Q000502:physiology*
D018797:Cell Cycle Proteins; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases