Quantitative Proteomics Reveals a “Poised Quiescence” Cellular State after Triggering the DNA Replication Origin Activation Checkpoint

Abstract

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 © 2010 American Chemical Society * To whom correspondence should be addressed: j.godovac-zimmermann@ucl.ac.uk, k.stoeber@ucl.ac.uk. Supporting Information Available: Figure S1: Linear regression comparison of three SILAC biological replicate experiments. Figure S2: Western blot validation of SILAC proteins. Figure S3: NRF2-mediated oxidative stress response pathway. Figure S4: Synthesis and degradation of ketone bodies pathway. Figure S5: Butanoate metabolism pathway. Figure S6: Bile acid biosynthesis pathway. Figure S7: Valine, leucine, and isoleucine degradation pathway. Figure S8: Biosynthesis of steroids pathway. Figure S9: Granzyme A signaling pathway. Figure S10: Oxidative phosphorylation pathway. Figure S11: Glutathione metabolism pathway. Figure S12: Propanoate metabolism pathway. Table S1: Protein information for each of the 315 differentially expressed proteins. Table S2: Protein information for all 1730 proteins that were quantified but found to be unchanged in abundance following Cdc7 depletion. Table S3: Protein names, HUGO gene symbols, fold changes, and p-values for all molecules mapped to each of the 10 top-scoring IPA pathways. Table S4: GO enrichment annotation data for up-and down-regulated protein groups.

Spatial Cross-Talk between Oxidative Stress and DNA Replication in Human Fibroblasts

“…Joint MS data processing for all OAC and OXS biological samples using MaxQuant (version 1.5.3.28) 18,19 with a common UniProt human proteome set (ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/proteomes/ downloaded 01-25-2013) was performed as described previously. 8,9,11 The acceptance of proteins as adequately quantified, which was based on minimum numbers of SILAC ratio counts, at least two replicates and MaxQuant Significance B scores, as well as the selection of the significant sets of proteins (401-set, 49-set, see text), is described in detail in the Supplementary Text.…”

“…The connection to MCM5 is of interest because suppression of the CDC7 kinase (for OAC) induces cell cycle arrest at the origin activation checkpoint for DNA replication by reducing phosphorylation of the MCM complex. 8,10 The data contains clear evidence that analyses based only on changes in total abundance of proteins may miss important functional relationships. other interaction is substantial ( ; for co-expression/binding respectively) and the other pathway SET-FTH1-HMOX1 is also substantial (Fig.…”

Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

“…To elucidate the mechanisms involved in regulation of this checkpoint, we have previously used a combination of cell biology, transcriptomics and a global quantitative proteomics approach. 5,22 Specifically, we triggered the activation of this novel checkpoint by RNAi depletion of CDC7 in IMR90 normal human diploid fibroblasts. We have shown that this checkpoint-activated cell cycle arrest is coordinated by the transcription factor FOXO3a, which undergoes subcellular redistribution to the nucleus for activation of the p53-p21 axis and for upregulation of p15-INK4B and p21.…”

The proteome of Toll-like receptor 3–stimulated human immortalized fibroblasts: Implications for susceptibility to herpes simplex virus encephalitis

“…Response to TLR3 activation was defined by altered levels of protein abundance in human SV40-fibroblasts when stimulated with poly(I:C) (used as TLR3 agonist 6–9 ) and was measured by SILAC/MS 12–14 monitoring of heavy/light isotope labelling ratios for peptides from proteins of stimulated/unstimulated cells (Figure 1A). …”