Molecular architecture of the DNA replication origin activation checkpoint

Abstract

Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-|Hdm2-|p53-p21 pathway and mediates p15 INK4B upregulation; p53 in turn activates expression of the Wnt/b-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.

Downregulation of Dickkopf-3 disrupts prostate acinar morphogenesis through TGF-β/Smad signaling

“…Moreover, although ectopic expression of Dkk-3 induces cancer cell apoptosis (Veeck and Dahl, 2012), we are not aware of any reports that silencing of Dkk-3 prevents apoptosis. In fact, silencing of Dkk-3 in growtharrested fibroblasts leads to apoptosis (Tudzarova et al, 2010). Our results therefore favour a model in which increased cell proliferation accounts for the phenotypes observed upon loss of Dkk-3 in mice and in 3D cultures.…”

Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

“…The almost 1000 proteins that we identified as being present in both the nucleus and mitochondria of MCF7 cells suggests that the set of proteins common to different subcellular sites may often be large and that complementary high throughput approaches that detect dynamic changes in the abundance or form of proteins in different sites as a response to cellular stimulation may provide the most direct way to select the most interesting proteins associated with particular cellular functions. 38 For example, using methods similar to those described here with augmentation by SILAC labeling, we have recently been able to show in IMH90 cells that engagement of the "origin activation checkpoint" for DNA translation initiation that is associated with cell cycle arrest 88,89 involves shuttling between the nucleus and cytoplasm of at least 50 proteins, including a few of the mitochondrial proteins identified in the nucleus in the present study of MCF7 cells (Mulvey et al, in preparation). Such dynamic studies are likely to be particularly important for dealing with cytosolic proteins.…”

The cell cycle and cancer

“…[122][123][124]. However, molecular tools to measure activation of the signalling pathways in tumour [129]. CDC7 knock-down by RNAi or, alternatively, inhibition of Cdc7 kinase activity with small molecule inhibitors (SMIs) triggers a cellular response that is dependent on three checkpoint axes coordinated through the cell stress transcription factor FoxO3a.…”

“…We discovered that the molecular architecture of the underlying cell cycle checkpoint is critically dependent on several tumour suppressor proteins, including p53, p21, Dkk3, ARF, Hdm2, FoxO3a, p15, p27 and RB [129] (Figure 4A). This suggests that loss of the protective checkpoint mechanism through inactivating mutations in checkpoint proteins will render most common solid tumours sensitive to anti-cancer agents targeting the DNA replication initiation machinery [129].…”