Cdc7 Inhibition Reveals a p53-Dependent Replication Checkpoint That Is Defective in Cancer Cells

Abstract

Cdc7 is an evolutionarily conserved kinase that regulates S phase by promoting replication origin activation. Down-regulation of Cdc7 by small interfering RNA in a variety of tumor cell lines causes an abortive S phase, leading to cell death by either p53-independent apoptosis or aberrant mitosis. Unlike replication fork blockade, Cdc7-depleted tumor cells do not elicit a robust checkpoint response; thus, inhibitory signals preventing additional cell cycle progression are not generated. In normal fibroblasts, however, a p53-dependent pathway actively prevents progression through a lethal S phase in the absence of sufficient Cdc7 kinase. We show that in this experimental system, p53 is required for the lasting maintenance of this checkpoint and for cell viability. With this work we reveal and begin to characterize a novel mechanism that regulates DNA synthesis in human cells, and we suggest that inhibition of Cdc7 kinase represents a promising approach for the development of a new generation of anticancer agents.

The cell cycle and cancer

“…In striking contrast, normal cells avoid entering S phase with a reduced number of replication-competent origins by engaging a recently described cell cycle checkpoint, the 'origin activation checkpoint'. Several studies have shown that following impairment of the DNA replication initiation machinery, normal cells arrest at the G 1 -S boundary with unreplicated DNA, elevated p53 levels and induction of CDKI p21 [111,125,126]. 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 would translate into low therapeutic indices often found for conventional chemotherapeutic drugs targeting the cell cycle. However, potent cancer cell-specific killing has been demonstrated in preclinical models after inhibition of origin licensing [125] or, alternatively, origin activation through targeting Cdc7 kinase [111,118,126,127]. Tumour cell specificity is thought to result from transformed cells entering S phase with inadequate numbers of competent origins to complete chromosomal replication.…”

“…This results in an abortive S phase with incompletely and/or abnormally replicated DNA. Tumour cells with a functional intra-S phase checkpoint appear to undergo rapid death after replication fork stalling/collapse, whereas more transformed cancer cells appear to survive longer but eventually face mitotic catastrophe as a result of partially replicated chromosomes [125,126,128]. In striking contrast, normal cells avoid entering S phase with a reduced number of replication-competent origins by engaging a recently described cell cycle checkpoint, the 'origin activation checkpoint'.…”

5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

“…32 However, other experimental evidence suggested that p53 is required for the lasting maintenance of the replication checkpoint. 33 We used HCT116 p53−/− , a p53-null HCT116-derived cell line 24 ( Figure 3B), to test whether p53 is required for the 5-ASA-induced S-phase accumulation ( Figure 3C, lower panels). Upon treatment with 5-ASA, p53-null cells showed a replication arrest very comparable with that of the p53 wild-type population (percentage S phase-HCT116 p53−/− : 0 mmol/L: 47.4% ± 2.9%; 10 mmol/L: 59.7% ± 2.1%, P <.0001), indicating that p53 does not have a causative role in the 5-ASA-mediated cell cycle arrest.…”

Azaindole-Based Inhibitors of Cdc7 Kinase: Impact of the Pre-DFG Residue, Val 195

“…10,11 Because Cdc7 overexpression promotes proliferation and survival of certain tumor cells, knockdown of Cdc7 by siRNA has led to p53-independent apoptosis in cancer cell lines. 12,13 Tumor cells with the often impaired S-phase checkpoints progress to mitosis without DNA repair. Meanwhile, upon Cdc7 depletion, normal cells are able to arrest in the G1 phase of the cell cycle and resume normal divisions once an initiation process is restored.…”