The role of Dbf4/Drf1-dependent kinase Cdc7 in DNA-damage checkpoint control.

The Dbf4/Drf1-dependent S-phase-promoting kinase Cdc7 (Ddk) is thought to be an essential target inactivated by the S-phase checkpoint machinery that inhibits DNA replication. However, we show here that the complex formation, chromatin association, and kinase activity of Ddk are not inhibited during the DNA-damage-induced S-phase checkpoint response in Xenopus egg extracts and mammalian cells. Instead, we find that Ddk plays an active role in regulating S-phase checkpoint signaling. Addition of purified Ddk to Xenopus egg extracts or overexpression of Dbf4 in HeLa cells downregulates ATR-Chk1 checkpoint signaling and overrides the inhibition of DNA replication and cell-cycle progression induced by DNA-damaging agents. These results indicate that Ddk functions as an upstream regulator to monitor S-phase checkpoint signaling. We propose that Ddk modulates the S-phase checkpoint control by attenuating checkpoint signaling and triggering DNA replication reinitiation during the S-phase checkpoint recovery.

The interaction between checkpoint kinase 1 (Chk1) and the minichromosome maintenance (MCM) complex is required for DNA damage-induced Chk1 phosphorylation.

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage.

JNK-mediated phosphorylation of Cdc25C regulates cell cycle entry and G(2)/M DNA damage checkpoint.

c-Jun NH(2)-terminal Kinases (JNKs) play a central role in the cellular response to a wide variety of stress signals. After their activation, JNKs induce phosphorylation of substrates, which control proliferation, migration, survival, and differentiation. Recent studies suggest that JNKs may also play a role in cell cycle control, although the underlying mechanisms are largely unexplored. Here we show that JNK directly phosphorylates Cdc25C at serine 168 during G(2) phase of the cell cycle. Cdc25C phosphorylation by JNK negatively regulates its phosphatase activity and thereby Cdk1 activation, enabling a timely control of mitosis onset. Unrestrained phosphorylation by JNK, as obtained by a cell cycle-stabilized form of JNK or as seen in some human tumors, results in aberrant cell cycle progression. Additionally, UV irradiation-induced G(2)/M checkpoint requires inactivation of Cdc25C by JNK phosphorylation. JNK phosphorylation of Cdc25C as well as Cdc25A establishes a novel link between stress signaling and unperturbed cell cycle and checkpoint pathways.

Essential role of phosphorylation of MCM2 by Cdc7/Dbf4 in the initiation of DNA replication in mammalian cells.

We report the identification of Cdc7/Dbf4 phosphorylation sites in human MCM2 and the determination of the role of Cdc7/Dbf4 phosphorylation of MCM2 in the initiation of DNA replication. Using immunoblotting, immunofluorescence, and high-speed automated cell-imaging analyses with antibodies specific against MCM2 and Cdc7/Dbf4 phosphorylated MCM2, we show that the chromatin recruitment and phosphorylation of MCM2 are regulated during the cell cycle in HeLa cells. Chromatin-bound MCM2 is phosphorylated by Cdc7/Dbf4 during G1/S, which coincides with the initiation of DNA replication. Moreover, we show that baculovirus-expressed purified MCM2-7 complex and its phosphomimetic MCM2E-7 complex display higher ATPase activity when compared with the nonphosphorylatable MCM2A-7 complex in vitro. Furthermore, suppression of MCM2 expression in HeLa cells by siRNA results in the inhibition of DNA replication. The inhibition can be rescued by the coexpression of wild type MCM2 or MCM2E but not MCM2A. Taken together, these results indicate that Cdc7/Dbf4 phosphorylation of MCM2 is essential for the initiation of DNA replication in mammalian cells.

The role of pre-replicative complex (pre-RC) components in oncogenesis.

Normal DNA replication is stringently regulated to ensure a timely occurrence no more than once per cell cycle. Abrogation of the exquisite control mechanisms that maintain this process results in detrimental gains and losses of genomic DNA commonly seen in cancer and developmental defects. Replication initiation proteins, known as prereplicative complex (pre-RC) proteins, serve as a primary level of regulation, controlling when DNA replication can begin. Unsurprisingly, several pre-RC proteins are overexpressed in cancer and serve as good tumor markers. However, their direct correlation with increasing tumor grade and poor prognosis has posed a long-standing question: Are pre-RC proteins oncogenic? Recently, a growing body of data indicates that deregulation of individual pre-RC proteins, either by overexpression or functional deficiency in several organismal models, results in significant and consistently perturbed cell cycle regulation, genomic instability, and, potentially, tumorigenesis. In this review, we examine this broad range of evidence suggesting that pre-RC proteins play roles during oncogenesis that are more than simply indicative of proliferation, supporting the notion that pre-RC proteins may potentially have significant diagnostic and therapeutic value.

CC-115, a dual inhibitor of mTOR kinase and DNA-PK, blocks DNA damage repair pathways and selectively inhibits ATM-deficient cell growth in vitro.

CC-115, a selective dual inhibitor of the mammalian target of rapamycin (mTOR) kinase and DNA-dependent protein kinase (DNA-PK), is undergoing Phase 1 clinical studies. Here we report the characterization of DNA-PK inhibitory activity of CC-115 in cancer cell lines. CC-115 inhibits auto-phosphorylation of the catalytic subunit of DNA-PK (DNA-PKcs) at the S2056 site (pDNA-PK S2056), leading to blockade of DNA-PK-mediated non-homologous end joining (NHEJ). CC-115 also indirectly reduces the phosphorylation of ataxia-telangiectasia mutated kinase (ATM) at S1981 and its substrates as well as homologous recombination (HR). The mTOR kinase and DNA-PK inhibitory activity of CC-115 leads to not only potent anti-tumor activity against a large panel of hematopoietic and solid cancer cell lines but also strong induction of apoptosis in a subset of cancer lines. Mechanistically, CC-115 prevents NHEJ by inhibiting the dissociation of DNA-PKcs, X-ray repair cross-complementing protein 4 (XRCC4), and DNA ligase IV from DNA ends. CC-115 inhibits colony formation of ATM-deficient cells more potently than ATM-proficient cells, indicating that inhibition of DNA-PK is synthetically lethal with the loss of functional ATM. In conclusion, CC-115 inhibits both mTOR signaling and NHEJ and HR by direct inhibition of DNA-PK. The mechanistic data not only provide selection of potential pharmacodynamic (PD) markers but also support CC-115 clinical development in patients with ATM-deficient tumors.

Reduced expression of the REIC/Dkk-3 gene by promoter-hypermethylation in human tumor cells.

The human REIC gene is a recently found mortalization-related gene and a candidate tumor suppressor gene expression of which is largely attenuated in many immortalized and tumor-derived cell lines (Biochem. Biophys. Res. Commun. 268 (2000) 20-24). To gain insight into the mechanisms of the down-regulation, we investigated the genomic structure and promoter activity of the human REIC gene. The gene, identical with the DKK-3 gene, resides on chromosome 11p15.1, consists of nine exons, and has two promoters. Methylation in the main promoter region was detected in 11 out of 21 cell lines tested (52%) derived from a variety of human tumors, in which the expression of the REIC gene was decreased. In ten of these 11 cell lines the minor promoter was also methylated. Similarly, the REIC gene expression was decreased in 14 of 24 fresh non-small cell lung cancer specimens (58%) compared to that in corresponding non-cancerous tissue, though allelic loss and tumor-specific mutation were rare. Of these 14 tumors, at least five tumors exhibited heavy methylation of the REIC promoter region. These results indicate that the down-regulation of the REIC gene expression is ascribed to the aberrant promoter hyper-methylation at least in a subset of human tumors. The expression was restored upon treatment of SQ5 cells with 5-aza-deoxycytidine, confirming DNA methylation as the mode of downregulation. A notable single nucleotide polymorphism in the coding region (cSNP) with an amino acid substitution of glycine (GGG) to arginine (AGG) was found at codon 335 of the REIC gene. However, the distribution of the cSNP showed no significant difference between lung cancer patients and healthy population.

Interplay between Cdh1 and JNK activity during the cell cycle.

The ubiquitin ligase APC/C(Cdh1) coordinates degradation of key cell cycle regulators. We report here that a nuclear-localized portion of the stress-activated kinase JNK is degraded by the APC/C(Cdh1) during exit from mitosis and the G1 phase of the cell cycle. Expression of a non-degradable JNK induces prometaphase-like arrest and aberrant mitotic spindle dynamics. Moreover, JNK phosphorylates Cdh1 directly, during G2 and early mitosis, changing its subcellular localization and attenuating its ability to activate the APC/C during G2/M. This regulatory mechanism between JNK and Cdh1 reveals an important function for JNK during the cell cycle.

Notch activates cell cycle reentry and progression in quiescent cardiomyocytes.

The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell-derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jkappa (RBP-Jkappa)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jkappa. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis.