The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spkl/Mec2/Rad53/Sadl, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spkl, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPKI. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfcS mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.The eukaryotic mitotic cell cycle consists of a temporally ordered series of events in which the initiation of late events is dependent on the completion of the early ones. This order is maintained by mechanisms called checkpoint controls that monitor completion of earlier events and control cell cycle progression (1-3). In eukaryotes, for example, incomplete DNA replication or DNA damage induces cell cycle arrest in G2 before mitosis. This dependency or checkpoint is important for ensuring that the cell does not divide unless the chromosomes have been completely duplicated.The budding yeast Saccharomyces cerevisiae has been one of the best model systems for the identification and genetic analysis of the cell cycle. In S. cerevisiae, a number of genes, such as RAD9, RAD17, RAD24, MECl/ESRI, SPKI/MEC2/ RAD53/SAD1, and MEC3, involved in the DNA damage checkpoint have been identified (4-7). MECI and SPKI are also necessary to inhibit the onset of mitosis in response to incomplete replication (6, 7). MEC1 is a homolog of the human ATM gene, which is mutated in patients with ataxia telangiectasia (8). SPK1 encodes a dual-specificity protein kinase (9). The mechanism that prevents mitosis until completion of S phase remains a central unanswered question.Active replication complexes have been proposed as potential sources of the S phase signal because DNA replication must be completed before initiation of mitosis (3). In support of this idea, Navas et al. (10) have demonstrated that DNA polymerase (pol) s of S. cerevisiae serves not only as an essential replication enzyme but also as a sensor in the S phase checkpoint, which is consistent with a role for active replication complexes in this process. However, it is not yet known how the replication apparatus senses the unreplicated DNA and sends the signal to the checkpoint machinery.To identify genes that genetically interact with Spkl, we have isolated temperature-sensitive (ts-) mutants that can be supp...

Aurora-A kinase is a one of the key regulators during mitosis progression. Aurora-A kinase is a potential target for anticancer therapies because overexpression of Aurora-A, which is frequently observed in some human cancers, results in aberrant mitosis leading to chromosomal instability and possibly tumorigenesis. MK-5108 is a novel small molecule with potent inhibitory activity against Aurora-A kinase. Although most of the Aurora-kinase inhibitors target both Aurora-A and Aurora-B, MK-5108 specifically inhibited Aurora-A kinase in a panel of protein kinase assays. Inhibition of Aurora-A by MK-5108 in cultured cells induced cell cycle arrest at the G2-M phase in flow cytometry analysis. The effect was confirmed by the accumulation of cells with expression of phosphorylated Histone H3 and inhibition of Aurora-A autophosphorylation by immunostaining assays. MK-5108 also induced phosphorylated Histone H3 in skin and xenograft tumor tissues in a nude rat xenograft model. MK-5108 inhibited growth of human tumor cell lines in culture and in different xenograft models. Furthermore, the combination of MK-5108 and docetaxel showed enhanced antitumor activities compared with control and docetaxel alone–treated animals without exacerbating the adverse effects of docetaxel. MK-5108 is currently tested in clinical trials and offers a new therapeutic approach to combat human cancers as a single agent or in combination with existing taxane therapies. Mol Cancer Ther; 9(1); 157–66

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae. We have previously shown that a temperature-sensitive (ts) rfc5-1 mutation is impaired in the S-phase checkpoint. In this report, we show that the rfc5-1 mutation is sensitive to DNA-damaging agents. RFC5 is necessary for slowing the S-phase progression in response to DNA damage. The phosphorylation of the essential central transducer, Rad53 protein kinase, is reduced in response to DNA damage in rfc5-1 mutants during the S phase. Furthermore, the inducibility of RNR3 transcription in response to DNA damage is dependent on RFC5. It has been shown that phosphorylation of Rad53 is controlled by Mec1 and Tel1, members of the subfamily of ataxiatelangiectasia mutated (ATM) kinases. We also demonstrate that overexpression of TEL1 suppresses the ts growth defect and DNA damage sensitivity of rfc5-1 mutants and restores phosphorylation of Rad53 and RNR3 induction in response to DNA damage in rfc5-1. Our results, together with the observation that overexpression of RAD53 suppresses the defects of the rfc5-1 mutation, suggest that Rfc5 is part of a mechanism transducing the DNA damage signal to the activation of the central transducer Rad53.In eukaryotic cells, successful mitotic division requires the events of the cell cycle to be ordered into dependent pathways in which the initiation of late cycle events is dependent on the completion of early events. The mechanisms which ensure that cell division does not occur before completion of such prerequisite steps have been termed checkpoint controls (8). Checkpoint controls ensure that cells remain in S phase before completion of DNA replication. DNA damage also activates checkpoint controls to provide enough time to complete DNA repair. Defects of these DNA-related checkpoints result in increased genomic instability and mutagenesis (5, 21).In the budding yeast Saccharomyces cerevisiae, checkpoint pathways induce cell cycle arrest in G 1 or G 2 /M and retard Sphase progression in response to DNA damage. Other checkpoints prevent cells with incompletely replicated DNA from exiting the S phase (5, 21). A number of genes that are involved in the DNA damage checkpoint and/or the replication checkpoint have been identified (5, 21). These include RAD9, RAD17, RAD24, POL2, MEC1/ESR1, RAD53/SPK1/MEC2/ SAD1, and MEC3 (1,10,17,28,[33][34][35]. Among these genes, RAD9, RAD17, RAD24, and MEC3 are involved not only in the G 2 /M-phase but also in the G 1 -and S-phase DNA damage checkpoints (12,20,(25)(26)(27)(33)(34)(35). POL2, which encodes a large subunit of DNA polymerase ε (pol ε), is proposed to sense DNA damage and replication block in S phase (16,17). MEC1 and RAD53 are necessary for checkpoints operating in response to both DNA damage and incomplete DNA replication (1, 35). RAD53 encodes a dual-specificity protein kinase (28), and Mec1 belongs to the phosphatidylinositol kinase family that includes S. cerevisiae Tel1 and human ATM proteins (10,15,24). MEC1 and TEL1 share some...

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosagedependent suppressor of rfc5-1. RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although the rad24⌬ mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect in rfc5-1 mutants. Furthermore, overexpression of RAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.The survival of eucaryotes depends on the accurate transmission of genetic information from one generation to the next. Successful mitotic division requires the events of the cell cycle to be ordered such that the initiation of late cycle events is dependent on the completion of early events. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls (7). When DNA replication is delayed and DNA damage occurs, checkpoint controls activate cell cycle arrest enough to complete DNA replication and repair DNA damage (4, 18).In the budding yeast Saccharomyces cerevisiae, checkpoint pathways induce cell cycle arrest in G 1 or G 2 /M and retard Sphase progression in response to DNA damage. Other checkpoints prevent cells with incompletely replicated DNA from exiting the S phase. A number of genes that are involved in the DNA damage checkpoint and/or the replication block checkpoint have been identified elsewhere (4, 18). These include RAD9, RAD17, RAD24, POL2, MEC1/ESR1, RAD53/SPK1/ MEC2/SAD1, RFC5, MEC3, and DDC1. Among these genes, RAD9, RAD17, RAD24, MEC3, and DDC1 are involved not only in the G 2 /M-phase but also in the G 1 -and S-phase DNA damage checkpoints (11,12,17,(22)(23)(24)(31)(32)(33). POL2, encoding a large subunit of DNA polymerase (Pol) ε, is required for the checkpoints responding to replication block and DNA damage in S phase (15, 16). MEC1 and RAD53 are necessary for the checkpoints operating in response to both DNA damage and incomplete DNA replication (1, 33). RAD53 encodes a dual-specificity protein kinase (25), and Mec1 belongs to the phosphatidylinositol kinase family that includes human ataxiatelangiectasia-mutated (ATM) proteins (9, 21). Rad53 is phosphorylated in response to DNA damage and DNA replication block in a MEC1-dependent manner (20,29).Replication factor C (RFC) is required for DNA replication and repair and consists of one large and four small...

MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Delta mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Delta mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Delta mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Delta mutants during S phase, but occurs in tel1Delta mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we therefore examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patients MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL-6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of CHOP, a fatal ER-stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24⌬ mutants are defective in all the G 1 -, S-, and G 2 /M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine 115 residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G 1 -and G 2 /M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24 K115R fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G 1 -, S-and G 2 /M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.Eukaryotic cells employ a set of surveillance mechanisms to coordinate cell cycle events by permitting the onset of one event only after the completion of the preceding event. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls (10). DNA damage triggers the activation of checkpoint pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. Other checkpoints are activated when DNA replication is blocked. Failure to respond properly to DNA alterations may result in genomic instability, a mutagenic condition that predisposes organisms to cancer (5, 24).The cell cycle is transiently arrested at different stages depending on the phase at which DNA damage occurs. Three responses have been characterized in the budding yeast Saccharomyces cerevisiae, known as the G 1 -, S-and G 2 /M-phase DNA damage checkpoints (16). Genetic studies have identified genes that are involved in all three checkpoints. These include RAD9, RAD17, RAD24, MEC3, DDC1, MEC1(ESR1), and RAD53 (SPK1 or MEC2) (1,17,18,22,23,(30)(31)(32)(33)(43)(44)(45). Several lines of genetic evidence have suggested that RAD17, RAD24, MEC3, and DDC1 operate in the same checkpoint pathway, while RAD9 functions separately (17,18,20). Indeed, Ddc1, Mec3, and Rad17 physically interact with each other, suggesting that they function as a complex (13). RAD53 encodes a dual-specificity protein kinase (35), and Mec1 belongs to the ATM protein family (12,28). Rad53 is phosphorylated in response to DNA damage in a MEC1-dependent manner (26, 39). DNA damage-induced Rad53 phosphorylation is also dependent on R...

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