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PubMed2006

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage.

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90-topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.

Apoptosis↗
PubMed2006

The DNA damage checkpoint is activated during residual tumour cell survival to methotrexate treatment as an initial step of acquired drug resistance.

In the process of acquired drug resistance, the absence of tumour cell subpopulations already resistant before treatment implies an initial adaptive stage of cell growth following drug exposure that, under the selective pressure of the drug, allows the emergence of stably resistant cell variants. Here, we show that p53-defective HT-29 colon cancer cells overcome methotrexate-induced cell death owing to DNA damage checkpoint-mediated cell survival at the adaptive stage that precedes stable resistance acquisition. HT-29 cell cycle progression was dramatically delayed in the presence of a lethal dose of methotrexate, leading to DNA damage during S-phase transition and to cell death as treated cells progressed to G2 and M phases. As a result, the DNA damage checkpoint was induced as indicated by the presence of activated phosphorylated forms of checkpoint proteins Chk1 and Rad9. As we recently described, in-vitro resistance to methotrexate occurs without cell subpopulations already resistant before treatment, hence resistance is acquired through a multistep process that includes an early stage of transient cell survival. Our present results showed that this acute cell survival stage was due to a minor percentage of cells that could complete the first division cycle after drug exposure. Cell survival was enhanced by drug withdrawal during S-phase transition and suppressed if drug withdrawal was followed by treatment with the checkpoint-inhibitor drug caffeine. These results thus point to checkpoint-mediated transient adaptation as a target to prevent the emergence of acquired resistance to methotrexate.

Antimetabolites, Antineoplastic↗
PubMed2004

Studies on the regulation of the two-component histidine kinase gene CHK1 in Candida albicans using the heterologous lacZ reporter gene.

The two-component histidine kinase Chk1p of Candida albicans has been implicated in the regulation of cell wall biosynthesis. Deletion of CHK1 results in avirulence that in part may be due to the increased sensitivity of mutant strains to polymorphonuclear leukocytes. The mutant also does not adhere to human oesophageal tissue in vitro, probably as a consequence of its altered cell wall. In the current study, a CHK1 promoter-lacZ reporter (CHK1prlacZ) construct was expressed in wild-type C. albicans strain CAI4 and in two-component signal transduction mutants to determine the effect of environmental stress conditions on the regulation of CHK1 and the co-regulatory activities among these proteins. It is shown that lacZ expression varied according to the type of growth conditions and incubation time; expression was also influenced by the strain background. lacZ expression in CAI4 was greater at 37 degrees C and at a pH of 3.5 and in the presence of 4 mM H2O2, 0.1 mM menadione, 10 % serum or 1.5 M NaCl compared to cells grown at 30 or 42 degrees C. The increases in expression were time-dependent and not observed until cells were incubated for 120 min in these conditions (P < 0.05). As a correlate of the increase in transcription of CHK1-lacZ in the presence of H2O2, the chk1 mutant was more sensitive than wild-type and revertant cells to H2O2 in vitro. In addition to strain CAI4, we also measured CHK1p-lacZ reporter activity of mutants deleted in genes encoding other two-component proteins such as the response regulator gene SSK1, the histidine kinases, SLN1 and NIK1, and the HOG1 MAP kinase. Of these proteins, Ssk1p and Sln1p are presumed to mediate phosphotransfer to the HOG1 [hyperosmotic glycerol] MAP kinase pathway during oxidative and perhaps osmotic stress in C. albicans. Compared to strain CAI4, lacZ reporter activity increased significantly in the ssk1 mutant under all growth conditions after a 10 and 120 min incubation (P < 0.0001). lacZ expression in the ssk1 mutant was less at 42 degrees C compared to all other growth conditions (P < 0.05). Furthermore, lacZ reporter activity also increased in the hog1 mutant of C. albicans. These data suggest that SSK1 and HOG1 indirectly or directly negatively regulate CHK1 under most growth conditions tested. In the sln1 mutant, downregulation of CHK1 was observed in all growth conditions compared to strain CAI4 (P < 0.05), while regulation of lacZ in the nik1 mutant was similar to strain CAI4 except when cells were incubated in the presence of 4 mM H2O2 for 120 min (P < 0.05). Western blot analysis was used to determine the role of Chk1p in phosphorylation of Hog1p under oxidative or osmotic stress. It was found that Hog1p was phosphorylated in the chk1 mutant similar to wild-type CAF2-1 cells, although the temporal events of phosphorylation differed slightly in mutant cells. These results show that transcription of CHK1, as measured by the lacZ reporter assay, is statistically increased when cells are exposed to several types of stress or when incubated in 10 % serum in a mutant-specific background and at a specific time point. Of importance, our data also suggest that lacZ expression is indirectly or directly regulated by the HOG1 MAP kinase pathway, although a determination of its position in this pathway or in a cross-talking pathway awaits additional studies.

Blotting, Western↗
PubMed2002

A requirement for replication in activation of the ATR-dependent DNA damage checkpoint.

Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polalpha accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase.

Animals↗
PubMed1997

Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1.

Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.

Amino Acid Sequence↗
PubMed1997

Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast.

A common cellular response to DNA damage is cell cycle arrest. This checkpoint control has been the subject of intensive genetic investigation, but the biochemical mechanism that prevents mitosis following DNA damage is unknown. In Schizosaccharomyces pombe, as well as vertebrates, the timing of mitosis under normal circumstances is determined by the balance of kinases and phosphatases that regulate inhibitory phosphorylation of Cdc2. In S. pombe, the phosphorylation occurs on tyrosine-15. This method of mitotic control is also used in S. pombe to couple mitosis with completion of DNA replication, but the role of Cdc2 tyrosine phosphorylation in the Chk1 kinase-mediated DNA damage checkpoint has remained uncertain. We show that, in contrast to recent speculation, the G2 DNA damage checkpoint arrest in S. pombe depends on the inhibitory tyrosine phosphorylation of Cdc2 carried out by the Wee1 and Mik1 kinases. Furthermore, the rate of Cdc2 tyrosine dephosphorylation is reduced by irradiation. This result implicates regulation of Cdc2 tyrosine dephosphorylation, mainly carried out by the Cdc25 tyrosine phosphatase, as an important part of the mechanism by which the DNA damage checkpoint induces Cdc2 inhibition and G2 arrest.

CDC2 Protein Kinase↗
PubMed2003

SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase.

Eukaryotic cells respond to DNA damage and stalled replication forks by activating protein kinase-mediated signaling pathways that promote cell cycle arrest and DNA repair. A central target of the cell cycle arrest program is the Cdc25A protein phosphatase. Cdc25A is required for S-phase entry and dephosphorylates tyrosine-15 phosphorylated Cdk1 (Cdc2) and Cdk2, positive regulators of cell division. Cdc25A is unstable during S-phase and is degraded through the ubiquitin-proteasome pathway, but its turnover is enhanced in response to DNA damage. Although basal and DNA-damage-induced turnover depends on the ATM-Chk2 and ATR-Chk1 pathways, how these kinases engage the ubiquitin ligase machinery is unknown. Here, we demonstrate a requirement for SCFbeta-TRCP in Cdc25A turnover during an unperturbed cell cycle and in response to DNA damage. Depletion of beta-TRCP stabilizes Cdc25A, leading to hyperactive Cdk2 activity. SCFbeta-TRCP promotes Chk1-dependent Cdc25A ubiquitination in vitro, and this involves serine 76, a known Chk1 phosphorylation site. However, recognition of Cdc25A by beta-TRCP occurs via a noncanonical phosphodegron in Cdc25A containing phosphoserine 79 and phosphoserine 82, sites that are not targeted by Chk1. These data indicate that Cdc25A turnover is more complex than previously appreciated and suggest roles for an additional kinase(s) in Chk1-dependent Cdc25A turnover.

Amino Acid Motifs↗
PubMed2005

PPM1D dephosphorylates Chk1 and p53 and abrogates cell cycle checkpoints.

The ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) kinases respond to DNA damage by phosphorylating cellular target proteins that activate DNA repair pathways and cell cycle checkpoints in order to maintain genomic integrity. Here we show that the oncogenic p53-induced serine/threonine phosphatase, PPM1D (or Wip1), dephosphorylates two ATM/ATR targets, Chk1 and p53. PPM1D binds Chk1 and dephosphorylates the ATR-targeted phospho-Ser 345, leading to decreased Chk1 kinase activity. PPM1D also dephosphorylates p53 at phospho-Ser 15. PPM1D dephosphorylations are correlated with reduced cellular intra-S and G2/M checkpoint activity in response to DNA damage induced by ultraviolet and ionizing radiation. Thus, a primary function of PPM1D may be to reverse the p53 and Chk1-induced DNA damage and cell cycle checkpoint responses and return the cell to a homeostatic state following completion of DNA repair. These homeostatic functions may be partially responsible for the oncogenic effects of PPM1D when it is amplified and overexpressed in human tumors.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1999

Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage.

The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage. We have found that the 14-3-3 proteins Rad24 and Rad25 physically interact with Chk1 in fission yeast. Association of Chk1 with 14-3-3 proteins is stimulated in response to DNA damage. DNA damage results in phosphorylation of Chk1 and the 14-3-3 proteins bind preferentially to the phosphorylated form. Genetic analysis has independently implicated both Rad24 and Rad25 in the DNA-damage checkpoint pathway. We suggest that DNA damage-dependent association of phosphorylated Chk1 with 14-3-3 proteins mediates an important step along the DNA-damage checkpoint pathway, perhaps by directing Chk1 to a particular substrate or to a particular location within the cell. An additional role for 14-3-3 proteins in the DNA-damage checkpoint has been suggested based on the observation that human Chk1 can phosphorylate Cdc25C in vitro creating a 14-3-3 binding site. Our results suggest that in fission yeast the interaction between the 14-3-3 proteins and Cdc25 does not require Chk1 function and is unaffected by DNA damage, in sharp contrast to the interaction between the 14-3-3 proteins and Chk1.

Base Sequence↗
PubMed2005

Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures.

The checkpoint mediator protein Claspin is indispensable for the ATR-dependent phosphorylation of Chk1 in response to stalled DNA replication forks in Xenopus egg extracts. We show that Claspin also participates in the detection of chromosomal double-stranded DNA breaks (DSBs) in this system. Significantly, removal of Claspin from egg extracts only partially abrogates the activation of Chk1 in response to chromatin with DSBs, whereas depletion of both Claspin and BRCA1 completely abolishes this activation. The function of Claspin in this DSB-triggered pathway depends on phosphorylation of T817 and S819 by ATR. Conversely, neither phosphorylation of Claspin on these sites nor the presence of BRCA1 is necessary for activation of Chk1 in response to stalled replication forks. Thus, site-specific phosphorylation of a checkpoint mediator protein is a crucial determinant in the discrimination between various checkpoint-inducing structures. Furthermore, checkpoint mediator proteins exhibit functional overlap that varies depending on the nature of the checkpoint-triggering DNA signal.

Adaptor Proteins, Signal Transducing↗
PubMed2002

EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants.

We have examined the role of checkpoint pathways in responding to a yku70Delta defect in budding yeast. We show that CHK1, MEC1, and RAD9 checkpoint genes are required for efficient cell cycle arrest of yku70Delta mutants cultured at 37 degrees C, whereas RAD17, RAD24, MEC3, DDC1, and DUN1 play insignificant roles. We establish that cell cycle arrest of yku70Delta mutants is associated with increasing levels of single-stranded DNA in subtelomeric Y' regions, and find that the mismatch repair-associated EXO1 gene is required for both ssDNA generation and cell cycle arrest of yku70Delta mutants. In contrast, MRE11 is not required for ssDNA generation. The behavior of yku70Delta exo1Delta double mutants strongly indicates that ssDNA is an important component of the arrest signal in yku70Delta mutants and demonstrates a link between damaged telomeres and mismatch repair-associated exonucleases. This link is confirmed by our demonstration that EXO1 also plays a role in ssDNA generation in cdc13-1 mutants. We have also found that the MAD2 but not the BUB2 spindle checkpoint gene is required for efficient arrest of yku70Delta mutants. Therefore, subsets of both DNA-damage and spindle checkpoint pathways cooperate to regulate cell division of yku70Delta mutants.

Antigens, Nuclear↗
PubMed2000

Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.

The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.

Amino Acid Motifs↗
PubMed2001

Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function.

In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways. Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry. Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo. Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1. Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact. Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage. We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage. This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered. Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry.

Amino Acid Sequence↗
PubMed2006

Distinct modes of DNA damage response in S. pombe G0 and vegetative cells.

Upon nitrogen-starvation, mostly G2 vegetative (VE) fission yeast cells promote two rounds of division and enter the G0 state with 1C DNA via an uncommitted G1. Whilst G0 cells are permanently arrested, they keep viability through recycling the intracellular nitrogen. We here show that, whilst the DNA damages are efficiently repaired in G0 cells, neither Chk1 activation nor Cdc2 implication for Crb2 (53BP1 like) do not occur. ATR-like Rad3 and non-hyperphosphorylated Crb2 participate the repair processes in G0 cells that are more sensitive to UV and gamma-ray than in VE cells. The sensitivity like in VE cells is restored after replication in the nitrogen-replenished medium, suggesting that the damage hyper-sensitive nature of G0 cells is due to the error-prone repair for single DNA duplex chromosome. The double-strand break (DSB) repair in G0 cells required Pku80, one of non-homologous end joining (NHEJ) proteins. S. pombe G0 cells upon DNA damages thus respond distinctively from VE cells in regard with regulation of checkpoint proteins and the mode of repair that is dependent upon the use of NHEJ.

Bacterial Proteins↗
PubMed2006

The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1.

ATR-dependent activation of the kinase Chk1 is the initial step in signal transduction in the DNA replication checkpoint, which allows a cell to enter mitosis only after the completion of DNA replication. TopBP1-related proteins in higher eukaryotes are implicated in the replication checkpoint, but their exact role remains elusive because of their requirements for replication initiation. Here we report that the initiation function of Xenopus Cut5/TopBP1 could be entirely separated from its checkpoint function: the N-terminal half fragment, a region of Cut5 conserved through evolution, is sufficient for initiation, but is incapable of activating the checkpoint; the C-terminal half fragment, which is unique in metazoan species, is by itself capable of activating the checkpoint response without initiating replication. Upon the activation of Chk1, the Ser1131 within the C-terminal region of Cut5 is phosphorylated, and this phosphorylation is critical for the checkpoint response. Furthermore, Cut5 directly stimulated Chk1 phosphorylation in the in vitro kinase assay reconstituted with recombinant proteins and ATR immunoprecipitated from extracts. On the basis of replication protein A (RPA)-dependent loading of Cut5 on to replicating and replication-arrested chromatin, we propose that Cut5 plays a crucial role in the initial amplification step of the ATR-Chk1 signaling pathway at the stalled replication fork.

Animals↗
PubMed2006

Benzo[a]pyrene-7,8-dihydrodiol promotes checkpoint activation and G2/M arrest in human bronchoalveolar carcinoma H358 cells.

Polycyclic aromatic hydrocarbons (PAHs) are potent carcinogens that require metabolic activation inside cells. The proximate carcinogens PAH-diols can be converted to o-quinones by aldo-keto reductases (AKRs) or to diol-epoxides by cytochrome P450 (P450) enzymes. We assessed the effect of benzo[a]pyrene-7,8-dihydrodiol (BPD) on proliferation in p53-null bronchoalveolar carcinoma H358 cells. BPD treatment led to a significant inhibition of proliferation and arrest in G2/M in H358 cells. The relative contribution of the AKR and P450 pathways to cell cycle arrest was assessed. Overexpression of AKR1A1 did not affect cell proliferation or cell cycle progression, and benzo[a]pyrene-7,8-dione did not cause any noticeable effect on cell growth, suggesting that AKR1A1 metabolic products were not involved in the antiproliferative effect of BPD. On the other hand, blockade of P450 induction or inhibition of P450 activity greatly impaired the effect of BPD. Moreover, P450 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly enhanced the antiproliferative effect of BPD. Mechanistic studies revealed that BPD caused a DNA damage response, Chk1 activation, and accumulation of phospho-Cdc2 (Tyr15) in H358 cells, effects that were impaired by an ataxia-telangectasia mutated (ATM)/ATM-related (ATR) inhibitor. Similar results were observed in human bronchoepithelial BEAS-2B cells, arguing for analogous mechanisms in tumorigenic and immortalized nontumorigenic cells lacking functional p53. Our data suggest that a p53-independent pathway operates in lung epithelial cells in response to BPD that involves P450 induction and subsequent activation of the ATR/ATM/Chk1 damage check-point pathway and cell cycle arrest in G2/M.

Adenocarcinoma, Bronchiolo-Alveolar↗