scholarly journals Surviving chromosome replication: the many roles of the S-phase checkpoint pathway

2011 ◽  
Vol 366 (1584) ◽  
pp. 3554-3561 ◽  
Author(s):  
Karim Labib ◽  
Giacomo De Piccoli
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Progression ◽  
Reductase Activity ◽  
Critical Role ◽  
Chromosome Replication ◽  
S Phase ◽  
Replication Forks ◽  
Checkpoint Pathway ◽  
S Phase Checkpoint

Checkpoints were originally identified as signalling pathways that delay mitosis in response to DNA damage or defects in chromosome replication, allowing time for DNA repair to occur. The ATR (ataxia- and rad-related) and ATM (ataxia-mutated) protein kinases are recruited to defective replication forks or to sites of DNA damage, and are thought to initiate the DNA damage response in all eukaryotes. In addition to delaying cell cycle progression, however, the S-phase checkpoint pathway also controls chromosome replication and DNA repair pathways in a highly complex fashion, in order to preserve genome integrity. Much of our understanding of this regulation has come from studies of yeasts, in which the best-characterized targets are the stimulation of ribonucleotide reductase activity by multiple mechanisms, and the inhibition of new initiation events at later origins of DNA replication. In addition, however, the S-phase checkpoint also plays a more enigmatic and apparently critical role in preserving the functional integrity of defective replication forks, by mechanisms that are still understood poorly. This review considers some of the key experiments that have led to our current understanding of this highly complex pathway.

scholarly journals Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins

2008 ◽  
Vol 180 (6) ◽  
pp. 1073-1086 ◽  
Author(s):  
Julie M. Caldwell ◽  
Yinhuai Chen ◽  
Kaila L. Schollaert ◽  
James F. Theis ◽  
George F. Babcock ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Replication Forks ◽  
Checkpoint Signaling ◽  
Checkpoint Pathway ◽  
Origin Licensing ◽  
Pause Site ◽  
S Phase Checkpoint

The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Δ cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Δ dun1Δ cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin–Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.

scholarly journals Critical Role for Mouse Hus1 in an S-Phase DNA Damage Cell Cycle Checkpoint

2003 ◽  
Vol 23 (3) ◽  
pp. 791-803 ◽  
Author(s):  
Robert S. Weiss ◽  
Philip Leder ◽  
Cyrus Vaziri
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Critical Role ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Null Mouse ◽  
Cycle Checkpoint ◽  
S Phase Checkpoint

ABSTRACT Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1− fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G2/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

scholarly journals A Role for Saccharomyces cerevisiae Chk1p in the Response to Replication Blocks

2004 ◽  
Vol 15 (9) ◽  
pp. 4051-4063 ◽  
Author(s):  
Kaila L. Schollaert ◽  
Julie M. Poisson ◽  
Jennifer S. Searle ◽  
Jennifer A. Schwanekamp ◽  
Craig R. Tomlinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Transcriptional Response ◽  
S Phase ◽  
Yeast Cells ◽  
Dna Damage Checkpoint ◽  
Checkpoint Kinases ◽  
Checkpoint Pathway ◽  
Dna Replication And Repair ◽  
S Phase Checkpoint

Replication blocks and DNA damage incurred during S phase activate the S-phase and intra-S-phase checkpoint responses, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1Δ cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1Δ dun1Δ cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

scholarly journals Topoisomerase III Acts Upstream of Rad53p in the S-Phase DNA Damage Checkpoint

2001 ◽  
Vol 21 (21) ◽  
pp. 7150-7162 ◽  
Author(s):  
Ronjon K. Chakraverty ◽  
Jonathan M. Kearsey ◽  
Thomas J. Oakley ◽  
Muriel Grenon ◽  
Maria-Angeles de la Torre Ruiz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cellular Response ◽  
Cell Cycle Progression ◽  
Uv Light ◽  
S Phase ◽  
Dna Damaging Agents ◽  
Topoisomerase Iii ◽  
Rad53 Kinase ◽  
S Phase Checkpoint

ABSTRACT Deletion of the Saccharomyces cerevisiae TOP3gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391–8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Δ strains. We show that top3Δ mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion,top3Δ strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Δ mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.

scholarly journals S-Phase Checkpoint Pathways Stimulate the Mobility of the Retrovirus-Like Transposon Ty1

2007 ◽  
Vol 27 (24) ◽  
pp. 8874-8885 ◽  
Author(s):  
M. Joan Curcio ◽  
Alison E. Kenny ◽  
Sharon Moore ◽  
David J. Garfinkel ◽  
Matthew Weintraub ◽  
...  
Keyword(s):  
Dna Damage ◽  
Reverse Transcriptase ◽  
Reverse Transcriptase Activity ◽  
S Phase ◽  
Dna Lesions ◽  
Restriction Factors ◽  
Transcriptase Activity ◽  
Checkpoint Pathway ◽  
Ty1 Mobility ◽  
S Phase Checkpoint

ABSTRACT The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101Δ mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101Δ mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

The Role of MLL in DNA Damage Checkpoint and Its Implication in Leukemogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5332-5332
Author(s):  
Han Liu ◽  
Todd D Westergard ◽  
David Y Chen ◽  
Emily H.-Y. Cheng ◽  
James J.-D. Hsieh
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Critical Role ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Chromosome Band ◽  
Human Leukemia ◽  
Hox Gene ◽  
S Phase Checkpoint

Abstract Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which results in human cancers. Hence, it is of great significance to discover and investigate novel key constituents of the mammalian DNA damage response network. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a transcription co-activator that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathway must be involved, which remains to be discovered. Our recent studies demonstrated an intimate relationship between MLL and the cell cycle(Takeda et al. 2006, Genes & Development, 20, 2397–2409; Liu et al. 2007, Genes & Development, 21, 2385–2398). More importantly, our studies uncovered a critical role of MLL in executing the S phase checkpoint. We showed: Over-expression of MLL induces an S phase block. MLL accumulates in the S phase upon DNA damage. MLL deficiency results in radioresistant DNA synthesis (RDS) and chromatid-type chromosomal abnormalities, two signature characteristics of S phase checkpoint defects. We further determined the underlying mechanisms concerning the DNA damage-induced MLL accumulation. Our data showed that MLL is phosphorylated after DNA damage, which in turn blocks its degradation by SCFSkp2 in the S phase and results in the ultimate accumulation. Our data revealed the link between MLL and the S phase checkpoint, which provides novel insights into the mammalian cell cycle checkpoint network and human leukemia pathogenesis. Future studies utilizing murine leukemia models will be performed to examine whether MLL translocation compromises the S phase checkpoint and if the resulted dysfunction contributes to MLL leukemogenesis.

BCR/ABL Requires Fanconi Anemia D2 (FANCD2) Protein to Transform Hematopoetic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3249-3249
Author(s):  
Tomasz Stoklosa ◽  
Mateusz Koptyra ◽  
Grazyna Hoser ◽  
Ilona Seferynska ◽  
Eliza Glodkowska ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Dna Repair ◽  
S Phase ◽  
Leukemia Cells ◽  
Growth Defect ◽  
Deficient Mutant ◽  
Abl Kinase ◽  
Clonogenic Potential ◽  
S Phase Checkpoint

Abstract Abstract 3249 Poster Board III-1 Fanconi D2 protein (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) and phosphorylated on S222 (FANCD2-phosphoS222) in response to DNA double-strand breaks (DSBs). FANCD2-Ub interacts with RAD51 to facilitate homologous recombination repair (HRR), and FANCD2-phosphoS222 activates the S phase checkpoint. We detected an increased amount of FANCD2-Ub in CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) patients and in BCR/ABL-positive cell lines in comparison to normal counterparts. This effect was not associated with up-regulation of FANCD2 ubiquitinase FANCL or down-regulation of FANCD2 deubiquitinase USP1, but was reversed after inhibition of BCR/ABL kinase with imatinib and reduction of reactive oxygen species (ROS) with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). In addition mitomycin C routinely used for diagnostic testing in Fanconi anemia, strongly elevated FANCD2-Ub in CD34+ CML cells. Therefore we postulate that FANCD2-Ub may play a role in BCR/ABL transformation. In support for this hypothesis, we observed that clonogenic potential of BCR/ABL-positive murine leukemia stem cells (LSCs)-enriched FANCD2-/- Sca1+Kit+lin- bone marrow cells was reduced by approximately 10-fold in comparison to BCR/ABL-positive FANCD2+/+ counterparts; non-transformed -/- and +/+ cells displayed similar clonogenic potential stimulated by SCF and GM-CSF. Restoration of FANCD2 expression “rescued” the impaired clonogenic activity of BCR/ABL-positive FANCD2-/- cells. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Negative effect of BCR/ABL kinase on FANCD2-/- cell growth was reversed by expression of exogenous FANCD2. The in vitro growth defect of BCR/ABL-positive FANCD2-/- cells was accompanied by delayed leukemogenesis in SCID mice. These results suggest that FANCD2, a key regulator of DNA damage response, may play an important role in the initiation and/or maintenance of BCR/ABL-positive leukemias. We showed before that CD34+ CML-CP and CML-BC cells contain higher number of ROS-induced DSBs in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that LSC-enriched (CD34+CD38-) CML-CP and CML-BC cells display more DSBs than normal counterparts. Thus, BCR/ABL-mediated leukemogenesis is associated with accumulation of an excess of ROS-induced DSBs, which if not repaired, may induce apoptosis. We hypothesize that FANCD2 is necessary to “protect” leukemia cells from potentially lethal effect of BCR/ABL-induced oxidative DNA damage (including DSBs) at early stages of transformation and possibly also during the progression to CML-BC. This suggestion is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DSBs in comparison to +/+ counterparts. This effect did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by VE and NAC reduced the number of DSBs and eliminated the growth defect of BCR/ABL-positive FANCD2-/- cells. Accumulation of excessive DNA damage (DSBs) and impairment of growth potential of BCR/ABL-positive FANCD2-/- cells could be prevented by expression of FANCD2 wild-type (proficient in DNA repair and S phase checkpoint) and S222A phosphorylation-deficient mutant (proficient in DNA repair, but deficient in S phase checkpoint), but not the K561R monoubiquitination-deficient mutant (deficient in DNA damage, but proficient in S phase checkpoint). Since FANCD2-Ub interacts with RAD51 to promote HRR and BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, it is plausible that elevated expression of FANCD2-Ub may facilitate DSB repair to protect leukemia cells from lethal effects of DSBs. In concordance, co-localization of FANCD2-Ub and RAD51 was readily detected in the nuclei of BCR/ABL-positive leukemia cells. In conclusion our results indicate that FANCD2 plays an important role during the induction and perhaps also progression of Philadelphia-chromosome positive leukemias due to its ability to facilitate the repair of numerous, potentially lethal DSBs. Disclosures: No relevant conflicts of interest to declare.

The Recql5 Helicase Is a Novel Downstream Target of Jak2V617F and Maintains Genome Stability in Response to Replication Stress

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 822-822
Author(s):  
Edwin Chen ◽  
Jong-Sook Ahn ◽  
Lawrence J Breyfogle ◽  
Anthony R Green ◽  
Benjamin L. Ebert ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Replication Stress ◽  
Therapeutic Window ◽  
Replication Forks ◽  
Erythroid Progenitor ◽  
Stalled Replication Forks

Abstract The JAK2V617F mutation is present in a majority of patients with chronic myeloproliferative neoplasms (MPNs). Mutant JAK2 induces hyperactivation of multiple downstream signaling processes with the net effect of conferring cells with a pro-survival advantage. In particular, JAK2V617F-expressing cells tolerate increased DNA damage and higher levels of intracellular reactive oxygen species. However, the mechanisms by which increased genotoxic tolerance is mediated remain unclear. Previously, we performed gene expression analysis on autologous wildtype and JAK2V617F-heterozygous erythroblasts from 36 MPN patients, and observed increased expression of the RECQL5 helicase in JAK2-mutant erythroblasts. Increased Recql5 transcript and protein levels were also validated in Hoxb8-immortalized, GMP-like cell lines derived from wildtype and Jak2V617F knock-in mice (WT-B8 and VF-B8 cells, respectively). Recql5 up-regulation was dependent on the Pi3k-Akt pathway, and was independent of Stat1/5 and Mapk/Erk activity. As the Recql family of helicases plays a critical role in replication fork stability, we tested whether Recql5 could modulate sensitivity of JAK2-expressing cells to agents which promote replication stress, such as hydroxyurea (HU) and aphidicolin (APH). Strikingly, VF-B8 cells transduced with two different Recql5 shRNAs were more susceptible to HU- and APH-induced apoptosis when compared to Recql5-deficient WT-B8 cells. Replication stress-induced cytotoxicity was accompanied by increased gamma-H2Ax-marked double stranded breaks (DSBs) and activation of DNA repair pathways. Importantly, re-introduction of an shRNA-resistant Recql5 cDNA successfully rescued Recql5-deficient VF-B8 cells from HU- and APH-cytotoxicity. Molecularly, we show that Recql5 plays two roles to protect against DSB formation and cell death in Jak2-mutant cells. First, we visualized replication tracts on individual DNA fibers by chromosome combing and observed that Recql5-deficient VF-B8 cells treated with HU exhibit increased numbers of stalled replication forks. Moreover, Recql5 deficiency also led to an inability to restart forks stalled by HU treatment. This indicates that the absence of Recql5 leads to replication forks which are unstably arrested upon HU treatment, leading to fork collapse and the generation of DSBs. Second, we quantified the rate of single-stranded annealing (SSA) repair following Recql5 knockdown. Consistent with previous reports, we observed increased rates of SSA repair in VF-B8 cells compared to WT-B8 cells. However, this difference in the rate of SSA repair is abrogated upon Recql5 knockdown, suggesting that Recql5 functions as a mediator for the SSA DNA repair pathway. Cumulatively, these findings suggest that Recql5 up-regulation in Jak2V617F-expressing cells plays a role in protecting against DNA damage-induced cell death through (1) stabilization of stalled replication forks thus preventing their collapse, and (2) promoting rapid (albeit error prone) SSA DNA repair to ameliorate genomic instability. Finally, we tested whether modulation of RECQL5 could also increase sensitivity of JAK2V617F-positive cells from primary MPN patients to HU. Following depletion with RECQL5, c-kit-enriched peripheral blood mononuclear cells from 2 essential thrombocythemia and 3 myelofibrosis patients were grown in semi-solid medium supplemented with HU for 14 days. Strikingly, we observed more specific eradication of JAK2V617F-positive erythroid progenitor colonies compared to autologous wildtype colonies from all patients examined. In contrast, no specific killing of JAK2V617F-positive erythroblasts was seen following transduction of control hairpins. This suggests that RECQL5 knockdown may potentially open a therapeutic window by sensitizing Jak2V617F-expressing cells to HU and other agents that induce replication stress. Disclosures No relevant conflicts of interest to declare.

scholarly journals RAD9, RAD17, and RAD24 Are Required for S Phase Regulation in Saccharomyces cerevisiae in Response to DNA Damage

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 45-62 ◽  
Author(s):  
A G Paulovich ◽  
R U Margulies ◽  
B M Garvik ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Repair ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Alkylation Damage ◽  
Checkpoint Pathway ◽  
Phase Regulation ◽  
Phase Progression

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9Δ, rad17Δ, and rad24Δ. Furthermore, the triple rad9Δ rad17Δ rad24Δ mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1Δ, mag1Δ, apn1Δ, rev3Δ, rad18Δ, rad16Δ, dun1-Δ100, sad4-1, tel1Δ, rad26Δ, rad51Δ, rad52-1, rad54Δ, rad14Δ, rad1Δ, pol30–46, pol30–52, mad3Δ, pds1Δ/esp2Δ, pms1Δ, mlh1Δ, and msh2Δ are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.

Enhanced Phosphorylation of Nbs1, a Member of DNA Repair/Checkpoint Complex RAD50-Mre11-Nbs1, Can Be Targeted Simultaneously with BCR/ABL Kinase To Eliminate Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2127-2127 ◽  
Author(s):  
Lori Rink ◽  
Tomasz Stoklosa ◽  
Margaret Nieborowska-Skorska ◽  
Artur Slupianek ◽  
Ilona Seferynska ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Kinase Activity ◽  
S Phase ◽  
Leukemia Cells ◽  
Abl Kinase ◽  
Transformed Cell Lines ◽  
S Phase Checkpoint

Abstract Growing evidence indicate that ABL kinase inhibitors may need partner drugs to cure BCR/ABL-positive leukemias. Genotoxic drugs have been successfully combined with imatinib mesylate to increase its anti-leukemia activity in vitro. Although BCR/ABL-positive cells may accumulate even higher levels of DNA damage in comparison to their normal counterparts the former cells repair the lesions more proficiently and eventually survive. Therefore, targeting the mechanisms responsible for survival of leukemia cells after genotoxic treatment may increase the chances to eradicate BCR/ABL-positive leukemias. Nbs1, a member of the Rad50/Mre11/Nbs1 complex, is phosphorylated by ATM on Serine 343 (S343) in response to DNA double strand breaks (DSBs) to regulate intra-S and G2/M cell cycle checkpoints and DNA repair. Here we show that BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC, BCR/FGFR, and NPM/ALK, stimulate Nbs1 expression by protection from caspase-dependent degradation and induction of c-Myc-dependent transactivation. Downregulation of Nbs1 in BCR/ABL positive cells using siRNA increased their sensitivity to mitomycin C (MMC). Enhanced phosphorylation of Nbs1 on S343 (pNbs1) was detected by Western analysis in BCR/ABL-positive leukemia cells (CD34+ CML patient cells and leukemic cell lines) treated with various cytotoxic drugs (MMC, hydroxyurea = HU, cisplatin - CPL) in comparison to normal counterparts. This effect is associated with increased ATM kinase activity in BCR/ABL cells treated with MMC. In addition, immunofluoresence studies demonstrated an increase of the pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. DNA damage-dependent enhancement of pNbs1 appears to be a broad phenomenon because it was also detected in MMC-treated tumor cells expressing other FTKs. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells and BCR/ABL-transformed cell lines displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase, indicating an intact intra-S phase checkpoint. Expression of the Nbs1-S343A phosphorylation-less mutant downregulated pNbs1 and disrupted intra-S phase checkpoint resulting in reduced accumulation of BCR/ABL leukemia cells in S phase after MMC treatment. This effect was associated with an increase of the sensitivity of leukemia cells to genotoxic treatment (MMC, HU, CPL). A combinatorial strategy was employed targeting enhanced Nbs1 phosphorylation and the deregulated BCR/ABL tyrosine kinase activity, using the Nbs1-S343A phosphorylation-less mutant and a sub-optimal concentration of STI571 eliminating ~50% of leukemia cells, respectively. Targeting both BCR/ABL kinase activity and Nbs1 phosphorylation in combination significantly sensitizes B/A-positive cells to MMC treatment, nearly eradicating all leukemia cells.

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