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.

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.

Enhanced Phosphorylation of Nbs1, a Member of the DNA Repair/Checkpoint Activation Complex Rad50/Mre11/Nbs1, Prolongs Cell Cycle S Phase and Contributes to Drug Resistance in BCR/ABL-Positive Leukemias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2867-2867 ◽  
Author(s):  
Lori Rink ◽  
Tomasz Stoklosa ◽  
Tomasz Skorski
Keyword(s):  
Dna Repair ◽  
Growth Factors ◽  
Dna Synthesis ◽  
Cell Lines ◽  
S Phase ◽  
Leukemia Cells ◽  
Checkpoint Activation ◽  
Normal Cells ◽  
Nuclear Foci ◽  
S Phase Checkpoint

Abstract Nbs1, a member of the DNA repair/checkpoint activation complex Mre11/Rad50/Nbs1, is phosphorylated by ATM in response to the presence of DNA double strand breaks (DSBs) resulting in the activation of the S phase checkpoint. Here we show that the BCR/ABL tyrosine kinase, as well as growth factors (IL-3, GM-CSF), stimulate the expression of Mre11 and Nbs1, but not Rad50. This effect is dependent on the kinase activity of BCR/ABL protecting Mre11 and Nbs1 from caspase-dependent, but not proteasome-dependent degradation. Cells expressing the BCR/ABL kinase are resistant to chemotherapeutic agents, including mitomycin C (MMC). Western analysis showed enhanced phosphorylation of Nbs1 on Serine 343 in MMC-treated BCR/ABL leukemia cells (CML patient cells and leukemic cell lines) in comparison to normal cells in the presence of growth factors. Immunofluoresence studies demonstrated an increase of γ-H2AX nuclear foci (DSBs indicator) and pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. In addition, BCR/ABL-positive cells displayed a higher percentage of colocalization of γ-H2AX and p-Nbs1 implicating the presence of p-Nbs1 at the DSBs. Clonogenic assays performed after down regulation of Nbs1 in BCR/ABL positive cells using siRNA showed increased sensitivity to MMC. Specifically, the expression of the Nbs1-S343A phosphorylation-less mutant also decreased resistance in BCR/ABL cells to MMC. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells, BCR/ABL-transformed cell lines and normal counterparts displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase. Expression of Nbs1-S343A mutant caused a significant decrease in the accumulation of BCR/ABL leukemia cells in S phase after MMC treatment, whereas cells transfected with both the empty construct and wild-type Nbs1 displayed S phase accumulation. Surprisingly, Nbs1-S343A mutant did not affect the ability of normal cells to accumulate in S phase in response to MMC. Altogether, we hypothesize that enhanced phosphorylation of Nbs1 on S343 leads to increased resistance to genotoxic agents in BCR/ABL leukemia cells by prolonging the S phase checkpoint and allowing longer time for the repair of excessive DNA damage.

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 Fission Yeast Gene,him1+/dfp1+, Encoding a Regulatory Subunit for Hsk1 Kinase, Plays Essential Roles in S-Phase Initiation as Well as in S-Phase Checkpoint Control and Recovery from DNA Damage

1999 ◽  
Vol 19 (8) ◽  
pp. 5535-5547 ◽  
Author(s):  
Tadayuki Takeda ◽  
Keiko Ogino ◽  
Etsuko Matsui ◽  
Min Kwan Cho ◽  
Hiroyuki Kumagai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Fission Yeast ◽  
Kinase Activity ◽  
S Phase ◽  
Regulatory Subunit ◽  
Checkpoint Control ◽  
Regulatory Subunits ◽  
Hydroxyurea Treatment ◽  
S Phase Checkpoint

ABSTRACT Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G1/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1+ from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1+ is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G1. The protein level is low at START in G1, increases at the G1/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G1/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1+ is identical to rad35+ . Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

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.

BCR/ABL Kinase Elevates ROS-Mediated Oxidative DNA Damage in CML Stem/Progenitor Cells and Affects the Efficiency and Fidelity of DNA Repair To Induce Genetic Instability.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 34-34
Author(s):  
Margaret Nieborowska-Skorska ◽  
Artur Slupianek ◽  
Tomasz Stoklosa ◽  
Tomasz Poplawski ◽  
Kimberly Cramer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genomic Instability ◽  
Chromosomal Aberrations ◽  
Oxidative Dna Damage ◽  
Point Mutations ◽  
Dna Lesions ◽  
Leukemia Cells ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract BCR/ABL kinase transforms hematopoietic stem cells (HSCs) to induce chronic myelogenous leukemia in chronic phase (CML-CP), which eventually evolves into fatal blast crisis (CML-BC). CML is a stem cell-derived but a progenitor-driven disease. In CML-CP leukemia stem (LSCs) and progenitor (LPCs) cells reside in CD34+CD38− and CD34+CD38+ populations, respectively, whereas in CML-BC LSCs are found also in CD34+CD38+ population. BCR/ABL kinase stimulates genomic instability causing imatinib-resistant point mutations and chromosomal aberrations associated with progression to CML-BC. Genomic instability may result from enhanced DNA damage and/or aberrant DNA repair mechanisms. We showed that CD34+ stem/progenitor CML cells contain higher levels of reactive oxygen species (ROS) than these from healthy donors (CML-BC>CML-CP>Normal). In addition, ROS were elevated in CD34+CD38− and CD34+CD38+ sub-populations isolated from CML-BC and CML-CP patients in comparison to cells from healthy donor. Higher ROS levels induced more oxidative DNA lesions such as oxidized bases (e.g., 8-oxoG) and DNA double-strand breaks (DSBs). ROS and oxidative DNA damage in CML stem/progenitor cells could be diminished by an antioxidant N-acetyl-cysteine. Moreover, inhibition of ROS by vitamin E reduced the frequency of imatinib-resistant BCR/ABL point mutants and chromosomal aberrations in leukemia cells in SCID mice. Cellular DNA repair systems act to remove DNA damage and ultimately preserve the informational integrity of the genome. Base excision repair (BER) and mismatch repair (MMR) are responsible for removal of oxidized bases. BER was assessed using single- and double-stranded DNA substrates containing 5-OH-U (a derivative of ROS-damaged hydroxy-deoxycytidine). MMR activity was measured by restoration of the expression of GFP from the construct containing T-G mismatch in the start codon. BCR/ABL kinase severely inhibited BER and MMR in cell lines and CD34+ CML cells, and promoted accumulation of point mutations in genes encoding BCR/ABL kinase and Na+/K+ ATPase. Inhibition of BCR/ABL kinase by imatinib restored BER and MMR activities. Oxidized bases, if not repaired, may lead to accumulation of DSBs observed in LSCs and LPCs. DSBs may be processed by homologous recombination (HR), non-homologous and-joininig (NHEJ), and single-strand annealing (SSA). HR represents faithful repair, NHEJ usually produces small deletions, and SSA causes very large deletions. Genome-integrated repair-specific reporter cassettes containing two disrupted fragments of the gene encoding GFP were used where a single DSB induced by I-SceI endonuclease in one of the fragments stimulated HR, NHEJ, or SSA. In general, BCR/ABL kinase enhanced DSBs repair activities, however at the expense of their fidelity. Numerous point mutations were introduced in HR repair products. NHEJ generated larger than usual deletions. SSA, rather rare but very unfaithful, was also induced in BCR/ABL-positive leukemia cells. In summary, BCR/ABL kinase enhanced ROS-mediated oxidative DNA damage in LSCs and LPCs. In addition, BCR/ABL inhibited BER and MMR of usually non-lethal oxidized DNA lesions leading to accumulation of point mutations. Moreover, BCR/ABL kinase stimulated HR, NHEJ and SSA of lethal DSBs, but compromised the fidelity of repair.

Mechanisms Generating free Radicals in CML Stem/Progenitor Cell Populations Causing DNA Damage and Genomic Instability

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 192-192 ◽  
Author(s):  
Margaret Nieborowska-Skorska ◽  
Mateusz Koptyra ◽  
Grazyna Hoser ◽  
Regina Ray ◽  
Danielle Ngaba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Free Radicals ◽  
Genomic Instability ◽  
Chromosomal Aberrations ◽  
Point Mutations ◽  
Leukemia Cells ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Transformed Cell Lines

Abstract BCR/ABL kinase is the founding member of a family of oncogenic tyrosine kinases (OTKs) also including TEL/JAK2, TEL/PDGFR, TEL/ABL, and JAK2V617F, which induce myeloproliferative disorders (MPDs). BCR/ABL transforms hematopoietic stem cells (HSCs) to induce chronic myelogenous leukemia in chronic phase (CML-CP), which eventually evolves into fatal blast crisis (CML-BC). CML is a stem cell-derived but progenitor-driven disease. In CML-CP, leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) reside in the CD34+CD38- and CD34+CD38+ populations, respectively, whereas in CML-BC, LSCs are also found in the CD34+CD38+ population. In addition, CD34+ CML cells belong to either proliferative or quiescent populations; the latter of which responds poorly to the ABL kinase inhibitors. BCR/ABL kinase stimulates genomic instability causing imatinib-resistant point mutations in the kinase domain and additional chromosomal aberrations associated with progression to CML-BC (Oncogene, 2007). Since genomic instability usually results from enhanced DNA damage, we investigated the mechanisms responsible for “spontaneous” DNA damage in cells transformed by BCR/ ABL and other OTKs. Much endogenous DNA damage arises from free radicals such as reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). We showed that CD34+ stem/progenitor CML cells contain higher levels of ROS (superoxide anion = ·O2−, hydrogen peroxide = H2O2 and hydroxyl radical = ·OH) and RNS (nitric oxide = NO·) than CD34+ cells from normal donors (CML-BC>CML-CP>Normal). Moreover, ROS levels were elevated in CD34+CD38- and CD34+CD38+ sub-populations isolated from CML-BC and CML-CP patients in comparison to the corresponding cells from healthy donor. In addition, both proliferative and quiescent CD34+ CML cell sub-populations contained more ROS than their normal counterparts. Interaction with the stromal cells further elevated ROS levels in BCR/ABL-positive cells. Higher ROS/RNS levels induced more oxidative/nitrative DNA lesions, such as 8-oxoG and DNA double-strand breaks (DSBs), in CML-CP cells resulting in induction of point mutations in BCR/ABL kinase causing imatinib resistance and accumulation of chromosomal aberrations characteristic of CML-BC. In addition, cells transformed by other OTKs also displayed elevated ROS/ RNS and oxidative/nitrative DNA damage, implicating their role in malignant progression of MPDs. Our previous studies showed that elevated levels of oxidative DNA damage in OTK-transformed cells could be diminished by scavenging of ROS with N-acetyl-cysteine and vitamin E, which reduced the frequency of imatinib-resistant BCR/ABL point mutants and chromosomal aberrations in leukemia cells cultured in vitro and growing in SCID mice (Blood, 2006; Leukemia, 2008). These studies highlighted the importance of identification of the sources of free radicals in CML and other MPDs. We found that elevated levels of ROS in BCR/ABL-transformed cell lines and CD34+ CML cells were generated by three major mechanisms: NADPH oxidase (NOX) complexes containing NOX1 and/or NOX2, complex III of the mitochondrial respiratory chain (MRC), and 5-lipoxygenase (LOX). In addition, inducible nitric oxygen synthase (iNOS) produced RNS in leukemia cells. Using selective inhibitors of NOX, MRC, LOX and iNOS we estimated the contribution of these pathways to accumulation of free radicals causing oxidative/nitrative DNA damage in CML cells. In summary, BCR/ABL kinase-dependent elevation of ROS/RNS depends on several mechanisms, which are now targeted to determine their actual role in genomic instability in CML.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

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