A novel DNA-dependent protein kinase inhibitor, NU7026, potentiates the cytotoxicity of topoisomerase II poisons used in the treatment of leukemia

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4659-4665 ◽  
Author(s):  
Elaine Willmore ◽  
Sarah de Caux ◽  
Nicola J. Sunter ◽  
Michael J. Tilby ◽  
Graham H. Jackson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Growth Inhibition ◽  
Topoisomerase Ii ◽  
Kinase Inhibitor ◽  
Cell Cycle Distribution ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Topo Ii

Abstract We report for the first time the use of a selective small-molecule inhibitor of DNA repair to potentiate topoisomerase II (topo II) poisons, identifying DNA-dependent protein kinase (DNA-PK) as a potential target for leukemia therapy. Topo II poisons form cleavable complexes that are processed to DNA double-strand breaks (DSBs). DNA-PK mediates nonhomologous end joining (NHEJ). Inhibition of this DSB repair pathway may sensitize cells to topo II poisons. We investigated the effects of a novel DNA-PK inhibitor, NU7026 (2-(morpholin-4-yl)-benzo[h]chomen-4-one), on the response to topo II poisons using K562 leukemia cells. NU7026 (10 μM) potentiated the growth inhibition of idarubicin, daunorubicin, doxorubicin, etoposide, amsacrine (mAMSA), and mitroxantrone with potentiation factors at 50% growth inhibition ranging from approximately 19 for mAMSA to approximately 2 for idarubicin (potentiation of etoposide was confirmed by clonogenic assay). In contrast, NU7026 did not potentiate camptothecin or cytosine arabinoside (araC). NU7026 did not affect the levels of etoposide-induced topo IIα or β cleavable complexes. NU7026 alone had no effect on cell cycle distribution, but etoposide-induced accumulation in G2/M was increased by NU7026. A concentration-dependent increase in etoposide-induced DSB levels was increased by NU7026. The mechanism of NU7026 potentiation of topo II poisons involves inhibition of NHEJ and a G2/M checkpoint arrest. (Blood. 2004;103:4659-4665)

scholarly journals DNA-dependent Protein Kinase Activity Is Not Required for Immunoglobulin Class Switching

2002 ◽  
Vol 196 (11) ◽  
pp. 1483-1495 ◽  
Author(s):  
Gayle C. Bosma ◽  
Jiyoon Kim ◽  
Teresa Urich ◽  
Donna M. Fath ◽  
Maria G. Cotticelli ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Kinase ◽  
Scid Mice ◽  
Nonhomologous End Joining ◽  
Protein Kinase Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Class switch recombination (CSR), similar to V(D)J recombination, is thought to involve DNA double strand breaks and repair by the nonhomologous end–joining pathway. A key component of this pathway is DNA-dependent protein kinase (DNA-PK), consisting of a catalytic subunit (DNA-PKcs) and a DNA-binding heterodimer (Ku70/80). To test whether DNA-PKcs activity is essential for CSR, we examined whether IgM+ B cells from scid mice with site-directed H and L chain transgenes were able to undergo CSR. Although B cells from these mice were shown to lack DNA-PKcs activity, they were able to switch from IgM to IgG or IgA with close to the same efficiency as B cells from control transgenic and nontransgenic scid/+ mice, heterozygous for the scid mutation. We conclude that CSR, unlike V(D)J recombination, can readily occur in the absence of DNA-PKcs activity. We suggest nonhomologous end joining may not be the (primary or only) mechanism used to repair DNA breaks during CSR.

scholarly journals DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

2018 ◽  
Author(s):  
Rajashree A. Deshpande ◽  
Logan R. Myler ◽  
Michael M. Soniat ◽  
Nodar Makharashvili ◽  
Linda Lee ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Homologous Recombination ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Dependent Protein ◽  
Non Homologous End Joining ◽  
Dna Ends

AbstractThe repair of DNA double-strand breaks occurs through non-homologous end joining or homologous recombination in vertebrate cells - a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from non-homologous end joining to homologous recombination by facilitating DNA-PK removal.One Sentence SummaryDNA-dependent protein kinase, an enzyme critical for non-homologous repair of DNA double-strand breaks, also stimulates end processing for homologous recombination.

scholarly journals DNA-Dependent Protein Kinase Catalytic Subunit: The Sensor for DNA Double-Strand Breaks Structurally and Functionally Related to Ataxia Telangiectasia Mutated

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1143
Author(s):  
Yoshihisa Matsumoto ◽  
Anie Day D. C. Asa ◽  
Chaity Modak ◽  
Mikio Shimada
Keyword(s):  
Protein Kinase ◽  
Ataxia Telangiectasia ◽  
Catalytic Subunit ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Dependent Protein

The DNA-dependent protein kinase (DNA-PK) is composed of a DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/Ku80 heterodimer. DNA-PK is thought to act as the “sensor” for DNA double-stranded breaks (DSB), which are considered the most deleterious type of DNA damage. In particular, DNA-PKcs and Ku are shown to be essential for DSB repair through nonhomologous end joining (NHEJ). The phenotypes of animals and human individuals with defective DNA-PKcs or Ku functions indicate their essential roles in these developments, especially in neuronal and immune systems. DNA-PKcs are structurally related to Ataxia–telangiectasia mutated (ATM), which is also implicated in the cellular responses to DSBs. DNA-PKcs and ATM constitute the phosphatidylinositol 3-kinase-like kinases (PIKKs) family with several other molecules. Here, we review the accumulated knowledge on the functions of DNA-PKcs, mainly based on the phenotypes of DNA-PKcs-deficient cells in animals and human individuals, and also discuss its relationship with ATM in the maintenance of genomic stability.

scholarly journals ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

1989 ◽  
Vol 94 (4) ◽  
pp. 693-717 ◽  
Author(s):  
B Ribalet ◽  
S Ciani ◽  
G T Eddlestone
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Insulin Secreting Cells ◽  
Dependent Protein ◽  
Inside Out

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

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