Cytotoxic signalling by inhibitors of DNA topoisomerase II

2001 ◽  
Vol 29 (6) ◽  
pp. 702-703 ◽  
Author(s):  
W. T. Beck ◽  
Y.-Y. Mo ◽  
U. G. Bhat
Keyword(s):  
Protein Interactions ◽  
Topoisomerase Ii ◽  
Fas Ligand ◽  
Regulatory Protein ◽  
Protein Distribution ◽  
Protein Protein Interactions ◽  
Dna Topoisomerase ◽  
Cell Cycle Regulatory Protein ◽  
Jun Kinase ◽  
Topo Ii

DNA topoisomerase (topo) II inhibitors either stabilize DNA–topo II complexes by blocking DNA religation (e.g. etoposide) or block the enzyme's catalytic activity (e.g. dexrazoxane). The former class of drugs causes direct DNA damage through topo II, while the latter class does not, but both classes cause apoptosis. We cloned the Fas ligand (FasL) promoter and coupled it to the luciferase gene. Treatment of cells transfected with this construct revealed that complex-stabilizing (DNA-damaging) agents induce FasL expression, but the catalytic inhibitors do not, suggesting that the FasL pathway may not be involved in all cases of topoisomerase-mediated apoptosis. Some topo II inhibitors activate a pathway involving stress-activated protein kinases, which include c-Jun N-terminal kinase-1 (JNK-1). We will discuss the effects of these agents on components of this pathway. Our earlier work revealed that topo IIα interacts with the cell cycle regulatory protein, retinoblastoma protein (Rb). This interaction and the subcellular distribution of these proteins are altered by topo II inhibitory drugs and lead to apoptosis. In addition, agents that affect Rb, such as E1A and E2F1/DP-1, when transfected into cells, also alter topo IIα-Rb localization, activate jun kinase pathways and cause apoptosis. This paper discusses current studies that are designed to determine the contributions of these signalling events to the alterations in subcellular protein distribution and apoptosis. We suggest that protein-protein interactions are important for mediation of cytotoxic signalling by anticancer drugs.

Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain

1994 ◽  
Vol 14 (10) ◽  
pp. 6962-6974
Author(s):  
Y S Vassetzky ◽  
Q Dang ◽  
P Benedetti ◽  
S M Gasser
Keyword(s):  
Chromosome Segregation ◽  
Protein Interactions ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Protein Protein Interactions ◽  
Dna Topoisomerase ◽  
Terminal Domain ◽  
Terminal Amino ◽  
In Vitro Effects

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.

scholarly journals Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase

2003 ◽  
Vol 371 (3) ◽  
pp. 957-964 ◽  
Author(s):  
Kui XU ◽  
Charles BELUNIS ◽  
Wei CHU ◽  
David WEBER ◽  
Frank PODLASKI ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Regulatory Protein ◽  
Protein Protein Interactions ◽  
Time Resolved ◽  
Cell Cycle Regulatory Protein ◽  
P27kip1 Protein ◽  
F Box Protein

The p27Kip1 protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCFSkp2 has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr187, thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr187. Using this assay, we have determined the dissociation constant of the Skp2–Cks1 complex (Kd 140 ± 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (Kd 37 ± 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2.

scholarly journals Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain.

1994 ◽  
Vol 14 (10) ◽  
pp. 6962-6974 ◽  
Author(s):  
Y S Vassetzky ◽  
Q Dang ◽  
P Benedetti ◽  
S M Gasser
Keyword(s):  
Chromosome Segregation ◽  
Protein Interactions ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Protein Protein Interactions ◽  
Dna Topoisomerase ◽  
Terminal Domain ◽  
Terminal Amino ◽  
In Vitro Effects

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.

scholarly journals Rearrangements of the MLL gene in therapy-related acute myeloid leukemia in patients previously treated with agents targeting DNA- topoisomerase II

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3705-3711 ◽  
Author(s):  
HJ Super ◽  
NR McCabe ◽  
MJ Thirman ◽  
RA Larson ◽  
MM Le Beau ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Topoisomerase Ii ◽  
De Novo ◽  
Chromosome Band ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mll Gene ◽  
Topo Ii ◽  
Acute Myeloid

Chromosome band 11q23 is frequently involved in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) de novo, as well as in myelodysplastic syndromes (MDS) and lymphoma. Five percent to 15% of patients treated with chemotherapy for a primary neoplasm develop therapy-related AML (t-AML) that may show rearrangements, usually translocations involving band 11q23 or, less often, 21q22. These leukemias develop after a relatively short latent period and often follow the use of drugs that inhibit the activity of DNA-topoisomerase II (topo II). We previously identified a gene, MLL (myeloid-lymphoid leukemia or mixed-lineage leukemia), at 11q23 that is involved in the de novo leukemias. We have studied 17 patients with t-MDS/t-AML, 12 of whom had cytogenetically detectable 11q23 rearrangements. Ten of the 12 t-AML patients had received topo II inhibitors and 9 of these, all with balanced translocations of 11q23, had MLL rearrangements on Southern blot analysis. None of the patients who had not received topo II inhibitors showed an MLL rearrangement. Of the 5 patients lacking 11q23 rearrangements, some of whom had monoblastic features, none had an MLL rearrangement, although 4 had received topo II inhibitors. Our study indicates that the MLL gene rearrangements are similar both in AML that develops de novo and in t-AML. The association of exposure to topo II- reactive chemotherapy with 11q23 rearrangements involving the MLL gene in t-AML suggests that topo II may play a role in the aberrant recombination events that occur in this region both in AML de novo and in t-AML.

Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue

2000 ◽  
Vol 279 (3) ◽  
pp. C860-C867 ◽  
Author(s):  
Kevin Strange ◽  
Thomas D. Singer ◽  
Rebecca Morrison ◽  
Eric Delpire
Keyword(s):  
Protein Interactions ◽  
Regulatory Protein ◽  
Phosphorylation Site ◽  
Fold Increase ◽  
Tyrosine Residue ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
Pharmacological Studies ◽  
Osmotic Homeostasis ◽  
Tyrosine Phosphorylation Site

K-Cl cotransporters (KCC) play fundamental roles in ionic and osmotic homeostasis. To date, four mammalian KCC genes have been identified. KCC2 is expressed exclusively in neurons. Injection of Xenopus oocytes with KCC2 cRNA induced a 20-fold increase in Cl−-dependent, furosemide-sensitive K+ uptake. Oocyte swelling increased KCC2 activity 2–3 fold. A canonical tyrosine phosphorylation site is located in the carboxy termini of KCC2 (R1081–Y1087) and KCC4, but not in other KCC isoforms. Pharmacological studies, however, revealed no regulatory role for phosphorylation of KCC2 tyrosine residues. Replacement of Y1087 with aspartate or arginine dramatically reduced K+ uptake under isotonic and hypotonic conditions. Normal or near-normal cotransporter activity was observed when Y1087 was mutated to phenylalanine, alanine, or isoleucine. A tyrosine residue equivalent to Y1087 is conserved in all identified KCCs from nematodes to humans. Mutation of the Y1087 congener in KCC1 to aspartate also dramatically inhibited cotransporter activity. Taken together, these results suggest that replacement of Y1087 and its congeners with charged residues disrupts the conformational state of the carboxy terminus. We postulate that the carboxy terminus plays an essential role in maintaining the functional conformation of KCC cotransporters and/or is involved in essential regulatory protein-protein interactions.

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