Resolution of DNA topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting.

Eukaryotic DNA Topoisomerase II (Topo II) has been studied using high-resolution two-dimensional polyacrylamide electrophoresis (2D-PAGE) and immunodetection of resolved proteins using specific antisera (Western blotting). Traditional methods of 2D-PAGE failed to resolve Topo II and neither nonequilibrium nor equilibrium pH gradients allowed Topo II to enter the first dimension gel. Exhaustive nuclease digestion and alternate protein solubilization strategies also produced negative results. We have developed altered first dimension pH gradient profiles and employed a more aggressive protein solubilization procedure which resulted in the resolution of Topo II. The 170-kDa polypeptide focuses with an apparent isoelectric point of approximately 6.5.     

Effects of irradiation on nuclear protein synthesis in G2 phase of the cell cycle

A method was developed to determine the synthesis of nuclear proteins throughout the cell cycle which was resolved into six compartments on the basis of DNA and nuclear protein content (i.e., early and late G1, early and late S, etc). Using this technique cell-cycle-specific synthesis of certain nuclear proteins was observed. Of particular interest was a 170-kDa protein(s) whose synthesis was initiated in early S phase and reached a maximum rate in late G2. Following irradiation with 6.8 Gy of 137Cs gamma rays the synthesis of the 170-kDa protein(s) declined in the G2 population with near total inhibition seen by 24 h. Synthesis of the 170-kDa protein(s) appeared to be slightly enhanced, and the postirradiation inhibition of its synthesis was reversed, in the presence of 3 mM caffeine. Also, the synthesis of 55-kDa nuclear protein(s) was stimulated throughout the cell cycle in the presence of 3 mM caffeine. These observations suggest new possibilities regarding the mechanism of the X-ray-induced G2 block and its reversal by caffeine. However, the exact role of these nuclear proteins in cellular events remains to be ascertained.     

Dynamic association of topoisomerase II to the mitotic chromosomes in live cells of Aspergillus nidulans

DNA topoisomerase II (Topo II) is an essential enzyme that catalyzes topological changes of DNA and consists of a major member of mitotic chromosomes. To investigate the dynamic localization of Topo II in nuclei, we engineered the strain of Aspergillus nidulans expressing Topo II fused with green fluorescent protein (GFP). Time-lapse microscopy revealed that the distribution of Topo II-GFP in nuclei varied depending on the cell cycle. In interphase, Topo II-GFP distributed evenly in the nucleoplasm and at the onset of G2 phase became concentrated into nucleolus. During mitosis, Topo II-GFP accumulated on chromosomes, when the chromosomes condensed. In the early mitosis, the Topo II also showed a single or two brighter spots among the fluorescence of clumped chromosomes. The spots once divided into several spots and then concentrated again into a spot per nucleus in the dividing nuclei of anaphase. Along with the subsequent decondensation of chromosomes, Topo II diffused back into nucleoplasm.     

Release of H2O2 and phosphorylation of 30 kilodalton proteins as early responses of cell cycle-dependent inhibition of DNA synthesis by transforming growth factor beta 1.

Transforming growth factor beta 1 (TGF-beta 1) and H2O2 both inhibited DNA synthesis of mouse osteoblastic (MC3T3) cells in the late G1 phase of the cell cycle. TGF-beta 1 stimulated cells to release H2O2 in the late G1 phase, but not in the G0 phase, even though TGF-beta 1 receptors were present in both phases. The inhibition of DNA synthesis caused by TGF-beta 1 was partly decreased by the addition of catalase. TGF-beta 1 and H2O2 increased the phosphorylation of the same proteins with a molecular weight of 30,000 in cells in the late G1 phase, and the increase by TGF-beta 1 was abolished at least partly by catalase. The results suggest that H2O2 is one of the mediators of inhibition of DNA synthesis by TGF-beta 1.     

Phylogenomics of type II DNA topoisomerases

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.     

Synthesis and characterization of defensin NP-1.

Defensins are a group of small, cationic, antimicrobial proteins found in the cytoplasmic granules of neutrophils and macrophages of a variety of mammalian species. One such defensin, NP-1, isolated from rabbit neutrophils, has been shown to consist of 33 amino acids rich in arginine and cysteine residues. We have synthesized NP-1 on an Applied Biosystems Model 431A peptide synthesizer using FastMoc chemistry involving HBtu [2-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] activation for coupling amino acids. The linear peptide was folded by air oxidation to the biologically active form containing three disulfide bonds and purified by reverse phase chromatography. The amino acid sequence of the synthetic peptide was confirmed by Edman degradation. Molecular weight determination by plasma desorption mass spectroscopy (PDMS) gave a value of 3898.6, in agreement with the expected molecular weight of 3898. The biological activity of the synthetic peptide, as measured by its antifungal activity against several pathogenic fungi, was indistinguishable from that of the natural NP-1. Also, the CD spectrum was equivalent to that of natural NP-1, indicating conformational identity of the two species.     

Mcl-1 depletion in apoptosis elicited by ionizing radiation in peritoneal resident macrophages of C3H mice

Remarkably, apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice, to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins, Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1, but not Bcl-2, Bcl-X(L), Bax, A1, or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand, Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis.     

In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies

Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.     

Depletion of the cap-associated isoform of translation factor eIF4G induces germline apoptosis in C. elegans

During mammalian programmed cell death, cleavage of the translation initiation factor 4G proteins (eIF4GI and eIF4GII) by caspase-3 induces the cap-independent synthesis of pro-apoptotic proteins. Apoptosis occurs naturally in the gonad to remove germ cells that are not selected to grow as oocytes and mature into eggs. Here, we describe two major isoforms of Caenorhabditis elegans eIF4G that are derived from a single gene (ifg-1) and their separate roles in germline homeostasis. Full length IFG-1 protein (170 kDa isoform) differs from the shorter isoform (130 kDa) by the inclusion of the N-terminal domain containing the putative eIF4E-binding site required for mRNA cap recognition. Depletion of the cap-associated p170 isoform induced CED-4 expression in oocytes and markedly increased germline apoptotic events, but did not prevent early mitotic germ cell proliferation. Loss of both p170 and p130 suppressed germ cell proliferation and arrested larval development. Evidence suggests that eIF4G isoforms are differentially utilized during oogenesis to regulate germ cell apoptosis. We propose that an alternative mechanism to eIF4G cleavage may be employed in germ cells by changing the availability of the p170 isoform.     

Topoisomerase II alpha is associated with the mammalian centromere in a cell cycle- and species-specific manner and is required for proper centromere/kinetochore structure

A study of the distribution of Topoisomerase II alpha (Topo II) in cells of six tissue culture cell lines, human (HeLa), mouse (L929), rat, Indian muntjac, rat kangaroo (PTK-2), and wallaby revealed the following features: (1) There is a cell cycle association of a specific population of Topo II with the centromere. (2) The centromere is distinguished from the remainder of the chromosome by the intensity of its Topo II reactivity. (3) The first appearance of a detectable population of Topo II at the centromere varies between species but is correlated with the onset of centromeric heterochromatin condensation. (4) Detectable centromeric Topo II declines at the completion of cell division. (5) The distribution pattern of Topo II within the centromere is species- and stage-specific and is conserved only within the kinetochore domain. In addition, we report that the Topo II inhibitor ICRF-193 can prevent the normal accumulation of Topo II at the centromere. This results in the disruption of chromatin condensation sub-adjacent to the kinetochore as well as the perturbation of kinetochore structure. Taken together, our studies indicate that the distribution of Topo II at the centromere is unlike that reported for the remainder of the chromosome and is essential for proper formation of centromere/kinetochore structure.     

Expression and localization of DNA topoisomerase II during rat spermatogenesis

The potential role(s) of DNA topoiosmerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immunofluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo IIβ mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo IIβ mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis. © 1996 Wiley-Liss, Inc.     

Molecular basis for premature senescence induced by surfactants in normal human cells

Sublethal doses of surfactants as exemplified by NP-40 clearly induce premature senescence in normal human cells. To understand molecular basis for this phenomenon, we tried to suppress it with use of various inhibitors. An inhibitor of p38 of the MAPK family almost completely suppressed growth arrest and morphological changes induced by surfactants; however, other inhibitors tested had no effect. Oleic acid, a weak inducer of premature senescence, was found to suppress the effect of NP-40. Fluorescein-labeled oleic acid rapidly bound to the cell surface, and this binding was clearly blocked by pre-treatment with surfactants, suggesting that surfactants and oleic acid compete for binding to the cell surface. Moderate concentrations of cycloheximide, an inhibitor of protein synthesis, also suppressed the senescent features induced by NP-40. These results suggest that surfactants activate p38 signaling pathway by binding to the cell surface, and induce cellular senescence.     

Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins.

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of approximately 100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact beta/alpha fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by site-directed mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg2+that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.     

Induction of drug metabolizing enzymes in human liver cell line Hep G2

Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20-30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on "Western blots' using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.Key words: topoisomerase II, mitosis, G₂, conditional knockdown, S phase, mitotic catastrophe     

Cdc14 protein phosphatase and topoisomerase II mediate rDNA dynamics and nucleophagic degradation of nucleolar proteins after TORC1 inactivation

Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicits nucleophagy degrading nucleolar proteins in budding yeast. After TORC1 inactivation, nucleolar proteins are relocated to sites proximal to the nucleus–vacuole junction (NVJ), where micronucleophagy occurs, whereas ribosomal DNA (rDNA encoding rRNA) escapes from the NVJ. Condensin-mediated rDNA condensation promotes the repositioning and nucleophagic degradation of nucleolar proteins. However, the molecular mechanism of TORC1 inactivation-induced chromosome condensation is still unknown. Here, we show that Cdc14 protein phosphatase and topoisomerase II (Topo II), which are engaged in rDNA condensation in mitosis, facilitate rDNA condensation after TORC1 inactivation. rDNA condensation after rapamycin treatment was compromised in cdc14-1 and top2-4 mutants. In addition, the repositioning of rDNA and nucleolar proteins and nucleophagic degradation of nucleolar proteins were impeded in these mutants. Furthermore, Cdc14 and Topo II were required for the survival of quiescent cells in prolonged nutrient-starved conditions. This study reveals that these factors are critical for starvation responses.