scholarly journals Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events.

1994 ◽  
Vol 126 (6) ◽  
pp. 1341-1351 ◽  
Author(s):  
R Ishida ◽  
M Sato ◽  
T Narita ◽  
K R Utsumi ◽  
T Nishimoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Topoisomerase Ii ◽  
Chromosome Condensation ◽  
S Phase ◽  
Cdc2 Kinase ◽  
Torsional Strain ◽  
Chromosome Dynamics ◽  
M Phase ◽  
Topo Ii

ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes.

Moxifloxacin Enhances the Antiproliferative and Apoptotic Effects of VP-16 but Inhibits Its Proinflammatory Effects in THP-1 and Jurkat Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4290-4290
Author(s):  
Ina Fabian ◽  
Debby Haite ◽  
Avital Levitov ◽  
Drora Halperin ◽  
Itamar Shalit
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
S Phase ◽  
Jurkat Cells ◽  
Cycle Arrest ◽  
Topo Ii ◽  
Number Of Cells

Abstract We previously reported that the fluoroquinolone moxifloxacin (MXF) inhibits NF-kB, mitogen-activated protein kinase activation and the synthesis of proinflammatory cytokines in activated human monocytic cells (AAC48:1974,2004). Since MXF acts on topoisomerase II (Topo II) in mammalian cells, we investigated its effect in combination with another Topo II inhibitor, VP-16, on cell proliferation (by the MTT method), cell cycle, caspase-3 activity and proinflammatory cytokine release in THP-1 and Jurkat cells. THP-1 cells were incubated for 24 h with 0.5–3 μg/ml VP-16 in the presence or absence of 5–20 μg/ml MXF. VP-16 induced a dose dependent decrease in cell proliferation. An additional 2.5-and 1.6-fold decrease in cell proliferation was observed upon incubation of the cells with 0.5 or 1 μg/ml VP-16 and 20 μg/ml MXF, respectively (up to 69% inhibition). To further elucidate the mechanism of the antiproliferative activity of MXF, its effect on cell cycle progression was investigated. In control cultures 1%, 45%,18% and 36% of cells were in G0, G1, S and G2/M phases at 24 h, respectively. In contrast, in cultures treated with 1 μg/ml VP-16 and VP-16+ 20 μg/ml MXF, the number of cells in G1 decreased to 5.4 and 6.5%, respectively, while the number of cells in S phase increased to 25.5 and 42%, respectively and the number of cells in G2/M cells increased to 60 and 44%, respectively. These data provide evidence for S-G2/M cell cycle arrest induced by VP-16 and that addition of MXF shifted the S-G2/M arrest more towards the S phase. Since the antiproliferative effects of MXF could also be attributed to apoptotic cell death in addition to cell cycle arrest, we investigated the effect of the drugs on apoptosis. Using the fluorogenic assay for caspse-3 activity, we show that incubation of THP-1 cells for 6 h with 1.5 μg/ml VP-16 resulted in 630±120 unit/50μg protein of caspase-3 activity while the combination of 1.5 μg/ml VP-16 and 20 μg/ml MXF enhanced caspase-3 activity up to 1700±340 units/50μg protein (vs.233±107 in control cells), indicating that MXF synergises with VP-16 in activation of caspase-3. In Jurkat cells, the addition of 0.5 or 1 μg/ml VP-16, did not affect cell proliferation while in the presence of 20 μg/ml MXF and 1 μg/ml VP-16 there was a 62% decrease in cell proliferation (p<0.05). Exposure of Jurkat cells to 3 μg/ml VP-16 alone resulted in 504±114 units/50μg protein of caspase-3 activity and the addition of 20μg/ml MXF enhanced caspase-3 activity up to 1676± 259 units/50μg protein (vs 226±113 units/50μg protein in control cells). We further examined pro-inflammatory cytokine secretion upon stimulation of THP-1 cells with VP-16, MXF or their combination. VP-16 alone at 3 μg/ml increased IL-8 and TNF-α secretion from THP-1 cells by 2.5 and 1.8-fold respectively. Addition of MXF (5–20 μg/ml) inhibited the two cytokines secretion by 72–77% and 58–72%, respectively. The above combined data indicate that MXF, at clinically attainable concentrations, demonstrates pronounced synergistic effect with VP-16 as an anti-proliferative agent mainly by enhancing caspase-3 activity and apoptosis. At the same time MXF inhibits the pro-inflammatory effects conferred by VP-16 in the tumor cells studied. The clinical significance of the above anti-proliferative and anti-inflammatory effects of MXF in combination with VP-16 should be further investigated in animal models.

scholarly journals Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome

2001 ◽  
Vol 154 (2) ◽  
pp. 331-344 ◽  
Author(s):  
Daniel Kornitzer ◽  
Rakefet Sharf ◽  
Tamar Kleinberger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mammalian Cells ◽  
Growth Arrest ◽  
Anaphase Promoting Complex ◽  
Reading Frame ◽  
Cycle Growth ◽  
M Phase ◽  
Dependent Growth ◽  
Synthetically Lethal

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)–dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4–PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.

scholarly journals The Paramyxovirus Simian Virus 5 V Protein Slows Progression of the Cell Cycle

2000 ◽  
Vol 74 (19) ◽  
pp. 9152-9166 ◽  
Author(s):  
Grace Y. Lin ◽  
Robert A. Lamb
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Simian Virus ◽  
S Phase ◽  
V Protein ◽  
Simian Virus 5 ◽  
C Terminus ◽  
M Phase ◽  
Infected Cells ◽  
Affect Cell Cycle Progression

ABSTRACT Infection of cells by many viruses affects the cell division cycle of the host cell to favor viral replication. We examined the ability of the paramyxovirus simian parainfluenza virus 5 (SV5) to affect cell cycle progression, and we found that SV5 slows the rate of proliferation of HeLa T4 cells. The SV5-infected cells had a delayed transition from G1 to S phase and prolonged progression through S phase, and some of the infected cells were arrested in G2 or M phase. The levels of p53 and p21CIP1were not increased in SV5-infected cells compared to mock-infected cells, suggesting that the changes in the cell cycle occur through a p53-independent mechanism. However, the phosphorylation of the retinoblastoma protein (pRB) was delayed and prolonged in SV5-infected cells. The changes in the cell cycle were also observed in cells expressing the SV5 V protein but not in the cells expressing the SV5 P protein or the V protein lacking its unique C terminus (VΔC). The unique C terminus of the V protein of SV5 was shown previously to interact with DDB1, which is the 127-kDa subunit of the multifunctional damage-specific DNA-binding protein (DDB) heterodimer. The coexpression of DDB1 with V can partially restore the changes in the cell cycle caused by expression of the V protein.

Synthesis and anticancer evaluation of sulfur containing 9-anilinoacridines

2021 ◽  
Vol 16 ◽  
Author(s):  
Pranav Gupta ◽  
Radhika V. Kumar ◽  
Chul-Hoon Kwon ◽  
Zhe-Sheng Chen
Keyword(s):  
Cell Cycle ◽  
Anticancer Activity ◽  
Topoisomerase Ii ◽  
Critical Role ◽  
K562 Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
In Vivo Models ◽  
Topo Ii ◽  
Sulfur Containing

Background: DNA topoisomerases are a class of enzymes that play a critical role in fundamental biological processes of replication, transcription, recombination, repair and chromatin remodeling. Amsacrine (m-AMSA), the best-known compound of 9-anilinoacridines series was one of the first DNA-intercalating agents to be considered as a Topoisomerase II inhibitor. Objective: A series of sulfur containing 9-anilinoacridines related to amsacrine were synthesized and evaluated for their anticancer activity. Methods: Cell viability was assessed by the MTT assay. The topoisomerase II inhibitory assay was performed using the Human topoisomerase II Assay kit and flow cytometry was used to evaluate the effects on cell cycle of K562 cells. Molecular docking was performed using Schrödinger Maestro program. Results: Compound 36 was found to be the most cytotoxic of the sulfide series against SW620, K562, and MCF-7. The limited SAR suggested the importance of the methansulfonamidoacetamide side chain functionality, the lipophilicity and relative metabolic stability of 36 in contributing to the cytotoxicity. Topoisomerase II α inhibitory activity appeared to be involved in the cytotoxicity of 36 through inhibition of decatenation of kinetoplast DNA (kDNA) in a concentration dependent manner. Cell cycle analysis further showed the Topo II inhibition through accumulation of K562 cells in G2/M phase of cell cycle. Docking of 36 into the Topo II α-DNA complex suggested that it may be an allosteric inhibitor of Topo II α. Conclusion: Compound 36 exhibits anticancer activity by inhibiting topoisomerase II and it could further be evaluated in in vivo models.

scholarly journals Posttranslational Phosphorylation and Ubiquitination of the Saccharomyces cerevisiae Poly(A) Polymerase at the S/G2 Stage of the Cell Cycle

2000 ◽  
Vol 20 (8) ◽  
pp. 2794-2802 ◽  
Author(s):  
Neptune Mizrahi ◽  
Claire Moore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Sorting ◽  
S Phase ◽  
Phase Arrest ◽  
Phosphatase Treatment ◽  
M Phase ◽  
64 Kda Protein ◽  
Mitotic Phosphorylation ◽  
Cycle Regulation

ABSTRACT The poly(A) polymerase of the budding yeast Saccharomyces cerevisiae (Pap1) is a 64-kDa protein essential for the maturation of mRNA. We have found that a modified Pap1 of 90 kDa transiently appears in cells after release from α-factor-induced G1 arrest or from a hydroxyurea-induced S-phase arrest. While a small amount of modification occurs in hydroxyurea-arrested cells, fluorescence-activated cell sorting analysis and microscopic examination of bud formation indicate that the majority of modified enzyme is found at late S/G2 and disappears by the time cells have reached M phase. The reduction of the 90-kDa product upon phosphatase treatment indicates that the altered mobility is due to phosphorylation. A preparation containing primarily the phosphorylated Pap1 has no poly(A) addition activity, but this activity is restored by phosphatase treatment. A portion of Pap1 is also polyubiquitinated concurrent with phosphorylation. However, the bulk of the 64-kDa Pap1 is a stable protein with a half-life of 14 h. The timing, nature, and extent of Pap1 modification in comparison to the mitotic phosphorylation of mammalian poly(A) polymerase suggest an intriguing difference in the cell cycle regulation of this enzyme in yeast and mammalian systems.

scholarly journals Fate of the M-phase-assembled centrioles during the cell cycle in the TP53;PCNT;CEP215-deleted cells

2020 ◽  
Author(s):  
Gee In Jung ◽  
Kunsoo Rhee
Keyword(s):  
Cell Cycle ◽  
Cancer Cells ◽  
Cell Lines ◽  
S Phase ◽  
M Phase ◽  
Dividing Cells ◽  
Centriole Assembly

ABSTRACTCancer cells frequently include supernumerary centrioles. Here, we generated TP53;PCNT;CEP215 triple knockout cell lines and observed precocious separation and amplification of the centrioles at M phase. Many of the triple KO cells maintained supernumerary centrioles throughout the cell cycle. The M-phase-assembled centrioles lack an ability to function as templates for centriole assembly during S phase. They also lack an ability to organize microtubules in interphase. However, we found that a fraction of them acquired an ability to organize microtubules during M phase. Our works provide an example how supernumerary centrioles behave in dividing cells.

scholarly journals 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.

1996 ◽  
Vol 134 (5) ◽  
pp. 1097-1107 ◽  
Author(s):  
J B Rattner ◽  
M J Hendzel ◽  
C S Furbee ◽  
M T Muller ◽  
D P Bazett-Jones
Keyword(s):  
Cell Cycle ◽  
Topoisomerase Ii ◽  
Chromatin Condensation ◽  
Culture Cell ◽  
Centromeric Heterochromatin ◽  
Tissue Culture Cell ◽  
Topoisomerase Ii Alpha ◽  
A Cell ◽  
Topo Ii ◽  
Species Specific

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.

The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

1998 ◽  
Vol 111 (12) ◽  
pp. 1751-1757 ◽  
Author(s):  
A. Abrieu ◽  
T. Brassac ◽  
S. Galas ◽  
D. Fisher ◽  
J.C. Labbe ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin A ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
M Phase ◽  
Egg Extracts ◽  
Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells

1985 ◽  
Vol 5 (12) ◽  
pp. 3443-3450
Author(s):  
J M Leeds ◽  
M B Slabaugh ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Ribonucleotide Reductase ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Cell Activity ◽  
S Phase ◽  
Whole Cell ◽  
Ribonucleotide Reductase Activity

Nuclear and whole-cell deoxynucleoside triphosphate (dNTP) pools were measured in HeLa cells at different densities and throughout the cell cycle of synchronized CHO cells. Nuclei were prepared by brief detergent (Nonidet P-40) treatment of subconfluent monolayers, a procedure that solubilizes plasma membranes but leaves nuclei intact and attached to the plastic substratum. Electron microscopic examination of monolayers treated with Nonidet P-40 revealed protruding nuclei surrounded by cytoskeletal remnants. Control experiments showed that nuclear dNTP pool sizes were stable during the time required for isolation, suggesting that redistribution of nucleotides during the isolation procedure was minimal. Examination of HeLa whole-cell and nuclear dNTP levels revealed that the nuclear proportion of each dNTP was distinct and remained constant as cell density increased. In synchronized CHO cells, all four dNTP whole-cell pools increased during S phase, with the dCTP pool size increasing most dramatically. The nuclear dCTP pool did not increase as much as the whole-cell dCTP pool during S phase, lowering the relative nuclear dCTP pool. Although the whole-cell dNTP pools decreased after 30 h of isoleucine deprivation, nuclear pools did not decrease proportionately. In summary, nuclear dNTP pools in synchronized CHO cells maintained a relatively constant concentration throughout the cell cycle in the face of larger fluctuations in whole-cell dNTP pools. Ribonucleotide reductase activity was measured in CHO cells throughout the cell cycle, and although there was a 10-fold increase in whole-cell activity during S phase, we detected no reductase in nuclear preparations at any point in the cell cycle.

Cell cycle variations of dinucleoside polyphosphates in synchronized cultures of mammalian cells

1987 ◽  
Vol 7 (7) ◽  
pp. 2444-2450
Author(s):  
G Orfanoudakis ◽  
M Baltzinger ◽  
D Meyer ◽  
N Befort ◽  
J P Ebel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
S Phase ◽  
Serum Deprivation ◽  
Specific Cell ◽  
Culture Cells ◽  
Mouse Embryo Fibroblasts ◽  
Atp Pool ◽  
Dinucleoside Polyphosphates ◽  
Synchronized Cultures

Zajdela hepatoma culture cells (ZHC) and mouse embryo fibroblasts (Swiss 3T3) were synchronized in G1 or S phase by serum deprivation and aphidicolin treatment, respectively, to study the variations in adenylyl nucleotide (Ap4X) pool size during the progress of the cell cycle. Only minor variations, which never exceeded a factor of 2, were observed when the Ap4X concentrations were expressed on a cellular basis. The variations were found to be strictly parallel to the ATP variations. Upon release from an aphidicolin block, the minor variations of Ap4X followed DNA synthesis and preceded cytokinesis. When the nucleotide content was compared with the amount of proteins, the faint specific cell cycle changes were almost completely damped when the cells were synchronized by serum deprivation, but remained practically unchanged in the case of aphidicolin synchronization. These results suggest that the observed variations could reflect the accumulation of some nucleotides before cell division. It is not clear yet whether the variation in Ap4X concentration is significant by itself or is simply a phenomenon resulting from changes in the ATP pool.

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