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Kirsch-Volders M, Mišík M, Fenech M. Tetraploidy in normal tissues and diseases: mechanisms and consequences. Chromosoma 2025; 134:3. [PMID: 40117022 PMCID: PMC11928420 DOI: 10.1007/s00412-025-00829-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2024] [Revised: 03/11/2025] [Accepted: 03/13/2025] [Indexed: 03/23/2025]
Abstract
Tetraploidisation plays a crucial role in evolution, development, stress adaptation, and disease, but its beneficial or pathological effects in different tissues remain unclear. This study aims to compare physiological and unphysiological tetraploidy in eight steps: 1) mechanisms of diploidy-to-tetraploidy transition, 2) induction and elimination of unphysiological tetraploidy, 3) tetraploid cell characteristics, 4) stress-induced unphysiological tetraploidy, 5) comparison of physiological vs. unphysiological tetraploidy, 6) consequences of unphysiological stress-induced tetraploidy, 7) nutritional or pharmacological prevention strategies of tetraploidisation, and 8) knowledge gaps and future perspectives. Unphysiological tetraploidy is an adaptive stress response at a given threshold, often involving mitotic slippage. If tetraploid cells evade elimination through apoptosis or immune surveillance, they may re-enter the cell cycle, causing genetic instability, micronuclei formation, aneuploidy, modification of the epigenome and the development of diseases. The potential contributions of unphysiological tetraploidy to neurodegenerative, cardiovascular and diabetes related diseases are summarized in schematic figures and contrasted with its role in cancer development. The mechanisms responsible for the transition from physiological to unphysiological tetraploidy and the tolerance to tetraploidisation in unphysiological tetraploidy are not fully understood. Understanding these mechanisms is of critical importance to allow the development of targeted nutritional and pharmacological prevention strategies and therapies.
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Affiliation(s)
- Micheline Kirsch-Volders
- Laboratory for Cell Genetics, Department Biology, Faculty of Sciences and Bio-Engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium
| | - Miroslav Mišík
- Center for Cancer Research, Medical University of Vienna, Borschkegasse 8a, 1090, Vienna, Austria.
| | - Michael Fenech
- Genome Health Foundation, North Brighton, SA, 5048, Australia
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2
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Kirsch-Volders M, Fenech M. Aneuploidy, inflammation and diseases. Mutat Res 2022; 824:111777. [PMID: 35358789 DOI: 10.1016/j.mrfmmm.2022.111777] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2021] [Revised: 02/22/2022] [Accepted: 03/15/2022] [Indexed: 05/23/2023]
Abstract
This review discusses how numerical aneuploidy may trigger inflammation in somatic cells and its consequences. Therefore we: i) summarized current knowledge on the cellular and molecular pathological effects of aneuploidy; ii) considered which of these aspects are able to trigger inflammation; iii) determined the genetic and environmental factors which may modulate the link between aneuploidy and inflammation; iv) explored the rôle of diet in prevention of aneuploidy and inflammation; v) examined whether aneuploidy and inflammation are causes and/or consequences of diseases; vi) identified the knowledge gaps and research needed to translate these observations into improved health care and disease prevention. The relationships between aneuploidy, inflammation and diseases are complex, because they depend on which chromosomes are involved, the proportion of cells affected and which organs are aneuploid in the case of mosaic aneuploidy. Therefore, a systemic approach is recommended to understand the emergence of aneuploidy-driven diseases and to take preventive measures to protect individuals from exposure to aneugenic conditions.
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Affiliation(s)
- Micheline Kirsch-Volders
- Laboratory for Cell Genetics, Department Biology, Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
| | - Michael Fenech
- Genome Health Foundation, North Brighton, SA 5048, Australia; Clinical and Health Sciences, University of South Australia, SA 5000, Australia.
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3
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Hong FU, Castro M, Linse K. Tumor-specific lytic path “hyperploid progression mediated death”: Resolving side effects through targeting retinoblastoma or p53 mutant. World J Clin Oncol 2020; 11:854-867. [PMID: 33312882 PMCID: PMC7701912 DOI: 10.5306/wjco.v11.i11.854] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Revised: 10/08/2020] [Accepted: 10/20/2020] [Indexed: 02/06/2023] Open
Abstract
A major advance was made to reduce the side effects of cancer therapy via the elucidation of the tumor-specific lytic path “hyperploid progression-mediated death” targeting retinoblastoma (Rb) or p53-mutants defective in G1 DNA damage checkpoint. The genetic basis of human cancers was uncovered through the cloning of the tumor suppressor Rb gene. It encodes a nuclear DNA-binding protein whose self-interaction is regulated by cyclin-dependent kinases. A 3D-structure of Rb dimer is shown, confirming its multimeric status. Rb assumes a central role in cell cycle regulation and the “Rb pathway” is universally inactivated in human cancers. Hyperploidy refers to a state in which cells contain one or more extra chromosomes. Hyperploid progression occurs due to continued cell-cycling without cytokinesis in G1 checkpoint-defective cancer cells. The evidence for the triggering of hyperploid progression-mediated death in RB-mutant human retinoblastoma cells is shown. Hence, the very genetic mutation that predisposes to cancer can be exploited to induce lethality. The discovery helped to establish the principle of targeted cytotoxic cancer therapy at the mechanistic level. By triggering the lytic path, targeted therapy with tumor specificity at the genetic level can be developed. It sets the stage for systematically eliminating side effects for cytotoxic cancer therapy.
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Affiliation(s)
- Frank-Un Hong
- Department of Research and Development, Bio-Synthesis, Lewisville, TX 75057, United States
| | - Miguel Castro
- Department of Research and Development, Bio-Synthesis, Lewisville, TX 75057, United States
| | - Klaus Linse
- Department of Research and Development, Bio-Synthesis, Lewisville, TX 75057, United States
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Kirsch-Volders M, Pacchierotti F, Parry EM, Russo A, Eichenlaub-Ritter U, Adler ID. Risks of aneuploidy induction from chemical exposure: Twenty years of collaborative research in Europe from basic science to regulatory implications. MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH 2018; 779:126-147. [PMID: 31097149 DOI: 10.1016/j.mrrev.2018.11.002] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 11/26/2018] [Indexed: 12/13/2022]
Abstract
Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980's until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.
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Affiliation(s)
- Micheline Kirsch-Volders
- Laboratory for Cell Genetics, Faculty of Sciences and Bioengineering, Vrije Universiteit Brussel, Brussels, Belgium.
| | | | | | - Antonella Russo
- Department of Molecular Medicine, University of Padova, Padova, Italy
| | - Ursula Eichenlaub-Ritter
- Institute of Gene Technology/Microbiology, Faculty of Biology, University of Bielefeld, Bielefeld, Germany
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5
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Brown A, Geiger H. Chromosome integrity checkpoints in stem and progenitor cells: transitions upon differentiation, pathogenesis, and aging. Cell Mol Life Sci 2018; 75:3771-3779. [PMID: 30066086 PMCID: PMC6154040 DOI: 10.1007/s00018-018-2891-z] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Revised: 07/22/2018] [Accepted: 07/25/2018] [Indexed: 01/30/2023]
Abstract
Loss of chromosome integrity is a major contributor to cancer. Checkpoints within the cell division cycle that facilitate the accuracy and outcome of chromosome segregation are thus critical pathways for preserving chromosome integrity and preventing chromosomal instability. The spindle assembly checkpoint, the decatenation checkpoint and the post-mitotic tetraploidy checkpoint ensure the appropriate establishment of the spindle apparatus, block mitotic entry upon entanglement of chromosomes or prevent further progression of post-mitotic cells that display massive spindle defects. Most of our knowledge on these mechanisms originates from studies conducted in yeast, cancer cell lines and differentiated cells. Considering that in many instances cancer derives from transformed stem and progenitor cells, our knowledge on these checkpoints in these cells just started to emerge. With this review, we provide a general overview of the current knowledge of these checkpoints in embryonic as well as in adult stem and progenitor cells with a focus on the hematopoietic system and outline common mis-regulations of their function associated with cancer and leukemia. Most cancers are aging-associated diseases. We will thus also discuss changes in the function and outcome of these checkpoints upon aging of stem and progenitor cells.
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Affiliation(s)
- Andreas Brown
- Institute of Molecular Medicine, Ulm University, Life Science Building N27, James Franck-Ring/Meyerhofstrasse, 89081, Ulm, Germany
| | - Hartmut Geiger
- Institute of Molecular Medicine, Ulm University, Life Science Building N27, James Franck-Ring/Meyerhofstrasse, 89081, Ulm, Germany.
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH, 45229, USA.
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6
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Bernal A, Zafon E, Domínguez D, Bertran E, Tusell L. Generation of Immortalised But Unstable Cells after hTERT Introduction in Telomere-Compromised and p53-Deficient vHMECs. Int J Mol Sci 2018; 19:ijms19072078. [PMID: 30018248 PMCID: PMC6073565 DOI: 10.3390/ijms19072078] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2018] [Revised: 07/11/2018] [Accepted: 07/13/2018] [Indexed: 01/08/2023] Open
Abstract
Telomeres, the natural ends of chromosomes, hide the linear telomeric DNA from constitutive exposure to the DNA damage response with a lariat structure or t-loop. Progressive telomere shortening associated with DNA replication in the absence of a compensatory mechanism culminates in t-loop collapse and unmasked telomeres. Dysfunctional telomeres can suppress cancer development by engaging replicative senescence or apoptosis, but they can also promote tumour initiation when cell cycle checkpoints are disabled. In this setting, telomere dysfunction promotes increasing chromosome instability (CIN) through breakage-fusion-bridge cycles. Excessive instability may hamper cell proliferation but might allow for the appearance of some rare advantageous mutations that could be selected and ultimately favour neoplastic progression. With the aim of generating pre-malignant immortalised cells, we ectopically expressed telomerase in telomere-compromised variant human mammary epithelial cells (vHMECs), proficient and deficient for p53, and analysed structural and numerical chromosomal aberrations as well as abnormal nuclear morphologies. Importantly, this study provides evidence that while immortalisation of vHMECs at early stages results in an almost stable karyotype, a transient telomere-dependent CIN period—aggravated by p53 deficiency—and followed by hTERT overexpression serves as a mechanism for the generation of immortal unstable cells which, due to their evolving karyotype, could attain additional promoting properties permissive to malignancy.
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Affiliation(s)
- Aina Bernal
- Unitat de Biologia Cel·lular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain.
| | - Elisenda Zafon
- Unitat de Biologia Cel·lular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain.
| | - Daniel Domínguez
- Unitat de Biologia Cel·lular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain.
| | - Enric Bertran
- Unitat de Biologia Cel·lular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain.
| | - Laura Tusell
- Unitat de Biologia Cel·lular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain.
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7
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Vassilev A, Lee CY, Vassilev B, Zhu W, Ormanoglu P, Martin SE, DePamphilis ML. Identification of genes that are essential to restrict genome duplication to once per cell division. Oncotarget 2018; 7:34956-76. [PMID: 27144335 PMCID: PMC5085202 DOI: 10.18632/oncotarget.9008] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Accepted: 04/07/2016] [Indexed: 12/02/2022] Open
Abstract
Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. All of them participate in one or more of eight cell cycle events. Seventeen of them have not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers.
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Affiliation(s)
- Alex Vassilev
- National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2753, USA
| | - Chrissie Y Lee
- National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2753, USA.,Current address: NantBioscience, Culver City, CA 90232, USA
| | - Boris Vassilev
- National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2753, USA
| | - Wenge Zhu
- Department of Biochemistry and Molecular Biology, George Washington University, Washington DC 20037, USA
| | - Pinar Ormanoglu
- National Center of Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA
| | - Scott E Martin
- National Center of Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.,Current Address: Genentech, Inc., South San Francisco, CA 94080, USA
| | - Melvin L DePamphilis
- National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2753, USA
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8
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Potapova T, Gorbsky GJ. The Consequences of Chromosome Segregation Errors in Mitosis and Meiosis. BIOLOGY 2017; 6:biology6010012. [PMID: 28208750 PMCID: PMC5372005 DOI: 10.3390/biology6010012] [Citation(s) in RCA: 108] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/10/2016] [Revised: 01/24/2017] [Accepted: 01/26/2017] [Indexed: 12/21/2022]
Abstract
Mistakes during cell division frequently generate changes in chromosome content, producing aneuploid or polyploid progeny cells. Polyploid cells may then undergo abnormal division to generate aneuploid cells. Chromosome segregation errors may also involve fragments of whole chromosomes. A major consequence of segregation defects is change in the relative dosage of products from genes located on the missegregated chromosomes. Abnormal expression of transcriptional regulators can also impact genes on the properly segregated chromosomes. The consequences of these perturbations in gene expression depend on the specific chromosomes affected and on the interplay of the aneuploid phenotype with the environment. Most often, these novel chromosome distributions are detrimental to the health and survival of the organism. However, in a changed environment, alterations in gene copy number may generate a more highly adapted phenotype. Chromosome segregation errors also have important implications in human health. They may promote drug resistance in pathogenic microorganisms. In cancer cells, they are a source for genetic and phenotypic variability that may select for populations with increased malignance and resistance to therapy. Lastly, chromosome segregation errors during gamete formation in meiosis are a primary cause of human birth defects and infertility. This review describes the consequences of mitotic and meiotic errors focusing on novel concepts and human health.
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Affiliation(s)
- Tamara Potapova
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
| | - Gary J Gorbsky
- Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
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9
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Links between DNA Replication, Stem Cells and Cancer. Genes (Basel) 2017; 8:genes8020045. [PMID: 28125050 PMCID: PMC5333035 DOI: 10.3390/genes8020045] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2016] [Revised: 01/02/2017] [Accepted: 01/12/2017] [Indexed: 12/31/2022] Open
Abstract
Cancers can be categorized into two groups: those whose frequency increases with age, and those resulting from errors during mammalian development. The first group is linked to DNA replication through the accumulation of genetic mutations that occur during proliferation of developmentally acquired stem cells that give rise to and maintain tissues and organs. These mutations, which result from DNA replication errors as well as environmental insults, fall into two categories; cancer driver mutations that initiate carcinogenesis and genome destabilizing mutations that promote aneuploidy through excess genome duplication and chromatid missegregation. Increased genome instability results in accelerated clonal evolution leading to the appearance of more aggressive clones with increased drug resistance. The second group of cancers, termed germ cell neoplasia, results from the mislocation of pluripotent stem cells during early development. During normal development, pluripotent stem cells that originate in early embryos give rise to all of the cell lineages in the embryo and adult, but when they mislocate to ectopic sites, they produce tumors. Remarkably, pluripotent stem cells, like many cancer cells, depend on the Geminin protein to prevent excess DNA replication from triggering DNA damage-dependent apoptosis. This link between the control of DNA replication during early development and germ cell neoplasia reveals Geminin as a potential chemotherapeutic target in the eradication of cancer progenitor cells.
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10
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De Santis Puzzonia M, Gonzalez L, Ascenzi S, Cundari E, Degrassi F. Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication. Cell Cycle 2016; 15:274-82. [PMID: 26693937 DOI: 10.1080/15384101.2015.1127469] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
Abstract
Tetraploidy has been proposed as an intermediate state in neoplastic transformation due to the intrinsic chromosome instability of tetraploid cells. Despite the identification of p53 as a major factor in growth arrest of tetraploid cells, it is still unclear whether the p53-dependent mechanism for proliferation restriction is intrinsic to the tetraploid status or dependent on the origin of tetraploidy. Substrate adherence is fundamental for cytokinesis completion in adherent untransformed cells. Here we show that untransformed fibroblast cells undergoing mitosis in suspension produce binucleated tetraploid cells due to defective cleavage furrow constriction that leads to incomplete cell abscission. Binucleated cells obtained after loss of substrate adhesion maintain an inactive p53 status and are able to progress into G1 and S phase. However, binucleated cells arrest in G2, accumulate p53 and are not able to enter mitosis as no tetraploid metaphases were recorded after one cell cycle time. In contrast, tetraploid metaphases were found following pharmacological inhibition of Chk1 kinase, suggesting the involvement of the ATR/Chk1 pathway in the G2 arrest of binucleated cells. Interestingly, after persistence in the G2 phase of the cell cycle, a large fraction of binucleated cells become senescent. These findings identify a new pathway of proliferation restriction for tetraploid untransformed cells that seems to be specific for loss of adhesion-dependent cytokinesis failure. This involves Chk1 and p53 activation during G2. Inhibition of growth and entrance into senescence after cytokinesis in suspension may represent an important mechanism to control tumor growth. In fact, anchorage independent growth is a hallmark of cancer and it has been demonstrated that binucleated transformed cells can enter a cycle of anchorage independent growth.
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Affiliation(s)
- Marco De Santis Puzzonia
- a Institute of Molecular Biology and Pathology, CNR National Research Council , Rome , Italy.,b Sapienza University , Department of Cellular Biotechnology and Hematology , Rome , Italy
| | - Laetitia Gonzalez
- c Vrije Universiteit Brussel, Laboratory of Cell Genetics , Brussels , Belgium
| | - Sonia Ascenzi
- a Institute of Molecular Biology and Pathology, CNR National Research Council , Rome , Italy
| | - Enrico Cundari
- a Institute of Molecular Biology and Pathology, CNR National Research Council , Rome , Italy
| | - Francesca Degrassi
- a Institute of Molecular Biology and Pathology, CNR National Research Council , Rome , Italy
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11
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Potapova TA, Seidel CW, Box AC, Rancati G, Li R. Transcriptome analysis of tetraploid cells identifies cyclin D2 as a facilitator of adaptation to genome doubling in the presence of p53. Mol Biol Cell 2016; 27:3065-3084. [PMID: 27559130 PMCID: PMC5063615 DOI: 10.1091/mbc.e16-05-0268] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Accepted: 08/16/2016] [Indexed: 01/12/2023] Open
Abstract
Tetraploidization, or genome doubling, is a prominent event in tumorigenesis, primarily because cell division in polyploid cells is error-prone and produces aneuploid cells. This study investigates changes in gene expression evoked in acute and adapted tetraploid cells and their effect on cell-cycle progression. Acute polyploidy was generated by knockdown of the essential regulator of cytokinesis anillin, which resulted in cytokinesis failure and formation of binucleate cells, or by chemical inhibition of Aurora kinases, causing abnormal mitotic exit with formation of single cells with aberrant nuclear morphology. Transcriptome analysis of these acute tetraploid cells revealed common signatures of activation of the tumor-suppressor protein p53. Suppression of proliferation in these cells was dependent on p53 and its transcriptional target, CDK inhibitor p21. Rare proliferating tetraploid cells can emerge from acute polyploid populations. Gene expression analysis of single cell-derived, adapted tetraploid clones showed up-regulation of several p53 target genes and cyclin D2, the activator of CDK4/6/2. Overexpression of cyclin D2 in diploid cells strongly potentiated the ability to proliferate with increased DNA content despite the presence of functional p53. These results indicate that p53-mediated suppression of proliferation of polyploid cells can be averted by increased levels of oncogenes such as cyclin D2, elucidating a possible route for tetraploidy-mediated genomic instability in carcinogenesis.
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Affiliation(s)
| | | | - Andrew C Box
- Stowers Institute for Medical Research, Kansas City, MO 64110
| | - Giulia Rancati
- Institute of Medical Biology, Agency for Science, Technology and Research, Singapore 138648, Singapore
| | - Rong Li
- Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205
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12
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Huang HL, Chao MW, Chen CC, Cheng CC, Chen MC, Lin CF, Liou JP, Teng CM, Pan SL. LTP-1, a novel antimitotic agent and Stat3 inhibitor, inhibits human pancreatic carcinomas in vitro and in vivo. Sci Rep 2016; 6:27794. [PMID: 27278358 PMCID: PMC4899784 DOI: 10.1038/srep27794] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2016] [Accepted: 05/23/2016] [Indexed: 01/05/2023] Open
Abstract
Pancreatic cancer is the leading cause of cancer death worldwide with a poor survival rate. The objective of this study was to determine the mechanism of action of a novel antimitotic and Stat3 inhibitor, LTP-1, on human pancreatic cancer in vitro and in vivo. We found that LTP-1 inhibited pancreatic cancer cell growth and viability with significant G2/M arrest and disruption of microtubule dynamics. LTP-1 also caused G2/M arrest-independent Stat3 dephosphorylation along with ERK activation, which indicated the possible dual function of LTP-1. Long-term treatment of LTP-1 also induced polyploidy, activated caspases, induced subG1 cell population, and therefore, triggered pancreatic cancer cell apoptosis. Finally, we used an in vivo xenograft model to demonstrate that LTP-1 suppressed the growth of pancreatic adenocarcinoma. In summary, our data suggest that LTP-1 may alter microtubule dynamics, which ultimately causes polyploidy and apoptosis, thereby inhibiting pancreatic cancer growth in vitro and in vivo. This study provides evidence that LTP-1 could be a potential therapeutic agent for further development of pancreatic cancer treatment.
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Affiliation(s)
- Han-Li Huang
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Min-Wu Chao
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Chung-Chun Chen
- Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Chun-Chun Cheng
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Mei-Chuan Chen
- Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
- Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Chao-Feng Lin
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Department of Internal Medicine, Division of Cardiology, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan
| | - Jing-Ping Liou
- School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Che-Ming Teng
- Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Shiow-Lin Pan
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan
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13
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Li P, Zhou L, Liu X, Jin X, Zhao T, Ye F, Liu X, Hirayama R, Li Q. Mitotic DNA damages induced by carbon-ion radiation incur additional chromosomal breaks in polyploidy. Toxicol Lett 2014; 230:36-47. [PMID: 25123929 DOI: 10.1016/j.toxlet.2014.08.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2014] [Revised: 08/07/2014] [Accepted: 08/08/2014] [Indexed: 12/23/2022]
Abstract
Compared with low linear energy transfer (LET) radiation, carbon-ion radiation has been proved to induce high frequency of more complex DNA damages, including DNA double strands (DSBs) and non-DSB clustered DNA lesions. Chemotherapeutic drug doxorubicin has been reported to elicit additional H2AX phosphorylation in polyploidy. Here, we investigated whether mitotic DNA damage induced by high-LET carbon-ion radiation could play the same role. We demonstrate that impairment of post-mitotic G1 and S arrest and abrogation of post-mitotic G2-M checkpoint failed to prevent mis-replication of damaged DNA and mis-separation of chromosomes. Meanwhile, mitotic slippage only nocodazole-related, cytokinesis failure and cell fusion collectively contributed to the formation of binucleated cells. Chk1 and Cdh1 activation was inhibited when polyploidy emerged in force, both of which are critical components for mitotic exit and cytokinesis. Carbon-ion radiation irrelevant of nocodazole incurred additional DNA breaks in polyploidy, manifesting as structural and numerical karyotype changes. The proliferation of cells given pre-synchronization and radiation was completely inhibited and cells were intensely apoptotic. Since increased chromosomal damage resulted in extensive H2AX phosphorylation during polyploidy, we propose that the additional γ-H2AX during polyploidy incurred by carbon-ion radiation provides a final opportunity for these dangerous and chromosomally unstable cells to be eliminated.
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Affiliation(s)
- Ping Li
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR China
| | - Libin Zhou
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China
| | - Xiongxiong Liu
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR China
| | - Xiaodong Jin
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR China
| | - Ting Zhao
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR China
| | - Fei Ye
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR China
| | - Xinguo Liu
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR China
| | - Ryoichi Hirayama
- Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba 263-8555, Japan
| | - Qiang Li
- Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, PR China; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR China.
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14
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Yamamoto M, Wakata A, Aoki Y, Miyamae Y, Kodama S. Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid. Mutat Res 2014; 762:10-16. [PMID: 24582839 DOI: 10.1016/j.mrfmmm.2014.02.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2013] [Revised: 12/19/2013] [Accepted: 02/18/2014] [Indexed: 06/03/2023]
Abstract
Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen). We analyzed the number of fluorescence in situ hybridization (FISH) signals targeted for a whole chromosome 2 in cytokinesis-blocked binucleated TK6 cells and WTK-1 cells treated with colcemid and MMS, and concurrently detected DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results revealed that DNA fragmentation occurred in 60% of all binucleated TK6 cells harboring colcemid-induced chromosome loss (30% of micronuclei and 30% of main nuclei). DNA fragmentation was observed in colcemid-induced micronuclei containing a whole chromosome but not in MMS-induced micronuclei containing chromosome fragments. In contrast, colcemid-induced nondisjunction had no effect on induction of DNA fragmentation, suggesting that DNA fragmentation was triggered by micronuclei containing a whole chromosome but not by micronuclei containing chromosome fragments or nondisjunction. In addition, the frequency of binucleated cells harboring chromosome loss with DNA fragmentation in micronuclei or main nuclei was higher in wild-type p53 TK6 cells than in mutated-p53 WTK-1 cells treated with colcemid. Taken together, these present and previous results suggest that colcemid-induced chromosome loss is caused by DNA fragmentation, which is triggered by a micronucleus with a whole chromosome and controlled by the p53-dependent pathway.
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Affiliation(s)
- Mika Yamamoto
- Drug Safety Research Laboratories, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan; Laboratory of Radiation Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan
| | - Akihiro Wakata
- Drug Safety Research Laboratories, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
| | - Yoshinobu Aoki
- Drug Safety Research Laboratories, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
| | - Yoichi Miyamae
- Drug Safety Research Laboratories, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
| | - Seiji Kodama
- Laboratory of Radiation Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan.
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15
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Novorol C, Burkhardt J, Wood KJ, Iqbal A, Roque C, Coutts N, Almeida AD, He J, Wilkinson CJ, Harris WA. Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression. Open Biol 2013; 3:130065. [PMID: 24153002 PMCID: PMC3814721 DOI: 10.1098/rsob.130065] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation. Nine genes are currently known to be associated with the condition, all of which encode centrosomal or spindle pole proteins. MCPH is associated with a reduction in proliferation of neural progenitors during fetal development. The cellular mechanisms underlying the proliferation defect, however, are not fully understood. The zebrafish retinal neuroepithelium provides an ideal system to investigate this question. Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size. Imaging studies reveal a dramatic rise in the fraction of proliferating cells in mitosis in all cases, and time-lapse microscopy points to a failure of progression through prometaphase. There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones.
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Affiliation(s)
- Claire Novorol
- Department of Physiology, Development and Neuroscience, Cambridge University, Cambridge CB2 3DY, UK
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16
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Yamamoto M, Wakata A, Aoki Y, Miyamae Y, Kodama S. Induction of a whole chromosome loss by colcemid in human cells elucidated by discrimination between FISH signal overlap and chromosome loss. Mutat Res 2013; 749:39-48. [PMID: 23792192 DOI: 10.1016/j.mrfmmm.2013.06.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2013] [Revised: 04/24/2013] [Accepted: 06/07/2013] [Indexed: 06/02/2023]
Abstract
Aneuploidy is a change in the number of chromosomes and an essential component in tumorigenesis. Therefore, accurate and sensitive detection of aneuploidy is important in screening for carcinogens. In vitro micronucleus (MN) assay has been adopted in the recently revised International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2 guideline and can be employed to predict both clastogenic and aneugenic chromosomal aberrations in interphase cells. However, distinguishing clastogens and aneugens is not possible using this assay. The Organization for Economic Co-operation and Development (OECD) guideline TG487 therefore recommends the use of centromere/kinetochore staining in micronuclei to differentiate clastogens from aneugens. Here, we analyzed numerical changes of a specific chromosome in cytokinesis-blocked binucleated cells by fluorescence in situ hybridization (FISH) using the specific centromere probe in human lymphoblastoid TK6 cells treated with aneugens (colcemid and vincristine) or clastogens (methyl methanesulfonate [MMS] and 4-nitroquinoline-1-oxide [4-NQO]). Colcemid and vincristine significantly increased the frequencies of nondisjunction and loss of FISH signals, while MMS and 4-NQO slightly increased only the frequency of loss of FISH signals. The loss of FISH signals of a specific chromosome from two to one per nucleus implies either a loss of a whole chromosome or an overlap of two signals. To distinguish a chromosome loss from signal overlap, we investigated the number of FISH signals and the fluorescent intensity of each signal per nucleus using a probe specific for whole chromosome 2 in binucleated TK6 cells and primary human lymphocytes treated with colcemid and MMS. By discriminating between chromosome loss and FISH signal overlap, we revealed that colcemid, but not MMS, induced a loss of a whole chromosome in primary lymphocytes and TK6 cells.
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MESH Headings
- Aneugens/pharmacology
- Aneuploidy
- Cells, Cultured
- Chromosome Aberrations/chemically induced
- Chromosome Segregation/drug effects
- Chromosomes, Human, Pair 2/drug effects
- Chromosomes, Human, Pair 2/genetics
- Chromosomes, Human, Pair 4/drug effects
- Chromosomes, Human, Pair 4/genetics
- Demecolcine/pharmacology
- Humans
- In Situ Hybridization, Fluorescence
- Lymphocytes/drug effects
- Micronucleus Tests
- Mutagens/pharmacology
- Reproducibility of Results
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Affiliation(s)
- Mika Yamamoto
- Drug Safety Research Laboratories, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan; Laboratory of Radiation Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan
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17
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Hashimoto K, Nakajima Y, Uematsu R, Chatani F. Difference in susceptibility to morphological changes in the nucleus to aneugens between p53-competent and p53-abrogated lymphoblastoid cell lines (TK6 and NH32 cells) in the in vitro micronucleus assay. Mutagenesis 2011; 27:287-93. [DOI: 10.1093/mutage/ger074] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
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18
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p53: guardian of ploidy. Mol Oncol 2011; 5:315-23. [PMID: 21852209 DOI: 10.1016/j.molonc.2011.07.007] [Citation(s) in RCA: 150] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2011] [Revised: 07/21/2011] [Accepted: 07/21/2011] [Indexed: 11/20/2022] Open
Abstract
Aneuploidy, often preceded by tetraploidy, is one of the hallmarks of solid tumors. Indeed, both aneuploidy and tetraploidy are oncogenic occurrences that are sufficient to drive neoplastic transformation and cancer progression. True to form, the tumor suppressor p53 obstructs propagation of these dangerous chromosomal events by either instigating irreversible cell cycle arrest or apoptosis. The tumor suppressor Lats2, along with other tumor inhibitory proteins such as BRCA1/2 and BubR1, are central to p53-dependent elimination of tetraploid cells. Not surprisingly, these proteins are frequently inactivated or downregulated in tumors, synergizing with p53 inactivation to establish an atmosphere of "tolerance" for a non-diploid state.
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19
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Androutsopoulos VP, Ruparelia KC, Papakyriakou A, Filippakis H, Tsatsakis AM, Spandidos DA. Anticancer effects of the metabolic products of the resveratrol analogue, DMU-212: structural requirements for potency. Eur J Med Chem 2011; 46:2586-95. [PMID: 21497957 DOI: 10.1016/j.ejmech.2011.03.049] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2010] [Revised: 03/17/2011] [Accepted: 03/22/2011] [Indexed: 01/17/2023]
Abstract
The methoxylated trans-stilbene resveratrol analogue, (E)-3,4,5,4'-tetramethoxystilbene (1), has shown promising antiproliferative activity in in vitro cell line and in vivo models. In vivo 1 gives rise to several metabolic products through demethylation or hydroxylation reactions at the stilbene moiety. In the present study we examined the anticancer activity of 1 and the metabolites (E)-3'-hydroxy-3,4,5,4'-tetramethoxystilbene (2), (E)-4'-hydroxy-3,4,5-trimethoxystilbene (3), (E)-4-hydroxy-3,5,4'-trimethoxystilbene (4) and (E)-3-hydroxy-4,5,4'-trimethoxystilbene (5) by means of cell viability testing, cell cycle analysis, immunostaining and Western blotting. Compounds 1 and 2 exhibited submicromolar toxicity in MCF-7 breast adenocarcinoma and HepG2 hepatoma cells, whereas 3, 4 and 5 were inactive in terms of inhibition of cellular proliferation. Incubation with 1 or 2 at 10 μM for 24h induced apoptosis and G2/M cell cycle arrest in MCF-7 and HepG2 cells. Immunostaining of MCF-7 cells for β-tubulin in the presence of either 1 or 2 revealed shorter localization of the protein around the nucleus, as compared to control cells. Western blot analyses further demonstrated that treatment with either 1 or 2 at concentrations between 30 and 50 μM for 24 h caused a downregulation in the levels of β-tubulin and cyclin D1 expression and an upregulation in the levels of p53 expression in MCF-7 and HepG2 cells. 2 further increased the ratio of mRNA levels of the apoptosis-related genes Bax/Bcl-xL in both MCF-7 and HepG2 cells in a dose-dependent manner. We conclude that 2 inhibits HepG2 and MCF-7 cellular proliferation by inducing apoptosis and G2/M arrest through p53 and Bax/Bcl-xL upregulation. Our findings further demonstrate that trimethoxy substitutions along with the presence of a methoxy group at position 4' are necessary for retaining the activity of 1.
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Affiliation(s)
- Vasilis P Androutsopoulos
- Laboratory of Clinical Virology, University of Crete, Medical School, Voutes, 71003 Heraklion, Crete, Greece.
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20
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A non-genetic route to aneuploidy in human cancers. Nat Cell Biol 2011; 13:324-30. [PMID: 21336303 DOI: 10.1038/ncb2174] [Citation(s) in RCA: 137] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2010] [Accepted: 12/17/2010] [Indexed: 12/18/2022]
Abstract
Aneuploidy is common in human tumours and is often indicative of aggressive disease. Aneuploidy can result from cytokinesis failure, which produces binucleate cells that generate aneuploid offspring with subsequent divisions. In cancers, disruption of cytokinesis is known to result from genetic perturbations to mitotic pathways or checkpoints. Here we describe a non-genetic mechanism of cytokinesis failure that occurs as a direct result of cell-in-cell formation by entosis. Live cells internalized by entosis, which can persist through the cell cycle of host cells, disrupt formation of the contractile ring during host cell division. As a result, cytokinesis frequently fails, generating binucleate cells that produce aneuploid cell lineages. In human breast tumours, multinucleation is associated with cell-in-cell structures. These data define a previously unknown mechanism of cytokinesis failure and aneuploid cell formation that operates in human cancers.
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21
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Zhu W, Lee CY, Johnson RL, Wichterman J, Huang R, DePamphilis ML. An image-based, high-throughput screening assay for molecules that induce excess DNA replication in human cancer cells. Mol Cancer Res 2011; 9:294-310. [PMID: 21257818 DOI: 10.1158/1541-7786.mcr-10-0570] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer.
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Affiliation(s)
- Wenge Zhu
- National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892-2753, USA
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22
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Abstract
Aurora kinases are serine and threonine kinases that function as key regulators of the mitosis process. There are three distinct human aurora kinases known as Aurora A, Aurora B, and Aurora C. Aurora A and Aurora B are overexpressed in a number of human cancers including non-small cell lung cancer, glioblastomas, and upper gastrointestinal adenocarcinomas. Given their association with tumorigenesis, both Aurora A and Aurora B have been targeted for cancer therapy. Currently, a number of selective and nonselective aurora kinase inhibitors are being tested in preclinical and clinical settings as anti-tumor agents. We review the biology of human aurora kinases, followed by an overview of inhibitors undergoing current clinical investigations.
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23
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Radiation-induced cell death mechanisms. Tumour Biol 2010; 31:363-72. [PMID: 20490962 DOI: 10.1007/s13277-010-0042-8] [Citation(s) in RCA: 466] [Impact Index Per Article: 31.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2010] [Accepted: 04/18/2010] [Indexed: 12/31/2022] Open
Abstract
The main goal when treating malignancies with radiation therapy is to deprive tumor cells of their reproductive potential. One approach to achieve this is by inducing tumor cell apoptosis. Accumulating evidences suggest that induction of apoptosis alone is insufficient to account for the therapeutic effect of radiotherapy. It has become obvious in the last few years that inhibition of the proliferative capacity of malignant cells following irradiation, especially with solid tumors, can occur via alternative cell death modalities or permanent cell cycle arrests, i.e., senescence. In this review, apoptosis and mitotic catastrophe, the two major cell deaths induced by radiation, are described and dissected in terms of activating mechanisms. Furthermore, treatment-induced senescence and its relevance for the outcome of radiotherapy of cancer will be discussed. The importance of p53 for the induction and execution of these different types of cell deaths is highlighted. The efficiency of radiotherapy and radioimmunotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation. Strategies to use specific inhibitors that will manipulate key molecules in these pathways in combination with radiation might potentiate therapy and enhance tumor cell kill.
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24
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Postactivation treatment with nocodazole maintains normal nuclear ploidy of cloned pig embryos by increasing nuclear retention and formation of single pronucleus. Theriogenology 2010; 73:429-36. [DOI: 10.1016/j.theriogenology.2009.09.026] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2009] [Revised: 08/04/2009] [Accepted: 09/30/2009] [Indexed: 11/22/2022]
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25
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Dupla D, Fraczek M, Woźniak Z, Krecicki T. [Relation between epidermal growth factor receptor (EGFR) and p53 expression and radiocurability of laryngeal squamous cell cancer]. Otolaryngol Pol 2009; 63:249-55. [PMID: 19886531 DOI: 10.1016/s0030-6657(09)70117-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
UNLABELLED Radiotherapy and surgery are the most important treatment modalities for the majority of laryngeal cancers. Because of high efficacy and better organ preservation radiotherapy is generally preferred for early and intermediate stage of the disease. Some of patients with more locally advanced cancers can still be cured by means of radiotherapy, but we have not got reliable prognostics factors for predicting radiocurability. THE AIM OF MY STUDY: was to investigate the value of p53 and EGFR expression for predicting clinical outcomes of laryngeal cancer patients treated with radiotherapy. METHODS AND MATERIALS The study included 50 patients with laryngeal cancer treated in Department of Radiotherapy of Silesian Oncology Center between the years 1998 and 2003. Paraffin sections from archival material were studied immunohistochemically for detection p53 and EGFR and correlated with clinical parameters and local tumor control and patient survival. RESULTS Accumulation of p53 and EGFR were detected in 65% and 50% of tumor respectively. No relationship was observed between immunostaining for investigated proteins and clinicopathologic factors. The TNM tumor stage was the most significant prognostic factor for local control and overall survival. p53 was favorable prognostic factor with 5-years disease free survival rate 82% for patients p53-positive and 75% for p53-negative patients (p = 0.04). CONCLUSION The TNM tumor stage is the most important prognostic factor for laryngeal cancer. Tumors accumulating p53 have better prognosis what indicates possibly role for p53 immunohistochemical analysis for predicting outcomes of radiotherapy in patients with laryngeal cancer.
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Affiliation(s)
- Dorota Dupla
- Zaklad Teleradioterapii, Dolnoślaskie Centrum Onkologii, Wrocław
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26
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Brito DA, Rieder CL. The ability to survive mitosis in the presence of microtubule poisons differs significantly between human nontransformed (RPE-1) and cancer (U2OS, HeLa) cells. ACTA ACUST UNITED AC 2009; 66:437-47. [PMID: 18792104 DOI: 10.1002/cm.20316] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
We used live cell imaging to compare the fate of human nontransformed (RPE-1) and cancer (HeLa, U2OS) cells as they entered mitosis in nocodazole or taxol. In the same field, and in either drug, a cell in all lines could die in mitosis, exit mitosis and die within 10 h, or exit mitosis and survive > or =10 h. Relative to RPE-1 cells, significantly fewer HeLa or U2OS cells survived mitosis or remained viable after mitosis: in nocodazole concentrations that inhibit spindle microtubule assembly, or in 500 nM taxol, 30% and 27% of RPE-1 cells, respectively, died in or within 10 h of exiting mitosis while 90% and 49% of U2OS and 78% and 81% of HeLa died. This was even true for clinically relevant taxol concentrations (5 nM) which killed 93% and 46%, respectively, of HeLa and U2OS cells in mitosis or within 10 h of escaping mitosis, compared to 1% of RPE-1 cells. Together these data imply that studies using HeLa or U2OS cells, harvested after a prolonged block in mitosis with nocodazole or taxol, are significantly contaminated with dead or dying cells. We also found that the relationship between the duration of mitosis and survival is drug and cell type specific and that lethality is related to the cell type and drug used to prevent satisfaction of the kinetochore attachment checkpoint. Finally, work with a pan-caspase inhibitor suggests that the primary apoptotic pathway triggered by nocodazole during mitosis in RPE-1 cells is not active in U2OS cells. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.
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Affiliation(s)
- Daniela A Brito
- Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York, USA
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27
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Staples OD, Hollick JJ, Campbell J, Higgins M, McCarthy AR, Appleyard V, Murray KE, Baker L, Thompson A, Ronseaux S, Slawin AMZ, Lane DP, Westwood NJ, Lain S. Characterization, chemical optimization and anti-tumour activity of a tubulin poison identified by a p53-based phenotypic screen. Cell Cycle 2008; 7:3417-27. [PMID: 18971638 DOI: 10.4161/cc.7.21.6982] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
A robust p53 cell-based assay that exploits p53's function as a transcription factor was used to screen a small molecule library and identify bioactive small molecules with potential antitumor activity. Unexpectedly, the majority of the highest ranking hit compounds from this screen arrest cells in mitosis and most of them impair polymerization of tubulin in cells and in vitro. One of these novel compounds, JJ78:1, was subjected to structure-activity relationship studies and optimized leading to the identification of JJ78:12. This molecule is significantly more potent than the original hit JJ78:1, as it is active in cells at two-digit nanomolar concentrations and shows clear antitumor activity in a mouse xenograft model as a single agent. The effects of nocodazole, a well established tubulin poison, and JJ78:12 on p53 levels are remarkably similar, supporting that tubulin depolymerization is the main mechanism by which JJ78:12 treatment leads to p53 activation in cells. In summary, these results identify JJ78:12 as a potential cancer therapeutic, demonstrate that screening for activators of p53 in a cell-based assay is an effective way to identify inhibitors of mitosis progression and highlights p53's sensitivity to alterations during mitosis.
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Affiliation(s)
- Oliver D Staples
- Department of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, UK
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28
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Wu YC, Yen WY, Yih LH. Requirement of a functional spindle checkpoint for arsenite-induced apoptosis. J Cell Biochem 2008; 105:678-87. [DOI: 10.1002/jcb.21861] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
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Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through a tragedy: mitotic catastrophe. Cell Death Differ 2008; 15:1153-62. [PMID: 18404154 DOI: 10.1038/cdd.2008.47] [Citation(s) in RCA: 485] [Impact Index Per Article: 28.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Mitotic catastrophe (MC) has long been considered as a mode of cell death that results from premature or inappropriate entry of cells into mitosis and can be caused by chemical or physical stresses. Whereas it initially was depicted as the main form of cell death induced by ionizing radiation, it is today known to be triggered also by treatment with agents influencing the stability of microtubule, various anticancer drugs and mitotic failure caused by defective cell cycle checkpoints. Although various descriptions explaining MC exist, there is still no general accepted definition of this phenomenon. Here, we present evidences indicating that death-associated MC is not a separate mode of cell death, rather a process ('prestage') preceding cell death, which can occur through necrosis or apoptosis. The final outcome of MC depends on the molecular profile of the cell.
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Affiliation(s)
- H Vakifahmetoglu
- Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden
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31
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Chan YW, On KF, Chan WM, Wong W, Siu HO, Hau PM, Poon RYC. The kinetics of p53 activation versus cyclin E accumulation underlies the relationship between the spindle-assembly checkpoint and the postmitotic checkpoint. J Biol Chem 2008; 283:15716-23. [PMID: 18400748 DOI: 10.1074/jbc.m800629200] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
Although cells can exit mitotic block aberrantly by mitotic slippage, they are prevented from becoming tetraploids by a p53-dependent postmitotic checkpoint. Intriguingly, disruption of the spindle-assembly checkpoint also compromises the postmitotic checkpoint. The precise mechanism of the interplay between these two pivotal checkpoints is not known. We found that after prolonged nocodazole exposure, the postmitotic checkpoint was facilitated by p53. We demonstrated that although disruption of the mitotic block by a MAD2-binding protein promoted slippage, it did not influence the activation of p53. Both p53 and its downstream target p21(CIP1/WAF1) were activated at the same rate irrespective of whether the spindle-assembly checkpoint was enforced or not. The accelerated S phase entry, as reflected by the premature accumulation of cyclin E relative to the activation of p21(CIP1/WAF1), is the reason for the uncoupling of the postmitotic checkpoint. In support of this hypothesis, forced premature mitotic exit with a specific CDK1 inhibitor triggered DNA replication without affecting the kinetics of p53 activation. Finally, replication after checkpoint bypass was boosted by elevating the level of cyclin E. These observations indicate that disruption of the spindle-assembly checkpoint does not directly influence p53 activation, but the shortening of the mitotic arrest allows cyclin E-CDK2 to be activated before the accumulation of p21(CIP1/WAF1). These data underscore the critical relationship between the spindle-assembly checkpoint and the postmitotic checkpoint in safeguarding chromosomal stability.
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Affiliation(s)
- Ying Wai Chan
- Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong
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32
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Decordier I, Cundari E, Kirsch-Volders M. Survival of aneuploid, micronucleated and/or polyploid cells: crosstalk between ploidy control and apoptosis. Mutat Res 2008; 651:30-39. [PMID: 18242119 DOI: 10.1016/j.mrgentox.2007.10.016] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2007] [Accepted: 10/28/2007] [Indexed: 05/25/2023]
Abstract
Microtubule inhibitors are known to block the cell cycle at M-phase, by damaging the mitotic spindle. However, under certain circumstances, cells can escape these effects and become aneuploid, polyploid and/or micronucleated. It is well known that aneuploidy can have adverse effects on human health such as pregnancy wastage, birth defects and the development of human tumours. The present paper aims at reviewing the data our laboratory has accumulated during the last years about the relation between aneuploidy/polyploidy/presence of micronuclei and the induction of apoptosis in human cells after in vitro exposure to the microtubule inhibitor nocodazole. Exposure to high doses of nocodazole results in polyploidy due to mitotic slippage in the absence of a functional spindle. Depending on their p53-status polyploid cells may eventually arrest, die or continue cycling. In these experimental conditions, our data showed that polyploidy does not constitute a strong apoptotic signal. In case of exposure to low concentrations of nocodazole, microtubule depolymerization is disturbed resulting in a spindle with damaged microtubules. This can give rise to chromosome loss and non-disjunction. Our data showed that in particular micronucleated cells, originating from chromosome loss can be eliminated by apoptosis. In addition, nocodazole-induced apoptosis involves the apical caspase-8 and -9 and the effector caspase-3. We show evidence that caspase-3, in addition to its function in apoptosis, plays a role in the formation of micronuclei.
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Affiliation(s)
- Ilse Decordier
- Vrije Universiteit Brussel, Laboratorium voor Cellulaire Genetica, Pleinlaan 2, 1050 Brussels, Belgium.
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33
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Decordier I, Cundari E, Kirsch-Volders M. Mitotic checkpoints and the maintenance of the chromosome karyotype. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2008; 651:3-13. [DOI: 10.1016/j.mrgentox.2007.10.020] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/16/2007] [Accepted: 10/28/2007] [Indexed: 01/07/2023]
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Kolomeichuk SN, Bene A, Upreti M, Dennis RA, Lyle CS, Rajasekaran M, Chambers TC. Induction of Apoptosis by Vinblastine via c-Jun Autoamplification and p53-Independent Down-Regulation of p21WAF1/CIP1. Mol Pharmacol 2007. [DOI: 10.1124/mol.107.039750] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
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35
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Greene LM, Kelly L, Onnis V, Campiani G, Lawler M, Williams DC, Zisterer DM. STI-571 (imatinib mesylate) enhances the apoptotic efficacy of pyrrolo-1,5-benzoxazepine-6, a novel microtubule-targeting agent, in both STI-571-sensitive and -resistant Bcr-Abl-positive human chronic myeloid leukemia cells. J Pharmacol Exp Ther 2007; 321:288-97. [PMID: 17202400 DOI: 10.1124/jpet.106.116640] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Interactions between the Bcr-Abl kinase inhibitor STI-571 (imatinib mesylate) and a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-6, were investigated in STI-571-sensitive and -resistant human chronic myeloid leukemia (CML) cells. Cotreatment of PBOX-6 with STI-571 induced significantly more apoptosis in Bcr-Abl-positive CML cell lines (K562 and LAMA-84) than either drug alone (P < 0.01). Cell cycle analysis of propidium iodide-stained cells showed that STI-571 significantly reduced PBOX-6-induced G2M arrest and polyploid formation with a concomitant increase in apoptosis. Similar results were obtained in K562 CML cells using lead MTAs (paclitaxel and nocodazole) in combination with STI-571. Potentiation of PBOX-6-induced apoptosis by STI-571 was specific to Bcr-Abl-positive leukemia cells with no cytoxic effects observed on normal peripheral blood cells. The combined treatment of STI-571 and PBOX-6 was associated with the down-regulation of Bcr-Abl and repression of proteins involved in Bcr-Abl transformation, namely the antiapoptotic proteins Bcl-x(L) and Mcl-1. Importantly, PBOX-6/STI-571 combinations were also effective in STI-571-resistant cells. Together, these findings highlight the potential clinical benefits in simultaneously targeting the microtubules and the Bcr-Abl oncoprotein in STI-571-sensitive and -resistant CML cells.
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Affiliation(s)
- Lisa M Greene
- School of Biochemistry and Immunology, Trinity College, Dublin 2, United Kingdom
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36
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Duan L, Sterba K, Kolomeichuk S, Kim H, Brown PH, Chambers TC. Inducible overexpression of c-Jun in MCF7 cells causes resistance to vinblastine via inhibition of drug-induced apoptosis and senescence at a step subsequent to mitotic arrest. Biochem Pharmacol 2007; 73:481-490. [PMID: 17126817 PMCID: PMC1829171 DOI: 10.1016/j.bcp.2006.10.026] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2006] [Revised: 10/11/2006] [Accepted: 10/25/2006] [Indexed: 11/17/2022]
Abstract
c-Jun is a major component of the AP-1 transcription factor and plays a key role in regulation of diverse biological processes including proliferation and apoptosis. Treatment of a wide variety of cells with the microtubule inhibitor vinblastine leads to a robust increase in c-Jun expression, JNK-mediated c-Jun phosphorylation, and activation of AP-1-dependent transcription. However, the role of c-Jun induction in the response of cells to vinblastine remains obscure. In this study we used MCF7 breast cancer cell lines that express the dominant-negative form of c-Jun, TAM-67, as well as cells that overexpress c-Jun, under the control of an inducible promoter. Vinblastine induced c-Jun protein expression, c-Jun phosphorylation, and AP-1 activation in MCF7 cells, and these parameters were strongly inhibited by inducible TAM-67 expression and strongly enhanced by inducible c-Jun expression. Vinblastine-induced cell death was not affected by TAM-67 expression whereas cells were protected by c-Jun overexpression. Further investigation revealed that apoptotic and senescent cells were observed after vinblastine treatment and that both outcomes were strongly inhibited by c-Jun overexpression. Although c-Jun expression inhibited cell death, it did not affect the ability of vinblastine to induce mitotic arrest. These results indicate that c-Jun expression plays a protective role in the cellular response to vinblastine and operates post-mitotic block to inhibit drug-induced apoptosis and senescence.
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Affiliation(s)
- Lingling Duan
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
| | - Kristen Sterba
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
| | - Sergey Kolomeichuk
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
| | - Heetae Kim
- Departments of Medicine and Molecular and Cellular Biology, Baylor Breast Center, Baylor College of Medicine, Houston, TX 77030
| | - Powel H. Brown
- Departments of Medicine and Molecular and Cellular Biology, Baylor Breast Center, Baylor College of Medicine, Houston, TX 77030
| | - Timothy C. Chambers
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
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37
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Grove LE, Ghosh RN. Quantitative characterization of mitosis-blocked tetraploid cells using high content analysis. Assay Drug Dev Technol 2007; 4:421-42. [PMID: 16945015 DOI: 10.1089/adt.2006.4.421] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
A range of cellular evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. Cancer research would benefit if these cellular properties could instead be measured by a single, quantitative, automated assay method, such as high content analysis (HCA). Thus, nocodazole-treated cells were fluorescently labeled for different cell cycle-associated properties, including DNA content, retinoblastoma (Rb) and histone H3 phosphorylation, p53 and p21(WAF1) expression, nuclear and cell sizes, and cell morphology, and automatically imaged, analyzed, and correlated using HCA. HCA verified that nocodazole-induced mitosis block resulted in tetraploid cells. Rb and histone H3 were maximally hyperphosphorylated by 24 h of nocodazole treatment, accompanied by cell and nuclear size decreases and cellular rounding. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and histone H3 dephosphorylation accompanied by cellular respreading. This was accompanied by increased p53 and p21(WAF1) expression levels. The range of effects accompanying nocodazole-induced block of mitosis and the resulting tetraploid cells' reversal to a pseudo-G1 state can be quantitatively measured by HCA in an automated manner, recommending this assay method for the large-scale biology challenges of modern cancer drug discovery.
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38
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Quignon F, Rozier L, Lachages AM, Bieth A, Simili M, Debatisse M. Sustained mitotic block elicits DNA breaks: one-step alteration of ploidy and chromosome integrity in mammalian cells. Oncogene 2006; 26:165-72. [PMID: 16832348 DOI: 10.1038/sj.onc.1209787] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Following prolonged mitotic spindle disruption by microtubule poisons, mammalian cells delay their entry into anaphase, then progressively slip out of mitosis and become tetraploid. Normal cells then stop cycling before S-phase onset, but the mechanisms underlying this arrest are still unclear. Here we show that a double block prevents endo-reduplication. First, cells that exit mitosis without a functional microtubule network are driven toward G0. Reconstitution of the network unmasks a second block that relies on DNA double-strand breaks occurring early in the G1 phase that follows the mitotic block. We propose that a stress signal elicited upon mitotic impairment triggers breakage, which couples the leaky spindle checkpoint to the stringent DNA damage response. Consistent with this finding, cells defective for the damage response continue cycling and acquire, within a single cell cycle, both chromosome rearrangements and abnormal chromosome numbers that remarkably mimic the complex genetic hallmark of tumorigenesis.
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Affiliation(s)
- F Quignon
- Institut Curie, Université Pierre et Marie Curie-Paris 6, CNRS UMR 7147, 26 Rue d'Ulm, 75248, Paris Cédex 05, France
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39
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Obey TB, Lyle CS, Chambers TC. Role of c-Jun in cellular sensitivity to the microtubule inhibitor vinblastine. Biochem Biophys Res Commun 2005; 335:1179-84. [PMID: 16111654 DOI: 10.1016/j.bbrc.2005.07.194] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2005] [Accepted: 07/30/2005] [Indexed: 11/22/2022]
Abstract
The role of c-Jun in the apoptotic response of cells to the microtubule inhibitor vinblastine was investigated using fibroblasts lacking or overexpressing c-Jun. c-Jun null cells were found to be more sensitive than wild-type cells at low (1-3 nM) concentrations of vinblastine, but showed essentially identical apoptotic responses as wild-type cells at a higher concentration of 10nM. In contrast, c-Jun overexpressing cells were highly vinblastine-resistant, with an IC50 of 12-fold greater than wild-type cells. The fate of cells exposed to lethal concentrations of vinblastine was examined by propidium iodide staining and flow cytometry. All cell types appeared to undergo mitotic arrest prior to apoptosis. Apoptosis of wild-type cells was associated with significant DNA re-replication. In contrast, DNA re-replication was much less prominent in vinblastine-treated c-Jun null cells and absent during apoptosis of c-Jun overexpressing cells. These results suggest that c-Jun plays a key role in the cellular sensitivity to vinblastine. In addition, c-Jun appears to regulate the pathway to cell death following mitotic arrest.
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Affiliation(s)
- Toria B Obey
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
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40
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Abstract
The well recognized activities of the mammalian centrosome--microtubule nucleation, duplication, and organization of the primary cilium--are under the control of the cell cycle. However, the centrosome is more than just a follower of the cell cycle; it can also be essential for the cell to transit G1 and enter S phase. How the centrosome influences G1 progression is a mystery.
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Affiliation(s)
- Greenfield Sluder
- Department of Cell Biology, University of Massachusetts Medical School, Worcester 01605, USA.
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41
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Duensing A, Duensing S. Guilt by association? p53 and the development of aneuploidy in cancer. Biochem Biophys Res Commun 2005; 331:694-700. [PMID: 15865924 DOI: 10.1016/j.bbrc.2005.03.157] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2005] [Indexed: 02/07/2023]
Abstract
Aneuploidy is one of the most frequent genetic alterations in solid tumors. It is commonly caused by cell division errors that are induced by oncogene activation or loss of tumor suppressor functions. In addition, certain viral oncoproteins have been implicated in the induction of chromosome copy number changes. Aneuploidy and inactivation of p53 frequently coincide in human cancers but there is increasing evidence that loss of p53 by itself is not a primary cause of aneuploidy. Nonetheless, p53 inactivation synergizes with additional oncogenic events to promote aneuploidy and may facilitate chromosomal imbalances through indirect mechanisms. This review summarizes the current knowledge about the association between aneuploidy and p53, and discusses two of the most controversial mechanisms that have been implicated in genomic instability associated with loss of p53: subversion of ploidy control and aberrant centrosome duplication.
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Affiliation(s)
- Anette Duensing
- Molecular Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA
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42
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Xiao Z, Xue J, Semizarov D, Sowin TJ, Rosenberg SH, Zhang H. Novel indication for cancer therapy: Chk1 inhibition sensitizes tumor cells to antimitotics. Int J Cancer 2005; 115:528-38. [PMID: 15688426 DOI: 10.1002/ijc.20770] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Paclitaxel (Taxol) is the most-prescribed anti-mitotic agent for a variety of advanced metastatic cancers. It induces mitotic arrest leading to apoptosis through microtubule stabilization. Chk1 is the major cell-cycle checkpoint kinase mediating S- and G2-arrests in response to various DNA-damages. Chk1 inhibitor is anticipated and has been demonstrated to potentiate the cytotoxicity of DNA-damaging agents through abrogation of cell-cycle checkpoints. Paclitaxel does not, however, induce Chk1 activation, and Chk1 has not been shown to function in mitotic checkpoint. Thus, Chk1 inhibitor is not expected to enhance the toxicity of paclitaxel. Here we show that downregulation of Chk1 sensitizes tumor cells to the toxicity of paclitaxel in cell proliferation assay. Fluorescence microscopy showed that Chk1 knockdown augments mitotic catastrophe and apoptosis in paclitaxel-treated cancer cells. Further, we elucidated the mechanism of this sensitization. Chk1 inhibition facilitates paclitaxel-induced M-phase entry by activation of Cdc2 kinase and accumulation of cyclin B1, the required cofactor for Cdc2 kinase activity. Moreover, Chk1 downregulation inhibits M phase exit through induction of the anaphase inhibitor, securin/PDS1. Collectively, Chk1 elimination sustains a more effective mitotic arrest as demonstrated by the more efficient accumulation of M-phase marker phospho-histone H3. We show that Chk1 elimination attenuates the paclitaxel-induced activation of the anti-apoptotic p42/p44 (ERK1/2) MAP kinase pathway, additionally contributing to the sensitization. Our results suggest that in addition to its well-established role as an enforcer of S and G2-checkpoints in response to genotoxic stress, Chk1 also plays a protective role in mitotic checkpoint to lessen mitotic catastrophe and thereby limits cell-death. Therefore Chk1 downregulation can not only potentiate DNA-damaging agents, but also enhance the toxicity of anti-microtubule agents, which significantly broadens its therapeutic applications.
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Affiliation(s)
- Zhan Xiao
- Cancer Research, Abbott Laboratories, Abbott Park, IL 60064-6101, USA
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43
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Wong C, Stearns T. Mammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure. BMC Cell Biol 2005; 6:6. [PMID: 15713235 PMCID: PMC554097 DOI: 10.1186/1471-2121-6-6] [Citation(s) in RCA: 108] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2004] [Accepted: 02/15/2005] [Indexed: 12/25/2022] Open
Abstract
Background Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest. Results Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed. Conclusions We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them.
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Affiliation(s)
- Connie Wong
- Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
| | - Tim Stearns
- Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
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44
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Sphyris N, Harrison DJ. p53 deficiency exacerbates pleiotropic mitotic defects, changes in nuclearity and polyploidy in transdifferentiating pancreatic acinar cells. Oncogene 2005; 24:2184-94. [PMID: 15735758 DOI: 10.1038/sj.onc.1208249] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
In a primary culture model for pancreatic acinar-ductal transdifferentiation, cells exhibited increased proliferation, changes in nuclearity and polyploidy. We identify the 'nucleus to centrosome' ratio of the progenitor cell, the dissemination of centrosomes at spindle poles and cytokinesis failure as critical determinants of mitosis outcome and centrosome inheritance. Abortive cytokinesis of mononuclear cells contributes to the binuclear cell pool, whereas enclosure of entire mitotic formations, within a single nuclear envelope, perpetuates polyploidization. Binuclear cell nuclei combine their genomes on a single metaphase plate, doubling descendant ploidy. Moreover, approximately 42% of binuclear and tetraploid cells assemble aberrant spindles with up to 8 centrosomes/poles. These phenotypes were exacerbated in p53-deficient cultures exhibiting increased S-phase entry, giant nuclei, multinucleation, multipolar mitoses and centrosome hyperamplification. The tendency of p53-proficient cells to spontaneously evade the tetraploidy checkpoint degenerates to uncontrolled polyploid progression in p53-deficient cultures, explaining why p53 abrogation alone rapidly descends to aneuploidy in this system. We detected constitutively nuclear mdm2, which may circumvent endogenous cell-cycle checkpoints, and pronounced accumulation of p21 and p27 in multinuclear cells and giant nuclei, consistent with roles in polyploidization. This in vitro model may recapitulate the processes underlying genomic instability in pancreatic tumours in vivo, and attests to the existence of a p53-dependent polyploidy checkpoint acting to limit the degree of polyploidization.
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Affiliation(s)
- Nathalie Sphyris
- Division of Pathology, School of Molecular and Clinical Medicine, The University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK
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45
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Abstract
Recent developments have highlighted the important role centrosomal defects play in the cellular changes associated with tumorigenesis. This article reviews recent developments addressing the impact of numerical centrosomal amplification on chromosomal segregational defects in the cancer cell. Probably, the most significant is the change to the structure of the spindle that leads to increased numbers of spindle poles and abnormal partitioning of the chromosomes in mitosis. I address how centrosomal changes are initiated and how they may lead to spindle multipolarity.
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Affiliation(s)
- William Saunders
- Department of Biological Sciences, 258 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260, USA.
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46
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Abstract
The three human homologues of Aurora kinases (A, B and C) are essential for proper execution of various mitotic events and are important for maintaining genomic integrity. Aurora-A is mainly localized at spindle poles and the mitotic spindle during mitosis, where it regulates the functions of centrosomes, spindles and kinetochores required for proper mitotic progression. Recent studies have revealed that Aurora-A is frequently overexpressed in various cancer cells, indicating its involvement in tumorigenesis. What are the normal physiological roles of Aurora-A, how are these regulated and how might the enzyme function during tumorigenesis?
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Affiliation(s)
- Tomotoshi Marumoto
- Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan
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47
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Rieder CL, Maiato H. Stuck in division or passing through: what happens when cells cannot satisfy the spindle assembly checkpoint. Dev Cell 2004; 7:637-51. [PMID: 15525526 DOI: 10.1016/j.devcel.2004.09.002] [Citation(s) in RCA: 509] [Impact Index Per Article: 24.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Cells that cannot satisfy the spindle assembly checkpoint (SAC) are delayed in mitosis (D-mitosis), a fact that has useful clinical ramifications. However, this delay is seldom permanent, and in the presence of an active SAC most cells ultimately escape mitosis and enter the next G1 as tetraploid cells. This review defines and discusses the various factors that determine how long a cell remains in mitosis when it cannot satisfy the SAC and also discusses the cell's subsequent fate.
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Affiliation(s)
- Conly L Rieder
- Division of Molecular Medicine, New York State Department of Health, Wadsworth Center, Albany, NY 12201, USA.
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48
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Vogel C, Kienitz A, Hofmann I, Müller R, Bastians H. Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy. Oncogene 2004; 23:6845-53. [PMID: 15286707 DOI: 10.1038/sj.onc.1207860] [Citation(s) in RCA: 117] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Treatment of cells with microtubule inhibitors results in activation of the mitotic spindle assembly checkpoint, leading to mitotic arrest before anaphase. Upon prolonged treatment, however, cells can adapt and exit mitosis aberrantly, resulting in the occurrence of tetraploid cells in G1. Those cells subsequently arrest in postmitotic G1 due to the activation of a p53-dependent G1 checkpoint. Failure of the G1 checkpoint leads to endoreduplication and further polyploidization. Using HCT116 and isogenic p53-deficient or spindle checkpoint compromised derivatives, we show here that not only p53 but also a functional spindle assembly checkpoint is required for postmitotic G1 checkpoint function. During transient mitotic arrest, p53 stabilization and activation is triggered by a pathway independent of ATM/ATR, Chk1 and Chk2. We further show that a prolonged spindle checkpoint-mediated mitotic arrest is required for proper postmitotic G1 checkpoint function. In addition, we demonstrate that polyploid cells are inhibited to re-enter mitosis by an additional checkpoint acting in G2. Thus, during a normal cell cycle, polyploidization and subsequent aneuploidization is prevented by the function of the mitotic spindle checkpoint, a p53-dependent G1 checkpoint and an additional G2 checkpoint.
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Affiliation(s)
- Celia Vogel
- Institute for Molecular Biology and Tumor Research, Philipps University Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany
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49
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Yan T, Desai AB, Jacobberger JW, Sramkoski RM, Loh T, Kinsella TJ. CHK1 and CHK2 are differentially involved in mismatch repair–mediated 6-thioguanine-induced cell cycle checkpoint responses. Mol Cancer Ther 2004. [DOI: 10.1158/1535-7163.1147.3.9] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Abstract
The DNA mismatch repair (MMR) system plays an important role in mediating a G2-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 μmol/L × 24 hours), activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G2 arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G1-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.
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Affiliation(s)
- Tao Yan
- Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio
| | - Anand B. Desai
- Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio
| | - James W. Jacobberger
- Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio
| | - R. Michael Sramkoski
- Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio
| | - Tamalette Loh
- Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio
| | - Timothy J. Kinsella
- Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio
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50
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Schneider Y, Fischer B, Coelho D, Roussi S, Gosse F, Bischoff P, Raul F. (Z)-3,5,4′-Tri-O-methyl-resveratrol, induces apoptosis in human lymphoblastoid cells independently of their p53 status. Cancer Lett 2004; 211:155-61. [PMID: 15219939 DOI: 10.1016/j.canlet.2004.02.017] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2003] [Revised: 12/30/2003] [Accepted: 02/23/2004] [Indexed: 10/26/2022]
Abstract
The pro-apoptotic ability of (Z)-3,5,4'-Tri-O-methyl-resveratrol (R3) was investigated in vitro on the human lymphoblastoid cell line TK6 and its p53-knockout counterpart (NH32). In both cell lines, R3 induced the stimulation of caspase-3. Although R3 induced growth inhibition and apoptosis of both cell lines, two distinct mechanisms were observed. The p53-knockout NH32 cells were shown to override the G2/M phase checkpoint with development of hyperdiploid cells, whereas TK6 cells accumulated at G2/M. As p53 function is often altered in human cancer cells, these results show that the pro-apototic effects of R3 against tumor cells are independent of their p53 status.
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Affiliation(s)
- Yann Schneider
- Laboratory of Nutritional Oncology, Inserm UMR S392, IRCAD, 1 place de l'Hôpital, BP 426, 67091 Strasbourg Cedex, France
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