scholarly journals Failure of cell cleavage induces senescence in tetraploid primary cells

2014 ◽  
Vol 25 (20) ◽  
pp. 3105-3118 ◽  
Author(s):  
Andreas Panopoulos ◽  
Cristina Pacios-Bras ◽  
Justin Choi ◽  
Mythili Yenjerla ◽  
Mark A. Sussman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
T Antigen ◽  
Primary Cells ◽  
Ki 67 ◽  
Tetraploid Cell ◽  
Cell Cleavage ◽  
Human Foreskin Fibroblasts ◽  
Interfering Rna

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen–transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

scholarly journals Cooperative effects of genes controlling the G2/M checkpoint

2000 ◽  
Vol 14 (13) ◽  
pp. 1584-1588
Author(s):  
Timothy A. Chan ◽  
Paul M. Hwang ◽  
Heiko Hermeking ◽  
Kenneth W. Kinzler ◽  
Bert Vogelstein
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Growth Arrest ◽  
Double Knockout ◽  
Specific Test ◽  
Cooperative Effects ◽  
Exogenous Expression ◽  
Induced Genes ◽  
Single Knockout

It is believed that multiple effectors independently control the checkpoints permitting transitions between cell cycle phases. However, this has not been rigorously demonstrated in mammalian cells. The p53-induced genes p21 and 14-3-3ς are each required for the G2 arrest and allow a specific test of this fundamental tenet. We generated human cells deficient in bothp21 and 14-3-3ς and determined whether the double knockout was more sensitive to DNA damage than either single knockout.p21−/−14-3-3ς−/− cells were significantly more sensitive to DNA damage or to the exogenous expression of p53 than cells lacking only p21 or only 14-3-3ς. Thus, p21 and 14-3-3ς play distinct but complementary roles in the G2/M checkpoint, and help explain why genes at the nodal points of growth arrest pathways, like p53, are the targets of mutation in cancer cells.

Abundance of cyclin B1 regulates γ-radiation–induced apoptosis

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2645-2650 ◽  
Author(s):  
Lisa A. Porter ◽  
Gurmit Singh ◽  
Jonathan M. Lee
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Hematopoietic Cells ◽  
Cyclin B1 ◽  
Regulatory Subunit ◽  
Potent Inducer ◽  
Γ Radiation ◽  
Radiation Induced ◽  
Induced Apoptosis

Abstract γ-Radiation is a potent inducer of apoptosis. There are multiple pathways regulating DNA damage-induced apoptosis, and we set out to identify novel mechanisms regulating γ-radiation–induced apoptosis in hematopoietic cells. In this report, we present data implicating the cyclin B1 protein as a regulator of apoptotic fate following DNA damage. Cyclin B1 is the regulatory subunit of the cdc2 serine/threonine kinase, and accumulation of cyclin B1 in late G2 phase of the cell cycle is a prerequisite for mitotic initiation in mammalian cells. We find that abundance of the cyclin B1 protein rapidly increases in several mouse and human hematopoietic cells (Ramos, DP16, HL60, thymocytes) undergoing γ-radiation–induced apoptosis. Cyclin B1 accumulation occurs in all phases of the cell cycle. Antisense inhibition of cyclin B1 accumulation decreases apoptosis, and ectopic cyclin B1 expression is sufficient to induce apoptosis. These observations are consistent with the idea that cyclin B1 is both necessary and sufficient for γ-radiation-induced apoptosis.

scholarly journals Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

2009 ◽  
Vol 284 (52) ◽  
pp. 36191-36201 ◽  
Author(s):  
Christopher A. Koczor ◽  
Inna N. Shokolenko ◽  
Amy K. Boyd ◽  
Shawn P. Balk ◽  
Glenn L. Wilson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mitochondrial Dna ◽  
Cell Cycle Arrest ◽  
Mammalian Cells ◽  
Mitochondrial Dna Damage ◽  
Cycle Arrest ◽  
A Cell

scholarly journals A Mouse PRMT1 Null Allele Defines an Essential Role for Arginine Methylation in Genome Maintenance and Cell Proliferation

2009 ◽  
Vol 29 (11) ◽  
pp. 2982-2996 ◽  
Author(s):  
Zhenbao Yu ◽  
Taiping Chen ◽  
Josée Hébert ◽  
En Li ◽  
Stéphane Richard
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Null Allele ◽  
Arginine Methylation ◽  
Genome Maintenance ◽  
Protein Arginine Methyltransferase 1 ◽  
Interfering Rna ◽  
Dna Damage Response Pathway

ABSTRACT Protein arginine methyltransferase 1 (PRMT1) is the major enzyme that generates monomethylarginine and asymmetrical dimethylarginine. We report here a conditional null allele of PRMT1 in mice and that the loss of PRMT1 expression leads to embryonic lethality. Using the Cre/lox-conditional system, we show that the loss of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to the loss of arginine methylation of substrates harboring a glycine-arginine rich motif, including Sam68 and MRE11. The loss of PRMT1 in MEFs leads to spontaneous DNA damage, cell cycle progression delay, checkpoint defects, aneuploidy, and polyploidy. We show using a 4-hydroxytamoxifen-inducible Cre that the loss of PRMT1 in MEFs leads to a higher incidence of chromosome losses, gains, structural rearrangements, and polyploidy, as documented by spectral karyotyping. Using PRMT1 small interfering RNA in U2OS cells, we further show that PRMT1-deficient cells are hypersensitive to the DNA damaging agent etoposide and exhibit a defect in the recruitment of the homologous recombination RAD51 recombinase to DNA damage foci. Taken together, these data show that PRMT1 is required for genome integrity and cell proliferation. Our findings also suggest that arginine methylation by PRMT1 is a key posttranslational modification in the DNA damage response pathway in proliferating mammalian cells.

scholarly journals Atm Is Dispensable for p53 Apoptosis and Tumor Suppression Triggered by Cell Cycle Dysfunction

1999 ◽  
Vol 19 (4) ◽  
pp. 3095-3102 ◽  
Author(s):  
Mai-Jing Liao ◽  
Chaoying Yin ◽  
Carrolee Barlow ◽  
Anthony Wynshaw-Boris ◽  
Terry van Dyke
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Tumor Suppression ◽  
Cancer Susceptibility ◽  
Tumor Model ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Checkpoint Pathway ◽  
Brain Tumor Model

ABSTRACT Both p53 and ATM are checkpoint regulators with roles in genetic stabilization and cancer susceptibility. ATM appears to function in the same DNA damage checkpoint pathway as p53. However, ATM’s role in p53-dependent apoptosis and tumor suppression in response to cell cycle dysregulation is unknown. In this study, we tested the role of murine ataxia telangiectasia protein (Atm) in a transgenic mouse brain tumor model in which p53-mediated apoptosis results in tumor suppression. These p53-mediated activities are induced by tissue-specific inactivation of pRb family proteins by a truncated simian virus 40 large T antigen in brain epithelium. We show that p53-dependent apoptosis, transactivation, and tumor suppression are unaffected by Atm deficiency, suggesting that signaling in the DNA damage pathway is distinct from that in the oncogene-induced pathway. In addition, we show that Atm deficiency has no overall effect on tumor growth and progression in this model.

scholarly journals ATM-dependent DNA Damage-independent Mitotic Phosphorylation of H2AX in Normally Growing Mammalian Cells

2005 ◽  
Vol 16 (10) ◽  
pp. 5013-5025 ◽  
Author(s):  
Kirk J. McManus ◽  
Michael J. Hendzel
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Small Population ◽  
Histone H2a ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Mammalian Cell Lines ◽  
Carboxy Terminal

H2AX is a core histone H2A variant that contains an absolutely conserved serine/glutamine (SQ) motif within an extended carboxy-terminal tail. H2AX phosphorylation at the SQ motif (γ-H2AX) has been shown to increase dramatically upon exogenously introduced DNA double-strand breaks (DSBs). In this study, we use quantitative in situ approaches to investigate the spatial patterning and cell cycle dynamics of γ-H2AX in a panel of normally growing (unirradiated) mammalian cell lines and cultures. We provide the first evidence for the existence of two distinct yet highly discernible γ-H2AX focal populations: a small population of large amorphous foci that colocalize with numerous DNA DSB repair proteins and previously undescribed but much more abundant small foci. These small foci do not recruit proteins involved in DNA DSB repair. Cell cycle analyses reveal unexpected dynamics for γ-H2AX in unirradiated mammalian cells that include an ATM-dependent phosphorylation that is maximal during M phase. Based upon similarities drawn from other histone posttranslational modifications and previous observations in haploinsufficient (H2AX-/+) and null mice (H2AX-/-), γ-H2AX may contribute to the fidelity of the mitotic process, even in the absence of DNA damage, thereby ensuring the faithful transmission of genetic information from one generation to the next.

scholarly journals Checkpoint non-fidelity induces a complex landscape of lineage fitness after DNA damage

2018 ◽  
Author(s):  
Callum J. Campbell ◽  
Ashok R. Venkitaraman ◽  
Alessandro Esposito
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Dna Lesions ◽  
Lineage Tracing ◽  
Cell Cycle Checkpoints ◽  
Complex Landscape ◽  
Cellular Dna

AbstractDNA damage in proliferating mammalian cells causes death1, senescence2 or continued survival, via checkpoints that monitor damage and regulate cell cycle progression, DNA repair and fate determination3. Cell cycle checkpoints facilitate tumour suppression by preventing the generation of proliferating mutated cells4, particularly by blocking passage of DNA lesions into replication and mitosis5. While checkpoint non-fidelity permits cells to carry genomic aberrations into subsequent cell cycle phases6, its long-term consequences on lineages descendant from damaged cells remains poorly characterised. Devising methods for microscopy-based lineage tracing, we unexpectedly demonstrate that transient DNA damage to single living cells bearing a negligent checkpoint induces heterogenous cell-fate outcomes in their descendant generations removed from the initial insult. After transiently damaged cells undergo an initial arrest, pairs of descendant cells without obvious cell-cycle abnormalities either divide or die in a seemingly stochastic way. Progeny of transiently damaged cells may die generations afterwards, creating considerable variability of lineage fitness that promotes overall persistence in a mutagenic environment. Descendants of damaged cells frequently form micronuclei, activating immunogenic signalling. Our findings reveal previously unrecognized, heterogenous effects of cellular DNA damage that manifest long afterwards in descendant cells. We suggest that these heterogenous descendant cell-fate responses may function physiologically to ensure the elimination and immune clearance of damaged cell lineages, but pathologically, may enable the prolonged survival of cells bearing mutagenic damage.

A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte

1997 ◽  
Vol 110 (24) ◽  
pp. 3083-3090 ◽  
Author(s):  
S.D. Gross ◽  
C. Simerly ◽  
G. Schatten ◽  
R.A. Anderson
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
T Antigen ◽  
Casein Kinase ◽  
Mouse Oocyte ◽  
Casein Kinase I ◽  
Sv40 Large T Antigen ◽  
Cycle Progression

Casein kinase I is a family of serine/threonine protein kinases common to all eukaryotes. In yeast, casein kinase I homologues have been linked to the regulation of growth, DNA repair and cell division. In addition, their subcellular localization to membraneous structures and the nucleus is essential for function. In higher eukaryotes, there exist seven genetically distinct isoforms: (alpha), ss, (gamma)1, (gamma)2, (gamma)3, (delta) and (epsilon). Casein kinase I(alpha) exhibits a cell cycle-dependent subcellular localization including an association with cytosolic vesicular structures and the nucleus during interphase, and the spindle during mitosis. casein kinase I has also been shown to modulate critical regulators of growth and DNA synthesis/repair in mammalian cells such as SV40 large T antigen and p53. These results suggest that casein kinase I may be involved in processes similar to those ascribed to the yeast casein kinase I homologues. To define a role for casein kinase I(alpha) in cell cycle regulation, the mouse oocyte was utilized because of its well-defined cell cycle and ease of micromanipulation. Immunofluorescence studies from meiosis I of maturation to the first zygotic cleavage demonstrated that the kinase was associated with structures similar to those previously reported. Microinjection of casein kinase I(alpha) antibodies at metaphase II-arrest and G2 phase, had no effect on the completion of second meiosis or first division. However, microinjection of these antibodies during the early pronucleate phase prior to S-phase onset blocked uptake of the kinase into pronuclei and interfered with proper and timely cell cycle progression to first cleavage. These results suggest that the kinase regulates the progression from interphase to mitosis during the first cell cycle.

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