Long Term Aggresome Accumulation Leads to DNA Damage, p53-dependent Cell Cycle Arrest, and Steric Interference in Mitosis

ABSTRACT Genomic instability, including the ability to undergo gene amplification, is a hallmark of neoplastic cells. Similar to normal cells, “nonpermissive” REF52 cells do not develop resistance toN-(phosphonacetyl)-l-aspartate (PALA), an inhibitor of the synthesis of pyrimidine nucleotides, through amplification of cad, the target gene, but instead undergo protective, long-term, p53-dependent cell cycle arrest. Expression of exogenous MYC prevents this arrest and allows REF52 cells to proceed to mitosis when pyrimidine nucleotides are limiting. This results in DNA breaks, leading to cell death and, rarely, to cad gene amplification and PALA resistance. Pretreatment of REF52 cells with a low concentration of PALA, which slows DNA replication but does not trigger cell cycle arrest, followed by exposure to a high, selective concentration of PALA, promotes the formation of PALA-resistant cells in which the physically linked cad and endogenous N-myc genes are coamplified. The activated expression of endogenous N-myc in these pretreated PALA-resistant cells allows them to bypass the p53-mediated arrest that is characteristic of untreated REF52 cells. Our data demonstrate that two distinct events are required to form PALA-resistant REF52 cells: amplification ofcad, whose product overcomes the action of the drug, and increased expression of N-myc, whose product overcomes the PALA-induced cell cycle block. These paired events occur at a detectable frequency only when the genes are physically linked, ascad and N-myc are. In untreated REF52 cells overexpressing N-MYC, the level of p53 is significantly elevated but there is no induction of p21 waf1 expression or growth arrest. However, after DNA is damaged, the activated p53 executes rapid apoptosis in these REF52/N-myc cells instead of the long-term protective arrest seen in REF52 cells. The predominantly cytoplasmic localization of stabilized p53 in REF52/N-myc cells suggests that cytoplasmic retention may help to inactivate the growth-suppressing function of p53.

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A novel mutation in DNA topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest.

Genetics ◽

10.1093/genetics/133.4.799 ◽

1993 ◽

Vol 133 (4) ◽

pp. 799-814 ◽

Cited By ~ 11

Author(s):

N A Levin ◽

M A Bjornsti ◽

G R Fink

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Arrest ◽

Topoisomerase I ◽

Novel Mutation ◽

Constitutive Expression ◽

Repair System ◽

Yeast Saccharomyces Cerevisiae ◽

Cycle Arrest ◽

Dependent Cell

Abstract DNA topoisomerases, enzymes that alter the superhelicity of DNA, have been implicated in such critical cellular functions as transcription, DNA replication, and recombination. In the yeast Saccharomyces cerevisiae, a null mutation in the gene encoding topoisomerase I (TOP1) causes elevated levels of mitotic recombination in the ribosomal DNA (rDNA), but has little effect on growth. We have isolated a missense mutation in TOP1 that causes mitotic hyper-recombination not only in the rDNA, but also at other loci, in addition to causing a number of other unexpected phenotypes. This topoisomerase I mutation (top1-103) causes slow growth, constitutive expression of DNA damage-inducible genes, and inviability in the absence of the double-strand break repair system. Overexpression of top1-103 causes RAD9-dependent cell cycle arrest in G2. We show that the Top1-103 enzyme nicks DNA in vitro, suggesting that it damages DNA directly. We propose that Top1-103 mimics the action of wild-type topoisomerase I in the presence of the anti-tumor drug, camptothecin.

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Methylation of histone H4 lysine 20 by PR-Set7 ensures the integrity of late replicating sequence domains in Drosophila

10.1101/028100 ◽

2015 ◽

Author(s):

Yulong Li () ◽

Robin L. Armstrong ◽

Robert J. Duronio ◽

David M. MacAlpine

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Cell Cycle Arrest ◽

Mammalian Cells ◽

Genome Stability ◽

Histone H4 ◽

Cycle Arrest ◽

Genomic Scale ◽

Dependent Cell

ABSTRACTThe methylation state of lysine 20 on histone H4 (H4K20) has been linked to chromatin compaction, transcription, DNA repair and DNA replication. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7. PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which has been partially attributed to defects in origin selection and activation. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 and H4K20 methylation impact the replication program on a genomic scale. We employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and genome stability in Drosophila cells. We find that deregulation of H4K20 methylation had no impact on origin activation throughout the genome. Instead, depletion of PR-Set7 and loss of H4K20me1 results in the accumulation of DNA damage and an ATR-dependent cell cycle arrest. Coincident with the ATR-dependent cell cycle arrest, we find increased DNA damage that is specifically limited to late replicating regions of the Drosophila genome, suggesting that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains.

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Design, Synthesis and Pharmacological Evaluation of Three Novel Dehydroabietyl Piperazine Dithiocarbamate Ruthenium (II) Polypyridyl Complexes as Potential Antitumor Agents: DNA Damage, Cell Cycle Arrest and Apoptosis Induction

Molecules ◽

10.3390/molecules26051453 ◽

2021 ◽

Vol 26 (5) ◽

pp. 1453

Author(s):

Haoran Wang ◽

Jianhua Wei ◽

Hong Jiang ◽

Ye Zhang ◽

Caina Jiang ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Arrest ◽

Antitumor Agents ◽

Mechanistic Study ◽

Ros Generation ◽

Polypyridyl Complexes ◽

Expression Levels ◽

Cycle Arrest ◽

Study Results

The use of cisplatin is severely limited by its toxic side-effects, which has spurred chemists to employ different strategies in the development of new metal-based anticancer agents. Here, three novel dehydroabietyl piperazine dithiocarbamate ruthenium (II) polypyridyl complexes (6a–6c) were synthesized as antitumor agents. Compounds 6a and 6c exhibited better in vitro antiproliferative activity against seven tumor cell lines than cisplatin, they displayed no evident resistance in the cisplatin-resistant cell line A549/DPP. Importantly, 6a effectively inhibited tumor growth in the T-24 xenograft mouse model in comparison with cisplatin. Gel electrophoresis assay indicated that DNA was the potential targets of 6a and 6c, and the upregulation of p-H2AX confirmed this result. Cell cycle arrest studies demonstrated that 6a and 6c arrested the cell cycle at G1 phase, accompanied by the upregulation of the expression levels of the antioncogene p27 and the down-regulation of the expression levels of cyclin E. In addition, 6a and 6c caused the apoptosis of tumor cells along with the upregulation of the expression of Bax, caspase-9, cytochrome c, intracellular Ca2+ release, reactive oxygen species (ROS) generation and the downregulation of Bcl-2. These mechanistic study results suggested that 6a and 6c exerted their antitumor activity by inducing DNA damage, and consequently causing G1 stage arrest and the induction of apoptosis.

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