DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.

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Inverse correlation between p53 protein levels and DNA repair efficiency in human fibroblast strains treated with 4-nitroquinoline 1-oxide: evidence that lesions other than DNA strand breaks trigger the p53 response

Carcinogenesis ◽

10.1093/carcin/20.6.941 ◽

1999 ◽

Vol 20 (6) ◽

pp. 941-946 ◽

Cited By ~ 14

Author(s):

R. Mirzayans

Keyword(s):

Dna Repair ◽

P53 Protein ◽

Inverse Correlation ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Protein Levels ◽

Repair Efficiency ◽

P53 Response ◽

Human Fibroblast Strains ◽

Dna Strand

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DNA Repair Functions That Control Sensitivity to Topoisomerase-Targeting Drugs

Eukaryotic Cell ◽

10.1128/ec.3.1.82-90.2004 ◽

2004 ◽

Vol 3 (1) ◽

pp. 82-90 ◽

Cited By ~ 39

Author(s):

Mobeen Malik ◽

John L. Nitiss

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Topoisomerase I ◽

Topoisomerase Ii ◽

Excision Repair ◽

Dna Strand Breaks ◽

Dna Topology ◽

Strand Breaks ◽

Wide Range ◽

Dna Strand

ABSTRACT DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.

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Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay)

Marine Environmental Research ◽

10.1016/s0141-1136(00)00040-4 ◽

2000 ◽

Vol 50 (1-5) ◽

pp. 405-409 ◽

Cited By ~ 27

Author(s):

J.P Shaw ◽

A.T Large ◽

J.K Chipman ◽

D.R Livingstone ◽

L.D Peters

Keyword(s):

Seasonal Variation ◽

Comet Assay ◽

Mytilus Edulis ◽

Digestive Gland ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Protein Levels ◽

Cytochrome P4501a ◽

Dna Strand

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ARF induction in response to DNA strand breaks is regulated by PARP1

Nucleic Acids Research ◽

10.1093/nar/gkt1185 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2320-2329 ◽

Cited By ~ 19

Author(s):

Giulia Orlando ◽

Svetlana V. Khoronenkova ◽

Irina I. Dianova ◽

Jason L. Parsons ◽

Grigory L. Dianov

Keyword(s):

Dna Damage ◽

Protein Function ◽

P53 Protein ◽

Dna Strand Breaks ◽

Vital Role ◽

Cellular Stress Response ◽

Strand Breaks ◽

Dna Strand ◽

Cellular Dna ◽

Arf Protein

Abstract The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating up-regulation of p53 protein and consequently promoting cell-cycle delay. Although p53 protein function has been clearly linked to the cellular DNA damage response, the role of ARF protein in this process is unclear. Here, we report that arf gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage. We discovered that poly(ADP-ribose) synthesis catalysed by PARP1 at the sites of unrepaired SBs activates ARF transcription through a protein signalling cascade, including the NAD+-dependent deacetylase SIRT1 and the transcription factor E2F1. Our data suggest that poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD+, thus down-regulating SIRT1 activity and consequently activating E2F1-dependent ARF transcription. Our findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.

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Prevention of base excision repair by TRC102 (methoxyamine) potentiates the anti-tumor activity of pemetrexed in vitro and in vivo

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.13005 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 13005-13005 ◽

Cited By ~ 2

Author(s):

L. Liu ◽

A. Bulgar ◽

J. Donze ◽

B. J. Adams ◽

C. P. Theuer ◽

...

Keyword(s):

Excision Repair ◽

Alkylating Agents ◽

Phase 1 ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Ap Sites ◽

Dna Strand ◽

Anti Tumor Activity

13005 Background: TRC102 (methoxyamine) reverses resistance to alkylating agents by inhibiting base excision repair (BER; a mechanism of DNA repair), thereby increasing DNA strand breaks and potentiating the anti-tumor activity of alkylating agents without additional toxicity, Based on these data, TRC102 is currently being studied in combination with temozolomide in a phase 1 trial. We hypothesized that inhibition of BER by TRC102 would also increase DNA strand breaks and improve the anti-tumor activity of anti-metabolite chemotherapeutics, including pemetrexed, because these agents also produce AP sites that are recognized and repaired by BER. Methods: Pemetrexed- induced AP sites and BER inhibition was quantified using an apurinic/apyrimidinic (AP) site assay in vitro. Single and double DNA strand breaks were quantified by the Comet assay in vitro and anti-tumor activity was assessed in an in vivo xenograft study of subcutaneously implanted H460 human lung cancer cells. Results: Pemetrexed induced and TRC102 reduced the number of available AP sites in pemetrexed- treated H460 cells (by 60–80%), indicating successful inhibition of BER. TRC102 treatment increased DNA strand breaks in pemetrexed-treated H460 cells (2 fold increase versus treatment with pemetrexed alone). Premetrexed treatment alone and in combination with TRC 102 delayed tumor growth in vivo (tumor growth delay of 4.7 days in the 150 mg/m2 pemetrexed alone group, 5.7 days in the 150 mg/m2 pemetrexed + 2 mg/m2 TRC102 group and 6.9 days in the 150 mg/m2 pemetrexed + 4 mg/m2 TRC102 group); in vivo systemic toxicity was not increased. TRC102 alone had no effect in vitro or in vivo. Conclusions: TRC102 effectively inhibits BER in lung cancer cells treated with pemetrexed. Inhibition of DNA repair by TRC102 results in an increase in DNA strand breaks and improved anti-tumor activity versus treatment with pemetrexed alone. Given its preclinical efficacy and safety profile, study of TRC102 combined with pemetrexed in a phase 1 trial is warranted. No significant financial relationships to disclose.

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Induction of CAF-1 Expression in Response to DNA Strand Breaks in Quiescent Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.26.5.1839-1849.2006 ◽

2006 ◽

Vol 26 (5) ◽

pp. 1839-1849 ◽

Cited By ~ 30

Author(s):

Arman Nabatiyan ◽

Dávid Szüts ◽

Torsten Krude

Keyword(s):

Excision Repair ◽

Dna Strand Breaks ◽

Significant Loss ◽

Human Cells ◽

Dna Breaks ◽

Strand Breaks ◽

Quiescent Cells ◽

Dna Strand

ABSTRACT Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks.

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MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy

Blood ◽

10.1182/blood-2005-02-0553 ◽

2005 ◽

Vol 106 (9) ◽

pp. 3150-3159 ◽

Cited By ~ 240

Author(s):

Kensuke Kojima ◽

Marina Konopleva ◽

Ismael J. Samudio ◽

Masato Shikami ◽

Maria Cabreira-Hansen ◽

...

Keyword(s):

Progenitor Cells ◽

Transcriptional Activation ◽

P53 Protein ◽

Negative Regulator ◽

Wild Type ◽

Protein Levels ◽

P53 Signaling ◽

P53 Activation ◽

Induction Of Apoptosis ◽

Wild Type P53

AbstractAlthough TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). Recently, small-molecule antagonists of MDM2, Nutlins, have been developed that inhibit the p53-MDM2 interaction and activate p53 signaling. Here, we study the effects of p53 activation by Nutlin-3 in AML cells. Treatment with MDM2 inhibitor triggered several molecular events consistent with induction of apoptosis: loss of mitochondrial membrane potential, caspase activation, phosphatidylserine externalization, and DNA fragmentation. There was a positive correlation in primary AML samples with wild-type p53 between baseline MDM2 protein levels and apoptosis induced by MDM2 inhibition. No induction of apoptosis was observed in AML samples harboring mutant p53. Colony formation of AML progenitors was inhibited in a dose-dependent fashion, whereas normal CD34+ progenitor cells were less affected. Mechanistic studies suggested that Nutlin-induced apoptosis was mediated by both transcriptional activation of proapoptotic Bcl-2 family proteins, and transcription-independent mitochondrial permeabilization resulting from mitochondrial p53 translocation. MDM2 inhibition synergistically enhanced cytotoxicity of cytosine arabinoside and doxorubicin in AML blasts but not in normal hematopoietic progenitor cells. p53 activation by targeting the p53-MDM2 interaction might offer a novel therapeutic strategy for AML that retain wild-type p53.

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