scholarly journals Characterization of a DNA-damage-recognition protein from F9 teratocarcinoma cells, which is inducible by retinoic acid and cyclic AMP

1993 ◽  
Vol 290 (1) ◽  
pp. 129-134 ◽  
Author(s):  
C C K Chao ◽  
N K Sun ◽  
S Lin-Chao
Keyword(s):  
Dna Damage ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Damage Recognition ◽  
Nuclear Extracts ◽  
Recognition Protein ◽  
Damaged Dna

A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

Biochemistry ◽  
1990 ◽  
Vol 29 (24) ◽  
pp. 5872-5880 ◽  
Author(s):  
Brian A. Donahue ◽  
Marianne Augot ◽  
Steven F. Bellon ◽  
Daniel K. Treiber ◽  
Jeffrey H. Toney ◽  
...  
Keyword(s):  
Dna Damage ◽  
Anticancer Drug ◽  
Dna Adducts ◽  
Mammalian Cells ◽  
Damage Recognition ◽  
Recognition Protein ◽  
Dna Damage Recognition

scholarly journals Induction, not associated with host-cell re-activation of damaged plasmid DNA, of damaged-DNA-recognition proteins by retinoic acid and dibutyryl cyclic AMP in mammalian cells

1996 ◽  
Vol 313 (2) ◽  
pp. 441-445 ◽  
Author(s):  
Nian-Kang SUN ◽  
Shang-Lang HUANG ◽  
Sue LIN-CHAO ◽  
Chuck C.-K. CHAO
Keyword(s):  
Dna Repair ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Cell Types ◽  
Dibutyryl Cyclic Amp ◽  
Nih3t3 Cells ◽  
Activation Assay ◽  
Cellular Dna ◽  
Damaged Dna

Our previous studies [Chao (1992) Biochem. J. 282, 203-207; C. C.-K. Chao, unpublished work] has suggested a correlation between the levels of constitutive UV-damaged-DNA-recognitionproteins (UVDRP) and cellular DNA repair in different cell types. In the present study, UVDRP were induced in F9 and NIH3T3 cells by 0.1 μM retinoic acid (RA) and 1 mM dibutyryl cyclic AMP (dbcAMP), which is sufficient to induce differentiation in murine F9 stem cells. The induction of UVDRP in F9 and NIH3T3 cells was optimized after 6 and 2 days incubation with RA/dbcAMP respectively. Since NIH3T3 cells were not induced to differentiate by RA/dbcAMP, the up-regulation of the UVDRP in mammalian cells would thus seem not to be mediated directly by differentiation. Using a plasmid re-activation assay to estimate DNA repair, we did not find a correlation between DNA repair and UVDRP in RA/dbcAMP-treated cells. The results suggest that UVDRP may have a function other than, or in addition to, its role in DNA repair.

scholarly journals Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC

2007 ◽  
Vol 120 (15) ◽  
pp. 2706-2716 ◽  
Author(s):  
M. S. Luijsterburg ◽  
J. Goedhart ◽  
J. Moser ◽  
H. Kool ◽  
B. Geverts ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Damage Recognition ◽  
Recognition Protein ◽  
Damaged Dna

Aberrant Non-Homologous End Joining in Multiple Myeloma: A Role in Genomic Instability and As Potential Prognostic Marker.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2932-2932 ◽  
Author(s):  
Teresa Calimeri ◽  
Mariateresa Fulciniti ◽  
Jianhong Lin ◽  
Mehmet Kemal Samur ◽  
Anne S. Calkins ◽  
...  
Keyword(s):  
Dna Damage ◽  
Board Of Directors ◽  
Genomic Instability ◽  
Cell Lines ◽  
Binding Activity ◽  
End Joining ◽  
Advisory Committees ◽  
Nhej Pathway ◽  
Dna Binding Activity ◽  
Nuclear Extracts

Abstract Abstract 2932 Multiple Myeloma (MM) is a hematologic malignancy characterized by a complex combination of structural and numerical chromosomal abnormalities. However, the underlying molecular basis of the genomic instability remains largely unknown. The ability to repair DNA damages, especially double-strand breaks (DSBs), is essential to suppress genetic instability. Non-homologous end joining (NHEJ) is one of the most important mechanisms responsible for repair of these breaks. Since both impaired and aberrant NHEJ seem to be linked to genomic instability in solid as well as other hematologic tumors, we have investigated its altered function in MM. To confirm involvement of an aberrant NHEJ pathway in MM genomic instability, we measured the end joining (EJ) capacity of 6 different MM cell lines using a plasmid based assay containing both the test gene (Luciferase - LUC) measuring end joining as well as a reporter gene (Alkaline Phosphatase - SEAP) to control for transfection efficiency. MM and normal control cells were transfected with this plasmid and the LUC and SEAP activity was detected directly in the supernatant of the cells at 24 h. Increased EJ activity was observed in all the MM cell lines tested compared to peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs) from healthy donor. To confirm the role of the NHEJ pathway in this increased DNA EJ activity, nuclear extracts from 9 different MM cell lines were used to determine the DNA-binding-activity of Ku86, a key protein of this repair mechanism involved in the recognition of the broken DNA ends and in the initiation of the DSBs repair process. As in EJ activity, all the MM cell lines showed an increased Ku86 binding respect to normal cells confirming the aberrant activation of the NHEJ pathway in MM cells. Interestingly, we did not observe significant differences in Ku86 level in nuclear extracts between PBMCs and MM cell lines suggesting that the difference in the Ku86 DNA-binding-activity was likely a functional and not due to disparity in the protein levels. We further investigated the link between this aberrant NHEJ activity and MM genomic instability using an immune-fluorescent based assay for DSBs. We observed an increased constitutive DNA damage in the absence of treatment with DSB-inducing agents in 5 of 6 MM cell lines compared with normal PBMCs. Most importantly, we noticed a direct correlation between the basal level of DSBs and the Ku86-binding-affinity. Furthermore, all the MM cell lines showed little or no ability to repair ionizing radiation (IR)-induced DNA damage compared to normal cells as well as no change in the Ku86-binding-affinity after stimulation suggesting that the aberrant NHEJ pathway in MM might represent a response to the constitutive endogenous DNA damage in these cells. We have also observed that 2 key NHEJ genes (Ku86 and Artemis) are overexpressed in MM compared to MGUS and normal plasma cells and their overexpression correlates with a shortened overall survival in MM suggesting that an hyper-activation of this pathway could have a potential role in MM progression and prognosis. Ongoing experiments are assessing the NHEJ activity in primary MM cells to correlate with clinical outcome. In conclusion, our data suggests that an aberrant NHEJ in the context of a constitutive endogenous DNA damage might contribute to the high frequency of chromosome abnormalities in MM cells, thus potentially playing a central role in the tumor progression and as an important prognostic marker in this disease. Disclosures: Anderson: Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

scholarly journals Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Agnieszka Siomek ◽  
Kamil Brzoska ◽  
Barbara Sochanowicz ◽  
Daniel Gackowski ◽  
Rafal Rozalski ◽  
...  
Keyword(s):  
Dna Damage ◽  
Superoxide Dismutase ◽  
Dna Binding ◽  
Urinary Excretion ◽  
Oxidative Dna Damage ◽  
Binding Activity ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Organ Specific ◽  
Damaged Dna

Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.

Regulation and mechanisms of gene amplification

1995 ◽  
Vol 347 (1319) ◽  
pp. 49-56 ◽  
Keyword(s):  
Dna Damage ◽  
Genetic Analysis ◽  
Mammalian Cells ◽  
Chromosome Breakage ◽  
Cellular Responses ◽  
Human Cells ◽  
Aspartate Transcarbamylase ◽  
Tumour Suppressor Function ◽  
Damage Recognition ◽  
Sister Chromatids

Amplification in rodent cells usually involves bridge-breakage-fusion (bbf) cycles initiated either by endto-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite A-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving bbf cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras , or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe . Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.

scholarly journals PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

2008 ◽  
Vol 183 (7) ◽  
pp. 1203-1212 ◽  
Author(s):  
Kazuto Sugimura ◽  
Shin-ichiro Takebayashi ◽  
Hiroshi Taguchi ◽  
Shunichi Takeda ◽  
Katsuzumi Okumura
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Parp Inhibitor ◽  
Small Interfering Rnas ◽  
Parp 1 ◽  
Dt40 Cells ◽  
Damaged Dna

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.

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