Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis

2001 ◽  
Vol 114 (20) ◽  
pp. 3771-3778 ◽  
Author(s):  
Damien D’Amours ◽  
Frédéric R. Sallmann ◽  
Vishva M. Dixit ◽  
Guy G. Poirier
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Catalytic Activity ◽  
Dna Binding ◽  
Energy Levels ◽  
Mouse Fibroblast ◽  
Dominant Negative ◽  
Energy Depletion ◽  
Parp 1

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.

scholarly journals Cleavage and Inactivation of ATM during Apoptosis

1999 ◽  
Vol 19 (9) ◽  
pp. 6076-6084 ◽  
Author(s):  
Graeme C. M. Smith ◽  
Fabrizio d’adda di Fagagna ◽  
Nicholas D. Lakin ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Caspase 3 ◽  
Cysteine Proteases ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Dna Damage Signalling ◽  
In Cells

ABSTRACT The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

Histone Deacetylase Inhibitors Promote Abnormal Binding of DNA Repair Proteins to DNA Double Strand Breaks: Consequences for DNA Repair and Genomic Instability?.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 186-186
Author(s):  
Carine Robert ◽  
Ivana Gojo ◽  
Feyruz V. Rassool
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Lines ◽  
Parp 1

Abstract Abstract 186 Histone Deacetylase inhibitors (HDi) affect gene expression through induction of histone acetylation and lead also to the acetylation of many other proteins which could affect their cellular activity. We have previously shown that HDi trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses, but actual DNA Double Strand Breaks (DSBs) which are significantly increased and persist for long periods of time compared with normal cells (Gaymes TJ. and al., Mol Cancer Res. 2006). This raises the hypothesis that HDi regulate the capacity of leukemic cells to repair DSBs, and the explanation for the increased and persistent DNA damage in leukemic cells may be that HDi directly acetylates proteins involved in DSB repair, thus decreasing repair activity. Non Homologous End-Joining (NHEJ) is one of the main pathways for the repair of DSBs in mammalian cells. While normal cells use NHEJ that is Ku and DNA-PKcs dependent, an alternative (Alt) NHEJ pathway (DNA-PKcs and Ku independent) involving Poly-ADP-Ribose Polymerase-1 (PARP-1), Werner syndrome helicase (WRN) and DNA LigaseIIIa proteins, has been identified and is responsible for deletions and translocations in cancer. We have recently reported that myeloid leukemia cells repair DSBs using this Alt NHEJ pathway (Sallmyr A. and al., Blood, 2008). Here we show that HDi treatment by Trichostatin A (300nM) results in differential acetylation of main NHEJ protein Ku70 in acute leukemia K562 cell line. In addition, PARP-1, active in several repair pathways, including single strand break repair and Alt NHEJ is also hyperacetylated in K562 cells after 1 and 6 hours of Trichostatin A treatment compared with control treatment. To investigate whether Trichostatin A treatment alters the binding of DNA repair proteins to DSBs, we used a chromatin immunoprecipitation (CHIP) assay in K562 cell line stably transfected with the DRNeo construct that can be induced to express a single DSB. Strikingly, CHIP analysis shows that PARP-1 is increased at the DSB after 1 hour of Trichostatin A treatment, compared with controls. Preliminary CHIP analysis for the protein XRCC1, necessary for the final step of Alt NHEJ repair, shows that it is decreased at the DSB site. Importantly, AML patients treated with the HDi MS-275 in vivo show significantly increased colocalization of PARP-1 and gH2A.x, a marker for DSBs, compared with pretreatment controls, confirming our in vitro data in leukemia cell lines. Altogether, these data suggest that HDi treatment leads to an increased presence of PARP-1 at DSBs, and that this may prevent subsequent critical repair steps, providing a possible explanation for the persistence of DNA damage. Finally, to determine whether DSB repair activity is indeed decreased with HDi treatment, we used an in vivo NHEJ repair assay in K562 and HL60 acute leukemia cell lines before and after treatment with Trichostatin A for 1 hour. Both leukemia cell lines demonstrate a significant decrease in the capacity of the cells to repair DSBs following Trichostatin A treatment. These results suggest that HDi result in both a physical and functional alteration of proteins participating in DNA repair pathways, leading to a decrease in NHEJ activity. The decrease in Alt NHEJ activity may have implications for genomic instability, diminishing abnormal repair following HDi treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rab7 Reduces α-Synuclein Toxicity in Rats and Primary Neurons

2021 ◽  
Author(s):  
Éva M. Szegõ ◽  
Eva M. Szegö ◽  
Chris Van den Haute ◽  
Lennart Höfs ◽  
Veerle Baekelandt ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rat Brain ◽  
Neuronal Degeneration ◽  
Degradation Pathway ◽  
Alpha Synuclein ◽  
Dominant Negative ◽  
Primary Neurons ◽  
Vicious Cycle ◽  
Axon Terminals

Abstract BackgroundDuring the pathogenesis of Parkinson’s disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo.MethodsThe disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or its dominant-negative T22N mutant as a control. The cellular integrity was quantified by morphological and biochemical analyses.ResultsIn primary neurons, WT Rab7 rescued the αSyn -induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn -induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain.ConclusionRab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.

scholarly journals Leukemia-Related Transcription Factor TEL Is Negatively Regulated through Extracellular Signal-Regulated Kinase-Induced Phosphorylation

2004 ◽  
Vol 24 (8) ◽  
pp. 3227-3237 ◽  
Author(s):  
Kazuhiro Maki ◽  
Honoka Arai ◽  
Kazuo Waga ◽  
Ko Sasaki ◽  
Fumihiko Nakamura ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Effect ◽  
Phosphorylation Sites ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser213 and Ser257. TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser213 and Ser257 functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.

scholarly journals Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2

Brain ◽  
2019 ◽  
Vol 142 (8) ◽  
pp. 2352-2366 ◽  
Author(s):  
Guo-zhong Yi ◽  
Guanglong Huang ◽  
Manlan Guo ◽  
Xi’an Zhang ◽  
Hai Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Progression Free Survival ◽  
Recurrent Glioblastoma ◽  
Dna Lesions ◽  
Dna Repair Proteins

Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

Drug-induced DNA damage and tumor chemosensitivity.

1990 ◽  
Vol 8 (12) ◽  
pp. 2062-2084 ◽  
Author(s):  
R J Epstein
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Tumor Cell ◽  
Malignant Disease ◽  
Dna Lesions ◽  
Drug Induced ◽  
Cell Subpopulations ◽  
Therapeutic Cell

Cytotoxic drugs act principally by damaging tumor-cell DNA. Quantitative analysis of this interaction provides a basis for understanding the biology of therapeutic cell kill as well as a rational strategy for optimizing and predicting tumor response. Recent advances have made it possible to correlate assayed DNA lesions with cytotoxicity in tumor cell lines, in animal models, and in patients with malignant disease. In addition, many of the complex interrelationships between DNA damage, DNA repair, and alterations of gene expression in response to DNA damage have been defined. Techniques for modulating DNA damage and cytotoxicity using schedule-specific cytotoxic combinations, DNA repair inhibitors, cell-cycle manipulations, and adjunctive noncytotoxic drug therapy are being developed, and critical therapeutic targets have been identified within tumor-cell subpopulations and genomic DNA alike. Most importantly, methods for predicting clinical response to cytotoxic therapy using both in vitro markers of tumor-cell sensitivity and in vivo measurements of drug-induced DNA damage are now becoming a reality. These advances can be expected to provide a strong foundation for the development of innovative cytotoxic drug strategies over the next decade.

scholarly journals The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

1999 ◽  
Vol 10 (11) ◽  
pp. 3583-3594 ◽  
Author(s):  
Robert M. Brosh ◽  
Adayabalam S. Balajee ◽  
Rebecca R. Selzer ◽  
Morten Sunesen ◽  
Luca Proietti De Santis ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Genetic Disorder ◽  
Cockayne Syndrome ◽  
Premature Aging ◽  
Atpase Domain ◽  
Acidic Region

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA andCSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, theCSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

Differential response of prostate cancer cells to ionizing radiation: The RB status.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 106-106
Author(s):  
Robert Benjamin Den ◽  
Steve Ciment ◽  
Ankur Sharma ◽  
Hestia Mellert ◽  
Steven McMahon ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Ionizing Radiation ◽  
Cell Survival ◽  
Hormone Sensitive ◽  
Clonogenic Cell ◽  
Castrate Resistant

106 Background: Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer death in U.S. men. The retinoblastoma tumor suppressor protein, RB, plays a critical role in cell cycle regulation and loss of RB has been observed in 25-30% of prostate cancers. We have previously shown that RB loss results in a castrate resistant phenotype, however the consequences of RB status with regard to radiation response are unknown. We hypothesized that RB loss would downregulate the G1-S cell cycle checkpoint arrest normally induced by irradiation, inhibit DNA repair, and subsequently sensitize cells to ionizing radiation. Methods: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAP-C4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of RB using shRNA). Gamma H2AX assays were used to quantitate DNA damage and PARP cleavage and Caspase 3 were used to quantitate apoptosis. FACS analysis with BrdU was used to assess the cell cycle. Cell survival was measured using the clonogenic cell survival assay. In vivo work was performed in nude mice with tumor xenografts. Results: We observed that loss of RB increased radioresponsiveness in both transient and clonogenic cell survival assays in both hormone sensitive and castrate resistant cell lines (p<0.05). Cell death was not mediated through increased apoptosis nor was perturbations in cell cycle noted. However, loss of RB effected DNA repair as measured by gamma H2AX staining as well as cellular senescence. In vivo xenografts of the RB deficient tumors exhibited diminished tumor mass, lower PSA kinetics and decreased tumor growth after treatment with single fraction of ionizing radiation in comparison to RB intact tumors (p<0.05). Conclusions: Loss of RB results in a differential response to ionizing radiation. Isogenic cells with RB knockdown are more sensitive to DNA damage and result in reduced cell survival. The underlying mechanism appears to be related to DNA damage repair and cellular senescence.

scholarly journals MicroRNA regulation of the MRN complex impacts DNA damage, cellular senescence and angiogenic signaling

2017 ◽  
Author(s):  
Cristina Espinosa-Diez ◽  
RaeAnna Wilson ◽  
Namita Chatterjee ◽  
Clayton Hudson ◽  
Rebecca Ruhl ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Targeted Delivery ◽  
Genotoxic Stress ◽  
Telomerase Activity ◽  
Loss Of Function ◽  
Mrn Complex ◽  
Vegf Signaling

AbstractMicroRNAs contribute to biological robustness by buffering cellular processes from external perturbations. Here we report an unexpected link between DNA damage response and angiogenic signaling that is buffered by two distinct microRNAs. We demonstrate that genotoxic stress-induced miR-494 and miR-99b inhibit the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Functionally, gain and loss of function experiments show that miR-494 and miR-99b affect telomerase activity, activate p21 and Rb pathways and diminish angiogenic sproutingin vitroandin vivo. Genetic and pharmacological disruption of VEGFR-2 signaling and the MRN complex reveal a surprising co-dependency of these pathways in regulating endothelial senescence and proliferation. Vascular-targeted delivery of miR-494 decreases both growth factor-induced and tumor angiogenesis in mouse models. Mechanistically, disruption of the MRN complex induced CD44, a known driver of senescence and regulator of VEGF signaling in addition to suppressing IL-13 a stimulator of VEGF signaling. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence and highlight the potential of targeting DNA repair to disrupt pathological angiogenesis.

scholarly journals Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Evi Goulielmaki ◽  
Anna Ioannidou ◽  
Maria Tsekrekou ◽  
Kalliopi Stratigi ◽  
Ioanna K. Poutakidou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Glucose Transporter ◽  
Ex Vivo ◽  
Disease Onset ◽  
Metabolic Reprogramming ◽  
Inflammatory Stimuli ◽  
Underlying Mechanisms ◽  
Repair Defect

AbstractDNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1F/−) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1F/− animal sera and are secreted in macrophage media after DNA damage. The Er1F/− EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging.

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