RESUMO
Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.
Assuntos
Ciclopropanos/farmacologia , Glicosídeo Hidrolases/toxicidade , Microssomos Hepáticos/efeitos dos fármacos , Administração Oral , Animais , Ciclopropanos/administração & dosagem , Ciclopropanos/química , Ciclopropanos/farmacocinética , Glicosídeo Hidrolases/administração & dosagem , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/farmacocinética , Meia-Vida , Humanos , Camundongos , Microssomos Hepáticos/metabolismoRESUMO
Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition.
Assuntos
Fluorescência , Glicosídeo Hidrolases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Medições Luminescentes/métodos , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/antagonistas & inibidores , Humanos , Relação Estrutura-Atividade , Fatores de TempoRESUMO
RNA-Seq is a recently developed sequencing technology, that through the analysis of cDNA allows for unique insights into the transcriptome of a cell. The data generated by RNA-Seq provides information on gene expression, alternative splicing events and the presence of non-coding RNAs. It has been realised non-coding RNAs are more then just artefacts of erroneous transcription and play vital regulatory roles at the genomic, transcriptional and translational level. Transcription of the DNA sense strand produces antisense transcripts. This is known as antisense transcription and often results in the production of non-coding RNAs that are complementary to their associated sense transcripts. Antisense tran-scription has been identified in bacteria, fungi, protozoa, plants, invertebrates and mammals. It seems that antisense tran-scriptional 'hot spots' are located around nucleosome-free regions such as those associated with promoters, indicating that it is likely that antisense transcripts carry out important regulatory functions. This underlines the importance of identifying the presence and understanding the function of these antisense non-coding RNAs. The information concerning strand ori-gin is often lost during conventional RNA-Seq; capturing this information would substantially increase the worth of any RNA-Seq experiment. By manipulating the input cDNA during the template preparation stage it is possible to retain this vital information. This forms the basis of strand-specific RNA-Seq. With an ability to unlock immense portions of new in-formation surrounding the transcriptome, this cutting edge technology may hold the key to developing a greater under-standing of the transcriptome.
RESUMO
Autophagy is believed to be an important process during tumorgenesis, and in recent years it has been shown to be modulated in response to a number of conventional anticancer agents. Furthermore, the development of targeted small molecule inhibitors, such as those to the PI3K-AKT-mTOR pathway, has presented a molecular link between the disruption of this signalling cascade and the process of autophagy. The cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. The field of autophagy is still evolving, and there is a constantly changing set of criteria for the assessment of the process in cells, tissues, and organs. The gold standard technique for analyzing autophagy in mammalian cells remains transmission electron microscopy, which has many limitations and is often difficult to perform on in vivo tissue including human tumor xenografts. In order to monitor autophagy in human tumor xenogaft tissue, we have taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagosome-associated protein, microtubule-associated protein 1 light chain 3 (LC3), in human tumor xenografts. After synthesis, LC3 is cleaved to form LC3-I, and upon induction of autophagy, LC3-I is conjugated to the lipid phosphatidylethanolamine to form LC3-II, which is tightly bound to the membrane of the autophagosome. It is thought that detection of endogenous LC3-II by IHC could be difficult because of the relatively low level of expression of the protein. Here we present the validation of an IHC method to detect LC3 in human tumor xenografts that we believe is able to distinguish LC3-I from LC3-II. It is hoped that this assay can become a useful tool for the detection of autophagy in preclinical xenograft models and determine the effects of anticancer therapies on the autophagic process.
Assuntos
Autofagia/efeitos dos fármacos , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Imunofluorescência/métodos , Humanos , Immunoblotting/métodos , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Transplante de Neoplasias , Transplante HeterólogoRESUMO
There is mounting evidence of androgen receptor signaling inducing genome instability and changing DNA repair capacity in prostate cancer cells. Expression of genes associated with base excision repair (BER) is increased with prostate cancer progression and correlates with poor prognosis. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are key enzymes in BER that elongate and degrade PAR polymers on target proteins. While PARP inhibitors have been tested in clinical trials and are a promising therapy for prostate cancer patients with TMPRSS2-ERG fusions and mutations in DNA repair genes, PARG inhibitors have not been evaluated. We show that PARG is a direct androgen receptor (AR) target gene. AR is recruited to the PARG locus and induces PARG expression. Androgen ablation combined with PARG inhibition synergistically reduces BER capacity in independently derived LNCaP and LAPC4 prostate cancer cell lines. A combination of PARG inhibition with androgen ablation or with the DNA damaging drug, temozolomide, significantly reduces cellular proliferation and increases DNA damage. PARG inhibition alters AR transcriptional output without changing AR protein levels. Thus, AR and PARG are engaged in reciprocal regulation suggesting that the success of androgen ablation therapy can be enhanced by PARG inhibition in prostate cancer patients.
Assuntos
Inibidores de Glicosídeo Hidrolases/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicosídeo Hidrolases/metabolismo , Humanos , Masculino , Terapia de Alvo MolecularRESUMO
The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.
Assuntos
DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerases/genética , Processamento de Proteína Pós-Traducional , Reparo de DNA por Recombinação , Telômero/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , Dano ao DNA , DNA de Neoplasias/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fase G2 , Glicosídeo Hidrolases/metabolismo , Células HeLa , Chaperonas de Histonas/antagonistas & inibidores , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Telômero/ultraestrutura , Homeostase do Telômero , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismoRESUMO
Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Chlorocebus aethiops , Citocromos c/metabolismo , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Mitochondrial fission ensures organelle inheritance during cell division and participates in apoptosis. The fission protein hFis1 triggers caspase-dependent cell death, by causing the release of cytochrome c from mitochondria. Here we show that mitochondrial fission induced by hFis1 is genetically distinct from apoptosis. In cells lacking the multidomain proapoptotic Bcl-2 family members Bax and Bak (DKO), hFis1 caused mitochondrial fragmentation but not organelle dysfunction and apoptosis. Similarly, a mutant in the intermembrane region of hFis1-induced fission but not cell death, further dissociating mitochondrial fragmentation from apoptosis induction. Selective correction of the endoplasmic reticulum (ER) defect of DKO cells restored killing by hFis1, indicating that death by hFis1 relies on the ER gateway of apoptosis. Consistently, hFis1 did not directly activate BAX and BAK, but induced Ca(2+)-dependent mitochondrial dysfunction. Thus, hFis1 is a bifunctional protein that independently regulates mitochondrial fragmentation and ER-mediated apoptosis.
Assuntos
Apoptose/fisiologia , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Respiração Celular/fisiologia , Análise Mutacional de DNA , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Humanos , Proteínas de Membrana , Camundongos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Permeabilidade , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína X Associada a bcl-2/deficiênciaRESUMO
Inhibitors of poly(ADP-ribose) polymerase (PARP) have demonstrated efficacy in women with BRCA-mutant ovarian cancer. However, only 15%-20% of ovarian cancers harbor BRCA mutations, therefore additional therapies are required. Here, we show that a subset of ovarian cancer cell lines and ex vivo models derived from patient biopsies are sensitive to a poly(ADP-ribose) glycohydrolase (PARG) inhibitor. Sensitivity is due to underlying DNA replication vulnerabilities that cause persistent fork stalling and replication catastrophe. PARG inhibition is synthetic lethal with inhibition of DNA replication factors, allowing additional models to be sensitized by CHK1 inhibitors. Because PARG and PARP inhibitor sensitivity are mutually exclusive, our observations demonstrate that PARG inhibitors have therapeutic potential to complement PARP inhibitor strategies in the treatment of ovarian cancer.
Assuntos
Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Feminino , Glicosídeo Hidrolases/antagonistas & inibidores , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Quinazolinonas/farmacologiaRESUMO
Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair-deficient cells compared with homologous repair-proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi. SIGNIFICANCE: PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Feminino , Inativação Gênica , Glicosídeo Hidrolases/genética , Humanos , Camundongos Nus , Terapia de Alvo Molecular , Oxaliplatina/farmacologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reparo de DNA por Recombinação , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Upon DNA binding the poly(ADP-ribose) polymerase family of enzymes (PARPs) add multiple ADP-ribose subunits to themselves and other acceptor proteins. Inhibitors of PARPs have become an exciting and real prospect for monotherapy and as sensitizers to ionising radiation (IR). The action of PARPs are reversed by poly(ADP-ribose) glycohydrolase (PARG). Until recently studies of PARG have been limited by the lack of an inhibitor. Here, a first in class, specific, and cell permeable PARG inhibitor, PDD00017273, is shown to radiosensitize. Further, PDD00017273 is compared with the PARP1/2/3 inhibitor olaparib. Both olaparib and PDD00017273 altered the repair of IR-induced DNA damage, resulting in delayed resolution of RAD51 foci compared with control cells. However, only PARG inhibition induced a rapid increase in IR-induced activation of PRKDC (DNA-PK) and perturbed mitotic progression. This suggests that PARG has additional functions in the cell compared with inhibition of PARP1/2/3, likely via reversal of tankyrase activity and/or that inhibiting the removal of poly(ADP-ribose) (PAR) has a different consequence to inhibiting PAR addition. Overall, our data are consistent with previous genetic findings, reveal new insights into the function of PAR metabolism following IR and demonstrate for the first time the therapeutic potential of PARG inhibitors as radiosensitizing agents.
Assuntos
Antineoplásicos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Histonas/metabolismo , Humanos , Micronúcleos com Defeito Cromossômico , Mitose/genética , Fenótipo , Poli(ADP-Ribose) Polimerases/metabolismo , Rad51 Recombinase/metabolismo , Tolerância a Radiação/genética , Radiação IonizanteRESUMO
Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically.
Assuntos
Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Camundongos da Linhagem 129 , Camundongos Knockout , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/efeitos dos fármacosRESUMO
DNA damage repair enzymes are promising targets in the development of new therapeutic agents for a wide range of cancers and potentially other diseases. The enzyme poly(ADP-ribose) glycohydrolase (PARG) plays a pivotal role in the regulation of DNA repair mechanisms; however, the lack of potent drug-like inhibitors for use in cellular and in vivo models has limited the investigation of its potential as a novel therapeutic target. Using the crystal structure of human PARG in complex with the weakly active and cytotoxic anthraquinone 8a, novel quinazolinedione sulfonamides PARG inhibitors have been identified by means of structure-based virtual screening and library design. 1-Oxetan-3-ylmethyl derivatives 33d and 35d were selected for preliminary investigations in vivo. X-ray crystal structures help rationalize the observed structure-activity relationships of these novel inhibitors.
Assuntos
Reparo do DNA , Desenho de Fármacos , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Quinazolinonas/química , Quinazolinonas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Domínio Catalítico , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Inibidores de Glicosídeo Hidrolases/farmacocinética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Células HeLa , Humanos , Masculino , Camundongos , Modelos Moleculares , Quinazolinonas/administração & dosagem , Quinazolinonas/farmacocinética , Relação Estrutura-AtividadeRESUMO
In response to many apoptotic stimuli, Bcl-2 family pro-apoptotic members, such as Bax and Bak, are activated. This results in their oligomerization, permeabilization of the outer mitochondrial membrane, and release of many proteins that are normally confined in the mitochondrial inter-membrane space. Among these proteins are cytochrome c, Smac/DIABLO, OMI/HtrA2, AIF and endonuclease G. Mitochondrial outer membrane permeabilization (MOMP) is also associated with fragmentation of the mitochondrial network. The mechanisms that lead to the oligomerization of proapoptotic members of the Bcl-2 family and to MOMP are still unclear and the role of mitochondrial fission in these events remains elusive.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , HumanosRESUMO
The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous BASP1 expression augments transcriptional activation by WT1. BASP1 is present in the developing nephron structures of the embryonic kidney and, coincident with that of WT1, its expression is restricted to the highly specialized podocyte cells of the adult kidney. Taken together, our results show that BASP1 is a WT1-associated factor that can regulate WT1 transcriptional activity.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Proteínas WT1/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular , DNA Complementar/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes do Tumor de Wilms , Células HeLa , Humanos , Técnicas In Vitro , Rim/crescimento & desenvolvimento , Rim/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas WT1/química , Proteínas WT1/genéticaRESUMO
Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the ß-nicotinamide adenine dinucleotide (ß-NAD+) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.
Assuntos
ADP-Ribosilação/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , ADP-Ribosilação/genética , Adenosina Difosfato Ribose/metabolismo , Animais , Reparo do DNA/genética , Reparo do DNA/fisiologia , Glicosilação , Humanos , Poli(ADP-Ribose) Polimerases/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.
Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Glicosídeo Hidrolases/antagonistas & inibidores , Recombinação Homóloga , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Transporte/metabolismo , DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/fisiologia , Humanos , Taninos Hidrolisáveis/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
We present IncucyteDRC, an R package for the analysis of data from live cell imaging cell proliferation experiments carried out on the Essen Biosciences IncuCyte ZOOM instrument. The package provides a simple workflow for summarising data into a form that can be used to calculate dose response curves and EC50 values for small molecule inhibitors. Data from different cell lines, or cell lines grown under different conditions, can be normalised as to their doubling time. A simple graphical web interface, implemented using shiny, is provided for the benefit of non-R users. The software is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation using the IncuCyte ZOOM.
RESUMO
After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.