Ribosome biogenesis is an essential, energy demanding process whose deregulation has been implicated in cancer, aging, and neurodegeneration. Ribosome biogenesis is therefore under surveillance of pathways including the p53 tumor suppressor. Here, we first performed a high-content siRNA-based screen of 175 human ribosome biogenesis factors, searching for impact on p53. Knock-down of 4 and 35 of these proteins in U2OS cells reduced and increased p53 abundance, respectively, including p53 accumulation after depletion of BYSL, DDX56, and WDR75, the effects of which were validated in several models. Using complementary approaches including subcellular fractionation, we demonstrate that endogenous human WDR75 is a nucleolar protein and immunofluorescence analysis of ectopic GFP-tagged WDR75 shows relocation to nucleolar caps under chemically induced nucleolar stress, along with several canonical nucleolar proteins. Mechanistically, we show that WDR75 is required for pre-rRNA transcription, through supporting the maintenance of physiological levels of RPA194, a key subunit of the RNA polymerase I. Furthermore, WDR75 depletion activated the RPL5/RPL11-dependent p53 stabilization checkpoint, ultimately leading to impaired proliferation and cellular senescence. These findings reveal a crucial positive role of WDR75 in ribosome biogenesis and provide a resource of human ribosomal factors the malfunction of which affects p53.

• MeSH
• buněčné jadérko genetika   metabolismus   MeSH
• DEAD-box RNA-helikasy metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• molekuly buněčné adheze metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• prekurzory RNA metabolismus   MeSH
• ribozomální proteiny * genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.

• MeSH
• chromatin MeSH
• DNA genetika   MeSH
• emetin * farmakologie   MeSH
• jednovláknová DNA * MeSH
• replikace DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET-a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.

• MeSH
• antitumorózní látky * farmakologie   terapeutické užití   MeSH
• bortezomib farmakologie   terapeutické užití   MeSH
• chemorezistence MeSH
• disulfiram farmakologie   MeSH
• inhibitory proteasomu farmakologie   terapeutické užití   MeSH
• lidé MeSH
• lokální recidiva nádoru farmakoterapie   MeSH
• mnohočetný myelom * patologie   MeSH
• nádorové buněčné linie MeSH
• přehodnocení terapeutických indikací léčivého přípravku MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The biosynthesis of ribosomes is a complex process that requires the coordinated action of many factors and a huge energy investment from the cell. Ribosomes are essential for protein production, and thus for cellular survival, growth and proliferation. Ribosome biogenesis is initiated in the nucleolus and includes: the synthesis and processing of ribosomal RNAs, assembly of ribosomal proteins, transport to the cytoplasm and association of ribosomal subunits. The disruption of ribosome biogenesis at various steps, with either increased or decreased expression of different ribosomal components, can promote cell cycle arrest, senescence or apoptosis. Additionally, interference with ribosomal biogenesis is often associated with cancer, aging and age-related degenerative diseases. Here, we review current knowledge on impaired ribosome biogenesis, discuss the main factors involved in stress responses under such circumstances and focus on examples with clinical relevance.

• MeSH
• biogeneze organel MeSH
• lidé MeSH
• nádory metabolismus   MeSH
• ribozomální proteiny metabolismus   MeSH
• ribozomy metabolismus   MeSH
• stárnutí metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway - RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.

• MeSH
• biogeneze organel * MeSH
• fyziologický stres MeSH
• genetická transkripce * MeSH
• jaderné proteiny metabolismus   MeSH
• kontrolní body buněčného cyklu MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• proliferace buněk MeSH
• proteiny vázající RNA metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• ribozomální proteiny metabolismus   MeSH
• ribozomy metabolismus   MeSH
• RNA ribozomální biosyntéza   MeSH
• RNA-polymerasa I genetika   MeSH
• signální transdukce MeSH
• vedlejší histokompatibilní antigeny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.

• MeSH
• alkoholismus farmakoterapie   epidemiologie   MeSH
• antitumorózní látky * farmakologie   terapeutické užití   MeSH
• cílená molekulární terapie MeSH
• disulfiram chemie   farmakologie   terapeutické užití   MeSH
• dospělí MeSH
• jaderné proteiny chemie   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• měď chemie   MeSH
• myši MeSH
• nádory farmakoterapie   metabolismus   mortalita   patologie   MeSH
• odvykací prostředky alkoholu * farmakologie   terapeutické užití   MeSH
• přehodnocení terapeutických indikací léčivého přípravku * MeSH
• proteinové agregáty MeSH
• proteolýza účinky léků   MeSH
• reakce na tepelný šok účinky léků   MeSH
• vazba proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Dánsko epidemiologie   MeSH

Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, pancreatic, gastric, and hepatocellular cancers. Despite its routine clinical use, little is known about the responses it induces in cancer cells. Therefore the whole-cell proteomics study was conducted to characterize the cellular response induced by oxaliplatin. Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3μM (5×IC50) for 240min (half-time to caspase activation). The proteomes of un-/treated cells were then compared by high-resolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in at least two replicates. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. BIOLOGICAL SIGNIFICANCE: We have performed a whole-cell proteomic study of cellular response to oxaliplatin treatment, which is the drug predominantly used in the treatment of colorectal cancer. Compared to its predecessors, cisplatin and carboplatin, there is only a small fraction of studies dedicated to oxaliplatin. From those studies, most of them are focused on modification of treatment regimens or study of oxaliplatin in new cancer diagnoses. Cellular response hasn't been studied deeply and to our best knowledge, this is the first whole-cell proteomics study focused exclusively to this important topic, which can help to understand molecular mechanisms of action.

• MeSH
• antitumorózní látky farmakologie   MeSH
• buněčné jadérko účinky léků   MeSH
• fyziologický stres MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádory farmakoterapie   patologie   MeSH
• organoplatinové sloučeniny farmakologie   MeSH
• poškození DNA * MeSH
• proteom analýza   účinky léků   metabolismus   MeSH
• proteomika metody   MeSH
• ribozomy účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1's role in HR, with potential clinical implications for cancer treatment.

• MeSH
• časové faktory MeSH
• chromatin metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• ftalaziny farmakologie   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• homologní rekombinace * MeSH
• interakční proteinové domény a motivy MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• nádory vaječníků farmakoterapie   enzymologie   genetika   patologie   MeSH
• PARP inhibitory farmakologie   MeSH
• piperaziny farmakologie   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• restrukturace chromatinu * MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• transfekce MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Based on a series of basic, preclinical and clinical studies, the Poly (ADP-ribose) Polymerase 1 (PARP1) inhibitor, olaparib, has recently been approved for use in ovarian cancer patients with BRCA1 or BRCA2 mutations. By identifying novel predictive biomarkers of tumour cell sensitivity to olaparib, it is possible that the utility of PARP inhibitors could be extended beyond this patient subgroup. Many of the known genetic determinants of PARP inhibitor response have key roles in DNA damage response (DDR) pathways. Although protein ubiquitylation is known to play an important role in regulating the DDR, the exact mechanisms by which this occurs are not fully understood. Using two parallel RNA interference-based screening approaches, we identified the E3 ubiquitin ligase, CBLC, as a candidate biomarker of response to olaparib. We validated this observation by demonstrating that silencing of CBLC causes increased sensitivity to olaparib in breast cancer cell line models and that defective homologous recombination (HR) DNA repair is the likely cause. This data provides an example of how defects in the ubiquitin machinery have the potential to influence the response of tumour cells to PARP inhibitors.

• MeSH
• časové faktory MeSH
• ftalaziny farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu farmakoterapie   enzymologie   genetika   patologie   MeSH
• oprava DNA MeSH
• PARP inhibitory farmakologie   MeSH
• piperaziny farmakologie   MeSH
• poly(ADP-ribosa)-polymerasy genetika   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• protein BRCA2 genetika   metabolismus   MeSH
• protoonkogenní proteiny c-cbl genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• rekombinace genetická MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• transfekce MeSH
• ubikvitinace MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH