RESUMO
PURPOSE: High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. METHODS AND MATERIALS: In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. RESULTS: We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. CONCLUSIONS: Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy.
Assuntos
Antineoplásicos , Neoplasias , Radiossensibilizantes , Feminino , Humanos , Ensaios de Triagem em Larga Escala/métodos , Detecção Precoce de Câncer , Radiossensibilizantes/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular TumoralRESUMO
Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml-/- mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.
Assuntos
Arsênio , Sumoilação , Animais , Elementos de DNA Transponíveis , Células-Tronco Embrionárias , Camundongos , Corpos Nucleares , Fatores de TranscriçãoRESUMO
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/patogenicidade , Transporte Proteico/fisiologia , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Receptores CCR1/metabolismo , Receptores CXCR4/metabolismo , Via Secretória/fisiologiaRESUMO
BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.
Assuntos
Precursores de RNA/genética , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Linhagem Celular , Análise por Conglomerados , Biologia Computacional/métodos , Éxons , Fibroblastos , Perfilação da Expressão Gênica , Humanos , Reprodutibilidade dos Testes , Transdução de Sinais , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteínas ras/metabolismoRESUMO
SIGNIFICANCE: Cellular metabolic activity impacts the production of reactive oxygen species (ROS), both positively through mitochondrial oxidative processes and negatively by promoting the production of reducing agents (including NADPH and reduced glutathione). A defined metabolic state in cancer cells is critical for cell growth and long-term self-renewal, and such state is intrinsically associated with redox balance. Promyelocytic leukemia protein (PML) regulates several biological processes, at least in part, through its ability to control the assembly of PML nuclear bodies (PML NBs). Recent Advances: PML is oxidation-prone, and oxidative stress promotes NB biogenesis. These nuclear subdomains recruit many nuclear proteins and regulate their SUMOylation and other post-translational modifications. Some of these cargos-such as p53, SIRT1, AKT, and mammalian target of rapamycin (mTOR)-are key regulators of cell fate. PML was also recently shown to regulate oxidation. CRITICAL ISSUES: While it was long considered primarily as a tumor suppressor protein, PML-regulated metabolic switch uncovered that this protein could promote survival and/or stemness of some normal or cancer cells. In this study, we review the recent findings on this multifunctional protein. FUTURE DIRECTIONS: Studying PML scaffolding functions as well as its fine role in the activation of p53 or fatty acid oxidation will bring new insights in how PML could bridge oxidative stress, senescence, cell death, and metabolism. Antioxid. Redox Signal. 26, 432-444.
Assuntos
Oxirredução , Estresse Oxidativo , Proteína da Leucemia Promielocítica/metabolismo , Transdução de Sinais , Animais , Autofagia , Metabolismo Energético , Humanos , Corpos de Inclusão Intranuclear/química , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica/química , Proteína da Leucemia Promielocítica/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Sumoilação , Proteína Supressora de Tumor p53/metabolismoRESUMO
Fasci et al proposed that a SENP1-mediated switch from SUMO2 to SUMO1 conjugation on Lys(65) in promyelocytic leukemia protein (PML) is required for arsenic-induced PML degradation, the basis for the antileukemic activity of arsenic. We found that PML or PML/RARA (retinoic acid receptor α) mutants that cannot be SUMO-conjugated on this specific site nevertheless underwent immediate arsenic-triggered SUMO modification. Moreover, these mutants were efficiently degraded in cells and even in vivo, demonstrating that SUMOylation of Lys(65) was dispensable for arsenic response. The existence and putative role of a SUMO switch on PML should thus be reassessed.
Assuntos
Arsênio , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Proteínas Supressoras de Tumor/genética , UbiquitinaçãoRESUMO
We previously examined the expression of Rbm5 during myoblast differentiation and found significantly more protein in the early stages of skeletal myoblast differentiation than during the later stages. We decided to determine if this elevated level was necessary for differentiation. Our hypothesis was that if high levels of Rbm5 protein expression were necessary for the initiation of skeletal myoblast differentiation, then inhibition of expression would prevent differentiation. Our long-term objective is to inhibit Rbm5 expression and examine the effect on H9c2 differentiation. Towards this end, stable knockdown clones and transient knockdown populations were generated. Expression analyses in H9c2 myoblasts demonstrated significant Rbm5 messenger RNA (mRNA) inhibition but, surprisingly, no effect on RBM5 protein levels. Expression of the Rbm5 paralogue Rbm10 was examined in order to (a) ensure no off-target knockdown effect, and (b) investigate any possible compensatory effects. RBM10 protein levels were found to be elevated, in both the clonal and transiently transfected populations. These results suggest that myoblast RBM5 expression is regulated by a process that includes RNA sequestration and/or controlled translation, and that (a) RBM5 function is compensated for by RBM10, and/or (b) RBM5 regulates RBM10 expression. We have developed a model to describe our findings, and suggest further experiments for testing its validity. Since upregulation of Rbm10 might compensate for downregulated Rbm5, and consequently might mask any potential knockdown effect, it could lead to incorrect conclusions regarding the importance of Rbm5 for differentiation. It is therefore imperative to determine how both RBM5 and RBM10 protein expression is regulated.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Mioblastos/citologia , Proteínas Nucleares/biossíntese , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Ativação Transcricional/genética , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: RBM10 is an RNA binding protein involved in the regulation of transcription, alternative splicing and message stabilization. Mutations in RBM10, which maps to the X chromosome, are associated with TARP syndrome, lung and pancreatic cancers. Two predominant isoforms of RBM10 exist, RBM10v1 and RBM10v2. Both variants have alternate isoforms that differ by one valine residue, at amino acid 354 (RBM10v1) or 277 (RBM10v2). It was recently observed that a novel point mutation at amino acid 354 of RBM10v1, replacing valine with glutamic acid, correlated with preferential expression of an exon 11 inclusion variant of the proliferation regulatory protein NUMB, which is upregulated in lung cancer. FINDINGS: We demonstrate, using the GLC20 male-derived small cell lung cancer cell line - confirmed to have only one X chromosome - that the two (+/-) valine isoforms of RBM10v1 and RBM10v2 result from alternative splicing. Protein modeling of the RNA Recognition Motif (RRM) within which the alteration occurs, shows that the presence of valine inhibits the formation of one of the two α-helices associated with RRM tertiary structure, whereas the absence of valine supports the α-helical configuration. We then show 2-fold elevated expression of the transcripts encoding the minus valine RBM10v1 isoform in GLC20 cells, compared to those encoding the plus valine isoform. This expression correlates with preferential expression of the lung cancer-associated NUMB exon 11 inclusion variant. CONCLUSIONS: Our observations suggest that the ability of RBM10v1 to regulate alternative splicing depends, at least in part, on a structural alteration within the second RRM domain, which influences whether RBM10v1 functions to support or repress splicing. A model is presented.
Assuntos
Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Carcinoma de Pequenas Células do Pulmão/genética , Linhagem Celular Tumoral , Éxons , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Motivos de Nucleotídeos , Mutação Puntual , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Valina/genética , Valina/metabolismoRESUMO
Centrosome amplification is a hallmark of human tumours. In flies, extra centrosomes cause spindle position defects that result in the expansion of the neural stem cell (NSC) pool and consequently in tumour formation. Here we investigated the consequences of centrosome amplification during mouse brain development and homeostasis. We show that centrosome amplification causes microcephaly due to inefficient clustering mechanisms, where NSCs divide in a multipolar fashion producing aneuploid cells that enter apoptosis. Importantly, we show that apoptosis inhibition causes the accumulation of highly aneuploid cells that lose their proliferative capacity and differentiate, thus depleting the pool of progenitors. Even if these conditions are not sufficient to halt brain development, they cause premature death due to tissue degeneration. Our results support an alternative concept to explain the etiology of microcephaly and show that centrosome amplification and aneuploidy can result in tissue degeneration rather than overproliferation and cancer.
Assuntos
Aneuploidia , Apoptose , Encéfalo/patologia , Centrossomo/patologia , Microcefalia/etiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Células Cultivadas , Centrossomo/metabolismo , Instabilidade Cromossômica , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Camundongos , Camundongos Knockout , Microcefalia/mortalidade , Microcefalia/patologia , Mitose , Células-Tronco Neurais , Taxa de SobrevidaRESUMO
Cordycepin (3' deoxyadenosine) has long been used in the study of in vitro assembled polyadenylation complexes, because it terminates the poly(A) tail and arrests the cleavage complex. It is derived from caterpillar fungi, which are highly prized in Chinese traditional medicine. Here we show that cordycepin specifically inhibits the induction of inflammatory mRNAs by cytokines in human airway smooth muscle cells without affecting the expression of control mRNAs. Cordycepin treatment results in shorter poly(A) tails, and a reduction in the efficiency of mRNA cleavage and transcription termination is observed, indicating that the effects of cordycepin on 3' processing in cells are similar to those described in in vitro reactions. For the CCL2 and CXCL1 mRNAs, the effects of cordycepin are post-transcriptional, with the mRNA disappearing during or immediately after nuclear export. In contrast, although the recruitment of RNA polymerase II to the IL8 promoter is also unaffected, the levels of nascent transcript are reduced, indicating a defect in transcription elongation. We show that a reporter construct with 3' sequences from a histone gene is unaffected by cordycepin, while CXCL1 sequences confer cordycepin sensitivity to the reporter, demonstrating that polyadenylation is indeed required for the effect of cordycepin on gene expression. In addition, treatment with another polyadenyation inhibitor and knockdown of poly(A) polymerase α also specifically reduced the induction of inflammatory mRNAs. These data demonstrate that there are differences in the 3' processing of inflammatory and housekeeping genes and identify polyadenylation as a novel target for anti-inflammatory drugs.