RESUMO
The TET (Ten-Eleven Translocation) dioxygenase enzyme family comprising 3 members, TET1-3, play key roles in DNA demethylation. These processes regulate transcription programs that determine cell lineage, survival, proliferation, and differentiation. The impetus for our investigations described here is derived from the need to develop illuminating small molecule probes for TET enzymes with cellular activity and specificity. The studies were done so in the context of the importance of TET2 in the hematopoietic system and the preponderance of loss of function somatic TET2 mutations in myeloid diseases. We have identified that 2-hydroxy-4-methylene-pentanedicarboxylic acid 2a reversibly competes with the co-substrate α-KG in the TET2 catalytic domain and inhibits the dioxygenase activity with an IC50 = 11.0 ± 0.9 µM at 10 µM α-KG in a cell free system and binds in the TET2 catalytic domain with Kd = 0.3 ± 0.12 µM.
Assuntos
Domínio Catalítico/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Ácidos Dicarboxílicos/síntese química , Ácidos Dicarboxílicos/farmacologia , Dioxigenases/metabolismo , Sistema Livre de Células , Metilação de DNA , Ácidos Dicarboxílicos/química , Humanos , Simulação de Acoplamento Molecular , Análise Espectral/métodos , Relação Estrutura-Atividade , Células THP-1RESUMO
Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.
Assuntos
Antígenos de Neoplasias/química , Pontos de Checagem do Ciclo Celular/fisiologia , Instabilidade Cromossômica/fisiologia , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/efeitos dos fármacos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiologia , Linhagem Celular , Dano ao DNA , DNA Topoisomerases Tipo II/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/fisiologia , DNA Complementar/genética , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Fibroblastos , Células HL-60 , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/farmacologiaRESUMO
Eltrombopag, an FDA-approved non-peptidyl thrombopoietin receptor agonist, is clinically used for the treatment of aplastic anemia, a disease characterized by hematopoietic stem cell failure and pancytopenia, to improve platelet counts and stem cell function. Eltrombopag treatment results in a durable trilineage hematopoietic expansion in patients. Some of the eltrombopag hematopoietic activity has been attributed to its off-target effects, including iron chelation properties. However, the mechanism of action for its full spectrum of clinical effects is still poorly understood. Here, we report that eltrombopag bound to the TET2 catalytic domain and inhibited its dioxygenase activity, which was independent of its role as an iron chelator. The DNA demethylating enzyme TET2, essential for hematopoietic stem cell differentiation and lineage commitment, is frequently mutated in myeloid malignancies. Eltrombopag treatment expanded TET2-proficient normal hematopoietic stem and progenitor cells, in part because of its ability to mimic loss of TET2 with simultaneous thrombopoietin receptor activation. On the contrary, TET inhibition in TET2 mutant malignant myeloid cells prevented neoplastic clonal evolution in vitro and in vivo. This mechanism of action may offer a restorative therapeutic index and provide a scientific rationale to treat selected patients with TET2 mutant-associated or TET deficiency-associated myeloid malignancies.
Assuntos
Anemia Aplástica , Benzoatos/farmacologia , Proliferação de Células , Proteínas de Ligação a DNA , Dioxigenases , Células-Tronco Hematopoéticas/enzimologia , Hidrazinas/farmacologia , Pirazóis/farmacologia , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/genética , Anemia Aplástica/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/antagonistas & inibidores , Dioxigenases/genética , Dioxigenases/metabolismo , Humanos , Camundongos , Camundongos KnockoutRESUMO
We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.
Assuntos
Antígenos de Neoplasias/metabolismo , Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Idelta/metabolismo , Clivagem do DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Serina/metabolismo , Antígenos de Neoplasias/química , Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase 1 épsilon/genética , Caseína Quinase I/genética , Caseína Quinase Idelta/antagonistas & inibidores , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Regulação para Baixo , Etoposídeo/farmacologia , Células HL-60 , Humanos , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
TET2 is frequently mutated in myeloid neoplasms. Genetic TET2 deficiency leads to skewed myeloid differentiation and clonal expansion, but minimal residual TET activity is critical for survival of neoplastic progenitor and stem cells. Consistent with mutual exclusivity of TET2 and neomorphic IDH1/2 mutations, here we report that IDH1/2 mutant-derived 2-hydroxyglutarate is synthetically lethal to TET-dioxygenase deficient cells. In addition, a TET-selective small molecule inhibitor decreased cytosine hydroxymethylation and restricted clonal outgrowth of TET2 mutant, but not normal hematopoietic precursor cells in vitro and in vivo. While TET-inhibitor phenocopied somatic TET2 mutations, its pharmacologic effects on normal stem cells were, unlike mutations, reversible. Treatment with TET inhibitor suppressed the clonal evolution of TET2 mutant cells in murine models and TET2-mutated human leukemia xenografts. These results suggest that TET inhibitors may constitute a new class of targeted agents in TET2 mutant neoplasia.
Assuntos
Dioxigenases , Leucemia , Animais , Proteínas de Ligação a DNA/genética , Hematopoese/genética , Humanos , Camundongos , Proteínas Proto-Oncogênicas/genéticaRESUMO
PURPOSE: The potentiation of topoisomerase (topo)-I-induced apoptosis by proteasome inhibitors is dependent on the treatment sequence, but not on NF-kappaB. In this study, alternate mechanisms modulating apoptosis induced with the topo I-targeting drug, SN-38, when followed by the proteasome inhibitor bortezomib (PS-341) were investigated. MATERIALS AND METHODS: Human non-small cell lung carcinoma (NSCLC-3) cells transfected with a control vector (NSCLC-3/neo) or a vector containing dominant negative IkappaBalpha (NSCLC-3/mIkappaBalpha) were treated with SN-38 for 1 h followed by PS-341 for 4 h (SN-38 --> PS-341), or with either drug alone. The functional role of the anti-apoptotic protein survivin was tested using NSCLC-3 transfected with myc-tagged wild-type (NSCLC-3/myc-survivin), or dominant negative mutant T34A survivin (NSCLC-3/myc-T34A). RESULTS: In NSCLC-3/neo or NSCLC-3/mIkappaBalpha cells, treatment with SN-38 --> PS-341 led to down-regulation of the survivin transcript and protein, enhanced apoptosis and reduced (> 3-fold) survival compared to SN-38 or PS-341 alone. In contrast to the cells transfected with wild-type survivin, or the control NSCLC-3/neo, those cells transfected with mutant survivin and treated with SN-38 --> PS-341 exhibited enhanced caspase 9 activity (> 2-fold), caspase 3 (> 2- to 3-fold) activity and cytotoxicity compared to the NSCLC-3/neo cells. CONCLUSION: In contrast to inhibition of NF-kappaB activity, down-regulation of the anti-apoptotic survivin was correlated with modulation of the sequence-dependent synergistic effects of PS-341 in SN-38-induced apoptosis.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácidos Borônicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Regulação para Baixo/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteassoma , Pirazinas/farmacologia , Inibidores da Topoisomerase I , Apoptose/efeitos dos fármacos , Apoptose/genética , Ácidos Borônicos/administração & dosagem , Bortezomib , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Dano ao DNA , Sinergismo Farmacológico , Humanos , Proteínas I-kappa B/genética , Proteínas Inibidoras de Apoptose , Irinotecano , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Inibidor de NF-kappaB alfa , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Inibidores de Proteases/farmacologia , Pirazinas/administração & dosagem , Survivina , TransfecçãoRESUMO
Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-kappaB-independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I-targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38-->PS-341). The p53-dependent sensitization of DNA damage-induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341-->SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341-->SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38-->PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38-->PS-341-treated cells compared with PS-341-->SN-38-treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3sigma and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3sigma or survivin in regulating the divergent function of p53 in response to SN-38-->PS-341 and PS-341-->SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage-induced apoptosis via a p53-dependent pathway.
Assuntos
Proteínas 14-3-3/fisiologia , Apoptose , Ácidos Borônicos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dano ao DNA , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias/fisiologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Sequência de Bases , Bortezomib , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Primers do DNA , Regulação para Baixo , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , SurvivinaRESUMO
Activation of signaling pathways following DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. NF-kappaB, a major regulator of the stress response and a negative regulator of apoptosis is often activated following treatment with topoisomerase poisons. Since activation of NF-kappaB is generally considered to relay an anti-apoptotic signal, inactivation of this signaling molecule is considered to represent an important strategy to improve therapeutic efficacy. Although this strategy seems to be effective in some model systems, our results in human non-small cell lung cancers differed. In this review we will discuss the role of NF-kappaB in mediating topoisomerase poison-induced DNA damage and apoptosis and the consequence of inhibiting its activity. Newer insights about the importance of proteasome inhibitors and anti-apoptotic genes in topoisomerase poison-induced signaling mechanisms leading to apoptosis will also be reviewed. The knowledge obtained from these studies may be useful for translation to a clinical setting for development of more effective therapeutic strategies.
Assuntos
Antineoplásicos/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Inibidores da Topoisomerase I , Inibidores da Topoisomerase II , Animais , Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisteína Endopeptidases , Dano ao DNA , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Complexo de Endopeptidases do ProteassomaRESUMO
BACKGROUND: We previously demonstrated that KN-62, an inhibitor of calcium calmodulin-dependent enzymes, sensitizes human leukemia HL-60 cells resistant to topoisomerase II-targeting drugs. The objective of this study was to determine pathways of apoptosis downstream of DNA damage induced by KN-62 co-treatment with VP-16. MATERIALS AND METHODS: HL-60/Y/DOX0.05 cells were treated with VP-16, KN-62, or VP-16 + KN-62. Following treatment, cells were assayed for c-IAP1, c-IAP2 and XIAP protein expression, as well as caspase activation, cytochrome c release and PARP cleavage. RESULTS: Baseline c-IAP1 protein levels were 2-fold higher in HL60 cells selected for resistance to doxorubicin compared to the parent sensitive line. VP-16 and KN-62 co-treatment was associated with caspase activation via the mitochondrial pathway and significant reductions (p = 0.002) in c-IAP1 protein expression but not with c-IAP2 or XIAP. CONCLUSION: These data suggest that KN-62 co-treatment sensitizes doxorubicin-resistant cells to VP-16-induced apoptosis by enhancing caspase activity and reducing c-IAP1 expression.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Biossíntese de Proteínas , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Antibióticos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Caspases/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Etoposídeo/administração & dosagem , Regulação Leucêmica da Expressão Gênica , Células HL-60 , Humanos , Proteínas Inibidoras de Apoptose , Proteínas/antagonistas & inibidores , Proteínas/genética , Ubiquitina-Proteína Ligases , Regulação para CimaRESUMO
BACKGROUND: Antiproliferative effects of proteasome inhibitors are suggested to be primarily due to effects on nuclear factor-kappaB (NF-kappaB)-dependent pathways and the induction of apoptosis. The objective of this study was to elucidate the mechanistic basis for the antiproliferative effects of the proteasome inhibitor, bortezomib, in human clear cell renal cell cancer cells (CCRCC). MATERIALS AND METHODS: von Hippel Lindau (VHL) mutation/methylation status and cytotoxic response to bortezomib was determined in a panel of CCRCC cell lines. Effects on target protein/gene expression and the role of p53 in bortezomib-mediated cytotoxicity, inhibition of proteasome activity, survivin transcript and protein expression as well as induction of p21 expression was determined in CCRCC that differed in their intrinsic sensitivity to bortezomib. RESULTS: VHL status was not associated with cytotoxic response to bortezomib treatment. Cytotoxicity in cell lines that differed in intrinsic sensitivity to bortezomib correlated with sustained inhibition of proteasome activity, survivin expression and induction of p21 expression. Stable down-regulation of p53 expression by siRNA led to attenuation of bortezomib effects, survivin down-regulation and p21 induction, suggesting that cellular effects are p53-dependent. CONCLUSION: These results demonstrate that the antiproliferative effects of bortezomib in CCRCC cells are VHL independent and dependent on pathways regulated by p53.
Assuntos
Ácidos Borônicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Apoptose/efeitos dos fármacos , Bortezomib , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Metilação de DNA/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Inibidores de Proteassoma , RNA Interferente Pequeno/farmacologia , Survivina , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.