RESUMO
Activation of endogenous retrotransposons frequently occurs in cancer cells and contributes to tumor genomic instability. To test whether inhibition of retrotranspositions has an anticancer effect, we used treatment with the nucleoside reverse transcriptase inhibitor (NRTI) stavudine (STV) in mouse cancer models, MMTV-HER2/Neu and Th-MYCN, that spontaneously develop breast cancer and neuroblastoma, respectively. In both cases, STV in drinking water did not affect tumor incidence nor demonstrate direct antitumor effects. However, STV dramatically extended progression-free survival in both models following an initial complete response to chemotherapy. To approach the mechanism underlying this phenomenon, we analyzed the effect of NRTI on the selection of treatment-resistant variants in tumor cells in culture. Cultivation of mouse breast carcinoma 4T1 in the presence of STV dramatically reduced the frequency of cells capable of surviving treatment with anticancer drugs. Global transcriptome analysis demonstrated that the acquisition of drug resistance by 4T1 cells was accompanied by an increase in the constitutive activity of interferon type I and NF-κB pathways and an elevated expression of LINE-1 elements, which are known to induce inflammatory responses via their products of reverse transcription. Treatment with NRTI reduced NF-κB activity and reverted drug resistance. Furthermore, the inducible expression of LINE-1 stimulated inflammatory response and increased the frequency of drug-resistant variants in a tumor cell population. These results indicate a mechanism by which retrotransposon desilencing can stimulate tumor cell survival during treatment and suggest reverse transcriptase inhibition as a potential therapeutic approach for targeting the development of drug-resistant cancers.
Assuntos
Retroelementos , Inibidores da Transcriptase Reversa , Animais , Camundongos , Inibidores da Transcriptase Reversa/farmacologia , Retroelementos/genética , NF-kappa B , Resistencia a Medicamentos Antineoplásicos/genética , Elementos Nucleotídeos Longos e DispersosRESUMO
Around 10% of acute leukemias harbor a rearrangement of the MLL/KMT2A gene, and the presence of this translocation results in a highly aggressive, therapy-resistant leukemia subtype with survival rates below 50%. There is a high unmet need to identify safer and more potent therapies for MLL-rearranged (MLL-r) leukemia that can be combined with established chemotherapeutics to decrease treatment-related toxicities. The curaxin, CBL0137, has demonstrated nongenotoxic anticancer and chemopotentiating effects in a number of preclinical cancer models and is currently in adult Phase I clinical trials for solid tumors and hematological malignancies. The aim of our study was to investigate whether CBL0137 has potential as a therapeutic and chemopotentiating compound in MLL-r leukemia through a comprehensive analysis of its efficacy in preclinical models of the disease. CBL0137 decreased the viability of a panel of MLL-r leukemia cell lines (n = 12) and xenograft cells derived from patients with MLL-r acute lymphoblastic leukemia (ALL, n = 3) in vitro with submicromolar IC50s. The small molecule drug was well-tolerated in vivo and significantly reduced leukemia burden in a subcutaneous MV4;11 MLL-r acute myeloid leukemia model and in patient-derived xenograft models of MLL-r ALL (n = 5). The in vivo efficacy of standard of care drugs used in remission induction for pediatric ALL was also potentiated by CBL0137. CBL0137 exerted its anticancer effect by trapping Facilitator of Chromatin Transcription (FACT) into chromatin, activating the p53 pathway and inducing an Interferon response. Our findings support further preclinical evaluation of CBL0137 as a new approach for the treatment of MLL-r leukemia.
Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Antineoplásicos/uso terapêutico , Apoptose/genética , Carbazóis/uso terapêutico , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Estimativa de Kaplan-Meier , Leucemia Aguda Bifenotípica/diagnóstico , Leucemia Aguda Bifenotípica/tratamento farmacológico , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/mortalidade , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Elongação da Transcrição/genética , Transcriptoma , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 9-aminoacridine (9AA) derivative quinacrine (QC) has a long history of safe human use as an antiprotozoal and antirheumatic agent. QC intercalates into DNA and RNA and can inhibit DNA replication, RNA transcription, and protein synthesis. The extent of QC intercalation into RNA depends on the complexity of its secondary and tertiary structure. Internal ribosome entry sites (IRESs) that are required for initiation of translation of some viral and cellular mRNAs typically have complex structures. Recent work has shown that some intercalating drugs, including QC, are capable of inhibiting hepatitis C virus IRES-mediated translation in a cell-free system. Here, we show that QC suppresses translation directed by the encephalomyocarditis virus (EMCV) and poliovirus IRESs in a cell-free system and in virus-infected HeLa cells. In contrast, IRESs present in the mammalian p53 transcript that are predicted to have less-complex structures were not sensitive to QC. Inhibition of IRES-mediated translation by QC correlated with the affinity of binding between QC and the particular IRES. Expression of viral capsid proteins, replication of viral RNAs, and production of virus were all strongly inhibited by QC (and 9AA). These results suggest that QC and similar intercalating drugs could potentially be used for treatment of viral infections.
Assuntos
Antivirais/farmacologia , Vírus da Encefalomiocardite/efeitos dos fármacos , Poliovirus/efeitos dos fármacos , Quinacrina/farmacologia , Replicação Viral/efeitos dos fármacos , Sítios de Ligação , Vírus da Encefalomiocardite/fisiologia , Células HeLa , Humanos , Conformação de Ácido Nucleico , Poliovirus/fisiologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Viral/metabolismo , Proteínas Virais/biossínteseRESUMO
Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor kappaB (NFkappaB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF-inducible genes that are largely regulated by NFkappaB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFkappaB-dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild-type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53-deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFkappaB, plays the role of a general "buffer" of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFkappaB in tumors.
Assuntos
Inflamação/prevenção & controle , NF-kappa B/antagonistas & inibidores , Proteína Supressora de Tumor p53/fisiologia , Animais , Ceco/cirurgia , Quimiocinas/genética , Citocinas/genética , DNA/metabolismo , Humanos , Inflamação/induzido quimicamente , Ligadura , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/análise , NF-kappa B/fisiologia , Neutrófilos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Peritonite/etiologia , Peroxidase/sangue , Fagocitose , Regiões Promotoras Genéticas/genética , Punções , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Séptico/mortalidade , Tioglicolatos , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/fisiologia , Transfecção , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Isolated limb perfusion (ILP) with the chemotherapeutic agent melphalan is an effective treatment option for extremity in-transit melanoma but is toxic and technically challenging to deliver locoregionally. CBL0137 is an experimental clinical drug with broad anticancer activity in animal models, owing to its ability to bind DNA in a nongenotoxic manner and inactivate the FACT chromatin modulator essential for tumor cell viability. Here, we report that CBL0137 delivered by ILP in a murine melanoma model is as efficacious as melphalan, displaying antitumor activity at doses corresponding to only a fraction of the systemic MTD of CBL0137. The ability to bind DNA quickly combined with a favorable safety profile made it possible to substitute CBL0137 in the ILP protocol, using an intra-arterial infusion method, to safely achieve effective tumor suppression. Our findings of a preclinical proof of concept for CBL0137 and its administration via intra-arterial infusion as a superior treatment compared with melphalan ILP allows for locoregional treatment anywhere a catheter can be placed. Cancer Res; 76(22); 6620-30. ©2016 AACR.
Assuntos
Extremidades/patologia , Bombas de Infusão , Melanoma/tratamento farmacológico , Animais , Feminino , Humanos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Resultado do Tratamento , Estudos de Validação como AssuntoRESUMO
Tumor necrosis factor (TNF) induces apoptosis in sensitive cells in culture when used in combination with inhibitors of transcription or translation. We applied the genetic suppressor element (GSE) methodology to search for the genetic elements protecting NIH3T3 cells from TNF-stimulated death. Ten putative GSEs were isolated from TNF-resistant cells, one of which (GSE0-1) corresponded to the cDNA sequence known as the mouse homolog of human serologically defined colon cancer antigen 3 (SDCCAG3). SDCCAG3 protein contains the region similar to the coiled-coil domain of the myosin tail. The same domain is present in the proteins related to the organelles/proteins trafficking, such as kinesin, Golgin-160, and dynein. We proposed that the SDCCAG3 function might be related to protein trafficking and secretion. The expression of the coiledcoil domain as the dominant negative mutant form of SDCCAG3 made the NIH3T3 and HeLa cells resistant to TNF-specific apoptosis. The presentation of TNFR1 at the surface of these cells was reduced, which affected the sensitivity of the cells to the TNF treatment. We recently showed that the inhibition of protein trafficking and secretion depleted the unstable TNFR1 from plasma membrane. The inhibition of SDCCAG3 activity by its dominant negative mutant suppressed the protein trafficking and secretion, and decreased TNFR1 presentation on the cell surface. Based on these results, we presume that SDCCAG3 is important for protein trafficking and presentation of TNFR1 on the cell surface. Therefore, SDCCAG3 can be viewed as a potential target for modulation of TNF response.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Apoptose/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Western Blotting , Primers do DNA , Citometria de Fluxo , Biblioteca Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases , Camundongos , Mutação/genética , Células NIH 3T3 , Transporte Proteico/imunologia , Supressão Genética/genética , Supressão Genética/imunologia , Proteínas de Transporte VesicularRESUMO
In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFkappaB activity differently; SHP-1 inhibits and SHP-2 stimulates NFkappaB activation. In epithelial cells the process of NFkappaB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPalpha) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPalpha and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPalpha suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPalpha, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expression of dominant negative mutant of SIRPalpha has an effect similar to the expression of dominant negative PTP SHP-1 in the process of NFkappaB activation.
Assuntos
Antígenos de Diferenciação , Glicoproteínas de Membrana/genética , NF-kappa B/metabolismo , Molécula L1 de Adesão de Célula Nervosa/genética , Proteínas Tirosina Fosfatases/metabolismo , Receptores Imunológicos/genética , Fibroblastos , Genes Dominantes , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Receptores Imunológicos/metabolismoRESUMO
Proteotoxic stress (PS) is generated in cells under a variety of conditions involving accumulation of misfolded proteins. To avoid the toxicity of unmitigated PS, cells activate the heat shock response (HSR). HSR involves upregulation of factors such as ubiquitin and the non-housekeeping chaperone Hsp70 which assist with metabolism of aberrant proteins. The PS-HSR axis is a potential anticancer treatment target since many tumor cells display constitutive PS and dependence on HSR due to their rapid rates of proliferation and translation. In fact, induction of PS via stimulation of protein misfolding (hyperthermia), inhibition of proteasomes (bortezomib) or inhibition of Hsp90 (geldanamycin) have all been considered or used for cancer treatment. We found that combination of bortezomib with an inducer of protein misfolding (hyperthermia or puromycin) resulted in enhanced PS. HSR was also induced, but could not mitigate the elevated PS and the cells died via largely p53-independent apoptosis. Thus, combination treatments were more cytotoxic in vitro than the component single treatments. Consistent with this, combination of non-toxic doses of puromycin with bortezomib significantly increased the antitumor activity of bortezomib in a mouse model of multiple myeloma. These results provide support for using combination treatments that disrupt the balance of PS and HSR to increase the therapeutic index of anticancer therapies.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/terapia , Inibidores de Proteassoma , Deficiências na Proteostase/metabolismo , Pirazinas/farmacologia , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácidos Borônicos/administração & dosagem , Bortezomib , Linhagem Celular Tumoral , Terapia Combinada , Sinergismo Farmacológico , Células HCT116 , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP90/biossíntese , Células HeLa , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Hipertermia Induzida , Camundongos , Camundongos Endogâmicos BALB C , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Deficiências na Proteostase/induzido quimicamente , Puromicina/administração & dosagem , Puromicina/farmacologia , Pirazinas/administração & dosagemRESUMO
The number of physical conditions and chemical agents induce accumulation of misfolded proteins creating proteotoxic stress. This leads to activation of adaptive pro-survival pathway, known as heat shock response (HSR), resulting in expression of additional chaperones. Several cancer treatment approaches, such as proteasome inhibitor Bortezomib and hsp90 inhibitor geldanamycin, involve activation of proteotoxic stress. Low efficacy of these therapies is likely due to the protective effects of HSR induced in treated cells, making this pathway an attractive target for pharmacological suppression. We found that the anti-malaria drugs quinacrine (QC) and emetine prevented HSR in cancer cells, as judged by induction of hsp70 expression. As opposed to emetine, which inhibited general translation, QC did not affect protein synthesis, but rather suppressed inducible HSF1-dependent transcription of the hsp70 gene in a relatively selective manner. The treatment of tumor cells in vitro with a combination of non-toxic concentrations of QC and proteotoxic stress inducers resulted in rapid induction of apoptosis. The effect was similar if QC was substituted by siRNA against hsp70, suggesting that the HSR inhibitory activity of QC was responsible for cell sensitization to proteotoxic stress inducers. QC was also found to enhance the antitumor efficacy of proteotoxic stress inducers in vivo: combinatorial treatment with 17-DMAG + QC resulted in suppression of tumor growth in two mouse syngeneic models. These results reveal that QC is an inhibitor of HSF1-mediated HSR. As such, this compound has significant clinical potential as an adjuvant in therapeutic strategies aimed at exploiting the cytotoxic potential of proteotoxic stress.
Assuntos
Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Resposta ao Choque Térmico/efeitos dos fármacos , Quinacrina/farmacologia , Apoptose , Benzoquinonas/farmacologia , Ácidos Borônicos/farmacologia , Bortezomib , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , Lactamas Macrocíclicas/farmacologia , Neoplasias/tratamento farmacológico , Pirazinas/farmacologia , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Flavonoid quercetin and its derivative, methylquercetin, inhibit the replication of poliovirus in several cell lines. Here, we show that replication of poliovirus is inhibited by quercetin and that the extent of this inhibition depends on the intracellular content of pirin, a quercetinase. HeLa cells contain higher content of pirin protein than normal kidney human epithelial (NKE) or 293 cells do. Poliovirus replication in HeLa cells is significantly more resistant to quercetin than its replication in NKE and 293 cells. Overexpression of pirin reduced antiviral inhibitory effect of quercetin, while siRNA-induced suppression of pirin level made poliovirus replication more sensitive to the flavonoid. The results suggest that quercetinase activity of pirin determines the resistance of poliovirus infection to quercetin.
Assuntos
Antioxidantes/farmacologia , Proteínas de Transporte/metabolismo , Farmacorresistência Viral , Proteínas Nucleares/metabolismo , Poliovirus/efeitos dos fármacos , Poliovirus/fisiologia , Quercetina/farmacologia , Replicação Viral/efeitos dos fármacos , Androstadienos/farmacologia , Linhagem Celular , Dioxigenases/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Células HeLa , Humanos , Inibidores de Proteínas Quinases/farmacologia , WortmaninaRESUMO
Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.
Assuntos
Poliovirus/metabolismo , Inibidores de Proteassoma , Vesiculovirus/metabolismo , Replicação Viral , Ácidos Borônicos/farmacologia , Bortezomib , DNA/metabolismo , Replicação do DNA , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HeLa , Humanos , Leupeptinas/farmacologia , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/farmacologia , Transfecção , Proteínas Virais/metabolismoRESUMO
Activation of NF-kappaB during viral infection is one of the critical elements in innate immune response. Several virus-specific factors, such as double-stranded RNA, can trigger host defense mechanisms by inducing NF-kappaB-mediated expression of cytokines and interferons. Early stages of poliovirus infection are also associated with degradation of IkappaB alpha and translocation of NF-kappaB into the nucleus. However, at later stages of poliovirus replication the p65-RelA component of the NF-kappaB complex undergoes a specific cleavage that coincides with the onset of intensive poliovirus protein synthesis and the appearance of the activity of poliovirus protease 3C. Indeed, the p65-RelA amino acid sequence contains the recognition site for 3C, and recombinant protein 3C was shown to be capable of proteolytic cleavage of p65-RelA, generating truncated product similar to that observed during poliovirus infection. Cleavage of p65-RelA occurs during replication of ECHO-1 and rhinovirus 14, suggesting that inactivation of NF-kappaB function by proteolytic cleavage of p65-RelA is the common mechanism by which picornaviruses suppress the innate immune response.
Assuntos
NF-kappa B/metabolismo , Poliomielite/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , NF-kappa B/química , Fator de Transcrição RelARESUMO
Many viruses encode anti-apoptotic proteins that have been used as valuable tools for identification and analysis of key cellular regulators of programmed cell death. Here we demonstrate that the poliovirus protein 3A, previously shown to exhibit anti-apoptotic activity, binds and inactivates LIS1, a component of the dynein/dynactin motor complex, encoded by the gene mutated in patients with type I lissencephaly ("smooth brain"), thereby causing deregulation of endoplasmatic reticilum-to-Golgi vesicular transport, resulting in rapid disappearance of short-living receptors from the plasma membrane and loss of cell sensitivity to TNF and interferon. Truncated derivatives of LIS1, acting in a dominant negative manner, cause similar effects. However, 3A, being an endoplasmic reticulum-bound protein, locks Golgi-targeted YFP in the endoplasmatic reticilum, while expression of LIS1 mutants results in a dispersed cytoplasmic localization of the reporter protein. LIS1 dysfunction caused by ectopic expressing 3A or LIS1 mutants, as well as by overexpression of wild type LIS1, leads to cell blocking at the postmitotic stage associated with inability to undergo cytokinesis. Thus, the use of poliovirus protein as a research tool allowed us to reveal the role of cellular protein LIS1 in membrane protein trafficking, maintenance of Golgi integrity, surface presentation of unstable receptors, cell sensitivity to TNF-induced apoptosis and cell cycle progression.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Core Viral/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Divisão Celular , Linhagem Celular , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Poliovirus , Ligação Proteica , Transporte Proteico , Proteínas do Core Viral/genéticaRESUMO
Screening of expression libraries for bioactive clones that modulate the growth of mammalian cells has been limited largely to positive selections incapable of revealing growth suppressive or lethal genetic elements. We have developed a technique, selection-subtraction approach (SSA), that allows growth-modulating clones to be isolated based on alterations in their relative abundance in growing cell populations that have been transduced with an expression library. SSA utilizes tagged retroviral libraries in bacteriophage lambda vectors (retrophages). Nylon prints from retrophage libraries are used to determine the relative abundance of tags in library-transduced cells to identify biological activity of individual clones. Applications of SSA for gene discovery, target discovery, and generation of mutant proteins have been demonstrated, by using p53 and ataxia telangiectasia mutated (ATM) as models to isolate growth inhibitory proteins, peptides and antisense RNAs, and temperature-sensitive mutant proteins.
Assuntos
Testes Genéticos/métodos , Polidesoxirribonucleotídeos/química , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Biblioteca Gênica , Vetores Genéticos , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Próstata , Proteínas/química , Proteínas/genética , RNA Antissenso/genética , Mapeamento por Restrição , Seleção GenéticaRESUMO
BACKGROUND: Cellular receptors play a significant role in pathogenesis of viral infections. Previously, we demonstrated that TNFa receptor (TNFR1) rapidly disappeared from the cell surface upon poliovirus infection, whereas FAS was much more stable [1]. We suggested that the rate of decrease in receptor presentation on the surface of infected cells might reflect its turnover rate on uninfected cells. MATERIAL/METHODS: To test this hypothesis, we estimated by FACS analysis the turnover rates of receptors for TRAIL (TRAILR1 and TRAILR2), signal regulatory protein SIRPa, receptor for alpha/beta interferon (INFR1), and poliovirus receptor (CD155) on the surface of HeLa cells after the treatment with brefeldin A (to stop receptor replenishment through the Golgi-mediated trafficking) or poliovirus infection. RESULTS: A good correlation between turnover rates caused by the two interventions was observed, with the stability of receptor presentation changing in the following order: TRAILR1, TRAILR2, SIRPa (half-life on infected cells between 2-4 h) < INFR1 (4-6 h) < CD155 (>8 h, besides some early masking of the receptor by its binding of the virus). CONCLUSIONS: Our results suggest that disruption of the protein trafficking pathway during poliovirus infection leads to the diminished sensitivity of infected cells to pro-apoptotic factors, and thus represents one of the mechanisms by which virus modulates the host defense reactions.
Assuntos
Proteínas de Membrana , Poliomielite/metabolismo , Poliovirus/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/metabolismo , Animais , Apoptose/fisiologia , Brefeldina A/metabolismo , Linhagem Celular , Separação Celular , Citometria de Fluxo , Células HeLa , Humanos , Inibidores da Síntese de Proteínas/metabolismo , Transporte Proteico/fisiologiaRESUMO
Pifithrin alpha (PFTalpha) is a chemical compound isolated for its ability to suppress p53-mediated transactivation. It can protect cells from p53-mediated apoptosis induced by various stimuli and reduce sensitivity of mice to gamma radiation. Identification of molecular targets of PFTalpha is likely to provide new insights into mechanisms of regulation of p53 pathway and is important for predicting potential risks associated with administration of PFTalpha-like p53 inhibitors in vivo. We found that PFTalpha, in addition to p53, can suppress heat shock and glucocorticoid receptor signaling but has no effect on nuclear factor-kappaB signaling. PFTalpha reduces activation of heat shock transcription factor (HSF1) and increases cell sensitivity to heat. Moreover, it reduces activation of glucocorticoid receptor and rescues mouse thymocytes in vitro and in vivo from apoptotic death after dexamethasone treatment. PFTalpha affected both signaling pathways in a p53-independent manner. These observations suggest that PFTalpha targets some unknown factor that is common for three major signal transduction pathways.
Assuntos
Glucocorticoides/farmacologia , Resposta ao Choque Térmico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Benzotiazóis , Proteínas de Ligação a DNA/biossíntese , Fatores de Transcrição de Choque Térmico , Camundongos , NF-kappa B/metabolismo , Receptores de Glucocorticoides/fisiologia , Fatores de TranscriçãoRESUMO
Genetic suppressor element (GSE) methodology was applied to identify new genes controlling cell response to tumor necrosis factor (TNF). A retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts was screened for GSEs capable of protecting NIH3T3 cells from TNF-induced apoptosis. The most abundant among isolated GSEs represented a fragment of cDNA encoding the C-terminal cytoplasmic region of the immunoglobulin family inhibitory receptor, SHPS-1 (mouse homologue of human SIRPalpha). Ectopic expression of this fragment (both from human and mouse versions) increased the NF-kappaB-dependent transcription in three cell lines tested; this effect could be reduced by the expression of full-length SIRPalpha, suggesting that the isolated GSE acts through a dominant negative mechanism. GSE-mediated activation of NF-kappaB depended on the presence of serum, was abrogated by wortmannin, and was associated with phosphorylation of PKB/Akt, suggesting that Akt mediates it. These data indicate that SIRPalpha/SHPS-1 is involved in negative regulation of NF-kappaB signaling.