RESUMO
Specific types of human papillomaviruses (HPVs) cause cervical cancer, the second most common tumor in women worldwide. Both cellular transformation and the maintenance of the oncogenic phenotype of HPV-positive tumor cells are linked to the expression of the viral E6 and E7 oncogenes. To identify downstream cellular target genes for the viral oncogenes, we silenced endogenous E6 and E7 expression in HPV-positive HeLa cells by RNA interference (RNAi). Subsequently, we assessed changes of the cellular transcriptome by genome-wide microarray analysis. We identified 648 genes, which were either downregulated (360 genes) or upregulated (288 genes), upon inhibition of E6/E7 expression. A large fraction of these genes is involved in tumor-relevant processes, such as apoptosis control, cell cycle regulation, or spindle formation. Others may represent novel cellular targets for the HPV oncogenes, such as a large group of C-MYC-associated genes involved in RNA processing and splicing. Comparison with published microarray data revealed a substantial concordance between the genes repressed by RNAi-mediated E6/E7 silencing in HeLa cells and genes reported to be upregulated in HPV-positive cervical cancer biopsies.
Assuntos
Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/genética , Interferência de RNA , Ciclo Celular/efeitos dos fármacos , Análise por Conglomerados , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Células HeLa , Humanos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologiaRESUMO
Livin (alternatively called ML-IAP or KIAP) is a cancer-associated member of the antiapoptotic inhibitor of apoptosis protein family. Two splicing variants of Livin, designated Livin alpha and Livin beta, have been identified. The significance of these isoforms for Livin-mediated apoptosis inhibition is largely unclear. Using an isoform-specific RNA interference (RNAi) strategy, we silenced endogenous Livin expression in HeLa cells. We found that the targeted inhibition of Livin beta, but not of Livin alpha, blocked the growth of HeLa cells in clonogenic survival assays. In addition, silencing of Livin beta, but not of Livin alpha, sensitized HeLa cells to different proapoptotic stimuli such as UV irradiation, tumor necrosis factor alpha, or etoposide. These events were linked to activation of caspase-3 and increased poly(ADP-ribose) polymerase cleavage, specifically upon silencing of Livin beta. The proapoptotic sensitization of HeLa cells upon RNAi-mediated silencing of the endogenous livin gene was specifically reverted by ectopic expression of Livin beta but not of Livin alpha. We conclude that the Livin beta isoform plays the key role for the antiapoptotic protection of HeLa cells by the livin gene. Our results show that the Livin isoforms can strongly differ in their functional significance for the antiapoptotic resistance of tumor cells. Studies evaluating Livin as a novel diagnostic and prognostic tumor marker should benefit from isoform-specific expression analyses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose , Caspase 3 , Caspases/metabolismo , Ensaio de Unidades Formadoras de Colônias , Etoposídeo/farmacologia , Inativação Gênica , Células HeLa/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Isoformas de Proteínas , Interferência de RNA , Fator de Necrose Tumoral alfa/farmacologia , Raios UltravioletaRESUMO
Cancer cells are typically characterized by increased resistance towards apoptosis. Livin (alternatively called ML-IAP or KIAP) is an anti-apoptotic protein which is expressed in several cancer forms. Using real-time reverse transcription-polymerase chain reaction (RT-PCR), we confirmed livin expression in a significant portion of non-small cell lung cancer (NSCLC) tissue samples and, in addition, detected livin expression in a number of NSCLC cell lines. In order to elucidate whether livin contributes to the apoptotic resistance of lung cancer cells, we silenced endogenous livin expression in a panel of cancer-derived NSCLC cell lines by RNA interference (RNAi). We observed that the targeted inhibition of livin strongly sensitized NSCLC cells to different pro-apoptotic stimuli, such as UV-irradiation or the chemotherapeutic drug etoposide. In addition, long-term silencing of livin blocked the outgrowth of NSCLC cells in colony formation assays. These effects of small interfering (si)RNA were specific for livin-expressing tumor cells. Our results indicate that Livin is an important contributor to the apoptosis resistance of NSCLC cells and may serve as a novel molecular target for therapeutic inhibition in NSCLC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Proteínas Inibidoras de Apoptose/fisiologia , Neoplasias Pulmonares/fisiopatologia , Proteínas de Neoplasias/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Etoposídeo/farmacologia , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno , Transfecção , Ensaio Tumoral de Célula-Tronco , Raios UltravioletaRESUMO
Increased resistance to apoptosis is a hallmark of many tumor cells. The functional inhibition of specific antiapoptotic factors may provide a rational basis for the development of novel therapeutic strategies. We investigated here whether the RNA interference (RNAi) technology could be used to increase the apoptotic susceptibility of cancer cells. As a molecular target, we chose the antiapoptotic livin (ML-IAP, KIAP) gene, which is expressed in a subset of human tumors. We identified vector-borne small interfering (si)RNAs, which could efficiently block endogenous livin gene expression. Silencing of livin was associated with caspase-3 activation and a strongly increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin, UV-irradiation, or TNFalpha. The effects were specific for Livin-expressing tumor cells. Our results (i) provide direct evidence that the intracellular interference with livin gene expression resensitizes human tumor cells to apoptosis, (ii) define the livin gene as a promising molecular target for therapeutic inhibition, and (iii) show that the livin gene is susceptible to efficient and specific silencing by the siRNA technology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/genética , Inativação Gênica , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas Inibidoras de ApoptoseRESUMO
The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6. This ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. These findings show that RNAi provides a powerful molecular strategy to inactivate intracellular E6 function efficiently. Moreover, they define E6 as a most promising therapeutic target to eliminate HPV-positive tumour cells specifically by RNAi. Thus, by sequence-specific targeting of antiapoptotic genes, siRNAs may be developed into novel therapeutics that can efficiently correct the apoptosis deficiency of cancer cells.
Assuntos
Proteínas de Ligação a DNA , Proteínas Oncogênicas Virais/genética , RNA Interferente Pequeno/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Terapia Genética/métodos , Células HeLa/efeitos dos fármacos , Células HeLa/virologia , Humanos , Proteínas Oncogênicas Virais/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The transcription factor signal transducer and activator of transcription (Stat) 3 is activated through the interleukin-6 family of cytokines and by binding of growth factors to the epidermal growth factor (EGF) receptor. It plays an essential role in embryonic development and assumes specialized tasks in many differentiated tissues. Constitutively activated Stat3 has been found in tumor cell lines and primary tumors and plays a crucial role in tumor cell survival and proliferation. To inhibit the oncogenic action of Stat3 in tumor cells, we have selected short peptides, so-called peptide aptamers, which specifically interact with defined functional domains of this transcription factor. The peptide aptamers were selected from a peptide library of high complexity by an adaptation of the yeast two-hybrid procedure. Peptide aptamers specifically interacting with the Stat3 dimerization domain caused inhibition of DNA binding activity and suppression of transactivation by Stat3 in EGF-responsive cells. Similarly, a peptide aptamer selected for its ability to recognize the Stat3 DNA binding domain inhibited DNA binding and transactivation by Stat3 following EGF stimulation of cells. Peptide aptamers were expressed in bacteria as fusion proteins with a protein transduction domain and introduced into human myeloma cells. This resulted in dose-dependent growth inhibition, down-regulation of Bcl-x(L) expression, and induction of apoptosis. The inhibition of Stat3 functions through the interaction with peptide aptamers counteracts the transformed phenotype and could become useful in targeted tumor therapy.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Neoplasias/patologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Transativadores/química , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Caspase 3 , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dimerização , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Proteína bcl-XRESUMO
In recent years, peptide aptamers have emerged as novel molecular tools that are useful for both basic and applied aspects of molecular medicine. Due to their ability to specifically bind to and inactivate a given target protein at the intracellular level, they provide a new experimental strategy for functional protein analyses, both in vitro and in vivo. In addition, by using peptide aptamers as "pertubagens", they can be employed for genetic analyses, in order to identify biochemical pathways, and their components, that are associated with the induction of distinct cellular phenotypes. Furthermore, peptide aptamers may be developed into diagnostic tools for the detection of a given target protein or for the generation of high-throughput protein arrays. Finally, the peptide aptamer technology has direct therapeutic implications. Peptide aptamers can be used in order to validate therapeutic targets at the intracellular level. Moreover, the peptide aptamer molecules themselves should possess therapeutic potential, both as lead structures for drug design and as a basis for the development of protein drugs.
Assuntos
Fenômenos Fisiológicos Celulares , Vírus da Hepatite B/fisiologia , Oligopeptídeos/metabolismo , Papillomaviridae/fisiologia , Transdução de Sinais/fisiologia , Animais , Desenho de Fármacos , Humanos , Oligopeptídeos/genética , Oligopeptídeos/uso terapêutico , Análise Serial de Proteínas , Ligação Proteica/genética , Técnicas do Sistema de Duplo-Híbrido , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein is an emerging target for the development of anti-HIV drugs. We recently described a new approach for inhibiting IN by "shiftides"--peptides that inhibit the protein by shifting its oligomerization equilibrium from the active dimer to the inactive tetramer. In this study, we used the yeast two-hybrid system with the HIV-1 IN as a bait and a combinatorial peptide aptamer library as a prey to select peptides of 20 amino acids that specifically bind IN. Five non-homologous peptides, designated as IN-1 to IN-5, were selected. ELISA studies confirmed that IN binds the free peptides. All the five peptides interact with IN with comparable affinity (K(d approximately )10 microM), as was revealed by fluorescence anisotropy studies. Only one peptide, IN-1, inhibited the enzymatic activity of IN in vitro and the HIV-1 replication in cultured cells. In correlation, fluorescence anisotropy binding experiments revealed that of the five peptides, only the inhibitory IN-1 inhibited the DNA binding of IN. Analytical gel filtration experiments revealed that only the IN-1 and not the four other peptides shifted the oligomerization equilibrium of IN towards the tetramer. Thus, the results show a distinct correlation between the ability of the selected peptides to inhibit IN activity and that to shift its oligomerization equilibrium.
Assuntos
Técnicas de Química Combinatória , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Aptâmeros de Peptídeos/metabolismo , Morte Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Integrase de HIV/química , Integrase de HIV/fisiologia , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/toxicidade , Células HeLa , Humanos , Cinética , Linfócitos/efeitos dos fármacos , Linfócitos/virologia , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/toxicidade , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , Replicação Viral/efeitos dos fármacosRESUMO
Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.
Assuntos
Aptâmeros de Peptídeos/química , Vírus da Hepatite B/genética , Proteínas/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/farmacologia , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Vimentina/química , Replicação ViralRESUMO
The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the anti-growth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.
Assuntos
Papillomaviridae/isolamento & purificação , Interferência de RNA , Proteína Supressora de Tumor p53/fisiologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Neoplasias do Colo do Útero/terapia , Neoplasias do Colo do Útero/virologia , Regulação para Baixo , Feminino , Células HeLa , Humanos , RNA Interferente Pequeno/uso terapêutico , Ubiquitina-Proteína Ligases/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.