RESUMO
CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.
Assuntos
Benchmarking , Sistemas CRISPR-Cas , Edição de Genes , Endonucleases/genéticaRESUMO
Although essential for T cell function, the identity of the T cell receptor (TCR) "inside-out" pathway for the activation of lymphocyte function-associated antigen 1 (LFA-1) is unclear. SKAP1 (SKAP-55) is the upstream regulator needed for TCR-induced RapL-Rap1 complex formation and LFA-1 activation. In this paper, we show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway. A SKAP1 PH domain-inactivating mutation (i.e. R131M) markedly impaired RapL translocation to membranes for Rap1 and LFA-1 binding and the up-regulation of LFA-1-intercellular adhesion molecule 1 (ICAM-1) binding. Further, N-terminal myr-tagged SKAP1 for membrane binding facilitated constitutive RapL membrane and Rap1 binding and effectively substituted for PI3K and TCR ligation in the activation of LFA-1 in T cells.
Assuntos
Antígeno-1 Associado à Função Linfocitária/biossíntese , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Regulação para Cima/fisiologiaRESUMO
Polo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ativação Enzimática/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Técnicas de Cultura de Células , Dimerização , Humanos , Transfecção , Quinase 1 Polo-LikeRESUMO
Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
Assuntos
Marcação de Genes/métodos , Mutagênese , RNA Guia de Cinetoplastídeos/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes by post-transcriptional silencing of gene expression. In this study we wondered whether inducible RNAi-cassettes integrated into cellular DNA possess the power to trigger neoplastic growth. For this purpose inducible RNAi vectors containing tetracycline (Tet)-responsive derivatives of the H1 promoter for the conditional expression of short hairpin RNA (shRNA) were used to target human polo-like kinase 1 (Plk1), which is overexpressed in a broad spectrum of human tumors. In the absence of doxycycline (Dox) HeLa clones expressing TetR, that carry the RNAi-cassette stably integrated, exhibited no significant alteration in Plk1 expression levels. In contrast, exposure to Dox led to marked downregulation of Plk1 mRNA to 3% and Plk1 protein to 14% in cell culture compared to mismatch shRNA/Plk1-expressing cells. As a result of Plk1 depletion cell proliferation decreased to 17%. Furthermore, for harnessing RNAi for silencing disease-related genes in vivo we transplanted inducible RNAi-HeLa cells onto nude mice. After administration of Dox knockdown of Plk1 expression was observed correlating to a significant inhibition of tumor growth. Taken together, our data revealed that genomically integrated RNAi-elements are suitable to hamper tumor growth by conditional expression of shRNA.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Regulação da Expressão Gênica , Neoplasias/terapia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Interferência de RNA , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Doxiciclina/farmacologia , Vetores Genéticos , Genoma Humano , Células HeLa , Humanos , Masculino , Camundongos , Neoplasias/enzimologia , Neoplasias/patologia , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent Apc Min/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/enzimologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias do Colo/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/fisiopatologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Instabilidade Cromossômica , Neoplasias do Colo/genética , Neoplasias do Colo/fisiopatologia , Feminino , Humanos , Cinetocoros/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Quinase 1 Polo-LikeRESUMO
The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.
RESUMO
RNA interference (RNAi) is a powerful tool for studying gene function. We developed an inducible genetic element for short interfering RNA-mediated gene silencing. This system uses a tetracycline (Tet)-responsive derivative of the H1 promoter and the Tet repressor (TetR) for conditional expression of short hairpin RNA (shRNA) in HeLa cells. Promoter constructs were generated, which contain the Tet operator (TetO) derived from a prokaryotic Tet resistance transposon upstream and/or downstream of the TATA box. To quantify the response of controllable transcription units for shRNA expression, we examined the functional activity of polo-like kinase 1 (PLK1), a key component of mitotic progression, that is overexpressed in many human tumors. Cotransfection of plasmids for the expression of TetR and shRNA/PLK1 under the control of an H1 promoter-variant carrying TetO upstream of the TATA box did not alter PLK1 expression and proliferation properties of HeLa cells in the absence of doxycycline. Addition of the antibiotic led to marked downregulation of endogenous PLK1 accompanied by strong inhibition of cellular proliferation. Our data indicate that an inducible transcription system for shRNAs based on the human H1 promoter could be a versatile tool for controlled gene silencing in vitro.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Inativação Gênica , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tetraciclina/metabolismo , Doxiciclina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Serina-Treonina Quinases , Inibidores da Síntese de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA/metabolismo , Tetraciclina/farmacologia , Quinase 1 Polo-LikeRESUMO
Upon interaction of the CD95 receptor with its ligand, sequential association of the adaptor molecule FADD (MORT1), pro-forms of caspases-8/10, and the caspase-8/10 regulator c-FLIP leads to the formation of a death-inducing signaling complex. Here, we identify polo-like kinase (Plk) 3 as a new interaction partner of the death receptor CD95. The enzymatic activity of Plk3 increases following interaction of the CD95 receptor with its ligand. Knockout (KO) or knockdown of caspase-8, CD95 or FADD prevents activation of Plk3 upon CD95 stimulation, suggesting a requirement of a functional DISC for Plk3 activation. Furthermore, we identify caspase-8 as a new substrate for Plk3. Phosphorylation occurs on T273 and results in stimulation of caspase-8 proapoptotic function. Stimulation of CD95 in cells expressing a non-phosphorylatable caspase-8-T273A mutant in a rescue experiment or in Plk3-KO cells generated by CRISPR/Cas9 reduces the processing of caspase-8 prominently. Low T273 phosphorylation correlates significantly with low Plk3 expression in a cohort of 95 anal tumor patients. Our data suggest a novel mechanism of kinase activation within the Plk family and propose a new model for the stimulation of the extrinsic death pathway in tumors with high Plk3 expression.
Assuntos
Caspase 8/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor fas/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Marcação por Isótopo , Células Jurkat , Ligantes , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Supressoras de TumorRESUMO
We have recently shown that caspase-8 is a new substrate of Polo-like kinase 3 (Plk3) that phosphorylates the protein on residue T273 thereby promoting its pro-apoptotic function. In the present study we aimed to investigate the clinical relevance of Plk3 expression and phosphorylation of caspase-8 at T273 in patients with anal squamous cell carcinoma (SSC) treated with 5-fluorouracil and mitomycin C-based chemoradiotherapy (CRT). Immunohistochemical detection of the markers was performed in pretreatment biopsy specimens of 95 patients and was correlated with clinical/histopathologic characteristics including HPV-16 virus load/p16INK4a expression and cumulative incidence of local and distant failure, cancer specific survival (CSS), and overall survival (OS). We observed significant positive correlations between Plk3 expression, pT273 caspase-8 signal, and levels of HPV-16 virus DNA load/p16INK4a detection. Patients with high scores of Plk3 and pT273 caspase-8 showed increased local control (p = 0.011; p = 0.001), increased CSS (p = 0.011; p = 0.013) and OS (p = 0.024; p = 0.001), while the levels of pT273 caspase-8 were significantly associated (p = 0.033) with distant metastases. In multivariate analyses Plk3 expression remained significant for local failure (p = 0.018), CSS (p = 0.016) and OS (p = 0.023). Moreover, a combined HPV16 DNA load and Plk3 or pT273 caspase-8 variable revealed a significant correlation to decreased local failure (p = 0.001; p = 0.009), increased CSS (p = 0.016; p = 0.023) and OS (p = 0.003; p = 0.003). In conclusion these data indicate that elevated levels of Plk3 and pT273 caspase-8 are correlated with favorable clinical outcome in patients with anal SCC treated with concomitant CRT.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Ânus/terapia , Carcinoma de Células Escamosas/terapia , Caspase 8/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias do Ânus/complicações , Neoplasias do Ânus/metabolismo , Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/metabolismo , Quimiorradioterapia , DNA Viral/análise , Feminino , Fluoruracila/administração & dosagem , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mitomicina/administração & dosagem , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/terapia , Infecções por Papillomavirus/virologia , Fosforilação , Prognóstico , Análise de Sobrevida , Proteínas Supressoras de Tumor , Tirosina/metabolismoRESUMO
The Polo-like kinase 1 (Plk1) plays a key role in regulating a broad spectrum of critical cell cycle events. Plk1 is a marker of cellular proliferation and has prognostic potential in different types of human tumors. In a series of preclinical studies, Plk1 has been validated as a cancer target. This prompted many pharmaceutical companies to develop small-molecule inhibitors targeting the classical ATP-binding site of Plk1 for anticancer drug development. Recently, FDA has granted a Breakthrough Therapy designation to the Plk inhibitor BI 6727 (volasertib), which provided a survival benefit for patients suffering from acute myeloid leukemia. Remarkably, a new generation of Plk1 inhibitors that target the second druggable domain of Plk1, the Polo-box domain, is currently being tested preclinically. Since various ATP-competitive compounds of Plk1 inhibit also the activities of Plk2 and Plk3, which act as tumor suppressors, the roles of closely related Plk-family members in cancer cells need to be considered carefully. In this article, the authors highlight recent insights into the biology of Plks in cancer cells and discuss the progress in the development of small-molecule Plk1 inhibitors. The authors believe that the greatest therapeutic benefit might come through leukemic cells that are in direct contact with the inhibitor in the blood stream. The identification of biomarkers and studies that document Plk activities in treated patients would also be beneficial to better understand the role of Plk inhibition in tumor development and anticancer therapy.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Quinase 1 Polo-LikeRESUMO
Caspase-8 is crucial for cell death induction, especially via the death receptor pathway. The dysregulated expression or function of caspase-8 can promote tumor formation, progression and treatment resistance in different human cancers. Here, we show procaspase-8 is regulated during the cell cycle through the concerted inhibitory action of Cdk1/cyclin B1 and polo-like kinase 1 (Plk1). By phosphorylating S387 in procaspase-8 Cdk1/cyclin B1 generates a phospho-epitope for the binding of the PBD of Plk1. Subsequently, S305 in procaspase-8 is phosphorylated by Plk1 during mitosis. Using an RNAi-based strategy we could demonstrate that the extrinsic cell death is increased upon Fas-stimulation when endogenous caspase-8 is replaced by a mutant (S305A) mimicking the non-phosphorylated form. Together, our data show that sequential phosphorylation by Cdk1/cyclin B1 and Plk1 decreases the sensitivity of cells toward stimuli of the extrinsic pathway during mitosis. Thus, the clinical Plk1 inhibitor BI 2536 decreases the threshold of different cancer cell types toward Fas-induced cell death.
Assuntos
Apoptose , Caspase 8/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Quinase CDC2 , Caspase 8/genética , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Células HeLa , Humanos , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Quinase 1 Polo-LikeRESUMO
ERK 1/2 are found to be hyperactive in many cancers. Active ERK 1/2 (pERK 1/2) are known to protect cancer cells from undergoing death receptor-mediated apoptosis, although the mechanism(s) behind this is poorly understood. Through in vitro kinase assays and mass-spectrometry we demonstrate that pERK 1/2 can phosphorylate pro-Caspase-8 at S387. Also, in EGFR-overexpressing Type I and II ovarian and breast cancer cell lines respectively, ERK 1/2 remain active only during the interphase. During this period, pERK 1/2 could inhibit Trail-induced apoptosis, most effectively during the G1/S phase. By knocking-down the endogenous pro-Caspase-8 using RNAi and replacing it with its non-phosphorylatable counterpart (S387A), a significant increase in Caspase-8 activity upon Trail stimulation was observed, even in the presence of pERK 1/2. Taken together, we propose that a combination of Trail and an inhibitor of ERK 1/2 activities could potentially enhance of Trail's effectiveness as an anti-cancer agent in ERK 1/2 hyperactive cancer cells.
Assuntos
Apoptose , Neoplasias da Mama/enzimologia , Ciclo Celular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspase 8 , Linhagem Celular Tumoral , Feminino , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosforilação/genéticaRESUMO
High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.
Assuntos
Antineoplásicos/toxicidade , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Interferência de RNA/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Apoptose/genética , Northern Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Primers do DNA/genética , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Imunofluorescência , Dosagem de Genes/genética , Técnicas de Silenciamento de Genes , Engenharia Genética/métodos , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Quinase 1 Polo-LikeRESUMO
Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. Furthermore, RNA interference-mediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A nonphosphorylatable procaspase-8 (S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the cross talk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis.
Assuntos
Apoptose/fisiologia , Proteína Quinase CDC2/metabolismo , Caspase 8/metabolismo , Ciclina B1/metabolismo , Receptor fas/metabolismo , Animais , Neoplasias da Mama/metabolismo , Proteína Quinase CDC2/genética , Caspase 8/genética , Linhagem Celular , Ciclina B1/genética , Inibidores de Cisteína Proteinase/metabolismo , Feminino , Humanos , Leupeptinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Receptor fas/genéticaRESUMO
Polo-like kinase 1 (Plk1) is overexpressed in tumor tissues and its expression level is tightly associated with the malignancy of tumors and prognosis of tumor patients. Thus, Plk1 is considered as one of the most attractive molecular targets for anticancer therapy. Recently, several small molecule inhibitors of Plk1 have been identified and characterized, and the first generation of Plk1 inhibitors has been investigated in clinical trials. However, the long-term effect of the downregulation of Plk1 on tumor cells has not yet been studied. In this work we have investigated the phenotype of HeLa cells, in which Plk1 is continuously downregulated by constitutive expression of shRNA. The data demonstrate that the long-term suppression of Plk1 increases the levels of cyclindependent kinase inhibitor p21(WAF1/CIP), which is partially induced by the elevated tumor suppressor p73 in p53-inactivated HeLa cells. The increased kinase inhibitor p21(WAF1/CIP1) localizes in both cyctoplasm as well as in nucleus and interacts directly with Cdk1/cyclin B1. Moreover, the knockdown of Plk1 leads to a decreased oncoprotein MDM2 and an elevated pro-apoptotic protein Bax in HeLa cells. Importantly, HeLa cells with reduced level of Plk1, which induces an increase of p21, p73 and Bax, are more sensitive to some chemotherapeutic agents, such as cisplatin.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proliferação de Células , Aberrações Cromossômicas , Cromossomos/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Quinase 1 Polo-LikeRESUMO
Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.
Assuntos
Inativação Gênica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA/genética , Tetraciclina/farmacologia , Animais , Relação Dose-Resposta a DrogaRESUMO
BACKGROUND: RNA interference initiated by small interfering RNAs effectively suppresses gene expression, but the suppression is transient, which limits the therapeutic use of this technique. Polo-like kinase 1 (PLK1) is a key cell cycle regulator that is overexpressed in various human tumors. We used a xenograft mouse model to determine whether an RNA interference-based strategy that used short hairpin RNAs (shRNAs) to suppress PLK1 expression could inhibit tumor growth in vivo. METHODS: HeLa S3 cervical and A549 lung cancer cell lines were transfected with plasmids containing U6 promoter-driven shRNAs against human PLK1 or control (parental or scrambled) plasmids. Plasmids were treated with the nuclease inhibitor aurintricarboxylic acid (ATA) as protection against nucleases in murine blood. Nude mice carrying xenograft tumors were injected with shRNA plasmids, and their xenograft tumor growth was assessed. Northern and western blot analyses were used to measure PLK1 mRNA and protein expression, respectively, in transfected cultured cells and in xenograft tumors. All statistical tests were two-sided. RESULTS: Levels of PLK1 mRNA and protein were lower in HeLa S3 and A549 cancer cells transfected with PLK1 shRNA plasmids than in corresponding cells transfected with control parental or scrambled PLK1S shRNA plasmids. Proliferation of cells transfected with PLK1 shRNA was lower than that of cells transfected with either control plasmid, and proliferation of cells transfected with ATA-treated PLK1 shRNA plasmids was even lower. In mice with human xenograft tumors, PLK1 shRNA expression from ATA-treated plasmids reduced tumor growth to 18% (95% confidence interval [CI] = 12% to 26%; P =.03) and from untreated plasmids reduced tumor growth to 45% (95% CI = 26% to 64%; P =.1) of that of tumors in mice treated with scrambled control PLK1S shRNA plasmids. CONCLUSIONS: The combination of shRNA-mediated gene silencing with effective in vivo gene delivery strategies appears to generate a long-lasting silencing signal.