RESUMEN
INTRODUCTION: Patients with Human Papillomavirus (HPV+)-associated Laryngeal Squamous Cell Carcinoma (LSCC) exhibit dramatically improved survival relative to those with HPV-Negative (HPV-) tumors. In this study, the authors aimed to investigate the radiosensitivity of all available confirmed HPV+ and HPV-LSCC cells in vitro and in vivo. METHODS: Primary LSCC cells were generated from tumor specimens obtained from patients. Real-time PCR was performed to confirm HPV infection and the expression of HPV-related genes (E6 and E7), p53, and pRB. Clonogenic survival assays, western blotting, and flow cytometry were used to assess radiation sensitivity, apoptosis, and the expression of p53 and pRB. p53 and pRB knockout cells were generated using CRISPR/Cas9 technology. RESULTS: HPV+ LSCC cells displayed enhanced radiation sensitivity compared to HPV- cells. Radiation-induced apoptosis in HPV+ LSCC cells, accompanied by increased levels of p53 and pRB. Knockout of p53 or pRB led to radiation resistance and attenuated radiation-induced apoptosis in HPV+ LSCC cells. In vivo experiments showed similar results, where knockout of p53 or pRB decreased radiosensitivity in tumor-bearing mice. CONCLUSION: The present findings demonstrated that HPV+ LSCC cells displayed obvious inherent radiation sensitivity, corresponding to increased apoptosis following radiation exposure. Mechanism study showed that the expression of p53 and pRB in HPV+ cells are required for radiation sensitivity. These findings highlight a novel mechanism by which p53 and pRB play key roles in the radiation sensitivity of HPV+ LSCC compared to HPV-LSCC.
Asunto(s)
Apoptosis , Carcinoma de Células Escamosas , Neoplasias Laríngeas , Infecciones por Papillomavirus , Tolerancia a Radiación , Proteína p53 Supresora de Tumor , Humanos , Neoplasias Laríngeas/radioterapia , Neoplasias Laríngeas/virología , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/virología , Proteína p53 Supresora de Tumor/metabolismo , Infecciones por Papillomavirus/radioterapia , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/complicaciones , Apoptosis/efectos de la radiación , Animales , Línea Celular Tumoral , Reacción en Cadena en Tiempo Real de la Polimerasa , Masculino , Ratones , Citometría de Flujo , Western Blotting , Proteína de Retinoblastoma/metabolismoRESUMEN
The Human papillomaviruses type 16 E7 oncoprotein is a 98-amino-acid, 11-kilodalton acidic oncoprotein with three conserved portions. Due to its interaction with the pRb-E2F complex, CKII, CKI (mostly p21), and even HDAC1, it possesses strong transformative and carcinogenic qualities that inhibit normal differentiation and cell cycle regulation. Here, we target the E7 oncoprotein using two prior research active compounds: asarinin and thiazolo[3,2-a]benzimidazole-3(2H)-one,2-(2-fluorobenzylideno)-7,8-dimethyl (thiazolo), and valproic acid as a control. We are performing molecular docking followed by molecular dynamic analysis. By acting as competitive inhibitors in the binding site, it was hypothesized that both drugs would inhibit E7-mediated pRb degradation and E7-mediated p21 degradation, resulting in decreased cell cycle progression, immortalization, and proliferation. In addition, we expect that the direct inhibitory action of valproic acid in E7 will target the CKII-mediated phosphorylation pathway necessary for destabilizing p130 and pRb. According to the results of the dynamic simulation, stable interactions exist between every compound. Despite the instability of E7 protein, stability results indicate that both natural chemicals are preferable, with thiazolo outperforming valproic acid.
Asunto(s)
Proteínas Oncogénicas Virales , Neoplasias del Cuello Uterino , Femenino , Humanos , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Simulación del Acoplamiento Molecular , Virus del Papiloma Humano , Proteína de Retinoblastoma , Neoplasias del Cuello Uterino/tratamiento farmacológico , Ligandos , Ácido Valproico/farmacologíaRESUMEN
The colonization of land by a single streptophyte algae lineage some 450 million years ago has been linked to multiple key innovations such as three-dimensional growth, alternation of generations, the presence of stomata, as well as innovations inherent to the birth of major plant lineages, such as the origins of vascular tissues, roots, seeds and flowers. Multicellularity, which evolved multiple times in the Chloroplastida coupled with precise spatiotemporal control of proliferation and differentiation were instrumental for the evolution of these traits. RETINOBLASTOMA-RELATED (RBR), the plant homolog of the metazoan Retinoblastoma protein (pRB), is a highly conserved and multifunctional core cell cycle regulator that has been implicated in the evolution of multicellularity in the green lineage as well as in plant multicellularity-related processes such as proliferation, differentiation, stem cell regulation and asymmetric cell division. RBR fulfills these roles through context-specific protein-protein interactions with proteins containing the Leu-x-Cys-x-Glu (LxCxE) short-linear motif (SLiM); however, how RBR-LxCxE interactions have changed throughout major innovations in the Viridiplantae kingdom is a question that remains unexplored. Here, we employ an in silico evo-devo approach to predict and analyze potential RBR-LxCxE interactions in different representative species of key Chloroplastida lineages, providing a valuable resource for deciphering RBR-LxCxE multiple functions. Furthermore, our analyses suggest that RBR-LxCxE interactions are an important component of RBR functions and that interactions with chromatin modifiers/remodelers, DNA replication and repair machinery are highly conserved throughout the Viridiplantae, while LxCxE interactions with transcriptional regulators likely diversified throughout the water-to-land transition.
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Neoplasias de la Retina , Retinoblastoma , Animales , Proteína de Retinoblastoma/metabolismo , Diferenciación CelularRESUMEN
Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.
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Proteínas Intrínsecamente Desordenadas , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Secuencia de Aminoácidos , Proteínas Intrínsecamente Desordenadas/química , Unión Proteica , Dominios Proteicos , Proteína de Retinoblastoma/metabolismoRESUMEN
The retinoblastoma gene (RB1) was the first tumour suppressor cloned; the role of its protein product (RB) as the principal driver of the G1 checkpoint in cell cycle control has been extensively studied. However, many other RB functions are continuously reported. Its role in senescence, DNA repair and apoptosis, among others, is indications of the significance of RB in a vast network of cellular interactions, explaining why RB loss or its malfunction is one of the leading causes of a large number of paediatric and adult cancers. RB was first reported in retinoblastoma, a common intraocular malignancy in the paediatric population worldwide. Currently, its diagnosis is clinical, and in nondeveloped countries, where the incidence is higher, it is performed in advanced stages of the disease, compromising the integrity of the eye and the patient's life. Even though new treatments are being continuously developed, enucleation is still a major choice due to the late disease stage diagnosis and treatments costs. Research into biomarkers is our best option to improve the chances of good results in the treatment and hopes of patients' good quality of life. Here, we recapitulated the history of the disease and the first treatments to put the advances in its clinical management into perspective. We also review the different functions of the protein and the progress in the search for biomarkers. It is clear that there is still a long way to go, but we should offer these children and their families a better way to deal with the disease with the community's effort.
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Neoplasias de la Retina , Retinoblastoma , Adulto , Niño , Genes Supresores de Tumor , Humanos , Calidad de Vida , Neoplasias de la Retina/diagnóstico , Neoplasias de la Retina/genética , Neoplasias de la Retina/terapia , Retinoblastoma/diagnóstico , Retinoblastoma/genética , Retinoblastoma/terapia , Proteína de Retinoblastoma/genéticaRESUMEN
Glutamine metabolism is one of the hallmarks of cancers which is described as an essential role in serving as a major energy and building blocks supply to cell proliferation in cancer cells. Many malignant tumor cells always display glutamine addiction. The "kidney-type" glutaminase (GLS1) is a metabolism enzyme which plays a significant part in glutaminolysis. Interestingly, GLS1 is often overexpressed in highly proliferative cancer cells to fulfill enhanced glutamine demand. So far, GLS1 has been proved to be a significant target during the carcinogenesis process, and emerging evidence reveals that its inhibitors could provide a benefit strategy for cancer therapy. Herein, we summarize the prognostic value of GLS1 in multiple cancer type and its related regulatory factors which are associated with antitumor activity. Moreover, this review article highlights the remarkable reform of discovery and development for GLS1 inhibitors. On the basis of case studies, our perspectives for targeting GLS1 and development of GLS1 antagonist are discussed in the final part.
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Glutaminasa/antagonistas & inhibidores , Glutaminasa/metabolismo , Glutamina/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Apoptosis/fisiología , Benzofenantridinas/farmacología , Proliferación Celular/fisiología , Diazooxonorleucina/metabolismo , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Genes myc/fisiología , Humanos , MicroARNs/fisiología , FN-kappa B/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Oxidación-Reducción , Fosfatos/metabolismo , Pronóstico , Proteína de Retinoblastoma/metabolismo , Sulfuros/farmacología , Tiadiazoles/farmacologíaRESUMEN
In this study, the changes in oncogenic and tumor suppressor signaling pathways in liver and their association with serum and urinary biomarkers of aflatoxin exposure were evaluated in Wistar rats fed diets containing aflatoxin B1 (AFB1) for 90 days. Rats were divided into four groups (n = 15 per group) and assigned to dietary treatments containing 0 (control), 50 (AFB50), 100 (AFB100) and 200 µg AFB1 kg-1 diet (AFB200). Multiple preneoplastic foci of hepatocytes marked with glutathione-S-transferase-placental form (GST-P) were identified in AFB100 and AFB200 groups. Hepatocellular damage induced by AFB1 resulted in overexpression of cyclin D1 and ß-catenin. The liver expression of retinoblastoma (Rb) and p27Kip1 decreased in AFB100 and AFB200 groups, confirming the favorable conditions for neoplastic progression to hepatocellular carcinoma. All samples from rats fed AFB1-contaminated diets had quantifiable AFB1-lysine in serum or urinary AFM1 and AFB1-N7-guanine, with mean levels of 20.42-50.34 ng mL-1, 5.31-37.68 and 39.15-126.37 ng mg-1 creatinine, respectively. Positive correlations were found between AFB1-lysine, AFM1 or AFB1-N7-guanine and GST-P+, ß-catenin+ and cyclin D1+ hepatocytes, while Rb + cells negatively correlated with those AFB1 exposure biomarkers. The pathways evaluated are critical molecular mechanisms of AFB1-induced hepatocarcinogenesis in rats.
Asunto(s)
Aflatoxina B1/toxicidad , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína de Retinoblastoma/metabolismo , beta Catenina/metabolismo , Aflatoxina B1/análogos & derivados , Aflatoxina B1/sangre , Aflatoxina B1/metabolismo , Aflatoxina B1/orina , Aflatoxina M1/orina , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Biomarcadores/sangre , Biomarcadores/orina , Expresión Génica/efectos de los fármacos , Guanina/análogos & derivados , Guanina/orina , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Lisina/sangre , Masculino , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , Ratas WistarRESUMEN
The cancer phenotype is usually characterized by deregulated activity of a variety of cellular kinases, with consequent abnormal hyper-phosphorylation of their target proteins. Therefore, antibodies that allow the detection of phosphorylated versions of proteins have become important tools both preclinically in molecular cancer research, and at the clinical level by serving as tools in pathological analyses of tumors. In order to ensure reliable results, validation of the phospho-specificity of these antibodies is extremely important, since this ensures that they are indeed able to discriminate between the phosphorylated and unphosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. A recommended validation approach consists in dephosphorylating the target protein and assessing if such dephosphorylation abrogates antigen immunoreactivity when using the phospho-specific antibody. In this chapter, we describe a protocol to validate the specificity of a phospho-specific antibody that recognizes a phosphorylated variant of the Retinoblastoma (Rb) protein in lung cancer cell lines. The protocol consists in the dephosphorylation of the Rb-containing protein lysates by treating them with bovine intestinal phosphatase, followed by assessment of the dephosphorylation by immunoblot.
Asunto(s)
Anticuerpos Antineoplásicos/química , Anticuerpos Fosfo-Específicos/química , Immunoblotting , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , Proteína de Retinoblastoma/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patologíaRESUMEN
The Retinoblastoma protein (pRb) is a key cell cycle regulator conserved in a wide variety of organisms. Experimental analysis of pRb's functions in animals and plants has revealed that this protein participates in cell proliferation and differentiation processes. In addition, pRb in animals and its orthologs in plants (RBR), are part of highly conserved protein complexes which suggest the possibility that analogies exist not only between functions carried out by pRb orthologs themselves, but also in the structure and roles of the protein networks where these proteins are involved. Here, we present examples of pRb/RBR participation in cell cycle control, cell differentiation, and in the regulation of epigenetic changes and chromatin remodeling machinery, highlighting the similarities that exist between the composition of such networks in plants and animals.
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Proteínas de Ciclo Celular/fisiología , Ensamble y Desensamble de Cromatina , Epigénesis Genética , Proteínas de Plantas/fisiología , Proteína de Retinoblastoma/fisiología , Animales , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/fisiología , Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Diferenciación Celular/genética , Daño del ADN , Genes de Plantas , Genes de Retinoblastoma , Homeostasis , Mamíferos/genética , Mamíferos/metabolismo , Modelos Moleculares , Familia de Multigenes , Complejos Multiproteicos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiología , Proteínas de Plantas/química , Plantas/genética , Plantas/metabolismo , Conformación Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Proteína de Retinoblastoma/química , Especificidad de la Especie , Células Madre/metabolismoRESUMEN
Aim: Histone acetylation and methylation control gene expression. We investigated the impact of SET knockdown on histone methylation status and the consequences for the miRNAs levels in oral squamous cell carcinoma (OSCC). Methods: OSCC cells with and without SET knockdown were analyzed by quantitative real-time PCR to determine miRNA levels, and by immunoreactions to histone modifications. Results: The knockdown of SET increased the levels of histone H4K20me2 and miR-137. Still, SET protein binds to the miR-137 promoter region. The transfection of miR-137 mimic reduced the KI67 and Rb proteins and proliferation of OSCC cells. Conclusion: Our results show for the first time a relationship between SET and histone methylation associated with the control of miRNA expression and KI67 and Rb as targets of miR-137 in OSCC.
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Proteínas de Unión al ADN/fisiología , Chaperonas de Histonas/fisiología , Histonas/metabolismo , MicroARNs/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Chaperonas de Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Antígeno Ki-67/metabolismo , Metilación , MicroARNs/metabolismo , Neoplasias de la Boca/patología , Proteína de Retinoblastoma/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patologíaRESUMEN
PURPOSE: Meningiomas are common brain tumors, the majority of which are considered benign. Despite surgery and/or radiation therapy, recurrence rates are approximately 8-10%. One likely cause is the dysregulation of cyclin D-cyclin-dependent kinases 4 and 6 (CDK4/6)-retinoblastoma (Rb) pathway, which controls the cell cycle restriction point. This pathway is commonly dysregulated in anaplastic meningioma cell lines (AM) and radiation-induced meningioma cells (RIM), making it a rational target for anti-meningioma therapy. In this study, we investigate the effect of a CDK4/6 inhibitor, palbociclib, with radiation in relevant pre-clinical models. METHODS: In vitro cell culture, ex vivo slice culture and in vivo cell line-derived orthotopic xenograft animal models of AM/RIM were utilized to assess treatment efficacy with palbociclib plus radiation. Treatment effects were examined by immunoblot, cell viability, apoptosis, and cell cycle progression. RESULTS: The in vitro and ex vivo studies demonstrate that palbociclib plus radiation treatment reduced proliferation and has additional effects on cell cycling, including induction of an RB-associated G (1) arrest in Rb+ AM and RIM cells, but not in Rb- cells. Our results also demonstrated reduced CDK4 and CDK6 expression as well as reduced E2F target gene expression (CCNA2 and CCNE2) with the combination therapy. MRI results in vivo demonstrated reduced tumor size at 5 weeks when treated with 14 days palbociclib (10 mg/kg) plus 6 Gy radiation compared to saline-treated tumors. Finally, no hepatic toxicity was found after treatments. CONCLUSION: A pre-clinical murine model provides preclinical evidence for use of palbociclib plus radiation as a therapeutic agent for Rb+ meningiomas.
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Antineoplásicos/uso terapéutico , Neoplasias Meníngeas/terapia , Meningioma/terapia , Neoplasias Inducidas por Radiación/terapia , Piperazinas/uso terapéutico , Piridinas/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Terapia Combinada , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Humanos , Masculino , Ratones , Proteína de Retinoblastoma/metabolismoRESUMEN
Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/genética , Proteína de Retinoblastoma/metabolismo , Retinoblastoma/genética , Astemizol/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Preescolar , Canales de Potasio Éter-A-Go-Go/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Lactante , Masculino , Oncogenes , ARN Mensajero , Neoplasias de la Retina/genética , Retinoblastoma/metabolismo , Proteína de Retinoblastoma/genética , TransfecciónRESUMEN
Retinoblastoma (Rb) was the first tumour suppressor factor described, and it is dysfunctional in several types of cancers. Structurally, Rb is a very large, multifunctional protein organized in different domains connected by intrinsically disordered regions. Due to the complex structure of Rb, biochemical manipulation is difficult. The Rb protein has been implicated in many different cellular processes, such as the cell cycle control, senescence and even apoptosis. The activity of Rb is regulated by phosphorylation, and many different sites of phosphorylation have been described. However, the oncoprotein HDM2, can promote Rb degradation by the proteasome. This form of Rb regulation is largely unknown. Here we report the expression and purification of the full-length Rb protein and its phosphomimetic form, Rb(S567D), in a recombinant system. We also produced and purified the HDM2 protein and its phosphomimetic mutant, HDM2(S395D). The proteins interacted strongly when we used the phosphomimetic mutants, mimicking damaged DNA conditions. The expression of the proteins in E. coli allowed us to control the phosphorylation status of the proteins.
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Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína de Retinoblastoma/aislamiento & purificación , Proteína de Retinoblastoma/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/genéticaRESUMEN
Retinoblastoma is a childhood ocular tumor often caused by the biallelic inactivation of the RB1 gene affecting children up to 5 years of age. A retinoblastoma protein (pRB), encoded by the tumor suppressor gene RB1, is responsible for the regular progression of the G1 phase to the phase S of the cell cycle. This protein forms a complex with the transcriptional factor E2F causing the cell cycle to remain in the G0/G1 stage. With a phosphorylation of cyclin-dependent kinases (CDK), the phosphorylation of the RB protein is activated and the complex formed with E2F is disrupted, with the advancement of the cell cycle to an S phase and cell proliferation. All the control of cell proliferation is regulated not only by the complex formed by RB and E2F proteins, but also by other proteins that participate in and/or interfere in this cell division control mechanism, such as mdm2, mdm4 and p21 proteins.
O retinoblastoma é um tumor ocular infantil ocasionado, frequentemente, pela inativação bialélica do gene RB1 acometendo crianças até os 5 anos de idade. A proteína retinoblastoma (pRB), codificada pelo gene supressor tumoral RB1, é responsável por regular a progressão da fase G1 para a fase S do ciclo celular. Essa proteína forma um complexo com o fator transcricional E2F fazendo com que o ciclo celular permaneça no estágio G0/G1. Com a fosforilação de quinases dependentes de ciclinas, a fosforilação da proteína RB é ativada e o complexo formado com o E2F é desfeito, havendo o avanço do ciclo celular para a fase S e a proliferação celular. Todo esse controle da proliferação celular é regulado não só pelo complexo formado pela proteína RB e E2F, mas também por outras proteínas que participam e/ou interferem neste mecanismo de controle da divisão celular, como, por exemplo, as proteínas mdm2, mdm4, p21
Asunto(s)
Retinoblastoma , Proteína de Retinoblastoma , Proteínas de Ciclo Celular , Silenciador del GenRESUMEN
Prediction of lung cancer metastasis relies on post-resection assessment of tumor histology, which is a severe limitation since only a minority of lung cancer patients are diagnosed with resectable disease. Therefore, characterization of metastasis-predicting biomarkers in pre-resection small biopsy specimens is urgently needed. Here we report a biomarker consisting of the phosphorylation of the retinoblastoma protein (Rb) on serine 249 combined with elevated p39 expression. This biomarker correlates with epithelial-to-mesenchymal transition traits in non-small cell lung carcinoma (NSCLC) cells. Immunohistochemistry staining of NSCLC tumor microarrays showed that strong phospho-Rb S249 staining positively correlated with tumor grade specifically in the squamous cell carcinoma (SCC) subtype. Strong immunoreactivity for p39 positively correlated with tumor stage, lymph node invasion, and distant metastases, also in SCC. Linear regression analyses showed that the combined scoring for phospho-Rb S249, p39 and E-cadherin in SCC is even more accurate at predicting tumor staging, relative to each score individually. We propose that combined immunohistochemistry staining of NSCLC samples for Rb phosphorylation on S249, p39, and E-cadherin protein expression could aid in the assessment of tumor staging and metastatic potential when tested in small primary tumor biopsies. The intense staining for phospho-Rb S249 that we observed in high grade SCC could also aid in the precise sub-classification of poorly differentiated SCCs.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Proteína de Retinoblastoma/metabolismo , Biomarcadores de Tumor/genética , Cadherinas/biosíntesis , Cadherinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Adhesión Celular/genética , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Clasificación del Tumor , Metástasis de la Neoplasia , Fosforilación , Proteína de Retinoblastoma/genéticaRESUMEN
Sphingolipids are involved in the regulation of cell proliferation. It has been reported that diacylglycerol and sphingosine-1-phosphate generation, during the synthesis of phospho-sphingolipids, is necessary for both, G1-S transition of cell cycle during the sustained activation of protein kinase C in various cell models (MDCK, Saccharomyces and Entamoeba) and AKT pathway activation. During the estrous cycle of the rat, AKT signaling is the main pathway involved in the regulation of uterine cell proliferation. The aim of the present study was to investigate the role of sphingolipid synthesis during proliferation of uterine cells in the estrous cycle of the rat. On metestrus day, when both luminal and glandular uterine epithelia present the maximal BrdU-labeled cells (S phase cells), there was an increase in the relative abundance of total sphingomyelins, as compared to estrus day. Myriocin, a sphingolipid synthesis inhibitor administered on estrus day, before the new cell cycle of epithelial cells is initiated, decreased the abundance of sphingomyelin, accompanied by proliferation arrest in uterine epithelial cells on metestrus day. In order to study the sphingolipid signaling pathway affected by myriocin, we evaluated the activation of the PKC-AKT-GSK3b-Cyclin D3 pathway. We observed that total and phosphorylated protein kinase C diminished in uterine epithelial cells of myriocin treated animals. Interestingly, cyclin D3 nuclear localization was blocked by myriocin, concomitantly with a decrease in nuclear pRb expression. In conclusion, we demonstrate that sphingolipid synthesis and signaling are involved in uterine epithelial cell proliferation during the estrous cycle of the rat.
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Endometrio/fisiología , Células Epiteliales/fisiología , Metestro/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingolípidos/biosíntesis , Animales , Ciclina D3/metabolismo , Ácidos Grasos Monoinsaturados , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Ratas Wistar , Proteína de Retinoblastoma/metabolismoRESUMEN
Several microRNAs (miRNAs) have been reported as oncogenes or tumor suppressors in many cancers, including gastric cancer (GC). However, the role and molecular mechanism of miR-3129 in GC is largely unknown. We aimed to explore the function and the underlying molecular mechanism of miR-3129 in GC. Cancer tissues and corresponding adjacent tissues were collected from 50 patients with GC, and the expression of miR-3129 was detected by RT-qPCR. The expression of miR-3129 and pRb in human GC cell line SCG7091 was altered by transient transfection. Thereafter, MTT and flow cytometry assays were used to analyze cell viability and cell cycle. The expression of cyclin E, CDK2, CDK2 inhibitors (p16 and 21), and pRb were detected by RT-qPCR and western blot. A significant up-regulation of miR-3129 was observed in GC tissues compared to adjacent tissues. Overexpression of miR-3129 significantly improved cell viability after 4 days of post-transfection. Flow cytometry assay results showed that the miR-3129 overexpression arrested more SGC7901 cells at S phase. Moreover, overexpression of miR-3129 down-regulated the expression of CDK2 inhibitors while it up-regulated the expression levels of cyclin E, CDK2, and pRb. Interestingly, we found that pRb inhibition reversed the effect of miR-3129 inhibitor on cell proliferation in SGC7901 cells, increased cell viability, reduced cells at G0/1 phase, and modulated the expression of proliferation-related factors. Our results revealed that miR-3129 functioned as an oncogene through positive regulation of pRb and may prove to be a promising option for molecular therapy of GC.
Asunto(s)
Proliferación Celular/genética , MicroARNs/metabolismo , Proteína de Retinoblastoma/genética , Neoplasias Gástricas/genética , Adulto , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Transfección , Regulación hacia ArribaRESUMEN
BACKGROUND: Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role. METHODS: PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures. RESULTS: HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin. CONCLUSION: This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.
Asunto(s)
Papillomavirus Humano 16/genética , Proteínas E7 de Papillomavirus/genética , Fosfolipasa D/metabolismo , Proteína de Retinoblastoma/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Resistencia a Antineoplásicos/efectos de los fármacos , Expresión Génica , Humanos , Queratinocitos/metabolismo , Queratinocitos/virología , Modelos Biológicos , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Fosfolipasa D/genética , Unión Proteica , Sirolimus/farmacologíaRESUMEN
Pocket proteins retinoblastoma (pRb), p107 and p130 are negative regulators of cellular proliferation and multifunctional proteins regulating development, differentiation and chromatin structure. The retinoblastoma protein is a potent tumor suppressor mutated in a wide range of human cancers, and oncogenic viruses often interfere with cell cycle regulation by inactivating pRb. The LxCxE and pRb AB groove short linear motifs (SLiMs) are key to many pocket protein mediated interactions including host and viral partners. A review of available experimental evidence reveals that several core residues composing each motif instance are determinants for binding. In the LxCxE motif, a fourth hydrophobic position that might allow variable spacing is required for binding. In both motifs, flanking regions including charged stretches and phosphorylation sites can fine-tune the binding affinity and specificity of pocket protein SLiM-mediated interactions. Flanking regions can modulate pocket protein binding specificity, or tune the high affinity interactions of viral proteins that hijack the pRb network. The location of SLiMs within intrinsically disordered regions allows faster evolutionary rates that enable viruses to acquire a functional variant of the core motif by convergent evolution, and subsequently test numerous combinations of flanking regions towards maximizing interaction specificity and affinity. This knowledge can guide future efforts directed at the design of peptide-based compounds that can target pocket proteins to regulate the G1/S cell cycle checkpoint or impair viral mediated pRb inactivation.
Asunto(s)
Proteína de Retinoblastoma/química , Proteína de Retinoblastoma/metabolismo , Secuencias de Aminoácidos , Animales , Humanos , Modelos Moleculares , Unión Proteica , Ratas , Especificidad por SustratoRESUMEN
Several microRNAs (miRNAs) have been reported as oncogenes or tumor suppressors in many cancers, including gastric cancer (GC). However, the role and molecular mechanism of miR-3129 in GC is largely unknown. We aimed to explore the function and the underlying molecular mechanism of miR-3129 in GC. Cancer tissues and corresponding adjacent tissues were collected from 50 patients with GC, and the expression of miR-3129 was detected by RT-qPCR. The expression of miR-3129 and pRb in human GC cell line SCG7091 was altered by transient transfection. Thereafter, MTT and flow cytometry assays were used to analyze cell viability and cell cycle. The expression of cyclin E, CDK2, CDK2 inhibitors (p16 and 21), and pRb were detected by RT-qPCR and western blot. A significant up-regulation of miR-3129 was observed in GC tissues compared to adjacent tissues. Overexpression of miR-3129 significantly improved cell viability after 4 days of post-transfection. Flow cytometry assay results showed that the miR-3129 overexpression arrested more SGC7901 cells at S phase. Moreover, overexpression of miR-3129 down-regulated the expression of CDK2 inhibitors while it up-regulated the expression levels of cyclin E, CDK2, and pRb. Interestingly, we found that pRb inhibition reversed the effect of miR-3129 inhibitor on cell proliferation in SGC7901 cells, increased cell viability, reduced cells at G0/1 phase, and modulated the expression of proliferation-related factors. Our results revealed that miR-3129 functioned as an oncogene through positive regulation of pRb and may prove to be a promising option for molecular therapy of GC.