RESUMEN
Leucine Rich Repeat of Flightless-1 Interacting Protein 1/GC-binding factor 2 (LRRFIP1/GCF2) cDNA was cloned for a transcriptional repressor GCF2, which bound sequence-specifically to a GC-rich element of epidermal growth factor receptor (EGFR) gene and repressed its promotor. LRRFIP1/GCF2 was also cloned as a double stranded RNA (dsRNA)-binding protein to trans-activation responsive region (TAR) RNA of Human Immunodeficiency Virus-1 (HIV-1), termed as TAR RNA interacting protein (TRIP), and as a binding protein to the Leucine Rich Repeat (LRR) of Flightless-1(Fli-1), termed as Flightless-1 LRR associated protein 1 (FLAP1) and LRR domain of Flightless-1 interacting Protein 1 (LRRFIP1). Subsequent functional studies have revealed that LRRFIP1/GCF2 played multiple roles in the regulation of diverse biological systems and processes, such as in immune response to microorganisms and auto-immunity, remodeling of cytoskeletal system, signal transduction pathways, and transcriptional regulations of genes. Dysregulations of LRRFIP1/GCF2 have been implicated in the causes of several experimental and clinico-pathological states and the responses to them, such as autoimmune diseases, excitotoxicity after stroke, thrombosis formation, inflammation and obesity, the wound healing process, and in cancers. LRRFIP1/GCF2 is a bioregulator in multidisciplinary systems of the human body and its dysregulation can cause diverse human diseases.
Asunto(s)
Enfermedad , Proteínas de Unión al ARN/metabolismo , Citoesqueleto/metabolismo , Humanos , Modelos Biológicos , Transducción de Señal , Cicatrización de HeridasRESUMEN
Autosomal recessive primary microcephaly (MCPH) is a very rare neuro-developmental disease with brain size reduction. More than a dozen loci encoding proteins of diverse function have been shown to be responsible for MCPH1-13. Mutations in the D40/KNL1/CASC5 gene, which was initially characterized as a gene involved in chromosomal translocation in leukemia and as a member of the cancer/testis gene family, was later found to encode a kinetochore protein essential for mitotic cell division and to cause MCPH4. Although our previous studies showed that this gene is required for cell growth and division in vitro and in animal experiments, the revelation that mutations in this gene caused microcephaly provides in vivo evidence of a critical role in brain growth. In this review, we describe mutated gene targets responsible for MCPH1-13 and summarize clinical studies of, and molecular and biological aspects of the gene and encoded protein responsible for MCPH4.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Microcefalia/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Proteínas de Ciclo Celular/metabolismo , Expresión Génica , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Microcefalia/clasificación , Microcefalia/metabolismo , Microcefalia/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitosis , Neuronas/metabolismo , Neuronas/patología , Unión Proteica , Dominios Proteicos , Mapeo de Interacción de ProteínasRESUMEN
The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis. Short inhibitory RNA against D40, D40 siRNA, has been shown to deplete the D40 protein in the human cancer cell line HeLa, which harbors wild-type p53, and this activity was followed by the significant inhibition of cell growth and induction of apoptotic cell death. The p53-null cancer cell line, PC-3M-luc, is also sensitive to the significant growth inhibition and cell death induced by D40 siRNA. The growth of PC-3M-luc tumors transplanted into nude mice was inhibited by the systemic administration of D40 siRNA and the atelocollagen complex. Furthermore, D40 siRNA significantly inhibited growth and induced apoptotic cell death in a cell line with a gain-of-function (GOF) mutation in p53, MDA-MB231-luc, and also inhibited the growth of tumors transplanted into mice when administered as a D40 siRNA/atelocollagen complex. These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status. Therefore, D40 siRNA is a potential candidate anti-cancer reagent.
Asunto(s)
Técnicas de Silenciamiento del Gen , Marcación de Gen , Proteínas Asociadas a Microtúbulos/genética , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Ratones , Mutación , Neoplasias/metabolismo , Neoplasias/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor suppressor genes have received much attention for their roles in the development of human malignancies. Gelsolin has been found to be down-regulated in several types of human cancers, including leukemias. It is, however, expressed in macrophages, which are the final differentiation derivatives for the monocytic myeloid lineage, implicating this protein in the differentiation process of such cells. In order to investigate the role of gelsolin in leukaemic cell differentiation, stable clones over-expressing ectopic gelsolin, and a control clone were established from U937 leukaemia cells. Unlike the control cells, both gelsolin-overexpressing clones displayed retarded growth, improved monocytic morphology, increased NADPH and NSE activities, and enhanced surface expression of the ß-integrin receptor, CD11b, when compared with the parental U937 cells. Interestingly, RT- PCR and western blot analysis also revealed that gelsolin enhanced p21CIP1 mRNA and protein expression in the overexpressing clones. Moreover, transient transfection with siRNA silencing P21CIP1, but not the control siRNA, resulted in a reduction in monocytic differentiation, accompanied by an increase in proliferation. In conclusion, our work demonstrates that gelsolin, by itself, is capable of inducing monocytic differentiation in U937 leukaemia cells, most probably through p21CIP1 activation.
Asunto(s)
Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Gelsolina/metabolismo , Regulación Neoplásica de la Expresión Génica , Monocitos/metabolismo , Antígeno CD11b/metabolismo , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Esterasas/metabolismo , Gelsolina/genética , Vectores Genéticos , Humanos , Monocitos/patología , NADP/metabolismo , Fenotipo , Interferencia de ARN , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Transcripción/metabolismo , Transfección , Células U937 , Regulación hacia ArribaRESUMEN
We previously identified a novel human cancer/testis gene, D40, which is dominantly expressed in testicular germ cells, various cancer cell lines and primary human tumors. The expression of D40 mRNA and proteins in various testicular tissues was quantified using the conventional reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR and western blot analysis. The relationship between levels of D40 expression, serum follicle stimulating hormone (FSH) level and Johnsen's score was examined. D40 mRNA expression was observed in the testes of infertile men, except those with Sertoli-cell-only syndrome or Klinefelter's syndrome. The quantity of D40 mRNA and protein was correlated with Johnsen's score and inversely correlated with serum FSH level. The present results show that the expression levels of D40 mRNA and proteins decrease according to the degree of spermatogenesis impairment in male infertile patients.
Asunto(s)
Infertilidad Masculina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Testículo/metabolismo , Humanos , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Masculino , Proteínas Asociadas a Microtúbulos/genética , Testículo/patologíaRESUMEN
N116Y, H-RAS mutant, has dominant negative activity in the RAS function and a suppressive effect on the growth of various types of cancer cells. However, a replication error of N116Y is of potential concern for carcinogenesis in clinical application. To decease the concern, we constructed modified N116Y by deleting the carboxyl terminus, which is necessary for the oncogenic function of Ras. One of the C-terminal deletion mutants of N116Y, N116Y-Cdel2 showed a growth-suppressing effect on various human cancer cell lines in vitro: the cervical cancer cell line HeLa, the pancreatic cancer cell line PCI43, the colon cancer cell lines SW480 and LoVo, and the tongue cancer cell line SAS. In addition, the suppressive effect of N116Y-Cdel2 on LoVo cells was also observed in vivo using a nonviral gene transfer vector, HVJ envelope. Our experiments suggest that the modified N116Y is a potential candidate gene for human cancer gene therapy.
Asunto(s)
Técnicas de Transferencia de Gen , Genes ras , Neoplasias/genética , Terapia Genética , Vectores Genéticos , Humanos , Neoplasias/patologíaRESUMEN
Epithelial-mesenchymal transition (EMT) describes a process occurring during development and oncogenesis by which epithelial cells obtain fibroblast-like properties and show reduced cell adhesion and increased motility. In this report, we demonstrated typical EMT in human mammary epithelial MCF10A small interfering (si)RNA gelsolin-knockdown cells. EMT was characterized by fibroblastic morphology, loss of contact inhibition and focus formation in monolayer growth, enhanced motility and invasiveness in vitro, increased actin filaments, overexpression of RAC, activation of both extracellular signal-regulated kinase and AKT, inactivation of glycogen synthase kinase-3, conversion of cadherin from the E- to N-type and induction of the transcription factor Snail. These results suggested that gelsolin functions as a switch that controls E- and N-cadherin conversion via Snail, and demonstrated that its knockdown leads to EMT in human mammary epithelial cells and possibly to the development of human mammary tumors.
Asunto(s)
Transformación Celular Neoplásica , Células Epiteliales/fisiología , Gelsolina/biosíntesis , Glándulas Mamarias Humanas/citología , ARN Interferente Pequeño , Neoplasias de la Mama/fisiopatología , Cadherinas/metabolismo , Adhesión Celular , Línea Celular , Movimiento Celular , Epitelio , Femenino , Fibroblastos , Genes de Cambio , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Glándulas Mamarias Humanas/fisiología , Mesodermo , Proteínas Proto-Oncogénicas c-akt/fisiología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/fisiologíaRESUMEN
Amyloid-beta (A beta) peptides play a central role in the development of Alzheimer's disease. They are known to induce mitochondrial dysfunction and caspase activation, resulting in apoptosis of neuronal cells. Here we show that human cytoplasmic gelsolin inhibits A beta peptide-induced cell death of neuronally differentiated rat pheochromocytoma (PC-12) cells. We also show that the segment 5 but not 6 of human cytoplasmic gelsolin is the important region responsible for inhibition of A beta-induced cytotoxicity. Mitochondrial dysfunction associated with cell death, membrane potential loss and the release of cytochrome c are all abrogated in the presence of human full-length or segment 5 cytoplasmic gelsolin. Furthermore, RNA interference to reduce expression of endogenous gelsolin in PC-12 cells shows that rat gelsolin act as an inhibitor of A beta cytotoxicity. These results demonstrate that cytoplasmic gelsolin plays a important role in inhibiting Abeta-induced cytotoxicity by inhibiting apoptotic mitochondrial changes. The segment 5 of human cytoplasmic gelsolin is sufficient for the function.
Asunto(s)
Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/farmacología , Gelsolina/metabolismo , Potenciales de la Membrana/fisiología , Mitocondrias/fisiología , Neuronas/metabolismo , Envejecimiento/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Células PC12 , RatasRESUMEN
We have previously identified and cloned a human gene, D40, that is preferentially expressed in testis among normal organs, while it is widely expressed in various human tumor cell lines and primary tumors derived from different organs. In this report, we have examined the expression and localization of this protein in human testis with an antibody specific to D40 protein. In Western analyses, the anti-D40 antibody recognized a major band with a molecular mass of 300 kDa and a minor band of 250 kDa. These bands were not observed in the testis lysates from patients with Sertoli-cell-only syndrome and with Kleinfelter syndrome, who lack germ cells of the testis, indicating that D40 protein is expressed in the germ cells of normal testis. Immunohistochemical studies have revealed that D40 protein is highly expressed in spermatocytes and in the pre-acrosome of round spermatids. In the acrosome, D40 protein expression is observed not inside but outside the acrosome membrane. This is consistent with the finding that the amino-acid sequence at the amino terminal of the D40 protein lacks a hydrophobic signal peptide that is required for proteins to translocate to the membrane. Expression of D40 protein is observed in the acrosome of ejaculated spermatozoa as well, although the level is low compared with that in the pre-acrosome of spermatids. These results suggest that D40 protein plays important roles in spermatogenesis, especially in the formation and maintenance of the acrosome.
Asunto(s)
Acrosoma/química , Proteínas Portadoras/análisis , Espermatogénesis/fisiología , Espermatozoides/química , Anticuerpos Monoclonales , Western Blotting/métodos , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica/métodos , Masculino , Proteínas Asociadas a Microtúbulos , Espermátides , Espermatocitos/química , Neoplasias Testiculares/genéticaRESUMEN
We previously reported that gelsolin gene expression is reduced in various tumors. In an effort to gain further insights into the mechanism of gelsolin downregulation in tumors, we examined the in vivo properties of the gelsolin promoter in urinary bladder cancer cell lines. Neither mutation nor hypermethylation was responsible for gene silencing at the promoter. After exposure to trichostatin A (TSA), a histone deacetylase inhibitor, gelsolin promoter activity was markedly enhanced in the cancer cells, not in cells derived from normal tissue. Chromatin immunoprecipitation assays revealed that both histones H3 and H4 were hypoacetylated in the promoter region of the cancer cells, and the accumulation of acetylated histones was detected by TSA treatment. In vivo footprinting analysis revealed the presence of dimethylsulfate (DMS) hypersensitive site in the untranslated region around nucleotide--35 only in the cancer cells but not in cells derived from normal tissue, and analysis of KMnO4 reactive nucleotides showed that the stem loop structure could be formed in vivo of the cancer cells. This novel stem loop structure may play a part in regulating the transcription of the gelsolin gene in the cancer cells. These results suggest that nucleosome accessibility through histone deacetylation and structural changes (DMS hypersensitivity and stem loop structure) in the promoter region form the basis of the mechanism leading to the silencing of gelsolin gene in human bladder cancer.