RESUMEN
Telomerase is a ribonucleoprotein ribonucleic enzyme that elongates telomere repeat sequences at the ends of chromosomes and contributes to cellular immortalization. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been observed to be reactivated in immortalized cells. Notably, most cancer cells have been found to have active hTERT mRNA transcription, resulting in continuous cell division, which is crucial for malignant transformation. Therefore, discovering mechanisms underlying the regulation of hTERT transcription is an attractive target for cancer-specific treatments.Loss of heterozygosity (LOH) of chromosome 3p21.3 has been frequently observed in human oral squamous cell carcinoma (OSCC). Moreover, we previously reported that HSC3 OSCC microcell hybrid clones with an introduced human chromosome 3 (HSC3#3) showed inhibition of hTERT transcription compared with the parental HSC3 cells. This study examined whether hTERT transcription regulators are present in the 3p21.3 region. We constructed a human artificial chromosome (HAC) vector (3p21.3-HAC) with only the 3p21.3-p22.2 region and performed functional analysis using the 3p21.3-HAC. HSC3 microcell hybrid clones with an introduced 3p21.3-HAC exhibited significant suppression of hTERT transcription, similar to the microcell hybrid clones with an intact chromosome 3. In contrast, HSC3 clones with truncated chromosome 3 with deletion of the 3p21.3 region (3delp21.3) showed no effect on hTERT expression levels. These results provide direct evidence that hTERT suppressor gene(s) were retained in the 3p21.3 region, suggesting that the presence of regulatory factors that control telomerase enzyme activity may be involved in the development of OSCC.
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Carcinoma de Células Escamosas , Cromosomas Artificiales Humanos , Neoplasias de la Boca , Telomerasa , Humanos , Telomerasa/genética , Telomerasa/metabolismo , Carcinoma de Células Escamosas/genética , Cromosomas Artificiales Humanos/metabolismo , Neoplasias de la Boca/genética , Transcripción GenéticaRESUMEN
We previously generated fully human antibody-producing TC-mAb mice for obtaining potential therapeutic monoclonal antibodies (mAbs). In this study, we investigated 377 clones of fully human mAbs against a tumor antigen, epithelial cell adhesion molecule (EpCAM), to determine their antigen binding properties. We revealed that a wide variety of mAbs against EpCAM can be obtained from TC-mAb mice by the combination of epitope mapping analysis of mAbs to EpCAM and native conformational recognition analysis. Analysis of 72 mAbs reacting with the native form of EpCAM indicated that the EpCL region (amino acids 24-80) is more antigenic than the EpRE region (81-265), consistent with numerous previous studies. To evaluate the potential of mAbs against antibody-drug conjugates, mAbs were directly labeled with DM1, a maytansine derivative, using an affinity peptide-based chemical conjugation (CCAP) method. The cytotoxicity of the conjugates against a human colon cancer cell line could be clearly detected with high-affinity as well as low-affinity mAbs by the CCAP method, suggesting the advantage of this method. Thus, this study demonstrated that TC-mAb mice can provide a wide variety of antibodies and revealed an effective way of identifying candidates for fully human ADC therapeutics.
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Neoplasias del Colon , Inmunoconjugados , Humanos , Ratones , Animales , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Molécula de Adhesión Celular Epitelial , Antígenos de Neoplasias , Neoplasias del Colon/patología , Anticuerpos MonoclonalesRESUMEN
Bladder cancer is divided into two molecular subtypes, luminal and basal, which form papillary and nodular tumors, respectively, and are identifiable by gene expression profiling. Although loss of heterozygosity (LOH) of the long arm of human chromosome 9 (9q) has been observed in the early development of both types of bladder cancer, the functional significance of LOH remains to be clarified. The present study introduced human chromosome 9q into basal bladder cancer cell line, SCaBER, using microcell-mediated chromosome transfer to investigate the effect of LOH of 9q on molecular bladder cancer subtypes. These cells demonstrated decreased proliferation and migration capacity compared with parental and control cells. Conversely, transfer of human chromosome 4 did not change the cell phenotype. Expression level of peroxisome proliferator-activated receptor (PPAR)γ, a marker of luminal type, increased 3.0-4.4 fold in SCaBER cells altered with 9q compared with parental SCaBER cells. Furthermore, the expression levels of tumor suppressor PTEN, which regulates PPARγ, also increased in 9q-altered cells. These results suggested that human chromosome 9q may carry regulatory genes for PPARγ that are involved in the progression of neoplastic transformation of bladder cancer.
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INSTRUCTION: The human amphoterin-induced gene and open reading frame (AMIGO) was identified as a novel cell adhesion molecule of type I transmembrane protein. AMIGO2 is one of three members of the AMIGO family (AMIGO1, 2, and 3), and the similarity between them is approximately 40% at the amino acid level. We have previously shown that AMIGO2 functions as a driver of liver metastasis. Immunohistochemical analysis of AMIGO2 expression in colorectal cancer (CRC) using a commercially available anti-AMIGO2 mouse monoclonal antibody clone sc-373699 (sc mAb) correlated with liver metastasis and poor prognosis. However, the sc mAb was found to be cross-reactive with all three molecules in the AMIGO family. METHODS: We generated a rat monoclonal antibody clone rTNK1A0012 (rTNK mAb) for human AMIGO2. The rTNK mAb was used to re-evaluate the association between AMIGO2 expression and liver metastases/clinical outcomes using the same CRC tissue samples previously reported with sc mAb. RESULTS: Western blot analysis revealed that a rTNK mAb was identified as being specific for AMIGO2 protein and did not cross-react with AMIGO1 and AMIGO3. The rTNK mAb and sc mAb showed higher AMIGO2 expression, which correlates with a high frequency of liver metastases (65.3% and 47.5%, respectively), while multivariate analysis showed that AMIGO2 expression was an independent prognostic factor for liver metastases (p = 7.930E-10 and p = 1.707E-5). The Kaplan-Meier analyses showed that the rTNK mAb (p = 0.004), but not sc mAb (p = 0.107), predicted worse overall survival in patients with high AMIGO2 expression. The relationship between AMIGO2 expression and poor disease-specific survival showed a higher level of significance for rTNK mAb (p = 0.00004) compared to sc mAb (p = 0.001). CONCLUSIONS: These results indicate that the developed rTNK1A0012 mAb is an antibody that specifically recognizes AMIGO2 by immunohistochemistry and can be a more reliable and applicable method for the diagnostic detection of liver metastases and worse prognosis in patients with high AMIGO2-expressing CRC.
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Neoplasias Colorrectales , Neoplasias Hepáticas , Animales , Neoplasias Colorrectales/patología , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/secundario , Proteínas de la Membrana/genética , Ratones , Proteínas del Tejido Nervioso/genética , Pronóstico , RatasRESUMEN
Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPß signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from 'permissive' H3K9me3 regions in senescent cells.
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Heterocromatina , Histonas , Heterocromatina/genética , Histonas/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Senescencia Celular/genética , Expresión GénicaRESUMEN
Our previous study revealed that metastasisassociated protein 1 (MTA1), which is expressed in vascular endothelial cells, acts as a tube formation promoting factor. The present study aimed to clarify the importance of MTA1 expression in tube formation using MTA1knockout (KO) endothelial cells (MTA1KO MSS31 cells). Tube formation was significantly suppressed in MTA1KO MSS31 cells, whereas MTA1overexpression MTA1KO MSS31 cells regained the ability to form tubelike structures. In addition, western blotting analysis revealed that MTA1KO MSS31 cells showed significantly higher levels of phosphorylation of nonmuscle myosin heavy chain IIa, which resulted in suppression of tube formation. This effect was attributed to a decrease of MTA1/S100 calciumbinding protein A4 complex formation. Moreover, inhibition of tube formation in MTA1KO MSS31 cells could not be rescued by stimulation with vascular endothelial growth factor (VEGF). These results demonstrated that MTA1 may serve as an essential molecule for angiogenesis in endothelial cells and be involved in different steps of the angiogenic process compared with the VEGF/VEGF receptor 2 pathway. The findings showed that endothelial MTA1 and its pathway may serve as promising targets for inhibiting tumor angiogenesis, further supporting the development of MTA1based antiangiogenic therapies.
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Células Endoteliales/metabolismo , Neovascularización Patológica/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Células Cultivadas , China , Ratones , Morfogénesis/efectos de los fármacos , Cadenas Pesadas de Miosina/metabolismo , Metástasis de la Neoplasia/genética , Fosforilación , Proteínas Represoras/fisiología , Proteína de Unión al Calcio S100A4/metabolismo , Transactivadores/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Melanoma is one of the most aggressive types of cancer wherein resistance to treatment prevails. Therefore, it is important to discover novel molecular targets of melanoma progression as potential treatments. Here we show that paired-like homeodomain transcription factor 1 (PITX1) plays a crucial role in the inhibition of melanoma progression through regulation of SRY-box transcription factors (SOX) gene family mRNA transcription. Overexpression of PITX1 in melanoma cell lines resulted in a reduction in cell proliferation and an increase in apoptosis. Additionally, analysis of protein levels revealed an antagonistic cross-regulation between SOX9 and SOX10. Interestingly, PITX1 binds to the SOX9 promoter region as a positive regulatory transcription factor; PITX1 mRNA expression levels were positively correlated with SOX9 expression, and negatively correlated with SOX10 expression in melanoma tissues. Furthermore, transcription of the long noncoding RNA (lncRNA), survival-associated mitochondrial melanoma-specific oncogenic noncoding RNA (SAMMSON), was decreased in PITX1-overexpressing cells. Taken together, the findings in this study indicate that PITX1 may act as a negative regulatory factor in the development and progression of melanoma via direct targeting of the SOX signaling.
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Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Melanoma/metabolismo , Familia de Multigenes , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción SOX/genética , Animales , Apoptosis/genética , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Secuenciación de Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Melanoma/patología , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción SOX/metabolismoRESUMEN
Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene (hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.
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Carcinogénesis/genética , Quimera/genética , Cromosomas Humanos Par 3/química , Pérdida de Heterocigocidad , Neoplasias Pancreáticas/genética , Telomerasa/genética , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Cromosomas Humanos Par 3/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Invasividad Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , Telomerasa/metabolismo , Transcripción GenéticaRESUMEN
Fragile X-related tremor/ataxia syndrome (FXTAS) is a neurodegenerative disease caused by CGG triplet repeat expansions in FMR1, which elicit repeat-associated non-AUG (RAN) translation and produce the toxic protein FMRpolyG. We show that FMRpolyG interacts with pathogenic CGG repeat-derived RNA G-quadruplexes (CGG-G4RNA), propagates cell to cell, and induces neuronal dysfunction. The FMRpolyG polyglycine domain has a prion-like property, preferentially binding to CGG-G4RNA. Treatment with 5-aminolevulinic acid, which is metabolized to protoporphyrin IX, inhibited RAN translation of FMRpolyG and CGG-G4RNA-induced FMRpolyG aggregation, ameliorating aberrant synaptic plasticity and behavior in FXTAS model mice. Thus, we present a novel therapeutic strategy to target G4RNA prionoids.
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Síndrome del Cromosoma X Frágil , G-Cuádruplex , Enfermedades Neurodegenerativas , Animales , Ataxia/genética , Ataxia/metabolismo , Ataxia/patología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Ratones , Temblor/genética , Temblor/metabolismoRESUMEN
Fragile X syndrome (FXS) is the most common inheritable form of intellectual disability. FMR1, the gene responsible for FXS, is located on human chromosome Xq27.3 and contains a stretch of CGG trinucleotide repeats in its 5' untranslated region. FXS is caused by CGG repeats that expand beyond 200, resulting in FMR1 silencing via promoter hypermethylation. The molecular mechanism underlying CGG repeat expansion, a fundamental cause of FXS, remains poorly understood, partly due to a lack of experimental systems. Accumulated evidence indicates that the large chromosomal region flanking a CGG repeat is critical for repeat dynamics. In the present study, we isolated and introduced whole human X chromosomes from healthy, FXS premutation carriers, or FXS patients who carried disease condition-associated CGG repeat lengths, into mouse A9 cells via microcell-mediated chromosome transfer. The CGG repeat length-associated methylation status and human FMR1 expression in these monochromosomal hybrid cells mimicked those in humans. Thus, this set of A9 cells containing CGG repeats from three different origins (FXS-A9 panel) may provide a valuable resource for investigating a series of genetic and epigenetic CGG repeat dynamics during FXS pathogenesis.
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Cromosomas Humanos X/genética , Síndrome del Cromosoma X Frágil/genética , Animales , Células Cultivadas , Cromosomas Humanos X/metabolismo , Cricetulus , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/patología , Humanos , Ratones , Repeticiones de Trinucleótidos/genéticaRESUMEN
The fields of drug discovery and regenerative medicine require large numbers of adult human primary hepatocytes. For this purpose, it is desirable to use hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells (PSCs). Premature hepatoblast-like cells (HB-LCs) differentiated from PSCs provide an intermediate source and steady supply of newly mature HLCs. To develop an efficient HB-LC induction method, we constructed a red fluorescent reporter, CYP3A7R, in which DsRed is placed under the transcriptional control of CYP3A7 coding for a human fetus-type P450 enzyme. Before using this reporter in human cells, we created transgenic mice using mouse embryonic stem cells (ESCs) carrying a CYP3A7R transgene and confirmed that CYP3A7R was specifically expressed in fetal and newborn livers and reactivated in the adult liver in response to hepatic regeneration. Moreover, we optimized the induction procedure of HB-LCs from transgenic mouse ESCs using semi-quantitative fluorometric evaluation. Activation of Wnt signaling together with chromatin modulation prior to Activin A treatment greatly improved the induction efficiency of HB-LCs. BMP2 and 1.7% dimethyl sulfoxide induced selective proliferation of HB-LCs, which matured to HLCs. Therefore, CYP3A7R will provide a fluorometric evaluation system for high content screening of chemicals that induce HB-LC differentiation, hepatocyte regeneration, and hepatotoxicity when it is introduced into human PSCs.
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Citocromo P-450 CYP3A/genética , Hepatocitos/citología , Regeneración Hepática , Hígado/embriología , Proteínas Luminiscentes/metabolismo , Células Madre Embrionarias de Ratones/citología , Animales , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Citocromo P-450 CYP3A/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Hígado/fisiología , Proteínas Luminiscentes/genética , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Recombinantes/metabolismo , Vía de Señalización Wnt , Proteína Fluorescente RojaRESUMEN
OBJECTIVE: To develop a mouse artificial chromosome (MAC) carrying the mouse Xist gene (X-inactive specific transcript; Xist-MAC) as a systematic in vitro approach for investigating Xist RNA-mediated chromosome inactivation. RESULTS: Ectopic expression of the Xist gene in CHO cells led to the accumulation of Xist RNA in cis on the MAC. In addition, the introduction of Xist-MAC to embryonic stem cells from male mice via microcell-mediated chromosome transfer resulted in the accumulation of Xist RNA in cis on the MAC. Chromosomal inactivation was observed in the differentiated state. Moreover, this phenomenon was accompanied by the epigenetic modification of H3K27 trimethylation. CONCLUSIONS: We successfully generated a novel chromosome inactivation model, Xist-MAC, which will provide a valuable tool for the screening and functional analysis of X chromosome inactivation-related genes and proteins.
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Cromosomas Artificiales/genética , Células Madre Embrionarias/citología , Histonas/metabolismo , ARN Largo no Codificante/genética , Animales , Células CHO , Células Cultivadas , Cricetulus , Epigénesis Genética , Masculino , Ratones , Inactivación del Cromosoma XRESUMEN
Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.
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Cromosomas Artificiales Humanos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Células CHO , Cricetulus , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Regiones de Fijación a la Matriz/genética , Técnicas de Amplificación de Ácido Nucleico , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.
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Regulación Enzimológica de la Expresión Génica , Melanoma/metabolismo , Factores de Transcripción Paired Box/metabolismo , ARN Mensajero/metabolismo , Telomerasa/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc/genética , Humanos , Melanoma/genética , Factores de Transcripción Paired Box/genética , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/genética , Telomerasa/genética , Factores de Transcripción/genética , Transcripción Genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Adenoid cystic carcinoma (ACC) is a relatively rare malignant neoplasm that occurs in salivary glands and various other organs. Recent studies have revealed that a significant proportion of ACCs harbor gene alterations involving MYB or MYBL1 (mostly fusions with NFIB) in a mutually-exclusive manner. However, its clinical significance remains to be well-established. METHODS: We investigated clinicopathological and molecular features of 36 ACCs with special emphasis on the significance of MYBL1 alterations. Reverse-transcription polymerase-chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH) were performed to detect MYB/MYBL1-NFIB fusions and MYBL1 alterations, respectively. Immunohistochemistry was performed to evaluate MYB expression in the tumors. The results were correlated with clinicopathological profiles of the patients. RESULTS: RT-PCR revealed MYB-NFIB and MYBL1-NFIB fusions in 10 (27.8%) and 7 (19.4%) ACCs, respectively, in a mutually-exclusive manner. FISH for MYBL1 rearrangements was successfully performed in 11 cases, and the results were concordant with those of RT-PCR. Immunohistochemically, strong MYB expression was observed in 23 (63.9%) tumors, none of which showed MYBL1 alterations. Clinicopathologically, a trend of a better disease-specific survival was noted in patients with MYBL1 alterations than in those with MYB-NFIB fusions and/or strong MYB expression; however, the difference was not significant. Interestingly, we found tumors with MYBL1 alterations significantly frequently occurred in the mandibular regions (P = 0.012). Moreover, literature review revealed a similar tendency in a previous study. CONCLUSION: Our results suggest that there are some biological or etiological differences between ACCs with MYB and MYBL1 alterations. Moreover, the frequent occurrence of MYBL1-associated ACC in the mandibular regions suggests that MYB immunohistochemistry is less useful in diagnosing ACCs arising in these regions. Further studies are warranted to verify our findings.
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AIM: To generate sunitinib-resistant renal cell carcinoma (RCC) cell lines and identify miRNAs contributing to sunitinib resistance. MATERIALS AND METHODS: Two RCC cell lines, ACHN and RCC23, were cultured by continuous treatment with sunitinib for 3 months, with doses gradually increasing up to the 50% inhibitory concentration for each cell line. We performed microarray and quantitative real-time polymerase chain reaction analyses of sunitinib-resistant ACHN (SR-ACHN) and RCC23 (SR-RCC23) cells, as well of as sunitinib-sensitive ACHN and RCC23 cells. RESULTS: SR-ACHN and SR-RCC23 cells exhibited significantly higher resistance to sunitinib treatment compared to sunitinib-sensitive cells. SR-ACHN and SR-RCC23 cells were hypertrophic and contained granules in the cytoplasm. When SR-ACHN and SR-RCC23 cells were compared to ACHN and RCC23 cells, expression of miR-575, miR-642b-3p, and miR-4430 was significantly increased, while that of miR-18a-5p, miR-29b-1-5p, miR-431-3p, and miR-4521 was significantly decreased. CONCLUSION: These miRNAs may contribute to sunitinib resistance in humans.
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Antineoplásicos/farmacología , Carcinoma de Células Renales/genética , Resistencia a Antineoplásicos/genética , Indoles/farmacología , Neoplasias Renales/genética , MicroARNs/genética , Pirroles/farmacología , Línea Celular Tumoral , Humanos , SunitinibRESUMEN
The pituitary homeobox 1 (PITX1) protein is essential for developmental processes in humans. Previously, PITX1 was identified as a possible tumor suppressor gene in various types of human carcinoma. However, the association between PITX1 and human head and neck squamous cell carcinoma (HNSCC) remains to be elucidated. Immunohistochemical analysis was performed to examine the expression levels of PITX1 in 47 cases of HNSCC, and in 4 control cases. The expression of p53 was also examined in these cases. The labeling indices (LIs) were calculated, and the correlations between clinical factors (chemosensitivity, prognosis and the degree of differentiation) and the LIs were assessed. The PITX1 LI in HNSCC was 27.4±14.5%, which was significantly lower compared with the LIs of the control samples: 76.9±6.97% (P<0.05). Additionally, the PITX1 LIs were 39.9±6.2, 26.9±16.9 and 24.2±11.8% in the complete response (CR), partial response (PR), stable disease or progressive disease (SD/PD) groups, respectively. The PITX1 LI in the CR group revealed the highest result between the all groups, and it was significantly greater compared with that in the SD/PD group (P<0.01). The p53 LIs were 24.5±19.9, 25.7±16.9 and 19.8±13.8 in the CR, PR and SD/PD groups, respectively (P>0.05). Neither the PITX1 nor the p53 LIs were a statistically significant indicator of the prognosis. PITX1 is a candidate tumor suppressor gene and a possible predictive biomarker of chemosensitivity of human HNSCC.
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Long noncoding RNAs (lncRNAs) have been implicated in many biological processes through epigenetic mechanisms. We previously reported that KCNQ1OT1, an imprinted antisense lncRNA in the human KCNQ1 locus on chromosome 11p15.5, is involved in cis-limited silencing within an imprinted KCNQ1 cluster. Furthermore, aberration of KCNQ1OT1 transcription was observed with a high frequency in colorectal cancers. However, the molecular mechanism of the transcriptional regulation and the functional role of KCNQ1OT1 in colorectal cancer remain unclear. Here, we show that the KCNQ1OT1 transcriptional level was significantly increased in human colorectal cancer cells in which ß-catenin was excessively accumulated in the nucleus. Additionally, overexpression of ß-catenin resulted in an increase in KCNQ1OT1 lncRNA-coated territory. On the other hand, knockdown of ß-catenin resulted in significant decrease of KCNQ1OT1 lncRNA-coated territory and an increase in the mRNA expression of the SLC22A18 and PHLDA2 genes that are regulated by KCNQ1OT1. We showed that ß-catenin can promote KCNQ1OT1 transcription through direct binding to the KCNQ1OT1 promoter. Our evidence indicates that ß-catenin signaling may contribute to development of colorectal cancer by functioning as a novel lncRNA regulatory factor via direct targeting of KCNQ1OT1.
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Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genética , beta Catenina/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorrectales/genética , Regulación hacia Abajo/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas de Transporte de Catión Orgánico/genética , Canales de Potasio con Entrada de Voltaje/genética , Regiones Promotoras Genéticas , Unión Proteica/genética , Transcripción GenéticaRESUMEN
Human plasma arginine vasopressin (AVP) levels serve as a clinically relevant marker of diabetes and related syndromes. We developed a highly sensitive method for measuring human plasma AVP using high-performance liquid chromatography tandem mass spectrometry. AVP was extracted from human plasma using a weak-cation solid-phase extraction plate, and separated on a wide-bore octadecyl reverse-phase column. AVP was quantified in ion-transition experiments utilizing a product ion (m/z 328.3) derived from its parent ion (m/z 542.8). The sensitivity was enhanced using 0.02% dichloromethane as a mobile-phase additive. The lower limit of quantitation was 0.200 pmol/L. The extraction recovery ranged from 70.2 ± 7.2 to 73.3 ± 6.2% (mean ± SD), and the matrix effect ranged from 1.1 - 1.9%. Quality-testing samples revealed interday/intraday accuracy and precision ranging over 0.9 - 3% and -0.3 - 2%, respectively, which included the endogenous baseline. Our results correlated well with radioimmunoassay results using 22 human volunteer plasma samples.