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1.
Exp Eye Res ; 205: 108497, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33596443

RESUMEN

Nanophthalmos-4 is a rare autosomal dominant disorder caused by two known variations in TMEM98. An Austrian Caucasian pedigree was identified suffering from nanophthalmos and late onset angle-closure glaucoma and premature loss of visual acuity. Whole exome sequencing identified segregation of a c.602G > C transversion in TMEM98 (p.Arg201Pro) as potentially causative. A protein homology model generated showed a TMEM98 structure comprising α4, α5/6, α7 and α8 antiparallel helix bundles and two predicted transmembrane domains in α1 and α7 that have been confirmed in vitro. Both p.Arg201Pro and the two missense variations representing proline insertions identified previously to cause nanophthalmos-4 (p.Ala193Pro and p.His196Pro) are located in the charge polarized helix α8 (p.183-p210). Stability of the C-terminal alpha helical structure of TMEM98 is therefore essential to prevent the development of human nanophthalmos-4. Precise molecular diagnosis could lead to the development of tailored therapies for patients with orphan ocular disease.


Asunto(s)
Glaucoma de Ángulo Cerrado/genética , Hiperopía/genética , Proteínas de la Membrana/genética , Microftalmía/genética , Mutación Missense , Trastornos de la Visión/genética , Agudeza Visual/fisiología , Adulto , Anciano de 80 o más Años , Sustitución de Aminoácidos , Arginina , Femenino , Cirugía Filtrante , Glaucoma de Ángulo Cerrado/fisiopatología , Glaucoma de Ángulo Cerrado/cirugía , Humanos , Hiperopía/fisiopatología , Hiperopía/cirugía , Implantación de Lentes Intraoculares , Masculino , Microftalmía/fisiopatología , Microftalmía/cirugía , Microscopía Acústica , Persona de Mediana Edad , Linaje , Facoemulsificación , Prolina , Conformación Proteica en Hélice alfa/genética , Microscopía con Lámpara de Hendidura , Trastornos de la Visión/fisiopatología , Secuenciación del Exoma
2.
Clin Otolaryngol ; 46(5): 1044-1049, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33851515

RESUMEN

OBJECTIVE: Identification of variations in tumour suppressor genes encoding the tetrameric succinate dehydrogenase (SDHx) mitochondrial enzyme complex may lead to personalised therapeutic concepts for the orphan disease, familial paraganglioma (PGL) type 1-5. We undertook to determine the causative variation in a family suffering from idiopathic early-onset (22 ± 2 years) head and neck PGL by PCR and Sanger sequencing. DESIGN: Prospective genetic study. SETTING: Tertiary Referral Otolaryngology Centre. PARTICIPANTS: Twelve family members. MAIN OUTCOME MEASURES: Main outcomes were clinical analysis and SDH genotyping RESULTS AND CONCLUSIONS: A novel heterozygous c.298delA frameshift variation in exon 3 of SDH subunit D (SDHD) was associated with a paternal transmission pattern of PGL in affected family members available to the study. Family history over five generations in adulthood indicated a variable penetrance for PGL inheritance in older generations. The c.298delA variant would cause translation of a 34-residue C-terminus distal to lysine residue 99 in the predicted transmembrane domain II of the full-length sequence p.(Thr100LeufsTer35) and would affect the translation products of all protein-coding SDHD isoforms containing transmembrane topologies required for positional integration in the inner mitochondrial membrane and complex formation. These results underly the importance of genetic screening for PGL also in cases of unclear inheritance, and variation carriers should benefit from screening and lifelong follow-up.


Asunto(s)
Neoplasias de Cabeza y Cuello/genética , Paraganglioma/genética , Succinato Deshidrogenasa/genética , Adulto , Edad de Inicio , Anciano de 80 o más Años , Austria , Exones , Femenino , Mutación del Sistema de Lectura , Pruebas Genéticas , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Paraganglioma/diagnóstico por imagen , Linaje , Penetrancia , Fenotipo , Estudios Prospectivos , Adulto Joven
3.
Eur Arch Otorhinolaryngol ; 274(10): 3619-3625, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28821934

RESUMEN

Bi-allelic variations in the gap junction protein beta-2 (GJB2) gene cause up to 50% of cases of newborn hearing loss. Heterozygous pathogenic GJB2 variations are also fivefold overrepresented in idiopathic patient groups compared to the normal-hearing population. Whether hearing loss in this group is due to unidentified additional variations within GJB2 or variations in other deafness genes is unknown in most cases. Whole-exome sequencing offers an effective approach in the search for causative variations in patients with Mendelian diseases. In this prospective genetic cohort study, we initially investigated a family of Turkish origin suffering from congenital autosomal recessive hearing loss. An index patient and his normal-hearing father, both bearing a single heterozygous pathogenic c.262G>T (p.Ala88Ser) GJB2 transversion as well as the normal-hearing mother were investigated by means of whole-exome sequencing. Subsequently the genetic screening was extended to a hearing-impaired cohort of 24 families of Turkish origin. A homozygous missense c.5492G>T transversion (p.Gly1831Val) in the Myosin 15a gene, previously linked to deafness, was identified as causative in the index family. This very rare variant is not listed in any population in the Genome Aggregation Database. Subsequent screening of index patients from additional families of Turkish origin with recessive hearing loss identified the c.5492G>T variation in an additional family. Whole-exome sequencing may effectively identify the causes of idiopathic hearing loss in patients bearing heterozygous GJB2 variations.


Asunto(s)
Conexinas/genética , Pérdida Auditiva Sensorineural , Adulto , Austria/epidemiología , Conexina 26 , Femenino , Pruebas Genéticas/métodos , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Sensorineural/epidemiología , Pérdida Auditiva Sensorineural/genética , Humanos , Recién Nacido , Masculino , Mutación , Estudios Prospectivos , Turquía , Secuenciación del Exoma/métodos
4.
Int J Mol Sci ; 17(10)2016 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-27669225

RESUMEN

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is a rapidly growing, highly-vascularized cancer. NBs frequently express angiogenic factors and high tumor angiogenesis has been associated with poor outcomes. Placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family and is up-regulated mainly in pathologic conditions. Recently, PlGF was identified as a member of a gene expression signature characterizing highly malignant NB stem cells drawing attention as a potential therapeutic target in NB. In the present study, we sought to investigate the expression of PlGF in NB patients and the effect of PlGF inhibition on high-risk MYCN-non-amplified SK-N-AS NB xenografts. Human SK-N-AS cells, which are poorly differentiated and express PlGF and VEGF-A, were implanted subcutaneously in athymic nude mice. Treatment was done by intratumoral injection of replication-incompetent adenoviruses (Ad) expressing PlGF- or VEGF-specific short hairpin (sh)RNA, or soluble (s)VEGF receptor 2 (VEGFR2). The effect on tumor growth and angiogenesis was analyzed. High PlGF expression levels were observed in human advanced-stage NBs. Down-regulating PlGF significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. In contrast, blocking VEGF by administration of Ad(sh)VEGF and Ad(s)VEGFR2 reduced tumor growth associated with decreased tumor vasculature. These findings suggest that PlGF and VEGF-A modulate MYCN-non-amplified NB tumors by different mechanisms and support a role for PlGF in NB biology.


Asunto(s)
Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/patología , Factor de Crecimiento Placentario/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adenoviridae/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Preescolar , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Lactante , Masculino , Ratones , Ratones Desnudos , Neovascularización Patológica , Neuroblastoma/metabolismo , Neuroblastoma/prevención & control , Factor de Crecimiento Placentario/antagonistas & inhibidores , Factor de Crecimiento Placentario/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
5.
Eur Arch Otorhinolaryngol ; 272(1): 229-32, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25085637

RESUMEN

The objective of this study was to investigate the relevance of routine assessment of c.-259C>T in the Austrian newborn screening program. Homozygous and compound heterozygous mutations in the coding region of the human gene encoding gap junction protein GJB2 (Connexin 26) cause up to 50 % of neonatal autosomal recessive non-syndromic hearing impairment identified in Caucasian newborn screening programs. More recently, a null mutation in the GC box of the GJB2 basal promoter c.-259C>T has been described which causes hearing impairment by completely suppressing GJB2 promoter activity. We determined the occurrence of c.-259C>T in cases of non-syndromic hearing impairment lacking known pathogenic alterations in GJB2 (n = 43), a non-syndromic hearing impaired patient group (n = 15) bearing the heterozygous GJB2 mutations c.35delG, c.[79G>A];[341A>G] (p. [V27I];[E114G]), c.109G>A (p.V37I), c.154G>C (p.V52L), c.262G>T (p.A88S), c.269T>C (p.L90P) and c.551G>C (p.R184P) and in a normal hearing group lacking alterations in GJB2 (n = 50). In the analyzed groups, no occurrence of c.-259C>T was found. The c.-259C>T mutation, previously described as -3438C>T, is not a common cause of non-syndromic hearing impairment alone or together with heterozygous pathogenic GJB2 mutations that are statistically overrepresented in non-syndromic hearing impaired patient groups. Screening of newborns for c.-259C>T is therefore unlikely to be commonly found in Austrian NSHI patients but could make a significant contribution to non-syndromic hearing impairment in other populations.


Asunto(s)
Conexinas/genética , ADN/genética , Pérdida Auditiva/genética , Mutación , Austria/epidemiología , Conexina 26 , Conexinas/metabolismo , Femenino , Pérdida Auditiva/epidemiología , Humanos , Recién Nacido , Masculino , Reacción en Cadena de la Polimerasa , Prevalencia , Regiones Promotoras Genéticas
6.
Audiol Neurootol ; 19(3): 203-9, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24801666

RESUMEN

Norrie disease is a rare, X-linked genetic syndrome characterized by combined congenital blindness and progressive hearing impairment. Norrie disease is caused by alterations in the NDP gene encoding the growth factor norrin that plays a key role in vascular development and stabilization of the eye, inner ear and brain. We identified a family with 3 affected deafblind males and a single female carrier presenting with a serous retinal detachment but normal hearing. Genetic analysis revealed a novel c.277T>C missense mutation causing the substitution of a hydrophobic cysteine to a hydrophilic arginine [p.(Cys93Arg)] within the highly conserved cysteine knot domain of the norrin protein. These results should expand the scope for amniocentesis and genetic testing for Norrie disease which is gaining in importance due to novel postnatal therapeutic concepts to alleviate the devastating retinal symptoms of Norrie disease.


Asunto(s)
Ceguera/congénito , Proteínas del Ojo/genética , Mutación Missense , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/genética , Espasmos Infantiles/genética , Ceguera/genética , Familia , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X , Pruebas Genéticas , Humanos , Masculino , Linaje , Degeneración Retiniana
7.
Int J Mol Sci ; 15(1): 1538-53, 2014 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-24451137

RESUMEN

The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Fibroblastos/metabolismo , Regulación hacia Arriba , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Ventrículos Cardíacos/citología , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo
8.
J Transl Med ; 11: 295, 2013 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-24279335

RESUMEN

BACKGROUND: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential. METHODS: We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197. RESULTS: In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo. CONCLUSIONS: These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.


Asunto(s)
Neoplasias del Colon/patología , Regulación hacia Abajo/efectos de los fármacos , Indoles/farmacología , Terapia Molecular Dirigida , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Quinasas p21 Activadas/metabolismo , Células 3T3 , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Indoles/química , Indoles/uso terapéutico , L-Lactato Deshidrogenasa/metabolismo , Ratones , Invasividad Neoplásica , Unión Proteica/efectos de los fármacos , Pirimidinas/química , Pirimidinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Análisis de Supervivencia , Proteína de Unión al GTP cdc42/metabolismo
9.
Int J Mol Sci ; 14(9): 17958-71, 2013 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-24005860

RESUMEN

The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.


Asunto(s)
Proteínas Gestacionales/metabolismo , Neoplasias de la Próstata/metabolismo , Células del Estroma/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , Ratones , Factor de Crecimiento Placentario , Proteínas Gestacionales/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Otol Neurotol ; 42(6): e648-e657, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-33710140

RESUMEN

INTRODUCTION: Genetic hearing loss (HL) is often monogenic. Whereas more than half of autosomal recessive (AR) cases in Austria are caused by mutations in a single gene, no disproportionately frequent contributing genetic factor has been identified in cases of autosomal dominant (AD) HL. The genetic characterization of HL continues to improve diagnosis, genetic counseling, and lays a foundation for the development of personalized medicine approaches. METHODS: Diagnostic HL panel screening was performed in an Austrian multiplex family with AD HL, and segregation was tested with polymerase chain reaction and Sanger sequencing. In an independent approach, 18 unrelated patients with AD HL were screened for causative variants in all known HL genes to date and segregation was tested if additional family members were available. The pathogenicity of novel variants was assessed based on previous literature and bioinformatic tools such as prediction software and protein modeling. RESULTS: In six of the 19 families under study, candidate pathogenic variants were identified in MYO6, including three novel variants (p.Gln441Pro, p.Ser612Tyr, and p.Gln650ValfsTer7). Some patients carried more than one likely pathogenic variant in known deafness genes. CONCLUSION: These results suggest a potential high prevalence of MYO6 variants in Austrian cases of AD HL. The presence of multiple rare HL variants in some patients highlights the relevance of considering multiple-hit diagnoses for genetic counseling and targeted therapy design.


Asunto(s)
Sordera , Pérdida Auditiva , Austria/epidemiología , Humanos , Mutación , Linaje , Prevalencia
11.
Cancers (Basel) ; 13(9)2021 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-33923093

RESUMEN

Oral tongue squamous cell carcinomas (OTSCCs) have an increasing incidence in young patients, and many have an aggressive course of disease. The objective of this study was to identify candidate prognostic protein markers associated with early-onset OTSCC. We performed an exploratory screening for differential protein expression in younger (≤45 years) versus older (>45 years) OTSCC patients in The Cancer Genome Atlas (TCGA) cohort (n = 97). Expression of candidate markers was then validated in an independent Austrian OTSCC patient group (n = 34) by immunohistochemistry. Kaplan-Meier survival estimates were computed, and genomic and mRNA enrichment in silico analyses were performed. Overexpression of protein kinase C alpha (PRKCA) was significantly more frequent among young patients of both the TCGA (p = 0.0001) and the Austrian cohort (p = 0.02), associated with a negative anamnesis for alcohol consumption (p = 0.009) and tobacco smoking (p = 0.02) and poorer overall survival (univariate p = 0.02, multivariate p< 0.01). Within the young subgroup, both overall and disease-free survival were significantly decreased in patients with PRKCA overexpression (both p < 0.001). TCGA mRNA enrichment analysis revealed 332 mRNAs with significant differential expression in PRKCA-upregulated versus PRKCA-downregulated OTSCC (all FDR ≤ 0.01). Our findings suggest that PRKCA overexpression may be a hallmark of a novel molecular subtype of early-onset alcohol- and tobacco-negative high-risk OTSCC. Further analysis of the molecular PRKCA interactome may decipher the underlying mechanisms of carcinogenesis and clinicopathological behavior of PRKCA-overexpressing OTSCC.

12.
Int J Cancer ; 126(6): 1339-52, 2010 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-19711348

RESUMEN

The molecular mechanisms of tumor-host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony-stimulating factor-1 (CSF-1) production to recruit tumor-associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF-1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF-1-expressing SK-N-AS and CSF-1-negative SK-N-DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF-1. Significant suppression of both SK-N-AS and SK-N-DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)-12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF-1 blockade. Furthermore, Tie-2-positive and -negative TAMs recruited by host CSF-1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host-CSF-1 blockade prolonged survival only in CSF-1-negative SK-N-DZ NB. These studies demonstrated that increased CSF-1 production by host cells enhances TAM recruitment and NB growth and that the CSF-1 phenotype of NB tumor cells adversely affects survival.


Asunto(s)
Factor Estimulante de Colonias de Macrófagos/metabolismo , Neuroblastoma/patología , Interferencia de ARN , Células del Estroma/metabolismo , Animales , Western Blotting , Comunicación Celular , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Técnicas de Cocultivo , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Microscopía Confocal , Neuroblastoma/genética , Neuroblastoma/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Análisis de Supervivencia , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Trasplante Heterólogo
13.
Stem Cells ; 27(9): 2342-52, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19522014

RESUMEN

Prostate cancer tumor growth and neovascularization is promoted by an interplay between migratory tumor stromal cells such as specialized tumor-associated macrophages (TAMs) and circulating endothelial precursor cells (CEPs). As vehicles for tumor therapy, human CEPs are relatively easy to isolate from peripheral blood, are able to proliferate long-term in vitro, are amenable to viral manipulation, and preferentially home to regions of ischemia found in growing tumors. We show here that human peripheral blood CEPs expanded ex vivo migrate to prostate cancer cells in vitro and efficiently home to human prostate tumor xenografts in vivo. Infection of precursors ex vivo with an adenovirus constructed to secrete a soluble form of the colony-stimulating factor-1 receptor CD115 that inhibits macrophage viability and migration in vitro significantly decreases the number of TAMs in xenografts (p < .05), reduces proliferation (p < .01) and vascular density (p < .03), and suppresses the growth of xenografts (p < .03). These data show for the first time that targeting stromal cell processes with cellular therapy has the potential to retard prostate tumor growth.


Asunto(s)
Adenoviridae/genética , Células Endoteliales/metabolismo , Neoplasias de la Próstata/terapia , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Endoteliales/citología , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Trasplante de Células Madre/métodos , Ensayos Antitumor por Modelo de Xenoinjerto
14.
Front Cell Neurosci ; 14: 585669, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33281559

RESUMEN

Background: Hereditary hearing loss is a disorder with high genetic and allelic heterogeneity. Diagnostic screening of candidate genes commonly yields novel variants of unknown clinical significance. TBC1D24 is a pleiotropic gene associated with recessive DOORS syndrome, epileptic encephalopathy, myoclonic epilepsy, and both recessive and dominant hearing impairment. Genotype-phenotype correlations have not been established to date but could facilitate diagnostic variant assessment and elucidation of pathomechanisms. Methods and Results: Whole-exome and gene panel screening identified a novel (c.919A>C; p.Asn307His) causative variant in TBC1D24 in two unrelated Caucasian families with Autosomal dominant (AD) nonsyndromic late-onset hearing loss. Protein modeling on the Drosophila TBC1D24 ortholog Skywalker crystal structure showed close interhelix proximity (6.8Å) between the highly conserved residue p.Asn307 in α18 and the position of the single known pathogenic dominant variation (p.Ser178Leu) in α11 that causes a form of deafness with similar clinical characteristics. Conclusion: Genetic variants affecting two polar hydrophilic residues in neighboring helices of TBC1D24 cause AD nonsyndromic late-onset hearing loss. The spatial proximity of the affected residues suggests the first genotype-phenotype association in TBC1D24-related disorders. Three conserved residues in α18 contribute to the formation of a functionally relevant cationic phosphoinositide binding pocket that regulates synaptic vesicle trafficking which may be involved in the molecular mechanism of disease.

15.
Methods Mol Biol ; 487: 243-66, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19301651

RESUMEN

Tumors are composed of both malignant and normal cells, including fibroblasts, endothelial cells, mesenchymal stem cells, and inflammatory immune cells such as macrophages. These various stromal components interact with cancer cells to promote growth and metastasis. For example, macrophages, attracted by colony-stimulating factor-1 (CSF-1) produced by tumor cells, in turn produce various growth factors such as vascular endothelial growth factor, which supports the growth of tumor cells and their interaction with blood vessels leading to enhanced tumor cell spreading. The activation of autocrine and paracrine oncogenic signaling pathways by stroma-derived growth factors and cytokines has been implicated in promoting tumor cell proliferation and metastasis. Furthermore, matrix metalloproteinases (MMPs) derived from both tumor cells and the stromal compartment are regarded as major players assisting tumor cells during metastasis. Collectively, these recent findings indicate that targeting tumor-stroma interactions is a promising strategy in the search for novel treatment modalities in human cancer. This chapter summarizes our current understanding of the tumor microenvironment and highlights some potential targets for therapeutic intervention with small interfering RNAs.


Asunto(s)
Comunicación Celular/efectos de los fármacos , Neoplasias/terapia , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Células del Estroma/efectos de los fármacos , Matriz Extracelular/metabolismo , Terapia Genética/métodos , Humanos , Metaloproteinasas de la Matriz/metabolismo , Neoplasias/metabolismo
16.
Cardiovasc Res ; 79(3): 395-404, 2008 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-18436538

RESUMEN

AIMS: Skeletal myoblasts are used in repair of ischaemic myocardium. However, a large fraction of grafted myoblasts degenerate upon engraftment. Colony-stimulating factor-1 (CSF-1) accelerates myoblast proliferation and angiogenesis. We hypothesized that CSF-1 overexpression improves myoblast survival and cardiac function in ischaemia-induced heart failure. METHODS AND RESULTS: Three weeks following myocardial infarction, rats developed heart failure and received intramyocardial injections of mouse CSF-1-transfected or untransfected primary autologous rat myoblasts, recombinant human CSF-1, mouse CSF-1 expressing plasmids, or culture medium. Tissue gene and protein expression was measured by quantitative RT-PCR (reverse transcription-polymerase chain reaction) and western blotting. Fluorescence imaging and immunocytochemistry were used to analyse myoblasts, endothelial cells, macrophages, and infarct wall thickening. Electrocardiograms were recorded online using a telemetry system. Left ventricular function was assessed by echocardiography over time, and improved significantly only in the CSF-1-overexpressing myoblast group. CSF-1-overexpression enhanced myoblast numbers and was associated with an increased infarct wall thickness, enhanced angiogenesis, increased macrophage recruitment and upregulated matrix metalloproteases (MMP)-2 and -12 in the zone bordering the infarction. Transplantation of CSF-1-overexpressing myoblasts did not result in major arrhythmias. CONCLUSION: Autologous intramyocardial transplantation of CSF-1 overexpressing myoblasts might be a novel strategy in the treatment of ischaemia-induced heart failure.


Asunto(s)
Terapia Genética/métodos , Insuficiencia Cardíaca/terapia , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Mioblastos Esqueléticos/trasplante , Isquemia Miocárdica/terapia , Miocardio/metabolismo , Animales , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Factor Estimulante de Colonias de Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Macrófagos/genética , Macrófagos/metabolismo , Masculino , Metaloproteinasas de la Matriz Secretadas/metabolismo , Ratones , Mioblastos Esqueléticos/metabolismo , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Miocardio/enzimología , Miocardio/patología , Neovascularización Fisiológica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/biosíntesis , Factores de Tiempo , Transfección , Trasplante Autólogo , Función Ventricular Izquierda , Remodelación Ventricular
17.
Sci Rep ; 9(1): 18866, 2019 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-31827181

RESUMEN

Overexpression of LAPTM4B-35 (lysosomal-associated transmembrane protein 4ß-35) is associated with a poor prognosis in numerous malignant tumours. Expression patterns and effects of LAPTM4B-35 on head and neck squamous cell carcinomas (HNSCC) are unknown. The aim of this study was to investigate the prognostic relevance of LAPTM4B-35 in HNSCC. Tissue microarrays were constructed with primary tumours and associated lymph node metastases isolated from 127 patients. The expression of LAPTM4B-35 was investigated by immunohistochemistry and the results were correlated with survival data. LAPTM4B-35 in the primary tumour was highly expressed in 47.2% of the patients (60/127). LAPTM4B-35 expression was significantly associated with tumour stage. Moreover, overexpression of LAPTM4B-35 correlated with a significantly worse disease-free survival (10.23 years vs. not reached) and a higher recurrence rate (40.7% vs. 25%). High expression of LAPTM4B-35 in lymph node metastasis was found in 29.2% of cases. In 19.4% of cases, high LAPTM4B-35 expression was observed in both the primary tumour and corresponding lymph node metastases. In conclusion, our data indicates that overexpression of LAPTM4B-35 is associated with poor prognosis and may therefore serve as a new prognostic marker in HNSCC.


Asunto(s)
Proteínas de la Membrana/genética , Proteínas Oncogénicas/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Biomarcadores de Tumor , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Pronóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo
18.
Clin Exp Otorhinolaryngol ; 12(4): 405-411, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31220907

RESUMEN

OBJECTIVES: Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant genetic disorder characterized by pathogenic blood vessel development and maintenance. HHT type 1 (HHT1) and type 2 (HHT2) are caused by variants in endoglin (ENG) and activin receptor-like kinase-1 (ACVRL1), respectively. The aim of this study was to identify the spectrum of pathogenic variants in ENG and ACVRL1 in Austrian HHT families. METHODS: In this prospective study, eight Austrian HHT families were screened for variants in ENG and ACVRL1 by polymerase chain reaction amplification and sequencing of DNA isolated from peripheral blood. RESULTS: Heterozygous variants were identified in all families under study. HHT1 was caused by a novel c.816+1G>A splice donor variant, a novel c.1479C>A nonsense (p.Cys493X) variant and a published c.1306C>T nonsense (p.Gln436X) variant in ENG. Variants found in ACVRL1 were novel c.200G>C (p.Arg67Pro) and known c.772G>A (p.Gly258Ser) missense variants in highly conserved residues, a known heterozygous c.100dupT frameshift (p.Cys34Leufs*4) and the known c.1204G>A missense (p.Gly402Ser) and c.1435C>T nonsense (p.Arg479X) variants as causes of HHT2. CONCLUSION: Novel and published variants in ENG (37.5%) and ACVRL1 (62.5%) were exclusively identified as the cause of HHT in an Austrian patient cohort. Identification of novel causative genetics variants should facilitate the development of tailored therapeutical applications in the future treatment of autosomal dominant HHT.

19.
Front Biosci ; 13: 5580-8, 2008 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-18508607

RESUMEN

It is becoming increasingly clear that interactions between cancer cells, stroma cells and the extracellular matrix (ECM) are pivotal to the processes of neovascularization and tumorigenesis. As tumor stromal components known as tumor associated macrophages (TAMs), mononuclear phagocytes can play a key role in tumor type specific neoangiogenesis by promoting remodeling of the ECM through the production of matrix metalloproteases (MMPs), secreting pro-angiogenic growth factors and stabilizing the tumor vasculature. The growth factor colony-stimulating factor-1 is produced by a wide variety of cancer cells and tumor stroma cells and influences the migration, survival and phenotype of TAMs. Thus, understanding the relationships of the cancer cell with the host environment is key for specifically exploiting tumor growth promoting tumor host interactions for new therapeutic strategies. This review outlines the strategies for targeting CSF-1 in malignancies to influence TAMs in tumor development.


Asunto(s)
Macrófagos/fisiología , Neoplasias/patología , Animales , Matriz Extracelular/fisiología , Homeostasis , Humanos , Inmunofenotipificación , Factor Estimulante de Colonias de Macrófagos/fisiología , Macrófagos/patología , Ratones , Ratones SCID , Neovascularización Patológica/patología , Fagocitosis
20.
Front Biosci ; 13: 5571-9, 2008 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-18508606

RESUMEN

Tumor cells can stimulate matrix metalloproteinase (MMP) production by stromal cells through cell-cell interactions mediated by cell adhesion molecules such as extracellular matrix metalloproteinase inducer (human CD147/EMMPRIN, mouse CD147/Basigin). This study sought to characterize whether specific tumor-stromal cell interactions mediated by CD147 promote colon cancer growth by utilizing small interfering (si)RNAs directed against human CD147/EMMPRIN or mouse CD147/Basigin in co-cultures of cancer cells with macrophages and fibroblasts and established human SW620 colon cancer xenograft models in immune deficient mice. We show that blockade of host (mouse) CD147/Basigin expression, but not cancer cell-derived CD147/EMMPRIN, suppresses tumor growth in human colon cancer xenografts. Experiments in vitro indicated that colon cancer cell-stromal cell interactions mediated by CD147 lead to increased MMP-2 expression in fibroblasts but not macrophages. Furthermore, expression of host VEGF-A in both fibroblasts and macrophages is independent of CD147 in vitro and in vivo. Interestingly, inhibition of cancer cell-derived EMMPRIN leads to increased MMP-9 levels in vivo. Our findings provide new insights into CD147-mediated tumor-host interactions mediating colon cancer growth.


Asunto(s)
Basigina/genética , Neoplasias del Colon/patología , ARN Interferente Pequeño/genética , Células 3T3 , Animales , División Celular , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Macrófagos/fisiología , Ratones , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/fisiología , Transfección , Trasplante Heterólogo
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