RESUMEN
Energy metabolism reprogramming is becoming an increasingly important hallmark of cancer. Specifically, cancers tend to undergo metabolic reprogramming to upregulate a celldependent glutamine (Gln) metabolism. Notably, hepatocellular cell adhesion molecule (HepaCAM) has been previously reported to serve a key role as a tumour suppressor. However, the possible regulatory role of HepaCAM in Gln metabolism in prostate cancer (PCa) remains poorly understood. In the present study, bioinformatics analysis predicted a significant negative correlation among the expression of HepaCAM, phosphatidylinositol4,5bisphosphate 3kinase catalytic subunit α (PIK3CA), glutaminase (GLS) and solute carrier family 1 member 5 (SLC1A5), components of Gln metabolism, in clinical and genomic datasets. Immunohistochemistry results verified a negative correlation between HepaCAM and PIK3CA expression in PCa tissues. Subsequently, liquid chromatographytandem mass spectrometry (LCMS/MS) and gas chromatographymass spectrometry (GCMS) assays were performed, and the results revealed markedly reduced levels of Gln and metabolic flux in the blood samples of patients with PCa and in PCa cells. Mechanistically, overexpression of HepaCAM inhibited Gln metabolism and proliferation by regulating PIK3CA in PCa cells. In addition, Gln metabolism was discovered to be stressresistant in PCa cells, since the expression levels of GLS and SLC1A5 remained high for a period of time after Gln starvation. However, overexpression of HepaCAM reversed this resistance to some extent. Additionally, alpelisib, a specific inhibitor of PIK3CA, effectively potentiated the inhibitory effects of HepaCAM overexpression on Gln metabolism and cell proliferation through mass spectrometry and CCK8 experiments. In addition, the inhibitory effect of PIK3CA on the growth of tumor tissue in nude mice was also confirmed by immunohistochemistry in vivo. To conclude, the results from the present study revealed an abnormal Gln metabolic profile in the blood samples of patients with PCa, suggesting that it can be applied as a clinical diagnostic tool for PCa. Additionally, a key role of the HepaCAM/PIK3CA axis in regulating Gln metabolism, cell proliferation and tumour growth was identified. The combination of alpelisib treatment with the upregulation of HepaCAM expression may serve as a novel method for treating patients with PCa.
Asunto(s)
Proliferación Celular/genética , Glutamina/metabolismo , Transducción de Señal/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral/metabolismo , Proliferación Celular/fisiología , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Modelos Animales de Enfermedad , Glutamina/genética , Masculino , Ratones , Ratones Desnudos/genética , Ratones Desnudos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/prevención & controlRESUMEN
Although anti-programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy has achieved great success in some cancers, most colorectal cancer (CRC) patients remain unresponsive. Therefore, further clarification of the underlying mechanisms is needed to improve the therapy. In this study, we explored the distinct functions of different PD-L1 alternative splicing isoforms in CRC. We investigated the biological functions in PD-L1 knocked down/knockout cells, which were verified through overexpression of PD-L1 isoforms a, b, and c. The roles of PD-L1 isoforms in immune surveillance resistance was also analyzed. Meanwhile, we performed RNA-seq to screen the downstream molecules regulated by PD-L1 isoforms. Finally, we detected PD-L1 and PD-L1 isoforms levels in a cohort of serum samples, two cohorts of CRC tissue samples, and analyzed the correlation of PD-L1 isoforms with PD-1 blockade therapy response in two clinical CRC cases. The results indicated that PD-L1 knockout inhibited proliferation, migration, and invasion, and isoform b exerted a more significant inhibitory effect on T cells than the other two isoforms. Moreover, isoform c could promote CRC progression through regulating epithelial-mesenchymal transition. Clinical data showed that CRC patients with positive PD-L1 expression were associated with poorer overall survival. High serum PD-L1 level was associated with poor prognosis. The level of isoform b or c was negatively associated with prognosis, and a higher level of isoform b was associated with a good response to anti-PD-1 therapy. In conclusion, isoform b should be considered as a biomarker for clinical responsiveness to anti-PD-1/PD-L1 immunotherapy; isoform c had a prometastatic role and is a new potential target for CRC therapy.
Asunto(s)
Empalme Alternativo/genética , Antígeno B7-H1/genética , Neoplasias Colorrectales/genética , Isoformas de Proteínas/genética , Animales , Biomarcadores de Tumor/genética , Línea Celular , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal , Células HCT116 , Células HT29 , Humanos , Inmunoterapia/métodos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Desnudos/genética , Ratones SCID , PronósticoRESUMEN
BACKGROUND: Zinc-finger protein 471 (ZNF471) is a member of the Krüppel-associated box domain zinc finger protein (KRAB-ZFP) family. ZNF471 is methylated in squamous cell carcinomas of tongue, stomach and esophageal. However, its role in breast carcinogenesis remains elusive. Here, we studied its expression, functions, and molecular mechanisms in breast cancer. METHODS: We examined ZNF471 expression by RT-PCR and qPCR. Methylation-specific PCR determined its promoter methylation. Its biological functions and related molecular mechanisms were assessed by CCK-8, clonogenicity, wound healing, Transwell, nude mice tumorigenicity, flow cytometry, BrdU-ELISA, immunohistochemistry and Western blot assays. RESULTS: ZNF471 was significantly downregulated in breast cell lines and tissues due to its promoter CpG methylation, compared with normal mammary epithelial cells and paired surgical-margin tissues. Ectopic expression of ZNF471 substantially inhibited breast tumor cell growth in vitro and in vivo, arrested cell cycle at S phase, and promoted cell apoptosis, as well as suppressed metastasis. Further knockdown of ZNF471 verified its tumor-suppressive effects. We also found that ZNF471 exerted its tumor-suppressive functions through suppressing epithelial-mesenchymal transition, tumor cell stemness and AKT and Wnt/ß-catenin signaling. CONCLUSIONS: ZNF471 functions as a tumor suppressor that was epigenetically inactivated in breast cancer. Its inhibition of AKT and Wnt/ß-catenin signaling pathways is one of the mechanisms underlying its anti-cancer effects.
Asunto(s)
Neoplasias de la Mama/genética , Metástasis de la Neoplasia/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Represoras/genética , Vía de Señalización Wnt/genética , Animales , Apoptosis/genética , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral/metabolismo , Proliferación Celular/genética , Metilación de ADN , ADN-Citosina Metilasas/metabolismo , Regulación hacia Abajo , Epigenómica , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Ratones , Ratones Desnudos/genética , Modelos Animales , Metástasis de la Neoplasia/patología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Represoras/farmacología , Dedos de Zinc/genéticaRESUMEN
WW domain-binding protein 2 (WBP2) has been demonstrated as oncogenic in breast cancer. Many studies have revealed the WBP2 gene as a high-risk gene for leukoariaosis and cerebral white matter lesions is important in the pathologic stage of glioma development. This study aimed to illustrate the underlying mechanism by which WBP2 regulates the process of glioma development. The expression pattern of WBP2 in several tumor cells was determined, clarifying the carcinogenic action of WBP2 in glioma cells. Overexpression of WBP2 in glioma cells promoted cell proliferation and migration, and the number of S-phase cells, whereas the depletion of WBP2 by RNAi-mediated knockdown restrained cell growth and cell cycle progression. Upregulation of WBP2 significantly enhanced the tumorigenic ability of U251 cells in vivo. MS/GST pulldown assay identified α-enolase (ENO1) and Homer protein homolog 3 (Homer3) as novel potent interaction partners of WBP2. Knockdown of ENO1 or Homer3 allowed cell growth and migration to return to normal levels. Furthermore, in vitro and in vivo experiments indicated that the oncogenic role of WBP2 in glioma was through modulating ENO1 and glycolysis activity via the ENO1-PI3K/Akt signaling pathway. Collectively, these results reveal that WBP2 plays a vital role in the occurrence and development of glioma, indicating a target gene for glioblastoma treatment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Glioma/metabolismo , Ratones Desnudos/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatología , Ciclo Celular , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/genética , Femenino , Glioma/genética , Glioma/fisiopatología , Glucólisis , Proteínas de Andamiaje Homer/genética , Proteínas de Andamiaje Homer/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos/genética , Persona de Mediana Edad , Oncogenes , Fosfopiruvato Hidratasa/genética , Unión Proteica , Transactivadores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Breast cancer is a leading cause of cancer death in women, with the majority of these deaths caused by metastasis to distant organs. The most common site of breast cancer metastasis is the bone, which has been shown to provide a rich microenvironment that supports the migration and growth of breast cancer cells. Additionally, growing evidence suggests that breast cancer cells that do successfully metastasize have a stem-like phenotype including high activity of aldehyde dehydrogenase (ALDH) and/or a CD44+CD24- phenotype. In the current study, we tested the hypothesis that these ALDHhiCD44+CD24- breast cancer cells interact with factors in the bone secondary organ microenvironment to facilitate metastasis. Specifically, we focused on bone-derived osteopontin and its ability to promote the migration and stem-like phenotype of breast cancer cells. Our results indicate that bone-derived osteopontin promotes the migration, tumorsphere-forming ability and colony-forming ability of whole population and ALDHhiCD44+CD24- breast cancer cells in bone marrow-conditioned media (an ex vivo representation of the bone microenvironment) (p≤0.05). We also demonstrate that CD44 and RGD-dependent cell surface integrins facilitate this functional response to bone-derived osteopontin (p≤0.05), potentially through activation of WNK-1 and PRAS40-related pathways. Our findings suggest that soluble bone-derived osteopontin enhances the ability of breast cancer cells to migrate to the bone and maintain a stem-like phenotype within the bone microenvironment, and this may contribute to the establishment and growth of bone metastases.
Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular/fisiología , Osteopontina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Animales , Neoplasias de la Mama/genética , Antígeno CD24/genética , Antígeno CD24/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Citometría de Flujo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Desnudos/genética , Ratones Desnudos/fisiología , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Osteopontina/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
Diabetes is induced in mice by using streptozotocin (STZ), a compound that has a preferential toxicity toward pancreatic ß cells. We evaluated nude male mice from various sources for their sensitivity to a single high dose (160 to 240 mg/kg) of STZ. Diabetes was induced in male mice (age: median, 12 wk; interquartile range, 11 to 14 wk; body weight, about 30 g) from Taconic Farms (TAC), Jackson Laboratories (JAX), and Charles River Laboratories (CRL). Mice were monitored for 30 d for adverse side effects, blood glucose, and insulin requirements. In CRL mice given 240 mg/kg STZ, more than 95% developed diabetes within 4 to 5 d, and loss of body weight was relatively low (mean, 0.4 g). In comparison, both TAC and JAX mice were more sensitive to STZ, as evidenced by faster development of diabetes (even at a lower STZ dose), greater need for insulin after STZ, greater body weight loss (mean: TAC, 3.5 g; JAX, 3.7 g), and greater mortality. We recommend conducting exploratory safety assessments when selecting a nude mouse source, with the aim of limiting morbidity and mortality to less than 10%.
Asunto(s)
Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Ratones Desnudos/genética , Ratones Desnudos/metabolismo , Estreptozocina/toxicidad , Análisis de Varianza , Animales , Masculino , Ratones , Especificidad de la EspecieRESUMEN
The purpose of tumorigenicity testing, as applied not only to cell substrates used for viral vaccine manufacture but also stem cells used for cell-based therapy, is to discriminate between cells that have the capacity to form tumors and cells that do not. Therefore, tumorigenicity testing is essential in assessing the safety of these biological materials. Recently developed NOD/Shi-scid IL2Rg(null) (NOG) mice have been shown to be superior to NOD/Shi-scid (SCID) mice for xenotransplantation of both normal and cancerous cells. To select a suitable mouse strain as a xenogenic host for tumorigenicity testing, we compared the susceptibility of NOG (T, B, and NK cell-defective), SCID (T and B cell-defective), and the traditionally used nude (T cell-defective) mice to tumor formation from xenotransplanted HeLa S3 cells. When 10(4) HeLa S3 cells were subcutaneously inoculated into the flanks of these mice, the tumor incidence on day 22 was 10/10 (100%) in NOG, 2/10 (20%) in SCID, and 0/10 (0%) in nude mice. The subcutaneous tumors formed reproducibly and semiquantitatively in a dose-dependent manner. Unexpectedly, half of the NOG mice (5/10) that had been inoculated with a mere 10(1) HeLa S3 cells formed progressively growing subcutaneous tumors on day 78. We confirmed that the engrafted tumors originated from inoculated HeLa S3 cells by immunohistochemical staining with anti-HLA antibodies. These data suggest that NOG mice may be the best choice as a suitable strain for testing tumorigenicity.
Asunto(s)
Predisposición Genética a la Enfermedad , Trasplante de Neoplasias , Animales , Pruebas de Carcinogenicidad/métodos , Femenino , Células HeLa/trasplante , Humanos , Masculino , Ratones , Ratones Endogámicos NOD/genética , Ratones Desnudos/genética , Ratones SCID/genética , Neoplasias Experimentales/patología , Tejido Subcutáneo/patología , Factores de Tiempo , Trasplante HeterólogoRESUMEN
In this study, we addressed the hypothesis that transcriptional suppression of erythroblastosis virus E26 oncogene homolog 1 (ETS-1) is an efficient therapeutic approach to pancreatic adenocarcinoma by investigating the effect of ETS-1 suppression in human pancreatic cancer cells. We accomplished this by using an adenoviral vector encoding only the DNA-binding domain of wild-type ETS-1 (ETS-1 dominant negative, ETS-1-DN). ETS-1-DN decreases ETS-1-binding by competing for its binding to DNA. Adenoviral-mediated transfer of ETS-1-DN (adenoviral ETS-1-DN construct, AdETS-1-DN) into pancreatic tumor cell lines did not affect their proliferation rate in vitro but did significantly inhibit their in vivo growth in nude mice. Furthermore, to test the efficacy of ETS-1-DN in vivo, we injected the AdETS-1-DN into established human pancreatic adenocarcinomas grown in nude mice. This treatment significantly reduced tumor size as compared to saline injection, without any detectable side effects. Microvessel density in mouse xenografts displayed significantly lower values in tumors in which ETS-1 was downregulated. In addition, expression of the ETS-1-DN in the pancreatic cancer cells resulted in downregulation of urokinase-type plasminogen activator (u-PA) and metalloproteinase-1 (MMP-1) expression. Taken together, these data suggest that transcriptional inactivation of ETS-1 is able to significantly affect angiogenesis and growth of pancreatic cancer. This effect may be due in part to downregulation of MMP-1 and u-PA expression. Our results suggest that ETS-1-DN is a promising candidate for antiangiogenic gene therapy in pancreatic cancer.
Asunto(s)
Adenocarcinoma/terapia , Silenciador del Gen , Neovascularización Patológica/terapia , Neoplasias Pancreáticas/terapia , Proteína Proto-Oncogénica c-ets-1/metabolismo , Transcripción Genética/fisiología , Adenocarcinoma/irrigación sanguínea , Adenoviridae/genética , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Terapia Genética , Humanos , Masculino , Ratones , Ratones Desnudos/genética , Ratones Desnudos/metabolismo , Ratones SCID , Neoplasias Pancreáticas/irrigación sanguínea , Trasplante HeterólogoRESUMEN
OBJECTIVE: To investigate the effect of antisense VEGF165 infection on the growth of A375 cells in nude mice. METHODS: A375 cells were injected s.c into the axilla of the nude mouse. After the tumor formed, we cut it into 16 pieces equally, then transplanted into another 15 nude mice. There were three groups: Group PBS, Group Ad-GFP, and Group Ad-aVEGF. Four weeks after interfere, the mice were sacrificed and their tumors were excised for naked eye and histological observation. The VEGF expression was checked with ISH and immunohistochemistry staining. The micro-vessel density (MVD) in tumor mass was counted by VIII factor immunohistochemistry staining. RESULTS: The visible and palpable nodules had developed at all the injected sites. Tumor growth speed was more slowly in Group Ad-aVEGF than that in other groups. GFP gene could express effectively in tumor mass. Ad-aVEGF infection could suppress the growth of tumors, and there were no obvious side effects. Ad-aVEGF resulted more tissue necrosis, but it had no obvious effect on cell apoptosis. VEGF expression was inhibited significantly in Group Ad-aVEGF, and MVD was decreased accordingly. CONCLUSIONS: Ad-aVEGF interfere may be a new method against human malignant melanoma, whose main mechanism is to induce ischemia, but not apoptosis.
Asunto(s)
Terapia Genética , Melanoma Experimental/genética , Melanoma/genética , Factor A de Crecimiento Endotelial Vascular/genética , Adenoviridae , Animales , Vectores Genéticos , Humanos , Masculino , Melanoma/patología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos/genética , Neoplasias Cutáneas/genéticaRESUMEN
In mice, rats, and humans, loss of function of Foxn1, a member of the winged helix/forkhead family of transcription factors, leads to macroscopic nudity and an inborn dysgenesis of the thymus. Nude (Foxn1(nu)/Foxn1(nu)) mice develop largely normal hair follicles and produce hair shafts. However, presumably because of a lack of certain hair keratins, the hair shafts that are generated twist and coil in the hair follicle infundibulum, which becomes dilated. Since hair shafts fail to penetrate the epidermis, macroscopic nudity results and generates the - grossly misleading - impression that nude mice are hairless. Here, we provide an overview of what is known on the role of Foxn1 in mammalian skin biology, its expression patterns in the hair follicle, its influence on hair follicle function, and onychocyte differentiation. We focus on the mechanisms and signaling pathways by which Foxn1 modulates keratinocyte differentiation in the hair follicle and nail apparatus and summarize the current knowledge on the molecular and functional consequences of a loss of function of the Foxn1 protein in skin. Foxn1 target genes, gene regulation of Foxn, and pharmacological manipulation of the nude phenotype (e.g. by cyclosporine A, KGF, and vitamin D3) are discussed, and important open questions as well as promising research strategies in Foxn1 biology are defined. Taken together, this review aims at delineating why enhanced research efforts in this comparatively neglected field of investigative dermatology promise important new insights into the controls of epithelial differentiation in mammalian skin.
Asunto(s)
Factores de Transcripción Forkhead/fisiología , Folículo Piloso/metabolismo , Uñas/citología , Fenómenos Fisiológicos de la Piel , Animales , Diferenciación Celular/fisiología , Factores de Transcripción Forkhead/genética , Humanos , Ratones , Ratones Desnudos/anatomía & histología , Ratones Desnudos/genética , Ratones Desnudos/fisiología , Mutación , RatasRESUMEN
Arsenic and cadmium (Cd(+2)) are human carcinogens, and epidemiological studies have implicated both pollutants in the development of urinary bladder cancer. Despite this epidemiological base, it is unknown if either Cd(+2) or arsenite (As(+3)) can directly cause the malignant transformation of human urothelial cells. The goal of this study was to determine if Cd(+2) and/or As(+3) are able to cause the malignant transformation of human urothelial cells. The strategy employed was to expose the nontumorigenic urothelial cell line UROtsa to long-term in vitro exposure to Cd(+2) and As(+3), with the endpoint being the ability of the cells to form colonies in soft agar and tumors when heterotransplanted into nude mice. It was demonstrated that a long-term exposure to either 1 M Cd(+2) or 1 M As(+3) resulted in the selection of cells that were able to form colonies in soft agar and tumors when heterotransplanted into nude mice. The histology of the tumor heterotransplants produced by UROtsa cells malignantly transformed by Cd(+2) had epithelial features consistent with those of a classic transitional-cell carcinoma of the bladder. The histology of the tumor heterotransplants produced by cells malignantly transformed by As(+3) was unique in that the cells displayed a prominent squamoid differentiation.
Asunto(s)
Arsenicales/efectos adversos , Compuestos de Cadmio/efectos adversos , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Experimentales/inducido químicamente , Vejiga Urinaria/patología , Urotelio/patología , Agar , Animales , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , Línea Celular , Transformación Celular Neoplásica/patología , Transformación Celular Neoplásica/ultraestructura , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Desnudos/genética , Trasplante de Neoplasias/métodos , Trasplante de Neoplasias/patología , Neoplasias Experimentales/patología , Neoplasias Experimentales/ultraestructura , Factores de Tiempo , Urotelio/efectos de los fármacosRESUMEN
PURPOSE: Animal models that mimic this hormone refractory prostate cancer may be useful for developing and testing novel treatment strategies. MATERIALS AND METHODS: Using the prostate of the 12T-10 transgenic mouse an allograft model was established by transplantation into a nude mouse. To our knowledge we describe the first allograft model derived from the primary prostate tumor of a transgenic mouse. RESULTS: The primary tumor progressed from high grade prostatic intraepithelial neoplasm to invasive, undifferentiated and metastatic cancer with loss of androgen receptor expression. After 10 passages in nude mice the allograft retained the same histological and immunohistochemical features as the primary tumors, including neuroendocrine differentiation. The allograft demonstrated androgen independent growth and metastases to liver and lung, paralleling tumor behavior in the original transgenic line. Cytogenetic characterization of the allograft revealed consistent chromosomal abnormalities for multiple in vivo passages. CONCLUSIONS: This allograft model may give insight into the mechanism by which human prostate cancer progresses to an androgen independent state and provide a system for testing drugs that can inhibit this disease.
Asunto(s)
Proteína de Unión a Andrógenos/genética , Carcinoma Neuroendocrino/genética , Neoplasias de la Próstata/genética , Transglutaminasas/genética , Andrógenos , Animales , Animales Modificados Genéticamente , Antígenos Virales de Tumores , Carcinoma Neuroendocrino/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inmunohistoquímica , Cariotipificación , Masculino , Ratones , Ratones Desnudos/genética , Trasplante de Neoplasias , Neoplasias de la Próstata/patologíaRESUMEN
In this paper we describe the production and analysis of mice carrying a 110-kb transgene that encompasses the wild-type Foxn1 genomic locus. Mutations in Foxn1 cause the nude phenotype. We show that in the hair follicles, transgenic mice with increased Foxn1 gene dosage exhibited increased Foxn1 expression that was restricted correctly to the nascent, post-mitotic cells of the differentiating hair cortex and hair cuticle lineages. We also demonstrate for the first time that a Foxn1 transgene rescues completely both the hair follicle and the thymus defects in animals that are also homozygous for the nude mutation at the endogenous Foxn1 locus, causing the development of a full coat of hair and a normal population of peripheral blood T lymphocytes. We conclude that sufficient cis-acting regulatory information resides within this 110-kb transgene to direct reliable and appropriate tissue-specific expression of the Foxn1 gene.
Asunto(s)
Proteínas de Unión al ADN/genética , Ratones Desnudos/genética , Ratones Transgénicos/genética , Factores de Transcripción/genética , Animales , ADN/química , ADN/genética , Proteínas de Unión al ADN/fisiología , Femenino , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica/genética , Folículo Piloso/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos/fisiología , Ratones Transgénicos/fisiología , Microscopía Confocal , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Mensajero/química , ARN Mensajero/genética , Piel/metabolismo , Factores de Transcripción/fisiología , TransgenesRESUMEN
Although several genes affected by the nude mutation inactivating the transcription factor Whn have recently been identified, a comprehensive molecular analysis of the nude phenotype is still missing. Gene expression profiling of wildtype and nude mice back skin reveals several so far unknown differences in mRNA levels and demonstrates that microarray hybridization is ideal to identify even quantitative changes in expression. Some genes are upregulated in the absence of Whn. Most of the differentially expressed genes are downregulated in nude skin. Our results identify metallothionein IV among these genes. This is the first report on metallothionein IV expression in the murine hair follicle; its expression domain almost completely overlaps that of Whn.
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Perfilación de la Expresión Génica , Ratones Desnudos/genética , Piel/metabolismo , Animales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Factores de Transcripción Forkhead , Hibridación in Situ , Metalotioneína/biosíntesis , Metalotioneína/genética , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Estructura Terciaria de Proteína , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
The nude mutation has been known for a long time. Nevertheless, the gene responsible for the defect has been identified only recently. It encodes a transcriptional activator of the family of forkhead proteins mainly expressed in thymic epithelium and distinct keratinocyte populations in the epidermis and hair follicles. The present review focuses on the molecular and functional characterization of the nude gene and its product and gives an overview as to its role in skin biology and the first identified target genes in the skin. In addition, evolutionary aspects are highlighted stressing the importance of such investigations for a comprehensive understanding of the nude gene product and the regulation of its expression. Furthermore, these studies give a hint as to when the nude gene has occurred first and how it has developed in molecular and functional terms since then.
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Ratones Desnudos/genética , Piel/fisiopatología , Empalme Alternativo , Secuencia de Aminoácidos/genética , Animales , Evolución Biológica , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Factores de Transcripción Forkhead , Expresión Génica , Genoma , Ratones , Datos de Secuencia Molecular , Mutación , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
T cell development requires the interaction of developing thymocytes with thymic epithelial cells. Thymic epithelial cells acquire their unique phenotype under the control of the winged-helix transcription factor Whn, which is lacking in the nude mouse. Whn-dependent genes may therefore be important regulators of lympho-epithelial interactions. To identify Whn target genes we isolated RNA populations of wild-type and nude thymic anlagen from embryonic day 12.5 embryos by laser capture microdissection and compared them by gene expression profiling on microarrays representing 22,000 transcripts. All cDNA with expression differences confirmed by quantitative RT-PCR using RNA from individual anlagen and by in situ hybridization were found to be present in wild-type and absent in nude samples. Three of eight confirmed transcripts were of hematopoietic origin; these transcripts emanate from hematopoietic precursors which have just entered the thymic anlage. Five transcripts were of epithelial origin; one of these corresponds to the recently identified PD-1 ligand (PD-L1), the receptor of which is known to modulate positive selection and to play a role in the control of autoimmunity, and the remaining transcripts code for novel genes. The presented results support our prediction that this systematic approach by gene expression profiling yields regulators of thymopoiesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Rayos Láser , Ratones Desnudos/genética , Factores de Transcripción/metabolismo , Algoritmos , Animales , Proteínas de Unión al ADN/genética , Etiquetas de Secuencia Expresada , Factores de Transcripción Forkhead , Hibridación in Situ , Ligandos , Ratones , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/embriología , Timo/metabolismo , Factores de Transcripción/genéticaRESUMEN
Inhibitors of topoisomerases are widely used in the treatment of cancer, including inhibitors of topoisomerase I (camptothecin analogs such as irinotecan and topotecan) and topoisomerase II (etoposide and doxorubicin). The novel bis-phenazine, XR5944, is a joint inhibitor of topoisomerase I and II as shown by the stabilization of topoisomerase-dependent cleavable complexes. XR5944 demonstrated exceptional activity against human and murine tumor cells in vitro and in vivo. In a range of cell lines XR5944 (IC50 0.04-0.4 nM) was significantly more potent than TAS-103, originally proposed as a joint topoisomerase I and II inhibitor, as well as agents specific for topoisomerase I or II (topotecan, doxorubicin and etoposide). In addition, XR5944 was unaffected by atypical drug resistance and retained significant activity in cells overexpressing P-glycoprotein or multidrug resistance-associated protein. Antitumor efficacy of XR5944 was demonstrated in human carcinoma xenograft models (H69 small cell lung cancer and HT29 colon). In the HT29 model, which is relatively unresponsive to chemotherapy, XR5944 (15 mg/kg i.v., q4dx3) induced tumor regression in the majority of animals (six of eight), whereas TAS-103, dosed at its maximum tolerated dose (45 mg/kg i.v., q7dx3), only induced a delay in tumor growth compared with control animals. In the H69 model, low doses of XR5944 (5 mg/kg i.v., qdx5/week for 2 weeks or 10-15 mg/kg i.v., q4dx3), induced complete tumor regression in the majority of animals. In contrast, topotecan (20 mg/kg i.v., q4dx3) or etoposide (30 mg/kg i.v., q5dx5) only slowed the tumor growth rate. These studies show that XR5944 is a highly active novel anticancer agent that is well tolerated at efficacious doses.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , ADN-Topoisomerasas de Tipo II , Isoenzimas/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Fenazinas/farmacología , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa II , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacología , Animales , Antígenos de Neoplasias , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , ADN/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Etopósido/metabolismo , Etopósido/farmacología , Femenino , Humanos , Indenos/metabolismo , Indenos/farmacología , Concentración 50 Inhibidora , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Isoenzimas/metabolismo , Ratones , Ratones Desnudos/genética , Ratones Desnudos/metabolismo , Fenazinas/metabolismo , Fenazinas/toxicidad , Inducción de Remisión , Topotecan/metabolismo , Topotecan/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nude mice are not bald but instead show an 'abortive' reduced hair growth on different sites of the integument. An albino (NMRI-nu) and a pigmented (C57BL/6-nu) strain of nude mice were examined as to whether the regional distribution pattern of this anagen hair proliferation is subject to the same ontogenetic development as in hairy mice. Hairy mice of both strains served as a comparison. Hair distribution was documented macroscopically by drawing and photography in a total of 415 mice of both sexes up to 421 days of age. Because of the pigmentation of the growing anagen hair follicles, the growth areas in the pigmented nude mice were distinctly visible whereas in the albino mice they were roughly recognisable from the boundaries of hair covering. The regional distribution of the 'abortive' anagen hair pattern in both nude strains corresponded to the wave-like course of the adult hair generations of hairy mice. As in older hairy mice, the hair cycle duration in nude mice was prolonged from an age of 121-180 days, the hair growth areas appeared reduced and less symmetrically orientated. Differences of up to 33% in body mass between the lighter nude and +/nu mice made ontogenetic comparison impossible so that all information is based on direct pattern or age comparison. The significance of experiments on the skin and hair follicles of nude mice is further increased if litters are examined comparatively and the temporal and spatial dimension of the follicle proliferation is considered more carefully than has been the case until now.
Asunto(s)
Cabello/crecimiento & desarrollo , Ratones Desnudos/fisiología , Pigmentación de la Piel/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos/genética , Especificidad de la Especie , Factores de TiempoRESUMEN
Regional variations in intraepithelial lymphocytes (IELs) in the small intestine were examined in BALB/c +/+, nu/+, and nu/nu mice. The small intestine was obtained from 11- to 12-week-old mice and divided equally into three (proximal, middle, and distal) parts. The IELs were isolated from each part of the intestine, and the total numbers of IELs in nu/+ and nu/nu mice were about a fifth of those in +/+ mice. Regional variations in the distribution of the IEL alphabeta, but not the gammadelta T-cell subset were found by use of flow cytometry in +/+ and nu/+ mice. On the other hand, such differences were not found in nu/nu mice, suggesting that thymus-independent development of T cells is not different among regions. Different local expansion of thymus-dependent alphabeta T cells may cause the regional variations seen in the distribution of alphabeta T cell IELs in +/+ and nu/+ mice.