RESUMEN
Anemia is associated with increased morbidity and mortality in older adults. Diagnostic cutoff values for defining anemia vary with age, sex, and possibly race. Anemia is often asymptomatic and discovered incidentally on laboratory testing. Patients may present with symptoms related to associated conditions, such as blood loss, or related to decreased oxygen-carrying capacity, such as weakness, fatigue, and shortness of breath. Causes of anemia in older adults include nutritional deficiency, chronic kidney disease, chronic inflammation, and occult blood loss from gastrointestinal malignancy, although in many patients the etiology is unknown. The evaluation includes a detailed history and physical examination, assessment of risk factors for underlying conditions, and assessment of mean corpuscular volume. A serum ferritin level should be obtained for patients with normocytic or microcytic anemia. A low serum ferritin level in a patient with normocytic or microcytic anemia is associated with iron deficiency anemia. In older patients with suspected iron deficiency anemia, endoscopy is warranted to evaluate for gastrointestinal malignancy. Patients with an elevated serum ferritin level or macrocytic anemia should be evaluated for underlying conditions, including vitamin B12 or folate deficiency, myelodysplastic syndrome, and malignancy. Treatment is directed at the underlying cause. Symptomatic patients with serum hemoglobin levels of 8 g per dL or less may require blood transfusion. Patients with suspected iron deficiency anemia should be given a trial of oral iron replacement. Lower-dose formulations may be as effective and have a lower risk of adverse effects. Normalization of hemoglobin typically occurs by eight weeks after treatment in most patients. Parenteral iron infusion is reserved for patients who have not responded to or cannot tolerate oral iron therapy.
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Anemia/diagnóstico , Examen Físico/métodos , Anciano , Anciano de 80 o más Años , Anemia/sangre , Anemia/etiología , Anemia/terapia , Femenino , Compuestos Ferrosos/administración & dosificación , Compuestos Ferrosos/efectos adversos , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Cripto-1, a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic family, is critical for early embryonic development. Together with its ligand Nodal, Cripto-1 has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Several studies have clearly shown that Cripto-1 is involved in regulating branching morphogenesis and epithelial-mesenchymal transition of the mammary gland both in vitro and in vivo and together with the cofactor GRP78 is critical for the maintenance of mammary stem cells ex vivo. Our previous studies showed that mammary-specific overexpression of human Cripto-1 exhibited dramatic morphological alterations in nulliparous mice mammary glands. The present study shows a novel mechanism for Cripto-1 regulation of mammary gland development through direct effects on progesterone receptor expression and pathways regulated by progesterone in the mammary gland. We demonstrate a strict temporal regulation of mouse Cripto-1 (mCripto-1) expression that occurs during mammary gland development and a stage-specific function of mCripto-1 signaling during mammary gland development. Our data suggest that Cripto-1, like the progesterone receptor, is not required for the initial ductal growth but is essential for subsequent side branching and alveologenesis during the initial stages of pregnancy. Dissection of the mechanism by which this occurs indicates that mCripto-1 activates receptor activator NF-κB/receptor activator NF-κB ligand, and NF-κB signaling pathways.
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Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Receptores de Progesterona/metabolismo , Transducción de Señal , Animales , Proliferación Celular , Chaperón BiP del Retículo Endoplásmico , Factor de Crecimiento Epidérmico/genética , Células Epiteliales , Transición Epitelial-Mesenquimal , Femenino , Humanos , Glándulas Mamarias Animales/citología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Modelos Biológicos , Subunidad p50 de NF-kappa B/genética , Proteínas de Neoplasias/genética , Especificidad de Órganos , Embarazo , Ligando RANK/genética , Receptor Activador del Factor Nuclear kappa-B/genética , Receptores de Progesterona/genéticaRESUMEN
Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.
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Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Células Epiteliales/química , Células Epiteliales/metabolismo , Humanos , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Receptor Notch4 , Receptores Notch/química , Receptores Notch/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Integration of mouse mammary tumor virus (MMTV) at the common integration site Int6 occurs in the gene encoding eIF3e, the p48 subunit of translation initiation factor eIF3. Integration is at any of several introns of the Eif3e gene and causes the expression of truncated Eif3e mRNAs. Ectopic expression of the truncated eIF3e protein resulting from integration at intron 5 (3e5) induces malignant transformation, but by an unknown mechanism. Because eIF3e makes up at least part of the binding site for eIF4G, we examined the effects of 3e5 expression on protein synthesis. We developed an NIH3T3 cell line that contains a single copy of the 3e5 sequence at a predetermined genomic site. Co-immunoprecipitation indicated diminished binding of eIF3 to eIF4G, signifying a reduction in recruitment of the mRNA-unwinding machinery to the 43 S preinitiation complex. Cell growth and overall protein synthesis were decreased. Translation driven by the eIF4G-independent hepatitis C virus internal ribosome entry sequence (HCV IRES) in a bicistronic mRNA was increased relative to cap-dependent translation. Endogenous mRNAs encoding XIAP, c-Myc, CYR61, and Pim-1, which are translated in a cap-independent manner, were shifted to heavier polysomes whereas mRNAs encoding GAPDH, actin, L32, and L34, which are translated in a cap-dependent manner, were shifted to lighter polysomes. We propose that expression of 3e5 diminishes eIF4G interaction with eIF3 and causes abnormal gene expression at the translational level. The correlation between up-regulation of cap-independent translation and MMTV-induced tumorigenesis contrasts with the well established model for malignant transformation involving up-regulation of highly cap-dependent translation.
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Factor 3 de Iniciación Eucariótica/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Virus del Tumor Mamario del Ratón , Biosíntesis de Proteínas , Caperuzas de ARN/genética , Integración Viral/genética , Animales , Transformación Celular Neoplásica , Factor 3 de Iniciación Eucariótica/metabolismo , Expresión Génica , Intrones , Ratones , Células 3T3 NIH , Polirribosomas , Subunidades de Proteína , ARN MensajeroRESUMEN
Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/ß-catenin pathway, or short hairpin RNA targeting ß-catenin expression demonstrated that the invasive activity was not mediated by ß-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the ß-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.
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Células Epiteliales/patología , Células Epiteliales/fisiología , Glándulas Mamarias Animales , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Trombospondinas/metabolismo , Animales , Línea Celular , Células Epiteliales/citología , Femenino , Proteína-1 Reguladora de Fusión/metabolismo , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Pulmonares/secundario , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica/patología , Trasplante de Neoplasias , Trombospondinas/genética , Vía de Señalización Wnt/fisiología , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context.
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Regulación del Desarrollo de la Expresión Génica , Glándulas Mamarias Animales/metabolismo , Receptores Notch/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al Calcio/genética , Línea Celular , Femenino , Hormonas Esteroides Gonadales/metabolismo , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Jagged-1 , Lactancia/genética , Ligandos , Glándulas Mamarias Animales/crecimiento & desarrollo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ovariectomía , Embarazo , Interferencia de ARN , ARN Mensajero/metabolismo , Receptor Notch2/genética , Receptor Notch3 , Receptores Notch/metabolismo , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Serrate-Jagged , TransfecciónRESUMEN
Direct-care Medical Readiness Training Exercises (MEDRETEs) are a popular method for conducting global health engagement. Unfortunately, direct-care MEDRETEs build partnerships without building partner nation capacity. This article proposes that global health engagement should instead focus on partner-led health engagements to accomplish both of these goals.
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Salud Global , Medicina Militar , Creación de Capacidad , HumanosRESUMEN
We have identified the transforming acidic coiled-coil protein-3 (Tacc3) as a binding partner for Notch4/Int3 and were able to show that it binds to the intracellular domain (ICD) of all members of the Notch receptor family. Members of the Tacc family reside at the centrosomes and associates with microtubules. Recent studies suggest that Tacc3 also contributes to the regulation of gene transcription. Tacc3 specifically interacts with the Notch4/Int3 CDC10/Ankyrin repeats and to a lesser extent, with residues C-terminal to these repeats in the ICD. Dual label immunofluorescence of mouse mammary tissue shows Tacc3 co-localizes with the Notch3 ICD. Co-immunoprecipitation of endogenous Notch and Tacc3 proteins from NIH3T3 cell extracts, lung and mammary gland confirms that these two proteins interact under physiological conditions. In addition, knock down of Tacc3 in NIH3T3 cells leads to the up-regulation of Hey2, a target gene for Notch signaling. The affinity of Tacc3 binding to Notch4/Int3 ICD is similar to that between Rbpj and Notch4/Int3 ICD. Notch4/Int3 ICD-Tacc3 interaction results in the inhibition of transcription from a Hes1-Luciferase reporter vector in COS-1 cells. The inhibition was reversed in these cells by increasing the levels of Rbpj. Taken together, these results suggest that Tacc3 is a negative regulator of the Notch signaling pathway.
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Repetición de Anquirina , Proteínas Portadoras/metabolismo , Proteínas Fetales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Notch/metabolismo , Septinas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células COS , Proteínas Portadoras/genética , Chlorocebus aethiops , Proteínas Fetales/genética , Regulación de la Expresión Génica , Genes Reporteros , Proteínas de Homeodominio/genética , Luciferasas , Ratones , Proteínas Asociadas a Microtúbulos , Células 3T3 NIH , Receptor Notch4 , Septinas/genética , Transducción de Señal , Factor de Transcripción HES-1 , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Owners of companion animals with serious illnesses are likely to experience "caregiver burden." This topic has not been fully evaluated in veterinary oncology. OBJECTIVES: To examine owners of a dog or cat with suspected cancer for relationships between early caregiver burden and (a) psychosocial factors: depression, stress, and quality of life; (b) owner communication behavior; and (c) specific pet treatment plan factors. ANIMALS: None. METHODS: This cross-sectional, observational study recruited 164 owners of a cat or dog presenting for evaluation by a veterinary oncology service at a single referral institution. Measures of caregiver burden, psychosocial function, treatment plan elements, and demographics were collected online via owner self-report. Medical records were reviewed to identify factors including diagnosis, medications, treatment schedules, and owner communications. RESULTS: Caregiver burden correlated with higher stress (rs = 0.40, P < .001), greater symptoms of depression (rs = 0.50, P < .001), and lower quality of life (rs = 0.39, P < .001). Pet treatment plan factors related to caregiver burden included changes in care routines, perception that compliance with new routines was challenging, and difficulty adhering to medication routines. There was low correlation between caregiver burden and owner-driven communications (rs = 0.15, P = .07). CONCLUSIONS AND CLINICAL IMPORTANCE: Findings suggest caregiver burden is similar in owners of pets with cancer and owners of pets with other diseases. Caregiver burden is present in the earliest stages of disease. Major correlates of burden including life-disruptive treatments and schedules provide key areas for potential intervention by veterinary teams.
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Carga del Cuidador , Enfermedades de los Gatos , Enfermedades de los Perros , Neoplasias , Animales , Enfermedades de los Gatos/terapia , Gatos , Comunicación , Estudios Transversales , Enfermedades de los Perros/terapia , Perros , Humanos , Neoplasias/terapia , Neoplasias/veterinaria , Propiedad , Mascotas , Calidad de Vida , Encuestas y CuestionariosRESUMEN
The paradigm of mammary cancer induction by the mouse mammary tumor virus (MMTV) is used to illustrate the body of evidence that supports the hypothesis that mammary epithelial stem/progenitor cells represent targets for oncogenic transformation. It is argued that this is not a special case applicable only to MMTV-induced mammary cancer, because MMTV acts as an environmental mutagen producing random interruptions in the somatic DNA of infected cells by insertion of proviral DNA copies. In addition to disrupting the host genome, the proviral DNA also influences gene expression through its associated enhancer sequences over significant inter-genomic distances. Genes commonly affected by MMTV insertion in multiple individual tumors include, the Wnt, FGF, RSpo gene families as well as eIF3e and Notch4. All of these gene families are known to play essential roles in stem cell maintenance and behavior in a variety of organs. The MMTV-induced mutations accumulate in cells that are long-lived and possess the properties of stem cells, namely, self-renewal and the capacity to produce divergent epithelial progeny through asymmetric division. The evidence shows that epithelial cells with these properties are present in normal mammary glands, may be infected with MMTV, become transformed to produce epithelial hyperplasia through MMTV-induced mutagenesis and progress to frank mammary malignancy. Retroviral marking via MMTV proviral insertion demonstrates that this process progresses from a single mammary epithelial cell that possesses all of the features ascribed to tissue-specific stem cells.
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Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Animales/virología , Virus del Tumor Mamario del Ratón/metabolismo , Animales , ADN Viral/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Virus del Tumor Mamario del Ratón/genética , Ratones , Modelos Biológicos , Mutagénesis , Embarazo , Transducción de SeñalRESUMEN
Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.
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Factor de Crecimiento Epidérmico/metabolismo , Proteínas de Homeodominio/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glicoproteínas de Membrana/metabolismo , Morfogénesis/fisiología , Proteínas de Neoplasias/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Caseínas/metabolismo , Diferenciación Celular , Células Cultivadas , Dexametasona/metabolismo , Células Madre Embrionarias/citología , Factor de Crecimiento Epidérmico/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Glucocorticoides/metabolismo , Proteínas de Homeodominio/genética , Insulina/metabolismo , Lactancia , Masculino , Glándulas Mamarias Animales/anatomía & histología , Glándulas Mamarias Animales/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Proteína Homeótica Nanog , Proteínas de Neoplasias/genética , Factores de Crecimiento Nervioso/genética , Netrina-1 , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Embarazo , Prolactina/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
INTRODUCTION: Int6 has been shown to be an interactive participant with the protein translation initiation complex eIF3, the COP9 signalosome and the regulatory lid of the 26S proteasome. Insertion of mouse mammary tumor virus into the Int6 locus creates a C-terminally truncated form of the protein. Expression of the truncated form of Int6 (Int6sh) in stably transfected human and mouse mammary epithelial cell lines leads to cellular transformation. In addition, decreased expression of Int6/eIF3e is observed in approximately one third of all human breast carcinomas. METHODS: To validate that Int6sh has transforming activity in vivo, a transgenic mouse model was designed using the whey acidic protein (Wap) promoter to target expression of truncated Int6 to differentiating alveolar epithelial cells in the mammary gland. Microarray analyses were performed on normal, premalignant and malignant WapInt6sh expressing tissues. RESULTS: Mammary tumors developed in 42% of WapInt6sh heterozygous parous females at an average age of 18 months. In WapInt6sh mice, the contralateral mammary glands from both tumorous and non-tumorous tissues contained widespread focal alveolar hyperplasia. Only 4% of WapInt6sh non-breeding females developed tumors by 2 years of age. The Wap promoter is active only during estrus in the mammary tissue of cycling non-pregnant mice. Microarray analyses of mammary tissues demonstrated that Int6sh expression in the alveolar tissue altered the mammary transcriptome in a specific manner that was detectable even in the first pregnancy. This Int6sh-specific transcriptome pattern subsequently persisted in both the Int6sh-expressing alveolar hyperplasia and mammary tumors. These observations are consistent with the conclusion that WapInt6sh-expressing alveolar cells survive involution following the cessation of lactation, and subsequently give rise to the mammary tumors that arise in aging multiparous females. CONCLUSION: These observations provide direct in vivo evidence that mammary-specific expression of the Int6sh truncation leads to persistence of alveolar hyperplasia with the accompanying increased predisposition to mammary tumorigenesis.
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Transformación Celular Neoplásica , Epitelio/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Hiperplasia/etiología , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/etiología , Animales , Biomarcadores de Tumor/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Hiperplasia/patología , Masculino , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Proteínas de la Leche/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Plásmidos/genética , Embarazo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Transfección , Células Tumorales CultivadasRESUMEN
Wap-Int3 transgenic females expressing the Notch4 intracellular domain (designated Int3) from the whey acidic protein promoter exhibit two phenotypes in the mammary gland: blockage of lobuloalveolar development and lactation, and tumor development with 100% penetrance. Previously, we have shown that treatment of Wap-Int3 tumor bearing mice with Imatinib mesylate (Gleevec) is associated with complete regression of the tumor. In the present study, we show that treatment of Wap-Int3 mice during day 1 through day 6 of pregnancy with Gleevec leads to the restoration of their lobuloalveolar development and ability to lactate in subsequent pregnancies in absence of Gleevec treatment. In addition, these mice do not develop mammary tumors. We investigated the mechanism for Gleevec regulation of Notch signaling and found that Gleevec treatment results in a loss of Int3 protein but not of Int3 mRNA in HC11 mouse mammary epithelial cells expressing Int3. The addition of MG-132, a proteasome inhibitor, shows increased ubiquitination of Int3 in the presence of Gleevec. Thus, Gleevec affects the stability of Int3 by promoting the degradation of Int3 via E3 ubiquitin ligases targeting it for the proteasome degradation. Gleevec is a tyrosine kinase inhibitor that acts on c-Kit and PDGFR. Therefore, we investigated the downstream substrate kinase GSK3ß to ascertain the possible role that this kinase might play in the stability of Int3. Data show that Gleevec degradation of Int3 is GSK3ß dependent. We have expanded our study of the effects Gleevec has on tumorigenesis of other oncogenes. We have found that anchorage-independent growth of HC11-c-Myc cells as well as tumor growth in nude mice is inhibited by Gleevec treatment. As with Int3, Gleevec treatment appears to destabilize the c-Myc protein but not mRNA. These results indicate that Gleevec could be a potential therapeutic drug for patients bearing Notch4 and/or c-Myc positive breast carcinomas.
Asunto(s)
Antineoplásicos/farmacología , Mesilato de Imatinib/farmacología , Glándulas Mamarias Animales/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Notch/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Masculino , Glándulas Mamarias Animales/efectos de los fármacos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratones Desnudos , Ratones Transgénicos , Embarazo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Receptores Notch/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Transgenic mice expressing the Notch-4 intracellular domain (designated Int3) in the mammary gland have two phenotypes exhibited with 100% penetrance: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch-4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. Interestingly, WAP-Int3/Rbpj knockout mice have normal mammary gland development but still developed mammary tumors with a slightly longer latency than the WAP-Int3 mice. Thus, Notch-induced mammary tumor development is Rbpj-independent. Here, we show that Int3 activates NF-κB in HC11 cells in absence of Rbpj through an association with the IKK signalosome. Int3 induced the canonical NF-κB activity and P50 phosphorylation in HC11 cells without altering the NF-κB2 pathway. The minimal domain within the Int3 protein required to activate NF-κB consists of the CDC10/Ankyrin (ANK) repeats domain. Treatment of WAP-Int3 tumor bearing mice with an IKK inhibitor resulted in tumor regression. In a soft agar assay, treatment of HC11-Int3 cells with P50-siRNA caused a significant decrease in colony formation. In addition, Wap-Int3/P50 knockout mice did not develop mammary tumors. This data indicates that the activation of NF-κB canonical signaling by Notch-4/Int3 is ANK repeats dependent, Rbpj-independent, and is mediated by IKK activation and P50 phosphorylation causing mammary tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/deficiencia , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , FN-kappa B/metabolismo , Receptor Notch4/metabolismo , Animales , Repetición de Anquirina , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratones Noqueados , Receptor Notch4/genética , Transducción de SeñalRESUMEN
We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Receptores de Superficie Celular/fisiología , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Secuencias de Aminoácidos , Animales , Western Blotting , Neoplasias de la Mama/patología , Femenino , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Receptor Notch4 , Receptores de Superficie Celular/metabolismo , Receptores Notch , Transducción de Señal/fisiología , Proteína smad3 , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales CultivadasRESUMEN
PURPOSE: The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a multifaceted protein involved in the regulation of protein translation and degradation through binding with three complexes: the eukaryotic translation initiation factor 3, the proteasome regulatory lid, and the constitutive photomorphogenesis 9 signalosome. This study aimed to investigate the prognostic role of Int6 in a large series of stage I non-small cell lung cancers (NSCLC) patients with long-term follow-up. EXPERIMENTAL DESIGN: We determined the methylation status of Int6 DNA by methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung tissues. RESULTS: In 27% of the tumors, Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 expression, the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P <0.000001). Low levels of Int6 RNA were found a significant predictor of overall and disease-free survival (P=0.0004 and P=0.0020, respectively). A multivariate analysis confirmed that low Int6 expression was the only independent factor to predict poor prognosis, for both overall (P=0.0006) and disease-free (P=0.024) survival. CONCLUSIONS: Our results suggest that Int6 expression, evaluated by quantitative real-time PCR, may represent a new prognostic factor in patients with stage I NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Factor 3 de Iniciación Eucariótica/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Adulto , Anciano , Análisis de Varianza , Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/genética , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de SupervivenciaRESUMEN
Recently, we have identified a novel 1.8 kb human Notch4/Int3 RNA species (designated h-Int3sh). The h-Int3sh RNA encodes a protein that is missing the CBF1-binding region (RAM23) of the Notch 4/Int3 intracellular domain (ICD). Expression of h-Int3sh in the MCF10A 'normal' human mammary epithelial cell line has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth in soft agar. To study the consequences of h-Int3sh expression in vivo on mammary gland development and tumorigenesis, three transgenic mouse lines were established, in which the transgene is the Whey acidic protein (WAP) promoter linked to h-Int3sh. Expression of WAP-Int3sh was detectable in the mammary gland at day 15 of pregnancy in each transgenic line. Mammary gland development in all founder lines is normal and the females can lactate. WAP-h-Int3sh females from each of the founder lines develop mammary tumors, but with a long latency (average age of 18 months). Tumor development was associated with activation of Notch pathway, as evidenced by upregulation of Hes-1. The long latency of mammary tumors in WAP-h-Int3sh mice could be due in part to the subcellular localization of h-Int3sh. Immunofluorescence analysis of transfected COS-1 cells showed that h-Int3sh is localized in the cytoplasm and nucleus, while Int3-ICD is detected only in the nucleus. We speculate that the Notch4/Int3 ICD-induced block to mammary gland development and tumorigenesis are consequences of an increasing gradient of CBF1-dependent Notch4/Int3 signaling.
Asunto(s)
Glándulas Mamarias Animales/crecimiento & desarrollo , Neoplasias Mamarias Experimentales/patología , Proteínas Proto-Oncogénicas/genética , Receptores de Superficie Celular/genética , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Femenino , Humanos , Inmunohistoquímica , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Transgénicos , Receptor Notch4 , Receptores NotchRESUMEN
Lipid-enhancement of activated sludges was conducted to increase the amount of saponifiable lipids in the sludges. The sludges were obtained from a conventional activated sludge (CAS) and an oxidation ditch process (ODP). Results showed 59-222% and 150-250% increase in saponifiable lipid content of the sludges from CAS and ODP, respectively. The fatty acid methyl ester (FAMEs) obtained from triacylglycerides was 57-67% (of total FAMEs) for enhanced CAS and 55-73% for enhanced ODP, a very significant improvement from 6% to 10% (CAS) and 4% to 8% (ODP). Regardless of the source, the enhancement resulted in sludges with similar fatty acid profile indicating homogenization of the lipids in the sludges. This study provides a potential strategy to utilize existing wastewater treatment facilities as source of significant amount of lipids for biofuel applications.