RESUMEN
Migration is a complex process for epithelial tissues, because the epithelium must move as an intact sheet to preserve its barrier function. The requirement for structural integrity is met by coupling cell-to-matrix and cell-to-cell adhesion at the cellular level, and by coordinating cell proliferation and cell migration in the tissue as a whole. Proliferation is suppressed at the migrating cell front, allowing cells in this region to remain tightly packed while advancing rapidly. At the same time, proliferation is enhanced in a region behind the advancing cell front to expand the epithelial cell sheet. This review considers the extracellular signals and intracellular signaling pathways that regulate these processes in the lens and corneal epithelium, with emphasis on the commonalities that link these tissues.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular , Córnea/fisiología , Cristalino/fisiología , Animales , Ácido Araquidónico/metabolismo , Córnea/enzimología , Córnea/metabolismo , Citocinas/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular/fisiología , Cristalino/enzimología , Cristalino/metabolismo , Metaloproteasas/genética , Metaloproteasas/metabolismo , Metaloproteasas/fisiología , Modelos Biológicos , Proteína Quinasa C/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Proteínas ras/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
The detection of disseminated tumor cells in bone marrow and blood is increasingly used for staging and therapeutic decisions in breast cancer and other solid tumors. Molecular biological methods improve the diagnostic accuracy. Limitations of the approach relate to the lack of disease-specific marker genes. The detection of tumor cells in the bone marrow after primary therapy is a negative prognostic parameter in many solid tumours. Axillary lymph node dissection and histopathology remain the standard staging procedure in breast cancer, but nodal negative patients exhibiting tumor cells in the bone marrow have an inferior outcome and may benefit from adjuvant therapy. The immunohistochemical and molecular detection of tumour cells in lymph nodes reduces the number of truly nodal-negative patients considerably. Tumour cells in bone marrow and blood may be used to directly monitor therapeutic responses.
Asunto(s)
Neoplasia Residual/diagnóstico , Neoplasia Residual/terapia , Neoplasias/terapia , Humanos , Neoplasia Residual/genética , Reacción en Cadena de la Polimerasa , PronósticoRESUMEN
PURPOSE: 12(S)-Hydroxyeicosatetraenoic acid (12(S)HETE), a 12-lipoxygenase metabolite of arachidonic acid, is required for epidermal growth factor (EGF)-dependent DNA synthesis and c-fos induction in lens epithelial cells. The present study was undertaken to identify signal transduction events upstream of c-fos induction that may be regulated by 12(S)HETE. METHODS: The rabbit lens epithelial cell line, N/N1003A, was cultured in serum-free medium, with or without EGF. Activation of PKC and other selected enzymes was examined in the presence of the lipoxygenase inhibitor baicalein and/or exogenous 12(S)HETE. Relative abundance of PKC isoforms in subcellular fractions was determined by immunoblot analysis with isoform-specific antibodies. PKC activity in subcellular fractions was measured by peptide substrate phosphorylation, with and without pseudosubstrate peptide inhibitor. Phosphorylated enzymes were detected by immunoblot analysis. Relative levels of c-fos mRNA were determined by RT/PCR with internal standard. RESULTS: Baicalein blocked EGF-dependent translocation and activation of PKC, without affecting phosphorylation of Erk1/2. Of several PKC isoforms investigated (alpha, betaI, betaII, and gamma), only PKCalpha and betaII were significantly activated by EGF and inhibited by baicalein. 12(S)HETE, in combination with EGF, countered the effect of lipoxygenase inhibitors on PKC activation, and 12(S)HETE in the absence of EGF stimulated PKC translocation. Also of note, 12(S)HETE alone activated PKCgamma, an isoform that was not significantly activated by EGF. Inhibiting PKC activation with GF109203X blocked induction of c-fos by EGF but did not affect EGF-stimulated phosphorylation of Erk1/2, indicating that the effect of PKC on c-fos induction is independent of the Erk1/2 pathway. CONCLUSIONS: In lens epithelial cells, 12(S)HETE-dependent activation of PKCalpha and betaII acts in concert with other EGF-dependent signals to induce c-fos mRNA.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Flavanonas , Regulación de la Expresión Génica , Cristalino/fisiología , Proteína Quinasa C/metabolismo , Animales , Transporte Biológico , Línea Celular , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Flavonoides/farmacología , Genes fos , Indoles/farmacología , Isoenzimas/metabolismo , Cristalino/citología , Cristalino/efectos de los fármacos , Maleimidas/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Conejos , Fracciones Subcelulares/enzimología , Distribución TisularRESUMEN
We describe in detail a 67-yr-old woman who was treated with a cytostatic combination chemotherapy for newly diagnosed common-acute lymphoblastic leukaemia. At the end of induction therapy, the patient acquired invasive mould infection affecting lung and brain. The patient entered complete remission of her leukaemia. Treatment with liposomal amphotericin B was initiated along with surgical excision of the fungal brain abscess. Intrathecal instillation of amphotericin B deoxycholate was started using an Ommaya reservoir because of an anatomical connection between the postoperative cavity and the ventricle. Full dose cytostatic chemotherapy was continued with little delay. A computerised tomography scan of the chest performed 2 months later revealed no fungal abscesses. Magnetic resonance imaging of the brain did not reveal any fungal manifestation. During maintenance therapy/week 69, the patient relapsed from leukaemia. High doses of intravenous liposomal amphotericin B were administered prophylactically. The patient's leukaemia proved refractory to reinduction chemotherapy and the patient died from pneumonia 8 wk later. Post mortem microbiological investigation and histopathological examination of lung and brain tissue did not reveal any macroscopical or microscopical fungal manifestations. This case underlines the feasibility and successful application of combined antileukaemic, antifungal and surgical therapy in a patient with acute leukaemia.
Asunto(s)
Anfotericina B/uso terapéutico , Antifúngicos/uso terapéutico , Absceso Encefálico/tratamiento farmacológico , Absceso Pulmonar/tratamiento farmacológico , Enfermedades Pulmonares Fúngicas/tratamiento farmacológico , Neuroaspergilosis/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/complicaciones , Anciano , Anfotericina B/administración & dosificación , Anfotericina B/efectos adversos , Antifúngicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Absceso Encefálico/diagnóstico , Absceso Encefálico/microbiología , Absceso Encefálico/cirugía , Terapia Combinada , Craneotomía , Ácido Desoxicólico/administración & dosificación , Ácido Desoxicólico/efectos adversos , Ácido Desoxicólico/uso terapéutico , Combinación de Medicamentos , Resultado Fatal , Femenino , Humanos , Huésped Inmunocomprometido , Infusiones Intravenosas , Inyecciones Espinales , Liposomas , Absceso Pulmonar/diagnóstico , Absceso Pulmonar/microbiología , Enfermedades Pulmonares Fúngicas/diagnóstico , Enfermedades Pulmonares Fúngicas/microbiología , Imagen por Resonancia Magnética , Recurrencia Local de Neoplasia , Neuroaspergilosis/diagnóstico , Neuroaspergilosis/microbiología , Neuroaspergilosis/cirugía , Neumonía Neumocócica/etiología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Inducción de Remisión , Tomografía Computarizada por Rayos XRESUMEN
The cyclin-dependent kinase member, Cdk5, is expressed in a variety of cell types, but neuron-specific expression of its activator, p35, is thought to limit its activity to neurons. Here we demonstrate that both Cdk5 and p35 are expressed in the human astrocytoma cell line, U373. Cdk5 and p35 are present in the detergent-insoluble cytoskeletal fraction of this cell line and Cdk5 localizes to filopodia and vinculin-rich regions of cell-matrix contact in lamellopodia. When exposed to a 46(o)C heat shock, U373 cells change shape, lose cell-matrix contacts and show increased levels of apoptosis. To test whether Cdk5 activation might play a role in these events, U373 cells were stably transfected with histidine-tagged or green fluorescent protein-tagged constructs of Cdk5 or a dominant negative mutation, Cdk5T33. Under normal growth conditions, growth characteristics of the stably transfected lines were indistinguishable from untransfected U373 cells and Cdk5 localization was not changed. However, when subjected to heat shock, cells stably transfected with Cdk5-T33 remained flattened, showed little loss of cell-matrix adhesion, and exhibited significantly lower levels of apoptosis. In contrast, cells that overexpressed wild-type Cdk5 showed morphological changes similar to those seen in untransfected U373 cells in response to heat shock and had significantly higher levels of apoptosis. Heat-shocked cells showed changes in p35 mobility and stability of the Cdk5/p35 complex consistent with endogenous Cdk5 activity. Together these findings suggest that endogenous Cdk5 activity may play a key role in regulating morphology, attachment, and apoptosis in U373 cells, and raise the possibility that Cdk5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells.
Asunto(s)
Apoptosis , Quinasas Ciclina-Dependientes/metabolismo , Respuesta al Choque Térmico/fisiología , Proteínas del Tejido Nervioso/metabolismo , Astrocitoma , Fraccionamiento Celular , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/biosíntesis , Quinasas Ciclina-Dependientes/genética , Citoesqueleto/metabolismo , Humanos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales CultivadasRESUMEN
42 breast cancer patients were treated by high-dose chemotherapy (HDC) and autologous peripheral stem-cell transplantation (ASTx) in the Donauspital between 1992 and 1999. 24 patients had stage II/III breast cancer with high risk for relapse. The other 18 patients underwent HDC and ASTx in chemosensitive stage IV. After previous conventional chemotherapy peripheral stem-cells were harvested by one cycle of mobilisation chemotherapy (epirubicin/taxol, FEC 120 or cyclophosphamide) followed by cytokine stimulation. 16 patients were treated by a tandem transplantation (conditioning protocol for 1st ASTx was melphalan 200 mg/m2 and for 2nd transplant it was CTC: cyclophosphamide 6 g/m2; thiotepa 500 mg/m2; carboplatin 800 mg/m2). The other 26 patients received one HDC with CTC as conditioning protocol. The HDC was well tolerated by all patients, there was no transplant-related mortality. The median survival and the progression-free survival (PFS) after HDC and ASTx in stage IV breast cancer patients were 28 and 11 months, respectively. The median survival and PFS were not yet reached in stage II/III patients after 55 months. The actuarial survival and PFS in that patient group were 70% after 55 months. Our data confirm the low risk and good efficacy of HDC and ASTx in breast cancer patients. Nevertheless randomised studies are necessary to evaluate the importance of HDC compared to intensified conventional protocols without ASTx.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/terapia , Trasplante de Células Madre Hematopoyéticas , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Terapia Combinada , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Neuroendocrine carcinoma and small-cell lung cancer (SCLC) are highly responsive to chemo- and radiotherapy. Nevertheless, most patients (pts.) experience relapse. At the 2nd department of medicine in the Donauspital, 4 pts. with neuroendocrine carcinomas of different primary sites underwent high-dose chemotherapy with autologous stem-cell transplantation (ASTx). Pt. 1 suffered from neuroendocrine lung cancer, pt. 2 from a small-cell carcinoma of the pancreas. Pt. 3 had a metastatic small-cell abdominal bulky tumor and pt. 4 presented with neuroendocrine carcinoma of the prostate. After 4-6 cycles induction chemotherapy pts. were consolidated with 1 cycle of HDCht and ASTx. Prior to HDCht pt. 1 and pt. 2 were in complete remission (CR) and pt. 3 and pt. 4 in partial remission. Pt. 3 converted in CR after HDCht. He is still in CR with a disease-free survival of 23 month after ASTx and 30 month after diagnosis. Pt. 1, 2 and 4 died from relapse 10, 16 and 5 month after ASTx and 16, 22 and 9 month after diagnosis. Pts. with neuroendocrine carcinomas might be suitable candidates for HDCht and ASTx.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Pequeñas/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas , Neoplasias Pulmonares/tratamiento farmacológico , Tumores Neuroendocrinos/tratamiento farmacológico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma de Células Pequeñas/mortalidad , Carcinoma de Células Pequeñas/patología , Relación Dosis-Respuesta a Droga , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Tumores Neuroendocrinos/mortalidad , Tumores Neuroendocrinos/patología , Tasa de SupervivenciaRESUMEN
Between 1992 and 1999 15 patients (pts.) suffering from multiple myeloma (MM) were treated with high-dose chemotherapy and consecutive autologous stem-cell transplantation (ASTx). 10/15 pts underwent two courses of ASTx (tandem- or double ASTx). So 25 ASTx were performed in these 15 pts. in total. All pts. were under 60 a. of age. 13/15 pts. received 6 cycles of chemotherapy on an average according to the VAD-protocol (Vincristin, Adriamycin, Dexamethason). Mobilisation of peripheral hematopoietic stem cells was performed with high-dose cyclophosphamide and hematopoietic growth-factors (CSFs). The conditioning protocol consisted of high-dose melphalan (200-225 mg/m2) in 24/25 ASTx. In one single case total body irradiation (TBI) plus melphalan 140 mg/m2 was used. 2/15 pts. died within 30 days from ASTx; one patient from interstitial pneumonia after TBI, and the other, who was in a very advanced stage of his disease with multiple pretreatment courses before ASTx. The overall survival (OS) was in the mean 68 months, the progression-free survival (PFS) after ASTx 21 m respectively. In pts. with MM high-dose melphalan (up to 225 mg/m2) without TBI plus ASTx is a safe and effective procedure when performed in the early course of the disease.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Mieloma Múltiple/terapia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Doxorrubicina/efectos adversos , Esquema de Medicación , Etopósido/administración & dosificación , Etopósido/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/mortalidad , Mieloma Múltiple/patología , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
PURPOSE: Cultured rat lenses and primary human lens epithelial cells (HLECs) express12-lipoxygenase (12-LOX) and require a 12-LOX metabolite of arachidonic acid for growth in response to EGF and insulin. This study seeks to identify an established cell line with these characteristics. METHODS: Immunoblotting was used to screen eight lens epithelial cell lines for 12-LOX expression: the human line, HLE-B3; mouse lines alphaTN4, 17EM15, 21EM15, and MLE6, and rabbit lines N/N1003A, LEP2 and B3. DNA synthesis was measured as incorporation of 3H-thymidine into DNA. Expression of c-fos mRNA was detected by RT-PCR. The involvement of 12-lipoxygenase metabolites was determined using the lipoxygenase inhibitors baicalein, cinnamyl 3,4-dihydroxy-alpha-cyanocinnamate (CDC), or nordihydroguiairetic acid (NDGA). RESULTS: 12-LOX was detected only in the rabbit lines N/N1003A, LEP2 and B3. N/N1003A cells were chosen for further study. 12-LOX inhibitors blocked DNA synthesis in response to EGF with or without insulin. Inhibition of EGF-stimulated DNA synthesis was reversed by 0.3 microM to 3 microM 12(S)hydroxyeicosatetraenoic acid (HETE), but not by equivalent concentrations of 5(S)HETE, 8(S)HETE, 15(S)HETE, or 12(R)HETE. Baicalein prevented EGF induction of c-fos mRNA. The transformed HLEC line, HLE-B3, showed little stimulation of DNA synthesis in response to EGF and was unaffected by the presence of 12-LOX inhibitors. CONCLUSIONS: N/N1003A cells, like primary cultured human lens epithelial cells or neonatal rat lenses, require 12-LOX activity for EGF dependent growth. This line will be useful for studies of the mechanism of action of 12(S)HETE.
Asunto(s)
Araquidonato 12-Lipooxigenasa/biosíntesis , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Flavanonas , Cristalino/enzimología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biosíntesis , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Animales , Ácidos Cafeicos/farmacología , Línea Celular , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Células Epiteliales/enzimología , Flavonoides/farmacología , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacología , Immunoblotting , Insulina/farmacología , Cristalino/citología , Inhibidores de la Lipooxigenasa , Masoprocol/farmacología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Conejos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previous studies from this laboratory have shown that differentiating lens fiber cells contain two active cyclin dependent kinases (Cdks), Cdk1 and Cdk5. The present study was undertaken to explore the expression and regulation of six additional members of the Cdk family (Cdk2, Cdk3, Cdk4, Cdk6, Cdk7 and Cdk8) during lens differentiation. Differentiating lens fiber cells were separated from lens epithelial cells by microdissection of developing rat lenses [embryonic day 16 (E16) to postnatal day 8 (P8)] and Cdk expression was assessed by RT-PCR and immunoblotting. Two Cdks (Cdk3 and Cdk6) were not expressed in lens fiber cells or epithelial cells during this developmental period. In the lens epithelium, we detected proteins and mRNAs corresponding to all other Cdks examined (Cdk2, Cdk4, Cdk7, Cdk8) throughout this developmental period. Epithelial cells showed significant Cdk2 activity, which decreased with developmental age, but no significant activity was detected for Cdk4, Cdk7, or Cdk8. Fiber cells contained all four Cdk proteins and the corresponding Cdk mRNAs except for Cdk2 mRNA. None of the Cdks examined showed significant kinase activity in fiber cells. Immunoprecipitates of Cdk2 and Cdk4 from fiber cells contained p57(kip2), supporting the view that this Cdk inhibitor blocks the activity of these Cdks in lens fibers. In contrast, p57(kip2)did not co-immunoprecipitate with Cdk5 from lens fibers. These findings suggest that the differential affinity of p57(kip2)for members of the Cdk family may provide a mechanism for specific regulation of individual Cdks during fiber cell differentiation.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/genética , Cristalino/embriología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas , Animales , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Inducción Embrionaria , Células Epiteliales/metabolismo , Expresión Génica , Immunoblotting , Cristalino/citología , Cristalino/metabolismo , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
PURPOSE: Human trabecular meshwork (HTM) cells were mechanically stretched in vitro as a potential model for the distension of this tissue that can occur in vivo in response to increased pressure gradients. Cell morphology and certain components of the signal transduction pathways, including the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK) pathways, were evaluated for stretch-induced alterations. METHODS: Primary HTM cells grown in tissue culture were subjected to a mechanical stretch lasting from 10 seconds to 4 days. The actin cytoskeletal network was visualized by phalloidin staining. Proteins phosphorylated on their tyrosine residues were isolated using an immunoaffinity system and were analyzed by gel electrophoresis and immunostaining. Mitogen-activated protein kinase activity was evaluated using an in-gel assay system, and the mRNA levels of c-fos and c-jun were determined by quantitation of competitive reverse transcription-polymerase chain reaction. In addition, the amount of c-Fos protein was estimated by chemiluminescent immunoblot analysis. RESULTS: On stretching, the HTM cells elongated but regained their normal morphologic characteristics within 24 hours. Unstretched HTM cells displayed a diffuse F-actin microfilament network, whereas stretched cells exhibited complex geodesic patterns. Ten seconds after stretching began, the level of tyrosine phosphorylation on the six major phosphoproteins significantly decreased between 80% and 100%, whereas the level of paxillin tyrosine phosphorylation significantly increased 39%. Stretching caused MAPK activity and the amount of mRNA and protein of the immediate-early gene c-fos to decrease more than 60% within 2 minutes, but within 15 to 30 minutes they increased above or equivalent to normal levels. The level of c-jun mRNA was unchanged by stretching. CONCLUSIONS: In response to a mechanical stretch, major cytoskeletal alterations occur in HTM cells, which involve changes in the levels of tyrosine phosphorylation. Mechanotransduction (signal transduction by mechanical stimulation) through the MAPK signaling pathway was significantly depressed immediately after stretching; however, the JNK pathway appeared to be unaffected. The data suggest that HTM cells adapt to mechanical stress by altering the cytoskeletal network and signaling cascades.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Transducción de Señal , Estrés Mecánico , Malla Trabecular/metabolismo , Adolescente , Adulto , Anciano , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Persona de Mediana Edad , Faloidina , Fosforilación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Malla Trabecular/citología , Tirosina/metabolismoRESUMEN
During growth arrest and differentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells differentiate into fiber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of newborn rats formed at least two specific complexes with an E2F consensus oligonucleotide. Proteins from lens fiber cells formed three specific complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody specific for p107 demonstrated that two fiber cell complexes and one epithelial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the fiber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identified p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the fiber cells. Thus, although p130/E2F complexes are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primary regulators of E2F activity.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Cristalino/metabolismo , Proteínas Nucleares/metabolismo , Proteínas , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F3 , Factor de Transcripción E2F5 , Immunoblotting , Técnicas para Inmunoenzimas , Cristalino/citología , Proteínas Nucleares/biosíntesis , Fosfoproteínas/biosíntesis , Fosfoproteínas/metabolismo , Ratas , Ratas Wistar , Proteína de Retinoblastoma/biosíntesis , Proteína 1 de Unión a Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Ubiquitinas/metabolismoRESUMEN
Previous work has shown that postmitotic, differentiating fiber cells of the embryonic chicken lens express cyclin B and Cdc2. The present study explores the possible physiological role of these proteins in lens differentiation by examining the developmental regulation of cyclin B/Cdc2 expression and activity in lens fiber cells of embryonic and newborn rats. Cyclin B mRNA and protein were detected not only in the lens epithelium, which contains proliferating cells, but also in postmitotic, differentiating fiber cells. In contrast, cyclin A mRNA and protein were detected only in epithelial cells. Immunoprecipitation with cyclin B antibody coprecipitated Cdc2 from both epithelial and fiber cell extracts. Immunoprecipitates of cyclin B from both epithelial cells and fiber cells showed H1 kinase activity when assayed in vitro, but the developmental pattern of cyclin B-associated kinase activity in these two lens fractions was markedly different. In the epithelium, H1 kinase activity decreased gradually with developmental age in parallel with the decrease in epithelial cell proliferation, whereas, in the fiber cells, kinase activity peaked sharply at embryonic day 18 (E18) and E19. Microscopic examination of rat lenses indicated that peak cyclin B/Cdc2 activity was correlated with changes in chromatin structure and nuclear envelope breakdown in the terminally differentiating primary lens fiber cells. These findings suggest that cyclin B/Cdc2 activity may play an active role in nuclear changes leading to primary fiber cell denucleation.
Asunto(s)
Proteína Quinasa CDC2/análisis , Ciclina B/análisis , Cristalino/embriología , Animales , Animales Recién Nacidos , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Diferenciación Celular , Extractos Celulares , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cromatina , Ciclina A/análisis , Ciclina A/genética , Ciclina B/genética , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Humanos , Cristalino/química , Cristalino/citología , Cristalino/crecimiento & desarrollo , Protamina Quinasa/metabolismo , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
Human trabecular meshwork (HTM) is distended and stretched with increases in intraocular pressure. During this stretching, there is a rearrangement of actin filaments. The HTM cells express alpha B-crystallin, a small heat shock protein that may have a role in the stabilization and regulation of the cytoskeleton in mammalian cells. The levels of alpha B-crystallin were examined in trabecular meshwork cells after mechanical stretch. Human TM primary cell cultures, plated onto silicone sheets, were subjected to a single 10% linear stretch and samples were prepared at various times after stretch for immunoblotting or Northern blotting. Immunoblots of total protein extracts with antibody specific for alpha B-crystallin detected a 26% decrease of cellular alpha B-crystallin levels within 2 minutes. After 1 hour alpha B-crystallin levels had decreased 90% compared to control cells. The levels of alpha B-crystallin began to recover in cells stretched for 2 hours and returned to initial levels by 24 hours. Northern blots probed with alpha B-crystallin exon III cDNA detected a transcript of 0.65 kb in human TM cells and the levels of the alpha B mRNA remained constant during alpha B-crystallin protein decrease. Later, levels of the 0.65 kb transcript of alpha B-crystallin increased during the cellular recovery. These results suggest that decreased levels of alpha B-crystallin after mechanical stretch were probably not due to transcriptional changes but rather to increased degradation of alpha B-crystallin protein. An increase in mRNA levels may play a role in the recovery of alpha B-crystallin during reorganization of the cytoskeleton and attachment to the substratum. These data raise the possibility of a specific proteolysis of alpha B-crystallin protein in cells after a physiological challenge.
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Cristalinas/metabolismo , Proteínas de Choque Térmico/metabolismo , Malla Trabecular/metabolismo , Northern Blotting , Western Blotting , Células Cultivadas , Cristalinas/genética , Citoesqueleto/metabolismo , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica , Proteínas de Choque Térmico/genética , Humanos , Focalización Isoeléctrica , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Mecánico , Malla Trabecular/citología , Transcripción GenéticaRESUMEN
Cyclin-dependent kinases and their regulatory subunits, the cyclins, are known to regulate progression through the cell cycle. Yet these same proteins are often expressed in non-cycling, differentiated cells. This review surveys the available information about cyclins and cyclin-dependent kinases in differentiated cells and explores the possibility that these proteins may have important functions that are independent of cell cycle regulation.
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Diferenciación Celular/fisiología , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/fisiología , Animales , Apoptosis/fisiología , Quinasas Ciclina-Dependientes/clasificación , Quinasas Ciclina-Dependientes/genética , Ciclinas/clasificación , Ciclinas/genética , Regulación del Desarrollo de la Expresión Génica , Modelos Biológicos , Familia de Multigenes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
We have investigated the expression of Cdk5 and its regulatory subunit, p35, in the developing rat lens from embryonic day 16 (E16) to postnatal day 8 (P8). Reverse transcription and polymerase chain reaction (RT/PCR) detected Cdk5 and p35 mRNA expression in lens epithelial cells and in differentiating lens fibers throughout this developmental period. Subsequent sequencing of the RT/PCR products confirmed their identifies. In sity hybridization with Cdk5 and p35 riboprobes showed especially high expression of both mRNAs in the newly formed lens fiber cells in the bow region of the lens. Immunocytochemistry at E18 showed that Cdk5 was present in the cytoplasm of lens epithelial cells and fiber cells, with especially strong immunostaining at the anterior ends of the fibers. Fiber cells in the final stages of maturation, immediately prior to nuclear degeneration, showed positive staining for Cdk5 in the nucleus. Immunoprecipitation of proteins with Cdk5 antibody followed by immunoblotting with either N-terminal specific or C-terminal specific p35 antibodies demonstrated that p35 is complexed with Cdk5 in lens epithelial cells and lens fibers. Immunoprecipitates of Cdk5 from epithelia and fibers showed kinase activity in vitro using histone H1 as a substrate. These findings demonstrate that p35/Cdk5 activity is not restricted to neurons and raise the possibility that this kinase may play a role in lens fiber cell differentiation.
Asunto(s)
Quinasas Ciclina-Dependientes , Cristalino/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Quinasa 5 Dependiente de la Ciclina , Cartilla de ADN , Femenino , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Cristalino/embriología , Ratones , Datos de Secuencia Molecular , ARN Mensajero , Ratas , Ratas WistarRESUMEN
A rat PCTAIRE-1 cDNA clone was isolated by immunoscreening of a PC12 cDNA library, followed by 5' RACE (rapid amplification of cDNA ends) to determine the 5' end. The rat PCTAIRE-1 cDNA sequence is 96% identical to mouse PCTAIRE-1 and contains an alternatively spliced exon of 131 bp near the 5' end. Although a mouse cDNA containing this exon has been reported, examination of several mouse cell lines provided no evidence for expression of the corresponding mRNA (Okuda et al., 1992). In contrast, reverse transcription and polymerase chain reaction (RT/PCR) across this region using RNA from proliferating, differentiated, and apoptotic PC12 cells demonstrated that alternatively spliced forms of PCTAIRE-1 mRNA with and without this exon are expressed. Both forms of PCTAIRE-1 mRNA are also expressed in vivo in neonatal rat brain, although other tissues examined contained only the form lacking the alternatively spliced exon. In the absence of the alternatively spliced exon PCTAIRE-1 mRNA contains an open reading frame of 1488 bp, corresponding to a 55-kDa protein that is 97% identical to mouse PCTAIRE-1 protein. When the alternatively spliced exon is present, this open reading frame is terminated by a stop codon and a second open reading frame is initiated, predicting a second PCTAIRE-1 protein of 52 kDa. The two predicted PCTAIRE-1 proteins are identical downstream of the splice site, but share no homology at their N-terminal ends.
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Empalme Alternativo , Quinasas Ciclina-Dependientes , Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Células PC12 , Biosíntesis de Proteínas , Ratas , Análisis de Secuencia de ADNRESUMEN
PURPOSE: To determine whether the 12-lipoxygenase pathway of arachidonic acid metabolism is present in the human lens and whether 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) plays a role in regulating proto-oncogene expression and DNA synthesis in human lens epithelial cells (HLECs). METHODS: Second- and third-passage primary cultures of HLECs were used for analysis. Human cataract epithelia were obtained from surgery. 12-lipoxygenase mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the PCR product was sequenced. The 12-lipoxygenase protein was detected by immunoblotting. 12(S)-HETE was detected in HLEC-conditioned medium by radioimmunoassay. For studies of growth factor-induced mitogenesis, HLECs were serum starved, then stimulated with 15 ng/ml epidermal growth factor (EGF) and 1 microgram/ml insulin or with 0.3 microM 12(S)-HETE. The 12-lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10 microM) was used to block endogenous 12-lipoxygenase activity. Expression of c-fos mRNA was determined by RT-PCR, and DNA synthesis was measured by 3H-thymidine incorporation. RESULTS: 12-lipoxygenase mRNA and protein were detected in HLECs and in human cataract tissues. 12(S)-HETE was released into the medium by HLECs in the presence of EGF-insulin. Stimulation of c-fos mRNA expression and DNA synthesis by EGF-insulin was inhibited when the 12-lipoxygenase pathway was blocked by CDC. This inhibition was reversed completely by exogenously added 12(S)-HETE. However, exogenous 12(S)-HETE was unable to stimulate HLEC DNA synthesis in the absence of growth factors. CONCLUSIONS: The 12-lipoxygenase pathway of arachidonic acid metabolism is present in human lens epithelial cells. 12(S)-HETE does not stimulate HLEC DNA synthesis in the absence of growth factors but enables the cellular response to EGF and insulin.
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Factor de Crecimiento Epidérmico/farmacología , Ácidos Hidroxieicosatetraenoicos/fisiología , Hipoglucemiantes/farmacología , Insulina/farmacología , Cristalino/fisiología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Adulto , Araquidonato 12-Lipooxigenasa/metabolismo , Secuencia de Bases , Ácidos Cafeicos/farmacología , Catarata/tratamiento farmacológico , Catarata/metabolismo , Células Cultivadas , ADN/biosíntesis , Cartilla de ADN/química , Epitelio/efectos de los fármacos , Epitelio/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Lactante , Cristalino/citología , Cristalino/efectos de los fármacos , Inhibidores de la Lipooxigenasa/farmacología , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fos/biosíntesis , ARN/aislamiento & purificación , ARN Mensajero/metabolismo , Radioinmunoensayo , Transcripción GenéticaRESUMEN
The present study examines whether cyclin B may be involved in apoptosis of neuronally differentiated PC12 cells following withdrawal of NGF. Cyclin B mRNA increased approximately 10-fold 4 days after NGF withdrawal, as indicated by competitive RT/PCR. Sequencing of the PCR product confirmed that it was derived from cyclin B mRNA. Cyclin B protein increased in parallel with cyclin B mRNA, as shown by immunoblotting. Immunoprecipitation with anti-cyclin B antibody demonstrated that cyclin B was associated with H1K activity, which reached a maximum 5 days after NGF withdrawal. When proteins immunoprecipitated with anti-cyclin B antibody were immunoblotted with anti-PSTAIR antibody, a protein with apparent molecular weight of 34 kDa was detected. This protein was identified as p34cdc2 on the basis of immunoreactivity with antibody against the C-terminal portion of mouse p34cdc2. Since cyclin B/p34cdc2 complexes are known to catalyze chromosomal condensation and nuclear envelope breakdown during mitosis, these results suggest that cyclin B/p34cdc2 may play some role in the nuclear changes accompanying apoptosis of PC12 cells.