RESUMEN
PURPOSE: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells. MATERIALS AND METHODS: Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized. RESULTS: The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post-mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double-strand break repair, radiation-induced increases in p53 and p21(WAF-1,CIP-1) expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen-induced mitotic arrest of fibroblasts was still possible in the hTERT-immortalized clones. CONCLUSIONS: Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.
Asunto(s)
Fibroblastos/enzimología , Fibroblastos/efectos de la radiación , Telomerasa/metabolismo , Adaptación Fisiológica/efectos de la radiación , Apoptosis/efectos de la radiación , División Celular/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de la radiación , Proteínas de Unión al ADN , Relación Dosis-Respuesta en la Radiación , Activación Enzimática/efectos de la radiación , Fibroblastos/citología , Humanos , Masculino , Pene/citología , Pene/enzimología , Pene/efectos de la radiación , Radiación Ionizante , Proteínas Recombinantes/metabolismo , Piel/citología , Piel/enzimología , Piel/efectos de la radiación , Telomerasa/genética , TransfecciónRESUMEN
Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.
Asunto(s)
Senescencia Celular/fisiología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Fibroblastos/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Telómero/metabolismo , Northern Blotting , Línea Celular , Tamaño de la Célula , Senescencia Celular/genética , Clusterina , Colagenasas/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Glicoproteínas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Fenotipo , ARN Ribosómico 18S/metabolismo , Telómero/ultraestructuraRESUMEN
Worldwideattention is presently focused on proliferating populations of neural precursor cells as an in vitro source of tissue for neural transplantation and brain repair. However, successful neuroreconstruction is contingent upon their capacity to integrate within the host CNS and the absence of tumorigenesis. Here we show that human neural precursor cells express very low levels of telomerase at early passages (less than 20 population doublings), but that this decreases to undetectable levels at later passages. In contrast, rodent neural precursors express high levels of telomerase at both early and late passages. The human neural precursors also have telomeres (approximately 12 kbp) significantly shorter than those of their rodent counterparts (approximately 40 kbp). Human neural precursors were then expanded 100-fold prior to intrastriatal transplantation in a rodent model of Parkinson's disease. To establish the effects of implanted cell number on survival and integration, precursors were transplanted at either 200,000, 1 million, or 2 million cells per animal. Interestingly, the smaller transplants were more likely to extend neuronal fibers and less likely to provoke immune rejection than the largest transplants in this xenograft model. Cellular proliferation continued immediately post-transplantation, but by 20 weeks there were virtually no dividing cells within any of the grafts. In contrast, fiber outgrowth increased gradually over time and often occupied the entire striatum at 20 weeks postgrafting. Transient expression of tyrosine hydroxylase-positive cells within the grafts was found in some animals, but this was not sustained at 20 weeks and had no functional effects. For Parkinson's disease, the principal aim now is to induce the dopaminergic phenotype in these cells prior to transplantation. However, given the relative safety profile for these human cells and their capacity to extend fibers into the adult rodent brain, they may provide the ideal basis for the repair of other lesions of the CNS where extensive axonal outgrowth is required.
Asunto(s)
Neuronas/citología , Neuronas/enzimología , Células Madre/enzimología , Telomerasa/biosíntesis , Animales , Axones/metabolismo , Axones/ultraestructura , Trasplante de Tejido Encefálico , Recuento de Células , Diferenciación Celular , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Femenino , Trasplante de Tejido Fetal , Factor 2 de Crecimiento de Fibroblastos/farmacología , Supervivencia de Injerto , Humanos , Fibras Nerviosas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/trasplante , Oxidopamina , Ratas , Trasplante de Células Madre , Células Madre/citología , Células Madre/efectos de los fármacos , Telómero/ultraestructura , Tiempo , Trasplante Heterólogo , Tubulina (Proteína)/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Telomere shortening has been causally implicated in replicative senescence in humans. To examine the relationship between telomere length and ageing in mice, we have utilized Mus spretus as a model species because it has telomere lengths of approximately the same length as humans. Telomere length and telomerase were analyzed from liver, kidney, spleen, brain and testis from >180 M.spretus male and female mice of different ages. Although telomere lengths for each tissue were heterogeneous, significant changes in telomere lengths were found in spleen and brain, but not in liver, testis or kidney. Telomerase activity was abundant in liver and testis, but weak to non-detectable in spleen, kidney and brain. Gender differences in mean terminal restriction fragment length were discovered in tissues from M.spretus and from M.spretus xC57BL/6 F1 mice, in which a M. spretus -sized telomeric smear could be measured. The comparison of the rank order of tissue telomere lengths within individual M. spretus showed that certain tissues tended to be longer than the others, and this ranking also extended to tissues of the M.spretus xC57BL/6 F1 mice. These data suggest that telomere lengths within individual tissues are regulated independently and are genetically controlled.
Asunto(s)
Envejecimiento/genética , Telómero , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Muridae , Caracteres Sexuales , Telomerasa/metabolismoRESUMEN
Classical radiographic features of patients presenting with silicosis are diffuse interstitial shadowing with subsequent enlargement of hilar nodes, sometimes with "eggshell" calcification. Five case histories are described of workers who were exposed to silica and presented initially with bilateral hilar lymphadenopathy without radiographic evidence of interstitial lung disease. One case progressed to show features of silicosis.
Asunto(s)
Enfermedades Linfáticas/etiología , Silicosis/complicaciones , Adulto , Humanos , Pulmón/diagnóstico por imagen , Enfermedades Linfáticas/diagnóstico por imagen , Enfermedades Linfáticas/patología , Masculino , Persona de Mediana Edad , Silicosis/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
It has been proposed that the biological clock underlying the limited division potential of eukaryotic cells is telomere length. We assayed telomerase activity in single cells of the hematopoietic and immune systems. We examined hematopoietic stem cells at four stages of differentiation, lineage-committed progenitors, and mature myeloid and lymphoid cells. The frequency of telomerase-expressing cells within each population was proportional to the frequency of cells thought to have self-renewal potential. Among bone marrow hematopoietic stem cells, 70% exhibited detectable telomerase activity. The telomerase-expressing somatic cells observed in this study are not thought to be immortal, and expression was not correlated with cell cycle distribution or differentiation state. This study demonstrates that the developmental characteristic most consistently associated with telomerase expression is self-renewal potential.
Asunto(s)
Células Madre Hematopoyéticas/enzimología , Telomerasa/metabolismo , Animales , Diferenciación Celular , Células Madre Hematopoyéticas/fisiología , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: The British Thoracic Society report on the diagnosis and treatment of the sleep apnoea/hypopnoea syndrome (SAHS) suggests that, if the pulse oximetry baseline oxygen saturation is above 90%, then 15 4% oxygen desaturation/hour in bed will diagnose SAHS requiring treatment. The diagnostic outcome of applying these guidelines has been studied. METHODS: One hundred patients referred to a district general hospital sleep clinic were recruited. After initial clinical assessment, overnight pulse oximetry measurements were performed, followed by full polysomnography at the regional laboratory. RESULTS: Sixty nine patients underwent both pulse oximetry and polysomnography. All 10 patients with more than 15 4% desaturations/hour on pulse oximetry had SAHS confirmed on polysomnography (specificity = 100%). Twenty two patients with SAHS were misdiagnosed using pulse oximetry alone (sensitivity = 31%). These patients had low apnoea scores but high hypopnoea scores. CONCLUSIONS: The BTS pulse oximetry criteria are highly specific when positive (specificity = 100%), but may miss patients with the SAHS who have hypopnoeic episodes which cause arousal but not significant oxygen desaturation (sensitivity = 31%). It should be emphasised that pulse oximetry alone does not always give sufficient information to discriminate between those patients with or without SAHS. Patients with "negative" pulse oximetry and symptoms of SAHS should undergo polysomnography or multi-channel recording.
Asunto(s)
Oximetría , Polisomnografía , Síndromes de la Apnea del Sueño/diagnóstico , Reacciones Falso Negativas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Guías de Práctica Clínica como Asunto , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
Telomere shortening and telomerase activation in human somatic cells have been implicated in cell immortalization and cellular senescence. To further study the role of telomerase in immortalization, we assayed telomere length and telomerase activity in primary mouse fibroblasts, in spontaneously immortalized cell clones, and in mouse tissues. In the primary cell cultures, telomere length decreased with increased cell doublings and telomerase activity was not detected. In contrast, in spontaneously immortalized clones, telomeres were maintained at a stable length and telomerase activity was present. To determine if telomere shortening occurs in vivo, we assayed for telomerase and telomere length in tissues from mice of different ages. Telomere length was similar among different tissues within a newborn mouse, whereas telomere length differed between tissues in an adult mouse. These findings suggest that there is tissue-specific regulation of mouse telomerase during development and aging in vivo. In contrast to human tissues, most mouse tissues had active telomerase. The presence of telomerase in these tissues may reflect the ease of immortalization of primary mouse cells relative to human cells in culture.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Piel/citología , Piel/enzimología , Telómero , Animales , Animales Recién Nacidos , Encéfalo/enzimología , División Celular , Células Cultivadas , Senescencia Celular , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , Cinética , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Muridae , Especificidad de Órganos , Especificidad de la Especie , Bazo/enzimología , Testículo/enzimologíaRESUMEN
Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Neoplasias/enzimología , Secuencia de Bases , División Celular , Línea Celular , Línea Celular Transformada/enzimología , Activación Enzimática , Represión Enzimática , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Ovario/enzimología , Reacción en Cadena de la Polimerasa , Testículo/enzimología , Células Tumorales CultivadasRESUMEN
Although expression of the haptoglobin (HP) as an acute phase reactant is evolutionarily conserved among mammals, there are differences among species with regard to the hormones required for stimulation. Using primary hepatocyte cultures, we show that in Mus caroli, as in rat, IL-1 and IL-6 are stimulatory, whereas in M. domesticus, as in humans, IL-1 response is diminished. In vivo, an acute inflammatory process increases hepatic HP expression in both mouse species up to 30-fold but minimally affects the low level HP expression in the lung. To define the species-specific differences in regulation, we isolated the hormone-responsive elements of the HP gene from the Mus species, M. domesticus, M. caroli, and M. saxicola. Functional studies in transfected hepatoma cells revealed an exceptionally strong dexamethasone response for all three murine HP gene elements. The IL-6 response was less prominent than in rat or human. A modest response to IL-1 was observed in M. caroli and M. saxicola. A mouse-specific insertion of a polypurine sequence led to a binding site for the PEA3 transcription factor in the HP gene promoter of M. domesticus and M. saxicola, but not M. caroli. The specific regulatory effects of glucocorticoid receptor, C/EBP beta, and Ets proteins were documented by co-transfection.
Asunto(s)
Secuencia Conservada , Regulación de la Expresión Génica/efectos de los fármacos , Haptoglobinas/genética , Hígado/metabolismo , Ratones/genética , Muridae/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Evolución Biológica , Carcinoma Hepatocelular , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Exones , Haptoglobinas/biosíntesis , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Interleucina-1/farmacología , Interleucina-6/farmacología , Lipopolisacáridos/toxicidad , Hígado/efectos de los fármacos , Neoplasias Hepáticas , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Datos de Secuencia Molecular , Familia de Multigenes , Ratas , Receptores de Glucocorticoides/metabolismo , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Transfección , Células Tumorales Cultivadas , Trementina/toxicidadAsunto(s)
Transformación Celular Neoplásica , Neoplasias/etiología , Telomerasa/metabolismo , Antineoplásicos/uso terapéutico , Senescencia Celular , Replicación del ADN , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Masculino , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Fenotipo , Telomerasa/antagonistas & inhibidoresRESUMEN
Telomerase activity was identified in extracts from several different mouse cell lines. Addition of telomeric TTAGGG repeats was specific to telomeric oligonucleotide primers and sensitive to pretreatment with RNase A. In contrast to the hundreds of repeats synthesized by the human and Tetrahymena telomerase enzymes in vitro, mouse telomerase synthesized only one or two TTAGGG repeats onto telomeric primers. The products observed after elongation of primers with circularly permuted (TTAGGG)3 sequences and after chain termination with ddATP or ddTTP indicated that mouse telomerase pauses after the addition of the first dG residue in the sequence TTAGGG. The short length of the products synthesized by mouse telomerase was not due to a diffusible inhibitor in the mouse extract, because the human telomerase continued to synthesize long products when mixed with mouse fractions. Primer challenge experiments showed that the human enzyme synthesized long TTAGGG repeats processively in vitro, whereas the mouse telomerase appeared to be much less processive. The identification of short telomerase reaction products in mouse extracts suggests that extracts from other organisms may also generate only short products. This knowledge may aid in the identification of telomerase activity in organisms where activity has not yet been detected.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Animales , Secuencia de Bases , Línea Celular , ADN Nucleotidilexotransferasa/aislamiento & purificación , Humanos , Ratones , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Especificidad de la Especie , Especificidad por Sustrato , Tetrahymena/enzimología , Células Tumorales CultivadasRESUMEN
Independent of de novo protein synthesis, interleukin-1, interleukin-6, and dexamethasone caused immediate stimulation of transcriptional activity of most major acute phase plasma protein genes in the rat hepatoma H-35 cells. However, activation of alpha 2-macroglobulin and alpha 1-acid glycoprotein genes were delayed by 2-4 h and required ongoing protein synthesis. The hormones also increased transiently the transcription of the junB gene and the amounts of JunB, C/EBP, and C/EBP-like mRNA. To identify whether JunB and C/EBP have the ability to control both the early and late acute phase reactants, expression vectors for mouse C/EBP and JunB together with reporter gene constructs containing recognized hormone-specific regulatory elements were introduced into hepatoma cells. C/EBP displayed prominent transactivation activity with the interleukin-1 and glucocorticoid regulatory elements of alpha 1-acid glycoprotein, the interleukin-1 regulatory element of haptoglobin gene, and the interleukin-6 regulatory element of beta-fibrinogen. The interleukin-6 regulatory elements of the first two genes and the glucocorticoid response element of the third gene were not affected by C/EBP. These data suggest that normal hormone activation of these three acute phase reactant genes might involve, in part, C/EBP-related factors which have a broad range of specificity. H-35 cells stably transformed with a mouse C/EBP expression vector showed an elevated basal level as well as cytokine inducible expression of some but not all acute phase reactants. Cotransfected JunB resulted in reduced activity of cytokine-responsive constructs and in lower transactivation by C/EBP. JunB appears to function as a modulator of plasma protein expression during the course of acute phase response.
Asunto(s)
Proteínas de Fase Aguda/genética , Proteínas Sanguíneas/genética , Citocinas/metabolismo , Glucocorticoides/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción , Transcripción Genética , Animales , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular Transformada , Cloranfenicol O-Acetiltransferasa/genética , Cicloheximida/farmacología , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Plásmidos , Ratas , TransfecciónRESUMEN
A single dose, single point method of predicting patients' oral maintenance theophylline dosage has been compared with a noninvasive method. Twenty patients with obstructive lung disease received an oral dose (6 mg kg-1) of micro-crystalline theophylline. The plasma theophylline concentration after 8-10 h was then used to calculate the optimum maintenance dose of sustained release aminophylline required to achieve steady state concentrations between 55 and 110 mumols l-1. The mean steady state plasma theophylline concentration for this dosage schedule was also predicted by a method using population average pharmacokinetic parameters (assumed clearance method). These predictions were then compared with observed concentration-time profiles at steady state. The mean difference between the observed values and those predicted from a morning test dose was -0.11 mumol l-1 (95% CI -7.0 to +7.2). A larger difference (-7.4 mumol l-1 95% CI -18.2 to +3.4) was found for the assumed clearance method. Since the confidence intervals contain zero, these differences are not significantly different from zero at the 5% level, although the morning test dose method allowed prediction of the whole concentration-time profile and was more precise. An evening test dose was also used in the study, but the mean difference between the observed values and those predicted from this method was larger at -24.8 mumol l-1 (95% CI -32.89 to -17.21) and was significantly different from zero. This study indicates that a morning test dose followed by a single blood sample can be used to establish maintenance theophylline therapy quickly and safely in selected patients.