RESUMEN
OBJECTIVE: The aims of this study were to develop a nomogram of umbilical cord diameter (UCD) for pathologic examination of the placenta, to identify the umbilical cord components responsible for variations in UCD, and to examine the relationship between UCD and other placental pathologic features and perinatal outcome. STUDY DESIGN: We prospectively collected 497 umbilical cords between 18 and 41 weeks' gestation over a 1-year period. Fresh-tissue UCD were grouped according to gestational age and compared to sonographic and histological measurements. Associations between UCD percentile and placental pathologic findings or obstetrical outcomes were examined. RESULTS: Mean UCD increased with gestational age until a plateau at 1.0 cm in the third trimester, a value that was 0.56 cm less than sonographic measurements prior to delivery and 0.17 cm greater than UCD measured histologically. Umbilical cord components varied with UCD percentile, with umbilical vessel area increased in thick cords (p < 0.001) and Wharton's jelly area reduced in thin cords (p = 0.002). Thin umbilical cords were associated with at least one pathologic histological placental finding (p = 0.02), low placental weight (p < 0.001), single umbilical artery (p = 0.02), marginal cord insertion (p = 0.01), and low infant birth weight (p < 0.001). CONCLUSIONS: This study provides reference curves for post-delivery UCD from 18 to 41 weeks' gestation for use by perinatal pathologists. We show that increased UCD is a function of increased umbilical blood vessel volume and decreased UCD is a function of decreased Wharton's jelly volume. UCD shows a strong association with placental and infant birth weight.
Asunto(s)
Peso al Nacer/fisiología , Enfermedades Placentarias/patología , Cordón Umbilical/anatomía & histología , Cordón Umbilical/patología , Estudios de Cohortes , Femenino , Edad Gestacional , Gráficos de Crecimiento , Humanos , Recién Nacido , Tamaño de los Órganos , Enfermedades Placentarias/etiología , Embarazo , Resultado del Embarazo , Pronóstico , Cordón Umbilical/crecimiento & desarrollo , Gelatina de Wharton/crecimiento & desarrollo , Gelatina de Wharton/patologíaRESUMEN
OBJECTIVE: The sonographic appearance of the placenta is normally homogenous throughout the second trimester. A variety of abnormalities in placental texture have been described, some of which may be pathologic and associated with adverse clinical outcomes. We characterized the pathologic basis of one lesion termed echogenic cystic lesions (ECLs) that may be a prognostic marker in intrauterine growth restriction (IUGR). STUDY DESIGN: We retrospectively correlated placental pathology in 50 pregnancies that had a total of 84 ECLs documented by ultrasound prior to delivery. Six additional women with placental ECLs prospectively underwent immediate post-delivery ultrasound-guided wire localization of 9 lesions followed by placental pathology. Obstetric outcome data were recorded. RESULTS: Severe pre-eclampsia (20%) and extreme IUGR (18%) were common outcomes. Of 93 ECLs identified, 46 (49%) gross lesions were found by placental pathology. Inter-villous thrombosis was the most significant lesion found (30/46, 65%) compared to all other lesions (35%; Z-Test, p = 0.007). Ultrasound guidance identified 8/9 (89%) lesions of which 6/8 (67%) were inter-villous thrombosis. Associated lesions (infarction, 36%; advanced villous maturation, 27%) and small placental weight (<10th centile, 38%) were present in 50%, but did not increase the risk of adverse perinatal outcome. CONCLUSIONS: ECLs are most commonly due to inter-villous thrombosis. The adverse clinical outcomes may be mediated by associated lesions not readily detectable by ultrasound. Ultrasound-guided wire localization is a promising research tool for future large-scale cohort studies needed to define the clinical utility of placental ultrasound findings.
Asunto(s)
Placenta/diagnóstico por imagen , Trombosis/diagnóstico por imagen , Adolescente , Adulto , Quistes/diagnóstico por imagen , Quistes/patología , Femenino , Humanos , Persona de Mediana Edad , Placenta/patología , Embarazo , Estudios Retrospectivos , Trombosis/patología , Ultrasonografía Prenatal , Adulto JovenRESUMEN
BACKGROUND: We have recently described two distinct pathways of intrauterine prostaglandin (PG) synthesis: a cortisol-dependent/estradiol-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an estradiol-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2(2alpha). We hypothesized that the differential effects of cortisol and estradiol on intrauterine PGH synthase-II (PGHS-II) expression and PG production may be because of the tissue specific expression of the glucocorticoid and estradiol receptors (GR and ER, respectively) within the intrauterine tissues. In addition, we suggest that these two pathways of PG production are linked through the expression of P450(C17hydroxylase) (P450(C17)) and subsequent increase in placental estradiol synthesis. METHODS: To test the hypotheses, we infused singleton, chronically catheterized fetal sheep beginning at day 125 of gestation (term 147 to 150 days) with (1) cortisol (0.45 mg/mL; n = 5); (2) cortisol and 4-hydroxyandrostenedione, a P450(aromatase) inhibitor (4-OHA: 1.44 mg/h; n = 5); (3) saline (n = 5); or (4) saline and 4-OHA (n = 5). PGHS-II, ER alpha, ER beta, and GR alpha were localized using immunohistochemistry. ER alpha, ER beta, P450(C17), and GR alpha protein expressions were determined by Western blot analysis. Data were analyzed by analysis of variance (ANOVA) (P < or =.05). RESULTS: Fetal cortisol infusion in the presence or absence of a rise in placental estrogen synthesis increased placental expression of GR alpha; both PGHS-II and GR alpha localized to the uninucleate trophoblast cells of the placentome and were excluded from the maternal stroma and binucleate cells. Both forms of ER were excluded from the trophoblast tissue of the placentome. ER alpha, ER beta, and PGHS-II showed a similar pattern of distribution within the luminal epithelium of the endometrium; there were no alterations in the level of the ER in the presence of cortisol +/- 4-OHA. Placental P450(C17) protein expression was increased in the presence of a rise in fetal cortisol independent of changes in placental estrogen synthesis. CONCLUSIONS: We concluded that the differential effects of cortisol and estradiol on intrauterine PGHS-II expression and PG production may be due to the tissue-specific expression of the GR and ER within the intrauterine tissues. Glucocorticoid effects on trophoblast PG production may be mediated in a positive feed-forward manner. We further suggest that either cortisol or a cortisol-stimulated intermediate, like PGE2, increased P450(C17) expression, leading to a rise in placental estradiol synthesis and triggering maternal intrauterine tissue PG production.
Asunto(s)
Ciclooxigenasa 2/biosíntesis , Preñez/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Dinoprost/biosíntesis , Dinoprostona/biosíntesis , Endometrio/metabolismo , Estradiol/metabolismo , Femenino , Embarazo , Ovinos , Trofoblastos/metabolismoRESUMEN
In the late-gestation sheep, increased fetal plasma cortisol concentration and placental oestradiol (E(2)) output contribute to fetal organ maturation, in addition to the onset of parturition. Both cortisol and E(2) are believed to regulate the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which interconverts bioactive 11-hydroxy glucocorticoids and their inactive 11-keto metabolites. 11beta-HSD1, abundantly expressed in fetal liver, operates primarily as a reductase enzyme to produce bioactive cortisol and thus regulates local hepatic glucocorticoid concentrations. Cortisol acts through the glucocorticoid receptor (GR) present in the liver. In this study, we examined the effects of cortisol and E(2) on hepatic 11beta-HSD1 and GR in the liver of chronically catheterized sheep fetuses treated with saline (n=5), cortisol (1.35 mg/h; n=5), saline+4-hydroxyandrostendione, a P450 aromatase inhibitor (4-OHA; 1.44 mg/h; n=5), or cortisol+4-OHA (n=5). Cortisol infusion resulted in increased plasma concentrations of fetal cortisol and E(2); concurrent administration of 4-OHA attenuated the increase in plasma E(2) concentrations. Using immunohistochemistry, we showed that fetal hepatocytes expressed both 11beta-HSD1 and GR proteins. Cortisol treatment increased GR in both cytosol and nuclei of hepatocytes; concurrent administration of 4-OHA was associated with distinct nuclear GR staining. Western blot revealed that cortisol, in the absence of increased E(2) concentrations, significantly increased concentrations of 11beta-HSD1 (34 kDa) and GR (95 kDa) proteins. 11beta-HSD1 enzyme activity was measured in the liver microsomal fraction in the presence of [(3)H]cortisone (10(-)(6) M) or [(3)H]cortisol (10(-)(6) M) and NADPH (reductase activity) or NADP(+) (dehydrogenase activity) respectively. 11beta-HSD1 reductase activity was significantly greater in the presence of cortisol. In summary, we found that, in sheep during late gestation, cortisol increased both 11beta-HSD1 and GR in the fetal liver, and these effects were accentuated in the absence of increased E(2).
Asunto(s)
Androstenodiona/análogos & derivados , Estradiol/farmacología , Hidrocortisona/farmacología , Hidroxiesteroide Deshidrogenasas/metabolismo , Hígado/embriología , Hígado/metabolismo , Receptores de Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasas , Análisis de Varianza , Androstenodiona/farmacología , Animales , Inhibidores de la Aromatasa , Western Blotting/métodos , Inhibidores Enzimáticos/farmacología , Femenino , Edad Gestacional , Hidroxiesteroide Deshidrogenasas/análisis , Inmunohistoquímica/métodos , Hígado/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Embarazo , Receptores de Glucocorticoides/análisis , Ovinos , Estimulación QuímicaRESUMEN
Recent evidence suggests that ovine placental output of prostaglandin (PG) E2 rises through late gestation partly because of a direct effect of cortisol on PGH2 synthase 2 (PGHS-2) expression and activity within trophoblast tissue. Synthesis of PGE2 is also dependent, however, on PGE2 synthase (PGES), which converts PGH2 to PGE2. We hypothesized that PGES is expressed in the ovine placenta, and that, similar to PGHS-2, expression increases through gestation and is regulated positively by cortisol. Placental tissues from pregnant ewes in mid and late gestation, at term, and during early and active labor were analyzed to determine the gestational profile of PGES. The regulation of PGES expression was assessed in placental tissues from pregnant ewes in which intrafetal cortisol infusion was administered in late gestation, in the presence or absence of an aromatase inhibitor, to block the cortisol-stimulated rise in estradiol. Expression of PGES was analyzed by in situ hybridization, Western blot analysis, and immunohistochemistry. In the placentome, PGES localized to fetal trophoblast cells and endothelial cells in maternal blood vessels, consistent with its contribution to the rise in placental PGE2 output toward the onset of labor and with a role of PGE2 in the local regulation of uteroplacental blood flow, respectively. Expression of PGES mRNA and protein increased with gestation. However, there was no significant further change with labor or during cortisol infusion in the presence or absence of a rise in fetal plasma estradiol, in contrast to reported changes in PGHS-2. These results suggest that PGES is not coregulated with PGHS-2 in the sheep placenta at term. The progressive increase in PGES, however, likely contributes to the rise in circulating PGE2 in the fetus in late pregnancy.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Oxidorreductasas Intramoleculares/genética , Placenta/enzimología , Animales , Western Blotting , Dinoprostona/sangre , Estradiol/sangre , Femenino , Sangre Fetal/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Edad Gestacional , Hidrocortisona/farmacología , Hibridación in Situ , Oxidorreductasas Intramoleculares/análisis , Trabajo de Parto , Embarazo , Prostaglandina-E Sintasas , ARN Mensajero/análisis , OvinosRESUMEN
We hypothesized that in the late-gestation sheep fetus there is an interaction between the prepartum rise in cortisol and the increase in placental estradiol production that allows expression of key components of the fetal hypothalamic-pituitary-adrenal (HPA) axis. Therefore, the goal of this study was to investigate the effects of cortisol on the fetal HPA axis in the presence and absence of increased placental estradiol production. We obtained fetal plasma samples and pituitary tissue from animals that had received an infusion of either cortisol, cortisol and 4-hydroxyandrostenedione (40HA, an aromatase inhibitor), saline, or saline + 40HA controls. Cortisol significantly decreased plasma adrenocorticotropic hormone concentrations, and in the presence of 40HA reduced pituitary proopiomelanocortin (POMC) mRNA levels in the pars distalis. There was no effect of any treatment on the expression of the key POMC processing enzymes, prohormone convertase-1 or -2 in the fetal pituitary. Conversely, levels of glucocorticoid receptor (GR) mRNA in the pituitary were increased with cortisol treatment in the absence of increased estradiol. We suggest that in the late-gestation sheep fetus, cortisol and estradiol have opposite effects on pituitary POMC and GR mRNA expression, and interact to regulate these key components of the fetal HPA axis.
Asunto(s)
Estradiol/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hidrocortisona/farmacología , Hipófisis/metabolismo , Proopiomelanocortina/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Glucocorticoides/biosíntesis , Subtilisinas/biosíntesis , Hormona Adrenocorticotrópica/sangre , Animales , Estradiol/sangre , Femenino , Furina , Hidrocortisona/sangre , Hibridación in Situ , Isoenzimas/biosíntesis , Hipófisis/enzimología , Embarazo , OvinosRESUMEN
Birth in many animal species and in humans is associated with activation of hypothalamic-pituitary-adrenal function in the fetus and the increased influence of glucocorticoids on trophoblast cells of the placenta and fetal membranes. We suggest that in ovine pregnancy glucocorticoids directly increase fetal placental prostaglandin production, and indirectly increase prostaglandin production by maternal uterine tissues through the stimulation of placental estradiol synthesis. The events of ovine parturition are compared with those of human parturition. In the latter, we suggest similar direct effects of glucocorticoids on prostaglandin synthesis and metabolism in fetal membranes and similar indirect effects mediated by glucocorticoid-stimulated increases in intrauterine corticotropin-releasing hormone expression.
Asunto(s)
Glándulas Suprarrenales/embriología , Glucocorticoides/fisiología , Hipotálamo/embriología , Trabajo de Parto/fisiología , Hipófisis/embriología , Prostaglandinas/biosíntesis , Glándulas Suprarrenales/fisiología , Animales , Hormona Liberadora de Corticotropina/fisiología , Femenino , Glucocorticoides/farmacología , Humanos , Hipotálamo/fisiología , Hipófisis/fisiología , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Prostaglandinas/metabolismo , Ovinos , Útero/metabolismoRESUMEN
A current hypothesis of ovine parturition proposes that fetal adrenal cortisol induces placental E2 production, which, in turn, triggers intrauterine PG production. However, recent evidence suggests that cortisol may directly increase PG production in trophoblast-derived tissues. To separate cortisol-dependent and estrogen-dependent PG production in sheep intrauterine tissues, we infused singleton, chronically catheterized fetuses beginning on day 125 of gestation (term, 147-150 days) with 1) cortisol (1.35 mg/h; n = 5); 2) cortisol and 4-hydroxyandrostendione, a P450aromatase inhibitor (4-OHA: 1.44 mg/h; n = 5); 3) saline (n = 5); or 4) saline and 4-OHA (n = 5). Fetal and maternal arterial blood samples were collected at 12-h intervals starting 24 h before infusion and continuing during treatment for 80 h or until active labor. Uterine contractility was measured by electromyogram recording of myometrial activity. Plasma E2, progesterone (P4), PGE2, and 13,14-dihydro- 15-keto-PGF2alpha were quantified by RIA. PGHS-II messenger RNA (mRNA) and protein expression were determined by in situ hybridization and Western blot analysis, respectively. Data were analyzed by ANOVA (P < or = 0.05). Labor-type uterine contractions were present after 68 h of cortisol infusion and had increased significantly by 80 h. Labor-type uterine contractions were induced after 68 h of cortisol plus 4-OHA infusion, but the contraction frequency remained less than that in the cortisol-treated animals. Fetal cortisol infusion increased fetal and maternal plasma E2 concentrations and decreased the maternal plasma P4 concentration significantly; concurrent 4-OHA infusion attenuated the increase in fetal and maternal plasma E2, but not the decrease in maternal plasma P4. The fetal plasma PGE2 concentration increased after both cortisol and cortisol plus 4-OHA infusion. The maternal plasma 13,14-dihydro-15-keto-PGF2alpha concentration rose after fetal cortisol infusion, but not after cortisol plus 4-OHA infusion. Placental trophoblast PGHS-II mRNA and protein expression were increased significantly after both cortisol and cortisol plus 4-OHA infusion. Endometrial PGHS-II mRNA and protein expression increased after cortisol infusion, but not after cortisol plus 4-OHA infusion. Plasma steroid and PG concentrations, uterine activity pattern, and intrauterine PGHS-II expression were not altered in either control group. We conclude that these data suggest distinct pathways of intrauterine PG synthesis: a cortisol-dependent/E2-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an E2-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2alpha and appears necessary for uterine activity and parturition.
Asunto(s)
Estrógenos/fisiología , Trabajo de Parto/metabolismo , Prostaglandinas/biosíntesis , Animales , Ciclooxigenasa 2 , Femenino , Sangre Fetal , Hormonas/sangre , Isoenzimas/genética , Isoenzimas/metabolismo , Trabajo de Parto/sangre , Embarazo , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/sangre , ARN Mensajero/metabolismo , Ovinos , Contracción Uterina/fisiologíaRESUMEN
The amnion, a single layer of epithelial cells (EC) overlying layers of mesenchymal cells (MC) has been identified as a source of intrauterine prostaglandins (PG). The objectives of the present study were: (1) to establish a technique for the isolation and culture of pure amnion EC and MC preparations, (2) to characterize the cellular expression of PGHS-II and PGHS activity within these separated amnion cells and (3) to characterize the pattern of glucocorticoid stimulation of these separated amnion cells. Term gestation human amnion was collected after elective caesarean section or vaginal delivery. A trypsin digestion was used to isolate EC and a mechanical digestion and collagenase dispersion was used to isolate MC. Following 48 or 96 h in culture, cells were incubated for 24 h in the presence or absence of 1 microm arachidonic acid and treated with cortisol (F: 10-1000 nm) or 1 microm dexamethasone (DEX). Cell types were identified by immunohistochemistry (IHC). Immunoreactive PGHS-II (ir-PGHS-II) and glucocorticoid receptor (ir-GR) were localized by IHC. PGHS activity was measured as PGE(2)output determined by radioimmunoassay. Mean PGE(2)production by MC at 72 h was 22-fold greater (P<0.05) and at 120 h was 32-fold greater (P<0.03) than PGE(2)output by EC. Administration of arachidonic acid stimulated a 5.0-fold increase in PGE(2)output (P<0.0002) by EC after 72 h and a 3.6-fold increase (P<0.05) after 120 h but did not alter MC PGE(2)output. Despite exogenous substrate, EC PGE(2)output remained significantly less than PGE(2)output by MC. There was no difference in PG production by EC and MC with the onset of labour. Ir-GR expression was found in both EC and MC. F and/or DEX with and without arachidonic acid (AA) stimulated PGE(2)output by EC. Only DEX and not F increased PGE(2)output by MC. These data suggest that relatively pure EC and MC preparations can be established from amnion. PG output and its regulation appears to differ within these two amnion cell types, dependent upon (1) substrate availability and (2) the regulation of PGHS activity.
Asunto(s)
Amnios/metabolismo , Células Epiteliales/enzimología , Glucocorticoides/farmacología , Isoenzimas/biosíntesis , Mesodermo/enzimología , Peroxidasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandinas/biosíntesis , Adulto , Amnios/citología , Ácido Araquidónico/farmacología , Técnicas de Cultivo de Célula/métodos , Separación Celular , Ciclooxigenasa 2 , Dexametasona/farmacología , Dinoprostona/biosíntesis , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Hidrocortisona/farmacología , Técnicas para Inmunoenzimas , Proteínas de la Membrana , Mesodermo/citología , Mesodermo/efectos de los fármacos , Embarazo , Prostaglandina H2 , Prostaglandinas H/biosíntesis , Receptores de Glucocorticoides/metabolismoRESUMEN
The synthesis of nuclear matrix components from human diploid fibroblasts of different in vitro ages was analyzed. Radiolabeled nuclear matrices were prepared from human diploid fibroblasts at various stages of the cell cycle, and their components were separated by two dimensional electrophoresis. The same general electrophoretic pattern was observed at all cell cycle points analyzed, regardless of in vitro age. However, several of the more than 150 peptides that were observed exhibited some cell cycle or age-related variation in radiolabeling. Ten of these were chosen for further analysis. One peptide, with an approximate molecular weight of 47 kDa and pI of 6.8 exhibited the most significant cell cycle and age-related alterations. In matrices from younger cells, incorporation into this peptide was very low in GO but increased as these cells moved through the cell cycle, with maximum incorporation occurring in S phase. As cells neared the end of their in vitro lifespan, labeling of this peptide was elevated at all stages of the cell cycle. Since many of the functional alterations observed in senescent human diploid fibroblasts are nuclear-matrix-associated activities, these results suggest that the inappropriate expression of nuclear matrix components contribute to the functional changes which characterize in vitro senescence.
Asunto(s)
Senescencia Celular/fisiología , Matriz Nuclear/metabolismo , Antígenos Nucleares , Ciclo Celular , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/metabolismo , Peso Molecular , Matriz Nuclear/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Péptidos/química , Péptidos/metabolismoRESUMEN
The purpose of the study was to examine the influence of culture media on prostaglandin (PG) production by amnion cells and their response to phospholipase A2. Cells were dispersed from term tissue obtained by elective C-section; the PGE2 output was studied during the first 24 h of culture. The basal PGE2 production from cells cultured in Media 199 (M199) supplemented by 10% horse serum (HS) was significantly greater than that of cells cultured in F12:DME supplemented by 10% fetal calf serum (FCS). Furthermore, phospholipase A2 stimulated PGE2 production in cells cultured with M199 + HS but had no influence on PGE2 output by cells cultured with F12:DME + FCS. This effect was dependent on the presence of HS. The factor(s) in HS responsible was not removed by heating at 56 degrees C for 3 min, treatment with dextran coated charcoal or by ultrafiltration through 10,000 MW filters. Thus, culture media alters the in vitro production of PG and response to phospholipase A2 by amnion cells.
Asunto(s)
Amnios/metabolismo , Medios de Cultivo , Fosfolipasas A/farmacología , Prostaglandinas/metabolismo , Amnios/citología , Células Cultivadas , Técnicas Citológicas , Femenino , Humanos , Fosfolipasas A2 , EmbarazoRESUMEN
Nucleosome spacing (DNA repeat length) was determined in human diploid fibroblast-like cells (HDF) of different in vitro ages following the electrophoretic separation of micrococcal nuclease digestion products. The results indicate that a heterogeneity of DNA repeat lengths is present in HDF of all in vitro ages. In older cells the organization of part of the DNA is conserved, but a greater proportion of shorter repeats is evident. The shorter repeat lengths are not due to nucleosome sliding, but result from the presence of shorter linker regions which are reduced by as much as 25% in part of the chromatin of high PDL cells.
Asunto(s)
Supervivencia Celular , ADN/fisiología , Fibroblastos/fisiología , Nucleosomas/fisiología , Secuencias Repetitivas de Ácidos Nucleicos , División Celular , Células Cultivadas , ADN/análisis , Fibroblastos/análisis , Fibroblastos/citología , Humanos , Recién Nacido , Masculino , Nucleasa Microcócica , Nucleosomas/análisis , Tamaño de la PartículaRESUMEN
Nuclei from confluent and mitotically arrested populations of human diploid fibroblast-like cells were subjected to digestion by micrococcal nuclease and deoxyribonuclease (DNase I) following the removal of various histone components by salt extraction. There was no age or culture state variation in the susceptibility of DNA to micrococcal nuclease digestion. There was an age related inhibition of DNA digestion by DNase I in nuclei from older confluent cells before and after the removal of H1 histone but not after the removal of core particle histones. This inhibition was not detected in older arrested populations. These results indicate that an age-related masking by nucleosome core histones may limit the accessibility of DNA to enzymatic activities in older confluent cells. Since this inhibition was absent in older arrested populations, the importance of limited DNA accessibility as a primary cause of cellular senescence is questionable.
Asunto(s)
ADN/metabolismo , Endodesoxirribonucleasas/farmacología , Histonas/metabolismo , Nucleasa Microcócica/farmacología , Supervivencia Celular , Células Cultivadas , Desoxirribonucleasa I , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , PielRESUMEN
The effect of low dose UV irradiation on the reinitiation of proliferative activity and on the life span of human diploid fibroblast-like cells is described. Cells were exposed to UV at confluence or after maintenance in an arrested state. Cell division was stimulated immediately after UV irradiation or after an additional post-UV incubation period. Arrested populations of all in vitro ages exhibited a greater sensitivity to UV and the reinitiation of proliferation was enhanced by post-UV incubation before stimulation. Ultraviolet light had no effect on life span regardless of in vitro cell age, culture state at the time of exposure, or the presence of a postirradiation period of arrest.
Asunto(s)
División Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Fibroblastos/efectos de la radiación , Rayos Ultravioleta , Células Cultivadas , Diploidia , Humanos , Factores de TiempoRESUMEN
Excision repair after ultraviolet (UV) irradiation was estimated in human diploid fibroblast-like cells (HDF) by treating DNA with a crude extract from Micrococcus luteus that contained pyrimidine dimer-specific endonucleases. The assays were done before and after the cells had been arrested in an essentially nonmitotic state. When low and high population doubling level (PDL) cells were assayed under these conditions, no age-related difference in the number of UV-induced endonuclease sensitive sites was observed, but the removal of these sites was more rapid in high PDL arrested cells. There was a difference between the calculated number average molecular weights (Mn) of DNA from unirradiated low and high PDL arrested cells. The lower Mn of the DNA from high PDL cells indicated that other enzymes present in the M. luteus extract were acting upon non-UV-induced DNA distortions and that these were present to a greater extent in the chromatin associated regions of older cells. These results support the hypothesis that DNA damage accumulates as HDF progress through their in vitro life span.
Asunto(s)
ADN/efectos de la radiación , Fibroblastos/metabolismo , División Celular , Células Cultivadas , ADN/biosíntesis , Reparación del ADN , Desoxirribonucleasa (Dímero de Pirimidina) , Desoxirribonucleasas/farmacología , Diploidia , Endonucleasas/farmacología , Humanos , Masculino , Micrococcus/enzimología , Peso Molecular , Rayos UltravioletaRESUMEN
Unscheduled DNA synthesis, used as a measure of excision repair following exposure to ultra-violet irradiation, was determined in confluent and arrested human diploid fibroblasts and correlated with in vitro age. Confluent cultures exhibited identical levels of unscheduled DNA synthesis at all in vitro ages. Cells arrested by lowering the serum concentration of the incubation medium exhibited similar levels of unscheduled DNA synthesis as did confluent cells during the first one-third of the cells' characteristic in vitro lifespan. During the last two-thirds of the lifespan, however, arrested populations exhibited a 30 to 50% increase in the amount of detectable DNA repair. This apparent increase in ability to perform unscheduled DNA synthesis was not time or dose dependent and could not be attributed to alterations in precursor pools. It was postulated that the increase may be correlated with changes in DNA structure.
Asunto(s)
ADN/biosíntesis , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Técnicas In Vitro , Mitosis , Factores de Tiempo , Rayos UltravioletaRESUMEN
Human diploid fibroblasts can be maintained in vitro in an arrested, essentially nonmitotic state for extended periods of time by reducing the serum concentration in the medium from 10 to 0.5%. Arrested cells can be induced to re-enter the proliferative state by subcultivation in medium containing 10% serum. Fine structure, acid phosphatase, cytochrome oxidase, and extracellular carbohydrates in arrested cells were examined and compared to cultures growing in 10% serum and to cells transferred to 10% serum after 21 days in 0.5% serum. Cells in 10% serum posessed a well-developed Golgi complex, extensive rough endoplasmic reticulum, mitochondria containing transverse cristae, and many free ribosomes in the cytoplasm. In arrested cells, Golgi complexes were rarely observed, the number of both free and membrane-bound ribosomes was reduced, the number of cristae per mitochondria was decreased and the amount of demonstrable cytochrome oxidase activity was diminished. There was an accumulation of intercellular carbohydrate components. After subcultivation with medium containing 10% serum, arrested cells regained the ultrastructural characteristics of cells continuously cultured at this serum level; however, the amount of intercellular carbohydrate remained elevated. These results indicate that distinct yet reversible changes occur in the subcellular morphology and organization of cells maintained in an essentially nonmitotic state. This arrested state may be a close approximation to the situation as it occurs in vivo in expanding cell populations.
Asunto(s)
Células Cultivadas/ultraestructura , Mitosis , Fosfatasa Ácida/análisis , Sangre , Carbohidratos/análisis , Membrana Celular/ultraestructura , Medios de Cultivo , Complejo IV de Transporte de Electrones/análisis , Retículo Endoplásmico/ultraestructura , Espacio Extracelular/análisis , Aparato de Golgi/ultraestructura , Humanos , Lisosomas/enzimología , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Ribosomas/ultraestructuraRESUMEN
Human diploid fibroblasts cultured in vitro weremonitored for amino acid uptake in preconfluent and confluent cultures under conditions amenable to growth and in confluent cultures arrested in an essentially nonmitotic state. Preconfluent and confluent cultures in growth medium showed similar uptake patterns for leucine and alpha-aminoisobutyrate; arrested cultures exhibited a reduced uptake of both amino acids. Kinetic measurements revealed a 4-fold reduction in apparent Vmax for alpha-aminoisobutyrate influx in arrested cultures. These results suggest that the culture conditions used in this study to produce restrictions in mitotic activity likewise influence amino acid accumulation.