RESUMEN
Study Objective: Cold Pressor Testing (CPT) is a known stimulus of the sympathetic nervous system (SNS). To better understand sympathetic contribution to coronary blood flow regulation in women with suspected ischemia and no obstructive coronary arteries (INOCA), we compared myocardial perfusion reserve during CPT stress cardiac magnetic resonance (CMR) imaging between women with suspected INOCA and reference subjects. Design: Prospective cohort. Setting: Academic hospital. Participants: 107 women with suspected INOCA and 21-age-matched reference women. Interventions: CPT stress CMR was performed with measurement of myocardial perfusion reserve index (MPRI), adjusted for rate pressure product (MPRIRPP). Invasive coronary function testing in a subset of INOCA women (n=42) evaluated for endothelial dysfunction in response to acetylcholine, including impaired coronary diameter response ≤0% and coronary blood flow response (ΔCBF) <50%. Main Outcome Measure: MPRIRPP. Results: Compared to reference women, the INOCA group demonstrated higher resting RPP (p=0.005) and CPT MPRIRPP (1.09±0.36 vs 0.83±0.18, p=0.002). Furthermore, INOCA women with impaired ΔCBF (n=23) had higher CPT MPRIRPP (p=0.044) compared to reference women despite lower left ventricular ejection fraction (64±7 % vs 69±2 %, p=0.005) and mass-to-volume ratio (0.79±0.15 vs 0.62±0.09, p<0.0001). These differences in CPT MPRIRPP did not persist after adjusting for age, body mass index, and history of hypertension. CPT MPRIRPP among INOCA women did not differ based on defined acetylcholine responses. Conclusions: Myocardial perfusion reserve to CPT stress is greater among women with INOCA compared to reference subjects. CPT induced a higher MPRIRPP also in women with coronary endothelial dysfunction, suggesting a greater contribution of the SNS to coronary flow than endothelial dysfunction. Further investigation in a larger cohort is needed.
RESUMEN
Hyperoxic rats treated with inosine during oxygen exposure have increased levels of active transforming growth factor (TGF)-beta in the bronchoalveolar lavage (BAL), yet alveolar epithelial type 2 cells (AEC2) isolated from these animals demonstrate less hyperoxia-induced DNA damage and increased expression of active Smad2. To determine whether TGF-beta1 signaling per se protected AEC2 against hyperoxic damage, freshly isolated AEC2 from hyperoxic rats were incubated with TGF-beta1 for 24 h and assayed for DNA damage by fluorescein-activated cell sorter analysis of TdT-mediated dUTP nick end labeling. TGF-beta1 was protective over a concentration range similar to that in BAL of inosine-treated hyperoxic animals (50-5,000 pg/ml). TGF-beta1 also augmented hyperoxia-induced DNA repair activity and cell migration, stimulated autocrine secretion of fibronectin, accelerated closure of a monolayer scratch wound, and restored hyperoxia-depleted VEGF secretion by AEC2 to normoxic levels. The TGF-beta receptor type I activin-like kinase-4, -5, and -7 inhibitor peptide SB-505124 abolished the protective effect of TGF-beta on hyperoxic DNA damage and increased TdT-mediated dUTP nick end labeling in normoxic cells. These data suggest that endogenous TGF-beta-mediated Smad signaling is required for AEC2 homeostasis in vitro, while exogenous TGF-beta1 treatment of hyperoxia-damaged AEC2 results in a cell that is equipped to survive, repair, migrate, secrete matrix, and induce new blood vessel formation more efficiently than AEC2 primed by hyperoxia alone.
Asunto(s)
Hiperoxia/fisiopatología , Alveolos Pulmonares/citología , Mucosa Respiratoria/citología , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/fisiología , Animales , Benzodioxoles/farmacología , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Supervivencia Celular , Imidazoles/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Alveolos Pulmonares/efectos de los fármacos , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Mucosa Respiratoria/efectos de los fármacosRESUMEN
Inosine, a naturally occurring purine with anti-inflammatory properties, was assessed as a possible modulator of hyperoxic damage to the pulmonary alveolar epithelium. Rats were treated with inosine, 200 mg/kg ip, twice daily during 48-h exposure to >90% oxygen. The alveolar epithelial type 2 cells (AEC2) were then isolated and cultured. AEC2 isolated from inosine-treated hyperoxic rats had less DNA damage and had increased antioxidant status compared with AEC2 from hyperoxic rats. Inosine treatment during hyperoxia also reduced the proportion of AEC2 in S and G2/M phases of the cell cycle and increased levels of the DNA repair enzyme 8-oxoguanine DNA glycosylase. Bronchoalveolar lavage (BAL) recovered from hyperoxic, inosine-treated rats contained threefold higher levels of active transforming growth factor-beta than BAL from rats exposed to hyperoxia alone, and Smad2 was activated in AEC2 isolated from these animals. ERK1/2 was activated both in freshly isolated and 24-h-cultured AEC2 by in vivo inosine treatment, whereas blockade of the MAPK pathway in vitro reduced the protective effect of in the vivo inosine treatment. Together, the data suggest that inosine treatment during hyperoxic exposure results in protective signaling mediated through pathways downstream of MEK. Thus inosine may deserve further evaluation for its potential to reduce hyperoxic damage to the pulmonary alveolar epithelium.
Asunto(s)
Citoprotección , Daño del ADN , Hiperoxia/fisiopatología , Inosina/farmacología , Sistema de Señalización de MAP Quinasas , Alveolos Pulmonares/fisiopatología , Animales , Líquido del Lavado Bronquioalveolar/química , Ciclo Celular , Células Cultivadas , Daño del ADN/efectos de los fármacos , ADN Glicosilasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonoides/farmacología , Glutatión/sangre , Hiperoxia/genética , Hiperoxia/metabolismo , Hiperoxia/patología , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Ratas , Ratas Sprague-Dawley , Proteína Smad2 , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Ácido Úrico/farmacologíaRESUMEN
The immortalized murine stromal cell line AFT024 has been reported to maintain human hematopoietic progenitors in an undifferentiated state in vitro. In the current studies the beige/nude/xid (bnx) mouse in vivo xenograft model was used to examine the engraftment and multilineage generative potential of human hematopoietic progenitors after 2-3 weeks growth on AFT024 stroma, in comparison to primary stromal monolayers derived from post-natal human bone marrow. Eight to 12 months after transplantation of human CD34+CD38- cells from umbilical cord blood, cultured on AFT024 vs human stroma for 2-3 weeks, the murine bone marrow was harvested and analyzed for the presence of human myeloid and lymphoid cells. The mean percent engraftment of total human hematopoietic cells in the murine marrow was significantly higher after co-cultivation on AFT024 than on human stroma. Human myeloid and lymphoid lineage cells were detected in all mice. However, engraftment of myeloid lineage cells (CD33+), B lymphoid (CD19+), and T lymphoid cells (CD4+and CD8+) were significantly higher after co-cultivation of the human cells on AFT024 than on human stroma, prior to transplantation. Interestingly, the length of time in culture did not significantly affect the engraftment of the myeloid and T lymphoid lineage progenitors, but the percentage of B lymphoid lineage engraftment decreased significantly between 2 and 3 weeks of co-cultivation on both types of stroma. Cells with a primitive phenotype (CD45+/CD34-/CD38- and CD45+/CD34-/lin-) and cells with the capacity to generate secondary human CFU after recovery from the bnx bone marrow were maintained at significantly higher levels during culture on AFT024 stroma than on human stroma. The current studies demonstrate that the AFT024 murine stromal cell line supports the ex vivo survival and maintenance of human hematopoietic progenitors that are capable of long-term multilineage reconstitution for 2-3 weeks ex vivo, to levels superior to those that can be obtained using human stromal cells.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Hematopoyesis/inmunología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD/sangre , Antígenos de Diferenciación , Linfocitos B/inmunología , Médula Ósea/inmunología , Células de la Médula Ósea , Células Cultivadas , Sangre Fetal/citología , Supervivencia de Injerto , Humanos , Inmunofenotipificación , Glicoproteínas de Membrana , Ratones , Ratones SCID , NAD+ Nucleosidasa , Células del Estroma/inmunología , Linfocitos T/inmunología , Trasplante HeterólogoRESUMEN
Murine studies implicate Ikaros proteins as regulators of hemopoiesis, particularly in the lymphoid lineages. High homology between murine and human Ikaros suggests that Ikaros expression in the two might be similar. However, initial human studies that focused on leukemia detected novel Ikaros transcripts in patient samples. Thus, novel Ikaros splice forms and DNA nonbinding isoforms were linked with malignancy. We undertook an extensive analysis of normal human Ikaros expression to determine whether novel mRNAs are expressed as proteins and the extent to which these splice variants are unique to leukemia. Here we show that both mRNA and protein for DNA nonbinding Ikaros isoforms and splice variants previously linked to leukemia are expressed in normal human cells. However, our studies identify a new Ikaros isoform not previously described in mouse or human. This isoform is the predominant Ikaros protein in normal human cells, but not in leukemia cell lines.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Factores de Transcripción/biosíntesis , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Sangre Fetal/citología , Sangre Fetal/inmunología , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/inmunología , Humanos , Factor de Transcripción Ikaros , Células Jurkat , Leucemia/inmunología , Leucemia/metabolismo , Mutagénesis Insercional/inmunología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Eliminación de Secuencia/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/inmunología , Células Tumorales CultivadasRESUMEN
Interleukin-7 (IL-7) is the major thymopoietic cytokine. Injections of IL-7 after murine bone marrow transplantation (BMT) correct defects in thymic differentiation, including thymic hypocellularity, abnormal differentiation of CD3- CD4- CD8- (triple-negative [TN]) thymocytes into CD4+ CD8+ (double-positive [DP]) cells, and antigen-specific mature T-lymphocyte proliferation. To determine whether IL-7 production is decreased in BMT recipients, BMT was performed with congenic murine donor-recipient strains and escalating doses of pre-BMT conditioning. Increasing doses of radiation resulted in decreased thymic cellularity and maturation from the TN to the DP stage. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that intrathymic production of IL-7 was significantly decreased in irradiated mice than in nonirradiated controls. Decline in IL-7 transcript levels was correlated with the dose of radiation administered. Analyses of the numbers of CD45- major histocompatibility complex class II+ thymic stromal cells suggested that the mechanism for the decreased IL-7 production was loss of IL-7-producing thymic stromal cells. Experiments indicated that pre-BMT conditioning with radiation led to decreased stromal production of IL-7 and consequent blocks in the maturation of thymocytes. They provided a mechanism for both the abnormal thymopoiesis observed after BMT and the previously observed beneficial effects of IL-7 administration in murine models. Impaired production of IL-7 by thymic stroma may be a general model for the clinically observed adverse effects of cytotoxic therapy on thymopoiesis.
Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Interleucina-7/biosíntesis , Linfocitos T/patología , Timo/efectos de la radiación , Acondicionamiento Pretrasplante/efectos adversos , Irradiación Corporal Total/efectos adversos , Animales , Animales Congénicos , Trasplante de Médula Ósea , Recuento de Células , Diferenciación Celular/efectos de la radiación , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Antígenos de Histocompatibilidad Clase II/análisis , Inmunofenotipificación , Interleucina-7/deficiencia , Interleucina-7/genética , Interleucina-7/fisiología , Ratones , Ratones Endogámicos C57BL , Tolerancia a Radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismo , Células del Estroma/efectos de la radiación , Timo/metabolismo , Timo/patologíaRESUMEN
The earliest stages of lymphoid commitment from human pluripotent hematopoietic stem cells have not been defined. A clonogenic subpopulation of CD34(+)CD38(-) cord blood cells were identified that expressed high levels of the CD7 antigen and possessed only lymphoid potential. CD34(+)CD38(-)CD7(+) (CD7(+)) cells uniformly coexpressed CD45RA and HLA-DR; c-kit and Thy-1 expression was absent to low. Clonal analysis demonstrated that single CD7(+) cells could generate B cells, natural killer cells, and dendritic cells but were devoid of myeloid or erythroid potential. In contrast, control CD34(+)CD38(-)CD7(-) (CD7(-)) cells generated both lymphoid and myelo-erythroid cells. The lymphoid potential (generation of lymphoid progeny in bulk and single cell cultures) of CD7(+) cells was equivalent to that of the pluripotent CD7(-) cells. RNA expression studies showed that CD7(+) cells expressed PU.1 and GATA-3, but did not express Pax-5, terminal deoxynucleotide transferase, or CD3epsilon. In contrast to the previously described murine common lymphoid progenitor, the alpha chain of the receptor for interleukin-7 was not detected by fluorescence-activated cell sorting analysis or RNA polymerase chain reaction in CD7(+) cells. These studies identify a clonogenic lymphoid progenitor with both B-cell and natural killer cell lineage potential with a molecular profile that suggests a developmental stage more primitive than previously identified lymphoid progenitors. The CD7(+) phenotype distinguishes primitive human lymphoid progenitors from pluripotent stem cells, thus allowing the study of regulation of early human lymphopoiesis and providing an alternative to pluripotent stem cells for genetic manipulation and transplantation. (Blood. 2001;97:3683-3690)
Asunto(s)
Antígenos CD , Linaje de la Célula , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Subgrupos Linfocitarios/inmunología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Antígenos CD34/sangre , Antígenos CD7/sangre , Antígenos de Diferenciación/sangre , Linfocitos B/citología , Diferenciación Celular , Células Clonales/citología , Células Clonales/inmunología , Células Dendríticas/citología , Sangre Fetal/inmunología , Hematopoyesis , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunofenotipificación , Células Asesinas Naturales/citología , Linfocitos/citología , Glicoproteínas de Membrana , NAD+ Nucleosidasa/sangre , ARN Mensajero/análisisRESUMEN
The cyclin-dependent kinase (CDK)-activating kinase (CAK) is involved in cell cycle control, transcription, and DNA repair (E. A. Nigg, Curr. Opin. Cell. Biol. 8:312-317, 1996). However, the mechanisms of how CAK is integrated into these signaling pathways remain unknown. We previously demonstrated that abrogation of MAT1 (ménage à trois 1), an assembly factor and targeting subunit of CAK, induces G(1) arrest (L. Wu, P. Chen, J. J. Hwang, L. W. Barsky, K. I. Weinberg, A. Jong, and V. A. Starnes, J. Biol. Chem. 274:5564-5572, 1999). This result led us to investigate how deregulation of CAK by MAT1 abrogation affects the cell cycle G(1) exit, a process that is regulated most closely by phosphorylation of retinoblastoma tumor suppressor protein (pRb). Using mammalian cellular models that undergo G(1) arrest evoked by antisense MAT1 abrogation, we found that deregulation of CAK inhibits pRb phosphorylation and cyclin E expression, CAK phosphorylation of pRb is MAT1 dose dependent but cyclin D1/CDK4 independent, and MAT1 interacts with pRb. These results suggest that CAK is involved in the regulation of cell cycle G(1) exit while MAT1-modulated CAK formation and CAK phosphorylation of pRb may determine the cell cycle specificity of CAK in G(1) progression.
Asunto(s)
Fase G1 , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , División Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosforilación , Unión Proteica , Proteínas Recombinantes de Fusión , Proteína de Retinoblastoma/metabolismo , Especificidad por Sustrato , Transducción Genética , Transfección , Células Tumorales Cultivadas , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.
Asunto(s)
Antígenos CD34/biosíntesis , Antígenos CD , Antígenos de Diferenciación/biosíntesis , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Interleucina-3/fisiología , NAD+ Nucleosidasa/biosíntesis , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adyuvantes Inmunológicos/fisiología , Antígenos CD19/biosíntesis , Subgrupos de Linfocitos B/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/inmunología , Células Cultivadas , Niño , Preescolar , Combinación de Medicamentos , Células Madre Hematopoyéticas/metabolismo , Humanos , Interleucina-7/fisiología , Ligandos , Glicoproteínas de Membrana , Proteínas de la Membrana/fisiología , Células del Estroma/inmunología , Factores de TiempoRESUMEN
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, and a progressive deterioration of immune function. WAS is caused by mutations in an intracellular protein, WASP, that is involved in signal transduction and regulation of actin cytoskeleton rearrangement. Because immune dysfunction in WAS may be due to an accelerated destruction of lymphocytes, we examined the susceptibility to apoptosis of resting primary lymphocytes isolated from WAS patients in the absence of exogenous apoptogenic stimulation. We found that unstimulated WAS lymphocytes underwent spontaneous apoptosis at a greater frequency than unstimulated normal lymphocytes. Coincident with increased apoptotic susceptibility, WAS lymphocytes had markedly attenuated Bcl-2 expression, whereas Bax expression did not differ. A negative correlation between the frequency of spontaneous apoptosis and the level of Bcl-2 expression was demonstrated. These data indicate that accelerated lymphocyte destruction by spontaneous induction of apoptosis may be one pathogenic mechanism by which the progressive immunodeficiency in WAS patients develops.
Asunto(s)
Apoptosis , Linfocitos/patología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Síndrome de Wiskott-Aldrich/patología , Adolescente , Muerte Celular , Preescolar , Humanos , Lactante , Linfocitos/metabolismo , Masculino , Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L714-L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-D-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1beta-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-D-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 microM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Daño del ADN/fisiología , Hiperoxia/genética , Hiperoxia/fisiopatología , Alveolos Pulmonares/fisiología , Animales , Caspasa 1/metabolismo , Adhesión Celular/fisiología , Línea Celular , Activación Enzimática/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Flavonoides/farmacología , Glutatión/metabolismo , Hiperoxia/metabolismo , Hiperoxia/patología , Laminina/antagonistas & inhibidores , Laminina/farmacología , Masculino , Mitocondrias/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
Cyclin D1 antisense (D1AS)-transfected lung epithelial cell lines were serum deprived and then analyzed for three hallmarks of apoptosis: appearance of single-strand DNA breaks, alteration of apoptosis-related protein expression, and induction of chromatin condensation. Single-strand DNA breaks appeared at significant levels 24 h after serum deprivation, whereas induction of chromatin condensation was observed after 72 h. The antioxidants dimethyl sulfoxide, ascorbate, and glutathione, as well as insulin-like growth factor-I, inhibited induction of DNA damage in this assay. Additionally, proliferating cell nuclear antigen expression is completely suppressed in the D1AS cells, indicating a mechanism to explain the reduced capacity for DNA repair. Increased expression of cyclin D1, which is a common lesion in lung cancer, may thus prevent induction of apoptosis in an oxidizing and growth factor-poor environment. Reducing cyclin D1 expression in lung cancer cells by expression of D1AS RNA disrupted these protective pathways.
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Antioxidantes/farmacología , Apoptosis , Ciclina D1/genética , Regulación Neoplásica de la Expresión Génica , Oligodesoxirribonucleótidos Antisentido/farmacología , Apoptosis/efectos de los fármacos , Ácido Ascórbico/farmacología , Medio de Cultivo Libre de Suero , Ciclina D1/fisiología , Daño del ADN , Reparación del ADN/efectos de los fármacos , Dimetilsulfóxido/farmacología , Glutatión/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Neoplasias Pulmonares , Antígeno Nuclear de Célula en Proliferación/análisis , Células Tumorales CultivadasRESUMEN
The conformation and activity of pRb, the product of the retinoblastoma susceptibility gene, is dependent on the phosphorylation status of one or more of its 16 potential cyclin-dependent kinase (cdk) sites. However, it is not clear whether the phosphorylation status of one or more of these sites contributes to the determination of the various conformations and activity of pRb. Moreover, whether and how the conformation of pRb may regulate the phosphorylation of the cdk sites is also unclear. In the process of analyzing the function and regulation of pRb, we uncovered the existence of an unusual structural motif, m89 (amino acids 880-900), the mutation of which confers upon pRb a hypophosphorylated conformation. Mutation of this structural domain activates, rather than inactivates, the growth suppressor function of pRb. In order to understand the effect of the mutation of m89 on the phosphorylation of cdk sites, we identified all the cdk sites (Thr-356, Ser-807/Ser-811, and Thr821) the phosphorylation of which drastically modify the conformation of pRb. Mutation of each of these four sites alone or in combinations results in the different conformations of pRb, the migration pattern of which, on SDS-polyacrylamide gel electrophoresis, resembles various in vivo hypophosphorylated forms. Each of these hypophosphorylated forms of pRb has enhanced growth suppressing activity relative to the wild type. Our data revealed that the m89 structural motif controls the exposure of the cdk sites Ser-807/Ser-811 in vitro and in vivo. Moreover, the m89 mutant has enhanced growth suppressing activity, similar to a mutant with alanine substitutions at Ser-807/Ser-811. Our recent finding, that the m89 region is part of a structural domain, p5, conserved antigenically and functionally between pRb and p53, suggests that the evolutionarily conserved p5 domain may play a role in the coordinated regulation of the activity of these two tumor suppressors, under certain growth conditions.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Ciclo Celular , Ciclina A/genética , Ciclina A/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Conformación Proteica , Proteína de Retinoblastoma/química , Proteína de Retinoblastoma/genética , Serina/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
The human MAT1 gene (ménage à trois 1) is an assembly factor and a targeting subunit of cyclin-dependent kinase (CDK)-activating kinase. The novel mechanisms by which MAT1 forms an active CDK-activating kinase and determines substrate specificity of CDK7-cyclin H are involved in the cell cycle, DNA repair, and transcription. Hyperplasia of vascular smooth muscle cells (SMC) is a fundamental pathologic feature of luminal narrowing in vascular occlusive diseases, and nothing is yet known regarding the cell cycle phase specificity of the MAT1 gene in its involvement in SMC proliferation. To investigate such novel regulatory pathways, MAT1 expression was abrogated by retrovirus-mediated gene transfer of antisense MAT1 RNA in cultured rat aortic SMCs. We show that abrogation of MAT1 expression retards SMC proliferation and inhibits cell activation from a nonproliferative state. Furthermore, we have demonstrated that these effects are due to G1 phase arrest and apoptotic cell death. Our studies indicate a link between cell cycle control and apoptosis and reveal a potential mechanism for coupling the regulation of MAT1 with G1 exit and prevention of apoptosis.
Asunto(s)
Apoptosis/genética , Fase G1 , Músculo Liso Vascular/citología , Proteínas de Neoplasias/genética , ARN sin Sentido/farmacología , Animales , Aorta/citología , Aorta/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Ratas , Transducción GenéticaRESUMEN
Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.
Asunto(s)
Adenosina Desaminasa/inmunología , Antígenos CD34/inmunología , Trasplante de Células Madre Hematopoyéticas , Linfocitos T/inmunología , Inmunología del Trasplante/inmunología , Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Animales Recién Nacidos , Línea Celular , Citometría de Flujo , Frecuencia de los Genes , Granulocitos/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Recuento de Linfocitos , Ratones , Ratones SCID , Polietilenglicoles , Linfocitos T/efectos de los fármacos , Transformación Genética , Trasplante Autólogo , Cordón UmbilicalAsunto(s)
Eritroblastos/citología , Eritropoyesis , Células Madre Hematopoyéticas/fisiología , Hemoglobinopatías/fisiopatología , Antígenos CD34/sangre , Células de la Médula Ósea/citología , Diferenciación Celular , Células Cultivadas , Eritroblastos/fisiología , Eritrocitos/citología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Hemoglobinopatías/sangre , Humanos , Modelos BiológicosRESUMEN
Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.
Asunto(s)
Apoptosis/fisiología , Daño del ADN/fisiología , Factores de Crecimiento de Fibroblastos , Hiperoxia/fisiopatología , Proteínas Proto-Oncogénicas c-bcl-2 , Alveolos Pulmonares/fisiopatología , Animales , Células Cultivadas , Colágeno/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Daño del ADN/efectos de los fármacos , Combinación de Medicamentos , Células Epiteliales/fisiología , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Hiperoxia/patología , Laminina/farmacología , Masculino , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteoglicanos/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Alveolos Pulmonares/patología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2RESUMEN
Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.
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Técnicas de Cultivo de Célula , Eritrocitos/citología , Eritropoyesis , Células Madre Hematopoyéticas/citología , Adulto , Antígenos CD34 , Diferenciación Celular , Citometría de Flujo , Marcadores Genéticos , HumanosRESUMEN
Human hematopoietic stem cells are pluripotent, ie, capable of producing both lymphoid and myeloid progeny, and are therefore used for transplantation and gene therapy. An in vitro culture system was developed to study the multi-lineage developmental potential of a candidate human hematopoietic stem cell population, CD34+CD38- cells. CD34+CD38- cells cocultivated on the murine stromal line S17 generated predominantly CD19(+) B-cell progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions ("switch culture") showed that a fraction of the immunophenotypically uncommitted CD19- cells generated on S17 stroma had myeloid potential (defined by expression of CD33 and generation of colony-forming unit-cells). Using the switch culture system, single CD34+CD38- cells were assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single CD34+CD38- cells possessed both B-lymphoid and myeloid potential. 94.7% of the CD34+CD38- cells with lympho-myeloid potential were late-proliferating (clonal appearance after 30 days), demonstrating that pluripotentiality is detected significantly more often in quiescent progenitors than in cytokine-responsive cells (P = .00002). The S17/switch culture system permits the in vitro assessment of the pluripotentiality of single human hematopoietic cells.
Asunto(s)
Antígenos CD34/análisis , Antígenos CD , Antígenos de Diferenciación/análisis , Células Madre Hematopoyéticas/química , Linfocitos/citología , NAD+ Nucleosidasa/análisis , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD19/análisis , Linfocitos B/citología , Diferenciación Celular , División Celular , Línea Celular , Células Cultivadas , Células Clonales , Técnicas de Cocultivo/métodos , Sangre Fetal , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Glicoproteínas de Membrana , RatonesRESUMEN
A novel technique that can be used for reacting toxic carbon dioxide (CO2) emissions from power plants and other combustion wastes with sea water is described. A chemical interaction between CO2 and the cations in sea water, with the pH electrolytically regulated, can precipitate almost all the calcium and magnesium ions, as well as some sodium and potassium ions, as carbonates and bicarbonates. The carbonates and bicarbonates thus prepared can then be mixed with ash to yield a building material. Sulfur ions will be neutralized with calcium and magnesium, and the remaining ions can be removed using reverse osmosis or some other method. The technology and equipment for purification are based on modules that can be used for industrial waste-water, sea water, solutions, and otherwise. The module for separation of sand and suspended coarse substances consists of a tank for flocculation, coagulation, and precipitation of solid particles; and a low-pressure hydrocyclone. The module for purification from oil and fine suspensions is based on column flotation, flotation with a special ejector, and adhesion flotation. The module for ions and colloids consists of an absorbing filter with zeolite, fly ash, and other absorbing materials. Using a laboratory model consisting of a special mini-plant, we processed 10 L of factory-waste water containing more than 20 g/L organic content (compare with the upper limit of 0.02 g/L allowed by the Ministry of Environmental Protection in Israel). After the experimental solution was treated and evaporated to a small bulk, the water obtained was almost clear. On the basis of the results in the model, we present a scaled-up process for the design, development, and production of equipment for and the assembly of a large installation for drainage and water purification.