RESUMEN
Biocompatible inorganic matrices have been used to enhance bone repair by integrating with endogenous bone architecture. Hypothesizing that a three-dimensional framework might support reconstruction of other tissues as well, we assessed the capacity of a tantalum-coated carbon matrix to support reconstitution of functioning thymic tissue. We engineered a thymic organoid by seeding matrices with murine thymic stroma. Co-culture of human bone marrow-derived hematopoietic progenitor cells within this xenogeneic environment generated mature functional T cells within 14 days. The proportionate T-cell yield from this system was highly reproducible, generating over 70% CD3+ T cells from either AC133+ or CD34+ progenitor cells. Cultured T cells expressed a high level of T-cell receptor excision circles (TREC), demonstrating de novo T lymphopoiesis, and function of fully mature T cells. This system not only facilitates analysis of the T-lymphopoietic potential of progenitor cell populations; it also permits ex vivo genesis of T cells for possible applications in treatment of immunodeficiency.
Asunto(s)
Órganos Artificiales , Organoides/fisiología , Linfocitos T/fisiología , Timo/fisiología , Antígeno AC133 , Animales , Antígenos CD , Antígenos CD34/biosíntesis , Células de la Médula Ósea/citología , Carbono/metabolismo , Materiales Biocompatibles Revestidos , Técnicas de Cocultivo , Técnicas de Cultivo/métodos , Citometría de Flujo , Glicoproteínas/metabolismo , VIH-1/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Organoides/ultraestructura , Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Linfocitos T/citología , Timo/citología , Timo/ultraestructura , Factores de TiempoRESUMEN
The ability to culture multipotent hematopoietic progenitor cells for extended periods is of practical importance to both clinical and research efforts involving these cells. Conventional techniques for the extended culture of hematopoietic progenitor cells (HPCs) have proven largely ineffective in sustaining these cells and preserving their multipotency over protracted periods. To overcome barriers to extended HPC culture, numerous alternative approaches, including cytokine augmentation and co-culture with bone marrow stroma, have been explored to enhance HPC maintenance but have generally yielded mixed results. The present study examined the ability of a novel, three-dimensional, tantalum-coated porous biomaterial (TCPB) to support HPC maintenance and multipotency in long-term cultures to which no exogenous cytokines have been added. As a follow-up to previously published short-term HPC cultures in TCPB, we examined the maintenance, phenotype and multipotency of HPCs cultured for up to 6 weeks in the TCPB matrix compared to control systems, including fibronectin-coated plastic, bone marrow stroma cocultures and other three-dimensional materials. These studies indicated that TCPB supports the maintenance of immature progenitors for up to 6 weeks in the absence of supplemented cytokines. Further, the results demonstrate that the TCPB matrix facilitates and enhances HPC maintenance and leads to a 1.5-fold expansion of HPC numbers following 1 week in culture and a 6.7-fold increase in colony-forming ability following 6 weeks in culture in the absence of exogenous cytokines. Under the same conditions, control systems were less able to support progenitor viability and multipotency. These findings point to new approaches that may improve the in vitro preservation of progenitors and may have important implications in clinical areas such as progenitor expansion, bone marrow transplantation and gene therapy.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Células Madre Hematopoyéticas/citología , Materiales Biocompatibles , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , División Celular , Linaje de la Célula , Células Cultivadas , Cerámica , Ensayo de Unidades Formadoras de Colonias , Estudios de Evaluación como Asunto , Fibronectinas , Humanos , Inmunofenotipificación , Microesferas , Linfocitos T/citología , Tantalio , Factores de TiempoRESUMEN
Gliomas are part of a subset of tumors in which overexpression of p53 protein in the absence of p53 gene mutation has been described. We have utilized a series of glioma cell lines to study the effects of ionizing radiation on the regulation of p53, p21, mdm2 and Rb proteins. The induction of p53 protein in glioma cell lines that overexpress wild-type p53 differs from normal control cells and glioma cell lines containing mutant p53. Alterations in the accumulation of p53 and p21 proteins are associated with diminished Rb hypophosphorylation. Gliomas that overexpress wild-type p53 also express high levels of mdm2 protein and exhibit a radiosensitivity that is intermediate between normal cells and cells with mutant p53. These findings suggest that, at least in certain glioma cell lines that over-express p53 which is wild-type in sequence, the function of p53 protein is abnormal.
Asunto(s)
Glioma/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas Nucleares , Proteína Oncogénica p21(ras)/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteína de Retinoblastoma/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Astrocitos/metabolismo , Astrocitos/efectos de la radiación , Western Blotting , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteína de Retinoblastoma/metabolismo , Células Tumorales Cultivadas/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Meningiomas represent a common class of tumors of the central nervous system. However, the molecular events underlying their formation are poorly understood. Because altered expression of the p53 tumor suppressor gene and the mdm2 proto-oncogene have been demonstrated in a wide variety of tumors, we carried out studies to assess the possible involvement of these two genes in meningioma tumorigenesis. We used Western blot analysis to examine the level of expression of the mdm2 and p53 proteins in a series of sixteen primary meningiomas and four meningioma cell lines. The data obtained from these studies suggest that elevated expression of the p53 or mdm2 protein products does not represent a common event in the development of human meningiomas.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Genes p53/genética , Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas/biosíntesis , Proto-Oncogenes/genética , Western Blotting , Humanos , Neoplasias Meníngeas/genética , Meningioma/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales CultivadasRESUMEN
The mammalian cellular response to ionizing radiation results in delays in progression through the cell cycle at several checkpoints and includes alterations in the activity of cyclin-dependent kinases. The product of the CDC2 gene is a key kinase involved in cell cycle progression. The signaling events that regulate its expression after exposure to DNA-damaging agents are not known. We show that cdc2 mRNA and protein are down-regulated after irradiation of normal human and mouse fibroblasts with doses as low as 0.5 Gy. This down-regulation is preceded by induction of p53 and p21Waf1 proteins. In human cells in which p53 was nonfunctional and in p53-/- or p21-/- mouse embryo fibroblasts, no effect of ionizing radiation on p34cdc2 expression levels was observed. These findings indicate that CDC2 down-regulation after irradiation is p53-dependent and involves the cyclin-dependent kinase inhibitor p21Waf1 as a negative factor in the control of CDC2 expression. Correspondence between the delay in initiation of DNA synthesis in irradiated cells and the down-regulation of CDC2 is described.
Asunto(s)
Proteína Quinasa CDC2/efectos de la radiación , Regulación hacia Abajo/efectos de la radiación , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteína Quinasa CDC2/biosíntesis , Proteína Quinasa CDC2/genética , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Ciclinas/efectos de la radiación , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/efectos de la radiación , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Ratones , ARN Mensajero/metabolismo , ARN Mensajero/efectos de la radiaciónRESUMEN
To better understand the molecular mechanisms responsible for meningioma tumorigenesis we previously utilized subtractive hybridization protocols to identify genes the expression or structure of which is altered in these common brain tumors. Here we show that a CA dinucleotide repeat element present in one complementary DNA isolated by this approach has undergone a contraction in size in a meningioma cell line. Extension of this initial observation has revealed widespread genetic alterations affecting simple repeat sequences in this and other meningiomas. These data indicate that genetic instability may play a previously unrecognized role in the etiology of meningiomas.
Asunto(s)
ADN de Neoplasias/genética , ADN Satélite/genética , Neoplasias Meníngeas/genética , Meningioma/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
Cytogenetic and molecular analyses of human breast cancer cells have identified consistent losses of specific chromosomal regions in these tumors, suggesting that such regions harbor tumor suppressor genes whose homozygous loss or inactivation directly contributes to tumorigenesis. To date, deletions of chromosome 8 sequences have been described infrequently and only in low percentages of breast carcinomas. We report the identification of a new DNA marker on chromosome 8p that is deleted in 6 (75%) of 8 breast carcinoma cell lines and in 1 primary breast carcinoma examined. No deletion of this marker was detected in any normal or nonbreast carcinoma cell lines analyzed. Southern blot and fluorescence in situ hybridization studies indicate that this clone maps to chromosome 8 between bands p12 and p21. These observations suggest that a new gene, whose loss or inactivation may foster breast carcinoma tumorigenesis, may reside in this chromosome 8p region.
Asunto(s)
Neoplasias de la Mama/genética , Cromosomas Humanos Par 8 , Eliminación de Secuencia , Secuencia de Bases , Mapeo Cromosómico , Sondas de ADN , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
Neurofibromatosis type 2 (NF2) is an autosomal dominantly-inherited disorder predisposing affected individuals to tumors of multiple cell types in the central nervous system, including meningiomas. A candidate tumor suppressor gene for this disorder has recently been cloned; the protein product of this gene has a predicted role in linking integral membrane proteins with the cytoskeleton. Utilizing reverse transcription-polymerase chain reaction (RT-PCR) analyses, we have identified a number of alternatively spliced transcription products encoded by the NF2 gene. These alternative splice variants were detected in RNA isolated from several sources, including primary leptomeningeal tissue and an established line of leptomeningeal cells (LMC). Several of these variants delete previously identified coding regions of this gene. Moreover, two of these splice variants add previously unrecognized exons to the NF2 coding region. These identified splice forms will serve as natural reagents for the functional dissection of the NF2 protein product(s). They also should be considered in studies investigating mutations of this gene in members of NF2 families and in tumor analyses.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes de la Neurofibromatosis 2 , Proteínas de la Membrana/genética , Neoplasias Meníngeas/genética , Meningioma/genética , Proteínas de Neoplasias/genética , Empalme del ARN , ARN Mensajero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Análisis Mutacional de ADN , ADN de Neoplasias/genética , ADN de Cadena Simple/genética , Exones , Humanos , Proteínas de la Membrana/biosíntesis , Neoplasias Meníngeas/metabolismo , Meninges/metabolismo , Meningioma/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Neurofibromatosis 2/genética , Neurofibromina 2 , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Células Tumorales CultivadasRESUMEN
Meningiomas are common tumours derived from the thin membrane that surrounds the brain and spinal cord. Currently, the molecular mechanisms responsible for the initiation and progression of these tumors are largely unknown. Toward the elucidation of such mechanisms, we have formulated an experimental design utilizing the technique of subtractive hybridization that is aimed at identifying the changes in gene expression between intracranial meningiomas and their normal precursor cells, leptomeningeal cells. We report here the identification and initial characterization of three genes whose expression is altered or aberrant in meningioma cell lines and tumours relative to cultures of normal leptomeningeal cells. Complementary DNA probes from one of these genes detect transcripts of altered size in several meningiomas relative to normal leptomeningeal cells. Another of these genes demonstrates decreased expression in meningiomas and in tumours associated with the disorder neurofibromatosis 2. A third gene isolated by this procedure is differentially expressed in both meningiomas and breast carcinomas. Therefore, the decreased expression of these genes may play roles in growth-regulatory pathways that are abrogated not only in meningiomas, but in other tumor types as well.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Meníngeas/genética , Meningioma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Células Cultivadas , Código Genético , Inhibidores de Crecimiento , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Datos de Secuencia Molecular , Neurofibromatosis 2/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
Steroid hormones and peptide growth factors promote growth and development of normal mammary tissues and some types of breast cancer. Ovarian steroids may influence mammary growth directly or indirectly. The epidermal growth factor (EGF) family of proteins may also regulate mammary growth. These two pathways may function independently of each other or they may act in concert, with steroids inducing transcription of genes that encode growth factors or growth factor receptors. We used a feline mammary adenocarcinoma cell line (K12) to address whether there was an interrelation between progesterone (PGN) and EGF-associated growth pathways. K12 cells responded to EGF by a dose-dependent increase in proliferation. PGN or promegestone (R5020, a synthetic progestagen) alone did not stimulate K12 growth, but when EGF and PGN, or EGF and R5020 were combined, they were synergistic. This synergistic response was abrogated by the PGN receptor antagonist RU486 or by antibodies that blocked binding of EGF to its receptor. K12 cells expressed characteristic double-affinity EGF receptors, as well as p185 (a functionally and structurally related protein, product of the neu gene) on their surface. PGN receptors were also found on intact cells and in cleared cytosols. Stimulation of K12 cells by PGN or by R5020 induced a two- to threefold increase in the number of high-affinity surface EGF receptors after 24 h. Stimulation of these cells by PGN also affected the relative levels of phosphorylation of the EGF receptor and p185 within minutes, but not of other cellular phosphoproteins. Our results show that PGN enhances the EGF-induced growth of K12 cells and suggest that this effect may be mediated at least partly via an increase in the number or function of high-affinity EGF receptors.
Asunto(s)
Adenocarcinoma/patología , Enfermedades de los Gatos/patología , Factor de Crecimiento Epidérmico/farmacología , Neoplasias Mamarias Animales/patología , Neoplasias Hormono-Dependientes/patología , Progesterona/farmacología , Animales , Gatos , División Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Femenino , Mifepristona/farmacología , Fosforilación , Progestinas/farmacología , Promegestona/farmacología , Receptores de Progesterona/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patologíaRESUMEN
We have investigated the therapeutic efficacy of a single injection of 131I-labeled murine mouse monoclonal antibody (NP-4) against carcinoembryonic antigen using the human colonic tumor xenograft, GW-39, grown in the cheek pouches of adult hamsters. Therapeutic efficacy was dependent on the dose of radioactivity, the specificity of the antibody for the tumor, and the size of the tumor when the radioantibody was administered. A dose of 1 mCi of 131I-labeled NP-4 given 1 day after tumor transplantation completely inhibited the growth of 6 of 11 tumors over a 12-week period, and histological evidence indicated that viable tumor was absent in the tissue remaining at the injection site. Lower doses (0.5 mCi) of 131I-labeled NP-4 inhibited tumor growth over 90% in comparison to untreated animals, but the tumors eventually resumed growth. Delaying the administration of radioantibody for 4 or 7 days after tumor transplantation significantly reduced the therapeutic efficacy. Although the same dose of 131I-labeled irrelevant immunoglobulin G also inhibited tumor growth, 131I-labeled NP-4 was generally 2-3 times more effective in reducing tumor growth than was the control IgG. There was a 13% loss in body weight within 7 days after treatment with 1 mCi, but all the animals regained their weight by day 14, indicating that the level of radioactivity was tolerated well. Dosimetric calculations predicted that over 14 days a dose of nearly 2400 rads was delivered to the tumors with 131I-labeled NP-4. These results confirm our previous studies that 131I-labeled antibody can effectively inhibit tumor growth, but suggest that radioantibody therapy is most effectively administered when there is a low tumor burden.