RESUMEN
Retroviral constructs were designed to express the novel cytokine interleukin 18 (IL-18), also known as interferon-gamma-inducing factor, in a murine neuroblastoma cell line [neuro-2a (N-2a)] to examine the effects of IL-18 expression on tumorigenicity. N-2a cells expressing proIL-18 (N-2a/IL-18p) were as tumorigenic as parental N-2a cells, whereas N-2a cells engineered to secrete mature IL-18 (N-2a/IL-18m) were nontumorigenic. Inoculation of mice with N-2a/IL-18m generated immediate immunity to parental N-2a. N-2a/IL-18m formed tumors in mice depleted of CD4+ and CD8+ T cells, suggesting that the antitumor immune response was T cell mediated. The resulting T-helper (Th) immune response was also characterized in vitro and had a large Th1 component based on in vitro production of the cytokines IFN-gamma and granulocyte macrophage colony-stimulating factor in response to tumor cells and IL-18.
Asunto(s)
Citocinas/inmunología , Huésped Inmunocomprometido/inmunología , Interleucina-18/inmunología , Interleucina-1/inmunología , Neuroblastoma/inmunología , Precursores de Proteínas/inmunología , Animales , Antígenos CD/metabolismo , Secuencia de Bases , Citocinas/análisis , Citocinas/biosíntesis , Femenino , Vectores Genéticos/biosíntesis , Inmunoterapia/métodos , Interleucina-1/biosíntesis , Interleucina-18/biosíntesis , Subunidad alfa del Receptor de Interleucina-18 , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/biosíntesis , Radioinmunoensayo , Receptores de Interleucina/antagonistas & inhibidores , Receptores de Interleucina/genética , Receptores de Interleucina-18 , Valores de Referencia , Retroviridae/genética , Bazo/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Transducción Genética , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/metabolismoRESUMEN
Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.
Asunto(s)
Catalasa/genética , Amplificación de Genes , Estrés Oxidativo , Adenina Fosforribosiltransferasa/genética , Animales , Línea Celular , Humanos , Hibridación in Situ , ConejosRESUMEN
Efficient retroviral gene transfer into hematopoietic stem and progenitor cells can be achieved by co-localizing retrovirus and target cells on specific adhesion domains of recombinant fibronectin (FN) fragments. In this paper, we further optimize this technology for human CD34+ cells. Investigating the role of cytokine prestimulation in retrovirus-mediated gene transfer on plates coated with the recombinant FN CH-296 revealed that prestimulation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) CD34+ cells was essential to achieve efficient gene transfer into clonogenic cells. The highest gene transfer occurred by prestimulating PB CD34+ cells for 40 hr with a combination of stem cell factor (SCF), G-CSF, and megakaryocyte growth and development factor (MGDF) prior to retroviral infection on CH-296. Surprisingly, a prolonged simultaneous exposure of primary CD34+ PB cells to retrovirus and cytokines in the presence of CH-296 lowered the gene transfer efficiency. Gene transfer into cytokine prestimulated CD34+ bone marrow (BM) cells was not influenced by increasing the coating concentrations of a recombinant FN fragment, CH-296, nor was it adversely influenced by increasing the number of CD34+ target cells, suggesting that the amount of retroviral particles present in the supernatant was not a limiting factor for transduction of CD34+ BM cells on CH-296-coated plates. The polycation Polybrene was not required for efficient transduction of hematopoietic cells in the presence of CH-296. Furthermore, we demonstrated that repeated exposure of CH-296 to retrovirus containing supernatant, called preloading, can be employed to concentrate the amount of retroviral particles bound to CH-296. These findings establish a simple and short clinically applicable transduction protocol that targets up to 68% of BM or G-CSF-mobilized PB CD34+ cells and is capable of genetically modifying up to 17% of CD34+CD38-/dim PB cells.
Asunto(s)
Antígenos CD , Fibronectinas/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Células Madre Hematopoyéticas/metabolismo , Receptores de Complemento 3b , Retroviridae , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Antígenos de Diferenciación , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Fibronectinas/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Bromuro de Hexadimetrina , Humanos , Interleucina-6/farmacología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Glicoproteínas de Membrana , NAD+ Nucleosidasa , Factor de Células Madre/farmacología , Factores de Tiempo , Transformación Genética , ViriónRESUMEN
The T cell co-stimulatory molecule B7-1 was transduced into a poorly immunogenic murine neuroblastoma cell line (Neuro-2a, N-2a) alone or in combination with MHC class II genes to test the ability of these genes to stimulate antitumor immunity. N-2a cells transduced with B7-1 exhibited reduced tumorigenicity, whereas N-2a cells overexpressing both MHC class II (syngeneic, I-Ak) and B7-1 totally abrogated tumorigenicity. Rejection of I-Ak/B7-1 cells was dependent on both CD4+ and CD8+ T cells. The ability of both vaccines to induce protection against parental N-2a was temporally dependent on the time of secondary N-2a challenge. To investigate the immunity generated by N-2a/B7-1 and N-2a/I-Ak/B7-1 vaccines, we tested the ability of these modified cells to stimulate in vitro the proliferation of syngeneic splenocytes from naive mice. A significant increase in splenocyte proliferation was observed with N-2a/I-Ak/B7-1 cells compared to N-2a cells. We also determined that vaccination with N-2a/I-Ak/B7-1 cells was able to generate cytotoxic T cell responses to unmodified N-2a cells. The introduction of B7-1 and I-Ak into N-2a was able to convert a poorly immunogenic tumor to a highly immunogenic one; however, mice bearing large established unmodified tumors had little response to vaccination with N-2a/I-Ak/B7-1 cells. Our results emphasize the importance of tumor immunogenicity in the treatment of established tumors with MHC class II/B7-1 tumor cell vaccines.
Asunto(s)
Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Neuroblastoma/genética , Neuroblastoma/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Vectores Genéticos , Activación de Linfocitos , Ratones , Recombinación Genética , Retroviridae/genética , Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Malignant tumors express tumor-related antigens, but effective antitumor immunity does not occur in the primary host. One hypothesis is that there is insufficient stimulation of T-cell responses due to ineffective antigen presentation. An approach to overcome these deficiencies is to modify tumor cells to express major histocompatibility complex (MHC) class II genes and thus facilitate the presentation of antigens directly by tumor cells. Our experiments with a murine neuroblastoma cell line (neuro-2a) transduced with DR (xenogeneic), 1-Ab (allogeneic), or 1-Ak (syngeneic) MHC class II genes support this notion. The relative potencies of the modified neuro-2a to induce immunity to unmodified neuro-2a were neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak. Modified neuro-2a also could stimulate naive splenocyte proliferation in vitro. The relative magnitude of the proliferative responses seen after stimulation with modified tumor cells was neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak > unmodified neuro-2a. Hence, the tumor cell-induced splenocyte proliferative responses observed in vitro correlate with the effectiveness of the tumor cell vaccines to induce antitumor immunity in vivo. These data show that the expression of exogenous MHC class II on tumor cells is a potent stimulus for specific antitumor immunity. Because of the correlation of the in vivo and in vitro immune responses to modified tumor cells, the tumor-induced lymphocyte proliferation assay may be useful in evaluating tumor cell vaccines produced by additional genetic modifications of tumor cells.
Asunto(s)
Técnicas de Transferencia de Gen , Genes MHC Clase II/genética , Retroviridae/genética , Vacunas/inmunología , Animales , División Celular/genética , Vectores Genéticos/genética , Humanos , Inmunización , Interleucina-2/farmacología , Ratones , Ratones Endogámicos , Mitomicina/farmacología , Neuroblastoma/inmunología , Bazo/metabolismo , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
Immunological recognition of transformed cells is critically important to limit tumor development and proliferation. Because established tumors have escaped immune recognition and elimination, novel strategies to enhance antitumor immunity have been developed. A unique approach has used the introduction of genes encoding major histocompatibility complex (MHC) antigens into tumor cells. Experiments in mice have shown that the expression of syngeneic class II MHC antigens in tumor cells completely abrogates tumorigenicity and induces tumor-specific immunity. In this study we sought to determine whether a more effective antitumor immune response would be generated by introducing xenogeneic class II MHC genes into tumor cells. To address this question we used recombinant retroviruses to express human class II MHC genes in a highly malignant murine neuroblastoma cell line, Neuro-2a. We found that normal mice inoculated with Neuro-2a expressing the human class II MHC antigen did not develop tumors and were immune to subsequent challenge with unmodified Neuro-2a cells. In addition, mice bearing small established Neuro-2a tumors were cured by vaccination with Neuro-2a expressing human class II MHC. We hypothesize that a similar approach using retroviral-mediated transduction of class II MHC genes into human tumor cells may be an effective alternative to current cancer treatment.
Asunto(s)
Genes MHC Clase II , Neuroblastoma/genética , Transfección/inmunología , Adyuvantes Inmunológicos , Animales , Modelos Animales de Enfermedad , Antígenos HLA-D/análisis , Humanos , Inmunidad Celular , Inmunoterapia , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Neuroblastoma/secundario , Neuroblastoma/terapia , Subgrupos de Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
Three retroviral vectors, containing a human adenosine deaminase (ADA) cDNA linked to either the simian virus 40 (SV40) early promoter, the human cytomegalovirus (CMV) immediate early promoter, or the Moloney murine leukemia virus (MoMLV) promoter, were tested for their ability to express ADA following infection and transplantation of murine bone marrow. Virus was produced by using PA317 amphotropic retrovirus packaging cells. The titer of each of the vectors was similar and no helper virus was detected. Human ADA was expressed in the blood of some animals for 6 months after transplantation of infected marrow, and vector DNA was found in the spleen and in bone marrow from these animals. The percentage of animals expressing human ADA (33%) and the amount of human ADA in blood (1-5% of total ADA) was similar for each of the vectors. These results show that amphotropic vectors are capable of infecting pluripotent hematopoietic stem cells having long-term repopulating ability, and that a variety of promoters allow gene expression following differentiation of these early cells.
Asunto(s)
Adenosina Desaminasa/genética , Trasplante de Médula Ósea , Vectores Genéticos , Retroviridae/genética , Adenosina Desaminasa/biosíntesis , Animales , Secuencia de Bases , Médula Ósea/enzimología , Médula Ósea/microbiología , ADN Viral/análisis , Femenino , Virus Helper/genética , Virus Helper/aislamiento & purificación , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Factores de TiempoRESUMEN
Adenosine deaminase (ADA) deficiency is associated with a fatal severe combined immunodeficiency. Because most patients do not have a suitable marrow donor, the introduction of a normal ADA gene into the patient's marrow cells is a potentially useful alternative therapy. To identify vectors that provide optimal gene expression in human hematopoietic cells, we investigated retroviral vectors containing the ADA gene under the transcriptional control of the promoter/enhancers of Moloney murine leukemia virus, the simian virus 40 early region, the cytomegalovirus immediate-early gene, the lymphotropic papovavirus, and the human beta-globin gene. ADA expression from these vectors was monitored in the ADA- human histiocytic lymphoma cell line DHL-9, and in the multipotential chronic myeloid leukemia cell line K562. ADA expression in infected K562 cells was also measured after induction of megakaryoblastic differentiation by phorbol ester, and after induction of erythroid differentiation by sodium n-butyrate or hemin. In these hematopoietic cell lines, the vectors that contained ADA controlled by either the Moloney murine leukemia virus promoter (LASN) or the cytomegalovirus promoter (LNCA) expressed ADA at much higher levels than the other vectors tested. Furthermore, in K562 cells infected with LASN and LNCA vectors, induction of terminal differentiation resulted in the same or higher level expression of ADA. These cell lines have permitted the evaluation of transduced gene expression in proliferating and differentiating hematopoietic cells that provide a model for bone marrow-targeted gene therapy.
Asunto(s)
Adenosina Desaminasa/genética , Células Madre Hematopoyéticas/enzimología , Nucleósido Desaminasas/genética , Regiones Promotoras Genéticas , Transfección , Animales , Línea Celular , Clonación Molecular , Genes , Vectores Genéticos , Humanos , Leucemia Eritroblástica Aguda/enzimología , Leucemia Eritroblástica Aguda/genética , Linfoma/enzimología , Linfoma/genética , Ratones , Células Tumorales Cultivadas/enzimologíaRESUMEN
Retroviral vectors with an internal cytomegalovirus major immediate-early gene enhancer/promoter regulating HLA class II gene expression were used to transfer HLA cDNA into human EBV-transformed B-lymphoblastoid cell lines. HLA-DQ2 beta and DQ3.2 beta cDNA were transferred into DQ3.2 and DQ2 homozygous lymphoblastoid cell lines, respectively. Serologic analysis of the infected cell lines with allospecific mAb demonstrated surface expression of these exogenous DQ molecules implying that DQ alpha-chains from DR3, DQ2-positive cells can pair with DQ3.2 beta-chains and, similarly, DQ alpha-chains from DR4, DQ3.2-positive cells can pair with DQ2 beta-chains. Immunoprecipitation of the introduced DQ3.2 beta molecule resulted in co-purification of the allotype-mismatched endogenous DQ2 alpha polypeptide. We also show that vectors with a cytomegalovirus major immediate-early gene enhancer/promoter result in higher levels of expression of the transduced gene compared to previously described HLA vectors with either the SV-40 early enhancer/promoter or the lymphotropic papovavirus-enhanced SV-40 promoter. Although deletion of HLA cDNA did sometimes occur in the process of generating virus-producing clones, the HLA cDNA is stably maintained in virus-producing clones, once it is generated. This retroviral expression system is a highly efficient way to study HLA gene function.
Asunto(s)
Antígenos HLA-DQ/genética , Haplotipos , Línea Celular , Deleción Cromosómica , Elementos Transponibles de ADN , Vectores Genéticos , Antígenos HLA-DQ/aislamiento & purificación , Homocigoto , Humanos , Sustancias Macromoleculares , Pruebas de Precipitina , Retroviridae/genética , TransfecciónRESUMEN
Hemophilia B is an X-chromosome-linked bleeding disorder resulting from lack of clotting factor IX activity and affects about 1 in 30,000 males. Current therapy involves injection of crude factor IX prepared from pooled human plasma. Treatment is complicated by viral contaminants in factor IX preparations, such as non A-non B hepatitis and the AIDS virus, and by the practical difficulties of chronic injections. An alternative therapy might include the insertion of a factor IX expression vector into the somatic cells of affected individuals to allow continued production of factor IX. Toward this end, we have constructed a retrovirus vector for transfer and expression of factor IX. Despite the fact that factor IX is normally synthesized in hepatocytes and requires extensive post-translational modification for activity, we have shown that fully active factor IX can be made by human skin-derived fibroblasts. These results open the way to testing the use of skin grafts for gene therapy of hemophilia B.
Asunto(s)
Factor IX/genética , Hemofilia B/terapia , Bioensayo , Células Cultivadas , Ingeniería Genética , Vectores Genéticos , Hemofilia B/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genéticaRESUMEN
Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. We have studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA-fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.
Asunto(s)
Adenosina Desaminasa/genética , ADN Recombinante , Nucleósido Desaminasas/genética , Retroviridae/genética , Adenosina/metabolismo , Adenosina Desaminasa/deficiencia , Línea Celular , Desoxiadenosinas/metabolismo , Diploidia , Resistencia a Medicamentos , Fibroblastos/enzimología , Ingeniería Genética , Humanos , Piel/citologíaRESUMEN
Patients with certain genetic disorders can be cured by bone marrow transplantation. However, as prospective donors do not exist for most patients with potentially curable genetic abnormalities, an alternative treatment for such patients involves the transfer of cloned genes into the patient's haematopoietic stem cells followed by re-infusion of the treated cells. Retroviral vectors provide an efficient means for transferring genes into mammalian cells and have been used to transfer genes into mouse haematopoietic cells. We have now produced amphotropic retroviral vectors containing either the bacterial gene for neomycin resistance or a mutant dihydrofolate reductase gene that confers resistance to methotrexate and have used these vectors to infect and confer drug resistance to human haematopoietic progenitor cells in vitro. Transfer could be demonstrated in the absence of helper virus by using an amphotropic retrovirus packaging cell line, PA12 (ref. 9). These studies are an important step towards the eventual application of retrovirus-mediated gene transfer to human gene therapy and for molecular approaches to the study of human haematopoiesis.
Asunto(s)
Resistencia a Medicamentos , Células Madre Hematopoyéticas , Retroviridae/genética , Transfección , Células de la Médula Ósea , Células Cultivadas , Eritrocitos , Granulocitos , Humanos , Metotrexato , Monocitos , Neomicina , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
The historical view of masculinity/femininity posited essentially bipolar opposites, with the presence of one set of characteristics precluding the other. More recent studies of sex-role stereotypes have defined sexual orientation within clusters of socially desirable attributes which males and females perceive as differentiating males from females. This view negates the contention that psychological sex roles are composed of bipolar opposites, and concludes that the constructs of masculinity and femininity are independent dimensions rather than a single bipolar dimension. Little is known about the sex-role functioning of adolescents, yet it is during adolescence that qualitative shifts occur in interpersonal relationships and concurrent changes occur in cognitive functioning, with adolescents shifting toward hypothetical thinking and abstract ideal notions. In view of these changes, much can be learned about adult functioning by studying the sex-role perceptions of adolescents related to familial and social variables. This study examines the sex-role perceptions that adolescents hold of fathers, mothers, ideal males, ideal females, and selves. Differences exist between male and female adolescents, and significant linkages exist between sex-role identification and academic achievement.
RESUMEN
The evaluation of a behavior and development training program that was integrated into a general pediatric training residency is reported. Thirty-two residents who participated in the program were evaluated over one year on measures designed to assess residents' opinions about the relevance of behavior and development concepts, residents' perceptions of their own competence in behavior and development knowledge and skills, supervisors' ratings of residents' general clinical performance, and residents' attitudes about 10 specific illnesses. Results suggest that residents perceive a high relevance for psychosocial concepts and an increasing competence with these concepts after training. Establishing rapport with patients emerged as an important variable in supervisors' ratings of resident performance and in residents' self-ratings of competence. Attitudes were initially negative toward the more behavioral and psychophysiological disease entities but showed some positive change over the year. These results and their implications are discussed.
Asunto(s)
Competencia Clínica , Internado y Residencia , Pediatría/educación , Actitud , Humanos , Relaciones Médico-PacienteRESUMEN
The binding of mouse epidermal growth factor-urogastrone (EGF-URO) to membranes from term human placenta is peptide-specific, saturable (about 20 pmol of EGF-URO bound maximally/mg of protein), reversible, and of high affinity (KD about 400 pM). Optimal binding is observed at pH 7.6. At low pH (3.5 to 5.0). EGF-URO can be reversibly dissociated from the receptor; however, exposure to pH < 3 irreversibly inactivates the receptor. The binding, which does not exhibit ligand cooperativity, exhibits an association rate constant of 6.1 x 10(-4) s-1 and a dissociation rate constant of 6.1 x 10(-4) s-1. The dissociation constant determined from the rate constants, 240 pM, is in reasonable agreement with the constant estimated by equilibrium methods. Both monovalent and divalent cations augment EGF-URO binding 2- to 3-fold. Although in general, divalent cations enhance binding at lower concentrations (optimum, 5 mM) than do monovalent cations (optimum, approximately 80 mM), there is no cation-specific effect. Neither guanine nor adenine nucleotides affect EGF-URO binding. Whereas the proteolytic enzymes (trypsin, chymotrypsin, papain, and pepsin) inactivate the receptor, neuraminidase and phospholipases A2, C, and D augment EGF-URO binding. Neuraminidase increases the number of available sites without affecting ligand affinity. Wheat germ agglutinin, concanavalin A, and phytohemagglutinin all compete for the binding of EGF-URO. The data complement previous observations of EGF-URO binding obtained in intact cells and provide a basis for the solubilization, characterization, and isolation of this receptor from a rich tissue source.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Hormonas Gastrointestinales/metabolismo , Péptidos/metabolismo , Placenta/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Cationes Bivalentes , Cationes Monovalentes , Membrana Celular/metabolismo , Receptores ErbB , Femenino , Humanos , Cinética , Lectinas/farmacología , Ratones , EmbarazoRESUMEN
We describe the solubilization and purification of both the photoaffinity-labeled and unlabeled human placenta receptor for epidermal growth factor-urogastrone (EGF-URO). The photolabeled receptor can be purified 300- to 500-fold from membrane extracts by combined immunoaffinity and lectin-agarose affinity chromatography. This isolation approaches theoretical purity. Upon gel filtration and wheat germ agglutinin-Sepharose chromatography, the photolabeled receptor and the EGF-URO binding activity co-migrate, as measured by a newly developed lectin-agarose immobilization assay of receptor binding. Sequential ion exchange, Cibacron blue-Sepharose, wheat germ agglutinin-Sepharose, and gel filtration chromatography yield a 110-fold purification of the EGF-URO binding activity; this purified fraction contains protein constituents that exhibit electrophoretic mobilities parallel to those of the photolabeled receptor constituents, that have apparent molecular weights of 180,000 and 160,000. This molecular weight range is consistent with a measured apparent Stokes radius for both the photolabeled and unlabeled receptor of 5.1 nm (Sephacryl S-200 gel filtration), although an apparent radius of 4.3 nm is estimated by gel filtration on Sepharose-6B. The apparent molecular weight of the photolabeled receptor is unaffected by 2-mercaptoethanol. This work provides a basis for further detailed studies of this growth factor receptor.