RESUMEN

DNA constructs based on bacterial artificial chromosomes (BACs) are frequently used to generate transgenic animals as they reduce the influence of position effects and allow predictable expression patterns for genes whose regulatory sequences are not fully identified. Despite these advantages BAC transgenics suffer from drawbacks such as complicated vector construction, low efficiency of transgenesis, and some remaining expression variegation. The recent development of transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) has resulted in new transgenic techniques which do not have the drawbacks associated with BAC transgenesis. Initial reports indicate that such designer nucleases (DNs) allow the targeted insertion of transgenes into endogenous loci by direct injection of the targeting vector and mRNA/DNA encoding the predesigned nucleases into oocytes. This results in the transgene being inserted at a specific locus in the mouse genome, thus circumventing the drawbacks associated with BAC transgenesis.

Asunto(s)

Cromosomas Artificiales Bacterianos/genética , Desoxirribonucleasas/metabolismo , Técnicas de Transferencia de Gen , Animales , Desoxirribonucleasas/química , Genómica , Ratones , Estructura Terciaria de Proteína , Transgenes/genética

RESUMEN

Separating rat pups from their mothers during the early stages of life is an animal model commonly used to study the development of psychiatric disorders such as anxiety and depression. The present study investigated how soon after the termination of the maternal separation period behavioural and neuroendocrine abnormalities relevant to above-mentioned illnesses would manifest. Sprague Dawley rat pups were subjected to maternal separation (3 h per day from postnatal day 2 through 14) and their behaviour and HPA axis activity determined 7 d later. We also measured nerve growth factor levels in their hippocampi and assessed the DNA methylation status of the promoter region of exon 1(7) of the glucocorticoid receptor in this brain region. As early as 7 d after the termination of the adverse event, a change in behaviour was observed that was associated with increased plasma corticosterone release and elevated nerve growth factor levels in the hippocampus. No alteration in the methylation status of the exon 1(7) glucocorticoid receptor promoter region was observed. Our data indicate that early life adversity may lead to the rapid development of abnormal behaviours and HPA axis dysregulation though no epigenetic changes to the exon 1(7) glucocorticoid receptor promoter region occurred. We further propose that the observed increased neurotrophin levels reflect compensatory mechanisms that attempt to combat the long-term deleterious effects of maternal separation.

Asunto(s)

Corticosterona/sangre , Privación Materna , Trastornos del Humor/metabolismo , Factor de Crecimiento Nervioso/sangre , Receptores de Glucocorticoides/metabolismo , Estrés Psicológico/metabolismo , Animales , Secuencia de Bases/genética , Conducta Animal/fisiología , Corticosterona/análisis , Metilación de ADN/fisiología , Modelos Animales de Enfermedad , Epigénesis Genética/fisiología , Exones/genética , Femenino , Hipocampo/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipotálamo-Hipofisario/fisiopatología , Masculino , Datos de Secuencia Molecular , Trastornos del Humor/genética , Trastornos del Humor/fisiopatología , Factor de Crecimiento Nervioso/análisis , Sistemas Neurosecretores/fisiología , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/genética , Estrés Psicológico/genética , Estrés Psicológico/fisiopatología

RESUMEN

Antibody-dependent cellular cytotoxicity (ADCC) is one means by which macrophages (as well as natural killer cells and granulocytes) elicit a cytotoxic response. This is achieved via interaction of the Fc-gamma-receptor (CD64) with the Fc portion of antibody bound to target cells. We have created a chimeric CD64 molecule that incorporates a single chain Fv molecule, targeted against human carcinoembryonic antigen (CEA), fused to the membrane spanning and cytosolic domains of human CD64. Following adenoviral transfer to primary human monocytes, this chimeric CD64 receptor induced antigen-specific cytokine secretion during culture on immobilised CEA protein or on CEA-expressing tumour cells. Moreover, CEA targeted, but not control, monocytes effectively retarded CEA-positive tumour cell growth in vitro. Importantly, targeted monocyte cultures significantly reduced in vivo tumour growth rates in xenograft studies resulting in improved survival rates over that of control monocyte cultures. These data suggest that genetically directing monocytes against tumour antigens may be a useful means of achieving an immunotherapeutic response.

Asunto(s)

Antígeno Carcinoembrionario/inmunología , Terapia Genética/métodos , Inmunoterapia/métodos , Monocitos/inmunología , Neoplasias/terapia , Receptores de IgG/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Citocinas/inmunología , Ingeniería Genética , Proteínas Fluorescentes Verdes/genética , Humanos , Células Asesinas Naturales/inmunología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/inmunología , Proteínas Recombinantes/metabolismo , Linfocitos T/inmunología , Trasplante Heterólogo

RESUMEN

This study investigates P-gp activity in placental villous fragments and the possibility of upregulating its expression and function by retroviral transduction. In fresh fragments, cyclosporin A caused a significant increase in 3H-vinblastine accumulation (187+/-48% at 180 min n=4), consistent with multi-drug resistance activity. After 7 days in culture, villous fragments showed a similar increase in 3H-vinblastine accumulation (143+/-10% at 180 min n=4), which was not significantly different from that in fresh tissue. Following transduction, immunohistochemistry revealed increased P-gp expression. However, the distribution of the protein differed from that in controls, with P-gp being located throughout the tissue as opposed to the normal specific location on the maternal facing plasma membrane. Transduced explants showed a significantly larger increase in 3H-vinblastine accumulation in the presence of cyclosporin A than control explants (245+/-15.5% at 180 min, n=4), suggesting reduced capacity to efflux vinblastine. This study demonstrates P-gp activity in intact placental tissue which is maintained in explant culture. Retroviral transduction of P-gp to such tissue leads to increased but undirected expression of the protein. The consequent increased activity at sites such as the basal, fetal facing, plasma membrane probably explains the increased substrate accumulation within the tissue.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Vellosidades Coriónicas/metabolismo , Retroviridae/genética , Transducción Genética , Adulto , Vellosidades Coriónicas/efectos de los fármacos , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Expresión Génica , Humanos , Técnicas de Cultivo de Órganos , Embarazo , Regulación hacia Arriba , Vinblastina/metabolismo

Asunto(s)

Células de la Médula Ósea/citología , Diferenciación Celular/genética , Eritrocitos/citología , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Técnicas In Vitro , Factores de Transcripción

RESUMEN

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

Asunto(s)

Células de la Médula Ósea/citología , Proliferación Celular , Células Madre Hematopoyéticas/citología , Mucopolisacaridosis I/genética , Células del Estroma/citología , Adolescente , Antígenos CD34/biosíntesis , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Adhesión Celular , Células Cultivadas , Niño , Preescolar , Colágeno/metabolismo , Dermatán Sulfato/metabolismo , Células Madre Hematopoyéticas/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Iduronidasa/metabolismo , Lactante , Células Madre/metabolismo , Factores de Tiempo

RESUMEN

Fluticasone propionate (FP) and mometasone furoate (MF) are potent synthetic corticosteroids that are widely used as anti-inflammatory agents to treat respiratory diseases. As part of the assessment of the potential for side-effects associated with their use, their activities, not only at the glucocorticoid receptor (GR) but also at the other members of the steroid nuclear receptor family, have been compared. Cell-based functional systems were established to measure different aspects of GR function, as well as the activity at all the other steroid nuclear receptors. The effects of MF and FP on the GR were potent and indistinguishable. Neither corticosteroid showed any activity at the oestrogen receptor, while both were weak antagonists of the androgen receptor. FP was a relatively weak agonist of the progesterone receptor but MF was a very potent agonist of the progesterone receptor, giving activity at similar concentrations to those that stimulate the GR (concentration generating 50% maximal effect (EC50)=50 pM). Moreover, while FP was a weak antagonist of the mineralocorticoid receptor (concentration generating 50% maximal inhibitory effect=80 nM), MF displayed potent partial agonist activity (EC50=3 nM, 30%). Mometasone furoate is considerably less specific for the glucocorticoid receptor than fluticasone propionate, showing significant activity at other nuclear steroid receptors.

Asunto(s)

Androstadienos/farmacología , Antiinflamatorios/farmacología , Pregnadienodioles/farmacología , Receptores de Glucocorticoides/efectos de los fármacos , Animales , Línea Celular , Células Cultivadas , Femenino , Fluticasona , Humanos , Furoato de Mometasona

RESUMEN

One of the main barriers to more efficacious use of modern chemotherapeutic agents, is the collateral toxicity exhibited in normal, highly proliferative tissues, primarily the haemopoietic, gastrointestinal and pulmonary tissues. Drug resistance of tumours to these drugs compounds this problem. This review discusses the role of O6-alkylguanine-DNA alkyltransferase (ATase) in conferring protection against O6-alkylating agents in normal tissue, focusing mainly on the haemopoietic compartment. The development of mutant forms of ATase, which are resistant to the effects of soluble analogues of O6-alkylation such as O6-benzylguanine, is examined and the gene therapy approach of combining these two strategies to confer chemoprotection to vulnerable tissues whilst sensitising malignant tissue is reviewed.

Asunto(s)

Técnicas de Transferencia de Gen , Mutación , O(6)-Metilguanina-ADN Metiltransferasa/genética , Animales , Plaquetas/metabolismo , Humanos , Leucocitos/metabolismo , Factores de Tiempo

RESUMEN

In view of the recent report of a myeloproliferative syndrome in mice that had received an MDR-1-transduced haemopoietic graft, we have investigated the potential effects of MDR-1 expression on primitive haemopoietic cell growth and differentiation. Retroviral gene transfer was used to achieve exogenous expression of either MDR-1 or truncated nerve growth factor receptor (tNGFR) in the multipotent murine haemopoietic progenitor cell line, FDCP-mix. Following gene transfer, clonal lines were derived and FACS analysis confirmed appropriate expression of each transgene. MDR-1 (but not tNGFR) expression was associated with verapamil-sensitive rhodamine efflux and resistance to killing by etoposide. When growth factor responsiveness, proliferative capacity and differentiation capacity were examined, MDR-1 expressing FDCP-mix cells exhibited a normal phenotype and mimicked the response of tNGFR-expressing or untransduced FDCP-mix cells. Thus, in the model system we have used, MDR-1 does not perturb haemopoietic cell growth and development and our data do not support a myeloproliferative role for MDR-1.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Células Madre Hematopoyéticas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Antineoplásicos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Diferenciación Celular , División Celular , Células Clonales/citología , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Medios de Cultivo Condicionados/farmacología , Resistencia a Antineoplásicos , Etopósido/farmacología , Colorantes Fluorescentes/metabolismo , Genes MDR , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Ratones , Fragmentos de Péptidos/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Rodaminas/metabolismo , Transfección , Verapamilo/farmacología

RESUMEN

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.

Asunto(s)

Etopósido/toxicidad , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Adenina Fosforribosiltransferasa/genética , Animales , Antineoplásicos Fitogénicos/toxicidad , Ensayo Cometa , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Hibridación Fluorescente in Situ , Masculino , Ratones , Pruebas de Micronúcleos , Bazo/efectos de los fármacos , Bazo/enzimología , Inhibidores de Topoisomerasa II

RESUMEN

OBJECTIVE: Bone mineral density (BMD) in later life is a major determinant of osteoporotic fracture risk and has been shown to be under strong genetic influence. Transforming growth factor beta 1 (TGF-beta 1) is an important regulatory cytokine, is found in high concentrations in the bone matrix, and is a plausible candidate for the genetic regulation of BMD. METHODS: This study investigated whether a novel polymorphism within the TGF-beta 1 gene is associated with BMD in a large normal female population of 1706 dizygotic (DZ) twins (age range 18-76 yr). RESULTS: A C--->T [corrected] polymorphism was identified in intron 5, the T [corrected] allele having a frequency of 0.25. Subjects homozygous for the presence of the TGF-beta 1 T [corrected] allele had a 4% reduction in femoral neck BMD compared with the other two genotype groups (P<0.025). No effect was seen at the lumbar spine or ultradistal radius, or with calcaneal ultrasound measurements. Results were unaffected after adjustment for potential confounders. These findings were predominantly seen in pre-menopausal subjects, suggesting that this locus has an effect on the attainment of peak BMD. In pre-menopausal women, subjects who were homozygous for the T [corrected] allele had a 5-fold excess risk of having osteoporosis at the femoral neck compared with the other genotype groups. A within-pair analysis using the sibling transmission disequilibrium test confirmed these findings in pre-menopausal women and supported the candidacy of the TGF-beta 1 locus in the genetic regulation of hip BMD. CONCLUSIONS: These results indicate that allelic variation at the TGF-beta 1 gene contributes to the development of osteoporosis at the hip. The study also highlights the power of candidate gene analysis in twins, in whom loci having modest effects on disease risk can be identified.

Asunto(s)

Densidad Ósea/genética , Desequilibrio de Ligamiento/genética , Factor de Crecimiento Transformador beta/genética , Adolescente , Adulto , Anciano , Femenino , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Cadera , Humanos , Persona de Mediana Edad , Osteoporosis/genética , Premenopausia/genética , Factor de Crecimiento Transformador beta1 , Estudios en Gemelos como Asunto

RESUMEN

Gene therapy has thus far promised much and delivered little. Much of this has been due to deficiencies in the reagents and methodologies employed in early clinical trials. Recent technological advances in vectors and haemopoietic stem cell manipulation, coupled with improved pre-clinical assays of gene transfer and expression in re-populating stem cells give cause for greater optimism. Here we review these advances and indicate areas requiring further development before clinical gene therapy in the haemopoietic system becomes a widely applicable treatment modality.

Asunto(s)

Terapia Genética/métodos , Células Madre Hematopoyéticas/metabolismo , Animales , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Humanos , Transducción Genética/métodos , Transducción Genética/tendencias

RESUMEN

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.

Asunto(s)

Glucosafosfato Deshidrogenasa/metabolismo , Leuconostoc/enzimología , Arginina/genética , Sitios de Unión , Catálisis , Secuencia Conservada , Dimerización , Glucosa-6-Fosfato/metabolismo , Glucosafosfato Deshidrogenasa/genética , Glutamina/genética , Cinética , Lisina/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , NAD/metabolismo , NADP/metabolismo , Prolina , Termodinámica

RESUMEN

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos Fitogénicos/farmacología , Médula Ósea/efectos de los fármacos , Etopósido/farmacología , Transferencia de Gen Horizontal , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Vías de Administración de Medicamentos , Esquema de Medicación , Etopósido/administración & dosificación , Femenino , Masculino , Ratones , Pruebas de Micronúcleos

RESUMEN

Genetic transfer and expression of drug-resistance functions into haematopoietic stem and progenitor cells is a promising means to overcome both the acute and longterm side-effects of cytotoxic drugs in bone marrow. Here, we describe a functional analysis of a retroviral vector that co-expresses human cDNAs for multidrug resistance 1/P-glycoprotein (MDR1) and a double mutant of O(6)-alkylguanine-alkyltransferase (hATPA/GA) to high levels. The hATPA/GA protein contains two amino acid substitutions that render it resistant to compounds such as O(6)-benzylguanine that inhibit the wild-type protein which is often overexpressed in resistant tumour cells. Evidence for simultaneous drug resistance of genetically modified primary murine progenitor cells to colchicine or the podophyllotoxin etoposide, both covered by MDR1-mediated efflux activity, and the nitrosourea BCNU, which is counteracted by hATPA/GA, is presented using in vitro colony assays.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Animales , Antineoplásicos Alquilantes/efectos adversos , Carmustina/efectos adversos , Resistencia a Múltiples Medicamentos/genética , Genes MDR , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Compuestos de Nitrosourea/efectos adversos , O(6)-Metilguanina-ADN Metiltransferasa/genética , Podofilotoxina/efectos adversos , Transducción Genética

RESUMEN

Many of the problems with current anti-tumour therapies stem from a lack of specificity for tumour as opposed to normal tissues. To address the problem of collateral toxicity during anti-tumour chemotherapy we have been developing a gene therapy approach to protect normal tissues from the toxic and potentially mutagenic effects of chemotherapeutic agents. As a paradigm for this we have been examining the potential of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) to confer genetic chemoprotection to the bone marrow. By transfer and expression of a mutant form of this protein, which is resistant to inactivation by the tumour sensitising agent O6-benzylguanine (O6-beG), we have been able to demonstrate protection of murine bone marrow in vitro from the cytotoxic and clastogenic effects of O6-beG in combination with the anti-tumour agent temozolomide. This protection is seen in multiple lineages, including erythroid and granulocyte/macrophage progenitors, as well as more primitive cells. Importantly, significant protection of the platelet lineage is also seen, with faster recovery of platelets. The multi-lineage protection seen has encouraged us to take this approach forward to clinical trial in the near future.

Asunto(s)

Resistencia a Medicamentos/genética , Ingeniería Genética , Animales , Antineoplásicos/efectos adversos , Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Reparación del ADN/genética , Femenino , Terapia Genética/métodos , Humanos , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/terapia , O(6)-Metilguanina-ADN Metiltransferasa/genética

RESUMEN

BACKGROUND AND OBJECTIVE: As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. DESIGN AND METHODS: To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). RESULTS: An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. INTERPRETATION AND CONCLUSIONS: A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.

Asunto(s)

Glicoproteínas de Membrana/genética , NADPH Oxidasas , Retroviridae/crecimiento & desarrollo , Células 3T3 , Animales , Northern Blotting , Southern Blotting , Western Blotting , ADN Complementario/genética , Técnicas de Transferencia de Gen/normas , Vectores Genéticos/química , Humanos , Glicoproteínas de Membrana/farmacología , Ratones , NADPH Oxidasa 2 , ARN Viral/biosíntesis , Retroviridae/efectos de los fármacos , Retroviridae/genética , Proteínas Virales/análisis

RESUMEN

Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, approximately 50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P < .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P < .05) and 37% (P < .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of transgene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P < .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis.

Asunto(s)

Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Células Madre Hematopoyéticas/efectos de los fármacos , Compuestos de Mostaza Nitrogenada/farmacología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Ensayo de Unidades Formadoras de Colonias , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Mutágenos/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/biosíntesis , O(6)-Metilguanina-ADN Metiltransferasa/genética , Retroviridae/genética

RESUMEN

The effect of expression of an O6-benzylguanine (O6-beG)-resistant mutant (hATPA/GA) of human O6-alkylguanine-DNA alkyltransferase (ATase) on the in vivo toxicity and clastogenicity of the anti-tumour agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) to murine bone marrow has been investigated. When compared with control animals, the bipotent granulocyte-macrophage colony-forming (GM-CFC) progenitor population of the hATPA/GA transduced mice were somewhat more resistant to BCNU (1.4-fold, P = 0.047) and this effect was more significant in the presence of the ATase inactivator O6-beG (3. 5-fold, P = 0.001). The polychromatic erythrocytes were also significantly protected against BCNU-induced clastogenicity both in the presence (P < 0.001) and absence of O6-beG (P < 0.05). The primitive, multipotent spleen colony-forming cells (CFU-S) in these animals also showed moderate (1.6-fold, P = 0.034) protection in the absence of O6-beG but in the presence of the inactivator they remained as sensitive to BCNU toxicity as those in the control animals (P = 0.133). This result contrasts with previous findings demonstrating significant hATPA/GA-mediated, O6-beG-resistant protection against the toxicity and clastogenicity of a number of O6-alkylating agents, including temozolomide, fotemustine and chlorozotocin. The possibility that our strategy for protective gene therapy may be highly agent and cell-type specific is unexpected and has possible implications for clinical trials of this approach using BCNU or related agents.

Asunto(s)

Carmustina/toxicidad , Terapia Genética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Nucleotidiltransferasas/metabolismo , Bazo/citología , Animales , Antineoplásicos/toxicidad , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Resistencia a Antineoplásicos , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Granulocitos/citología , Granulocitos/efectos de los fármacos , Granulocitos/enzimología , Guanina/análogos & derivados , Guanina/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Inmunohistoquímica , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ratones , Pruebas de Micronúcleos , Mutágenos/toxicidad , Mutación , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/genética , Bazo/efectos de los fármacos , Bazo/enzimología , Bazo/metabolismo , Transducción Genética

RESUMEN

Following transduction with a retrovirus (SF1MIH) expressing both the multidrug resistance 1 (MDR1) and O6-alkylguanine-DNA-alkyltransferase (ATase) proteins, human erythroleukaemic progenitor (K562) cells were isolated which were resistant to killing by the MDR1 substrate, colchicine. In colony-forming survival assays, K562-SF1MIH cells exhibited resistance to colchicine and doxorubicin, as well as to the O6-alkylating agents N-Methyl-N-nitrosourea (MNU) and temozolomide. Furthermore, the resistance to doxorubicin was abolished by preincubation with the MDR1 inhibitor verapamil while resistance to MNU was ablated by the specific ATase inactivator, O6-benzylguanine (O6-beG) confirming that resistance to doxorubicin and MNU was conferred by MDR1 and ATase, respectively. When K562-SF1MIH were exposed to combinations of colchicine and MNU or doxorubicin and temozolomide, simultaneous resistance to these agents was observed. Thus, transduction of K562 with SF1MIH conferred dual resistance to these cells. These data offer the prospect of designing vectors that will confer resistance to entire regimens of chemotherapy rather than just to individual components of such drug cocktails, thereby substantially increasing the efficacy of therapy. Furthermore, the use of such dual expression constructs is likely to be highly informative for the design of effective in vivo selection protocols, an issue likely to make a major impact in a clinical context in gene therapy in the near future.

Asunto(s)

Antineoplásicos/uso terapéutico , Resistencia a Múltiples Medicamentos/genética , Células Madre Hematopoyéticas/fisiología , O(6)-Metilguanina-ADN Metiltransferasa/genética , Retroviridae/genética , Transducción Genética/genética , Resistencia a Antineoplásicos/genética , Genes MDR/genética , Vectores Genéticos , Células Madre Hematopoyéticas/enzimología , Humanos , Células K562 , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Retroviridae/enzimología