Search PubMed⌕ Search

Biomedical subjects

A Bank

Publications and source records attributed to A Bank.

At least 55 records · Page 3Linked to original sources
PubMed1994

Retrovirus-mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells.

We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer. Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, efficiency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 micrograms/ml taxol. In uninfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotein was found on 17-25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

In vitro transcription from the human A gamma-globin gene promoter.

We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.

Base Sequence↗
PubMed1993

Long-term high-level expression of human beta-globin occurs following transplantation of transgenic marrow into irradiated mice.

When the human beta-globin gene is transferred into the bone marrow cells of live mice, its expression is very low. To investigate the reason for this, we transferred the bone marrow of transgenic mice containing and expressing the human beta-globin into irradiated recipients. We demonstrate that long-term high level expression of the human beta-globin gene can be maintained in the marrow and blood of irradiated recipients following transplantation. Although expression decreased over time in most animals because of host marrow reconstitution, the ratio of human beta-globin transgene expression to endogenous mouse beta-globin gene expression in donor-derived erythroid cells remained constant over time. We conclude that there is no inherent limitation to efficient expression of an exogenous human beta-globin gene in mouse bone marrow cells following marrow transplantation.

Animals↗
PubMed1992

Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells.

Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene. Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo. To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector. We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression. Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly. These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.

3T3 Cells↗
PubMed1992

Transfer and expression of the human multiple drug resistance gene into live mice.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Punctal dyeing: an adjunct to intraoperative lacrimal punctal identification.

During lid operations, identifying the lacrimal punctum is sometimes difficult. The difficulty may be increased following local anesthetic infiltration into the medial canthus and manipulation of the lid tissues. Punctal dyeing with a skin marking pen is a simple technique that improves visualization of the lacrimal punctum during surgery in this region. Preceded by punctal dilatation, it lasts for the duration of standard lid or lacrimal procedures.

Coloring Agents↗
PubMed1991

A DNA-binding factor in adult hematopoietic cells interacts with a pyrimidine-rich domain upstream from the human delta-globin gene.

To date, DNA-binding factors with a developmental pattern of expression have not been described in human erythroid cells to explain the switch from fetal (gamma-) to adult (delta- and beta-) globin gene expression. Here we describe a factor present in nuclear extracts from adult mouse and human hematopoietic cells that binds to an oligopyrimidine repeat approximately 960 base pairs upstream from the human delta-globin gene. The binding site for the factor is within an unusual 250-base-pair domain that is greater than 95% pyrimidines on one strand. This domain is preferentially sensitive to S1 nuclease in supercoiled plasmids, indicating that it can adopt an alternative non-B-DNA conformation. A number of S1-sensitive sites within the domain, including the factor-binding site, have sequence characteristics associated with the formation of a triple helix (H-DNA). The position of the binding site between the fetal and adult beta-globin-like genes, its potential for adopting an unusual secondary structure, and the restricted activity of the binding factor to adult hematopoietic tissues suggest possible roles in hematopoietic cell development and hemoglobin switching.

Adult↗
PubMed1991

The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562.

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3' enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3' enhancer is erythroid-specific but not developmental stage- or gene-specific.

Cloning, Molecular↗
PubMed1991

ABL oncogene expression during erythroleukemia cell differentiation.

The translocation between chromosome 9 and chromosome 22 which creates the Philadelphia chromosome moves the ABL oncogene from its normal location on chromosome 9 and fuses it with a portion of the BCR gene on chromosome 22. This new BCR/ABL fusion gene generates a unique 8.7 kilobase (kb) RNA which codes for a new 210 kilodalton (kd, p210) protein which has a protein tyrosine kinase activity that is greatly increased in comparison to the normal ABL protein. The human K562 cell line was derived from a patient with CML, and serves as one model for the regulation of expression of the ABL and BCR/ABL genes. This study examines the expression of the BCR/ABL fusion gene and the normal ABL gene in relation to differentiation and changes in proliferative state. The expression of both the normal ABL transcripts and the BCR/ABL fusion transcript decrease approximately ten-fold when the cells are induced to differentiate with hemin. In contrast, expression of the MYC oncogene is unaffected by hemin-induced differentiation. The results suggest that both ABL and BCR/ABL expression vary in proportion to the differentiation of the cells, but minimally if at all as a function of the cells' proliferative state.

Cell Differentiation↗
PubMed1991

Gene transfer of adenosine deaminase into primitive human hematopoietic progenitor cells.

The inherited deficiency in adenosine deaminase (ADA), which results in severe combined immunodeficiency, is generally regarded as an optimal model for the development of human somatic gene therapy. The ideal target for the correction of ADA deficiency and other lympho-hematopoietic disorders would be the hematopoietic stem cell. We have used a combination of recombinant human interleukins-3 and -6 to stimulate the proliferation of primitive human hematopoietic progenitor cells during a period of co-cultivation with irradiated cells producing high titers of an ADA-transducing retroviral vector packaged in amphotropic particles. In a series of nine experiments, an average of 83% of the clonogenic progenitors (CFU-E and CFU-GM) were found to have acquired the transferred sequence as determined by polymerase chain reaction analysis. In addition, in two experiments, 24-44% of the clonogenic progenitors derived from long-term myeloid cultures 9 weeks post-transduction were found to contain vector sequence. The latter cells are derived from so-called "long-term culture-initiating cells" (LTC-IC), which are primitive cells probably related to hematopoietic stem cells. Moreover, the transduced ADA enzyme was found to be expressed in both normal and ADA-deficient erythroid colonies, and in the nonadherent cells of long-term bone marrow culture for at least 2 weeks at levels that approximate the endogenous ADA levels of normal erythroid cells. These results indicate that the ADA coding sequence can efficiently be introduced by retroviral gene transfer into both committed and primitive human hematopoietic progenitor cells, and that this will result in adequate expression of the transduced enzyme in the progeny of committed hematopoietic progenitors.

Adenosine Deaminase↗
PubMed1991

Arteriovenous fistula complicating lumbar disc surgery. Case report.

Arteriovenous fistula is a rare complication of lumbar disc surgery. The diagnosis is often delayed and the condition erroneously treated as deep venous thrombosis, because of primary leg swelling. A case is presented in which the diagnosis was confirmed 18 months after a second lumbar disc operation.

Adult↗
PubMed1991

Somatic gene therapy.

Many problems obviously continue to exist in gene transfer using retroviruses as a means of inserting foreign DNA into hematopoietic stem cells, especially with regulated genes such as the human beta globin genes. First, it is unclear whether the available retroviral vectors will infect enough stem cells for gene transfer to be successful over the long term. Second, there may be sequences necessary for normal beta globin gene expression that may also inhibit the normal retroviral life cycle, thus decreasing the efficiency of gene transfer or gene expression. It seems clear that in order to optimize the success of gene transfer, the highest possible titer of viral production is necessary. New approaches are aimed at increasing viral titer. The transfect/infect method appears useful. Growth factors may also be useful by increasing stem cell proliferation. Single growth factors may not be sufficient to optimize stem cell cycling. To date, interleukin-3 seems to be the single most useful growth factor, although interleukin-3 with interleukin-6 or other combinations of growth factors including interleukin-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) appear to have potential. Future work also is required to optimize the number of marrow stem cells needed for successful transplantation. Long-term bone marrow culture and stromal cell cultures may provide new and improved marrow culture conditions for achieving this goal. Improvements in the efficiency of both gene transfer and gene expression are necessary before we can consider the concept of gene transfer for the treatment of various hematologic genetic diseases in humans.

Animals↗
PubMed1990

Protein-DNA interactions upstream from the human A gamma globin gene.

We have used DNAase I footprinting and the gel mobility shift assay to study proteins which bind to promoter elements located between -140 and -382 upstream of the human A gamma globin gene. Footprints are found with both erythroid and nonerythroid nuclear extracts at three sites: from -294 to -264, -242 to -227, and -189 to -172 from the transcription initiation site. An erythroid-specific footprint is identified from -194 to -189. We demonstrate that two known transcription factors, the ubiquitous octamer-binding protein OTF-1 and the erythroid regulatory factor NFE-1, bind to the -194 to -172 region and that their footprints overlap. Binding of OTF-1 to this region is reduced by a mutation at -175 associated with a form of non-deletion hereditary persistence of fetal hemoglobin. We conclude that OTF-1 may compete with NFE-1 for the -175 binding site, possibly functioning as a repressor of gamma globin transcription.

Base Sequence↗
PubMed1990

Efficient gene transfer in live mice using a unique retroviral packaging line.

Irradiated mice were transplanted with cells containing a foreign gene to evaluate gene transfer and expression as a model for gene therapy. Using a uniquely safe and efficient retroviral packaging line developed in this laboratory (GP + E86), we demonstrate efficient and safe long-term transfer of the neomycin resistance (neoR) gene into mice. By infusing cells obtained from spleen colonies of primary post-transplant mice marked with the neoR gene into irradiated recipients, secondary and tertiary generations of recipient mice were produced. There was very low reconstitution activity of single stem cells in these successive generations of mice. We conclude that many more than one stem cell is necessary for successful long-term bone marrow transplantation in mice, presumably as a result of the relatively low frequency of stem cell cycling.

Animals↗
PubMed1990

Stopping the biologic clock for globin gene switching.

The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if expression of the adult (beta) globin genes is defective and produces little or no protein, as in the beta-thalassemias. Preventing or reversing the globin gene switch could provide a way of keeping the abnormal globin genes "silent" and maintaining expression of the fetal globin gene. We have identified a class of agents which, when present in elevated plasma concentrations during gestation, inhibits the gamma----beta-globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma-globin and decrease beta-globin expression in cultured erythroid cells of patients with beta-thalassemia. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited and even reversed in some fetal lambs. Histone hyperacetylation, which maintains active chromatin structure, and an effect on the gamma-globin promoter appear to be mechanisms of action involved. These data suggest that inhibiting expression of abnormal beta-globin genes by pharmacologic means may in the future be possible for treatment of individuals with beta-globin disorders.

Anemia, Sickle Cell↗
PubMed1990

The regulation of gamma-globin gene expression.

In summary, our analysis indicates that important sequences for the proper initiation of fetal gene transcription in fetal cells are located in the gamma-globin [sequence: see text] promoter. These sequences are sufficient for tissue-specific expression but not induction in K562 cells. Sequences in the gamma-globin IVS-2 and the beta-globin 3' enhancer increase gamma beta and gamma-Neo transcripts when cells containing these genes undergo erythroid maturation as measured by induction with hemin. The mechanism by which these sequences exert their effect remains to be elucidated. [see text] Multiple protein factors bind to both the gamma promoter and the beta 3' enhancer. Both of these regions contain binding sites for the erythroid-specific factor NFE-1 and the octamer binding factor OTF-1. In the gamma upstream region, there may be a competition between OTF-1 binding and NFE-1 binding that affects gamma gene regulation. Our results indicate that the beta 3' enhancer interacts with the gamma gene promoter to permit increased gamma gene expression. We have developed a model for globin gene switching that takes into consideration the effect of cis-acting sequences on globin gene transcription. A similar model of hemoglobin switching in chickens has been proposed by Choi and Engel. In our model, competition for the beta-globin 3' enhancer is involved in stage-specific transcriptional activation of gamma-globin genes in fetal cells and beta-globin genes in adult cells. In adult cells the protein-protein interactions between adult cell-specific factors interacting with the beta-globin promoter and erythroid-specific factors interacting with the beta 3' enhancer would activate transcription of the beta-globin gene. In fetal cells protein-protein interactions between fetal cell-specific factors interacting with the gamma-globin promoter and erythroid-specific factors interacting with the beta 3' enhancer would activate the transcription of the gamma-globin genes.

Base Sequence↗