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Biomedical subjects

G D Ginder

Publications and source records attributed to G D Ginder.

At least 37 records · Page 2Linked to original sources
PubMed1991

Cell-type specificity of interferon-gamma-mediated HLA class I gene transcription in human hematopoietic tumor cells.

Major histocompatibility complex class I gene expression plays a central role in cellular immunity and tumor surveillance. A substantial proportion of spontaneous tumors are class I-deficient and numerous experiments have suggested that alterations in class I expression may alter oncogenicity and, as a result, have potential therapeutic impact. Interferons (IFNs) are able to upregulate class I expression by mechanisms that remain to be elucidated, but which appear to be IFN- and cell-type specific. We have characterized in detail the in vivo class I transcriptional response to IFN-gamma in two human hematopoietic tumor cell lines, the class I-deficient K562 cell line and the class I-positive Ramos cell line. In each, IFN-gamma induces a rapid increase in class I transcription, which is sustained in Ramos cells, but transient in K562 cells. In each, stimulation by IFN-gamma is dependent on ongoing protein synthesis, suggesting the requirement for production of a "primary response" protein. These data suggest that more than one type of IFN-gamma-induced signal is operative in the transcriptional response to IFN-gamma. Cycloheximide alone is also capable of inducing a rapid increase in class I transcription in both cell types, suggesting that constitutive attenuation of class I transcription may be a common phenomenon, and that IFN-gamma may act, in part, by interfering with such attenuation.

Antibodies, Monoclonal↗
PubMed1991

5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

Acetylation↗
PubMed1990

BVAC ablative chemotherapy followed by autologous bone marrow transplantation for patients with advanced lymphoma.

Forty-one consecutive patients with lymphoma resistant to conventional combination chemotherapy have been entered into a study in which chemo-ablative therapy and autologous marrow rescue were used with curative intent. The actuarial proportion of 20 patients with Hodgkin's lymphoma remaining alive and free of recurrent disease is 49%, while that for 21 patients with non-Hodgkin's lymphoma is 41%. Our clinical approach to these patients involved a strategy whereby lymphomatous nodes greater than 2 cm in diameter that persisted despite salvage chemotherapy were given boost radiation therapy immediately before chemo-ablation. However, patients with this variable had a significantly lower survival due to septic complications rather than recurrent disease. We conclude that the treatment strategy used in this study with some modification may improve further on the already high probability of long-term disease-free survival experienced by this group of patients.

Adolescent↗
PubMed1990

A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer.

Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA.

Animals↗
PubMed1990

Binding of HMG 17 to mononucleosomes of the avian beta-globin gene cluster in erythroid and non-erythroid cells.

The binding of HMG 17 to stripped core mononucleosomes containing DNA from the avian beta-globin gene cluster was examined to determine whether binding in vitro in this developmentally-regulated gene domain was associated with transcriptional activity or DNaseI-sensitivity in intact nuclei. Mononucleosomes were prepared from primitive and definitive stage embryonic red blood cells of chick embryos, adult reticulocytes, adult reticulocytes in which embryonic rho-globin transcription was induced, and adult thymus cells. Preferential binding by HMG 17 to mononucleosomes containing the beta-globin gene cluster was confined to erythroid-derived mononucleosomes that contain the embryonic rho-globin gene, the adult beta-globin gene, and DNA sequences located between these two genes, but not to those that contain the embryonic epsilon-globin gene. Comparison of these results to the known patterns of transcription and DNaseI-sensitivity within the beta-globin gene cluster shows that HMG 17 binding, although tissue-specific, does not correlate directly with either DNaseI-sensitivity or active gene transcription, but is dependent on other factors present in core mononucleosomes from this active gene domain.

Animals↗
PubMed1989

Heparin binds to intact mononucleosomes and induces a novel unfolded structure.

It has been previously shown that heparin can bind to chromatin and enhance transcriptional activity. To characterize this phenomenon further, we have studied the interaction of heparin with isolated core mononucleosomes from avian reticulocytes. The results of these studies suggest that heparin bound reversibly to intact core mononucleosomes to induce a new structure, identified by decreased electrophoretic mobility and altered circular dichroism spectra. This altered nucleosome conformation exhibits 3-5-fold increased sensitivity to digestion by the nuclease, DNase I, and allows more efficient passage of RNA polymerase. At higher concentrations of heparin, core histones were completely removed from DNA. The finding of a reversible nucleosome-heparin complex in which core DNA is readily accessible to both RNA polymerase and the nuclease DNase I is discussed in the context of transcriptionally active chromatin.

Animals↗
PubMed1988

Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells.

An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis-regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.

Acetylation↗
PubMed1988

Allogeneic marrow grafting with partially mismatched, unrelated marrow donors.

Forty patients with advanced hematologic malignancies or severe aplastic anemia received marrow grafts from partially mismatched, unrelated marrow donors. All patients were administered conventional prophylaxis for acute graft-v-host disease (GVHD) consisting of methotrexate and low-dose glucocorticoids. All but two patients who survived at least 30 days showed durable engraftment. Six patients survive 17+ to 36+ months following transplantation. Severe acute GVHD was seen in 47% of the patients; however, no direct correlation between GVHD and the degree of mismatching could be determined. Fatal infections were seen in 29 patients, and in the majority the infection occurred after the granulocyte count had risen to greater than 500 cells/microL. We conclude that the problems encountered in this pilot study can potentially be solved, and that further studies with this type of marrow grafting are warranted.

Adolescent↗
PubMed1987

Trans-activation of an upstream early gene promoter of bovine papilloma virus-1 by a product of the viral E2 gene.

The approximately 1000 nucleotide long upstream regulatory region (URR) of bovine papilloma virus-1 (BPV-1) contains a cis element which responds to trans-activation by a diffusible factor encoded in the viral E2 open reading frame (ORF). A series of URR DNA fragments have been linked to two heterologous genes, bacterial chloramphenicol acetyl transferase (cat) or herpes simplex virus-1 thymidine kinase (tk), and tested in transient transfection assays for transcription initiating at the authentic upstream early viral promoter, P89. Transcriptional activity of the P89 promoter was greatly elevated in the presence of the E2 trans-activator gene product. The E2-responsive cis element (E2R) of P89 has been mapped to sequences -277 to -131 nucleotides upstream from the transcription start site (BPV nucleotide 89). The E2R element functioned as a strong transcriptional enhancer in cis with the SV40 early or the tk promoter in the presence, but not in the absence, of the E2 gene product. However, several heterologous promoters which lack sequences related to the E2R element were also trans-activated in transient cotransfections by a function encoded in the E2 ORF of BPV-1, albeit to a much lesser extent. In addition to activation of early viral gene transcription, the E2 regulatory gene(s) may therefore have the potential to alter cellular gene expression.

Animals↗
PubMed1987

Negative and positive regulation of human leukocyte antigen class I gene transcription in K562 leukemia cells.

The mechanism of transcriptional activation of human leukocyte antigen class I genes by gamma interferon and 5-azacytidine was studied in K562 human leukemia cells. Nuclear run-on transcription assays with various protein and RNA synthesis inhibitors yield evidence for both stimulation of a positive regulatory factor and inhibition of an mRNA that codes for a labile repressor. A novel mechanism is proposed to explain how 5-azacytidine can activate repressed genes without affecting DNA methylation.

Azacitidine↗
PubMed1986

Preferential in vitro binding of high mobility group proteins 14 and 17 to nucleosomes containing active and DNase I sensitive single-copy genes.

High mobility group (HMG) proteins 14 and 17 bind to mononucleosomes in vitro, but the exact nature of this binding has not been clearly established. A new method was developed to allow direct membrane transfer of DNA from HMG 14/17 bound and unbound nucleosomes, which have been separated by acrylamide gel electrophoresis. Hybridization analysis of membranes obtained by this method revealed that the HMG 14/17 bound nucleosomes of avian erythrocytes and rat hepatic tumor (HTC) cells were enriched, about 2-fold, in actively transcribed genes and also inactive but DNase I sensitive genes. Nucleosomes containing inactive, DNase I resistant genes were bound by HMG 14/17, but not preferentially. Several factors that have been reported to greatly influence the binding of HMG 14/17 to nucleosomes in vitro were tested and shown to not account for the preferential binding to DNase I sensitive chromatin. These factors include nucleosomal linker DNA length, single-stranded DNA nicks, and DNA bulk hypomethylation. An additional factor, histone acetylation, was preferentially associated with the HMG 14/17 bound chromatin fraction of avian erythrocytes, but it was not associated with the HMG 14/17 bound chromatin fraction of metabolically active HTC cells. The latter finding was true for all kinetic forms of histone acetylation.

Acetylation↗
PubMed1986

Gamma interferon and 5-azacytidine cause transcriptional elevation of class I major histocompatibility complex gene expression in K562 leukemia cells in the absence of differentiation.

We studied the effects of gamma interferon (IFN-gamma) on HLA class I gene expression, differentiation, and proliferative capacity of K562 human leukemia cells. In the uninduced state, K562 cells show little or no class I gene expression but actively express the erythroid-specific gamma-globin gene as well as genes associated with cell proliferation, including the transferrin receptor, c-myc, and alpha-actin genes At both the surface protein and mRNA levels, IFN-gamma induces class I and beta 2-microglobulin gene expression, but does not alter the expression of the gamma-globin, transferrin receptor, c-myc, or alpha-actin genes. A 10-fold maximal induction of both class I surface protein and mRNA occurs at 48 h and is reversible upon withdrawal of IFN-gamma from the culture medium. In vitro nuclear run-on transcription assays were performed to directly establish that IFN-gamma exerts an early effect at the level of transcription, with maximal transcription rates occurring within 4 h. The difference between the time course of transcription induction and that of mRNA accumulation suggests that the regulation of class I gene expression in this human leukemic cell line also involves posttranscriptional mechanisms. Measurements of cell proliferation rates and cell cycle distribution, as well as the reversibility of the effects of IFN-gamma, demonstrate that the selective induction of class I genes in these cells occurs in the absence of differentiation.

Azacitidine↗
PubMed1985

The use of partially matched, unrelated donors in clinical bone marrow transplantation.

It is estimated that 60-70% of patients who might benefit from a bone marrow transplant will not have a suitably matched, related donor. We have, therefore, designed a clinical experiment to test the safety and feasibility of using marrow from partially matched, unrelated donors. This paper details our transplant experience in the first eight patients with leukemia. The first four patients had advanced leukemia at the time of transplantation. Each showed hematopoietic recovery, but all died from septic complications largely related to extended neutropenia encompassing both the pre-marrow-grafting and the post-marrow-grafting period. The next four patients were in remission at the time of transplantation. Each showed prompt and sustained hematopoiesis with variable graft-versus-host disease (GVHD). No acute or chronic GVHD was seen in two patients, grade II (skin only) was seen in one patient, and grade IV (skin, liver, and gut) was seen in one patient. One patient has died from sepsis five-and-one-half months following transplantation, and three are alive and well six-and-one-half to nine-and-one-half months postengraftment. This preliminary experience, together with several case reports in the literature, leads us to conclude that bone marrow transplantation with partially matched, unrelated marrow is a safe and feasible approach. If these results are confirmed by longer follow-up in a larger group of patients, the development of marrow donor pools would appear to be justified.

Adolescent↗
PubMed1985

Transcriptional activation of an embryonic globin gene in adult erythroid cells in vivo.

We have studied the mechanism and level of activation of the normally silent rho globin gene in adult erythroid cells of anemic chickens treated with 5-azacytidine and sodium butyrate. We have shown that while demethylation of the rho globin gene does not result in high levels of specific mRNA, demethylation does appear to be a prerequisite for sodium butyrate to cause such an effect. Runoff nuclear transcription assays were used to demonstrate that 5-azacytidine plus sodium butyrate treatment act to cause transcriptional activation of the rho gene in adult animals. However, the data also show that there may be post-transcriptional down-regulation of mRNA levels in adult red cells, since the level of rho gene nuclear runoff relative to beta exceeds the corresponding stable mRNA ratios for the two gene products. These data are consistent with a model in which developmental switching of the chicken globin genes may involve both transcriptional regulation and one or more levels of post-transcriptional regulation. Our studies of histone acetylation in erythroid cells from butyrate treated animals show that no bulk changes in histone acetylation can be detected. While transient localized changes in the fast kinetic form of histone acetylation cannot be excluded, these results are consistent with the hypothesis that the transcriptional activation of the rho gene by sodium butyrate in this animal model may be mediated by some trans-acting transcription regulating factor. Thus, the requirement for both 5-azacytidine and sodium butyrate for gene activation could reflect a mechanism which involves both an alteration in a specific regulatory DNA recognition sequence (by demethylation) and a change in either the specificity or amount of a stimulatory (or inhibitory) trans acting factor. These proposed mechanisms are directly testable with currently available techniques.

Acetylation↗
PubMed1984

Activation of a chicken embryonic globin gene in adult erythroid cells by 5-azacytidine and sodium butyrate.

Adult White Leghorn chickens were rendered anemic by injection with 1-acetyl-2-phenylhydrazine and then treated with parenteral 5-azacytidine, and levels of embryonic globin RNA in circulating reticulocytes were measured. A very small but detectable amount of correctly initiated embryonic p-type globin RNA was detected in reticulocytes from birds treated with 5-azacytidine, while none was detected in reticulocytes from those receiving only phenylhydrazine or phenylhydrazine plus 1-beta-D-arabinofuranosylcytosine (cytosine arabinonucleoside). An attempt to increase embryonic globin RNA induction by treatment with parenteral sodium butyrate after 7 days of 5-azacytidine administration resulted in a 5- to 10-fold increase in the level of embryonic globin RNA. However, sodium butyrate did not induce embryonic gene expression when given alone or after treatment with cytosine arabinonucleoside. Sodium butyrate treatment also caused a DNase I-hypersensitive site to be exposed at the 5' end of the rho-globin gene only after 5-azacytidine induced demethylation of several CpG sites in and around the gene. The implications of this model of gene activation in vivo are discussed in the context of multistep gene regulation.

Animals↗