Search PubMed⌕ Search

Biomedical subjects

D W Golde

Publications and source records attributed to D W Golde.

At least 37 records · Page 2Linked to original sources
PubMed1998

A phase I trial of a single high dose of idarubicin combined with high-dose cytarabine as induction therapy in relapsed or refractory adult patients with acute lymphoblastic leukemia.

Relapsed or refractory adult acute lymphoblastic leukemia (ALL) carries a grave prognosis. The most promising strategy for curing these patients is through re-induction chemotherapy followed by successful allogeneic transplant. We studied a new high-dose induction regimen in order to improve the outcome for these patients. Eighteen adult patients with relapsed/refractory ALL were treated on a phase I study of high-dose cytarabine combined with a single escalating dose of idarubicin. Five patients had primary refractory disease and 13 were treated in refractory relapse. Nine patients (50%) had Ph+ ALL. The induction regimen was cytarabine 3 g/m2/day intravenously days 1-5 and idarubicin as a single intravenous dose on day 3. G-CSF 5 microg/kg subcutaneously every 12 h was started on day 7. The initial idarubicin dose was 20 mg/m2 with dose escalations of 10 mg m2. Cohorts of three patients were treated at each idarubicin dose level. Unacceptable toxicity was encountered at 50 mg/m2 with one death from infection and one death from cardiotoxicity in a patient with significant prior anthracycline exposure. There were no instances of grade 4 non-hematologic toxicity encountered at idarubicin doses of 20 mg/m2, 30 mg/m2, or 40 mg/m2. The data suggest a dose-response relationship for increasing doses of idarubicin with 0/3 complete responses (CR) at 20 mg/m2, 1/3 CR at 30 mg/m2, and 7/12 (58%) CR at idarubicin doses > or = 40 mg/m2. We conclude that concomitant administration of cytarabine 3 g/m2/day x 5 and high-dose idarubicin at 40 mg/m2 as a single dose on day 3 can be administered safely to patients with refractory and relapsed ALL.

Adult↗
PubMed1998

Expression of the human GM-CSF receptor alpha subunit in Saccharomyces cerevisiae.

The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 45 kDa membrane protein with a higher apparent molecular weight of 50-85 kDa due to glycosylation. Previously, we had demonstrated that N-glycosylation plays a critical role in the GM-CSF receptor-ligand interaction. To assess the activity of the alpha subunit of the human GM-CSF receptor (GMRalpha) in a lower eukaryote, we expressed GMRalpha in the yeast S. cerevisiae and found that the protein has a lower apparent molecular weight compared with that expressed in mammalian cells. Using indirect immunofluorescence microscopy, we showed that GMRalpha protein expressed in yeast localizes to the plasma membrane. Although the yeast-expressed GMRalpha is able to interact with anti-GMRalpha antibody, the heterologously expressed receptor does not bind GM-CSF. Our results indicate that specific sites and/or forms of glycosylation of the GM-CSF receptor are crucial for ligand binding.

Gene Expression↗
PubMed1997

Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters.

Vitamin C concentrations in the brain exceed those in blood by 10-fold. In both tissues, the vitamin is present primarily in the reduced form, ascorbic acid. We identified the chemical form of vitamin C that readily crosses the blood-brain barrier, and the mechanism of this process. Ascorbic acid was not able to cross the blood-brain barrier in our studies. In contrast, the oxidized form of vitamin C, dehydroascorbic acid (oxidized ascorbic acid), readily entered the brain and was retained in the brain tissue in the form of ascorbic acid. Transport of dehydroascorbic acid into the brain was inhibited by d-glucose, but not by l-glucose. The facilitative glucose transporter, GLUT1, is expressed on endothelial cells at the blood-brain barrier, and is responsible for glucose entry into the brain. This study provides evidence showing that GLUT1 also transports dehydroascorbic acid into the brain. The findings define the transport of dehydroascorbic acid by GLUT1 as a mechanism by which the brain acquires vitamin C, and point to the oxidation of ascorbic acid as a potentially important regulatory step in accumulation of the vitamin by the brain. These results have implications for increasing antioxidant potential in the central nervous system.

Animals↗
PubMed1997

Increased facilitated transport of dehydroascorbic acid without changes in sodium-dependent ascorbate transport in human melanoma cells.

Many cell types transport vitamin C solely in its oxidized form, dehydroascorbic acid, through facilitative glucose transporters. These cells accumulate large intracellular concentrations of vitamin C by reducing dehydroascorbic acid to ascorbate, a form that is trapped intracellularly. Certain specialized cells can transport vitamin C in its reduced form, ascorbate, through a sodium-dependent cotransporter. We found that normal human melanocytes and human malignant melanoma cells are able to transport vitamin C using both mechanisms. Melanoma cell lines transported dehydroascorbic acid at a rate that was at least 10 times greater than the rate of transport by melanocytes, whereas both melanoma cells and melanocytes transported ascorbate with similar efficiency. Dehydroascorbic acid transport was inhibited by deoxyglucose and cytochalasin B, indicating the direct participation of facilitative glucose transporters in the transport of oxidized vitamin C. Melanoma cells accumulated intracellular vitamin C concentrations that were up to 100 times greater than the corresponding extracellular dehydroascorbic acid concentrations, whereas intracellular accumulation of vitamin C by melanocytes never exceeded the extracellular level of dehydroascorbic acid. Melanoma cells transported dehydroascorbic acid through at least two different transporters, each with a distinct K(m), a finding that agreed well with the presence of several glucose transporter isoforms in these cells. Only one kinetic component of ascorbate uptake was identified in both melanocytes and melanoma cells, and ascorbate transport was sodium dependent and inhibited by ouabain. Both cell types were able to accumulate intracellular concentrations of vitamin C that were greater than the extracellular ascorbate concentrations. The data indicate that melanoma cells and normal melanocytes transport vitamin C using two different transport systems. The transport of dehydroascorbic acid is mediated by a facilitated mechanism via glucose transporters, whereas transport of ascorbic acid involves a sodium-ascorbate cotransporter. The differential capacity of melanoma cells to transport the oxidized form of vitamin C reflects the increased expression of facilitative transporters associated with the malignant phenotype.

Ascorbic Acid↗
PubMed1997

Human erythrocytes express GLUT5 and transport fructose.

Although erythrocytes readily metabolize fructose, it has not been known how this sugar gains entry to the red blood cell. We present evidence indicating that human erythrocytes express the fructose transporter GLUT5, which is the major means for transporting fructose into the cell. Immunoblotting and immunolocalization experiments identified the presence of GLUT1 and GLUT5 as the main facilitative hexose transporters expressed in human erythrocytes, with GLUT2 present in lower amounts. Functional studies allowed the identification of two transporters with different kinetic properties involved in the transport of fructose in human erythrocytes. The predominant transporter (GLUT5) showed an apparent Km for fructose of approximately 10 mmol/L. Transport of low concentrations of fructose was not affected by 2-deoxy-D-glucose, a glucose analog that is transported by GLUT1 and GLUT2. Similarly, cytochalasin B, a potent inhibitor of the functional activity of GLUT1 and GLUT2, did not affect the transport of fructose in human erythrocytes. The functional properties of the fructose transporter present in human erythrocytes are consistent with a central role for GLUT5 as the physiological transporter of fructose in these cells.

Biological Transport↗
PubMed1997

Efficient transport and accumulation of vitamin C in HL-60 cells depleted of glutathione.

Human myeloid leukemia cells (HL-60) transport only the oxidized form of vitamin C (dehydroascorbic acid) and accumulate the vitamin in the reduced form, ascorbic acid. We performed a detailed study of the role of glutathione in the intracellular trapping/accumulation of ascorbic acid in HL-60 cells. Uptake studies using HL-60 cells depleted of glutathione by treatment with L-buthionine-(S,R) sulfoximine and diethyl maleate, revealed no changes in the cells' ability to transport dehydroascorbic acid and accumulate ascorbic acid. Similar transport and accumulation rates were obtained using HL-60 cells containing intracellular glutathione concentrations from 6 mM to 1 microM. HL-60 cells, containing as little as 5 microM glutathione, were able to accumulate up to 150 mM ascorbic acid intracellularly when incubated with dehydroascorbic acid. Glutathione was capable of reducing dehydroascorbic acid by a direct chemical reaction, but only when present in a greater than 10-fold stoichiometric excess over dehydroascorbic acid. The accumulation of ascorbic acid by HL-60 cells was strongly temperature-dependent and was very inefficient at 16 degrees C. On the other hand, the direct chemical reduction of dehydroascorbic acid by excess glutathione proceeded efficiently at temperatures of 16 degrees C. Our data indicate that glutathione-dependent reductases in HL-60 cells are not responsible for the ability of these cells to accumulate millimolar concentrations of ascorbic acid. These findings indicate that alternative enzymatic mechanisms are involved in the cellular reduction of dehydroascorbic acid.

Antimetabolites↗
PubMed1997

Increased uptake and accumulation of vitamin C in human immunodeficiency virus 1-infected hematopoietic cell lines.

Vitamin C (ascorbic acid) is required for normal host defense and functions importantly in cellular redox systems. To define the interrelationship between human immunodeficiency virus (HIV) infection and vitamin C flux at the cellular level, we analyzed vitamin C uptake and its effects on virus production and cellular proliferation in HIV-infected and uninfected human lymphoid, myeloid, and mononuclear phagocyte cell lines. Chronic or acute infection of these cell lines by HIV-1 led to increased expression of glucose transporter 1, associated with increased transport and accumulation of vitamin C. Infected cells also showed increased transport of glucose analogs. Exposure to vitamin C had a complex effect on cell proliferation and viral production. Low concentrations of vitamin C increased or decreased cell proliferation depending on the cell line and either had no effect or caused increased viral production. Exposure to high concentrations of vitamin C preferentially decreased the proliferation and survival of the HIV-infected cells and caused decreased viral production. These findings indicate that HIV infection in lymphocytic, monocytic, and myeloid cell lines leads to increased expression of glucose transporter 1 and consequent increased cellular vitamin C uptake. High concentrations of vitamin C were preferentially toxic to HIV-infected host defense cell lines in vitro.

Ascorbic Acid↗
PubMed1996

Expression of the fructose transporter GLUT5 in human breast cancer.

The primary metabolic characteristic of malignant cells is an increased uptake of glucose and its anaerobic metabolism. We studied the expression and function of the glucose transporters in human breast cancer cell lines and analyzed their expression in normal and neoplastic primary human breast tissue. Hexose uptake assays and immunoblotting experiments revealed that the breast carcinoma cell lines MCF-7 and MDA-468 express the glucose transporters GLUT1 and GLUT2, isoforms expressed in both normal and neoplastic breast tissue. We also found that the breast cancer cell lines transport fructose and express the fructose transporter GLUT5. Immunolocalization studies revealed that GLUT5 is highly expressed in vivo in human breast cancer but is absent in normal human breast tissue. These findings indicate that human breast cancer cells have a specialized capacity to transport fructose, a metabolic substrate believed to be used by few human tissues. Identification of a high-affinity fructose transporter on human breast cancer cells opens opportunities to develop novel strategies for early diagnosis and treatment of breast cancer.

Breast↗
PubMed1996

IGF-I resistance in virus-transformed B-lymphocytes from African Efe Pygmies.

To investigate IGF-I resistance in African Efe Pygmies, we examined clonal responsiveness to IGF-I in Epstein-Barr virus-transformed B-lymphocytes from three Efe Pygmies and three American control subjects. The Efe B-lymphoblasts did not increase clonal responsiveness when incubated with IGF-I (as high as 250 micrograms/liter) in contrast to the control B-lymphoblasts which showed a bimodal dose-response with a maximal stimulation of 50% above baseline. The proliferative response of Efe B-lymphoblasts was similar to that of control B-lymphoblasts when incubated with another growth factor, phorbol 12-myristate 13-acetate, which does not activate the IGF-I receptor. These findings indicate that Efe Pygmy B-lymphoblasts are resistant to IGF-I as measured by in vitro clonal proliferation assays. Coupled with our previous report of IGF-I unresponsiveness in Efe Pygmy HTLV-II-transformed T-lymphocytes, these data suggest that IGF-I resistance is generalized and may play a central role in the etiology of short stature in this population.

Adult↗
PubMed1996

IGF-I does not mediate T-lymphoblast colony formation in response to estradiol, testosterone, 1,25(OH)2 vitamin D3, and triiodothyronine: studies in control and pygmy T-cell lines.

The mechanism by which estradiol, testosterone, 1,25(OH)2 vitamin D3, and triiodothyronine promote tissue growth is unknown, although, in some tissues, a role for local IGF-I has been suggested. We previously showed that HTLV-II-transformed T-cell lines from healthy adults augmented basal colony formation in response to peptide (growth hormone, parathormone, and adrenocorticotrophin) and glycoprotein (thyroid-stimulating hormone) hormones through stimulation of local IGF-I. T-cell lines from African Efe Pygmies, however, were resistant to the direct growth-promoting action of IGF-I, as well as to the growth-promoting action of growth hormone, parathormone, adrenocorticotrophin, and thyroid-stimulating hormone. We, therefore, used these cell lines to determine the mechanism of T-cell growth in response to steroid and thyroid hormones. We quantified colony formation of American control T-cell lines in the presence and absence of alpha IR-3 antibody against the type 1 IGF receptor and Pygmy T-cell lines in response to estradiol (36.7-1835 pmol/ liter), testosterone (34.7-17,350 pmol/liter), 1,25(OH)2 vitamin D3 (2.4-24,000 pmol/liter), and triiodothyronine (1536-192,000 pmol/liter). There were no statistically significant differences by ANOVA in overall response curves for any of the four hormones comparing control clonal responses in the presence or absence of alpha IR-3 and no statistically significant difference in overall responsiveness between control and Pygmy T-cell lines. From these data, we conclude that (i) normal T-cell lines grow in response to estradiol, testosterone, 1,25(OH)2 vitamin D3, and triiodothyronine; (ii) these responses are not mediated through local IGF-I since they are not blocked by pretreatment with antibody to the type 1 IGF receptor; and (iii) Pygmy T-cell lines, which are genetically resistant to IGF-I, grow equivalently to control T-cell lines in response to estradiol, testosterone, 1,25(OH)2 vitamin D3, and triiodothyronine, further underscoring the IGF-I independence of this stimulation in our system.

Analysis of Variance↗
PubMed1996

Decreased insulin-like growth factor I receptor expression and function in immortalized African Pygmy T cells.

Efe Pygmies of northeast Zaire have the shortest mean adult stature of any population on earth. Although various alterations in the GH/insulin-like growth factor I (IGF-I) axis have been suggested, the basis for short stature in the Pygmy is unknown. We previously described IGF-I unresponsiveness in a T lymphoblast cell line derived from an Efe Pygmy, and studies in five additional lines have confirmed severe IGF-I resistance in these cells. We have now performed experiments to determine the molecular basis for the IGF-I resistance in these cells. We found markedly decreased cell surface expression of IGF-I receptors with normal ligand binding affinity. The Pygmy IGF-I receptors were not autophosphorylated and did not transmit a signal in response to physiological concentrations of IGF-I. There was a substantially decreased level of IGF-I receptor messenger ribonucleic acid in the Pygmy cells with a normal messenger ribonucleic acid half-life. The nucleotide sequence of the full-length IGF receptor complementary DNA in Pygmy 1 showed no significant variation. These results indicate decreased IGF-I receptor gene transcription and IGF-I receptor signaling as the primary variation in the Pygmy cell lines. The findings point to the IGF-I receptor as the locus governing short stature in the African Pygmy and suggest that human stature may be genetically controlled by expression of the IGF-I receptor.

Base Sequence↗
PubMed1996

Granulocyte colony-stimulating factor following chemotherapy in elderly patients with newly diagnosed acute myelogenous leukemia.

Given the high treatment-related mortality in elderly patients with acute myelogenous leukemia (AML), we undertook a study using granulocyte colony-stimulating factor (G-CSF) following chemotherapy in an effort to ameliorate toxicity. Patients ( > 60 years) received induction with idarubicin 12 mg/m2/day x 3 and cytosine arabinoside (Ara-C) 200 mg/m2/day x 5. A second course of chemotherapy consisting of mitoxantrone 12mg/m2/day x 3, etoposide (VP-16) 150 mg/m2/day x 3 and Ara-C 200 mg/m2/day x 4 was given approximately 1 month after achieving a complete remission (CR) or immediately if patients failed the first induction. Twenty-four hours following completion of the chemotherapy, G-CSF (10 micrograms/kg/day continuous i.v. infusion) was started. A historical control group of 28 patients treated without G-CSF was used for comparison. Twenty-six patients were evaluable for response. Following induction, the recovery of neutrophils to greater than 500/microliters and 1000/microliters was more raped in the responders who received G-CSF compared to historical controls (median 13 vs 17 days, P = 0.008; 14 vs 19 days, P = 0.005). The toxic death rate of 8% in the study group was significantly lower than the 32% mortality observed in the historical controls (P = 0.04). There was no difference in supportive care requirements or infectious complications. The complete remission (CR) rate was 58% in the entire study group with 71% of de novo AML patients achieving CR. Disease-free survival and overall survival were comparable between the study and historical control groups. These results indicate that G-CSF benefits elderly patients after intensive chemotherapy for AML by decreasing the duration of neutropenia. The reduced neutropenic period may have contributed to the small number of early toxic deaths.

Aged↗
PubMed1995

Metalloproteinase inhibition and erythroid potentiation are independent activities of tissue inhibitor of metalloproteinases-1.

Tissue inhibitor of metalloproteinases-1 (TIMP-1), the major physiological matrix metalloproteinase inhibitor and a potent antimetastatic factor, also stimulates the growth of erythroid progenitors (erythroid-potentiating activity). We analyzed the relationship between the growth factor activity and protease inhibition by preparing purified TIMP-1 "knockout" proteins lacking in vitro antiproteolytic activity. The growth-stimulatory effect of these N-terminal TIMP-1 point mutants, as tested in an in vitro assay using erythroid precursors (erythroid burst-forming units) was equal to that of unmutated TIMP-1. A fully antiproteolytic C-terminal TIMP-1 truncation also stimulated growth in the erythroid burst-forming unit assay. The results indicate that the influence of TIMP-1 on erythroid precursor growth is independent of its ability to inhibit metalloproteinases. TIMP-1 is analogous to proteins that have both proteolytic and growth factor activity, such as plasmin, thrombin, and urokinase. However, TIMP-1 is novel in this regard because it is a metalloproteinase inhibitor. We show that the antiproteolytic and growth factor activities of the TIMP-1 molecule are physically and functionally distinct.

Amino Acid Sequence↗
PubMed1995

N-glycosylation of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit is essential for ligand binding and signal transduction.

The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein containing 11 potential N-glycosylation sites in the extracellular domain. We examined the role of N-glycosylation on alpha subunit membrane localization and function. Tunicamycin, an N-glycosylation inhibitor, markedly inhibited GM-CSF binding, GM-CSF-induced deoxyglucose uptake, and protein tyrosine phosphorylation in HL-60(eos) cells but did not affect cell surface expression of the alpha subunit as detected by an anti-alpha subunit monoclonal antibody. In COS cells expressing the alpha subunit and treated with tunicamycin, N-unglycosylated alpha subunit was expressed and transported to the cell surface but was not capable of binding GM-CSF. High affinity binding in COS cells expressing both alpha and beta subunits was also blocked by tunicamycin treatment. These studies indicate that N-linked oligosaccharides are essential for alpha subunit ligand binding and signaling by the human GM-CSF receptor.

Animals↗
PubMed1995

Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid.

We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars. A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external Na+, whereas the intracellular accumulation of ascorbic acid was found to be a Na(+)-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na(+)-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic acid and its subsequent intracellular accumulation.

3-O-Methylglucose↗
PubMed1995

Membrane-associated and soluble granulocyte/macrophage-colony-stimulating factor receptor alpha subunits are independently regulated in HL-60 cells.

The effects of granulocyte/macrophage-colony-stimulating factor (GM-CSF) are mediated by interaction with its composite receptor (GMR), which consists of a unique alpha subunit (GMR alpha) and a beta subunit (GMR beta) that is common to the receptors for GM-CSF, interleukin 3, and interleukin 5. GMR beta is required for high-affinity binding, cell proliferation, and protein phosphorylation but has no intrinsic GM-CSF-binding activity. GMR alpha in isolation binds to GM-CSF with low affinity and can signal for increased glucose uptake. In addition to the membrane-bound receptor (mGMR alpha), there is a naturally occurring soluble isoform (sGMR alpha) that is released free into the pericellular milieu. Analysis of genomic sequences reveals that the soluble GMR alpha isoform comes about by alternative mRNA splicing. To examine GMR alpha expression, we developed a quantitative reverse transcription-polymerase chain reaction assay based on serial dilutions of in vitro transcribed GMR alpha RNA. This assay provides a strict log-log measure of GMR alpha RNA expression, distinguishes transcripts related to the soluble and membrane-associated isoforms, and quantitatively detects 0.1 fg of GMR alpha-related mRNA. There was little or no GMR alpha expression in two human lymphoid cell lines and in the erythroblastic leukemia cell line K562, but all myeloid cell lines tested expressed both the membrane-associated and soluble isoforms of GMR alpha. Baseline level of expression of both isoforms varied > 20-fold among the myeloid cell lines studied. Differentiation of HL-60 cells to neutrophils with dimethyl sulfoxide led to a 2-fold downregulation of sGMR alpha and a 20-fold upregulation of mGMR alpha. These differentiation-induced transcriptional changes were unrelated to changes in mRNA stability. These findings indicate that sGMR alpha is differentially expressed from mGMR alpha in human hematopoietic cells and that programmed downregulation of sGMR alpha may be important in myeloid maturation.

Amino Acid Sequence↗