Biomedical subjects

W C Hooper

Publications and source records attributed to W C Hooper.

At least 55 records · Page 3Linked to original sources

PubMed1994

Tumor necrosis factor-alpha downregulates protein S secretion in human microvascular and umbilical vein endothelial cells but not in the HepG-2 hepatoma cell line.

Protein S deficiency, which is associated with thrombosis, can either be inherited or acquired. Recently, we reported that a decrease in free protein S was observed in 19 of 25 persons with HIV/AIDS. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been reported to be elevated in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients and has been shown to induce a procoagulant state on the surface of endothelial cells. We report here that recombinant TNF-alpha (rTNF-alpha) downregulated protein S synthesis in the SV-40T transfected human microvascular endothelial cell line (HMEC-1) model system by approximately 70% and in primary human umbilical vein and dermal microvascular endothelial cell cultures by approximately 50%. Using the HMEC-1 model, Northern blot analysis showed a decrease in protein S RNA at 24 hours that was corroborated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) quantification. Evidence supporting the specificity of the TNF-alpha effect included the following: (1) TNF-alpha down-regulation of protein S was completely blocked by TNF neutralizing antibody; (2) the effect was transient, and protein S was restored to near normal levels after TNF was removed from cell cultures; (3) an antibody directed to the TNF RI (55-kD receptor) was shown to mimic the action of TNF-alpha on HMEC-1 cells; and (4) other proinflammatory cytokines, interleukin (IL)-1, IL-6, and TGF-beta, had no effect on protein S secretion. However, TNF-alpha showed no regulatory control over protein S synthesis in the human hepatocellular carcinoma cell line HepG-2. We suggest that TNF-alpha downregulation of protein S may be a mechanism for localized procoagulant activity and thrombosis recently reported in some AIDS patients with associated protein S deficiency.

Carcinoma, Hepatocellular↗

PubMed1994

Development of an ELISA for quantification of human protein S in cell culture fluids using commercial polyclonal antisera.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure protein S antigen released into cell culture fluids. We used readily available commercial polyclonal antisera to develop the assay. This assay was sensitive with a detection limit of about 0.086 ng/ml. Between-assay precision (coefficient of variation) at levels of 0.2, 1.1, and 13.9 ng/ml was 14%, 15%, and 11% respectively. Specificity and accuracy were demonstrated from the use of: 1) culture fluids from 3-primary endothelial cell cultures and 7-cell lines known to constitutively produce protein S; 2) 2-cell lines not synthesizing protein S; and 3) from selected samples of normal and protein S deficient plasma. The ELISA described here was about 12-fold more sensitive and 40-fold more cost effective when compared to a commercial ELISA kit. Thus the assay provided a sensitive, specific, precise and economical method useful for the measurement of the nanogram amounts of protein S commonly encountered in cell culture fluids.

Cell Line↗

PubMed1993

Infection with human T-lymphotropic viruses leads to constitutive expression of leukemia inhibitory factor and interleukin-6.

Leukemia inhibitory factor (LIF), similar to interleukin-6 (IL-6), is a glycoprotein growth factor and differentiation regulator that has pleiotropic activity in several cellular systems. Recent reports of constitutive IL-6 production from spontaneously proliferating cells from human T-cell leukemia virus (HTLV)-infected individuals led us to examine the expression of IL-6 and LIF during HTLV infection. In vitro infection of peripheral blood lymphocytes with HTLV-I was associated with production of both soluble LIF and IL-6 in conjunction with the increasing HTLV antigen concentration. Northern blot analysis of T-cell lines generated from individuals infected with HTLV-I (MT-2, HuT-102, FS, EG, SP) and HTLV-II (Mo-T, H2A, H2E) demonstrated a marked increase in constitutive expression of LIF and IL-6 transcripts, as compared with uninfected cell lines (HuT-78, Jurkat). The constitutive expression of LIF and IL-6 was independent of presence of IL-2 in the culture medium, as both IL-2-independent (MT-2, HuT-102, SP, Mo-T) and IL-2-dependent (FS, EG, H2A, H2E) cell lines expressed LIF and IL-6 transcripts. Furthermore, LIF and IL-6 RNA expression in an HTLV-I-infected cell line (MT-2) was enhanced by phorbol ester stimulation via mechanisms that appear to be dependent on the posttranscriptional regulatory controls. These results show that both LIF and IL-6 are produced by HTLV-I- and HTLV-II-infected cells, which could potentially alter the transcriptional regulation of HTLV gene expression by inducing certain early response genes.

Cell Line↗

PubMed1993

Expression of functional insulin-like growth factor-I receptors by human amnion cells.

OBJECTIVES: The purpose of the study was to investigate whether amnion cells contain functional insulin-like growth factor-I receptors. STUDY DESIGN: To test whether human amnion cells contain insulin-like growth factor-I receptors, radioligand binding studies, affinity cross-linking studies, and Northern blot analysis were conducted in primary amnion cells and in an immortal amnion cell line (WISH). To test whether the insulin-like growth factor-I receptors on amnion cells are functional, cytochalasin B-inhibitable 2-deoxyglucose uptake was measured after stimulating the cells with insulin-like growth factor-I. RESULTS: Radioligand binding studies demonstrated that primary amnion cells and WISH cells contained a single class of high-affinity receptors with an apparent dissociation constant of 0.18 +/- 0.04 nmol/L and a receptor concentration of 79 +/- 26.2 fmol/mg protein and dissociation constant of 0.44 +/- 0.03 nmol/L and a receptor concentration of 33.3 +/- 6.45 fmol per 106 cells, respectively. Affinity cross-linking studies revealed two major insulin-like growth factor-I binding sites, 135 and 270 kd. Both primary amnion cells and WISH cells exhibited cytochalasin B-inhibitable tritiated 2-deoxyglucose uptake in response to insulin-like growth factor-I treatment. Finally, treatment of WISH cells caused tyrosine phosphorylation of three proteins (molecular weight, 116, 95.4, and 83.5 kd) was observed by Western blotting with antiphosphotyrosine antibodies. CONCLUSION: These results provide the first evidence that human amnion epithelial cells contain functional high-affinity insulin-like growth factor-I receptors that mediate glucose transport.

Amnion↗

PubMed1993

Over expression of insulin-like growth factor receptor type-I in T-cell lines infected with human T-lymphotropic virus types-I and -II.

To determine if aberrant expression of tyrosine kinase growth factor receptors may be related to the cell transformation capabilities of human T-lymphotropic viruses (HTLVs), we examined the expression of the epidermal growth factor receptor (EGF-R), insulin receptor (INS-R), and insulin-like growth factor receptor type-I (IGFR-I) in cell lines infected with HTLV type I (MT-2, HuT-102) and HTLV type II (Mo-T). Levels of mRNA transcripts for IGFR-I were significantly higher in both MT-2, HuT-102 (HTLV-I) and Mo-T (HTLV-II) cell lines than in uninfected cell lines (HuT-78, Jurkat); no detectable levels of EGF-R or INS-R mRNA transcript were observed in HTLV-infected or uninfected cell lines. Southern blot analysis demonstrated that no amplification or rearrangement of the IGFR-I gene occurred in either the MT-2 or Mo-T cell line. Flow cytometry analysis demonstrated that while IGFR-I protein was constitutively expressed on the cell surface in both MT-2 and Mo-T cell lines, neither EGF-R nor INS-R proteins could be detected. Ligand binding studies with MT-2 and Mo-T cell lines demonstrating binding of 125I insulin-like growth factor type-1 (IGF-I) in a dose-dependent manner and this response could be inhibited by increasing concentrations of cold IGF-I. These data demonstrate that deregulated expression of functional IGFR-I, the regular component of the growth control machinery of normal cells, may contribute to cellular proliferation and eventual transformation in HTLV-I- and HTLV-II-infected cell lines.

Cell Transformation, Viral↗

PubMed1993

Protein S, an antithrombotic factor, is synthesized and released by neural tumor cells.

Protein S, an anticoagulant factor in the protein C antithrombotic pathway, was found to be synthesized and released by six tumor cell lines of neural origin by western blotting and ELISA. The rate of synthesis ranged from three- to 11-fold higher than that of a microvascular endothelial cell line and 36-144% that of a hepatoma cell line. The secreted protein S displayed specific anticoagulant activity similar to that of purified plasma protein S, implying that it was fully gamma-carboxylated. Ten primary brain tumor tissues also expressed protein S antigen, as shown by western blot analysis. Expression of anticoagulantly active protein S by neural cells raises important questions concerning possible physiologic roles for this multidomain protein beyond its function in control of thrombosis.

Blotting, Western↗

PubMed1992

Human lymphotropic retroviruses associated with mycosis fungoides: evidence that human T-cell lymphotropic virus type II (HTLV-II) as well as HTLV-I may play a role in the disease.

The human T-cell lymphotropic virus type I (HTLV-I) is causally associated with adult T-cell leukemia, but its role in mycosis fungoides (MF) has remained enigmatic. The virus is suspect because a small percentage of patients with MF have antibodies to it, the cells of others harbor deleted HTLV-I proviral sequences, and particles resembling HTLV-I emerge in cultured blood lymphocytes obtained from most patients. An alternative possibility is that disparate lymphotropic retroviruses may infect or affect a population of epidermotropic lymphocytes, leading to the same outcome, ie, MF. In studies designed to identify the particles detected in lymphocyte cultures of nine patients with a diagnosis of skin involvement characteristic of MF, this concept has gained support. While the cells of four patients provided evidence of HTLV-I infection, molecular hybridization with HTLV-II-specific pol probes showed HTLV-II in the cells of another patient. The 103-bp fragment amplified by the HTLV-II-specific probe was sequenced and proved to have greater than 90% homology with the same fragment amplified from cells known to be infected with HTLV-II. A role for HTLV-II in MF has not been suggested heretofore. Therefore, HTLV-I, HTLV-II, and their incomplete forms may be found in cells of MF patients, suggesting new theories regarding the pathogenesis of this disease.

Base Sequence↗

PubMed1992

Hodgkin disease mortality in the United States: 1979-1988.

Mortality trends for Hodgkin disease in the United States were examined from 1979 to 1988 with the use of mortality data for multiple causes of death, which were obtained from the National Center for Health Statistics. A progressive decrease in the death rate from Hodgkin disease was observed over this period. The decrease in death rate was greatest among white patients. Patients who were 55 years or older had the highest death rate. Analysis by geographic region showed decreases in each of the regions, with no significant difference among groups of states with high and low incidences of acquired immune deficiency syndrome. However, the decrease in the South was approximately 35% greater than that in the North-east. In 1988 the most significant difference in death rates between male and female patients was in the 35-54-year age group, whereas a significant difference in death rates between white and black patients was seen only in patients who were 55 years of age or older. In summary, although there has been a significant reduction in deaths resulting from Hodgkin disease between 1979 and 1988, the decreases observed have varied between sexes and among age groups, racial groups, and geographic regions.

Adolescent↗

PubMed1992

Use of the polymerase chain reaction to detect hypermethylation in the calcitonin gene. A new, sensitive approach to monitor tumor cells in acute myelogenous leukemia.

Based on the recent observations that, in a majority of patients with acute leukemia, the 5' end of the calcitonin gene was hypermethylated and abnormal DNA fragments were observed following HpaII restriction digestion, we have developed a PCR-based method to sensitively detect this abnormal methylation of the calcitonin gene in AML. Applying the concept of competitive PCR, a semi-quantitative correlation was obtained between the amount of hypermethylation and the amount of leukemic cells present. These results suggest that this method will be useful to monitor the amount of tumor cells in bone marrow from patients with AML.

Base Sequence↗

PubMed1992

PCR regimen for enhanced specificity and yield of targeted genomic DNA sequences: ras and p53.

By weighting the PCR reaction in favor of specificity for the target sequence in the beginning cycles and for continued efficient amplification of the sequence into later cycles, we were able to show an improvement in the specificity and quantity of amplified ras and p53 sequences. Increased purity and yield of specific products favorably enhanced post-PCR evaluation and interpretation of results using direct sequencing and single-stranded conformation polymorphism (SSCP) analysis when point mutations were present in DNA from tumor cell lines and tissues.

Base Sequence↗

PubMed1991

Constitutive expression of c-jun and jun-B in cell lines infected with human T-lymphotropic virus types I and II.

To better understand the transcriptional regulation of human T-lymphotropic viruses, expression of the nuclear proto-oncogenes, jun-B and c-jun were examined in cell lines infected with HTLV-I/II. Constitutive high levels of jun-B and c-jun expression were observed in HTLV-I (MT-2, Hut-102, IR, FS, SP) and HTLV-II infected cell lines (Mo-T, PAN). In contrast, the uninfected cell lines (Jurkat, Hut-78) expressed only basal levels of jun. This expression of jun was not dependent upon IL-2, as both IL-2 dependent (IR, FS, SP, and Pan) and IL-2 independent (MT-2, Hut-102, Mo-T) cell lines constitutively expressed transcripts for jun-B and c-jun. These data demonstrate that deregulated expression of nuclear protooncogenes such as jun may lead to cellular proliferation and the protein products of these nuclear oncogenes may potentially serve as transcriptional activators of HTLV-LTR by complexing with other nuclear proteins.

Blotting, Northern↗

PubMed1991

Effect of tumor necrosis factor-alpha on the proliferation of leukemic cells from children with B-cell precursor-acute lymphoblastic leukemia (BCP-ALL): studies of primary leukemic cells and BCP-ALL cell lines.

The effect of recombinant tumor necrosis factor-alpha (rTNF-alpha) on the primary leukemic blasts and leukemic cell lines derived from children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) was studied. The proliferation of leukemic cells from the bone marrow of 11 of 13 patients (seven at diagnosis, four in relapse) and from the 697 (BCP-ALL) cell line was significantly inhibited by rTNF-alpha at the lowest dose tested (0.1 ng/mL), as measured by 3H-TdR uptake. The degree of inhibition was variable, ranging from 17% to 78%. Furthermore, a dose-dependent inhibitory effect was observed, with approximately 70% mean inhibition of DNA synthesis detected when cells from 12 of 13 patients were incubated with 100 ng/mL of rTNF-alpha for 3 days. In contrast, rTNF-alpha did not inhibit another BCP-ALL cell line (EU-1/ALL) established recently in our laboratory. Studies indicated that the TNF-alpha gene was expressed by the primary leukemic blasts of one TNF-resistant case in his third relapse and by EU-1 cells. Also, TNF-alpha protein was detected by Western blot analysis and enzyme-linked immunoabsorbent assay in the supernatant of EU-1 cells; this is the first report of TNF production by a BCP-ALL cell lines. The production of TNF-alpha mRNA and protein was not detected in the 697 cell line and in the primary leukemic blasts from six patients (four at diagnosis, two in relapse) whose leukemic cells were inhibited by TNF. The partially purified TNF-alpha obtained from the EU-1 cell line also suppressed the proliferation of TNF-sensitive primary leukemic cells, and this inhibitory activity was abolished by an anti-TNF-alpha specific antibody. Our results demonstrate that TNF-alpha is an inhibitor of in vitro proliferation of BCP-ALL cells from most patients. The TNF-resistant leukemic cells from a few patients and the EU-1 cell line express TNF mRNA, suggesting that the induction of TNF gene expression is associated with the development of TNF resistance.

Blotting, Western↗

PubMed1991

Cellular kinetics of transforming growth factor-beta induced hemoglobin accumulation in the HEL erythroleukemia cell line.

Transforming growth factor-beta 1 (TGF beta 1) can induce hemoglobin accumulation in a clone of the human HEL erythroleukemia cell line. This clone has previously been designated as HEL-T. The effect of TGF beta 1 was reversible and it had to be continuously present for the maximal number of cells to become positive for hemoglobin. The TGF beta 1 effect was blocked by phorbol ester and partially blocked by the calmodulin antagonist W-7, but not by dexamethasone. Simultaneous exposure to gamma-interferon, IL-1, IL-6, IL-3 and GM-CSF had no significant effect on TGF beta induced hemoglobin accumulation. However, when TGF beta was combined with TNF alpha, it was observed that there was approximately a 10-15% reduction in benzidine-positive cells. Cell-cycle analysis revealed no significant long-term alterations in any of the compartments. Analysis of the TGF beta 1 effect on 10 different HEL-T-derived clones revealed that the number of benzidine-positive cells ranged from 12 to 70% after 5 days of continuous exposure. Cell proliferation was similarly differentially affected. Another HEL cell line, designated as W-HEL, did not accumulate hemoglobin in the presence of TGF beta 1, but did have an increase in alpha-globin RNA expression.

Cell Cycle↗

PubMed1991

The synergistic effect of hemin and transforming growth factor-beta on hemoglobin accumulation in HEL erythroleukemia cells.

Transforming growth factor-beta (TGF beta) and hemin can both independently induce hemoglobin accumulation in the human HEL erythroleukemia cell line. The combination of these two agents resulted in a synergistic effect in the production of hemoglobin. On day 1, following exposure to both hemin and TGF beta, approximately 35% of the cells had accumulated hemoglobin, as evidenced, by benzidine staining. Whereas, when treated alone with either agent, the percentage of benzidine-positive cells was less than 10%. By day 5, approximately 70-80% on the cells treated with the combination were benzidine-positive. Cell surface analysis showed that the combination of TGF beta and hemin increased the expression of CD34, CD64, glycophorin A, and GPIIb-IIIa(CDW41). Cell proliferation was decreased by the combination.

Antigens, Surface↗

PubMed1991

The role of transforming growth factor-beta in hematopoiesis. A review.

The role of TGF beta hematopoiesis is currently being defined. It does appear that TGF beta and related polypeptides can modulate and regulate bone marrow and thymic-derived cells throughout their functional life span. The effects of TGF beta are pleiotropic and may depend on (a) cell lineage, (b) specific cellular phenotype, (c) stage of differentiation, (d) immediate microenvironment, (e) other cytokines and (f) cellular function. The understanding of TGF beta and its relationship to other cytokines should not only increase our understanding of hematopoiesis but may also have therapeutic implications.

Cell Differentiation↗

PubMed1991

Epidemiology of progressive multifocal leukoencephalopathy in the United States: analysis of national mortality and AIDS surveillance data.

We analyzed progressive multifocal leukoencephalopathy (PML) mortality data from 1979 to 1987 and data on persons with acquired immunodeficiency syndrome (AIDS) reported to the Centers for Disease Control (CDC). Based on analyses of multiple-cause-of-death vital statistics, deaths related to PML have increased fourfold from 1.5/10,000,000 persons in 1979 to 6.1/10,000,000 persons in 1987. The increase in the PML annual death rate began in 1984, occurred primarily in men 20 to 49 years of age, and was greatest in states known to have a high incidence of AIDS. In 1987, 56% of death certificates that listed PML as a cause of death also listed human immunodeficiency virus (HIV) infection. Analysis of AIDS case reports to the CDC from 1981 through June 1990 demonstrated that 0.72% of persons with AIDS were reported as having PML. Although most persons with AIDS who had PML were 20 to 49 years of age (84.6%), PML was reported more frequently among persons with AIDS greater than or equal to 50 years old than less than 50 years old. In addition, PML was reported more frequently among persons with AIDS who were exposed to HIV by blood transfusion than those in all other exposure categories. These data demonstrate that the increase in PML mortality from 1979 to 1987 was associated with the large increase in immunosuppressed persons with AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Phorbol ester induces morphological changes and acidic fibroblast growth factor RNA expression in the RD/TE 671 cell line.

The TE 671 medulloblastoma cell line was induced by phorbol-12-myristate-13-acetate (PMA) to resemble neuronal cells by both morphological and immunohistochemical criteria. Five days following PMA treatment at a concentration of 10 ng/ml, the cells acquired long cellular processes and cell growth was markedly inhibited. Immunohistochemically, PMA-treated cells stained intensely for neuron-specific enolase and were positive for neurofilaments. Northern blot analysis revealed that PMA induced the transcript for acidic fibroblast growth factor. Our data suggest that the TE 671 cell may be useful not only in the study of acidic fibroblast growth factor regulation but also as a model for embryonic tumor cell differentiation.

Cell Differentiation↗

PubMed1990

Oncogene expression and immunoglobulin synthesis in a North American Burkitt (NAB-2) lymphoma cell line with a 8;22 chromosome translocation.

A Burkitt's lymphoma (BL) cell line, (NAB-2) deriving from a North American patient, exhibited a 8;22 (q22;q11-12) translocation involving the c-myc gene and lambda immunoglobulin genes. In addition, NAB-2 cells have two other consistent translocations: a 1;5 (p22;q23) and a 3;7 (p25;q22), with breakpoints close to the location of N-ras, fms, and raf-1 protooncogenes, respectively. In situ hybridization with myc, N-ras, and raf-1 radiolabeled DNA probes to NAB-2 chromosomes showed that none of these genes was relocated as a result of translocation. However, by Northern blot analysis, the myc mRNA was represented by two transcripts, one approximately 2.4 kb and the other considerably larger (74 kb). The raf-1 gene transcript was also detected in NAB-2 cells; however, its size and level were similar to those seen in two other BL lines. On the other hand, the N-ras, fms, and fgr genes, which are frequently activated in BL, were not actively transcribed. NAB-2 cells also have a chromatid defect visible as an achromatic region on the short arm of chromosome 2 near the locus of the kappa immunoglobulin gene. This alteration, which is a viral modification site caused by the Epstein-Barr virus or viral products, had no influence on immunoglobulin synthesis, as NAB-2 cells concordant to the 8;22 translocation were positive for cytoplasmic and surface lambda light chains. Although NAB-2 cells exhibit several chromosomal abnormalities, only translocation, 8;22 was associated with gene alterations relevant to the neoplastic development of this malignancy.

Adolescent↗