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W S Stillman

Publications and source records attributed to W S Stillman.

At least 19 recordsLinked to original sources
PubMed2001

Comparative toxicity of dithiocarbamates and butadiene metabolites in human lymphoid and bone marrow cells.

Apparent differences in the pattern of leukemia risk have been observed between workers employed in 1,3-butadiene (BD) monomer production and those working in styrene-butadiene rubber production (SBR). There are a number of possible explanations for these discrepancies, including differences in disease classification and diagnosis as well as possible quantitative and qualitative differences in occupational exposure between these two industries. This led us to evaluate the possibility that the pattern of disease observed in SBR might be influenced by the presence of an important class of biologically reactive chemicals, dithiocarbamates (DTC), that were present in SBR but not BD monomer production. Therefore, we compared the immunotoxic and hematotoxic activities of DTC and BD metabolites in human immune and hematopoietic cells. Relative to the mouse, human CD34+ bone marrow cells are relatively resistant to the direct effects of BD metabolites, with only the bis-oxide producing any evidence of suppression of clonogenic response at concentrations between 1 and 10 microM. Similarly, treatment of human CD4+ lymphocytes with known (2,3-epoxybutene) and putative BD metabolites (D,L-butane-bis-oxide, (2S,3R)-3-epoxybutane-1,2-diol) does not result in appreciable T-cell toxicity at concentrations likely to be encountered in vivo. In contrast, treatment of human cells with DTC at concentrations as low as 100 nM results in significant suppression of hematopoietic clonogenic response and T-lymphocyte function. Additional studies in our laboratory and others suggest a role for copper in DTC toxicity in both human lymphocytes and bone marrow cells, although the pattern of altered transcriptional regulation observed is markedly different in these two cell populations. These results are consistent with the pattern of DTC toxicity previously observed in clinical and molecular studies.

Animals↗
PubMed2000

Comparative toxicity of known and putative metabolites of 1, 3-butadiene in human CD34(+) bone marrow cells.

Species-specific susceptibility to the hematotoxic effects of 1, 3-butadiene (BD) is well known. Previous studies have revealed that murine bone marrow is uniquely susceptible to toxicity following exposure to the parent compound in vivo or exposure of bone marrow cells to the monoepoxide metabolite, 3,4-epoxybutane, in vitro. Studies described herein compare the relative ability of putative and known BD metabolites to produce concentration dependent suppression of colony formation and cytotoxicity in human CD34(+) bone marrow cells. Compounds evaluated included 3,4-epoxybutane, D, L-butane-bis-oxide, meso-butane-bis-oxide and (2S, 3R)-3-epoxybutane-1,2-diol. In contrast to results previously observed in mice, only the bis-oxides produced significant suppression of colony formation at potentially relevant concentrations (10(-8) to 10(-3) M). No enantiospecific differences were observed between the meso- and D,L-bis-oxides and no significant lineage-specific differences in susceptibility to inhibition of clonogenic response were observed among early multi-potential myeloid and erythroid hematopoietic progenitor cells. The relative potencies of the bis-oxides were found to be comparable to that of the prototype hematotoxic compound, hydroquinone. These results confirm previous studies that reveal marked species-specific differences in the susceptibility of bone marrow cells to 3,4-epoxybutane. Moreover, these results suggest that the bis-oxides of BD are capable of suppressing the clonogenic function of human hematopoietic progenitor cells, if, in fact, they are produced in human bone marrow in significant concentration. Further interpretation of these findings requires a better understanding of the metabolism of BD in humans.

Adult↗
PubMed2000

Dithiocarbamates inhibit hematopoiesis via a copper-dependent mechanism.

Dithiocarbamates (DTC), an important class of therapeutic and industrial chemicals, have alternatively been reported to be either beneficial or toxic to the hematopoietic and immune systems. In the present study, we investigated the potential of dimethyl- and diethyl-dithiocarbamate to alter clonogenic response of primary human CD34(+) bone marrow cells in vitro. Our results demonstrate that both compounds are potent inhibitors of clonogenic response in human CD34(+) bone marrow cells, suppressing cytokine-induced colony formation at concentrations between 100 and 500 nM. Pretreatment of bone marrow cells for 1 h with very high doses of DTC (30 microM) had no effect on colony formation. DTCs are known inhibitors of nuclear factor-kappa B (NF-kappa B); however, data presented herein demonstrate that DTC do not inhibit cytokine activation of NF-kappa B in CD34(+) bone marrow cells. Additional experiments demonstrate that DTCs induce a dose-related increase in apoptosis, potentially acting via a cytotoxic mechanism. We further demonstrate that the addition of copper sulfate greatly potentiates the hematotoxicity of DTC and that the addition of a copper-specific chelator completely abrogates DTC clonogenic suppression. These data support a role for copper in DTC-induced hematotoxicity.

Antigens, CD34↗
PubMed2000

The benzene metabolite, hydroquinone, selectively induces 5q31- and -7 in human CD34+CD19- bone marrow cells.

OBJECTIVE: Chronic exposure to high concentrations of benzene is associated with an increased incidence of myelodysplastic syndrome and acute myelogenous leukemia. Acute myelogenous leukemia developing in patients treated with alkylating agents for other cancers or occupationally exposed to benzene exhibit a pattern of cytogenetic aberrations predominantly involving loss of all or part of chromosomes 5 and/or 7. In contrast, trisomy 8 is observed equally in both de novo and secondary acute myelogenous leukemia. Studies using peripheral lymphocytes or lymphoblastoid cell lines have observed dose-dependent loss of chromosomes 5, 7, and 8 following treatment with the benzene metabolite, hydroquinone. The purpose of this study was to determine the dose response and specificity of hydroquinone-induced aberrations on chromosomes 5, 7, and 8 using human CD34+CD19 bone marrow cells. MATERIALS AND METHODS: Fluorescence in situ hybridization analysis was performed on CD34+CD19- bone marrow cells using the locus-specific probes, 5q31, 5p15.2, and centromeric probes specific for human chromosomes 7 and 8 following hydroquinone exposure. RESULTS: Hydroquinone exposure results in -7, selective deletion of 5q31 but not chromosome 5 and no loss or gain of chromosome 8 in human CD34+CD19- cells. CONCLUSION: CD34+ bone marrow cells are more susceptible and show a different pattern of cytogenetic aberrations as a result of hydroquinone exposure compared to lymphocytes. CD34+ bone marrow cells exhibit unique susceptibility to the development of specific chromosome aberrations that have been identified as the earliest structural changes occurring in the development of secondary myelodysplastic syndrome and acute myelogenous leukemia.

Benzene↗
PubMed2000

Hydroquinone inhibits PMA-induced activation of NFkappaB in primary human CD19+ B lymphocytes.

Hydroquinone (HQ), a reactive metabolite of benzene, is known to inhibit mitogen-stimulated activation of both T and B lymphocytes. Despite extensive study, the underlying mechanism for the immunotoxicity of the HQ is not clear. We have previously demonstrated that 1 micromol/L HQ inhibits TNF-induced activation of NFkappaB in CD4+ T cells, resulting in decreased IL-2 production. NFkappaB, known to be important in T lymphocytes, also plays a critical role in normal B cell development and activation. We therefore hypothesized that alterations in NFkappaB might be involved in HQ-induced B cell immunosuppression as well. In this study, we demonstrate that 1-10 micromol/L HQ inhibits PMA/ionomycin-induced activation of NFkappaB in primary human CD19+ B cells. Inhibition of NFkappaB is accompanied by a dose-dependent decrease in PMA-stimulated production of TNF with no corresponding loss in viability or increased apoptosis. HQ also does not appear to alter NFkappaB directly, as preincubation of B cell nuclear extracts with HQ does not diminish DNA binding activity of this protein. In contrast to T cells, inhibition of NFkappaB by HQ in B cells is not reversible after 72 h in culture, suggesting a long-term functional suppression. These data support our original findings in T cells and indicate that NFkappaB is particularly susceptible to inhibition by HQ. We further hypothesize that inhibition of NFkappaB in lymphocytes, and perhaps other cell types as well, may play a significant role in the observed toxicity of HQ.

Adult↗
PubMed1999

An essential role for NF-kappaB in human CD34(+) bone marrow cell survival.

The transcription factor, NF-kappaB, is important for T-cell activation, B-cell maturation, and human immunodeficiency virus transcription and plays a role in alternatively mediating and protecting against apoptosis in a variety of cell types. However, a role for NF-kappaB in human CD34(+) bone marrow cells has not been described. We provide evidence here that virtually all human CD34(+) bone marrow cells express NF-kappaB that can be activated by exposure to phorbol 12-myristate 13-acetate and a variety of cytokines, eg, tumor necrosis factor alpha, interleukin-3, and granulocyte-macrophage colony-stimulating factor. In addition, we demonstrate that NF-kappaB may be required for human CD34(+) bone marrow cell clonogenic function and survival. These results offer insight into a new role for NF-kappaB in maintaining survival and function in hematopoietic stem and progenitor cells and suggest that proposed strategies involving inhibition of NF-kappaB activation as an adjunct to cancer chemotherapy should be approached with caution.

Adult↗
PubMed1999

The benzene metabolites hydroquinone and catechol act in synergy to induce dose-dependent hypoploidy and -5q31 in a human cell line.

Chronic exposure to high concentrations of benzene is associated with an increased incidence of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Studies of patients occupationally exposed to benzene show a pattern of cytogenetic aberrations involving loss of all or part of chromosomes 5 and/or 7 as well as trisomy 8 and we have previously reported that hydroquinone (HQ) induces deletions of 5, 7 and 8. Benzene metabolism is a requirement for bone marrow toxicity and the phenolic metabolites, HQ and catechol (CAT), have been implicated in benzene hematotoxicity. A research project was designed to determine whether CAT by itself and in conjunction with HQ could directly induce loss of chromosome 5 and/or 7 and gain of chromosome 8. Using fluorescence in situ hybridization with chromosome-specific 5, 7, and 8 probes we demonstrate that 5 to 150 uM CAT does not produce chromosomal aberrations, however CAT and 25 uM HQ can act in synergy to induce dose dependent loss of these chromosomes. In addition HQ/CAT selectively induces -5q which is not observed for HQ only. These results demonstrate for the first time that CAT/HQ act in synergy to induce specific chromosome loss found in secondary MDS/AML.

Benzene↗
PubMed1998

Dimethyldithiocarbamate inhibits in vitro activation of primary human CD4+ T lymphocytes.

Dithiocarbamates (DTC), a diverse group of industrial and therapeutic chemicals, have been reported to inhibit, enhance or have no effect on the immune system. These apparent inconsistencies reflect the complexity of the DTCs biological activities and are probably due in part to differences in dose, route of exposure, animal species used and/or specific compound tested. The studies described herein were undertaken to investigate the immunotoxicity of one member of this family, dimethyldithiocarbamate (DMDTC). We demonstrate that 0.1-0.5 microM DMDTC inhibits TNF-alpha-induced activation of NF-kappaB in primary human CD4+ T cells. This inhibition is not accompanied by a loss in viability, and DMDTC-treated T cells retain other active signaling pathways throughout the exposure duration. The inhibition of NF-kappaB is apparently permanent as DMDTC-treated T cells did not regain normal TNF-alpha activation, even after 72 h in culture. DMDTC does not appear to alter NF-kappaB directly as pre-incubation of nuclear extracts with DMDTC does not diminish binding activity of this protein. We further demonstrate that 0.1-0.5 microM DMDTC inhibits intracellular IL-2 production and decreases surface expression of CD25 (the alpha subunit of the IL-2 receptor) in T cells stimulated with phorbol ester. These data demonstrate that DMDTC is a potent immunosuppressive compound in vitro.

CD4-Positive T-Lymphocytes↗
PubMed1998

Hydroquinone, a reactive metabolite of benzene, inhibits NF-kappa B in primary human CD4+ T lymphocytes.

Hydroquinone (HQ), a reactive metabolite of benzene, is present in cigarette smoke and is known to inhibit mitogen-stimulated activation of both T and B lymphocytes. Despite extensive study, the underlying mechanism for HQ's immunotoxicity is not clear. NF-kappa B is a transcription factor known to regulate the expression of a number of genes critical for normal T cell activation. We therefore hypothesized that NF-kappa B might be involved in HQ-induced immunosuppression. In this study, we demonstrate that 1 microM HQ inhibits tumor necrosis factor alpha induced activation of NF-kappa B in primary human CD4+ T cells. This inhibition is not accompanied by a loss in viability, and HQ-treated T cells maintain other active signaling pathways throughout the exposure duration. Additionally, the inhibition of NF-kappa B is reversible as HQ-treated T cells regain normal functioning after 72 h in culture. HQ does not appear to alter NF-kappa B directly as preincubation of nuclear extracts with HQ does not diminish activity of this protein. We further demonstrate that 1 microM HQ inhibits intracellular IL-2 production in T cells stimulated with phorbol ester but does not alter surface expression of CD25 (the alpha-subunit of the IL-2 receptor). These data suggest that NF-kappa B may be an important molecular mediator of HQ's (and benzene's) immunotoxicity.

Benzene↗
PubMed1997

The benzene metabolite, hydroquinone, induces dose-dependent hypoploidy in a human cell line.

Chronic exposure to high concentrations of benzene can result in the development of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Studies of patients occupationally exposed to benzene show a pattern of cytogenetic aberrations involving high frequency of loss of all or part of chromosomes 5 and/or 7 as well as trisomy 8. The pattern of reoccurring chromosome abnormalities associated with the development of leukemia can be used as a guide in understanding the etiology and pathogenesis of these diseases. Therefore, a research project was designed to determine whether a metabolite of benzene, hydroquinone (HQ), could directly induce loss of chromosome 5 and/or 7 and gain of chromosome 8. Using fluorescence in situ hybridization with chromosome-specific 5, 7 and 8 probes we demonstrate that 42, 49 and 26 microM HQ induces monosomy 5, 7 and 8, respectively, in the human lymphoblast cell line GM09948. These results demonstrate for the first time that HQ induces a specific chromosome loss found in secondary MDS/AML. The pattern of chromosome 5 and/or 7 loss in benzene-induced MDS/AML is probably due to selective cell survival after HQ exposure rather than specific targeting of HQ for chromosomes 5 or 7.

Cells, Cultured↗
PubMed1997

Characterization and phenotypic analysis of differentiating CD34+ human bone marrow cells in liquid culture.

Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.

Antigens, CD↗
PubMed1996

Murine thymic lymphoma is associated with a species-specific hematopoietic progenitor cell subpopulation.

Many strains of laboratory mouse are uniquely susceptible to the development of T cell lymphoma/leukemia, either spontaneously or as a result of chemical or radiation exposure. In contrast, T cell leukemias or lymphomas which are relatively uncommon in human populations, are not easily induced by radiation, and are not generally associated with chemotherapy or chemical exposure. Evidence is presented to suggest that differences in the susceptibility to the development of these malignancies is related to subtle but important variations in the regulation of hematopoietic stem cell differentiation between these two species.

Adult↗
PubMed1996

Inorganic lead activates NF-kappa B in primary human CD4+ T lymphocytes.

Inorganic lead (Pb) is a ubiquitous environmental contaminant that produces a variety of effects on humoral and cell mediated immune responses. The underlying molecular mechanism for Pb's complex effects on the immune system remain obscure. Many of Pb's effects on the immune system could be explained through activation of the transcription factor, NF-kappa B. NF-kappa B is critical for T lymphocyte function and is a strong inducer of HIV-LTR activation. We demonstrate that Pb at physiologically relevant concentrations activates NF-kappa B in primary human CD4+ T lymphocytes. Pb-induced activation of NF-kappa B is blocked by antibodies for p65 and p50 subunits but not cRel, indicating that the p65:p50 heterodimer (NF-kappa B) is involved. Functional activation of gene expression by Pb was confirmed using primary CD4+ T cells transfected with an NF-kappa B dependent reporter gene construct. Pb did not activate NF-kappa B in 4 different T cell lines, suggesting that lymphoid cell lines may not be reliable surrogates for the study of transcriptional activation in human T cells. These data suggest that NF-kappa B may be an important molecular mediator of Pb-induced immunotoxicity.

Base Sequence↗
PubMed1996

The effects of benzene and other leukaemogenic agents on haematopoietic stem and progenitor cell differentiation.

A characteristic shared by a diverse group of myelotoxic compounds and leukaemogens is the ability to act synergistically with granulocyte-macrophage colony stimulating factor (GM-CSF) in increasing clonogenic response. Pretreatment of murine or human bone marrow cells with the benzene metabolite, hydroquinone, but not phenol, catechol or trans, trans-muconaldehyde, results in a selective enhancement of GM-CSF but not an interleukin-3 (IL-3)-mediated clonogenic response. Clonal enhancement is preserved and magnified in enriched populations of CD34+ cells (> 95% purity), suggesting an intrinsic effect on haematopoietic progenitor cell (HPC) recruitment rather than a secondary effect involving accessory cytokines. Clonogenic enhancement of murine HPCs is not accompanied by alterations in GM-CSF receptor expression or ligand affinity and appears to be mediated via a p53-independent mechanism. These observations suggest that hydroquinone treatment alters recruitment and differentiation in a primitive subpopulation of CD34+ cells and are consistent with a role for altered stem cell differentiation in the development of chemically induced myelodysplasias.

Animals↗
PubMed1996

The process of leukemogenesis.

Leukemias are monoclonal diseases that arise from cells in the hematopoietic stem and progenitor cell compartment. Consistent with emerging models of carcinogenesis, leukemogenesis is an evolutionary process that involves multiple independent genetic and epigenetic events. Over the last half-century a predominant paradigm has emerged to describe leukemia developing secondary to alkylating drug therapy or exposure to benzene in which progressive dysplastic changes, accompanied by a distinct pattern of clonal cytogenetic abnormalities, give rise to acute myelogenous leukemia. Characterization of these clonal chromosomal aberrations, together with observed alterations in other growth-promoting genes, provides a useful framework for studying chemical leukemogenesis and for use in understanding the origins and development of leukemia in general.

Benzene↗
PubMed1996

Impact of benzene metabolites on differentiation of bone marrow progenitor cells.

Interleukin-3 (IL-3) and granulocyte/macrophage-colony-stimulating factor (GM-CSF) are responsible for maintaining survival and stimulating growth of early dormant hematopoietic progenitor cells (HPC). These cytokines exhibit extensive overlap, with GM-CSF supporting growth and differentiation of myeloid HPC. A characteristic shared by a diverse group of leukemogens is the ability to act synergistically with GM-CSF to increase clonogenic response. Previous studies have revealed that pretreatment of murine HPC with hydroquinone (HQ) but not phenol, catechol, or trans-trans-muconaldehyde results in a selective enhancement of GM-CSF but not IL-3-mediated clonogenic response. Pretreatment of murine bone marrow cells with these agents or their metabolites in vitro results in increased numbers of HPC dividing and forming colonies in response to GM-CSF but not IL-3. The present studies explored the molecular mechanisms associated with altered cytokine response in early HPC in murine bone marrow and extended our initial observations in murine bone marrow to human bone marrow cells. HQ pretreatment of murine HPC did not induce either an up- or a down-regulation of GM-CSF receptors or any change in receptor affinity. CD34+ cells, which represent between 1 and 5% of human bone marrow, contain virtually all clonogenic stem and HPC. Pretreatment of CD34+ cells (approximately 95% purity) with HQ also results in enhanced clonogenic response with GM-CSF but not IL-3. These findings suggest that an early step in chemical leukemogenesis may involve transient alterations in the regulation of cytokine response to GM-CSF.

Animals↗
PubMed1996

An epidemiologic study of early biologic effects of benzene in Chinese workers.

Benzene is a recognized hematotoxin and leukemogen, but its mechanisms of action in humans are still uncertain. To provide insight into these processes, we carried out a cross-sectional study of 44 healthy workers currently exposed to benzene (median 8-hr time-weighted average; 31 ppm), and unexposed controls in Shanghai, China. Here we provide an overview of the study results on peripheral blood cells levels and somatic cell mutation frequency measured by the glycophorin A (GPA) gene loss assay and report on peripheral cytokine levels. All peripheral blood cells levels (i.e., total white blood cells, absolute lymphocyte count, platelets, red blood cells, and hemoglobin) were decreased among exposed workers compared to controls, with the exception of the red blood cell mean corpuscular volume, which was higher among exposed subjects. In contrast, peripheral cytokine levels (interleukin-3, interleukin-6, erythropoietin, granulocyte colony-stimulating factor, tissue necrosis factor-alpha) in a subset of the most highly exposed workers (n = 11) were similar to values in controls (n = 11), suggesting that benzene does not affect these growth factor levels in peripheral blood. The GPA assay measures stem cell or precursor erythroid cell mutations expressed in peripheral red blood cells of MN heterozygous subjects, identifying NN variants, which result from loss of the GPA M allele and duplication of the N allele, and N phi variants, which arise from gene inactivation. The NN (but not N phi) GPA variant cell frequency was elevated in the exposed workers compared with controls (mean +/- SD, 13.9 +/- 8.4 mutants per million cells versus 7.4 +/- 5.2 per million cells, (respectively; p = 0.0002), suggesting that benzene produces gene-duplicating but not gene-inactivating mutations at the GPA locus in bone marrow cells of exposed humans. These findings, combined with ongoing analyses of benzene macromolecular adducts and chromosomal aberrations, will provide an opportunity to comprehensively evaluate a wide range of early biologic effects associated with benzene exposure in humans.

Adult↗