Biomedical subjects

D W Kufe

Publications and source records attributed to D W Kufe.

At least 109 records · Page 6Linked to original sources

PubMed1992

Genomic structure, induction, and production of TNF-alpha.

The TNF gene is located in close proximity to the HLA-B locus in both humans and mouse. TNF has a high degree of sequence observation across species, and this is reflected in its relatively high cross-species activity. The 5' flanking region of the TNF gene contains multiple potential regulatory sites, including consensus sequences for the AP-1 and AP-2 sites, the cAMP-responsive element, and sequences similar to the kappa B sequences found in immunoglobulin and cytokine regulatory elements. This sequence has been demonstrated to be responsive to LPS and TNF stimulation. The 3' untranslated region contains a sequence element affecting posttranslational control of TNF through mRNA stability and translation efficiency. The functional importance and interactions of these regulatory elements remain undefined. TNF is widely expressed in granulocytes, macrophages, fibroblasts, and epithelial cells. Induction of TNF can occur through a variety of stimuli, including LPS, TPA, cytokines, calcium flux, and oxygen free-radical mechanisms. A common pathway for these diverse agents remains unknown. In human monocytes, the regulation of TNF expression is regulated at both transcriptional and posttranscriptional levels. Experimental evidence suggests that phospholipase A2 and the lipoxygenase pathway may be central in the process of TNF induction in leukocytes. Down-regulation of TNF expression is better understood. The inhibition of expression appears to result from high levels of cAMP, frequently induced through the action of PGE2. TNF secretion is separately regulated and may involve the action of G binding proteins.

Amino Acid Sequence↗

PubMed1992

Activation of the transcription factor kappa B in human KG-1 myeloid leukemia cells treated with 1-beta-D-arabinofuranosylcytosine.

The present studies have examined the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on activation of the transcription factor kappa B (NF-kappa B). The results demonstrate that treatment of human KG-1 myeloid leukemia cells with ara-C is associated with induction of protein binding to the NF-kappa B consensus sequence. NF-kappa B binding was activated at 30 min and reached maximal levels of binding at 1-2 hr of ara-C treatment. The NF-kappa B consensus sequence was ligated to the heterologous thymidine kinase (TK) promoter and the human growth hormone (GH) reporter gene to determine whether ara-C-induced NF-kappa B activity includes an enhancer function. Ara-C treatment had little effect on transient expression of pTKGH in KG-1 cells but increased transcription of the p (NF-kappa B) TKGH vector by 8-fold. The results also demonstrate that ara-C transiently increases NF-kappa B mRNA levels. However, the finding that ara-C-induced binding of NF-kappa B to DNA occurs in the presence of cycloheximide indicates that this agent activates preexisting NF-kappa B protein. These results suggest that ara-C induces a cytoplasmic pathway that transduces signals to the nucleus by activation of NF-kappa B.

Base Sequence↗

PubMed1992

Transcriptional and posttranscriptional regulation of H1 histone gene expression by 1-beta-D-arabinofuranosylcytosine.

Recent studies have demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) activates the transcription of the jun/fos early response genes in human myeloid leukemia cells. The basis for ara-C-induced control of gene expression remains unclear. However, down-regulation of H1 histone mRNA levels has been reported as one of the earliest changes in specific gene expression associated with ara-C treatment. In this report, we describe the mechanisms responsible for H1 histone expression by this agent. Treatment of HL-60 cells with ara-C resulted in a decrease in H1 histone mRNA levels that was detectable by 15 min. In contrast, this down-regulation by ara-C was completely blocked by treatment of the cells with cycloheximide. Nuclear run-on analyses demonstrated that ara-C treatment is associated with inhibition of H1 histone gene transcription. The results also demonstrate that cycloheximide abrogates the transcriptional down-regulation by ara-C but alone has no detectable effect. We also show that ara-C treatment is associated with a decrease in stability of the H1 histone transcript and that this effect is also reversed by inhibition of protein synthesis. Taken together, these findings demonstrate that ara-C regulates H1 histone expression at both the transcriptional and posttranscriptional levels. The results also indicate that control of this gene by ara-C involves the activation of at least two signaling events that require de novo protein synthesis.

Cycloheximide↗

PubMed1992

Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis.

The infiltration of monocytes into the vascular wall and their transformation into lipid-laden foam cells characterizes early atherogenesis. Macrophages are also present in more advanced human atherosclerotic plaques and can produce many mediators that may contribute to lesion formation and progression. Macrophage colony-stimulating factor (MCSF) enhances the proliferation and differentiation of monocyte progenitors and is required for the survival and activation of mature monocytes and macrophages. The authors therefore examined the expression of the MCSF gene in cultured human vascular endothelial (EC) and smooth muscle cells (SMC) as well as in atheromatous lesions from rabbits and humans. Growth arrested EC and SMC contain a low level of MCSF mRNA. Bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) induced MCSF mRNA accumulation in a concentration-dependent manner in both EC and SMC. These stimuli induced large increases in MCSF mRNA with peak induction between 4-8 hours after treatment. LPS, IL-1 alpha, and TNF alpha stimulated EC and SMC also showed increased fluorescent antibody staining for MCSF protein and released immunoreactive MCSF in a time-dependent manner. In contrast, phorbol 12-myristate 13-acetate (PMA) was a less potent inducer of MCSF gene expression and iron-oxidized low-density lipoproteins (ox-LDL) did not increase consistently MCSF mRNA or the synthesis and secretion of immunoreactive protein. Northern analysis of mRNA isolated from the atheromatous aorta of rabbits fed a 1% cholesterol diet for 10 weeks showed elevated MCSF mRNA compared with controls. Immunostaining of atheromatous arterial lesions of rabbits demonstrated MCSF protein in association with intimal SMC as well as macrophages. Furthermore, polymerase chain reaction (PCR) analysis of MCSF mRNA in human atheromata showed higher levels than found in nonatherosclerotic arteries and veins. Since the authors found no mRNA for the MCSF receptor, c-fms, in cultured EC or SMC macrophages are likely the primary target for MCSF within atheromatous vessels. The authors therefore investigated the effects of MCSF on monocyte functions related to foam cell development. Treatment of cultured human monocytes with recombinant human MCSF (10(3) U/ml, 72 hr) led to the accumulation of mRNA for the acetyl-LDL (scavenger) receptor and apolipoprotein E (apo E). These studies establish that vascular EC and SMC produce substantial MCSF in response to a variety of stimuli. The local production of MCSF during atherogenesis may contribute to macrophage survival and proliferation or activate specific macrophage functions such as expression of the scavenger receptor and secretion of apo E.

Animals↗

PubMed1991

Inhibition of protein kinase C is associated with a decrease in c-myc expression in human myeloid leukemia cells.

Treatment of human myeloid leukemic cells with phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation and then partial down-regulation of protein kinase C activity. Previous work has suggested that the activation of protein kinase C by TPA contributes to the decrease in c-myc expression during differentiation of these cells. The present studies demonstrate that the decline in c-myc mRNA levels following exposure of HL-60 cells to TPA is preceded by an increase in expression of this gene. In contrast, exposure of HL-60 cells to inhibitors of protein kinase C activity is associated with down-modulation of c-myc expression. Similar findings have been obtained in U-937 myeloid leukemia cells. Taken together, these findings suggest that phorbol esters have a biphasic effect on c-myc expression. Whereas the activation of protein kinase C by phorbol esters may be associated with an increase in c-myc gene expression, the subsequent partial down-regulation of kinase activity may initiate a cascade of events resulting in the down-modulation of c-myc expression.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

A broadly neutralizing monoclonal antibody that recognizes the V3 region of human immunodeficiency virus type 1 glycoprotein gp120.

Previous studies have demonstrated that the principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) is located in the V3 loop of glycoprotein gp120. Antibodies prepared against this region using gp120 or peptides as immunogens have been predominantly HIV-1-isolate-specific. In the present studies, murine monoclonal antibodies (mAbs) were prepared against the HIV-1MN strain. One mAb, designated NM-01, was selected for its ability to neutralize both the MN and IIIB strains. Neutralization of H9-cell infectivity as determined by reverse transcriptase assay demonstrated an ID50 of less than 1 microgram/ml for both MN and IIIB. mAb NM-01 also blocked MN and IIIB infectivity in the MT-2 assay and inhibited their reactivity in syncytium formation. The results further demonstrate that mAb NM-01 binds to the V3 loop of gp120 at amino acids 312-326. This mAb reacted equally well with loop peptides from the MN, IIIB, RF, and CDC4 isolates. In contrast, there was less affinity with a similar peptide from the NY5 strain and little if any reactivity with loop peptides from the Z2, Z6, and ELI strains. We also demonstrate that peptides corresponding to the V3 loops of MN and IIIB, but not Z6, block neutralization of IIIB virus by mAb NM-01. These findings indicate that mAb NM-01 reacts with diverse HIV-1 isolates through the Gly-Pro-Gly-Arg sequence of the V3 loop.

Amino Acid Sequence↗

PubMed1991

Tumor necrosis factor gene expression is mediated by protein kinase C following activation by ionizing radiation.

Tumor necrosis factor (TNF) production following X-irradiation has been implicated in the biological response to ionizing radiation. Protein kinase C (PKC) is suggested to participate in TNF transcriptional induction and X-ray-mediated gene expression. We therefore studied radiation-mediated TNF expression in HL-60 cells with diminished PKC activity produced by either pretreatment with protein kinase inhibitors or prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Both treatments resulted in attenuation of radiation-mediated TNF induction. Consistent with these results, we found no detectable induction of TNF expression following X-irradiation in the HL-60 variant deficient in PKC-mediated signal transduction. The rapid activation of PKC following gamma-irradiation was established using an in vitro assay measuring phosphorylation of a PKC specific substrate. A 4.5-fold increase in PKC activity occurred 15 to 30 s following irradiation, which declined to baseline at 60 s. Two-dimensional gel electrophoresis of phosphoproteins extracted from irradiated cells demonstrated in vivo phosphorylation of the PKC specific substrate Mr 80,000 protein at 45 s following X-irradiation. These findings indicate that signal transduction via the PKC pathway is required for the induction of TNF gene expression by ionizing radiation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Effects of hexamethylene bisacetamide on induction of monocytic differentiation of human U-937 myeloid leukemia cells.

The present studies have examined the effects of hexamethylene bisacetamide (HMBA) on the human U-937 monocytic cell line. HMBA treatment was associated with: (1) decreases in U-937 cell proliferation, (2) increases in nonspecific esterase activity and cell surface antigen expression consistent with monocytic differentiation, (3) decreases in c-myc gene expression, and (4) induction of tumor necrosis factor (TNF) transcripts. Treatment of U-937 cells with HMBA was also associated with increases in phospholipase A2 activity and increases in the release of arachidonic acid and its metabolites. Dexamethasone, an agent previously shown to inhibit monocytic differentiation, had no detectable effect on the down-regulation of c-myc, but blocked the induction of TNF expression. Taken together, the results demonstrate that HMBA induces monocytic differentiation of U-937 cells and that this effect is sensitive, in part, to dexamethasone.

Acetamides↗

PubMed1991

Biological consequences of gene regulation after ionizing radiation exposure.

Ionizing radiation is a ubiquitous environmental mutagen and carcinogen widely used in cancer therapy. However, little is known about the induction of cellular signaling events and specific gene expression after radiation exposure. We review the accumulating evidence that ionizing radiation induces signal transduction pathways involving activation of protein kinase C and a program of genetic events that may contribute to the biological effects of x rays.

Base Sequence↗

PubMed1991

Regulation of tumor necrosis factor gene expression by ionizing radiation in human myeloid leukemia cells and peripheral blood monocytes.

Previous studies have demonstrated that ionizing radiation induces the expression of certain cytokines, such as TNF alpha/cachectin. However, there is presently no available information regarding the molecular mechanisms responsible for the regulation of cytokine gene expression by ionizing radiation. In this report, we describe the regulation of the TNF gene by ionizing radiation in human myeloid leukemia cells. The increase in TNF transcripts by x rays was both time- and dose-dependent as determined by Northern blot analysis. Similar findings were obtained in human peripheral blood monocytes. Transcriptional run-on analyses have demonstrated that ionizing radiation stimulates the rate of TNF gene transcription. Furthermore, induction of TNF mRNA was increased in the absence of protein synthesis. In contrast, ionizing radiation had little effect on the half-life of TNF transcripts. These findings indicate that the increase in TNF mRNA observed after irradiation is regulated by transcriptional mechanisms and suggest that production of this cytokine by myeloid cells may play a role in the pathophysiologic effects of ionizing radiation.

Cell Line↗

PubMed1991

Posttranscriptional regulation of the zinc finger-encoding EGR-1 gene by granulocyte-macrophage colony-stimulating factor in human U-937 monocytic leukemia cells: involvement of a pertussis toxin-sensitive G protein.

The EGR-1 gene is an immediate early response gene encoding a zinc finger DNA-binding protein. The present studies have examined the regulation of EGR-1 gene expression in human U-937 monocytic leukemia cells treated with granulocyte-macrophage colony-stimulating factor (GM-CSF). The results demonstrate that GM-CSF rapidly and transiently increases EGR-1 gene expression in U-937 cells. Similar findings were obtained in GM-CSF-treated human monocytes. We also show that the regulation of EGR-1 expression by GM-CSF is a pertussis toxin-sensitive event. The results of nuclear run-on assays further demonstrate that the EGR-1 gene is constitutively transcribed in untreated U-937 cells and that GM-CSF has little effect on this rate of transcription. Inhibition of protein synthesis with cycloheximide also had no detectable effect on EGR-1 gene transcription but was associated with superinduction of EGR-1 mRNA levels in GM-CSF-treated cells. Moreover, the half-life of GM-CSF-induced EGR-1 transcripts was prolonged from 33 to 70 min following inhibition of protein synthesis. Taken together, the results indicate that GM-CSF activates signaling pathways which regulate EGR-1 gene expression through a pertussis toxin-sensitive G protein and that these events increase EGR-1 mRNA levels by a posttranscriptional mechanism. This GM-CSF-dependent regulation of EGR-1 expression may provide a mechanism for transducing signals to the nucleus that are involved in the control of gene transcription.

Cell Line, Transformed↗

PubMed1990

Phospholipase A2 activation and autoinduction of tumor necrosis factor gene expression by tumor necrosis factor.

Tumor necrosis factor (TNF) acts via a cell surface receptor to induce a variety of cellular events including cytolysis, differentiation, and mitogenesis. The mechanisms underlying the cell specific actions of TNF are not known. In the present study, postreceptor events associated with the autoinduction of TNF expression were examined in HL-60 cells. There was no detectable alteration in phospholipase C activity as measured by inositol phosphate generation or release of choline metabolites following TNF stimulation. However, TNF increased the release of arachidonic acid metabolites from HL-60 cells. This increase in arachidonic acid metabolism was associated with a 40% increase in phospholipase A2 activity. Furthermore, the release of arachidonic acid metabolites was blocked by inhibitors of phospholipase A2. Taken together, these findings indicated that TNF stimulates phospholipase A2 and arachidonic acid metabolism in HL-60 cells. The results also demonstrate that TNF expression is induced 15-30 min after stimulation with TNF and that this effect is associated with an increase in the rate of TNF transcription. This autoinduction of TNF mRNA was blocked by inhibitors of phospholipase A2. While the cyclooxygenase inhibitor indomethacin had no detectable effect, ketoconazole and nordihydroguaiaretic acid, inhibitors of lipoxygenase, also blocked the induction of TNF expression by TNF. These findings suggest that phospholipase A2 and lipoxygenase activity are required for the transcriptional activation of TNF gene expression associated with TNF stimulation of HL-60 cells.

Acetophenones↗

PubMed1990

Inhibition of phorbol ester-induced monocytic differentiation and c-fms gene expression by dexamethasone: potential involvement of arachidonic acid metabolites.

The treatment of human U-937 leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation. However, the signaling pathways responsible for induction of the differentiated monocytic phenotype remain unclear. The present studies demonstrate that dexamethasone blocks TPA-induced U-937 cell growth inhibition, adherence, and alpha-naphthyl acetate esterase staining. The results also demonstrate that dexamethasone inhibits the appearance of c-fms transcripts associated with TPA treatment. Run-on transcription assays demonstrated that the c-fms gene is transcriptionally active in uninduced U-937 cells and that the rate of transcription is unchanged after dexamethasone and/or TPA treatment. These findings indicated that TPA increases c-fms expression by a dexamethasone-sensitive posttranscriptional mechanism. Treatment of U-937 cells with TPA was also associated with stimulation of arachidonic acid metabolism. Furthermore, dexamethasone, an inhibitor of phospholipase A2 activity, blocked TPA-induced increases in arachidonic acid release. These findings suggested that TPA may regulate certain features of monocytic differentiation, such as c-fms gene expression, through the formation of arachidonic acid metabolites. Indomethacin, an inhibitor of cyclooxygenase, had no detectable effect on c-fms gene expression. However, the cyclooxygenase metabolite, prostaglandin E2, inhibited the TPA-induced increases in c-fms mRNA levels. Taken together, the results indicate that TPA regulates c-fms gene expression by a dexamethasone-sensitive mechanism and that c-fms mRNA levels are controlled by metabolites of the arachidonic acid pathway.

Arachidonic Acids↗

PubMed1990

DF3 antigen, a human epithelial cell mucin, inhibits adhesion of eosinophils to antibody-coated targets.

Although mucins provide lubrication and physical protection for epithelial cell surfaces, other functional roles for these large glycoproteins are unknown. One human mucin, designated DF3 Ag, is detectable on apical surfaces of normal epithelial secretory cells and in normal milk, urine, and plasma. The present studies have examined the effects of DF3 Ag purified from both normal and malignant sources on the antibody-dependent cytotoxicity of Schistosoma mansoni by eosinophils (ADCC-E) and on the adherence of eosinophils to inert antibody-coated targets. DF3 Ag purified from tissue culture supernatant of a human breast carcinoma cell line or from human milk inhibited ADCC-E in a concentration-dependent manner, with half-maximal inhibitory activity at 3 to 10 micrograms/ml. Inhibition of ADCC-E was specific for the DF3 mucin, because no inhibition was observed with two other unrelated, circulating glycoproteins: carcinoembryonic Ag and alpha 1-acid glycoprotein. Inhibition was not a result of direct cytotoxicity of the DF3 Ag for eosinophils, as demonstrated by the lack of detectable effect of the mucin on cellular trypan blue exclusion or PMA-induced H2O2 release. The inhibitory effect was time dependent, requiring the presence of DF3 Ag in the ADCC-E culture for at least 4 h, beginning within the first 2 h of eosinophil-schistosomula interaction. Furthermore, inhibition was not a result of interaction between DF3 Ag and the activating lymphokine. These data suggest that inhibition of ADCC-E by DF3 Ag is a result of interference of adhesion of eosinophils to Ig-coated targets. In this regard, purified DF3 tumor Ag prevents eosinophil adherence to human Ig-conjugated Sepharose 4B beads. Preincubation of the inert Ig-coated targets with DF3 Ag did not inhibit subsequent eosinophil adherence, suggesting that DF3 Ag interacts with a moiety present on the eosinophil. Inhibition of adhesion occurred at 37 degrees C, but was also observed at 4 degrees C. These results suggest that DF3 Ag acts as an immunomodulating agent. Because activated eosinophils can damage surrounding normal tissues as well as infectious organisms, DF3 Ag may serve to protect secretory epithelium from the cytotoxic effects of activated inflammatory cells.

Animals↗

PubMed1990

Regulation of proto-oncogene and tumor necrosis factor gene expression by ethanol in HL-60 myeloid leukemia cells.

Previous studies have shown that certain low molecular weight polar solvents downregulate c-myc gene expression and induce terminal differentiation of human HL-60 myeloid leukemia cells. We have examined the effects of ethanol on gene expression in this cell line. The results show that while ethanol induces a more differentiated phenotype, this agent has little effect on the self-renewal capacity of HL-60 cells. Ethanol treatment was also associated with a concentration-dependent and transient downregulation of c-myc transcripts. Similar effects were observed for c-myb mRNA levels. The results further show that ethanol exposure is associated with induction of tumor necrosis factor gene expression. These findings indicate that ethanol induces changes in specific gene expression during non-terminal differentiation of HL-60 cells. The clinical effects of this agent could thus be related to altered patterns of gene expression in hematopoietic or other cells.

Cell Differentiation↗

PubMed1990

Transcriptional and post-transcriptional regulation of c-jun expression during monocytic differentiation of human myeloid leukemic cells.

AP-1, the polypeptide product of c-jun, recognizes and binds to specific DNA sequences and stimulates transcription of genes responsive to certain growth factors and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). We studied the effects of TPA on the regulation of c-jun gene expression in HL-60 cells during monocytic differentiation. Low levels of c-jun transcripts were detectable in untreated HL-60 leukemic cells, increased significantly by 6 h, and reached near maximal levels by 24 h of exposure to 32 nM TPA. Similar kinetics of c-jun induction by TPA were observed in human U-937 and THP-1 monocytic leukemia cells. Similar findings were obtained with bryostatin 1 (10 nM), another activator of protein kinase C and inducer of monocytic differentiation. Furthermore, 1,25-dihydroxyvitamin D3 (0.5 microM), a structurally distinct agent which also induces HL-60 monocytic differentiation, increased c-jun expression. TPA treatment of HL-60 cells in the presence of cycloheximide was associated with superinduction of c-jun transcripts. Run-on analysis demonstrated detectable levels of c-jun gene transcription in untreated HL-60 cells, and that exposure to TPA increases this rate 3.3-fold. Treatment of HL-60 cells with both TPA and cycloheximide had no effect on the rates of c-jun transcription. The half-life of c-jun RNA as determined by treating HL-60 cells with TPA and actinomycin D was 30 min. In contrast, the half-life of c-jun RNA in TPA-treated HL-60 cells exposed to cycloheximide and actinomycin D was greater than 2 h. These findings suggested that the increase in c-jun RNA observed during TPA-induced monocytic differentiation is mediated by both transcriptional and post-transcriptional mechanisms.

Calcitriol↗

PubMed1990

Evaluation of monoclonal antibody DF3 conjugated with ricin as a specific immunotoxin for in vitro purging of human bone marrow.

DF3 is an IgG1 monoclonal antibody (MAb) generated against a Mr 350,000-400,000 glycoprotein expressed by approximately 80% of human breast cancers. We have coupled MAb DF3 to ricin. Purification of the immunotoxin (DF3-IT) was obtained by affinity and size exclusion chromatography. DF3 antigen-positive breast cancer cell lines (ZR-75-1, BT-20, and MCF-7) and DF3 antigen-negative lung cancer cell lines (A549 and CALU6) were tested for cytotoxicity with metabolic labeling and clonogenic assays. The cells were exposed for 3 h to different concentrations of DF3-IT, MAb DF3, ricin, and a combination of unconjugated MAb DF3 and ricin. In the presence of 100 mM lactose, DF3-IT specifically inhibited protein synthesis of lines expressing DF3 antigen on their cell surface. Moreover, clonogenic survival experiments demonstrated that DF3-IT at a concentration of 1 x 10(-9) M specifically kills 2.6-2.8 log of ZR-75-1 and BT-20 cells and 1.6 log of MCF-7 cells. At the same concentration, nonspecific toxicity of DF3-IT resulted in a 30% reduction of bone marrow granulocyte and macrophage colony formation. In bone marrow-purging experiments, tumor cells were mixed with an excess of bone marrow cells and treated with DF3-IT or ricin. Tumor cell clonogenic survival assays demonstrated that the presence of bone marrow cells had no detectable effect on activity or specificity of the DF3-IT. These results thus indicate that MAb DF3 is an effective vehicle to specifically deliver toxins to cancer cells which express DF3 antigen on their surface and that DF3-IT may be useful for in vitro purging of bone marrow.

Antibodies, Anti-Idiotypic↗

PubMed1990

Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells.

Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5'-diphosphate (GDP) in the unstimulated state and guanosine 5' triphosphate (GTP) in the active state. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL-60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate-ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N-formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

Adenosine Diphosphate Ribose↗