Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates

The glycosylation pattern of the external envelope glycoproteinof human immunodeficiency virus type 2 (HIV-2) was studied independence on host cells and virus isolates. Strains HIV-2_ALT,HIV-2_ROD and HIV-2_D194, differing in their biological propertiesand in the amino acid sequences of their env genes, were propagatedin MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytesand monocytes/macrophages in the presence of [6-³] glucosamine.Radiolabelled viral glycoproteins were isolated from the cell-freesupernatants and digested with trypsin. Glycans were sequentiallyliberated by endo-ß-N-acetylglucosaminidase H andpeptide-N⁴-(N-acetyl-ß-glucosaminyl) asparagine amidaseF, and fractionated according to charge and size. Comparisonof the oligosaccharide profiles revealed that the envelope glycoproteinsof different virus isolates, propagated in the same host cells,yielded very similar glycan patterns, whereas cultivation ofan isolate in different host cells resulted in markedly divergentoligosaccharide maps. Variations concerned the proportion ofhigh-mannose-, hybrid- and complex-type substituents, as wellas the state of charge and structural parameters of the complex-typespecies. As a characteristic feature, complex-type glycans ofmacrophage-derived viral glycoprotein were almost exclusivelysubstituted by lactosamine repeats. Hence, glycosylation ofthe HIV-2 external envelope glycoprotein seems to be primarilygoverned by host cell-specific factors rather than by the aminoacid sequence of the corresponding polypeptide backbone. envelope glycoprotein glycosylation human immunodeficiency virus type 2     

Glycosylation pattern and processing of envelope gene products encoded by glycosylation mutants of Friend spleen focus-forming virus

The protein encoded by the envelope gene of Friend spleen focus-formingvirus is responsible for the acute leukaemogenicity of thisvirus. In order to correlate glycosylation and intracellularprocessing of this protein with viral pathogenicity, envelopegene products of pathogenic and apathogenic glycosylation mutantswere expressed in Rat-1 cells and metabolically labelled with[6-³H] glucosamine. Following immunoprecipitation, primary andsecondary gene products (gp55, gp65) were separated by preparativepolyacrylamide gel electrophoresis. Oligosaccharides were releasedfrom tryptic glycopeptides by treatment with endo-ß-N-acetylglucosaminidaseH (gp55), peptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagineamidase F (gp65) or by reductive ß-elimination. Resultingglycans were characterized by cochromatography with authenticoligosaccharide standards using different HPLC systems and digestionwith exoglycosidases. The results revealed that the primaryenvelope gene products of pathogenic glycosylation mutants were,in part, further processed in Rat-1 cells similar to wild-typeglycoprotein, resulting in polypeptides carrying complex-typeN-glycans as well as partially sialylated O-linked oligosaccharides.In contrast, corresponding glycoproteins encoded by apathogenicmutants were found to remain at the level of the primary translationproduct exclusively comprising high-mannose-type N-glycans.Hence, intracellular maturation of the envelope gene productsin this model cell line seems to correlate with the in vivopathogenicityof the glycosylation mutants studied. carbohydrate structure glycoprotein murine leukaemia virus oligosaccharide processing SFFV     

Light and heavy lysosomes: characterization of N-acetyl-{beta}-D-hexosaminidase isolated from normal and I-cell disease lymphoblasts

We previously reported that I-cell disease lymphoblasts maintainnormal or near-normal intracellular levels of lysosomal enzymes,even though N-acetylglucosamine-1-phosphotransferase activityis severely depressed or absent (Little et al., Biochem. J.,248, 151–159, 1987). The present study, employing subcellularfractionation on colloidal silica gradients, indicates thatboth light and heavy lysosomes isolated from I-cell diseaseand pseudo-Hurler polydystrophy lymphoblasts possess normalspecific activity levels of N-acetyl-ß-D-hexosaminidase,-D-mannosidase and ß-D-glucuronidase. These currentfindings are in contrast to those of cultured fibroblasts fromthe same patients, where decreased intralysosomal enzyme activitiesare found. Column chromatography on Ricinus communis revealedthat N-acetyl-ß-D-hexosaminidase in both heavy andlight I-cell disease lysosomal fractions from lymphoblasts possessesan increased number of accessible galactose residues (30–50%)as compared to the enzyme from the corresponding normal controls.Endo-ß-N-acetylglucos-aminidase H treatment of N-acetyl-ß-D-hexosaminidasefrom the I-cell lysosomal fractions suggests that the majorityof newly synthesized high-mannose-type oligosaccharide chainsare modified to complex-type carbohydrates prior to being transportedto lysosomes. This result from lymphoblasts differs from previousfindings with fibroblasts, where N-acetyl-ß-D-hexosaminidasefrom I-cell disease and pseudo-Hurler polydystrophy lysosomesexhibited properties associated with predominantly high-mannose-typeoligosaccharide chains. The current results imply that differentcell types may modify the carbohydrate side chains of lysosomalenzymes in a differential manner, and that selected cell typesmay also employ mechanisms other than the mannose-6-phosphatepathway for targeting lysosomal enzymes to lysosomes. I-cell disease lymphoblasts lysosomes mannose-6-phosphate oligosaccharide chains pseudo-Hurler polydystrophy     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked     

Processing of asparagine-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells

The levels of ß1,2-N-acetylglucosaminyltransferase(GlcNAc-T) I and II activities in cultured cells from Bombyxmori(Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda(Sf-9 and Sf-21) were investigated. Apart from initial experimentswith Man     

Characterization of asparagine-linked oligosaccharides on a mouse submandibular mucin

The asparagine-linked oligosaccharides from an adult femalemouse submandibular gland mucin were released by treatment withpeptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagine amidaseF or endo-ß-N-acetylglucosaminidase H. Endo-ß-N-acetylglucosaminidaseH appeared to be more effective at releasing the asparagine-linkedoligosaccharides from this mucin than was peptide-N⁴-(N-acetyl-ß-glucosaminyl)-asparagineamidase F. After quantitative reductive labelling with the fluorophore,8-aminonaphthalene-1, 3, 6-sulphonic acid, the oligosaccharideswere separated by polyacrylamide gel electrophoresis and isolated.The individual oligosaccharides were sequenced by a batteryof recombinant exoglycosidases. Approximately 50% of the oligosaccharideswere of the high-mannose type. The five-mannose member of thisfamily was the most prevalent. The second group of oligosaccharideswere of the non-bisected hybrid type. No complex asparagine-linkedoligosaccharides were detected. The hybrids exhibited both biantennaryand triantennary branching patterns. The triantennary hybridwas the most common hybrid at >30% of all oligosaccharides.With 98% of the hybrid oligosaccharides sialylated and all lackinga bisecting N-acetylglucosamine, these oligosaccharides as agroup have been only rarely observed in other glycoproteins.The fully sialylated triantennary hybrid may be unique. asparagine-linked oligosaccharides biantennary salivary mucin sialylated hybrid triantennary     

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human {beta}1,2-N-acetylglucosaminyltransferase I

Spodoptera frugiperda (Sf9)-cells differ markedly in their proteinglycosylation capacities from vertebrate cells in that theyare not able to generate complex type oligosaccharide side chains.In order to improve the oligosaccha ride processing propertiesof these cells we have used baculovirus vectors for expressionof human (ß1,2-N-acetylglucosaminyltransferase I (hGNT-I),the enzyme catalysing the crucial step in the pathway leadingto complex type N-glycans in vertebrate cells. One vector (Bac/GNT)was designed to express unmodified GNT-I protein, the secondvector (Bac/tagGNT) to express GNT-I protein with a tag epitopefused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virusa protein of about 50 kDa representing hGNT-I was detected withan antiserum directed against the tag epitope. HGNT-I activitywas increased at least threefold in lysates of infected cellswhen N-acetylglucosamine (GlcNAc)-free ovalbumine was used assubstrate. To monitor hGNT-I activity in intact Sf9-cells, theglycosylation of coexpressed fowl plague virus hemagglutinin(HA) was investigated employing a galactosylation assay andchromatographic analysis of isolated HA N-glycans. Coexpressionof hGNT-I resulted in an at least fourfold increase of HA carryingterminal GlcNAc-residues. The only structure detectable in thisfraction was GlcNAcMan₃GlcNAc₂. These results show that hGNT-Iis functionally active in Sf9-cells and that the N-glycans ofproteins expressed in the baculovirus/insect cell system areelongated by coexpression of glycosyltransferases of vertebrateorigin. Complete complex type oligosaccharide side chains werenot observed when hGNT-I was overexpressed, thus supportingthe concept that Sf9-cells do not contain glycosyltransferasesacting after hGNT-I. ß1,2-N-acetylglucosaminyltransferase I baculovirus expression of recombinant protiens N-glycosylation in Sf9-cells     

Relationship between Flower Induction by Benzoic Acid and its Metabolism in Lemna paucicostata 151

The flower-inducing activities in Lemna paucicostata 151 offour major metabolites of benzoic acid (N-benzoyl aspartate,benzyl 6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranoside,O-benzoyl isocitrate and O-benzoyl malate) were measured, andthe effects on the uptake and metabolism of benzoic acid dueto change in the level of the benzoic acid concentration orto the addition of plant hormones were investigated. N-Benzoylaspartate had weak activity, and O-benzoyl isocitrate and malatehad fairly strong activities, while benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosideshowed no activity. As the concentration of benzoic acid rose,the ratio of N-benzoyl aspartate increased and that of benzyl6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranosidedecreased. GA₃ and IAA, inhibitors of flower induction by benzoicacid, seemed to promote conversion to N-benzoyl aspartate insteadof to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranoside.The conversion to N-benzoyl aspartate was considered to be adetoxification process and that to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosidemay be directly related to flower induction in Lemna. (Received November 2, 1987; Accepted January 23, 1988)     

{beta}1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay

Lymphocytic ß1,4-galactosyltransferase (ß1,4-GalTase,EC 2.4.1.38 [EC] ) activity was measured in B cells using a neoglycoprotein,N-acetylglucosamine-phenylisothlocyanate-bovine serum albumin(GlcNAc-pITC-BSA), as an acceptor substrate in a novel enzyme-linkedimmunosorbent assay (ELISA)-based method. This assay provedto be much simpler to use than the lengthy and expensive radiochemicalassays commonly used, and has the additional advantage thatit specifically detects the enzyme mediating transfer via theGalß1,4GlcNAc linkage. A F(ab')₂ antibody againstGalTase was able to specifically inhibit the reaction. Greatersensitivity for ß1,4-GalTase activity was obtainedusing GlcNAc-pITC-BSA as an acceptor substrate rather than ovalbumin.Low levels of ß-galactosidase activity were detectablein lymphocyte cell lysates at acidic pH, although such activitywas not detectable at the neutral pH used in the ß1,4-GalTaseactivity assay. Using this assay with the GlcNAc-pITC-BSA acceptor,similar ß1,4-GalTase activities were observed in CD19⁺B cells from patients with rheumatoid arthritis (RA) to thoseseen in normal control individuals. ELISA ß1,4-galactosyltransferase lymphocyte neoglycoprotein radiochemical     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas

Enzymatic analysis of oligosaccharides using exoglycosidaseshas become a powerful tool for determining the sequence andstructure of sugar chains. The principal limitation to thesemethods has been the lack of highly purified and wellcharacterizedenzymes. Using fluorescently labelled carbohydrate substratesand TLC, we have developed a method to identify glycosidaseswith novel specificities. This screening method led to the discoverythat bacteria of the genus Xanthomonas are a rich source ofexoglycosidases. From Xanthomonas manihotis, eight novel exoglyccosidaseshave been isolated and characterized. A novel ß-N-acetylglucosaminidasehas been purified that, unlike those previously described, willcleave N-acetylglactosamine without cleaving N-acetylgalactosamineresidues. A novel ß-galactosidase has been isolatedthat preferentially hydrolyses ß(1     

Glycosylation in Lepidopteran insect cells: identification of a {beta}1->4-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains

The choice for a heterologous expression system to produce glycoproteintherapeutics highly depends on its potential to perform mammalian-likeposttranslational modifications such as glycosylation. To gainmore insight into the glycosylation potential of the baculovirusmediated insect cell expression system, we have studied theexpression of glycosyltransferases involved in complex-typeN-glycosylation. Lepidopteran insect cell lines derived fromTrichoplusia ni, Spodoptera frugiperda, and Mamestra brassicaewere found to express a ß1     

A subset of myeloid dendritic cells derived from peripheral blood monocytes represented a predominant subset characterized by their potential tumor-inhibiting activity

Besides their role as potent antigen-presenting cells, myeloid dendritic cells (MDCs), but not plasmacytoid dendritic cells (PDCs), have been reported to have cytotoxic or cytostatic activity on some tumor cells. In this article, we analyzed the tumoristatic potential of a distinct peripheral blood monocyte-derived MDC subset which co-expressed PDC-specific marker CD123. CD123⁺ MDCs represented a subset of small-sized DCs and accounted for 45–60% of peripheral blood monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukine-4 (IL-4) for 7 d. They exhibited more significant antiproliferative activity toward hematological tumor cell lines of Jurkat, HL60, and myelodysplastic syndromes over-leukemia than CD123⁻ MDCs even at a low effecter/target ratio. Pretreatment of MDC and their supernatant with TRAIL-R2:Fc significantly reduced the tumoristatic effect of CD123⁺ MDCs but not of CD123⁻ MDCs and their supernatant. CD123⁺ MDCs expressed higher level of cytoplasmic TNF-α-related apoptosis-inducing ligand (TRAIL) than CD123⁻ MDCs, whereas both expressed very little surface and soluble TRAIL. These results reveal that CD123⁺ cells represented a predominant subset of MDCs generated from peripheral blood monocytes in vitro, characterized by their potential tumoristic activity partially via cytoplasmic TRAIL.     

Effects of overexpression of {beta}1, 4-galactosyltransferase on glycoprotein biosynthesis in F9 embryonal carcinoma cells

ß1,4-Galactosyltransferase (GalTase) plays a centralrole in the biosynthesis of N-acetyllactosamine-containing oligo-saccharides.However, despite this seemingly important function, little isknown about how changes in the levels of GalTase affect oligosaccharidebiosynthesis. We have examined the effects of overexpressingGalTase on the glycosylation of endogenous glycoproteins inF9 mouse embryonal carcinoma cells. Cells transfected with eitherthe short form of the GalTase cDNA (encoding a protein of 386amino acids) or the long form of the GalTase cDNA (encodinga protein of 399 amino acids) had a 3-fold increase in totalGalTase activity, relative to control F9 cells. Analysis ofpronase-digested glycopeptides obtained from control and transfectedcells after metabolic labelling with [6-³H]galactose revealedno significant qualitative or quantitative differences, as assessedby Bio-Gel P-6 gel filtration chromatography and Tomato lectinaffinity chroma-tography. Furthermore, SDS-PAGE analysis ofimmuno-precipitated [³H]galactose-labelled lysosomal-associatedmembrane protein-1 (LAMP-1) glycoprotein showed no differencein amounts or mobility. Pronase digestion and subsequent analysisof the gel-fractionated LAMP-1 glycoproteins also indicatedno differences between the various cell lines. The inabilityof elevated GalTase activity to affect glycosylation was notdue to limiting levels of GalTase substrates, since an excessof substrates was detectable in lysed cells using either endogenousor exogenous GalTase and UDP-[³H]galactose. Finally, the subcellulardistribution of GalTase, as assessed by sucrose gradient fractionation,was similar between all cell types, thus suggesting that GalTasewas appropriately compartmentalized in the transfected cells.More importantly, GalTase specific activities in the Golgi membranesof the transfected cells were 3–4 times greater than incontrol cells. These results show that selectively increasingGalTase activity does not alter glycoprotein biosynthesis inF9 cells. F9 cells galactosyltransferase glycoprotein biosynthesis     

Interaction of the photosensitizer 13,15-<Emphasis Type="Italic">N</Emphasis>-(3′-hydroxypropyl)cycloimide chlorin p<Subscript>6</Subscript> with normal and cancerous blood cells

The interaction of 13,15-N-(3′-hydroxypropyl)cycloimide chlorin p₆ (CIC) with normal blood cells and human myeloid leukemia K562 and HL60 cells was studied. CIC was found to be bound by the erythrocyte membrane but did not penetrate into the cytoplasm. It is characterized by a diffuse distribution in the cytoplasm of normal leukocytes, whereas its diffuse distribution in K562 and HL60 cells is accompanied by perinuclear accumulation and binding to the plasma membrane. The average cytoplasmic concentration corresponding to the CIC accumulation in leukemic cells at saturation is 2.2 to 2.6 times higher than that in normal leukocytes. CIC is more intensely accumulated in granulocytes than in lymphocytes. The kinetics of the cellular uptake and efflux was characterized. The normal leukocytes and erythrocytes were found to be 1.5 times and 3 to 4 times less sensitive, respectively, to the photodynamic action of CIC than the K562 and HL60 cells.     

Alterations of O-glycan biosynthesis in human colon cancer tissues

Human colon cancer is associated with antigenic and structuralchanges in mucin-type carbohydrate chains (O-glycans). To elucidatethe control of the biosynthesis of these O-glycans in coloncancer, we have studied glycosyltransferase and sulphotransferaseactivities involved in the assembly of elongated O-glycan structures.We analysed homogenates prepared from cancer tissue, adjacentnormal and distal normal tissue from 20 patients. Several transferaseactivities showed pronounced changes in cancer tissue. The changescorrelate with previous findings of a loss of O-glycans in cancermucins, but did not always correlate with levels of Tn, sialyl-Tn,T and Le^x antigens in homogenates or with the differentiationstatus and Duke's stages of the cancer tissue or the patient'sblood type, sex and age. UDP-GlcNAc: Gal NAc-R ß3-N-acetylglucosaminyltransferase(where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetyl-D-galactosamine)synthesizing O-glycan core 3, GlcNAcß1-3GalNAc-, CMP-sialicacid: GalNAc-peptide     

Induction of (2′–5′) oligoadenylate synthetase activity during granulocyte and monocyte differentiation

Summary Variations in the (2′–5′) oligoadenylate synthetase (2–5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2–5 A synthetase was enhanced in both cell lines in response to α, β interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2–5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2–5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2–5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2–5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.