The response of three human leukemia cell lines, the proliferative promonocyte THP-1 and the promyeloid HL60 cells and the non-proliferative phorbol ester-treated HL60 cells (HL60/PMA), to oxidative stress induced by tert-butylhydroperoxide (t-BHP) treatment was analyzed by fluorescence microplate assay, anti-oxidant enzyme activity measurements, high performance liquid chromatography, yopro-1/PI incorporation, poly (ADP-ribose) polymerase and caspase 3 cleavages. After t-BHP treatment, the non-proliferative HL60/PMA cells exhibited a weak increase in reactive oxygen species (ROS) production, a better preservation of thiol content, a decrease of glutathione peroxidase activity and a high ability to undergo necrosis rather than apoptosis. Submitted to the same treatment, the proliferative HL60 and THP-1 cells exhibited a high increase of ROS production, a moderate thiol depletion and a high percentage of apoptosis. Under thiol depleting conditions, the oxidative treatment of the HL60/PMA cells resulted in a high ROS production that reached levels similar to those of the two other cell lines and in cell death mainly by necrosis. In conclusion, these results that show proliferative phenotype is essential for cell response towards oxidative stress, are of particular interest in chemotherapy involving an oxidative mechanism.

A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tent-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.

Changes in beta1-integrin expression have been involved in abnormal cellular interactions between malignant lymphocytes from Sézary (Sz) patients and keratinocytes. In this paper, we compare the activity of both distal and proximal promoters of the beta1-integrin gene in malignant lymphocytes from Sz patients with human normal lymphocytes. Activity of both beta1-integrin promoters was also analysed in human normal keratinocytes. Northern blot analysis shows that beta1-integrin mRNA expression is higher in malignant Sz lymphocytes than in normal lymphocytes. CAT assays show that the activity of proximal beta1-integrin promoter is markedly increased (up to 6-fold) in malignant lymphocytes from Sz patients, in comparison to normal lymphocytes. These results suggest that changes in activity of the proximal promoter of beta1-integrin subunit could be, in part, responsible for the abnormal cellular interactions between malignant lymphocytes and keratinocytes observed in Sz syndrome.

The vascular endothelium represents an important interface between the blood vessel lumen and the surrounding tissues, and regulates vasotonicity, permeability, homeostasis and angiogenesis. The adhesive interactions between endothelial cells (EC) and their surrounding extracellular matrix (ECM) regulate growth, differentiation and migration1. The localization of endothelial cells at the contact with leukocytes lead to their exposure to peroxides during local inflammatory reactions. It has been reported on that many inflammatory mediators, including reactive oxygen species (ROS) and cytokines modulate EC-leukocyte interaction,2 In contrast little reports have focused on EC-ECM interactions. The purpose of this study was to set-up an in-vitro model for studying the effects of oxidative stress on EC-ECM interactions, using two immortalized cell lines derived from human umbilical vein cord (HUVEC), and classified as endothelial. ECV304 spontaneously transformed HUVEC,3 and EA.hy926 a hybridoma of the epithelial cell line A549 and HUVEC,4 were used to study the effects of ter-Butyl Hydroperoxide (t-BHP) on cell adhesion to collagen I, intracellular glutathione (GSH) depletion, ROS production, integrin expression and cytoskeleton organization.

We previously showed that HL 60 leukemia cells exhibit various changes in their cellular glycans after phorbol 12-myristate 13-acetate (PMA) treatment. These changes could originate largely from changes in one or several glycosyltransferases. In this report, we show using enzymatic measures, fluorescence microscopy, immunoblotting and Northern blot that β-(1→4)-galactosyltransferase I (GalT I) activity was higher (>×2) in PMA-treated compared with untreated HL 60 cells. Immunoblotting showed an increased intensity of the GalT I band at 49 kDa and Northern blot a weak increase of the GalT I transcript band, after PMA treatment. Moreover, Northern blot performed after actinomycin-D treatment of the cells, which inhibits transcription, suggests that the observed increase of GalT I expression could originate, in part, from increase of the stability of GalT I transcripts.

Development of microspectrofluorometric methods using specific fluorogenic probes has provided precious help in studying in situ oxidative stress and cellular protective systems. The aim of this study was to determine ROS production concomitantly with a modification of the intracellular thiol pool after applying an oxidative stress to a nonadherent cell model represented by the HL60 cell line. The dichlorodihydrofluorescein diacetate (H2DCFDA) probe assessed the kinetic production of ROS by cells submitted to the chemical oxidant t-butylhydroperoxide with a high signal/noise ratio. The probe sensitivity permitted us to detect endogenous ROS production in HL60 cells and the protective effect of N-acetyl cysteine against ROS. The chloromethylfluorescein diacetate probe (CMFDA) permitted us to evaluate the thiol depleting effect of N-ethyl maleimide. Complete thiol depletion was associated with a moderate increase in ROS production. The cell viability was determined with calcein-AM, which gave results similar to those with the tetrazolium dye. This probe was not affected by intracellular pH and did not required an extraction step, unlike tetrazolium dye. In conclusion, cell-permeant fluorogenic probes are useful and sensitive tools to determine in situ ROS production concomitantly with consecutive change in the thiol system in a living and non-adherent cell model.

Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4+, CD45 RO+ T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Szbeta(1-6)+] expressed high levels of beta(1-6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Szbeta(1-6)-] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Szbeta(1-6)+ patients adhered less to normal keratinocyte monolayers than T lymphocytes from Szbeta(1-6)- patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Szbeta(1-6)-, especially alpha4, alpha5, beta1 and beta2, than on Szbeta(1-6)+ and normal lymphocytes. Although these results suggest that beta1- and beta2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of beta(1-6)-GlcNAc-branched N-linked oligosaccharides on beta1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the beta1 integrin subunit expressed by T lymphocytes from Szbeta(1-6)+ patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.

The circulating forms of malignant cells from patients with Sezary syndrome exhibit on their glycoproteins a high level of beta (1-6)GlcNAc-branched N-linked oligosaccharides, a particular species of glycans related to the metastatic potential of several tumors and T lymphocytes activation. An increased activity of the N-acetylglucosaminyltransferase V and of the beta (1-4)galactosyltransferase, two enzymes implicated in beta (1-6)GlcNAc-branching is also found. Nevertheless, contrary to activated normal T lymphocytes, Sezary lymphocytes in agreement with their non-proliferating state, do not exhibit increased thymidine uptake. This result suggests that expression of the beta (1-6)GlcNAc-branched N-linked carbohydrates could be related to some of the malignant properties of Sezary lymphocytes.

Human HL 60 myeloid leukaemia cells have the potential to differentiate into either macrophage-like cells or granulocyte-like cells under the stimulus of chemical treatments, Using glycotechnology procedures, the glycosylation patterns of differentiated and undifferentiated HL 60 cells were analysed and compared with those of normal human peripheral monocytes, Both in vitro differentiations result in significant morphologic and functional changes, but we observed that the glycosylation patterns of undifferentiated and differentiated HL 60 cells exhibit several common glycosidic features that are absent in normal peripheral monocytes: the presence of (i) bisecting beta-N-acetylglucosamine attached at the C-4 position of the beta-mannose of polyantennary complex-type carbohydrate chains and (ii) complex-type carbohydrate chains enriched with non-reducing terminal beta-N-acetylglucosamine residues. Moreover, the three populations of HL 60 cells express small amounts of biantennary complex-type structures (<6%), whereas normal peripheral monocytes expressed >20% of such structures, Thus, the cell glycosylation pattern could reflect the pathological state of the HL 60 cells.

Activation of human T lymphocytes by phorbol 12-myristate 13-acetate and leukoagglutinin from Phaseolus vulgaris (L-PHA) results in important changes in N-glycosylation. The most important event is the increase, in both T4 and T8 cells (especially the latter), of L-PHA+ structures characterized by beta 1-6-branching of complex-type oligosaccharides. Moreover, the existence of a CD4-mediated increase of these beta 1-6-branched structures on positively selected T4 cells, as compared with the negatively selected ones, suggests that the presence of these structures, not detectable on T8 resting cells, could be related to stimulation events triggered by both selection methods. This beta 1-6-branching on N-glycans, strongly associated with a metastatic phenotype in human and rodent tumors, is exhibited by numerous glycoproteins on stimulated cells, as shown by blot analysis.

Tリンパ球が活性化すると細胞膜糖タンパク質の糖鎖に重要な変化が誘導されるという報告が最近複数なされている。N-およびO-結合糖鎖のいずれにもみられるこの変化は、β1-6結合のラクトサミン分岐の著明な増加を中心としたものである。他の結合様式のラクトサミン分枝と異なり、β1-6ではこの分枝がタンパク質の骨格に向かって折れ曲がりやすいためにタンパク質の立体構造や活性に影響を与える可能性がある。この総説は人のTリンパ球の活性化によって誘導される糖構造の変化についての最新の知見を総括し、それらの変化がリンパ球の生理や免疫応答においてどのような機能をはたしていると考えられるかを議論する。

Robinia pseudoacacia seeds contain lectins which are closely related. Pronase digestion of the dimeric and tetrameric lectins, RPA1 and RPA3, gave glycopeptides. The structure of the oligosaccharide was determined by 1H NMR spectroscopy and carbohydrate determination as alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-[alpha-D-Manp+ ++-(1-->6)]-beta- D-Manp-(1-->4)-beta-D-GlcpNAc-(1-->4)-[alpha-L-Fucp-(1-->3)] -beta-D-GlcpNAc - (1-->4)-Asn. It appears that the 34-kDa constitutive polypeptide of RPA1 contains 4-5 carbohydrate chains whereas the 30.5-kDa and 29-kDa subunits of RPA3 contain two and one oligosaccharide chains, respectively.

Glycobiology, the study of the biology of carbohydrates and glycoconjugates, has recently benefited from a renewed interest. Indeed, results of structural studies performed during the past years can now be used for investigating of the physiological roles of glycoconjugates, particulary in the field of immunobiology and hematology. Recent studies on leukocyte adhesion have led to the characterization of three lectins of the same family, the selectins or LEC-CAMs (L for lectin, E for epidermal growth factor-like, C for complement-like short consensus repeat, CAM for cell adhesion molecule). L-selectin (LEC-CAM 1) is involved in lymphocyte homing while E-selectin (LEC-CAM 2 or ELAM 1) and P-selectin (LEC-CAM 3, GMP 140 or CD 62) play an important role in leukocyte adhesion to endothelia and platelets during inflammation and blood clotting. Another adhesion molecule termed H-CAM (CD44 or Hermes antigen) plays also a role in lymphocyte homing by binding to carbohydrate determinants on extracellular matrix. Moreover, some hematological or immunological diseases as Wiskott-Aldrich syndrom or congenital dyserythropoietic anemia type 2 are related to glycosylation abnormalities or defects.

Glucose transport activity was determined on erythrocytes from healthy adult and children. A large variability of values was found between each donor, but transport rate was significantly higher in erythrocytes from adults than those from children. In opposite, the same number of glucose transport sites was found on erythrocytes from adult and children, suggesting that transporters from adult erythrocytes exhibited an higher own activity than transporters from child erythrocytes. Involvement of various factors in glucose transport activity was discussed.

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the V(max) In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.