Biomedical subjects

F Malavasi

Publications and source records attributed to F Malavasi.

At least 73 records · Page 4Linked to original sources

PubMed1996

Post-translational modification of CD38 protein into a high molecular weight form alters its catalytic properties.

Human CD38 is a 45-kDa transmembrane protein that acts as a bifunctional ectoenzyme, catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the hydrolysis of cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that RA-induced CD38 protein from human myeloid (HL-60) leukemia cells coimmunoprecipitates with another protein of molecular mass approximately190 kDa (p190). The p190 protein is localized exclusively in the membranes and is a consequence of post-translational cross-linking of CD38 protein. This conclusion was based on the observations that purified CD38 effectively competes with p190, its accumulation is preceded by the accumulation of CD38, it immunoreacted with three different monospecific anti-CD38 antibodies on immunoblots, and its peptide map revealed several peptides in common with CD38. Furthermore, CD38 could serve as a suitable substrate for transglutaminase (TGase)-catalyzed cross-linking reactions in vitro, and the accumulation of p190 in RA-treated HL-60 cells is effectively blocked by the presence of TGase-specific inhibitor. The purified p190 showed at least three times more cyclase activity than CD38. Conversely, p190 was at least 2.5-fold less active than CD38 in hydrolyzing cADPR to ADPR. These results suggest that post-translational modification of CD38 may represent an important mechanism for regulating the two catalytic activities of this bifunctional enzyme.

ADP-ribosyl Cyclase↗

PubMed1996

Expression of cyclic ADP-ribose-synthetizing CD38 molecule on human platelet membrane.

CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP-ribose from NAD+, and hydrolysis of cyclic ADP-ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP-ribose hydrolase activities. This finding indicates that the calcium-mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation.

ADP-ribosyl Cyclase↗

PubMed1996

Human CD38 ligand. A 120-KDA protein predominantly expressed on endothelial cells.

Human CD38, a pleiotropic molecule with ADP-ribosyl cyclase activity, regulates activation and growth of several cell types. Its in vivo function is incompletely determined, mainly due to the lack of evidence concerning the existence of a single or multiple ligands. We recently observed that CD38 rules a selectin-type adhesion between lymphoid cells and HUVECs. A panel of murine mAbs raised against HUVEC included one (Moon-1) constantly blocking the CD38-mediated adhesion of several cell lines to HUVEC. Tissue distribution studies and an extended immunohistochemical analysis on the majority of normal human tissues revealed that the Moon-1 molecule displays a unique pattern of expression, being present at high levels on resting and activated vascular endothelium, on the majority of monocytes, platelets, NK cells, and to a lesser extent on T, B, and myeloid cells. The Moon-1 structure of an apparent molecular mass of 120 kDa proved to be a ligand for human CD38, as inferred by the direct binding observed when using a chimeric mouse CD8 alpha-human CD38 (mCD8 alpha-hCD38) molecule as a probe in Western blot experiments. Furthermore, Ab-induced modulation experiments highlighted an association between the Moon-1 molecule and human CD38 on the surface of cell lines coexpressing the two structures, which also share a common regulation system of surface expression. Finally, direct ligation of Moon-1 on T cell lines caused a relevant increase in the cytoplasmic concentration of calcium ([Ca2+]i).

ADP-ribosyl Cyclase↗

PubMed1996

CD38 signal transduction in human B cell precursors. Rapid induction of tyrosine phosphorylation, activation of syk tyrosine kinase, and phosphorylation of phospholipase C-gamma and phosphatidylinositol 3-kinase.

Ligation of CD38 inhibits proliferation and induces apoptosis of human immature B cells, but the molecular mechanisms underlying this function are unknown. We found that CD38 dimerization with the specific mAbs T16 and IB4 induces rapid and transient tyrosine phosphorylation of several intracellular proteins in the immature B cell lines RS4;11, REH, 380, Nalm6, and OP-1. This effect could be markedly reduced by incubating cells with the tyrosine kinase inhibitors genistein, staurosporine, and herbimycin A. CD38 dimerization induced tyrosine phosphorylation of the protein kinase syk and increased syk kinase activity. CD38 dimerization also induced tyrosine phosphorylation of phospholipase C-gamma and of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). The latter was accompanied by a distinct increase in PI 3-kinase activity in the immunoprecipitates obtained with an anti-phosphotyrosine Ab. In contrast to the signaling triggered by surface Ig engagement in B lymphocytes, CD38 ligation did not appear to induce tyrosine phosphorylation of the src-like protein tyrosine kinases lyn, fyn, and btk, or of vav- and ras-GTPase-activating protein, nor did it induce detectable changes in cytosolic CA2+ concentrations. CD38 signaling also differed from cytokine-induced signaling in that it did not cause tyrosine phosphorylation of Jak1 and Jak2. Finally, CD38 ligation did not inhibit IL-3-induced tyrosine phosphorylation of Jak2. These results identify CD38 as a cell surface receptor with signal transduction properties activated by dimerization. Induction of signal transduction by CD38 ligation implies the existence of a yet unidentified natural ligand of CD38.

ADP-ribosyl Cyclase↗

PubMed1996

Identification and characterization of an active soluble form of human CD38 in normal and pathological fluids.

Human CD38 is a transmembrane glycoprotein involved in lymphocyte activation and adhesion to endothelium. The ectocellular domain of the molecule possesses properties of a bifunctional enzyme catalyzing both the synthesis from NAD+ and the hydrolysis of the calcium-releasing metabolite cyclic ADP-ribose (cADPR). Surface expression of CD38 (mCD38) is rapidly and almost completely down-modulated upon ligation by specific mAb in cells from different lineages. The data presented here also show that, in addition to the existence of a mCD38, a soluble form of CD38 (sCD38) is detectable in the cell culture supernatant of allo-activated T lymphocytes and of several tumor cell lines. sCD38 is also present in vivo and is assayable in normal (fetal serum and amniotic fluid) and pathological (serum and ascites from patients with multiple myeloma, and serum from patients with AIDS) biological fluids. Immunoaffinity chromatography, SDS-PAGE and Western blot analyses with mAb and polyclonal antibodies, along with metabolic labeling, yield a body of data concerning the structure of sCD38, which displays a M(r) of 39 kDa. Native sCD38 maintains the ability to inhibit the binding activity of different anti-CD38 mAb and still catalyzes the synthesis and the hydrolysis of cADPR at the same ratio observed with mCD38. Furthermore, cross-linking experiments indicate that the purified soluble molecule binds a 120 kDa molecule expressed by monocytoid cells and identified as a candidate ligand for human mCD38.

ADP-ribosyl Cyclase↗

PubMed1996

Human CD38, a cell-surface protein with multiple functions.

Human CD38 is a nonlineage-restricted type II transmembrane glycoprotein that has emerged as a multifunctional protein in recent years. It can serve as an ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose, a recently identified Ca2+ mobilizing agent that acts independently of inositol triphosphate. The enzymatic functions of CD38 probably contribute to an array of its immunoregulatory functions. The release of soluble CD38 and the ability of membrane-bound CD38 to become internalized in response to appropriate stimuli suggest that extracellular and intracellular roles for this protein are equally plausible. Ligation of CD38 with agonistic antibodies induces diverse effects in hematopoietic cells that range from growth stimulation to induction and prevention from apoptosis, induction of cytokines, activation of kinases, and phosphorylation of certain proteins. These observations suggest that CD38 may serve as receptor for an as yet unidentified ligand. Other molecules that share significant structural and functional homology to CD38 have been identified in humans and mice, suggesting that these molecules may represent a new family of proteins. Understanding the role of CD38 in certain pathological conditions such as myeloma, X-linked agammaglobulinemia, and HIV infection may provide insight into its physiological functions.

ADP-ribosyl Cyclase↗

PubMed1995

Ligation of CD38 suppresses human B lymphopoiesis.

CD38 is a transmembrane glycoprotein expressed in many cell types, including lymphoid progenitors and activated lymphocytes. High levels of CD38 expression on immature lymphoid cells suggest its role in the regulation of cell growth and differentiation, but there is no evidence demonstrating a functional activity of CD38 on these cells. We used stroma-supported cultures of B cell progenitors and anti-CD38 monoclonal antibodies (T16 and IB4) to study CD38 function. In cultures of normal bone marrow CD19+ cells (n = 5), addition of anti-CD38 markedly reduced the number of cells recovered after 7 d. Cell loss was greatest among CD19+ sIg- B cell progenitors (mean cell recovery +/- SD = 7.2 +/- 11.7% of recovery in control cultures) and extended to CD19+CD34+ B cells (the most immature subset; 7.6 +/- 2.2%). In contrast, CD38 ligation did not substantially affect cell numbers in cultures of normal peripheral blood or tonsillar B cells. In stroma-supported cultures of 22 B-lineage acute lymphoblastic leukemia cases, anti-CD38 suppressed recovery of CD19+ sIg- leukemic cells. CD38 ligation also suppressed the growth of immature lymphoid cell lines cultured on stroma and, in some cases, in the presence of stroma-derived cytokines (interleukin [IL] 7, IL-3, and/or stem cell factor), but did not inhibit growth in stroma- or cytokine-free cultures. DNA content and DNA fragmentation studies showed that CD38 ligation of stroma-supported cells resulted in both inhibition of DNA synthesis and induction of apoptosis. It is known that CD38 catalyzes nicotinamide adenine dinucleotide (NAD+) hydrolysis into cyclic ADP-ribose (cADPR) and ADPR. However, no changes in NAD+ hydrolysis or cADPR and ADPR production after CD38 ligation were found by high-performance liquid chromatography; addition of NAD+, ADPR, or cADPR to cultures of lymphoid progenitors did not offset the inhibitory effects of anti-CD38. Thus, anti-CD38 does not suppress B lymphopoiesis by altering the enzymatic function of the molecule. In conclusion, these data show that CD38 ligation inhibits the growth of immature B lymphoid cells in the bone marrow microenvironment, and suggest that CD38 interaction with a putative ligand represents a novel regulatory mechanism of B lymphopoiesis.

ADP-ribosyl Cyclase↗

PubMed1995

Modulation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-1 gp120.

CD4, a lymphocyte surface glycoprotein, serves as co-receptor for antigen with the T cell receptor (TCR). It is also the lymphocyte receptor for HIV by binding the gp120 viral envelope protein. Interaction of gp120 with CD4 is crucial for viral infection, but is not sufficient to allow viral entry into cells. Recombinant gp120 alters CD4+ T cell responsiveness to activation stimuli. To express its co-receptor function fully, CD4 must be laterally associated with the TCR and CD45 to form multi-receptor complexes competent to transduce potent activation signals. Here, we examine the possibility that gp120/CD4 binding alters lateral associations of CD4 with other lymphocyte surface molecules, and that assembly of abnormal multi-molecular complexes is involved in the gp120-induced CD4+ T cell dysfunction and in viral entry. In the absence of gp120, CD4 displayed high association with CD3, CD5, CD45RC, CD25, CD28, CD44, and CD53; weak association with CD2, CD38, CD45RB, CD62L, and CD26; and no association with CD45RA, CD45RO, CD11b, CD11a, CD54, CD7, CD48, CD98, CD59 CD55, HLA class I and class II molecules. Treatment with gp120 significantly increased CD4 association with CD3, CD45RA, CD45RB, CD59, CD38, CD26 and HLA class I, and decreased that with CD45RC. Specificity of these results were assessed at various levels. First, gp120 did not influence lateral associations displayed by other molecules, such as HLA class II. Second, the Leu3 mAb which binds CD4 on a site overlapping the gp120 binding site, did not elicit the same CD4 lateral associations as gp120, and finally, a direct gp120/CD4+ interaction was needed to induce the lateral associations, as shown by the observation that blocking the gp120/CD4 binding by the Leu3 mAb inhibited the gp120-induced associations. These results can be interpreted in several ways gp120/CD4 interaction could trigger an inside-out signal responsible for the associations, or gp120 could induce steric modifications of CD4 that increase its affinity for the associating molecules. Alternatively, these molecules may interact directly with gp120, bridging them with CD4. It is also possible that th e associations may be mediated by additional components, interacting with both gp120 and the associating surface molecule. The last hypothesis is likely for CD59, whose gp120-induced association with CD4 required the presence of serum in the co-capping assay. Since both CD59 and gp120 bind complement, the observed association could be mediated by complement components.

Antibodies, Monoclonal↗

PubMed1995

CD38 ligation induces discrete cytokine mRNA expression in human cultured lymphocytes.

Human CD38 is a surface glycoprotein expressed by different immuno-competent cells such as immature and activated lymphocytes, plasma cells and natural killer cells. It has recently been reported that the CD38 molecule exerts adenosine diphosphate ribosyl cyclase activity and is associated with distinct transmembrane signaling molecules. This study reports that ligation of CD38 by specific monoclonal antibodies (mAb) induces multiple cytokine mRNA expression in cultured peripheral blood mononuclear cells (PBMC). The mRNA for tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-12 were always detected, whereas interferon-gamma and IL-10 mRNA expression were seen in most, but not all PBMC cultures. Low levels of IL-2, IL-4 and IL-5 mRNA were also found. The key observation of this work is that CD38 ligation in PBMC induces a large spectrum of cytokines, many of which overlap with those induced via CD3 activation. The main differences between CD38 and CD3 activation are the low to undetectable levels of IL-2 mRNA, and the sustained IL-1 beta and IL-6 mRNA accumulation found in PBMC cultures following treatment with anti-CD38 mAb. Furthermore, PBMC proliferation was not found to be a prerequisite for CD38-mediated cytokine induction. Together, these results suggest that human CD38 activates a signaling pathway which leads to the induction of a discrete array of cytokines, and that this pathway only partially overlaps with that controlled by T cell receptor CD3.

ADP-ribosyl Cyclase↗

PubMed1995

Lymphocyte adhesion to endothelium.

The different homing and recirculation behaviors of lymphocytes depend on expression of specific adhesion receptors by lymphocytes and endothelial cells. Expression of these receptors is finely regulated according to cell type, functional state, and anatomical location, and builds up a complex network of interactions that simultaneously involve several of these receptors working as "traffic signals" or "postcodes" for lymphocyte migration and homing. There are five main families of adhesion molecules: immunoglobulin superfamily, integrins, selectins, cadherins, and mucin-like molecules. Together with these "classified" receptors, other molecules, such as CD44 and CD38, have been shown to be involved in lymphocyte migration and homing. Leukocytes have evolved a intracellular system that allows them to maintain these receptors in an inactive state during transit in the bloodstream and extracellular-fluids and activate them only when proper specific stimuli are delivered. This activity has been called "inside-out" signaling. Most receptor/ligand systems regulating lymphocyte migration are not selectively dedicated to this function. They are involved in lymphocyte interaction with several cell types and play a key role in both the afferent and efferent branches of immune responses by mediating lymphocyte interaction with APC and target cells. They not only "passively" anchor lymphocytes to these cells but also exert an active "outside-in" signaling function that modulates the cell response to activation stimuli. In this review, we briefly describe the major features of these molecules, survey what is known about their role in lymphocyte/endothelium interactions both in vitro and in vivo, and discuss their possible therapeutical application.

Cell Adhesion↗

PubMed1994

Interaction between endothelium and CD4+CD45RA+ lymphocytes. Role of the human CD38 molecule.

CD38 is a type II transmembrane glycoprotein, which is widely used as a marker for immature and activated lymphocytes, as well as plasma cells. Although its functional role and natural ligand are not known, CD38 has been shown to transduce activation signals to lymphocytes. Our work shows that CD38 is preferentially expressed by CD4+CD45RA+ cells, but not by CD4+CD45R0+ cells. CD4+CD45RA+ cells are reported to respond poorly to stimuli acting through the CD3/TCR in vitro and to display unique migration pathways in vivo. Cross-linking of CD38 by mAb did not overcome the hyporesponsiveness of CD4+ resting/naive cells to several activation stimuli; in contrast, CD38 engagement by mAb specifically inhibited their binding with human vein endothelial cells. These data suggest that CD38 may play a role in lymphocyte migration. The same inhibitory effect was detected on the (human x mouse) hybrid cell line CP410.A10, which expresses human CD38, but not on its CD38- subclone CP14. CD38 mAb did not inhibit the conventional binding assay between endothelium and several human CD38+ T and B cell lines. However, the inhibition was apparent when the binding assay was performed at 4 degrees C on a rocking shelf, conditions that minimized integrin function. These data suggest that CD38 mediates weak cell binding to endothelium, which is effective even in dynamic conditions. These features are reminiscent of those exerted by selectins, which are adhesion molecules that account for leukocyte rolling on vascular endothelial cells and play an important role in lymphocyte homing.

ADP-ribosyl Cyclase↗

PubMed1994

Retinoic acid-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid receptor-alpha.

CD38 is a leukocyte differentiation antigen that has been thought to be a phenotypic marker of different subpopulations of T- and B-lymphocytes. In myeloid cells, CD38 is expressed during early stages of differentiation. Virtually no information is available on regulation and functions of CD38. Recently we reported that all-trans-retinoic acid (ATRA) is a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells. Here we report that ATRA-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid-alpha receptor (RAR alpha). ATRA failed to induce CD38 expression in a mutant subclone of the HL-60 myeloid leukemia cell line (designated HL-60R) that is relatively resistant to ATRA-induced granulocytic differentiation. Retroviral vector-mediated transduction of RA receptor (RAR alpha) into this HL-60R subclone completely restored the sensitivity of these cells to ATRA in terms of their ability to express CD38. In contrast, CD38 expression was not inducible by ATRA in HL-60R cells, transfected with a functional RAR beta, RAR gamma, or RXR alpha receptor. Induction of CD38 in acute promyelocytic and acute myeloblastic leukemia cells was independent of ATRA-induced cytodifferentiation. Following culture with ATRA, increased CD38 protein levels were also observed in normal CD34+ bone marrow cells, but not on normal circulating granulocytes. From these results, we conclude that CD38 is ATRA inducible in myeloid leukemia cells and normal CD34+ bone marrow cells. This effect is independent of differentiation and is mediated by RAR alpha in HL-60 cells, suggesting a similar role for RAR alpha in CD38 expression in other hematopoietic cells.

ADP-ribosyl Cyclase↗

PubMed1994

CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells.

The present study demonstrates that an agonistic anti-CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa-stained cytospin preparations or flow cytometry on propidium iodide-stained cells. Two other anti-CD38 mAb (Leu-17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up-regulates the bcl-2 proto-oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti-CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti-CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti-CD40, CD40L or recombinant interleukin-4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.

ADP-ribosyl Cyclase↗

PubMed1994

Biosensor analysis of antigen-antibody interactions as a priority step in the generation of monoclonal bispecific antibodies.

A biosensor system aimed at real-time measuring molecular interactions among label-free reactants has been used for a comparative analysis of the binding features (i.e., association-dissociation rates and affinity constants) as well as epitope mapping between bivalent monoclonal antibodies and the derived monovalent bispecific monoclonal antibody. The results show that observed different affinities between parental and derived bispecific antibodies concern the association rate constant, whereas the dissociation rate constants are unaltered. The apparent affinity-constant values determined by solid-phase radioimmunoassay yielded figures almost overlapping with those obtained with the biosensor instrument. The results of the present work indicate that the biosensor system has gained a key role not only as a tool for the study of antigen-antibody interactions, but also for setting up the reference parameters for the selection of the best candidates in the generation of bispecific monoclonal antibodies.

Antibodies, Monoclonal↗

PubMed1994

Cytosolic free calcium concentration in the mitogenic stimulation of T lymphocytes by anti-CD3 monoclonal antibodies.

The effects of anti-CD3 monoclonal antibodies on cytosolic free Ca2+ concentration, [Ca2+]i, were investigated in freshly isolated lymphocytes, T cell lines, T clones and the leukemic T cell line Jurkat with three different methodologies, i.e. classical cuvette experiments, cytofluorimetry and videoimaging. With any technique, concentrations of anti-CD3 antibodies optimal for stimulation of DNA synthesis were completely ineffective at inducing early increases of [Ca2+]i in freshly isolated lymphocytes. At supraoptimal mitogenic concentrations: (i) anti-CD3 mAb induced negligible increases of [Ca2+]i when tested in suspensions of freshly isolated lymphocytes, but the response increased progressively during in vitro culturing with IL2; (ii) most, but not all, T clones, when tested in suspension, were responsive to these concentrations of anti-CD3 antibodies in terms of [Ca2+]i; (iii) using the videoimaging technique at the single cell level, it was demonstrated that the anti-CD3 antibodies induced large increases of [Ca2+]i in lymphocytes only under conditions which allowed adherence of the antibodies (and of the cells) to the glass surface. In all T cell types investigated, the [Ca2+]i increases were most often composed by multiple, asynchronous oscillations. The buffering of [Ca2+]i increases, obtained by loading the cells with membrane permeant esters of Quin-2 and Fura-2, inhibited anti-CD3 mAb induced DNA synthesis, but this appeared entirely attributable to a toxic side effect of the ester hydrolysis. The relevance of these data is discussed in terms of their methodological and functional implications for the understanding of the role of Ca2+ in mitogenic stimulation of T cells.

Aminoquinolines↗

PubMed1994

Human CD38: a glycoprotein in search of a function.

The human CD38 molecule appears to mediate several diverse activities, including signal transduction, cell adhesion and cyclic ADP-ribose synthesis. In this article, the authors consolidate the information available on this highly interesting, multifunctional protein.

ADP-ribosyl Cyclase↗

PubMed1994

Stimulation of T cells via CD44 requires leukocyte-function-associated antigen interactions and interleukin-2 production.

This article reports the results of the analysis of the activation signals delivered to T and B cells by means of the CD44 molecule and an agonistic mAb, i.e., CB05 mAb, which is able to induce cell activation and aggregation upon binding. The functional effects culminate in T-cell proliferation in the presence of autologous accessory cells. Such effects are barely detectable in thymocytes, while B cells prove refractory to the action of the agonistic mAb. All of these events have been followed by the expression of surface activation markers, by the transcription of selected cytokine genes (IFN-gamma, IL-4, and GM-CSF), and by the secretion of IL-2. Cell activation via CD44 has been evaluated as to its relationship with CD3 and CD2 activation pathways, proving synergistic with the latter. The CD44 signaling is protein kinase dependent. Furthermore, the role of surface molecules as cosignals in the CD44 pathway has been analyzed, showing that CD11a (and its ligand CD54), HLA class I, and CD25 are instrumental in the implementation of (a) efficient activation/proliferation signals and (b) a potent cytotoxic potential.

Carrier Proteins↗

PubMed1994

Retrovirus-mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells.

We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer. Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, efficiency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 micrograms/ml taxol. In uninfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotein was found on 17-25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗