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M Allouche

Publications and source records attributed to M Allouche.

At least 19 recordsLinked to original sources
PubMed1997

Influence of Bcl-2 overexpression on the ceramide pathway in daunorubicin-induced apoptosis of leukemic cells.

We have previously demonstrated that daunorubicin (DNR) induces apoptosis in some leukemic myeloid cell lines. We investigated a potential protective role for Bcl-2 in apoptosis induced by DNR in two leukemic cell lines, one myeloid and one lymphoid, overexpressing the anti-apoptotic gene Bcl-2. Parental cells treated with DNR exhibited classical features of apoptosis 6 h after drug exposure, all the cells being dead after 30-48 h. In contrast, overexpression of Bcl-2 significantly delayed, but did not prevent the occurrence of DNR-induced apoptosis, with no surviving cells 96 h after drug exposure. To elucidate the mechanism of the protection mediated by Bcl-2, we explored the signaling pathway which initiates DNR-induced apoptosis. In this report, we show that, in both the myeloid and lymphoid parental cell lines, DNR triggered a sphingomyelin (SM) hydrolysis after 10-15 min with a concomitant ceramide generation. Moreover, exogenous ceramide induced DNA fragmentation in these cells, with levels similar to those observed with DNR treatment. In contrast, Bcl-2 overexpression protected the cells against apoptosis induced by ceramide treatment, without preventing the early SM hydrolysis nor the ceramide generation in these cells. Our results strongly suggest that Bcl-2-mediated protection of DNR-induced apoptosis is effected downstream of the SM-ceramide signaling pathway.

Antibiotics, Antineoplastic↗
PubMed1997

Increased gadd153 messenger RNA level is associated with apoptosis in human leukemic cells treated with etoposide.

Treatment of leukemic cells with topoisomerase inhibitors can lead to growth arrest and subsequent apoptotic cell death. The relationships between cell cycle regulation and apoptosis triggering remain poorly understood. The gadd153 gene encodes the nuclear protein CHOP 10 that acts as a negative modulator of CCAAT/enhancer binding protein transcriptional factors and inhibits cell cycle progression. We have investigated the relationships between gadd153 gene expression and apoptosis induction in four human leukemic cell lines with different sensitivities to apoptosis induced by etoposide (VP-16), a topoisomerase II inhibitor. The gadd153 gene was constitutively expressed in the four studied cell lines. In U937 and HL-60 cells that were very sensitive to apoptosis induction by the drug, VP-16 induced a time- and dose-dependent increase of gadd153 gene mRNA expression. Using agarose gel electrophoresis and a quantitative filter elution assay, apoptotic DNA fragmentation was observed to begin when gadd153 gene expression increased. Equitoxic doses of VP-16 (as defined using a 96-h 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide assay) did not increase the gadd153 mRNA level in K562 and KCL22 cell lines that were more resistant to apoptosis induction by the drug. Nuclear run-on and mRNA stability experiments demonstrated that VP-16 treatment increased gadd153 gene transcription in the sensitive U937 cells. Cycloheximide did not prevent gadd153 expression increase. Both gadd153 mRNA level increase and internucleosomal DNA fragmentation were inhibited by N-tosyl-L-phenylalanine chloromethylketone, a serine threonine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal, an inhibitor of calpain, N-acetylcysteine, an inhibitor of oxidative metabolism, and overexpression of Bcl-2. Z-VAD and Z-DEVD peptides that inhibit interleukin 1beta-converting enzyme-like proteases suppressed DNA fragmentation without preventing gadd153 mRNA increase in VP-16-treated U937 cells. These results indicate that gadd153 gene expression increase occurs downstream of events sensitive to N-tosyl-L-phenylalanine chloromethylketone, calpain inhibitor I, and Bcl-2 and upstream of interleukin 1beta-converting enzyme-related proteases activation in leukemic cells in which treatment with VP-16 induces rapid apoptosis.

Antineoplastic Agents↗
PubMed1997

Autocrine IL-2-dependent growth of a newly established CD3+, CD16-, CD56+, CD57+, J(H)-, TCRbeta-, TCRgamma- leukemia cell line (NOI-90).

Leukemic cells from a 45-year-old male patient with a CD3+, CD56+, CD57+, CD7+ acute lymphoblastic leukemia were cultured in vitro in the absence of any added growth factor for up to 6 years and a continuous lymphoblastoid cell line (NOI-90) was established. NOI-90 cells have the same phenotype and karyotype as initial leukemic cells. Southern blot of DNA from NOI-90 cells showed that TCRbeta, TCRgamma, and J(H) were in germ line. Two and 25% of NOI-90 cells were positive when stained with the IOT14 and 7G7/B6 moAbs, which recognize the CD25 molecule (IL-2R alpha chain); moreover, 4% and 13% of the cells were positive when stained with the TU-27 and mik beta3 moAbs which recognize the CD122 molecule (IL-2Rbeta chain). Equilibrium binding experiments with radiolabelled IL-2 revealed the presence of a small number of high affinity IL-2R on both fresh and continuously growing cells. Media conditioned by NOI-90 cells could induce proliferation of an IL-2-dependent cell line and this IL-2 activity could be detected by a sensitive immunoenzymatic assay using antibodies recognizing distinct epitopes of IL-2. Moreover, IL-2 activity could be adsorbed by immunoaffinity on anti-IL-2 polyclonal purified IgG and the retained molecule displayed a m.w. of 14.5 kDa in SDS-PAGE. In addition, IL-2 immunoreactive molecules could be revealed in the cytoplasm of the cells. Finally, IL-2 fixed on the cell membrane could be detected by indirect immunofluorescence. Although added IL-2 could not induce cell proliferation, monoclonal antibodies against CD25, CD122 and IL-2 could specifically inhibit spontaneous cell proliferation in a dose-dependent manner. NOI-90 cells failed to demonstrate any cytotoxic activity against the K-562, Raji or Daudi cells. These findings indicate that NOI-90 cells are of non-T, non-B, origin lacking NK activity but proliferate under an autocrine pathway which involves, at least partly, the IL-2/IL-2R system.

CD3 Complex↗
PubMed1996

Daunorubicin-induced internucleosomal DNA fragmentation in acute myeloid cell lines.

The study was designed to evaluate the implication of apoptosis in myeloid leukemic cell death induced by daunorubicin (DNR) and to identify the possible factors which may influence this process. DNR-induced apoptosis was characterized by morphology and DNA fragmentation in six leukemic myeloid cell lines which expressed different differentiation phenotypes. In phenotypically mature HL-60 and U937 cells, DNR induced typical apoptosis with characteristic morphological changes and intense internucleosomal DNA fragmentation within a narrow concentration range (0.5-2 microM). When these cells were treated with higher doses of DNR, large DNA fragments (100 kbp), but not internucleosomal fragments, were identified. DNR-induced DNA fragmentation in HL-60 and U937 was inhibited by antioxidants such as N-acetylcysteine (N-ac) or pyrrolidine-dithiocarbamate (PDTC). In the phenotypically immature KG1a, KG1, HEL and ML1 cell lines DNR induced no characteristic apoptotic morphological features as well as very low levels of internucleosomal DNA fragmentation, whereas large DNA fragments (200 kbp) were observed in KG1a treated with 7 microM DNR. Since the latter expressed P-glycoprotein (P-gp), the role of P-gp in the lack of apoptotic response to DNR was investigated. One P-gp inhibitor (verapamil) slightly improved DNR-induced DNA fragmentation in KG1a cells whereas the combination of verapamil and buthionine-sulfoximine (BSO), which depletes glutathion store, further increased internucleosomal DNA fragmentation. In conclusion, DNR induced internucleosomal DNA fragmentation in some but not all AML cells; the magnitude of this process being influenced by both intracellular drug concentration and oxidative balance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis.

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic growth factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and is thought to play an important role in the mesoderm induction. Although hematopoietic cells derive from the mesoderm, relatively few studies have, until recently, addressed the role of FGF-2 in hematopoiesis. FGF-2 is expressed in cells of the bone marrow including stromal cells, and possibly cells from several hematopoietic cell lineages. It is stored in the bone marrow extra-cellular matrix and released by enzymes such as heparanase, plasmin, or phospholipase C and D. FGF-receptors (FGF-Rs) are expressed in leukemic cell lines and in hematopoietic cells. FGF-2 positively regulates hematopoiesis, by acting on stromal cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. The action of FGF-2 is most likely indirect since its action, on megakaryocytopoiesis for example, is abrogated by anti-IL6 antibodies. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of TGF-beta. Taken together, these results demonstrate that FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.

Animals↗
PubMed1995

Basic fibroblast growth factor and hematopoiesis.

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and plays an important role in the mesoderm induction, together with transforming growth factor-beta (TGF-beta). Although hematopoietic cells derive from the mesoderm, relatively few studies have addressed the role of bFGF in the hematopoietic system until recently. It appears that bFGF is expressed and produced by bone marrow stromal cells, as well as by cells from several mature peripheral blood lineages. It is released and stored in the bone marrow extra-cellular matrix. FGF-receptors (FGF-Rs) are expressed on nearly every cell of hematopoietic origin tested so far. Growing evidence shows that bFGF can positively regulate hematopoiesis, by acting on various cellular targets: stromal cells, early and committed hematopoietic progenitors, and possibly some mature blood cells. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of another factor, TGF-beta, thus potentially playing a central role in hematopoiesis.

Animals↗
PubMed1995

Expression of basic fibroblast growth factor (bFGF) and FGF-receptors in human leukemic cells.

Recent reports have suggested that basic fibroblast growth factor (bFGF) could play a permissive role in hematopoiesis, in combination with specific colony-stimulating factors. We investigated the expression of bFGF and FGF-receptors (FGF-Rs) in leukemic cell lines of various hematopoietic lineages. Three protein isoforms of bFGF of approximately 18, 22 and 24 kDa were detected in the myeloid cell line K562, but not in myelomonocytic or lymphoid (T or B) cell lines. In vitro-induced differentiation of K562 cells did not change the pattern of expression of the different bFGF isoforms. Accordingly, the mRNA of bFGF was found expressed in K562, but not in other leukemic lines tested, as assayed by reverse transcript amplification (RT-PCR). Using the same technique, we searched for the presence of high affinity FGF-Rs on these cells: in eight out of ten cell lines tested, mRNA for at least one FGF-R among FGF-R1, FGF-R3 or FGF-R4 was expressed, whereas FGF-R2 was never detected. We found that two cell lines were responsive to bFGF in different biological assays: (i) in K562 myeloid cells induced to differentiate by hemin, preincubation with bFGF and heparin increased cell viability and decreased hemin-induced DNA fragmentation, without affecting erythroid differentiation; and (ii) in U937 monocytic cells, the production of plasminogen activator was increased by bFGF or aFGF in combination with heparin. Binding experiments with 125I-bFGF (up to 200 pM) in the presence of heparin revealed high affinity receptors on the K562 and U937 cell lines (1177 +/- 440 and 392 +/- 184 sites/cell, Kd = 61.7 +/- 8.6 and 43.1 +/- 13.5 pM, respectively). Thus our results strongly suggest that cells of hematopoietic origin could express functional FGF-receptors.

Base Sequence↗
PubMed1992

Abnormal expression of IL-2R beta (p70)-binding polypeptide on HIV-infected patients' cells.

The constitutively expressed IL-2R beta (p70) chain participates in the formation of high-affinity (h.a.) IL-2R and transduces IL-2-mediated signals to normal cells. Its expression on HIV-infected patients' PBMC was evaluated and was found to be decreased in both nonstimulated CD4+ and CD8+ cells. Mitogenic cell stimulation induced IL-2R beta chain expression on both CD4+ and CD8+ cells from asymptomatic and persistent generalized lymphadenopathy patients but not on those from AIDS patients. Comparison of mean fluorescence intensity of IL-2R beta positively stained cells from normals and patients did not reveal significant differences. Cross-linking of 125I-rIL-2 on patients' PHA-blasts revealed decreased signals corresponding to both IL-2-binding chains and, in some cases, neither IL-2R alpha nor IL-2R beta chains could be detected. Decreased expression of IL-2R beta polypeptide was associated with impaired accumulation of the corresponding mRNA transcripts. Binding experiments with 125I-rIL-2 under h.a. conditions showed a decreased number of IL-2-binding sites/cell which was more pronounced in patients with AIDS than in patients with less advanced clinical stages. In vitro HIV infection of normal PHA-blasts also resulted in a decreased number of h.a. IL-2R/cell. High concentrations of rIL-2 in the absence of other mitogenic stimuli induced a decreased cell proliferation and expression of the IL-2R alpha chain and did not enhance the constitutive NK and the generation of LAK activity in several patients, suggesting an impaired IL-2R beta chain-mediated cell activation.

Antibodies, Monoclonal↗
PubMed1992

Interleukin 2 production and interleukin 2 receptor expression by human immature leukemic T cells.

In order to determine the role of interleukin 2 (IL2) on the proliferation of leukemic cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) we studied the production of IL2, the function of IL2 receptors (IL2R) expressed on T-ALL cells and their IL-2-dependent in vitro proliferation. Leukemic cells from six out of 17 T-ALL/T-cell non-Hodgkin's lymphoma patients with a prothymocyte (stage I) or a mature thymocyte (stage III), but not with a common thymocyte (stage II) phenotype, could proliferate, in a dose-dependent manner, in response to recombinant IL2 (rIL2) and anti-Tac and TU27 moAbs as well as polyclonal anti-IL2 purified immunoglobulin G could inhibit this IL2-induced cell proliferation. Both crude or/and Amicon-concentrated media conditioned by T-ALL cells from 10 out of 13 tested patients contained IL2 activity as assessed by colorimetric biological and immunoenzymatic assays; this biologic activity was due to a 14.5 kDa molecule adsorbed by anti-IL2 antibodies in an immunoaffinity assay. Although less than 10% of fresh leukemic cells expressed IL2R alpha (Tac) chain, a 24 h cell incubation in the absence of any mitogenic stimulation induced IL2R alpha chain expression in five out of 13 patients (11-83% Tac+ cells). Morever, Tac mRNA transcripts could be detected in fresh cells from all 10 patients tested. Staining of fresh leukemic cells with an IL-2R beta-chain-specific monoclonal antibody and flow cytometry analysis revealed that 4-13% of leukemic cells were positive. Binding experiments with 125I-rIL2 showed a small number of high affinity IL2R on fresh cells from three T-ALL patients (114-200 sites/cell, dissociation constant = 101-181 pm). Finally, antibodies against IL2R alpha, IL2R beta and IL2 could inhibit both IL2 driven and spontaneous cell proliferation of most patients' T-ALL cells, although in some cases an heterogenous pattern of inhibition was observed. Taken together, these findings strongly suggest that an IL2/IL2R-dependent mechanism could be involved in the proliferation of some T-ALL cells.

Antigens, CD↗
PubMed1992

Phorbol myristate acetate-induced expression of high-affinity interleukin 2 receptors and production of interleukin 2 by human acute lymphoblastic leukemia T cells.

The effect of phorbol myristate acetate (PMA) on the expression of interleukin 2 receptors (IL-2R), production of IL-2 and IL-2-dependent proliferation of acute lymphoblastic leukemia T cells (T-ALL cells) from 10 patients was studied. First, the effect of PMA on the expression of cell surface markers was assessed: a decrease of CD3 and CD4, and an enhanced expression of CD8 molecule were observed on T-ALL cells. Moreover, PMA exhibited an heterogenous effect on various activation-associated molecules such as a decreased expression of transferrin receptor and T10 molecule and an induced expression of 4F2 and CD9 molecules. It is known that functional high-affinity IL-2R are composed of at least two IL2 binding molecules, the alpha (p55) and beta (p70) chains. We found that PMA induced the expression of both IL-2R alpha and IL-2R beta chains, as well as IL-2 production by T-ALL cells. These effects were time- and dose-dependent. Cross-linking experiments with 125I-labelled recombinant IL-2 (125I-rIL-2) revealed both p55 (IL-R alpha) and p70 (IL-2R beta) IL-2-binding polypeptides, whereas binding equilibrium assays on PMA-treated cells demonstrated the presence of a low number (31-413) of high-affinity binding sites/cell in five out of six cases analysed, as well as intermediate affinity IL-2R (1234-3919 sites/cell) in four out of six cases, according to the time of incubation with PMA. In two cases tested high-affinity IL-2R on PMA-treated T-ALL cells could internalize 125I-rIL-2 at 37 degrees C. PMA alone enhanced the spontaneous proliferation of T-ALL cells in three cases, whereas a clear synergy between IL-2 and PMA could be detected in three patients' cells. Moreover, exogenous rIL-2 enhanced cell proliferation of PMA-preincubated T-ALL cells in four cases studied. Taken together, these observations indicate that a short-term incubation of T-ALL cells with PMA can activate the IL-2/IL-2R system on these cells without inducing strong modifications of their differentiation status. These results thus suggest that this system may be involved in the proliferation process of some activated immature T cells.

Cell Division↗
PubMed1990

Autocrine growth of leukemic cells.

Autocrine growth is a process whereby a cell both secretes and responds to a growth factor. This paper describes the stepwise malignant progression of leukemic cells which has been demonstrated in many experimental models of autocrine leukemic growth. In contrast, autocrine growth has not been proven as a major physiopathological mechanism for the growth of leukemic cells in vivo in human myeloid and lymphocytic leukemias. Growth-factor independency of human leukemic cell lines may be due to clonal selection.

Cell Division↗
PubMed1990

The role of growth-factor receptors (excluding IL-2 receptors) in the proliferation and differentiation of normal and leukemic hematopoietic cells.

Receptors (R) are considered as allosteric enzymes whose action on metabolic chains is modulated through binding to the ligand. They play an essential role in the transduction of the multiple signals (e.g. interleukins or CSF) which intervene in the regulation of hematopoiesis. Their ordered interactions are necessary to regulate the growth and differentiation of normal hematopoietic precursors. This paper summarizes recent data concerning the structure-action relationship of growth-factor receptors in the signal transduction and alterations of growth-factor receptors which may play an important role in leukemic transformation. Some therapeutic modulations of growth-factors cascades are also discussed.

Cell Differentiation↗
PubMed1990

Interleukin 2 receptors.

Interleukin 2 receptors (IL-2-R) are composed of (at least) two chains: the alpha chain (or p55 or Tac) and the beta chain (or p70 or p75). The association of both chains constitutes high affinity IL2-R complexes (Kd congruent to 10-100 pM), whereas the alpha-chain or the beta chain alone bind IL-2 with low (Kd congruent to 5-50 nM) and intermediate (Kd congruent to 1 nM) affinities, respectively. The beta chain (but not the alpha chain) mediates IL-2 internalization and signal transduction (proliferation or differentiation) to the cell, thus only high and intermediate affinity IL-2-R are functional. High affinity IL-2-R are expressed on activated normal and leukemic T and B lymphoid cells. Low affinity IL-2-R (Tac alone) are seen on stimulated T cells, B cells, and monocytes, as well as on various leukemic cells. The p70 chain alone is constitutively expressed by subsets of normal resting T and B cells, LGL and NK cells and we have recently demonstrated its presence on leukemic cells from various hemopoietic lineages. The finding of IL-2-R on non-lymphoid cells may disclose new functions for IL-2.

B-Lymphocytes↗
PubMed1990

Phorbol myristate acetate induces both high affinity and low affinity interleukin 2-receptors on a pre-B leukemic cell line.

The human pre-B leukemic cell line Reh6 does not constitutively express the Tac molecule (p55), which is one of the two polypeptide chains of the interleukin 2-receptor (IL2-R). Cell incubation with Phorbol Myristate Acetate (PMA) induces expression of the Tac molecule in a dose and time-dependent manner. Binding experiments with radiolabelled recombinant IL2 (rIL2) revealed both high and low affinity IL2-R (225 +/- 105 sites/cell with a Kd of 130 +/- 51 pM and 24060 sites/cell with a Kd of 17.3 nM respectively), whereas unstimulated Reh6 cells only expressed intermediate affinity Reh6 cells revealed the presence of two polypeptide chains of mol. wts 55,000 (Tac molecule) and 70,000, as in normal activated T cells, while the 70,000 mol. wt chain alone was observed in unstimulated Reh6 cells. IL2-R-bearing Reh6 cells could absorb rIL2 in a dose-dependent manner and this absorption was inhibited by a monoclonal antibody against the Tac molecule (anti-Tac). Moreover, partial internalization of IL2 bound under high affinity conditions occurred at 37 degrees C. IL2-R expressed on PMA-induced cells were functional since rIL2 specifically enhanced the proliferation in vitro of PMA-treated cells in semi-solid but not liquid cultures. These findings thus demonstrate an IL2-dependent mechanism of proliferation in vitro of pre-B leukemic cells induced by PMA, which can express high affinity, functional IL2-R.

Cell Division↗
PubMed1990

Effect of phorbol myristate acetate on T cell colony formation, interleukin-2 (IL-2) receptor expression and IL-2 production by cells from patients at all stages of HIV infection.

We and others have shown that several T cell responses induced by the mitogen phytohaemagglutinin (PHA), including T cell colony formation, IL-2 receptor (IL-2R) expression, and IL-2 production are impaired in patients with AIDS and lymphadenopathy syndrome (LAS). We investigated whether phorbol myristate acetate (PMA) could act in synergy with PHA (as it does in healthy subjects) to enhance in vitro T cell responses of patients at all stages of infection by HIV. In AIDS patients with opportunistic infections (AIDS/OI), PHA + IL-2 + PMA led to a total disappearance of T cell colonies in 10/11 patients, among whom six already displayed very low numbers of colonies induced by PHA + IL-2 (less than 50 colonies/5 x 10(4) cells). In contrast, T cell colony formation induced by PHA + IL-2 + PMA was maintained or increased, compared with that induced by PHA + IL-2, in five out of six AIDS patients with Kaposi's sarcoma (AIDS/KS), 10/14 LAS and six out of seven HIV-seropositive asymptomatic (HIV+/AS) homosexuals. In these three groups of patients, a low percentage of colony cells induced by PHA + IL-2 + PMA expressed CD3 and CD4 molecules, but 50-89% of cells were IL-2R (Tac) positive, as in healthy controls. Studies on T cell activation and IL-2 production were performed on a selected group of 12 HIV-infected patients for whom sufficient numbers of lymphocytes could be obtained. PMA induced CD4 down-modulation in controls and in HIV-infected patients. However, CD3 down-modulation and induction of the Tac chain of IL-2R by PMA were significantly impaired in patients, compared with controls, and these two parameters were correlated. Although PHA alone induced virtually normal levels of Tac antigen on patients' cells, Tac induction by PHA + PMA was significantly decreased in patients versus controls. Cells from five out of 10 patients tested failed to produce detectable amounts of IL-2 after PHA stimulation, whereas IL-2 production increased significantly in all patients tested (n = 9) after PHA + PMA, with a level of IL-2 activity significantly higher than in controls. No correlation was found in this group of patients between the effects of PMA + PHA on T cell colony formation, Tac expression, or IL-2 production, as compared with PHA alone. Taken together, our results indicate that in vitro T cell functional studies with PMA may be useful to evaluate better the defects of T cell activation in HIV-infected patients.

Antigens, CD↗
PubMed1990

Intracytoplasmic detection of the Tac (p55) chain of interleukin-2 receptor in pre-B leukemic cells associated with a constitutive expression of Tac mRNA.

The pre-B Reh-6 leukemic cells do not express membrane interleukin-2 (IL-2)-R alpha (Tac or p55) chain; however, their incubation with PMA induces the expression of both high and low affinity IL-2-R. Northern analysis of nonstimulated Reh-6 as well as leukemic cells from patients with acute B cell precursor lymphoblastic leukemia displayed a constitutive expression of p55 mRNA transcripts, which could be enhanced by PMA. Both actinomycin-D and cycloheximide could abrogate PMA-induced p55 membrane expression, suggesting the need for de novo mRNA and protein synthesis. The increased PMA-induced p55 mRNA accumulation was an early event (4 hr) and could be enhanced, specifically, by rIL-2 because anti-Tac moAb inhibited this rIL-2-mediated effect. Immunofluorescence and cross-linking studies using 125I-rIL-2 failed to reveal membrane-associated p55 protein on both Reh-6 and patients' leukemic cells. Conversely, immunogold staining and electron microscopy studies, revealed p55 immunoreactive molecules in the cytoplasm but not in the nucleus of all Reh-6 cells. Using a sensitive EIA, p55 molecules could be detected in cell lysates but not the culture supernatants of Reh-6 cells, suggesting that p55 was not released into the culture medium. These results indicate that constitutively expressed p55 mRNA on pre-B leukemic cells is translated into a relative immunoreactive protein that cannot be expressed on cell surface for unknown, yet, reasons.

Blotting, Northern↗
PubMed1989

Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages.

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.

Cell Differentiation↗