Biomedical subjects

M Lanotte

Publications and source records attributed to M Lanotte.

At least 91 records · Page 5Linked to original sources

PubMed1991

NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3).

Acute promyelocytic leukemia (APL) is a well-defined entity among acute leukemia, cytogenetically characterized by a t(15;17) (q22;q11-12) translocation. In vitro and in vivo studies suggest that all-trans retinoic acid (RA) treatment restores cell maturation. We have isolated the first permanent cell line with t(15;17), derived from the marrow of a patient with APL in relapse. The establishment of the cell line, its morphologic, karyotypic, and immunohistochemical features are reported. RA induced cell line maturation. Cells strongly expressed myeloid markers, but also some T-cell markers. Additional karyotypic abnormalities, a 12p rearrangement and the possible presence of a homogeneous staining region (HSR) on 19q+ are discussed both in relation to T-cell (CD2, CD4) and monocyte (CD9) markers, and to the acquired cell growth autonomy. The cell line represents a remarkable tool for biomolecular studies.

Adult↗

PubMed1991

Programmed cell death (apoptosis) is induced rapidly and with positive cooperativity by activation of cyclic adenosine monophosphate-kinase I in a myeloid leukemia cell line.

Programmed death (apoptosis) of the rat myelocytic leukemic cell line IPC-81 was triggered by cyclic adenosine monophosphate (cAMP) analogs or by agents (cholera toxin, prostaglandins) increasing the endogenous cAMP level. The induction of cell death by cholera toxin was preceded by increased activation of cAMP-kinase. Cell lysis started already 5 hr after cAMP challenge and was preceded by internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. The cell suicide could be prevented by inhibitors of macromolecular synthesis. cAMP analogs induced cell death in a positively cooperative manner (apparent Hill coefficient of 2.9), indicating that triggering of the apoptotic process was under stringent control. There was a strong synergism between cAMP analogs complementing each other in the activation of cAMP-dependent protein kinase I (cAKI). No such synergism was noted for analogs complementing each other in the activation of cAKII. It is concluded that apoptosis can be induced solely by activation of cAKI. The IPC-81 cells expressed about four times more cAKI than cAKII. The expression of cAK subunits, on the protein and mRNA levels, was only minimally affected by cholera toxin treatment.

Animals↗

PubMed1991

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells.

Okadaic acid, a specific and potent inhibitor of protein phosphatases 2A and 1, was tested for its effect on the morphology of a number of cell types: freshly isolated rat hepatocytes in suspension or in primary culture, the human mammary carcinoma cell line MCF-7, the human neuroblastoma cell line SK-N-SH, rat pituitary adenoma GH3 cells, and rat promyelocytic IPC-81 cells. All the cell types reacted within a few hours to okadaic acid in the concentration range 0.1 to 1 microM with profound morphological alterations. Among the changes noted were: condensation of chromatin, shedding of cell contents via surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. In some cells nuclear fragmentation was noted. In addition, cells growing as monolayers rounded up and detached from the substratum. The treated cells had no swollen mitochondria and retained the ability to exclude trypan blue until the final stage of dissolution, supporting the hypothesis that the changes were apoptotic rather than necrotic. In hepatocytes the action of okadaic acid was mimicked by another phosphatase inhibitor, microcystin, and was accompanied by shrinkage of the cell volume, as judged by Coulter counter analysis. The action of phosphatase inhibitor was not abolished by protein synthesis inhibitors, Ca(2+)-depleted medium, or phorbol ester. Although hepatocyte DNA replication was very sensitive to inhibition by okadaic acid, DNA fragmentation was less pronounced in response to okadaic acid than other agents inducing the morphological appearance of apoptosis.

Animals↗

PubMed1990

The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus.

Retinoic acid is a vitamin A derivative with striking effects on development and cell differentiation. Several nuclear retinoic acid receptors (RARs), acting as ligand-inducible transcription factors, have been characterized and indirect evidence suggests that they have distinct roles. One of the most intriguing properties of retinoic acid is its ability to induce in vivo differentiation of acute promyelocytic leukaemia (APL) cells into mature granulocytes, leading to morphological complete remissions. Because the RAR alpha gene maps to chromosome 17q21 (ref. 14), close to the t(15;17) (q21-q11-22) translocation specifically associated with APL, we analysed RAR alpha gene structure and expression in APL cells. We report here that, in one APL-derived cell line, the RAR alpha gene has been translocated to a locus, myl, on chromosome 15, resulting in the synthesis of a myl/RAR alpha fusion messenger RNA. Using two probes located on either side of the cloned breakpoint, we have found genomic rearrangements of one or other locus in six patients out of eight, demonstrating that the RAR alpha and/or myl genes are frequently rearranged in APL and the breakpoints are clustered. These findings strongly implicate retinoic acid receptor alpha in leukaemogenesis.

Base Sequence↗

PubMed1990

Reduction of adenylyl cyclase activity by cholera toxin in myeloid cells. Long-term down-regulation of Gs alpha subunits by cholera toxin treatment.

In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane. The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.

Adenosine Diphosphate Ribose↗

PubMed1990

A new Ph1+, bcr cell line derived from a patient with ALL-L1 gained autonomy in culture concomitant to CD23 expression.

A permanent cell line, LEF1, has been established from the cells of an adult suffering from a Philadelphia positive acute lymphoblastic leukemia. The LEF1 cell line was obtained by maintaining peripheral blood cells from the patient in culture on a fibroblast feeder; subsequently, an autonomously growing cell population, independent of that feeder layer, developed. The karyotype of the cell line, 46, t(9;22)(q34;q11), was different from the karyotype at diagnosis which had 53 chromosomes and two Philadelphia chromosomes. Furthermore, compared with the initial leukemic blasts, the immortalized cell had three differences in surface phenotype (CD23+, CD11b-, CD10-). However, molecular studies indicated that the breakpoint in the 3' part of the first intron of the BCR gene was unchanged, confirming the leukemic origin of LEF1. The cell line was shown to be Epstein-Barr virus negative.

Adult↗

PubMed1990

Towards large-scale purification of natural CSF-1 from human placenta tissue extracts.

The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.

Blotting, Western↗

PubMed1989

A subline of the Brown Norway myeloid leukemia in the Lewis x Brown Norway rat: in vivo growth characteristics and development of an in vitro clonogenic assay.

A subline of Brown Norway (BN) acute myelocytic leukemia (AML) which can be propagated in suspension culture (designated IPC-81) is described. Injection into Lewis x BN F1 hybrid (LBN) rats resulted in a log-linear correlation between tumor cell dose and time till death from the onset of leukemia even after multiple (greater than 16) passages in vitro. An in vitro clonogenic assay for IPC-81 colony formation (CFU-leuk) was developed with excellent cloning efficiency (55-82%). Colonies grew without the addition of specific growth factors; syngeneic spleen-conditioned medium inhibited CFU-leuk by 40%, but co-culture with untreated normal LBN rat bone marrow cells had no effect on CFU-leuk. CFU-leuk could be detected in the bone marrow 7 to 10 days before morphologic detection of leukemia in injected animals. This cell line should prove useful in the preclinical evaluation of new strategies for treating AML and evaluating new bone marrow purging methods.

Animals↗

PubMed1989

The fate of the centrosome-microtubule network in monocyte-derived giant cells.

Avian monocyte-derived giant cells in vitro, which are in many respects similar to osteoclasts, display a complex microtubule array that plays a prominent role in cell spreading. It is organized by a polygonal row of regularly spaced centrosomes surrounding an irregular cluster of nuclei. The immediate progenitor cells are binucleate cells with a single microtubule-organising center (MTOC), the result of the congregation of the two individual centrosomes. The one-to-one correspondence between numbers of centrosomes and nuclei in giant cells suggests that the centrosome of each precursor cell has been conserved through the fusion process. This is in marked contrast to the absence of centrosomes in myotubes, another example of a differentiated cell derived from the fusion of progenitor cells.

Animals↗

PubMed1989

Cholera toxin resistance associated with deficient adenylate-cyclase activity in a subclone of the rat promyelocytic leukemia (BNML).

The rat promyelocytic leukemia cell line BNML is highly sensitive to cAMP elevating agents, and to cholera toxin (CT) in particular: 99.9% of the cells are killed in less than 48 hr of toxin treatment. We described here a subclone of the same leukemia, which, in contrast, is completely resistant to CT but still sensitive to other cAMP inducers. This locates the defect responsible for CT resistance at the membrane, somewhere between surface CT receptors and adenylate cyclase. CT-resistant BNML cells (CTR-BNML) do have surface CT receptors (several thousands per cell). Adenylate cyclase activity in CTR-BNML cells is not stimulated by cholera toxin. Other GS mediated stimulation of adenylate cyclase (by PGE, isoproterenol, histamine, NaF) remains relatively high, though 25-60% lower than in CTS-BNML cells. These results suggest that a specific adenylate cyclase defect is involved in the resistance of CTR-BNML cells to cholera toxin.

Adenylyl Cyclases↗

PubMed1988

In vitro patterns of enzymic tetrahydrofolate dehydrogenase (EC 1.5.1.3) expression in bone marrow stromal cells.

The toxicity of antifolate drug therapy on microenvironmental function of hemopoietic marrow stromal cells may be critical enough to justify the development of a simple cell culture model to appraise the dihydrofolate reductase (DHFR)/tetrahydrofolate dehydrogenase (EC 1.5.1.3) (4HFDH) enzymic system in individual stromal cells. The localization and pattern of expression of 4HFDH/DHFR in cultured bone marrow stromal cells were studied in situ in morphologically identifiable cells. The 4HFDH/DHFR expression varies quantitatively and qualitatively according to cell types and thus constitutes specific phenotypic patterns for fibroblastic stromal cells, endothelial cells, monocyte-macrophages, and osteoclast-like cells. Data indicate a good correlation between the labeling of the enzymic expression and the resistance of cells to methotrexate treatment. This conclusion is also supported by similar features shown by cell sublines bearing multiple DHFR gene copies. Levels of 4HFDH/DHFR expression in marrow stromal cells should provide useful markers underlining the effects of cytostatic drugs used in chemotherapy (for example, methotrexate injuries or resistance). The method can be applied to isolated cells, stromal colonies (fibroblast CFU), or whole adherent layers from marrow cultures. Moreover, with this method, enzymic reactions can be carried out in situ and visual correlations between a supportive microenvironment (hemopoietic foci) and the 4HFDH/DHFR levels on the stromal adherent layer are possible.

Animals↗

PubMed1987

[Human placental tissue: a source of natural hematopoietic regulators].

In this paper, it has been shown that human placental tissular extracts are a potent source of natural haemopoietic growth factors. The colony-stimulating activities (CSA) recovered by extraction from washed placental pulp were active both on human and murine haemopoietic progenitors, from monocytic and granulocytic lineages. Crude tissular extracts contained CSA titers at least ten fold the titers usually found in placenta culture media. Placenta is the only human tissue easily available for the study of natural tissue-bound haemopoietic regulators. Extraction on an industrial scale, as proposed for the first time in this paper, should also benefit the identification and purification of new minor molecular classes of growth and maturation factors or inhibitors involved in human haemopoiesis.

Animals↗

PubMed1986

Murine basophil-mast differentiation: toward optimal conditions for selective growth and maturation of basophil-mast or allied cells.

Recent investigations revealed that basophil-mast cells were related to the hemopoietic system. Strikingly, murine bone marrow showed a singular paucity in cells with basophil-mast features; moreover in clonogenic assays (methylcellulose, agarose) bone marrow was found to be manifestly poor in basophil-mast progenitor cells. Our work brought to light several new facts concerning the culture and differentiation of this cell type: 1 degree pure and mixed mast clones can be derived in large numbers from bone marrow, provided progenitors are cultured in collagen matrix. Up to 1,382 hemopoietic clones were analysed in situ after staining: 30% contained mast cells (34 per 10(5) cells), thus the basophil-mast lineage was one of the most frequent. We concluded that other cloning media were noticeably nonoptimal for the growth and/or maturation of mast cells. We suggested that collagen and the molecular edifices derived from it, both found in variable amounts in the natural mast environments, should play essential roles in mast phenotype expression. 2 Degrees cholera toxin (CT) selectively eradicated nonmast progenies: mast progenitors and mast progenies were resistant. In this way, pure and rapidly expanding mast cell clones were obtained at a frequency never reported before. CT possibly acts both directly, as a stimulator of mast cell proliferation, or indirectly on marrow subpopulations which repress basophil-mast cell growth and maturation. In vitro culture conditions, specifically designed for basophil-mast lineage, should prove of interest in the search for an unifying hypothesis concerning the multiple forms of mast cells found in various tissues.

Animals↗

PubMed1986

On growth regulation of the rat promyelocytic leukemia (BNML): growth inhibition and eradication of clonogenic cells by cholera toxin.

Our recent establishment of several permanent in-vitro cell lines from Brown Norway rat leukemia (BNML) and the development of a clonogenic assay prompted us to undertake detailed studies on the growth control mechanism of a cell type which for several years has served as an animal model for human AML and preclinical studies. So far, these cells have no defined biological regulators but require intricate cellular interactions to sustain their growth. The effects on cell growth and clonogenicity, of agents known to modify the intracellular levels of cyclic nucleotides, were analysed. Here we report that CT binding strongly inhibited cell growth at a wide range of concentrations (10(-6)-10(-14) M) while beta chain pentameric subunits or alpha chain had no effects. Cell growth was inhibited in a dose-dependent manner. The ligand-receptor interactions mediated the alpha chain's transit through the membrane; the adenylate cyclase activation and the rise in c-AMP levels (60 min) resulted in DNA synthesis arrest (5 h), then finally ended in cell death (24-48 h). A significant decrease in the clonal ability of treated cultures was seen. A decrease of up to five logs in the clonogenic cell number was observed after 48 h of toxin treatment (10(-7) M). The growth inhibition of CT were reproduced by several agents (PGE, theophylline, isobutylmethylxanthine) known to raise intracellular c-AMP levels. Data are commented from a biochemical approach to intracellular events controlling the cell growth of this leukemia. The potential interests of c-AMP inducing agents on the eradication of this leukemia by ex-vivo marrow treatments are also considered.

1-Methyl-3-isobutylxanthine↗

PubMed1986

Selective inhibition of the proliferation of various murine hemopoietic progenitor cells by cholera toxin.

We have studied in detail the effects of cholera toxin (CT), its pentameric B-chain subunit (toxoid) and the A-promoter chain on the differentiation of hemopoietic progenitor cells. Murine marrow cells were treated either with CT or its subunits. After stimulation with either the multilineage growth factor (multi-CSF; also called interleukin 3 or HCGF) or other hemopoietic regulators (colony-stimulating factors, or CSF), the clones grown in semisolid collagen cultures were scored in situ. Pluripotent stem cells (CFU-S) and multilineage (mixed CFU) or lineage-restricted progenitors (CFU-c) were estimated. We found that CT sensitivity is gradually gained by cells through the stepwise differentiation processes (i.e., CFU-S less than multilineage CFC less than committed CFC less than maturing cells). CT also has a selective, dose-dependent inhibitory effect (1 microM to 1 pM) on hemopoietic lineages (basophil-mast cells less than megakaryocytes less than neutrophils less than monomacrophages). These phenomena were obvious when the clonal growth was supported by multi-CSF but, interestingly, were not observed when lineage-restricted CSF were used. They furnished additional evidence that multi-CSF activates cells in a specific manner. This growth factor involved in progenitor cell self-renewal control may contribute to maintaining, on maturing cells, characteristics that are normally the attributes of progenitors.

Animals↗

PubMed1985

Low or undetectable levels of surface high affinity cholera toxin receptors on normal hemopoietic growth factor-dependent cells.

The membrane monosialoganglioside GM1, the high affinity receptor for cholera toxin, is generally considered ubiquitous on normal cells. It was found to be abundant both on normal mature hemopoietic cells and on leukemic cells. By contrast, the normal factor-dependent cell lines, which achieve indefinite proliferation in the presence of the multilineage hemopoietic growth factor apparently displayed the unique character of having low or undetectable levels of surface membrane and cytoplasmic cholera toxin receptors. These results were obtained by the Scatchard analysis of 125iodinated toxin binding, immunofluorescence studies and gel electrophoresis autoradiography. This corroborated the fact that these cells were highly resistant to growth inhibition by cholera toxin (microM to fM) while normal mature cells and leukemic cells of similar phenotype were sensitive.

Animals↗

PubMed1984

Evaluation of the clonogenic cell population (Leuk-CFU) in the marrow of BN rats during development of a promyelocytic leukemia (BNML): an in vitro assay.

We report a reproducible in vitro clonogenic assay for the transplantable BN rat promyelocytic leukemia (BNML). Colony growth required a feeder activity elaborated by normal rat marrow cells. This stimulating activity is ascribed to the stromal elements. The in vitro maintained BNML cell line IPC-81 [Lacaze et al., Leukemia Research 7, 145 (1983)] also exhibited stimulating activities at high cell concentrations, confirming the autocrine capacities previously described. The nature of the stimulating activity is unknown but it is probably not of CSF type, and is not transferred to culture supernatants. This in vitro clonal assay permits the quantification of the clonogenic cells present in leukemic marrow during the early stage of the disease, when BNML cells are not yet distinguishable morphologically. Leukemic Cell Forming Unit (L-CFU) response was linear; 5-10(3) clones can be scored reproducibly. The plating efficiency obtained with cultured IPC-81 cells was high (60-90%), whereas marrow transplanted leukemia cells had reduced clonogenic capacities. These results are discussed.

Animals↗

PubMed1984

Terminal differentiation of hemopoietic cell clones cultured in tridimensional collagen matrix: in situ cell morphology and enzyme histochemistry analysis.

Collagen, a major component of the extracellular matrix in vivo, has been used as a tridimensional gel matrix for cultured hemopoietic clones. Its resemblance to the natural matrix produced by cells makes it ideal for studies on proliferation and differentiation of hemopoietic lineages. Every lineage, including granulocytes (basophilic, eosinophilic and neutrophilic polymorphs) monocyte-macrophages, megakaryocytes, erythroid and lymphoid lineages could be grown using a standardized collagen medium, provided that specific stimulators were added in the culture. Clones were scored on either live or fixed cultures. Compared to other gel substrates, collagen matrix proved superior for cell proliferation and maturation. Additional advantages (in situ clonal analysis by histological staining, enzyme cytochemistry), and other possibilities of the method are reported and discussed. The system offers great potential for cellular immunology, hematology and molecular biology with peculiar reference to differentiation of normal hemopoietic cells, viral transformation and leukemogenesis in vitro. These applications are reviewed.

Animals↗