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S Hermouet

Publications and source records attributed to S Hermouet.

33 records · Page 2Linked to original sources
PubMed1996

G-CSF alone mobilizes sufficient peripheral blood CD34+ cells for positive selection in newly diagnosed patients with myeloma.

We have evaluated CD34+ cell positive selection from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in 26 patients with either multiple myeloma (MM, n = 18) or follicular non-Hodgkin's lymphoma (NHL, n = 8). 26 PBPC were collected with two leukaphereses: 16 contained sufficient numbers of CD34+ cells and were elected. The absolute number of CD34+ cells in the leukapheresis products was found to be significantly related to the duration of underlying disease and exposure to prior treatment. CD34+ cell positive selection allowed recovery of a median of 35% of CD34+ cells, the selected fraction containing a median number of 1.43 x 10(6)/kg CD34+ cells/kg (range 0.48-41.5). 10 patients were transplanted and received a median dose of 1.51 x 10(6) CD34+ cells (range 0.48-4.2). The median time to granulocyte ( > 0.5 x 10(9)/l) and platelet ( > 20 x 10(9)/l) engraftment was 12 and 13 d respectively (ranges 10-13 and 0-95). Lymphoma cells were found by a sensitive polymerase chain reaction technique in four out of five CD34+ cell fractions tested.

Adult↗
PubMed1996

Ex vivo expansion of hematopoietic progenitors from CD34+ cells selected from leukapheresis products of lymphoma and myeloma patients: feasibility and enhancement by fibronectin.

The feasibility of ex vivo expansion of hematopoietic progenitors selected from leukapheresis products of patients treated for multiple myeloma (MM) was studied and compared with progenitor expansions from patients with nodular non-Hodgkin's lymphoma (NHL) or healthy donors. After positive selection, CD34+ cells from leukapheresis products of 4 MM and 5 NHL patients and CD34+ cells from bone marrow (BM) of 3 healthy donors were grown in IMDM plus 12.5% horse serum, 12.5% fetal calf serum, IL-1alpha, IL-3, IL-6, SCF, GM-CSF, G-CSF (10 ng/ml each), and EP (4 UI/ml). Outputs of CD34+ cell cultures from MM and NHL patients were similar. Day 14 mean increases in CD34+, CFU-GM, and total cell numbers were, respectively, 5.3-fold, 19.8-fold, and 1173-fold for MM patients and 4.3-fold, 15.6-fold, and 1659-fold for NHL patients, with at least 40% of day 14 cells being of granulocytic lineage. Patient CD34+ cell culture output was found to be related to the CFU-GM/CD34+ cell ratio of selected CD34+ cells, not to underlying pathology. When the initial CFU-GM/CD34+ cell ratio was above 0.025, MM and NHL CD34+ cell culture outputs were always above 1000-fold. Moreover, in all but one CD34+ cell culture, the use of fibronectin (FN)-coated dishes improved CFU-GM and total cell expansion. In patient CD34+ cultures carried out in FN-coated dishes, mean day 14 CFU-GM and total cell outputs were increased, respectively, 2.1-fold and 1.9-fold. We conclude that if the CFU-GM/CD34+ cell ratio is sufficient (>0.025), ex vivo expansion of hematopoietic progenitors from CD34+ cells selected from leukapheresis products is possible for both MM and NHL patients and that using FN-coated flasks is a simple and reliable way to improve both CFU-GM and total cell output.

Blood Cell Count↗
PubMed1995

Regulation of colony-stimulating factor 1-induced proliferation by heterotrimeric Gi2 proteins.

Receptors for hematopoietic cytokines possess intrinsic tyrosine-kinases or are associated with tyrosine-kinases; interactions between metabolic pathways activated by tyrosine-kinases and heterotrimeric G proteins are suspected, but not yet proven. To investigate whether alteration of G protein function affects signal transduction of hematopoietic cytokines, we expressed mutant Gi2 proteins in BAC 1.2F5 cells, a murine macrophage cell line that is dependent from monocyte-macrophage colony-stimulating factor (CSF 1) for its proliferation. Mutations made in alpha subunits constitutively activate (alpha i2-Q205L) or inactivate (alpha i2-G204A) Gi2 heterotrimers. We show that expression of alpha i2-Q205L in BAC 1.2F5 cells does not induce independence from CSF 1, but reduces the cells' requirement in CSF 1, shortens the length of the G1 phase and the cell doubling time in response to CSF 1, and protects cells from death by apoptosis induced by CSF 1 withdrawal, exposure to H2O2 or heat shock, but not mitoxantrone. More importantly, expression of alpha i2-G204A, a dominant negative mutant, inhibits BAC 1.2F5 cell proliferation in response to CSF 1, increases the length of the G1 phase and the cell doubling time, and accelerates apoptotic cell death after withdrawal of CSF 1, exposure to H2O2 or heat shock. We conclude that the metabolic pathways regulated by Gi2 proteins and CSF 1 tyrosine-kinase receptors converge on a common effector necessary for the regulation of macrophage survival and proliferation.

Animals↗
PubMed1995

In vitro expansion of CD34+ cells from peripheral blood of myeloma and lymphoma patients.

We studied the feasibility of in vitro expansion of CD34+ cells from patients with multiple myeloma (MM) or follicular non Hodgkin lymphoma (NHL). CD34+ cells were selected from peripheral blood (PB) using avidinbiotin immunoadsorption columns: purified CD34+ cells from three MM and five NHL patients were expanded. First, CD34+ cells (2 MM, 4 NHL) were grown for 14 days in 5 ml of IMDM plus 12.5% horse serum (HS), 12.5% fetal calf serum (FCS) and a commonly used combination of cytokines: IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (10 ng/ml each) and EP (4 UI/ml). In these conditions, at day 14, average increase in CD34+, CFU-GM and total cell numbers were, respectively: x 6.0 x 23 and x 2,113 fold with 20 to 35% of granulocytic cells. In terms of CD34+ cell, CFU-GM and total cell outputs, MM cultures were comparable to NHL cultures, but MM cultures seemed to produce less granulocytic cells than NHL cultures. Next, in vitro expansion of PB CD34+ cells was tested in culture media suitable for clinical use. Two cultures (1 MM, 1 NHL) were carried out for 14 days in 20 ml of X-Vivo 10 medium, 2% human serum, IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (6 ng/ml each) and EP (2 UI/ml). Increase in CD34+, CFU-GM and total cell numbers in these conditions were, respectively: x 5.7 and x 19.7, x 11.9 and x 40.9, x 424 and x 408 fold, with at least 75% of granulocytic cells in both cultures. We conclude that, although further improvements are necessary, in vitro expansion of PB CD34+ cells can presumably be carried out successfully for MM patients as well as for NHL patients, including in conditions suitable for clinical use.

CD4 Antigens↗
PubMed1993

Stable changes in expression or activation of G protein alpha i or alpha q subunits affect the expression of both beta 1 and beta 2 subunits.

G proteins consist of three subunits: alpha, beta and gamma. Four beta subunits have been cloned: beta 1 and beta 4 (36 kDa), and beta 2 and beta 3 (35 kDa). We studied endogenous beta subunits in mouse NIH 3T3 fibroblasts stably expressing high levels of G protein alpha subunits after transfection with cDNAs encoding alpha i1, alpha i2, alpha i3 and alpha q. Immunoblots showed that NIH 3T3 cells express beta 36 and beta 35 subunits; in these cells, beta 35 subunits are four times more abundant than beta 36 subunits. We could detect beta 1 and beta 2 mRNA, but neither beta 3 nor beta 4 mRNA. We found that a stable increase in expression of wild-type alpha i1, alpha i2, alpha i3 or alpha q subunits is always accompanied by an increase in beta 1 and beta 2 mRNA and protein levels. There was no evidence of selectivity for an increase in beta 1 rather than beta 2 subunits depending on the type of alpha subunit overexpressed. However, constitutive activation or inactivation of alpha subunits induced specific changes in beta subunits. Expression of constitutively inactivated alpha i2 subunits was accompanied by an increase in mRNA and protein levels of both beta subunits. In contrast, cells expressing constitutively activated alpha i2 subunits did not show any change in the amount of beta proteins expressed in membranes, despite a significant increase in beta 1 and beta 2 mRNA. We conclude that stable changes in the levels of expression or degree of activation of G alpha subunits affect the level of expression, and possibly the turn-over, of beta subunits, without selectivity among beta 1 and beta 2 subunits.

3T3 Cells↗
PubMed1993

Mitogenic effects of pertussis toxin-sensitive G-protein alpha subunits: the mitogenic action of alpha i2 in NIH 3T3 cells is mimicked by alpha i1, but not alpha i3.

In fibroblasts and other cell types, pertussis toxin (PTX) inhibits DNA synthesis in response to serum and certain growth factors. GTPase deficient forms of the PTX-sensitive G-protein alpha i2 subunit have been shown to induce partial transformation in fibroblasts. In order to determine whether other PTX-sensitive G-proteins can stimulate mitogenic pathways, we stably expressed constitutively activated G-protein alpha i1 and alpha i3 subunits in NIH 3T3 cells. Expression of activated alpha i1, alpha i2 or alpha i3 results in inhibition of forskolin-stimulated cAMP accumulation in intact cells. Constitutively activated alpha i1, but not alpha i3, induces a loss of contact inhibition, a loss of anchorage-dependence, a reduced serum requirement and a decreased doubling time in NIH 3T3 cells. We conclude that alpha i1 and alpha i2 are both capable of transducing mitogenic signals, but that alpha i3 is not involved in the regulation of fibroblast growth. Furthermore, adenylyl cyclase inhibition is clearly not sufficient to explain the effect of alpha i2 on fibroblast growth.

3T3 Cells↗
PubMed1992

High level expression of transfected G protein alpha i3 subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts.

The alpha subunits of pertussis toxin-sensitive G proteins Gi1, Gi2 and Gi3 have been shown to inhibit adenylyl cyclase in transfected cells. However, Gi3 has recently been associated with protein transport and localized to the Golgi apparatus in a number of cell lines, rather than to the plasma membrane. We studied NIH 3T3 clones stably expressing different levels of a constitutively activated mutant of the alpha subunit of Gi3 (alpha i3-Q204L). Transfected alpha i3 subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing alpha i3-Q204L at high levels, alpha i3 subunits were also localized to the plasma membrane. Those clones which demonstrated expression of alpha i3 at the plasma membrane showed a 40% to 60% inhibition of forskolin-induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane alpha i3 was undetectable, did not show inhibition of forskolin-induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, alpha i3 is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.

3T3 Cells↗
PubMed1992

Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.

3T3 Cells↗
PubMed1991

Activating and inactivating mutations of the alpha subunit of Gi2 protein have opposite effects on proliferation of NIH 3T3 cells.

Previous studies have demonstrated that mutations of highly conserved residues in the alpha subunit of Gs (alpha s) can inhibit either the intrinsic GTPase activity (glutamine-227 to leucine, Q227L) or the ability of the protein to be activated by GTP (glycine-226 to alanine, G226A). We stably transfected NIH 3T3 cells with cDNAs encoding Gi2 alpha subunit (alpha i2) containing either wild-type sequence or the homologous mutations Q205L and G204A. High expression of wild-type alpha i2, Q205L alpha i2, and G204A alpha i2 was confirmed in transfected cells by immunoblot analysis. The overexpression of all three alpha i2 proteins was accompanied by an increase in beta-subunit expression. Q205L alpha i2 was a poor substrate for ADP-ribosylation by pertussis toxin as compared with wild-type alpha i2. Expression of Q205L alpha i2 markedly decreased forskolin- or cholera toxin-stimulated intracellular cAMP levels in intact cells, confirming the constitutively activated state of the protein. In contrast, G204A alpha i2 increased intracellular cAMP and was resistant to guanosine 5'-[gamma-thio]triphosphate-induced inhibition of ADP-ribosylation by pertussis toxin, as expected for an inactive alpha i2. Transfection of wild-type, Q205L, or G204A alpha i2 cDNA did not induce focus formation of NIH 3T3 cells. However, overexpression of Q205L alpha i2 induced a decreased serum requirement, a reduced doubling time, and an 8- to 10-fold increase in [3H]thymidine incorporation. Q205L alpha i2 cells formed small colonies in soft agar, demonstrating some degree of anchorage-independent proliferation. Expression of G204A alpha i2 slowed the growth of NIH 3T3 cells. We conclude that alpha i2 plays an important role in regulation of fibroblast growth.

3T3 Cells↗
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↗
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↗
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↗
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↗