Accumulation of p21ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity.

The ras gene product (p21) is a GTP-binding protein and has been thought to transduce signals regulating proliferation or differentiation of cells. Like other GTP-binding proteins, p21.GTP is an active conformation, which can transduce the signals downstream, whereas p21.GDP is an inactive one. Recently, we have shown that p21.GTP levels increased in cells treated with fetal bovine serum or platelet-derived growth factor to initiate DNA synthesis. In this paper, we report that epidermal growth factor can also increase the amounts of p21.GTP in the cells. Effects of epidermal growth factor and platelet-derived growth factor are not additive. In contrast, mutant [Val12]p21, which has transforming activity, responded neither to platelet-derived growth factor nor to epidermal growth factor. We also found that the ratio of p21.GTP to p21.GDP increased 3- to 4-fold in transformants carrying activated erbB-2/neu or v-src oncogenes. These results strongly suggest an important role of p21 in transduction of signals for both normal proliferation and malignant transformation through growth factor receptors with tyrosine kinase activity or related oncogene products.     

Identification of a nucleotide exchange-promoting activity for p21ras.

The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21ras has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchange-promoting protein acts reversibly and does not cause degradation of p21ras. It is inactive towards the alpha subunit of a heterotrimeric GTP-binding protein (Go alpha) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchange-promoting), has the characteristics expected of a physiological activator of p21ras in cellular growth-signal-transduction pathways.     

Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity.

Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. We were able to detect a weak GTPase activity associated with the purified proteins. The normal cellular ras protein (p21N) exhibits approximately equal to 10 times higher GTPase activity than the "activated" proteins. Even though the turnover rate of the reaction is very low (0.02 mol of GTP hydrolyzed per mol of p21N protein per minute), the reaction appears to be catalytic; one molecule of p21N hydrolyzes more than one molecule of GTP. The GTPase and the GDP binding activities both have been recovered from a Mr 23,000 protein eluted following NaDodSO4/polyacrylamide gel electrophoresis, suggesting that these two activities are associated with the same protein. Mg2+ ions and dithiothreitol are required for GTPase activity and the optimal pH is between 7 and 8. Guanidine X HCl, which is required for solubilizing bacterially expressed ras protein, is strongly inhibitory to GTPase activity at concentrations higher than 0.5 M.     

Purification of a factor capable of stimulating the guanine nucleotide exchange reaction of ras proteins and its effect on ras-related small molecular mass G proteins.

We have previously identified a membrane factor capable of stimulating guanine nucleotide exchange activity for ras p21 proteins. The ras guanine nucleotide exchange factor (rGEF) was purified from bovine brain to near homogeneity by successive chromatographies on DE52 DEAE-cellulose, Sepharose 6B, hydroxylapatite, and FPLC phenyl-Superose resins. SDS/polyacrylamide gel electrophoresis of the purified rGEF showed a single major protein with a molecular mass of 35 kDa. rGEF increased the exchange rate of GDP in normal [Gly12]p21 or oncogenic [Val12]p21 up to 30- to 40-fold under physiological concentrations of Mg2+. Since the factor was free from GDP/GTP binding activity and nonspecific GDP hydrolytic activity, we propose that rGEF may regulate GDP/GTP exchange reaction of ras proteins in response to external growth signals. Moreover, rGEF enhanced the dissociation of bound GDP from some of ras-like G proteins, R-ras, rap1-A, rab1-B, and rho proteins, raising the possibility that rGEF may affect the activities of these proteins.     

Antibodies specific for amino acid 12 of the ras oncogene product inhibit GTP binding.

An antibody (anti-p21ser) was raised against a ras p21-related synthetic peptide and was able to recognize specifically the substitution of serine for glycine at amino acid 12 of p21. This substitution causes oncogenic activation of p21. Anti-p21ser was found to immunoprecipitate v-Ki-ras p21 and to strongly inhibit its ability to autophosphorylate and to bind GTP in an immunoabsorption assay. Furthermore, binding of the antibody to p21 was specifically inhibited by GTP or GDP, suggesting that amino acids around position 12 are part of the GTP/GDP binding site. These results, taken together with the observation that the microinjection of anti-p21ser into cells transformed by v-Ki-ras p21 causes a transient reversion of the cells to a normal phenotype [Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R. & McCormick, F. (1985) Nature (London) 314, 639-642], support the idea that interaction of p21 with guanine nucleotides is crucial to the transforming function of this protein.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.     

Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells.

The ras gene product (p21) is a GTP-binding protein and is thought to play an important role in signal transduction of growth and differentiation in many types of mammalian cells. The p21.GTP complex is an active conformation, as described previously for polypeptide chain elongation factors (EF-Tu and EF-G) and heterotrimeric GTP-binding proteins (G proteins). In the study reported here, we measured the amounts of p21-bound guanine nucleotides under various conditions in the G54 cell line, a derivative of Swiss 3T3 cells that overexpresses normal c-Ha-ras. More p21.GTP complexes were present in growing cells than in quiescent cells. When quiescent cells were stimulated with fetal bovine serum to promote DNA synthesis, p21.GTP increased approximately 2-fold. Among a number of purified growth factors, platelet-derived growth factor enhanced the formation of p21.GTP, whereas the combination of bombesin and insulin, which also induces DNA synthesis, did not. These results strongly suggest that p21 is a transducer of the growth signal from the platelet-derived growth factor receptor in Swiss 3T3 cells and that the signal is transmitted through a p21.GTP complex.     

GTPase domains of ras p21 oncogene protein and elongation factor Tu: analysis of three-dimensional structures, sequence families, and functional sites.

GTPase domains are functional and structural units employed as molecular switches in a variety of important cellular functions, such as growth control, protein biosynthesis, and membrane traffic. Amino acid sequences of more than 100 members of different subfamilies are known, but crystal structures of only mammalian ras p21 and bacterial elongation factor Tu have been determined. After optimal superposition of these remarkably similar structures, careful multiple sequence alignment, and calculation of residue-residue interactions, we analyzed the two subfamilies in terms of structural conservation, sequence conservation, and residue contact strength. There are three main results. (i) A structure-based alignment of p21 and elongation factor Tu. (ii) The definition of a common conserved structural core that may be useful as the basis of model building by homology of the three-dimensional structure of any GTPase domain. (iii) Identification of sequence regions, other than the effector loop and the nucleotide binding site, that may be involved in the functional cycle: they are loop L4, known to change conformation after GTP hydrolysis; helix alpha 2, especially Arg-73 and Met-67 in ras p21; loops L8 and L10, including ras p21 Arg-123, Lys-147, and Leu-120; and residues located spatially near the N and C termini. These regions are candidate sites for interaction either with the GTP/GDP exchange factor, with a GTPase-affected function, or with a molecule delivered to a destination site with the aid of the GTPase domain.     

Multifactor stimulation of megakaryocytopoiesis: effects of interleukin 6.

We have previously demonstrated that interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) stimulate various aspects of megakaryocytopoiesis. We have investigated the capacity of interleukin 6 (IL-6) to stimulate megakaryocyte colony formation from both normal Balb/C marrow and light-density marrow extensively depleted of adherent, pre-B, B and T cells. Human recombinant IL-6 (167 ng/ml) stimulated megakaryocyte colony formation from normal marrow (8.6 +/- 1 megakaryocyte colony-forming units [CFU-meg]/10(5) cells) as compared to control (1.5 +/- 4 CFU-meg/10(5) cells) in 16 determinations (p less than 0.01). IL-6 (167 ng/ml) also stimulated CFU-meg formation from depleted marrow (control, 10.8 +/- 4 CFU-meg/10(5) cells versus IL-6, 68 +/- 19 CFU-meg/10(5) cells in 12 determinations, p less than 0.01). IL-6 synergistically augmented IL-3-induced colony formation (139% IL-3 control, 120% calculated IL-3 plus IL-6 control, n = 11, p less than 0.01) in normal marrow and showed an additive effect in depleted marrow (133% IL-3 control, p less than 0.01, 114% of IL-3 plus IL-6, value not significant [NS] at 0.05 level). Studies with recombinant murine IL-6 gave similar results. There was an increasing level of megakaryocyte colony-stimulating activity from G-CSF (16,667 U/ml, 2.47 +/- 0.6 CFU-meg/10(5) cells, n = 17), to IL-6 (167 ng/ml, 8.47 +/- 0.96 CFU-meg/10(5) cells, n = 19), to GM-CSF (52 U/ml, 23 +/- 4 CFU-meg/10(5) cells, n = 14), to IL-3 (167 U/ml, 48 +/- 5 CFU-meg/10(5) cells, n = 20) as compared to media-stimulated marrow (range 1.29-1.86 CFU-meg/10(5) cells). A similar hierarchy was seen with depleted marrow. Combinations of factors (including IL-3, GM-CSF, G-CSF, and IL-6) tested against normal unseparated murine marrow did not further augment CFU-meg numbers over IL-3 plus IL-6 but did increase colony size. These data suggest that IL-6 is an important megakaryocyte regulator, that at least four growth factors interact synergistically or additively to regulate megakaryocytopoiesis, and that combinations of growth factors, possibly in physical association, might be critical in stimulating megakaryocyte stem cells.     

Priming effects of granulocyte-macrophage colony-stimulating factor are coupled to cholera toxin-sensitive guanine nucleotide binding protein in human T lymphocytes

In addition to the mobilization of neutrophils and monocytes, granulocyte-macrophage colony-stimulating factor (GM-CSF) also mobilizes lymphocytes into peripheral blood. We examined the ability of GM-CSF to induce the proliferation of purified human T cells (CD3+ CD4+ CD56- CD16- B1- MO2-) in two major aspects: (1) the mechanisms of GM- CSF interaction with interleukin-2 (IL-2) causing T-cell proliferation, and (2) the intracellular signals transmitted by GM-CSF in T lymphocytes. We observed that concentrations of GM-CSF between 0.01 ng/mL and 10 ng/mL had a synergistic effect with concentrations of IL-2 between 1 U/mL and 10 U/mL in stimulating T-cell proliferation. This effect of GM-CSF was maximal when it was added at the start of the culture. In situ hybridization showed the presence of mRNA for GM-CSF receptors in T cells. Further analysis showed that GM-CSF induced the expression of IL-2 receptor (IL-2R) on the surface of T lymphocytes. These events coincide with the ability of GM-CSF to increase the intracellular levels of both cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) in T cells, to increase the binding of (gamma-35S) GTP to T-cell membranes, and to enhance GTPase activity as determined by increased hydrolysis of 32P- GTP. IL-2 also induced IL-2R expression, cyclic nucleotide secretion, and G-protein activation. However, the presence of IL-2 reduced GM-CSF induction of these activities. Addition of antibodies to the alpha and beta subunits of IL-2R permitted the activation of G protein by GM-CSF even when IL-2 was present. Furthermore, GTP binding and GTPase activity induced by GM-CSF or IL-2 were inhibited by the addition of cholera toxin (CT), but not pertussis toxin (PT). Cumulatively, these results suggest that in T lymphocytes, receptors for GM-CSF or IL-2 may be coupled to the same CT-sensitive G protein, although other possibilities may exist. The role that G proteins play in mediating the intracellular signaling pathways induced by GM-CSF or IL-2 in human T cells is supported by adenosine diphosphate-ribosylation of a 44-kD or a 39-kD G protein in T-cell membranes by CT and PT, respectively.     

A stimulatory GDP/GTP exchange protein for smg p21 is active on the post-translationally processed form of c-Ki-ras p21 and rhoA p21.

We have purified a stimulatory GDP/GTP exchange protein for smg p21A and -B, ras p21-like small GTP-binding proteins (G proteins), cloned its cDNA, and named it GDP dissociation stimulator (smg p21 GDS). We show here that smg p21 GDS is active not only on smg p21A and -B but also on c-Ki-ras p21 and rhoA p21, all of which are post-translationally processed. Furthermore, we show that smg p21 GDS is inactive on the post-translationally unprocessed form of these proteins and on the post-translationally unprocessed form of c-Ha-ras p21 and smg p25A. All of the small G proteins recognized by smg p21 GDS have a cDNA-predicted C-terminal "CAAX" motif (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid) and a polybasic region upstream of this motif. These results suggest that smg p21 GDS is at least active on a group of small G proteins having these unique C-terminal structures. Moreover, they suggest that the C-terminal post-translational processing of these small G proteins, by farnesylation or geranylgeranylation of the C-terminal cysteine residue, removal of amino acids in positions denoted "AAX", and carboxyl methylation of the exposed cysteine residue, is important for the smg p21 GDS action.     

Haemoglobin inhibits GTP-hydrolysis and GDP/GTP-exchange activities of a low Mr GTP-binding protein, ras p21

Summary Haemoglobin was observed to inhibit the GDP/GTP-exchange activity of ras protein ( ras p21) by measurement of [³H]GDP-dissociation activity in time- and dosedependent manners. Haemoglobin also inhibited the [³²P]GTP-hydrolysis activity of ras p21 time- and dose-dependently. These inhibitory effects of haemoglobin were lost after incubation of haemoglobin at 80°C for 3 min. Globin showed limited inhibition on the [³²P]GTP-hydrolysis activity of ras p21, and haemin had no effect, indicating that the ternary tetrameric structure of haemoglobin is essential for the inhibitory effects on ras p21 activities. Methaemoglobin also inhibited both [³H]GDP-dissociation and [³²P]GTP- hydrolysis activities of ras p21 in a very similar manner to that by haemoglobin. The obtained results strongly suggest that haemoglobin suppresses the physiological functions(s) of ras p21 in vivo inhibiting both [³²P]GTP-hydrolysis and GDP/GTP-dissociation of ras p21 in erythrocytes.     

Isolation of ras GTP-binding mutants using an in situ colony-binding assay.

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p21s were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.     

Purification of ras GTPase activating protein from bovine brain.

In cytosolic extracts of bovine brain, we detected ras GTPase activating protein (GAP) activity that stimulated the GTP hydrolytic activity of normal c-Ha-ras p21 but not that of the oncogenic [Val12]p21 variant. GAP was purified 19,500-fold by a five-column procedure involving DEAE-Sephacel, Sepharose 6B, orange dye and green dye matrices, and Mono Q resins. A single major protein band of 125 kDa was observed on NaDodSO4/polyacrylamide gels that correlated with the elution of GAP activity on Mono Q. Purified GAP was devoid of inherent GTP hydrolytic activity, suggesting that it was a regulator of ras intrinsic GTPase activity. Under submaximal velocity conditions, the second-order rate constant of GTP hydrolysis at 24 degrees C for p21-GTP + GAP (4.5 X 10(6) M-1.sec-1) was at least 1000-fold greater than that for [Val12]p21-GTP + GAP (less than 3 X 10(3) M-1.sec-1).     

A product of yeast RAS2 gene is a guanine nucleotide binding protein.

Yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, which show remarkable homology to mammalian ras genes. To characterize these gene products, we have expressed the RAS2 gene in yeast using an inducible GAL10 promoter. After labeling with [35S]methionine and immunoprecipitating with a monoclonal antibody Y13-259, which reacts with p21 encoded by mammalian ras genes, a major band having an apparent molecular weight of 41,000 is detected. This band has also been identified in cell-free translation products of polyadenylated RNA extracted from yeast cells grown in the presence of galactose. Crude extracts of cells expressing the RAS2 gene exhibit guanine nucleotide binding activity. This is detected by incubation with [3H]GDP followed by immunoprecipitation with the antibody Y13-259. The binding of labeled GDP is inhibited by a 20-fold excess of GDP, GTP, and, to a lesser extent, by UTP, a characteristic similar to that possessed by the mammalian ras proteins. However, the activity of the yeast protein differs from that of the mammalian proteins in its strong dependence on temperature. The guanine nucleotide binding activity provides an assay to purify the yeast protein.     

p21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase-activating protein.

Products of the ras gene family, termed p21ras, are GTP-binding proteins that have been implicated in signal transduction via receptors encoding tyrosine kinase domains. Recent findings have defined a superfamily of hemopoietin receptors that includes receptors for a number of interleukins and colony-stimulating factors. The intracellular portions of these receptors show only restricted homologies, have no tyrosine kinase domain, and provide no clues to the mode of signal transduction. However, in most cases the factors stimulate tyrosine phosphorylation. We demonstrate here that ligand-induced activation of the interleukin (IL)-2, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors resulted in activation of p21ras in various hemopoietic cell lines. The only cytokine tested that binds to a hemopoietin receptor and that did not activate p21ras was IL-4. Activation of p21ras was also observed in response to Steel factor, which stimulates the endogenous tyrosine kinase activity of the c-kit receptor, as well as with phorbol esters, which activate protein kinase C. Experiments with protein kinase inhibitors implicated tyrosine kinase activity, but not protein kinase C activity, as the upstream signal in p21ras activation via these growth factor receptors. Attempts to demonstrate tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP) were negative, suggesting that phosphorylation of GAP may not be the major mechanism for regulation of p21ras activity by tyrosine kinases.     

Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library.

Several oligonucleotide mixtures corresponding to a 6-amino acid sequence that is strictly conserved in all the ras and ras-related proteins (from various organisms) were tested for their ability to hybridize to 11 cloned members of the ras gene superfamily. Among these mixtures, a combination of two sets of partially complementary oligomers were able to hybridize to all the tested sequences. To identify members of the ras superfamily, we screened a rat brain cDNA library with these probes and isolated four genes, denoted rab1, -2, -3, and -4, encoding proteins homologous to the yeast YPT protein. Amino acid homology scores with YPT range from 75% for rab1 to 37% for rab4, whereas the homologies with p21 ras and other ras-related proteins are approximately equal to 30%, and homologous residues were clustered in the regions involved in GTP/GDP binding. Another striking similarity shared by the rab and the other ras-related proteins is the conservation of at least one cysteine residue near the carboxyl-terminal end involved in the membrane binding of the ras proteins. rab1 is a mammalian homolog of the yeast YPT gene, and the four rab genes constitute an additional branch of the ras gene superfamily that to our knowledge has not been described in higher eukaryotes.     

Heterogeneity in therapeutic response of genetically altered myeloma cell lines to interleukin 6, dexamethasone, doxorubicin, and melphalan

Because there is no known genetic abnormality common to all patients with myeloma, it is important to understand how genetic heterogeneity may lead to differences in signal transduction, cell cycle, and response to therapy. Model cell lines have been used to study the effect that mutations in p53 and ras can have on growth properties and responses of myeloma cells. The U266 cell line has a single mutant p53 allele. Stable expression of wild-type (wt) p53 in U266 cells results in a significant suppression of interleukin (IL)-6 gene expression and in the concomitant suppression of cell growth that could be restored by the addition of exogenous IL-6. Expression of wt p53 also leads to cell cycle arrest and protection from doxorubicin (Dox)- and melphalan (Mel)-induced apoptosis. The addition of IL-6 resulted in cell cycle progression and blocked p53-mediated protection from apoptosis. ANBL6 is an IL-6-dependent cell line that is sensitive to dexamethasone (Dex), Dox, and Mel. IL-6 is able to protect ANBL6 cells from Dex- and Mel- but not Dox-induced apoptosis. To study the effect of an activating mutation in ras, the ANBL6 cell line transfected with either a constitutively activated N- or K-ras gene was used. Both N-ras12 and K-ras12 genes were able to protect ANBL6 cells from apoptosis induced by Dex, Dox, and Mel. These data show that changes in ras or p53 can alter the myeloma cell response to IL-6 and demonstrate that the genetic background can alter therapeutic responses.     

Establishment and characterization of a new human leukemia cell line derived from M4E0

A new human leukemia cell line, designated as ME-1, was established from the peripheral blood leukemia cells of a patient with acute myelomonocytic leukemia with eosinophilia (M4E0). This cell line has the characteristic chromosome abnormality of M4E0, inv(16) (p13q22). When cultured in RPMI 1640 medium containing 10% fetal calf serum, ME-1 cells were monoblastoid, but with the addition of cytokines such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, or medium conditioned by phytohemagglutinin-stimulated human peripheral leukocytes (PHA-LCM), the cells exhibited differentiation to macrophage-like cells. PHA-LCM also promoted eosinophilic-lineage differentiation of this cell line, although IL-5 did not do so. To elucidate the mechanism of proliferation and differentiation of ME-1 cells, we studied the effect of a potent inhibitor of protein kinase C, 1-(5-isoquinolinyl-sulfonyl)-2- methylpiperazine (H-7), on colony formation of ME-1 cells. H-7 inhibited colony formation of ME-1 cells by IL-3 or GM-CSF dose dependently, but had little inhibitory effect on colony formation by IL- 4. These results indicate that the proliferation and differentiation of ME-1 cells by IL-3 or GM-CSF were related to the activation of protein kinase C, while those by IL-4 involved other regulatory systems. ME-1 cells should be useful for studying the pathogenesis of M4E0 and the mechanisms of proliferation and differentiation of leukemic and normal progenitors by cytokines.     

Differential regulation of colony-stimulating factors and interleukin 2 production by cyclosporin A.

Stimulation of T lymphocytes with mitogens or antigens is followed by proliferation and lymphokine production. Although cyclosporin A (CsA), an immunosuppressive drug, has been shown to inhibit the production of certain lymphokines, including interleukin 2 (IL-2), interleukin 3 (IL-3), and gamma-interferon, its effect on the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) has not been evaluated. In the current study, concanavalin A (Con A)-stimulated murine spleen cells secreted GM-CSF, IL-3, and IL-2, and in the presence of CsA (0.1-1.0 micrograms/ml), IL-2 and IL-3 activities were inhibited. In contrast, significant activity was detected when the CsA-treated culture supernatants were assayed on a cell line that is dependent on GM-CSF and/or IL-3. Similar CsA-resistant activity was observed when the EL-4 thymoma cells were stimulated with a phorbol ester [phorbol 12-myristate 13-acetate (PMA)] in the presence of CsA. The activity resistant to CsA was identified as GM-CSF by the ability of specific antibodies against murine recombinant GM-CSF to neutralize its activity. These findings indicate that GM-CSF, in contrast to IL-2 and IL-3, was not inhibited by CsA. In additional experiments, transfer blot of poly(A)+ RNA isolated from PMA-induced EL-4 cells in the presence or the absence of CsA was hybridized with GM-CSF and IL-2 cDNA probes. Expression of the GM-CSF gene in EL-4 cells was detected independent of CsA, whereas CsA inhibited the expression of the IL-2 gene. The present data show that production of IL-2 and IL-3, but not that of GM-CSF, is inhibited by CsA and suggest a differential control mechanism for lymphokine synthesis in T lymphocytes.