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*
Institut National de la Santé et de la Recherche Médicale, U.365, Institut Curie, Paris, France; and
Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or PMA results in a
dephosphorylation and consequent activation of 4E-BP1.
Dephosphorylation of 4E-BP1 was also observed when U-937 cells were
induced to differentiate into monocytes/macrophages following treatment
with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with
retinoic acid or DMSO, which results in a granulocytic differentiation
of these cells, decreases 4E-BP1 amount without affecting its
phosphorylation and strongly increases 4E-BP2 amount. Taken together,
these data provide evidence for differential regulation of the
translational machinery during human myeloid differentiation, specific
to the monocytic/macrophage pathway or to the granulocytic
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The translation rate in an important determinant of cell proliferation and differentiation, and generally varies in response to treatment with growth factors, cytokines, hormones, and mitogens (reviewed in Refs. 2) and 3 . Most of the control of translation occurs at the level of initiation. Translation initiation entails the process leading to the positioning of the ribosome at the AUG initiation codon. Cellular mRNAs contain a cap structure (m7G(5')ppp(5')N, in which N is any nucleotide) at their 5' termini 4 . The multisubunit translation initiation factor eIF4F binds to the cap structure via the eIF4E subunit to promote ribosome binding 2 . Two repressors of cap-mediated translation, termed 4E-BP1 and 4E-BP2 (eIF4E-binding proteins-1 and -2), also known as PHAS, have been characterized 5, 6, 7 . 4E-BP1 and 4E-BP2 are heat- and acid-stable proteins whose activity is regulated by phosphorylation 5, 6, 7, 8 . Dephosphorylated 4E-BP1 and 4E-BP2 interact with eIF4E, and these interactions result in the specific inhibition of cap-dependent translation, both in vivo and in vitro 5, 8 . Furthermore, overexpression of 4E-BPs reduces cell proliferation 9 . We have also recently reported that expression of 4E-BP2 is down-regulated during human thymocyte maturation, and that may be a determinant in their different proliferation potential during maturation in response to activation 10 .
Our goal was to investigate translation rates of 4E-BPs function during differentiation of human myeloid cells. Many of the myeloid cell lines can be induced to terminally differentiate along one or several pathways to mature into differentiated cells, and therefore, they provide a useful experimental model to study the expression of 4E-BP1 and 4E-BP2 during monocytopoiesis and granulopoiesis 11 . In particular, the HL-60 cell line has been of interest because of its ability to undergo both granulocytic maturation and monocyte/macrophage differentiation following exposure to various agents. The monoblastic U-937 cells have a clear monocytic lineage derivation and are a widely used model for monocytic differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rIFN- was a gift from Roussel-Uclaf (Romainville,
France), PMA was from Sigma (St. Quentin-Fallavier, France),
all-trans RA2 was
kindly provided by Hoffman-La Roche (Basel, Switzerland), and DMSO was
from Merck (Nogent sur Marne, France). PMA and RA were dissolved in
absolute ethanol at an initial concentration of 1 mg/ml and
10-2 M, respectively. Dilutions were performed in RPMI
1640 medium. The final concentration of ethanol had no effect on cell
growth and differentiation.
Cell and culture conditions
The human myeloid cell lines HL-60 and U-937 were obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in the presence of 5% heat-inactivated FCS (Life Technologies, Sarl, France), using RPMI 1640 medium supplemented with 2 mM L-glutamine, 10 mM HEPES buffer, and gentamicin (20 µg/ml). The cultures were incubated in humidified air with 5% CO2 at 37°C and subcultivated twice per week.
Induction of differentiation
Twelve to fifteen hours before induction, cells were harvested
and resuspended in fresh medium. HL-60 cells and U-937 cells (2 x
105 cells/ml) were induced to differentiate for 1 to 5
days, by treatment with 5 ng/ml PMA, 200 U/ml IFN-, 1 µM
all-trans RA, or 1% DMSO, without any change of media.
Differentiation was monitored after 3 and 5 days by determining cell
growth with a Coulter (Margency, France) counter ZM equipped
with a Coultronic 256 channelizer, and viability was estimated by
trypan blue dye exclusion. Cell morphology was examined by staining of
cytocentrifuged cells with May-Grünwald and subsequent
light-microscope examination.
Cell surface Ag analysis
The appearance of various cellular markers normally associated with the maturation of the granulocytic and monocytic elements was also determined. The analysis of cell surface Ag expression was performed by direct immunofluorescence using flow cytometry (FACScan; Becton Dickinson, Mountain View, CA). Briefly, control and induced cells were collected, washed twice in PBS at 4°C, and incubated with 1 µg/ml monoclonal mouse anti-human FITC-conjugated anti-CD11b (Mac-1; BEAR 1; mIgG1; Immunotech, Luminy, France) and with 0.5 µg/ml monoclonal mouse anti-human FITC-conjugated anti-HLA-DR (B8.12.2, mIgG2b; Immunotech) for 30 min at 4°C. The cells were then washed twice with PBS containing 1% BSA, 0,1% sodium azide, fixed in 1% paraformaldehyde, and analyzed for fluorescence. Data analysis was based on examination of 5000 cells/sample. The results are given as percent positive cells, and mean fluorescence intensity was obtained by subtracting the peak channel number of the negative control from the peak channel number of the corresponding experimental sample.
SDS-PAGE and Western blotting
Cells were rinsed twice with ice-cold PBS and lysed by successive freeze-thaw cycles, in 20 mM Tris-HCl, pH 7.5, buffer containing 5 mM EDTA and 100 mM KCl. The homogenate was centrifugated at 6000 x g for 10 min, and the supernatant was collected. To analyze for 4E-BP1 and 4E-BP2, 100 µg of protein was dissolved in Laemmli sample buffer 12 , and the samples were loaded onto a 15% polyacrylamide gel. Proteins were transferred onto a 0.22-µm nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), which was blocked in 5% milk for 2 h, followed by incubation for 2 h with rabbit polyclonal antiserum against 4E-BP1 (11208) and 4E-BP2 (11211) in 10 mM Tris-HCl, pH 8, buffer containing 150 mM NaCl at a dilution 1/1000. The membrane was then incubated for 2 h with horseradish peroxidase-labeled conjugate Ab at a dilution 1/2000. Immunodetection was realized by enhanced chemoluminescence (ECL) reagents and autoradiography.
RNA extraction and Northern blot
Total RNA was extracted by guanidium isothiocyanate lysis and CsCl centrifugation 13 and separated by electrophoresis through 1% agarose, 6% formaldehyde gels. Twenty micrograms of denaturated total RNA were used for each lane. After transfer onto nylon membrane, RNA blots were UV cross-linked (UV Stratalinker, 120,000 µJ; Stratagene, La Jolla CA), prehybridized in 50% formamide, 5x SSPE, 1x Denhardt’s solution, and 0.5% SDS for 24 h at 42°C, and then hybridized for 24 h at 42°C in the same solution containing 200 µg/ml of heat-denatured salmon sperm DNA and 32P-labeled cDNA probe (adding approximatively 2 x 106 cpm of the labeled probe/ml). Following washes of increase stringency, membranes were analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and autoradiographed. The blots were rehybridized with an actin probe to ensure that equal amounts of RNA were present in each lane.
Probes
The 4E-BP1 and 4E-BP2 probes were a 0.8-kb human 4E-BP1 and a 3.5-kb human 4E-BP2 cDNA fragment. The control probe was an actin probe made of a 2.1-kb human cDNA fragment. For Northern blot analysis, cDNA inserts were purified and labeled with [32P]ATP using a random primer kit (Multiprime labeling kit; Amersham, Orsay, France), according to the manufacter’s instructions.
Metabolic labeling
Undifferentiated and differentiated HL-60 cells were preincubated for 1 h in methionine-free RPMI 1640 medium. [35S]Methionine (100 µCi) was added for 4 h, and cells were lysed in 20 mM Tris-HCl, pH 7.5, buffer containing 5 mM EDTA and 100 mM KCl. The radioactivity incorporated into TCA-precipitable material was measured.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differentiation of the promyelocytic HL-60 cells into
monocytes/macrophages or into granulocytes can be induced in vitro by
treating the cells with different agents, including IFN- or PMA for
monocytes/macrophages and RA or DMSO for granulocytes 14 . We treated
HL-60 cells with IFN-, PMA, RA, or DMSO for 3 and 5 days and
determined their differentiation stage by three criteria: cell
proliferation, morphology, and cell surface expression of specific
markers. The cells were seeded at an initial density of 2 x
105/ml and cultured in the presence of IFN- (200 U/ml),
PMA (5 ng/ml), RA (1 µM), or DMSO (1%). Following treatment with
each agent, cell proliferation was strongly reduced at 3 and 5 days of
culture, as shown in Fig. 1
A.
The PMA-treated cells ceased to proliferate after 3 days. The cells
were more than 80% viable at the end of the culture period, except for
the PMA-treated cells, in which cell viability at 5 days was about
65%. Morphology of the cells was analyzed by the method of
May-Grünwald staining (Fig. 1B). In untreated cells,
cultured HL-60 cells are predominantly promyelocytes with
characteristic cytoplasmic granules, large nuclei, and prominent
nucleoli. HL-60 cells treated with either IFN- or PMA for 3 days
acquired monocyte/macrophage morphology, with a high
nuclear/cytoplasmic ratio, vacuolization, and blebbing, whereas HL-60
cells treated with RA or DMSO for 3 days acquired granulocyte
morphology, with chromatin condensation, loss of nucleoli, and nuclear
lobulation. The PMA-treated cells became adherent to the substrate,
with almost 100% cell attachment observed by 48 h. Similar
morphologies were observed after 5 days of treatment (data not shown).
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). The CD11b surface Ag is restricted to
cells of the myelomonocytic lineage, and its expression is up-regulated
during granulocytic and monocytic differentiation 15 . The MHC class
II determinant, HLA-DR, is expressed transiently during early
development of myelomonocytic precursors and later in monocytes 11 .
In the absence of any stimuli, HL-60 cells express very low levels of
the two Ags. Exposure of the cells to the four inducers (IFN-, PMA,
RA, and DMSO) for 3 days results in an increase of CD11b surface Ag
expression. Upon differentiation of HL-60 cells by IFN- or PMA
treatment, the expression of HLA-DR was increased significantly. In
contrast, the HLA-DR determinant was not induced upon treatment with RA
or DMSO, consistent with the differentiation of these cells into
granulocytes 11 .
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As protein synthesis rate is a major determinant of cell
proliferation, we analyzed protein synthesis rates during
differentiation of the HL-60 cells. The translation rate was determined
by metabolic labeling of cells with [35S]methionine, and
incorporation rates were measured 3 and 5 days after stimulation.
Protein synthesis was strongly inhibited in HL-60 cells induced to
differentiate into monocytes/macrophages after IFN- and PMA
treatments for 5 days (75 and 90% inhibition, respectively) (Fig. 2A). Slight inhibition of
protein synthesis was observed with IFN- after 3 days of culture,
whereas maximal inhibition was already obtained after 3 days of
treatment with PMA. A similar strong inhibition of protein synthesis
was observed when the cells were induced to differentiate into
granulocytes in response to RA or DMSO for 5 days (85% inhibition)
(Fig. 2B). Therefore, reduction of protein synthesis is a
general mechanism, common to the two pathways of HL-60 cell
differentiation. It correlates well with the proliferation status of
these cells during their differentiation, with a faster and stonger
effect of PMA treatment as compared with IFN-, RA, or DMSO
treatments.
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To study the mechanisms by which IFN- and PMA inhibit protein
synthesis and induce monocytic differentiation of HL-60 cells, the
effect of these agents on 4E-BP1 and 4E-BP2 expression was examined.
Three isoforms of 4E-BP1 (indicated by the arrows; Fig. 3) were detected following immunoblotting
of extracts from untreated HL-60 cells. We previously reported that
these isoforms represent various phosphorylation states of 4E-BP1, the
fastest migrating electrophoretic isoform corresponding to the
unphosphorylated 4E-BP1 5, 6, 8 . Both IFN- and PMA induced a
dephosphorylation of 4E-BP1. Whereas slight dephosphorylation of 4E-BP1
was observed after 3 days of IFN- or PMA treatments, complete
dephosphorylation was obtained after 5 days. No significant
modification was observed in 4E-BP2 expression during the treatment of
the cells by both inducers.
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4E-BP1 and 4E-BP2 expression during differentiation of HL-60 cells along the granulocytic pathway
To study the mechanisms by which RA and DMSO inhibit protein
synthesis as they trigger another differentiation pathway, the effect
of these agents on 4E-BP1 and 4E-BP2 expression was also examined. In
contrast to the dephosphorylation of 4E-BP1 observed during monocytic
differentiation of HL-60 cells, no variation of 4E-BP1 phosphorylation
occurred after exposure to RA and DMSO for 3 and 5 days, but 4E-BP1
protein expression was reduced significantly by two- to threefold after
3 days of treatment (Fig. 4A).
Furthermore, 4E-BP2 protein expression was strongly induced (five- to
sixfold) after 3 days of treatment with both agents. Because strong
variations of 4E-BP1 and 4E-BP2 protein expression were already
observed after 3 days of treatment, we performed a kinetic of RA and
DMSO treatment from 4 h to 3 days. Maximal induction of 4E-BP2
protein expression was observed after 2 days of RA (Fig. 4B)
and DMSO (data not shown) treatment. A decrease in 4E-BP1 protein
expression was observed after 3 days of treatment with both agents
(data not shown).
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show no variation of 4E-BP1 and 4E-BP2
mRNA expression (Fig. 5). Exposure to RA
and DMSO for 3 days results in a dramatic decrease in 4E-BP1 mRNA to a
hardly detectable level, whereas no apparent modification of 4E-BP2
mRNA level was detected, as quantified by densitometry (Fig. 5).
Hybridization of the blot with an actin cDNA probe demonstrates that
equivalent amounts of RNA were loaded in each lane.
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or PMA and RA or
DMSO regulate 4E-BP1 and 4E-BP2 functions differently. During
differentiation of HL-60 cells along the granulocytic pathway,
expression of 4E-BP2 protein is strongly increased with no modification
of its mRNA level, whereas expression of 4E-BP1 protein and mRNA is
decreased. These results also suggest that the regulation of 4E-BP1
expression is controlled primarily at a transcriptional level, whereas
4E-BP2 expression is regulated at a posttranscriptional level. Assessment of U-937 cell differentiation
To confirm that the regulation of 4E-BP and 4E-BP2 expression
observed during differentiation of HL-60 cells is specific to the
differentiation pathway and not to the particular inducers or cells
used in our experiments, we analyzed the expression of 4E-BP1 and
4E-BP2 during differentiation of U-937 cells. The monoblastic U-937
cells are arrested in an advanced stage of differentiation, and
therefore, treatment of these cells with any of the four inducers,
IFN-, PMA, RA, or DMSO, carries their differentiation into
monocytes/macrophages 17, 18 . We treated the U-937 cells for 3 and 5
days with IFN-, PMA, or RA, and monitored their differentiation
stage by the same three criteria as used for HL-60 cells: cell
proliferation, morphology, and cell surface expression of specific
markers. Following treatment with each agent, cell proliferation was
strongly reduced at 3 and 5 days of culture (Fig. 6A). Morphology of the cells
was analyzed by the method of May-Grünwald staining (Fig. 6B). The different treatments resulted in the induction of
U-937 cell differentiation toward a more mature state with major
characteristic of monocytes/macrophages: large cytoplasms with
vacuolizations, particularly in the PMA-induced cells, nuclear
chromatin not condensed, and persistence of nucleoli. Similar
morphologies were observed after 5 days of treatment (data not shown).
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). Differentiation of HL-60 cells
induced by RA, IFN-, and PMA results in an increase of the two
surface Ag expression, consistent with the differentiation of the cells
into monocytes/macrophages 19 .
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We analyzed protein synthesis rates during differentiation of the
U-937 cells by metabolic labeling of cells with
[35S]methionine, and incorporation rates were measured 3
and 5 days after stimulation. Protein synthesis was strongly inhibited
in U-937 cells induced to differentiate into monocytes/macrophages
after IFN- and PMA treatments for 5 days (75 and 90% inhibition,
respectively) (Fig. 7). A lesser extent
of inhibition was obtained following RA treatment for 5 days (35%
inhibition). Slight inhibition of protein synthesis was observed with
IFN- and RA after 3 days of culture, whereas inhibition was already
very strong after 3 days of treatment with PMA.
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We analyzed 4E-BP1 and 4E-BP2 proteins by Western blotting during
U-937 cell differentiation induced by IFN-, PMA, or RA. As in HL-60
cells, three isoforms of 4E-BP1 (indicated by the arrows; Fig. 8) were detected following immunoblotting
of extracts from untreated U-937 cells. RA as well as IFN- and PMA
induced a dephosphorylation of 4E-BP1. The dephosphorylation of 4E-BP1
was strong after 3 days and complete after 5 days. 4E-BP2 expression is
low in U-937 cells, and no increase, but rather a slight decrease, was
observed during the treatment of the cells by the three inducers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have suggested that 4E-BP1 is a major target of
mitogen-activated protein kinases 6 . However, it has also been
reported that the mitogen-activated protein kinase pathway is not
necessary to induce 4E-BP1 phosphorylation 20, 21, 22, 23 , and we have shown
that dephosphorylation of 4E-BP1 occurs following rapamycin treatment
of cells 8 , indicating that phosphorylation of 4E-BP1 is mediated by
mTOR (mammalian target of rapamycin, also called FRAP or RAFT1), a
member of the family of phosphoinositide 3-kinase-related kinases
(reviewed in 24 . Despite the fact that dephosphorylation of
4E-BP1 induced by IFN- is slow, the involvement of mTOR in IFN-
signaling should be investigated.
Up-regulation of 4E-BP2 during granulocyte differentiation is of particular interest, as it occurs after 2 days of treatment, preceding the morphology changes of the cells. Furthermore, we did not observe an increase in 4E-BP2 protein expression in cellular clones resistant to the granulocytic differentiation induced by RA (A.G. and L.B., personnal observations). The up-regulation of 4E-BP2 protein is not due to a RA-response element in the promoter of the 4E-BP2 gene, as no induction of 4E-BP2 mRNA was observed, suggesting a posttranscriptional regulation of the gene. Because the effect of RA on differentiation of promyelocytic leukemic cells is now used clinically in the therapy of these leukemias 25, 26 , it would be of great interest to analyze expression of 4E-BP2 in leukemias and to investigate the consequence of modifying 4E-BP2 gene expression in these tumor cells.
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Acknowledgments |
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Footnotes |
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2 Abbreviation used in this paper: RA, retinoic acid.
Received for publication July 22, 1998. Accepted for publication December 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by human leukemia cells (HL-60 and U-937) during monocytic differentiation. Leukemia 9:671.[Medline]
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