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Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 1957-1966
By
From the Fels Institute for Cancer Research and Molecular Biology,
and Department of Biochemistry, Temple University School of Medicine,
Philadelphia, PA.
We previously have shown that the zinc finger transcription factor
Egr-1 blocked granulocytic differentiation of HL-60 cells, restricting
differentiation along the monocytic lineage. Egr-1 also was observed to
block granulocyte colony-stimulating factor (G-CSF)-induced
differentiation of interleukin-3 (IL-3)-dependent 32Dcl3 hematopoietic
precursor cells, endowing the cells with the ability to be induced by
granulocyte-macrophage colony-stimulating factor (GM-CSF) for terminal
differentiation along the macrophage lineage. To better understand the
function of Egr-1 as a positive modulator of monocytic differentiation,
in this work we have studied the effect of ectopic expression of Egr-1
on the murine myeloblastic leukemic cell line M1, which is induced for
differentiation by the physiological inducer IL-6. It is shown that,
unlike in HL-60 and 32Dcl3 cells, ectopic expression of Egr-1 in M1
cells resulted in activation of the macrophage differentiation program
in the absence of differentiation inducer. This included the appearance of morphologically differentiated cells, decreased growth rate in mass
culture, and cloning efficiency in soft agar, and expression of
endogenous c-myb and c-myc mRNAs was markedly
downregulated. Untreated M1Egr-1 cells also exhibited cell adherence,
expression of Fc and C3 receptors, and upregulation of the myeloid
differentiation primary response genes c-Jun, junD, and
junB and the late genetic markers ferritin light-chain
and lysozyme. Ectopic expression of Egr-1 in M1 cells also
dramatically increased the sensitivity of the cells for IL-6-induced
differentiation, allowed a higher proportion of M1 cells to become
terminally differentiated under conditions of optimal stimulation for
differentiation, and decreased M1 leukemogenicity in vivo. These
findings demonstrate that the functions of Egr-1 as a positive
modulator of macrophage differentiation vary, depending on the state of
lineage commitment for differentiation of the hematopoietic cell type.
© 1998 by The American Society of Hematology.
THE COMPLEX PROCESS of blood cell
formation provides a profound example of cell homeostasis that is
regulated throughout life, whereby a hierarchy of hematopoietic
progenitor cells in the bone marrow proliferate and terminally
differentiate along multiple, distinct cell lineages, including the
proliferation and differentiation of myeloid precursor cells into
mature granulocytes and macrophages.1-5 The murine M1
myeloid leukemic cell line proliferates autonomously and can be induced
with the physiological inducers interleukin-6 (IL-6), leukemia
inhibitory factor (LIF),6 or conditioned media of mouse
lungs (LUCM), containing both IL-6 and LIF,7 to undergo terminal differentiation and growth arrest, which culminates in programmed cell death.8
To identify genes that may play a role in the regulation of
hematopoietic cell differentiation, we have isolated cDNA clones of
myeloid differentiation primary response (MyD) genes, activated in the
absence of de novo protein synthesis, in HL-60 and M1 cells after
induction for macrophage or granulocyte
differentiation.7,9,10 In the course of this work, the gene
encoding the zinc finger transcription factor Egr-1 (Krox24, NGIF-A, or
Zif268 Tis8) has been identified as a myeloid differentiation primary
response gene, specifically induced upon HL-60 macrophage
differentiation.10
Egr-1 was initially identified as an early growth response gene in
cultured fibroblasts11,12 and was subsequently shown to be
induced in response to B-cell maturation as well as during differentiation of nerve, bone, myeloid, and erythroleukemic
cells.10,13-17 Recently, Egr-1 also has been shown to be
involved in cell proliferation,18-20 negative regulation of
cell growth,21,22 and apoptosis.23 The Egr-1
protein has been localized to the nucleus and shown to bind
specifically to the consensus sequence 5 Egr-1 was found by us to be a macrophage differentiation primary
response gene that restricts the differentiation of human HL-60 cells
along the monocytic lineage10 and potentiates macrophage differentiation of the hematopoietic precursor cell line
32Dcl3.30 Egr-1 is induced in M1 cells immediately after
stimulation with physiological inducers IL-6, LIF, or LUCM, and
blocking Egr-1 expression by antisense oligonucleotides results in
repression of monocytic differentiation.10 These findings
raised the possibility that deregulated expression of Egr-1 in M1 cells
may predispose the cells for terminal differentiation. To test this
hypothesis, we studied the effect of deregulated expression of Egr-1 on
macrophage differentiation of the murine myeloblastic leukemic cell
line M1. It was shown that ectopic expression of Egr-1 in M1 cells activated the macrophage differentiation program and included the
appearance of morphologically differentiated cells. In addition, the
cells were sensitized for further induction of terminal differentiation by IL-6, and a higher proportion of M1 cells became terminally differentiated under conditions of optimal stimulation for
differentiation. Ectopic expression of Egr-1 in M1 cells decreased the
leukemogenicity in vivo.
Cells, mice, and cytokines.
The differentiation competent murine myeloid leukemic cell line M1 has
been described previously.31 The cells were cultured in
Dulbecco's modified Eagle's medium (DMEM; Cellgro, Mediatech Inc,
Heyndon, VA) supplemented with 10% heat-inactivated horse serum (HS; GIBCO-BRL, Grand Island, NY) plus 1%
penicillin and streptomycin (Cellgrow) in a humidified atmosphere with
10% CO2 at 37°C. Conditions to stimulate the cells for
terminal differentiation were described in detail
previously.31 Briefly, the cells were seeded at 0.15 × 106 cells/mL with or without IL-6 at 1 or 50 ng/mL,
as indicated. For RNA extractions, cell concentrations were adjusted to
give a final density of greater than 0.25 × 106
cells/mL at the time of extraction. Viable cell numbers were determined
by trypan blue dye exclusion. Experiments were repeated at least three
times. PA317 (American Type Culture Collection, Rockville, MD), a
retrovirus packaging cell line,32 was cultured in DMEM
supplemented with 10% heat-inactivated newborn calf serum (GIBCO-BRL)
plus 1% penicillin and streptomycin in a humidified atmosphere with
10% CO2 at 37°C. PA317 cells were periodically selected in HAT (hypoxanthine, aminopterin, and thymidine)
medium to maintain their packaging function. For M1 leukemogenicity
assays, 4- to 6-week-old CD-1 homozygous nude mice were obtained from Charles River Laboratories (Wilmington, MA). In the leukemogenicity assay, nude mice were intravenously injected (tail vein) with 104 or 105 cells prepared in 200 µL of
1× phosphate-buffered saline (PBS) for each cell type. Control
animals were injected with the same volume of 1× PBS. Recombinant
human IL-6 was a generous gift from Amgen Inc (Thousand Oaks, CA).
General recombinant DNA techniques, expression vectors, and DNA
probes.
Plasmid preparations, restriction enzyme digestions, DNA fragment
preparations, and agarose gel electrophoresis were performed as
previously described.31,33,34 The retroviral plasmid
expression vector, MSCV EB neo, used in this study was a gift from Dr
Robert G. Hawley (University of Toronto, Toronto, Ontario,
Canada).35 The 2.3-kb BamHI and Sal I
fragment of the full-length murine Egr-1 cDNA10 was cloned
into the Xho I site of the MSCV EB neo retroviral vector by
blunt end ligation. DNA probes for murine c-Jun, junD,
junB, MyD88, c-fos, Icam-1,
c-myc, c-myb, b-actin, ferritin, and
lysozyme have been previously described.30,31,33,34 stat3 cDNA was excised from pRcCMV-stat3 plasmid by ApaI/NotI digestion (James E. Darnell Jr, Invitrogen Inc, Carlsbad,
CA). The probes were labeled by random priming (GIBCO-BRL;
RadPrime DNA labeling, catalogue no. 18428-011) to a specific activity equal to or greater than 109 cpm/µg. Genomic DNA
extraction and Southern blot analysis were performed as described
previously.10,30
Establishment of M1 cells that ectopically express the Egr-1
transgene.
Virus was generated from the plasmid forms of retroviral vectors, MSCV
EB neo (as a control) and MSCV EB neo Egr-1, by transfection of the
packaging cell line PA317 using calcium phosphate-DNA
precipitation.36 Transfected PA317 cells were selected
using 800 µg/mL G418 (Geneticin; 400 µg/mL; GIBCO-BRL) in growth
medium. Several clones were expanded and the viral titer of the
supernatants (viral conditioned medium [VCM]) was determined to be
0.8 × 105/mL by infecting NIH 3T3
cells.37 The VCM was passed through a 0.4-µm
filter and immediately used to infect M1 cells. Infection of M1 cells
was accomplished by resuspending pellet of cells (0.5 × 106 cells) in 4 mL of VCM in the presence of 8 mg/mL
polybrene for 4 hours. After infection, the cells were washed once with
growth medium and incubated for 48 hours. For neomycin-resistant colony selection, infected cells were seeded at 100 cells/mL in growth media
containing G418, and 1-mL aliquots were dispensed into 24-well trays.
After 10 to 15 days, cultures from wells containing surviving cells
were expanded. The infectants were maintained in growth media
containing 200 µg/mL of G418. Four independent clones with different
integration sites were detected using Southern blot analysis as
described previously10,34 and used throughout the study.
All experiments in this study were initiated with nonadherent M1Egr-1
cells, and the results of all experiments represent the mean of at
least three independent determinations, with standard deviations
indicated in the appropriate figure legend.
Assays for differentiation-associated properties.
Morphological differentiation was determined by counting at least 300 cells on May-Grunwald-Giemsa-stained cytospin smears and scoring the
proportion of immature blast cells, cells at intermediate stages of
differentiation, and mature macrophages.7,9 Immature blast
cells are characterized by scant cytoplasm and round or oval nuclei;
cells at intermediate monocyte stages of differentiation are flattened,
with a larger cytoplasm to nucleus ratio, and contain irregularly
shaped nuclei and few interspersed or no vacuoles; mature
macrophage-like cells are flattened; and spread out cells are
interspersed with numerous vacuoles in a greatly enlarged cytoplasm. Fc
and C3 receptor assays7 and cell adherence were determined
as previously described.9 Agar colony assays were performed
as previously described.33 Colonies in soft agar were examined after 7 days and scored after 14 days.
RNA extraction, Northern blotting, and hybridization.
RNA was extracted using Trizol (GIBCO-BRL) reagent according to the
manufacturer's specifications. Total RNA (10 µg/lane, equal amounts
of RNA in each lane were confirmed by equal intensity of ethidium
bromide staining of ribosomal RNA bands) was electrophoresed on 1%
agarose formaldehyde gels. Northern blots, using Duralon-UV membranes
(Stratagene, La Jolla, CA), were prepared and UV
cross-linked (Stratalinker; Stratagene) before baking at 60°C under
vacuum for 2 hours. Blots were hybridized in 50% deionized formamide, 10% dextran sulfate, 1 mol/L NaCl, 1% sodium dodecyl sulfate (SDS), and 100 µg/mL sheared salmon sperm DNA at 42°C with
106 cpm/mL of probe for 12 to 16 hours. Blots were washed
at room temperature twice for 5 minutes in 2× SSC, 0.1% SDS and
at 60°C twice for 30 minutes in 0.1× SSC, 1% SDS and exposed
to x-ray film at Reverse transcriptase-polymerase chain reaction
(RT-PCR)8.
Primers for murine IL-6 and gp130 genes were selected with the aid of
the program PCRPLAN (PCGENE; Intelligenetics Inc, Mountain View,
CA). The primers corresponded to bases 1946 to 1966 (5 Establishment of M1Egr-1 cells ectopically expressing the Egr-1
transgene.
M1Egr-1 and M1neo cell lines were established via infection of M1 cells
with the retrovirus derived expression vectors MSCV EB neoEgr-1 and
MSCV EB neo, as described in Materials and Methods. As shown in
Fig 1, the M1Egr-1 clones expressed
exogenous Egr-1 transcripts, whereas parental M1 cells or M1 cells
infected with the MSCV EB neo vector carrying the selectable neo marker
did not. Southern blot analysis of M1Egr-1 cell lines confirmed that each clone was an independent isolate, as evident by the distinct integration sites of the Egr-1 transgene in different M1Egr-1 clones (Fig 1B). Four distinct clones of M1Egr-1 and four clones of
M1neo have been established.
Ectopic expression of Egr-1 in M1 leukemic myeloblasts activates the
macrophage differentiation program.
Ectopic expression of Egr-1 in untreated M1 cells was observed to
markedly inhibit the growth of the cells.
Figure 2A depicts the growth kinetics of
untreated M1, M1neo, and M1Egr-1 cells analyzed in mass culture. M1
cells expressing an exogenous Egr-1 transgene exhibited a
significantly slower growth rate than the control parental M1 and M1neo
cells (Fig 2A). Starting with a culture of nonadherent M1Egr-1 cells,
the cells grew in aggregates and after 3 days about 50% of the cells
adhered to the culture dish and exhibited an elongated morphology,
characteristic of monocytic differentiation
(Table 1 and Fig 2C). Analysis of
morphology by May Grunwald-Giemsa-stained cytospin smears showed that
greater than 90% of the untreated M1Egr-1 cells differentiated into
either intermediate or mature macrophages, with fewer than 10%
retaining blast morphology (Table 1). In contrast, none of the
uninduced M1 or M1neo clones grew in aggregates, adhered to the culture plate, or exhibited the morphology of differentiated cell types (Table
1 and Fig 2C). It should be noted that, while establishing the M1Egr-1
cell lines, some of the clones could not be expanded in culture,
because most of the cell population underwent differentiation.
Ectopic expression of Egr-1 increases the propensity of M1 cells to
be induced for macrophage differentiation by IL-6.
The growth response of M1, M1neo, and M1Egr-1 cells after treatment
with varying concentrations of IL-6 for 3 days is depicted in Fig 2B.
It can be seen that low concentrations of IL-6 (up to 1 ng/mL) did not
inhibit and even somewhat stimulated the proliferation of control M1
and M1neo cell lines; in contrast, proliferation of M1Egr-1 cell
lines, which was markedly inhibited in the absence of IL-6, was further
suppressed (compare Fig 2A and B). Consistent with the effect of low
concentrations of IL-6 on growth inhibition of M1Egr-1, low
concentrations of IL-6 (1 ng/mL) further increased the percentage of
M1Egr-1 cells that adhered to the culture dish compared with untreated
cells (60% to 65% compared with 52% to 57%), whereas at most 10%
of M1 and M1neo cell lines were adherent (Table 1). Analysis of cell
morphology showed that, at low concentrations of IL-6 (1 ng/mL),
control M1 and M1neo cells retained predominantly blast-like morphology
(94% to 96%). In sharp contrast, low concentrations of IL-6
further increased the percentage of M1Egr-1 cells that differentiated
into mature cell types compared with untreated cells (65% to 69%
compared with 33% to 39%) and decreased the percentage of cells at
intermediate stages of differentiation, with not more than 5% of the
cells retaining the blast morphology (Table 1). These data are
consistent with the notion that ectopic expression of Egr-1, in
addition to allowing M1 cells to undergo differentiation in the absence
of any exogenous stimuli, also renders M1 cells responsive to low
levels of IL-6 that have no or a minimal effect on parental M1 or M1neo
cells.
Ectopic expression of Egr-1 decreases the leukemogenicity of M1
cells.
M1 cells are leukemogenic when injected into syngeneic or nude mice,
and their leukemogenicity is lost after induction of differentiation in
vitro or in vivo.33,43 It has been shown that constitutive
expression of Egr-1 in M1 cells activated the monocytic differentiation
program, in which a substantial portion of the cells underwent
differentiation in the absence of external stimuli, reduced the growth
rate, and allowed the cells to be highly responsive to low levels of
the differentiation inducer IL-6. Therefore, to better understand the
relationship between these acquired traits in vitro and leukemogenicity
in vivo, the leukemogenicity of the M1Egr-1 cells was compared with the
parental M1 cells. As shown in Fig 5, 12 control nude mice injected with PBS survived for the observed 12 weeks,
whereas all nude mice that were injected with 104 M1 cells
died within 7 weeks. In contrast, only 2 of 12 nude mice injected with
the same number of M1Egr-1 cells died within this time period. Even
when the nude mice were injected with 105 M1Egr-1 cells,
only 4 of 12 animals died during the 7-week time period, in which the
surviving nude mice showed no sign of leukemogenicity. Also, 5 weeks
after injection, no myeloid leukemic cells were recovered from bone
marrow of animals injected with M1Egr-1 cells, as determined by growth
autonomy and differentiation characteristics.8 In contrast,
during the same period of time, leukemic myeloid cells were recovered
from the bone marrow of nude mice injected with M1 cells. Thus,
constitutive expression of Egr-1 decreased the leukemogenicity of the
cells in vivo.
Effect of deregulated Egr-1 on the expression of IL-6 and its
receptor subunit gp130.
Multiple cytokine- and second messenger-responsive elements have been
located within the 5 Egr-1 was found by us to be a macrophage differentiation primary
response gene that is essential for and restricts differentiation along
the macrophage lineage.10 More recently, it has been shown that Egr-1 potentiates macrophage differentiation of the hematopoietic precursor cell line 32Dcl3.30 In the present work, it is
shown that ectopic expression of Egr-1 in the myeloblastic leukemic cell line M1 resulted in activation of the macrophage differentiation program, rendered M1 cells responsive to low levels of IL-6, allowed a
higher proportion of M1 cells to become terminally differentiated under
conditions of optimal stimulation for differentiation, and decreased
the leukemogenicity of M1 cells.
Submitted December 3, 1997;
accepted April 17, 1998.
The authors thank Dr Arthur G. Balliet for his critical evaluation and
comments.
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Abstract
Introduction
Abstract
-GCGGGGGCG-3
80°C for 48 to 72 hours. Stripping blots of
probe to rehybridize was performed as described
previously.
-actin transcripts were amplified using murine
) PBS; (
) M1 (104 cells); (
)
M1Egr-1 (104 cells); (
) M1Egr-1 (105
cells).