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Division of Medical Oncology and Hematology, Departments of Internal Medicine [X. H., A. B. F., K. S. Z.], Biochemistry and Molecular Biology [K. S. Z.], and Pathology [L. C. M., N. I. V., B. J. H.], University of South Florida, and H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612
Abstract
The role of the mitogen-activated protein kinase (MAPK) signal
transduction pathway in the proliferation of mammalian cells has been
well established. However, there are relatively few reports concerning
cell differentiation being mediated by MAPK. The effect of phorbol
12-myristate 13-acetate (PMA) on cell differentiation and signal
transduction in a human myeloid leukemia cell line, TF-1a, was
investigated. When TF-1a cells were treated with 10-6,
10-7, 10-8, and 10-9
M PMA for 24 h, they underwent 98, 93, 91, and 51%
macrophage-like differentiation, respectively. PMA treatment rapidly
(10 min) induced phosphorylation of MAPK kinase (MEK and p44/42 MAPK),
which persisted for at least 24 h. p44/42 MAPK immunoprecipitates
from lysates of PMA-treated cells had increased ability to
phosphorylate the transcription factor Elk-1. This is important because
phosphorylated Elk-1 can be considered an "end-product" of the MAPK
pathway. In contrast, treatment of TF-1a cells with
granulocyte/macrophage-colony stimulating factor induced only transient
activation of MEK and p44/42 MAPK (10–20 min) and an increase
(
50%) in cell proliferation, without any change in cellular
differentiation. These results suggest that macrophage-like
differentiation may be dependent on prolonged activation of the MAPK
pathway. Additional support for this conclusion was obtained from
experiments showing that treatment of TF-1a cells with antisense
oligonucleotides for MEK1 coding sequences prior to adding PMA
inhibited macrophage-like differentiation. Furthermore, transient
transfection with an inactive, dominant-negative MEK mutant also
inhibited PMA-induced differentiation, whereas transient transfection
with a plasmid coding for constitutively activated MEK led to
macrophage-like differentiation in the absence of PMA.
Introduction
Activation of the MAPK3 pathway after ligand binding to various receptors has been correlated with numerous cellular responses, including proliferation, differentiation, and regulation of specific metabolic pathways in different cell types. It is now well established that MAPK is a key regulator of cell proliferation (1, 2, 3) . Studies from fibroblasts and hematopoietic cells suggest that the activation of Ras, Raf, MEK, and MAPK appears to be a linear pathway that is stimulated by growth factor receptors and leads to cell proliferation (4, 5, 6, 7) . The 44 kDa MAPK (ERK1) and 42 kDa MAPK (ERK2) are phosphorylated and activated by highly specific MEK1 and MEK2 (8, 9, 10, 11, 12) . It has been argued that it is the duration of MAPK activation that determines whether a stimulus induces proliferation or differentiation (2 , 13) . Thus far, MAPK-dependent differentiation has been demonstrated in only two cell types. Studies of rat PC 12 neuroblastoma cells (14, 15, 16) suggest that cell differentiation requires prolonged activation of MAPK (lasting hours to days), in contrast to transient activation of MAPK occurring in response to proliferative signals. Megakaryocytic differentiation in the K562 cell line, obtained from a patient with an acute transformation of chronic myeloid leukemia, is also associated with activation of MAPK (17) . In K562 cells, active MAPK remained elevated for 2 h and returned to near-basal level by 24 h in response to PMA. However, megakaryocytic differentiation in the human erythroid/megakaryocytic cell line, HEL, appears to be dependent on PKC activation (18) . Since PKC has been reported to direct phosphorylation of Raf (19) , which is upstream of MEK, it is possible that PKC-induced differentiation may be through the activation of the MAPK pathway. However, direct evidence of cell differentiation induced by the PKC-MAPK pathway has not been reported. A recent report shows that activation of PKC is associated with the MEK kinase 1/stress-activated protein kinase pathway (20) . BCL-XL has also been proposed to be involved in human myeloid cell differentiation (21) . Thus, the role of MAPK in the control of cell differentiation, particularly in hematopoietic cells, is not well established. Further evidence is needed to reveal whether prolonged activation of MAPK is required for cell differentiation. In the study reported here, we investigated the induction of macrophage-like differentiation and its possible linkage with prolonged activation of the MAPK pathway in TF-1a myeloid leukemia cells (22) . TF-1a is a factor-independent cell line derived from the human factor-dependent erythroid leukemia TF-1 cell line (23) . The TF-1 cell line was isolated from a patient with erythroleukemia, which is dependent on GM-CSF or IL-3 for its growth in vitro. Erythropoietin also sustains the short-term growth of TF-1 cells but does not induce erythroid differentiation. TF-1 cells can be induced to differentiate into two different pathways, dependent on the type of an inducer. Hemin and delta-aminolevulinic acid induces erythroid differentiation with hemoglobin synthesis in TF-1 cells, whereas PMA brings about dramatic macrophage-like differentiation of the cells (23) . The TF-1a cells retain the ability to increase their proliferation in response to GM-CSF and IL-3 (22) . Compared with its parent TF-1 cells, this subline can survive longer in serum-free medium and has the ability to resist apoptosis as it exits from the cell cycle and undergoes differentiation. These characteristics of TF-1a cells enabled us to study the kinetics of MAPK activation over a relatively long period of time. To determine the role of the MAPK pathway in macrophage-like differentiation of TF-1a cells, we investigated the ability of MEK1 antisense oligonucleotides and dominant-negative MEK1 to block PMA-induced differentiation and the ability of a constitutively form of MEK1 to induce macrophage-like differentiation of TF-1a cells. These results suggest that prolonged activation of the MAPK pathway is necessary and possibly sufficient for macrophage-like differentiation of TF-1a cells.
Results
Macrophage-like Differentiation of TF-1a Cells.
TF-1a and parental TF-1 cells appear to be cytologically identical,
consisting of a relatively homogeneous population of medium-size cells
with the appearance of blasts. They contain moderate amounts of dark
basophilic, agranular cytoplasm with frequent small cytoplasmic
vacuoles. They have a smooth cytoplasmic border. The nuclei are oval
with fine chromatin and 1–3 macronucleoli. Many binucleated and
occasional large multinucleated forms are present (Fig. 1A)
. The addition of PMA to the cells results in typical macrophage-like
changes, characterized as a decrease in nuclear size and an increase in
the degree of nuclear chromatin condensation. The majority of the cells
have moderately abundant light basophilic, agranular to finely granular
cytoplasm with irregular cytoplasmic borders (Fig. 1B)
. CD14
remained undetectable. These results are in agreement with reports
published previously from the study on TF-1 cells (23)
.
Morphological differentiation is dose dependent. At least 50% of TF-1a
cells had macrophage-like alterations in response to 10-6
to 10-9 M PMA. Compared with TF-1 cells, TF-1a
showed a higher sensitivity to PMA treatment (Table 1)
.
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, proliferating TF-1a cells expressed very limited
IL-1ß, and the addition of PMA markedly increased the IL-1ß levels.
This effect is dose dependent, with maximal expression of IL-1ß being
detected at a concentration of 10-6 M PMA. A
similar increase in the level of IL-1ß was also observed in TF-1
cells in response to PMA treatment.
Prolonged Activation of MAPK Pathway by PMA versus
Transient Activation of the Signal by GM-CSF.
To determine whether the macrophage-like differentiation is correlated
with MAPK pathway activation, the expression of phosphorylated MEK and
MAPK in response to PMA was first investigated. The cells were also
exposed to GM-CSF, which has been reported to be a potent stimulator of
p44/42 MAPK phosphorylation (7)
. Western blot analysis was
performed, using an antiphosphorylated MAPK antibody. Fig. 2
illustrates that both GM-CSF (10 ng/ml) and PMA (10-7
M) rapidly induced phosphorylation of p44/42 MAPK after
exposure to GM-CSF or PMA for 5 min. Since the signal duration has been
linked to the decision to undergo either proliferation or
differentiation, the time course of the phosphorylation of p44/42 MAPK
and MEK was examined. As shown in Fig. 3A
, phosphorylation of p44/42 MAPK induced by PMA was rapid and persistent.
Peak activation occurs at 3 h after PMA treatment, after which the
signal slightly decreases but remains at a high level up to 36 h,
as compared with control cells. The kinetics of MEK phosphorylation
nearly parallel those seen with p44/42 MAPK. (Fig. 3A)
. PMA
had no effect on levels of total MAPK and MEK protein, because the
levels of these two proteins detected by anti-MAPK and MEK antibodies
remained constant at all time points tested up to 36 h after
initiating PMA treatment (Fig. 3A)
. To determine the
specificity of PMA-induced phosphorylation of MEK, PD098059, an
inhibitor of MEK activity (28)
, was added to cells 1
h before the addition of PMA. After another 1 h of culture in the
presence of PMA at 37°C, the cells were collected, lysed, and
subjected to Western blotting for detection of MEK phosphorylation. PMA
induced phosphorylation of MEK because preincubation of cells with
PD098059 at a concentration of 40 µM or more completely
suppressed the activation of MEK (Fig. 3B)
. The PMA-induced
activation of the MAPK pathway is dose dependent, with maximum
activation being observed at a concentration of 10-6
M PMA. Low concentrations of PMA (<10-10
M) did not induce phosphorylation of p44/42 MAPK or MEK
(Fig. 3C)
. Western blot experiments using the nuclear
extracts made from TF-1a cells showed that PMA rapidly induced p44/42
MAPK (Fig. 3D)
but not MEK (data not shown) nuclear
translocation (see next section). PMA had no effect on the
proliferation of TF-1a cells, as measured by BrdUrd incorporation (Fig. 4)
or MTT (data not shown) assays. In contrast, GM-CSF only induced a
transient phosphorylation of MAPK and MEK. The peak signal induced by
GM-CSF was at 10 min, which then returned to basal level within 2
h (Fig. 4B)
. GM-CSF is a major growth factor capable of
stimulating the proliferation of several factor-dependent leukemic cell
lines, such as TF-1 (23)
and megakaryoblastic Mo7e cell
lines (29)
. GM-CSF can also increase the proliferation
rate of several factor-independent cell lines (22
, 30)
.
The addition of GM-CSF increased proliferation of TF-1a cells by
60% (P < 0.01; Fig. 4A
) but failed to
induce macrophage-like differentiation in the cells (data not shown).
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, it is clear that phosphorylated p44/42 MAPK was present
at a low (basal) level in the nuclei of TF-1a cells grown in the
absence of PMA, whereas the level of phosphorylated p44/42 MAPK rapidly
increased (within 15 min) in the nuclei of the cells treated with PMA.
Again, the translocation was persistent and remained up to 6 h,
after which the signal decreased and returned to basal level by 24
h. To directly observe nuclear translocation of MAPK, an indirect
immunofluorescence assay was carried out. TF-1a cells were treated with
10-7 M PMA for various times, after which the
cells were incubated with antibody specific for phosphorylated MAPK and
FITC-labeled secondary antibody, and translocation of MAPK was
visualized on the images captured by an Axiovezt 100 M Zeiss
microscope. As shown in Fig. 5A
, a low level of phosphorylated p44/42 MAPK is present in untreated
cells, and this was primarily in the cytoplasm. This appears to
represent a basal level (22)
. After 10 min of PMA
treatment, the MAPK started to migrate into nuclei (Fig. 5B)
, and the level of phosphorylated MAPK in the nuclei was
markedly increased at 1 and 3 h (Fig. 5, C and D)
. By 6 h, the nuclear staining was significantly
decreased (Fig. 5E)
, and most of this staining reappeared in
the cytoplasm 24 h after treatment (Fig. 5F)
. These
kinetics were consistent with the results obtained from Western
blotting analysis of nuclear extracts described above.
|
. The levels of phosphorylation of Elk-1 after treatment with PMA for 10
min to 24 h were approximately the same. The phosphorylation was
dose dependent within the range of 10-9 to
10-6 M PMA (Fig. 6, bottom panel)
.
|
, PMA induced macrophage-like differentiation in 98% of cells by
24 h; however, the degree of differentiation decreased
dramatically to 16% of cells after the addition of the MEK1 antisense
oligonucleotides. The sense or nonsense oligonucleotides had no
significant effect on blocking cell differentiation. Of note, TF-1a
cells treated with antisense oligonucleotides exhibited nuclear
budding, which was not present in the original control cells (Fig. 7)
. The inhibition of differentiation by MEK-1 antisense oligonucleotides
appeared to correlate with the inhibition of the MAPK pathway (Fig. 8)
, because they significantly suppressed both MEK and p44/42 MAPK
phosphorylation (60–70% at 12 and 24 h, respectively),
whereas the sense oligonucleotides did not inhibit the phosphorylation
of these substrates. To confirm that the antisense
oligonucleotide-induced blocking effect was not from nonspecific
cytotoxicity, cell viability was examined by trypan blue staining. The
cells treated with sense, antisense, or no oligonucleotides had similar
viability (>90%) after 12 and 24 h of incubation.
|
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. These results demonstrated that neither the plasmid alone or WT MEK
interfered with the effect of PMA on cell differentiation. However,
transfection with a dominant-negative MEK (8E) greatly diminished the
efficiency of PMA-induced differentiation, with only 32%
macrophage-like differentiation (a decrease of 50%;
P < 0.05 compared with the cells treated with PMA). In
contrast, the constitutively active mutant (MEK1-R4F) induced
macrophage-like differentiation from a basal level of 1–2% to a
MEK1-R4F-stimulated level of 35% in the absence of PMA
(P < 0.01; compared with that of PCEP4+, PMA-).
Constitutively active MEK at least partially mimics the morphological
response of the cells to PMA.
|
Despite the recent advances in understanding signaling pathways, the role of the MAPK pathway in mediating cell differentiation has not been well established, and many gaps remain to be filled. The fact that MAPK becomes activated after mitogenic or nonmitogenic stimulation does not prove that it is necessary for proliferation or differentiation. In this study, data obtained suggest that prolonged MAPK is required for macrophage-like differentiation of human myeloid leukemic TF-1a cells. The results reported here demonstrate that PMA induced macrophage-like differentiation of TF-1a cells, characterized by a decrease in nuclear size, an increase in the amount of nuclear chromatin condensation, absence of nucleoli, increased cytoplasm, and up-regulation of IL-1ß. PMA rapidly induced phosphorylation of MEK and MAPK. These phosphorylations persisted for at least 24 h, which suggests that the MAPK pathway might play an important role in the control of differentiation. To test this hypothesis, a number of approaches were used to delineate the possible contribution of the MAPK pathway to TF-1a cell macrophage-like differentiation. We examined whether MAPK was translocated into the nucleus after the addition of PMA, because this reagent is not able to induce p44mapk nuclear translocation in fibroblasts (31) . By examining the activity of p44/42 MAPK in nuclear extracts and the presence of p44/42 MAPK in nuclei by immunofluorescence, we demonstrated that MAPK is primarily cytoplasmic before stimulation. After PMA treatment, MAPK rapidly translocated into the nucleus. This translocation persisted, at least for 6 h, after which the signal declined to the basal level. This finding is consistent with previous studies in K562 cells (17) . Because phosphorylated MAPK-specific antibody was used to visualize translocated MAPK, the translocated MAPK observed was phosphorylated and, therefore, activated. This raises the possibility that at least some of nuclear transcription factors such as c-Jun (34 , 35) , c-myc (36) , p62 (37) , and Elk-1 (38) that are known to be activated by phosphorylation might be activated by MAPK. The duration of activation of MAPK that was observed in these studies should be sufficient for MAPK to activate a nuclear substrate. To determine this, Elk-1 was selected as a test substrate for MAPK function. Elk-1 is a member of the Ets family of transcription factors that has been reported to be an important physiological substrate of ERK, mediating serum-induced expression of immediate-early genes and resulting neuronal differentiation of PC12 cells (39) . Our results showed that immunoprecipitates of p44/42 MAPK from all time periods after treatment with PMA phosphorylated Elk-1, and that the levels of phosphorylation of Elk-1 from 10 min to 24 h were approximately the same.
The direct role of the prolonged activation of the MAPK pathway in
TF-1a cell differentiation was demonstrated by using MEK antisense
oligonucleotides. The levels of the MEK and p44/42 MAPK were found to
be similarly suppressed by the MEK antisense oligonucleotides (Fig. 8)
,
which is consistent with the role of the MEK as a regulator working
upstream of p44/42 MAPK. At a concentration of 7.5 µM of
the antisense oligonucleotides, we obtained a 60–70% reduction in
the level of p44/42 MAPK and MEK, respectively. Under these conditions,
PMA-induced macrophage-like differentiation of TF-1a cells was greatly
diminished (80%) by the antisense oligonucleotides (Table 2)
.
Neither the sense nor nonsense oligonucleotides had any significant
effect on PMA-induced differentiation.
If prolonged activation of the MAPK pathway is essential for macrophage-like differentiation, as discussed above, then it would be expected that constitutively active mutants of MEK would induce macrophage differentiation in the absence of PMA and that the presence of a dominant-negative MEK would inhibit the ability of PMA to induce macrophage-like differentiation of TF-1a cells. Therefore, transient transfection with these plasmid cDNAs was carried out. As controls, the vector pCEP4, as well as the WT MEK, were also expressed in these cells. Indeed, the MEK1-dominant negative mutant (8E) was found to inhibit the ability of PMA to induce differentiation. In contrast, the constitutively active MEK mutant induced differentiation in the absence of PMA, although to a lesser degree than that induced by PMA alone. These experiments provide strong evidence that prolonged activation of the MAPK signaling pathway is essential for PMA-induced differentiation of TF-1a cells, and they suggest that prolonged MAPK activation may even be sufficient for inducing the macrophage-like differentiation.
It has been argued that it is the duration of MAPK activation that determines whether proliferation or differentiation will happen after stimulation (2 , 14) . Studies with PC12 cells suggest that prolonged activation of ERK leads to nuclear translocation, which has been proposed to explain the correlation between the kinetics of ERK activation and that of differentiation versus proliferation responses. In K562 cells (17) , the time of expression of phosphorylated MAPK is somewhat shorter than in PC12 cells, with a peak at 2 h after addition of an inducer and return to basal levels by 24 h. In TF-1a cells, phosphorylation of MAPK and MEK was more prolonged, lasting up to 36 h after initiating PMA treatment. In contrast, GM-CSF, a well-known proliferation stimulator for human hematopoietic cells including TF-1 and TF-1a cells, only caused transient phosphorylation of MEK and MAPK. These findings suggest that prolonged activation of MAPK may be important for induction of differentiation. However, there are differences in the duration of phosphorylation/activation of MAPK in the nucleus versus the findings in whole-cell lysates. The shorter time of expression of activated MAPK in the nucleus suggests that not all activated MAPK in the cytoplasm is translocated to the nucleus. It is important to note that the ability of MAPK to activate Elk-1 is stable over at least the first 24 h after PMA treatment. It is possible that some activated MAPK molecules may return to the cytoplasm after they finish their kinase job in the nucleus (as seen from 24 h of fluorescent staining in the nucleus) and maintain an active status, following which these molecules may easily re-enter the nucleus as the level of MAPK in the nucleus decreases. This presumed "shuttle" may be important for maintaining the differentiation process. Thus far, the mechanism of nuclear translocation of phosphorylated MAPK is obscure because MAPK does not seem to possess any canonical nuclear localization signal in its sequence.
In summary, it was shown here that PMA induces prolonged activation of the MAPK pathway, nuclear translocation of phosphorylated MAPK, and macrophage-like differentiation of TF-1a myeloid leukemic cells. By using antisense MEK oligonucleotides and constitutively active and dominant-negative MEK mutants, it was demonstrated that the prolonged activation of MAPK is required for macrophage-like differentiation of TF-1a cells. The data reported here also demonstrate that the time of expression of activated MAPK in the nucleus is different from that in the cytoplasm. Similar observations have been found in KG-1 human myeloid leukemia cell line (Ref. 40 ; data not shown).
Materials and Methods
Reagents.
Recombinant human GM-CSF was purchased from Immunex (Seattle, WA),
recombinant human IL-3 from R&D Systems (Minneapolis, MN), and PMA from
Sigma Chemical Co. (St. Louis, MO). PD098059, polyclonal
antiphosphorylated MEK and MAPK antibody kits, and the MAP Kinase Assay
kit were purchased from New England BioLabs (Beverly, MA). An anti-MAPK
antibody was a gift of Dr. J. Wu, H. Lee Moffitt Research Institute,
Tampa, FL. Anti-IL-1ß antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The kits
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, BrdUrd)
for proliferation assays were purchased from Boehringer Mannheim
(Indianapolis, IN). Several plasmid cDNAs were used in this study. WT
MEK1, catalytically inactive MEK1 (8E) and constitutively active MEK1
(R4F) were kindly provided by Dr. Natalie G Ahn
(17)
. The kit for transient transfection was purchased
from Pharmacia Biotech (Piscataway, NJ). NP40, BSA, and goat antirabbit
FITC-labeled IgG were obtained from Sigma.
Maintenance of TF-1a Cells.
TF-1a (CD34+, CD38-; Ref. 22
) is a factor-independent subline isolated
from the human factor-dependent myeloid leukemic cell line, TF-1
(CD34+, CD38+; Ref. 22
). TF-1a cells were maintained in RPMI 1640
supplemented with 10% FBS at 37°C in humidified air containing 5%
CO2. All the media and sera were purchased from Life
Technologies, Inc. (Gaithersburg, MD).
Cell Cytology and Induction of Differentiation.
Cells were cultured in the medium described above in the presence or
absence of GM-CSF. During log-phase growth, PMA was added to the cells
and incubated for 24–48 h. Cytospins were prepared by spinning
aliquots of harvested cells at 500 rpm for 4 min, after which the cells
were stained with Wright-Giemsa stain, and the level of cell
differentiation was determined by morphological examination under a
light microscope at 10 x 100 magnification.
Assays of Cell Proliferation.
Cell proliferation was examined by directly counting cells with a
hemocytometer and by indirect colorimetric immunoassay (BrdUrd
incorporation). The BrdUrd assay is based on the incorporation of the
pyrimidine analog BrdUrd instead of thymidine into the DNA of
proliferating cells. Briefly, cells were incubated in 96-well
microplates in a final volume of 100 µl/well in the presence or
absence of PMA and GM-CSF (5 ng/ml). After 48 h
incubation, BrdUrd was added to the cells for another 12 h.
Subsequently, the culture medium was removed, and anti-BrdUrd antibody
was added to the cells. The immune complexes were then detected by a
subsequent substrate reaction and measured using an ELISA reader at a
wavelength of 450 nM as recommended by the manufacturer.
Preparation of Cell Lysates and Nuclear Extracts.
Exponentially growing TF-1a cells were washed free of serum and growth
factors and incubated in serum-free RPMI 1640 for 24 h at 37°C
in a humidified atmosphere of air containing 5% CO2.
Before stimulation, TF-1a cells were pelleted and resuspended in
serum-free medium without growth factors. These cells were then exposed
to PMA at 37°C for 0–36 h, after which the cells were washed once
with cold PBS and lysed in 1 x SDS sample buffer (62.5
mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT,
and 0.1% bromphenol blue). Aliquots of each lysate containing the same
cell number were subjected to Western blotting and were probed with
anti-p44/42 MAPK and antiphosphorylated MAPK antibodies. To prepare
nuclear extracts, TF-1a cells (1 x 107) were washed
twice with PBS and once with the PBS containing 1 mM sodium
orthovanadate (Na3VO4) and 5 mM
NaF. Subsequently, the cells were washed with 2 ml of 1x hypotonic
buffer [20 mM HEPES (pH 7.9), 1 mM EDTA, and 1
mM EGTA] and lysed in 1x hypotonic buffer supplemented
with 0.2% NP40 (41)
. Thereafter, the nuclear pellets were
collected by centrifugation at 15,000 x g for 10 min
and resuspended in 50–100 µl of 1x high salt buffer (420
mM NaCl, 20 mM HEPES (pH 7.9), 1 mM
EDTA, 1 mM EGTA, and 20% glycerol), after which they were
incubated at 4°C for 30 min with constant rotation. Subsequently, the
nuclear extracts (the supernatants) were collected by centrifugation
and were stored at -80°C. Protein concentrations of the nuclear
extracts were determined by colorimetric assay using DC protein assay
kit (Bio-Rad, Hercules, CA), following the manufacturer’s instruction.
Western Blotting Analysis.
An aliquot of each lysate was heated at 100°C for 4 min before
SDS-PAGE. Proteins were separated on a 10% SDS-PAGE and
electrophoretically transferred to a nitrocellulose membrane (Amersham,
Arlington Heights, IL). The proteins in the membrane were then
immunoblotted with anti-IL-1ß, antiphosphorylated p44/42 MAPK, or MEK
antibody overnight at 4°C. The first antibody-containing solution was
then removed, and the blot was washed three times in TBST buffer [20
mM Tris, 137 mM NaCl (pH 7.6), and 0.1% Tween
20]. To detect the antibody reaction, the blot was incubated with
horseradish peroxidase-conjugated antirabbit secondary antibody at room
temperature for 1 h, and the product was detected by
chemiluminescence (NewEngland BioLabs, Beverly, MA), as recommended by
the manufacturer.
Immunofluorescent Analysis of Fixed Cells.
TF-1a cells untreated or treated with PMA were plated in six-well
plates for 0–24 h at 37°C in humidified air containing 5%
CO2, after which the cells were collected by centrifugation
and resuspended in PBS. About 5 x 104 cells were
cytospun on slides with a Shandon cytocentrifuge (Pittsburg, PA) for 3
min at 500 rpm and were immediately fixed with 4% paraformaldehyde at
4°C for 20 min. The cross-linking was stopped with 1% glycine in
PBS, and the cells were permeabilized for 2 h by several changes
of PBS containing 1% glycine and 0.5% Triton X-100. Rabbit polyclonal
antibody against phosphorylated MAPK was diluted 1:250 with PBS
containing 0.1% NP40 and 1% BSA and incubated with the cells for
1 h at room temperature. After several washes with PBS for 2
h, the cells on the slides were incubated with 1:125 diluted goat
antirabbit FITC-labeled IgG in 0.1% NP40, 1% BSA in PBS for 25 min at
room temperature. Subsequently, the slides were washed, dried, and
covered with coverslips in Vectashield mounting media of
antifade/4',6-diamidino-2-phenylindole (1:1) (Vector Laboratories,
Inc., Burlingame, CA). Immunofluorescent confocal laser scanning
microscopy was performed with an Axiovert 100 M microscope (Zeiss,
Germany), and images were captured with the LSM 510 programme (Zeiss,
Germany).
Functional MAPK Activity Assay.
Untreated or PMA-treated serum-free TF-1a cells were chilled on ice,
washed with cold PBS, and lysed in cold lysis buffer (20 mM
Tris, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1
mM ß-glycerophosphate, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate,
and 1 µg/ml leupeptin). Cell lysates were centrifuged at 13,000
x g for 10 min at 4°C, and the supernatant fluid
fractions were transferred to fresh tubes. Protein concentrations of
the lysates were determined by colorimetric assay using the DC protein
assay kit, as described in "Preparation of Cell Lysates and Nuclear
Extracts." Lysates containing 200 µg of total protein in 500 µl
were immunoprecipitated by incubating at 4°C overnight with
antiphosphorylated p44/42 MAPK monoclonal antibody. Subsequently,
protein A-Sepharose (Pharmacia Biotech, Inc., Piscataway, NJ) was
added. The immunoprecipitates were washed three times with cell lysis
buffer and twice with kinase buffer (25 mM Tris, 5
mM ß-glycerophosphate, 2 mM DTT, 0.1
mM sodium orthovanade, and 10 mM
MgCl2). The kinase activity assay was performed by
incubating the immunoprecipitates in 50 µl of kinase buffer
supplemented with 200 µM ATP and 2 µg of Elk1 fusion
protein (NewEngland Biolab). After 30 min at 30°C, the reaction was
stopped by adding 30 µl of 3x SDS sample buffer and loaded
onto 10% SDS-PAGE. Phosphorylation of Elk-1 was analyzed by Western
blotting using an antiphosphorylated Elk-1 antibody (NewEngland
Biolab), following the manufacturer’s protocol.
Oligonucleotide Uptake.
Phosphorothioate antisense (5'-GCTTCTTCTTGGGCATCT-3', corresponding to
the start codon of MEK1), sense (5'-AGATGCCCAAGAAGAAGC-3'), and
nonsense (5'-AAATTCGTGGACGTTCGC-3') oligonucleotides were purchased
from Bio-Synthesis, Inc. (Lewisville, TX). The oligonucleotides were
purified by polyacrylamide gel electrophoresis. Briefly, cells were
incubated with the oligonucleotides (5–7.5 µM) described
above for 2 h at 37°C, 5% CO2. Subsequently, the
cells were incubated in the presence or absence of PMA
(10-7 M) for another 24 h, after which
they either were harvested by centrifugation and stained with
Wright-Giemsa for the examination of macrophage-like differentiation or
lysed in 1x sample buffer for Western blot analysis.
Transient Transfection.
TF-1a cells in log phase were washed once with Tris-HCl (pH 7.5) and
resuspended in Tris-HCl at the concentration of 2 x
106/ml. A DEAE-Dextran-DNA mixture containing 5 µg of
plasmid DNA was prepared following the manufacturer’s instructions,
and 200 µl of the mixture were added, drop by drop, to the 1 ml of
cell suspension described above. The cells were then incubated for 15
min at 37°C and rocked every 5 min to spread the solution evenly
across the cells. Then cells were collected by centrifugation, washed
once in Tris-HCl (pH 7.5), and resuspended in complete culture medium.
The next day, PMA was added, and the mixture was incubated for another
24 or 48 h. Subsequently, the cells were harvested and stained
with Wright-Giemsa for the examination of differentiation as described
previously. Transfection efficiencies were measured indirectly by
expression of green fluorescent protein (Clonetech, Palo Alto,
CA) in paralleled transfection assays for each experiment.
Acknowledgments
We gratefully thank Dr. J. Wu for helpful discussions and critical review of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by National
Cancer Institute Grant P30CA76292. 
2 To whom requests for reprints should be
addressed, at Division of Medical Oncology and Hematology, University
of South Florida College of Medicine, H. Lee Moffitt Cancer Center and
Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. Phone: (813)
979-6721; Fax: (813) 979-3893; E-mail: hu{at}moffitt.usf.edu
3 The abbreviations used are: MAPK,
mitogen-activated protein kinase; MEK, MAPK kinase; PKC, protein kinase
C; GM-CSF, granulocyte/macrophage-colony stimulating factor; IL,
interleukin; PMA, phorbol 12-myristate 13-acetate; WT, wild type;
BrdUrd, bromodeoxyuridine.
Received for publication 8/ 9/99. Revision received 11/15/99. Accepted for publication 3/14/00.
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