From the Picower Institute for Medical Research, Manhasset, New York 11030
Received for publication, November 25, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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Macrophage migration inhibitory factor (MIF) has
been shown to functionally inactivate the p53 tumor suppressor and to
inhibit p53-responsive gene expression and apoptosis. To better
understand the role of MIF in cell growth and tumor biology, we
evaluated MIF-null embryonic fibroblasts with respect to their
immortalization and transformation properties. Although minor
deviations in the growth characteristics of MIF Macrophage migration inhibitory factor
(MIF)1 was originally
identified for its ability to inhibit the random migration of macrophages in vitro (1, 2). Subsequent work defined MIF as
a potent cytokine with mitogenic and pro-inflammatory functions (3, 4).
However, efforts aimed toward identifying a cellular receptor for MIF
have been unsuccessful to date; thus, the process by which
extracellular MIF may exert its effect on target cells is not understood.
Early evidence suggesting a role for MIF in cell growth and/or
differentiation came from the observations of its expression in
developing mouse embryos. The MIF gene is expressed at early embryonic
stages prior to implantation (5); and at mid-gestation, its expression
pattern parallels tissue specification and organogenesis (6-8).
However, MIF appears to be dispensable for normal development because
MIF-null mice reproduce and grow normally (9, 10). Among cytokines, MIF
is unique in terms of its abundant expression by various cell types
(4), as well as storage within the cytoplasm (11). A recent study (12)
proposed an intracellular function for MIF in cell cycle regulation via
modulation of the transcription factor AP-1. MIF deficiency has also
been associated with decreased NF- To better understand the role of MIF in cell growth and tumor biology,
we analyzed MIF-null embryonic fibroblasts with respect to their
immortalization and transformation properties. The data presented here
implicate MIF as a mediator of normal and malignant cell growth.
Cells and Tissue Culture--
MIF-knockout mice were kindly
provided by Dr. John R. David (Harvard School of Public Health,
Boston). Animals were maintained on a mixed 129Sv × C57Bl/6
background (F1). Mouse embryonic fibroblasts (MEFs) were
prepared from day 14.5 embryos using standard techniques, and passage
4-6 MEFs were used in most experiments. Unless specified, cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Invitrogen).
Fibroblast proliferation was measured by plating equal cell numbers
onto six-well dishes in duplicate, followed by direct counting of
trypsinized cells using a Coulter counter. Each growth curve represents
at least two independent experiments. As an alternative, cell
proliferation was assessed by [3H]thymidine incorporation
into DNA. Briefly, 3 × 104 cells were plated per well
in 96-well plates in DMEM containing 10% FBS and 0.5 µCi/well
[3H]thymidine (PerkinElmer Life Sciences). Following
48 h of incubation, the cells were harvested, and radioactive
incorporation was determined by
Cell viability was assessed using MEFs that were grown until confluent
and either kept in serum-containing DMEM or placed in serum-free DMEM
for 48 h. Cells were harvested using enzyme-free dissociation
solution (Invitrogen) and analyzed using the TACSTM annexin
V/propidium iodide apoptosis kit (R&D Systems) according to the
manufacturer's protocol.
Retroviral Constructs--
The replication-defective retroviral
expression vector REBNA has been described previously (21). Retroviral
vector REBNA/IRESGFP contains a poliovirus internal ribosomal
entry site element followed by green fluorescent protein cDNA
(kindly provided by Dr. Sergei Zolotukhin, University of Florida,
Gainesville, FL) placed downstream of the multiple-cloning site in
REBNA. Retroviral vector REBNApuro contains a puromycin resistance
cassette and a minimal cytomegalovirus promoter placed upstream of the
multiple-cloning site in REBNA. The following cDNAs were used for
retroviral expression: mouse c-myc (a gift from Dr. Michael
J. Cole, Princeton University); a dominant-negative human
p53H175 mutant (a gift from Dr. Dan Notterman, Princeton
University); SV40 large T-antigen (a gift from Dr. Robert Kalejta,
Princeton University); E1A12S and E1A mutants E1A Transformation Assays--
Soft agar colony formation was
examined in 0.25% Noble agar/DMEM supplemented with 10% FBS
and antibiotic/antimycotic (Invitrogen). For focus formation assays,
3 × 103 virus-infected fibroblasts were mixed with
105 uninfected MEFs and plated onto 6-cm dishes. Cells were
maintained in DMEM supplemented with 5% FBS and
antibiotic/antimycotic. The growth medium was changed every 3 days. In
12-14 days, transformation efficiency was evaluated by counting
individual colonies. All transformation assays were repeated at least
three times. Data are shown as the average number ± S.D. of
foci/plate using triplicate plates. Representative plates were stained
with Giemsa and photographed.
Protein Expression Analysis--
Aliquots of whole-cell lysates
(50-100 µg of protein) were separated on an 8% SDS-acrylamide gel
and blotted onto a Protran BA83 nitrocellulose membrane (Schleicher & Schüll). They were then incubated with antibodies specific for
p16 (M-156), p21 (C-19), p53 (FL-393), p107 (C-18), cyclin A (C-19),
cyclin D1 (C-20), cyclin E (M-20), CDC6 (180.2), E2F1 (KH-95), E1A
(135-5), B-Myb (N-19), and c-Myc (N-262) (Santa Cruz Biotechnology);
CDK2 (C18520), CDK4 (C18720), and Ras (R02120) (Transduction
Laboratories); p19ARF (ab80; Novus Biologicals); MAPK (3A7;
Cell Signaling), Rb (554136; Pharmingen); and SV40 T-antigen (Ab-2;
Calbiochem) to detect the respective proteins. Secondary antibodies
were horseradish peroxidase-conjugated (Amersham Biosciences). The ECL
protein detection system (PerkinElmer Life Sciences) was used as
recommended by the manufacturer. Nuclear cell extracts were prepared
using the NE-PER nuclear extraction reagent (Pierce).
Cell Cycle Analysis--
Trypsinized MEFs were washed with
phosphate-buffered saline and fixed in 70% ethanol on ice for at least
24 h. The cells were washed with phosphate-buffered saline
containing 1% FBS, resuspended in phosphate-buffered saline containing
1% FBS and 25 µg/ml RNase A, and incubated for 30 min at 37 °C.
Prior to flow cytometry, 100 µl of propidium iodide solution (50 µg/ml propidium iodide and 0.1% sodium citrate) was added to the
cell suspension and incubated for 1 h on ice. The staining was
assessed using the FACSCalibur flow cytometer and CellQuest
software following calibration using DNA QCTM beads (BD
Biosciences). Data were analyzed using ModFit software (BD Biosciences).
Growth Properties of MIF
To test the possibility that the observed growth differences in cells
lacking MIF were related to p53 function, we investigated whether
interference with p53 activity can alter these effects. For this
purpose, a dominant-negative p53H175 allele was introduced
into fibroblasts using a replication-defective retrovirus. The
functionality of this p53 mutant, including its ability to
interfere with p53-dependent transcription, has been documented in previous studies (22, 23). Contrary to expectations, the
expression of p53H175 failed to reverse the difference in
the growth phenotype between the wild-type and MIF
Because the oncogenic capacity of LT depends in large part on its
ability to block the function of p53 (24), we examined wild-type and
MIF MIF
To exclude the possibility that the observed difference in
transformation between wild-type and MIF Viral Oncogenes Cooperate in the Transformation of
MIF
MIF Myc Induces a Growth Inhibitory Response in
MIF
The growth properties of MIF E2F Contributes to the Suppression of the ras-induced Transformed
Phenotype in MIF Delayed Induction of E2F Target Genes in MIF
The delay in E2F-responsive gene expression observed in
MIF Interference with E2F DNA-binding Activity Overcomes the Effect of
MIF Deficiency on ras-mediated Transformation--
Because
activated Ras can increase E2F1 protein levels through both
Rb-dependent and Rb-independent mechanisms (29, 41, 42), we
examined whether interference with the E2F DNA-binding activity can
affect ras-mediated transformation in the absence of
exogenous Myc expression. Several attempts to express a DNA binding-defective E2F1E132 mutant in MIF
Next, we established wild-type and MIF We have used a genetic approach to compare MIF-deficient and
wild-type fibroblasts with respect to their growth, immortalization, and transformation properties. Several recent studies suggested a role
for MIF in cell growth regulation through the modulation of the AP-1,
NF- Carcinogenesis is a multistep process involving the activation of
oncogenes and inactivation of tumor suppressor genes (47-49). The
dominant-negative p53H175 allele used in this study has
been well characterized with respect to its ability to cooperate with
the activated ras mutants in transformation of rodent cells
(50, 51). In our experiments, abrogation of p53-dependent
transactivation was sufficient to promote ras-induced
transformation of wild-type MEFs. However, MIF In rodent cells, p53 controls both the anti-immortalizing and
anti-tumorigenic pathways (15). Several studies suggested a role for
the Rb family proteins as the downstream effectors of p53 action
(52-54), whereas E2F1, a downstream target of Rb regulation, has been
identified as a major cellular regulator of p53 (32, 34, 55). The
E2F1374 mutant utilized in this study interferes with both
E2F-dependent transactivation (35, 36) and recruitment of
the Rb-E2F repressor complexes to the corresponding promoters (37). The
incorporation of E2F1374 into either wild-type or
MIF E2F1 is the only E2F family member that so far has displayed properties
of both a tumor suppressor and an oncogene in different model systems.
Thus, E2f1 loss has been found to reduce tumor development
in Rb+/ Given the complexity of the E2F-regulated function, we can only
speculate as to how MIF deficiency might influence its
tumor-suppressive properties. Our data suggest that the binding of E2F1
to Rb, although important for the stability of Rb-E2F repressor
complexes during normal cell cycle progression, probably cannot account
for the E2F-mediated growth inhibition in MIF It has been well documented that cancers frequently accumulate
mutations that impair the p53 pathway (47). Our results demonstrate that MIF plays a role in the generation of the transformed phenotype and implicate MIF as a mediator of malignant cell growth. Of note, the
MIF gene is located on chromosomes 10 (mouse) and 22 (human) and does
not overlap with other transcriptionally active
loci.2 The probability is low
that the observed effects on cell growth and oncogenic transformation
were inconsequent of MIF loss, but rather due to interference with
other genes essential for the proper function of either the p53 or
Rb/E2F pathway. Additional experiments including the analysis of
compound mouse mutants may clarify the remaining questions regarding
the mechanism of MIF involvement in the modulation of p53-specific
cellular response.
/
fibroblasts were observed under normal culture conditions,
MIF-deficient cells were growth-impaired following the introduction of
immortalizing oncogenes. The growth retardation by the immortalized
MIF
/
cultures correlated with their reduced
susceptibility to Ras-mediated transformation. Our results identify E2F
as part of the restraining mechanism that is activated in response to
oncogenic signaling and show that the biological consequences of E2F
induction in MIF
/
fibroblasts vary depending on the p53
status, inducing predominantly G1 arrest or apoptosis in
p53-positive cells. This E2F activity is independent of Rb binding, but
contingent on binding DNA. Resistance to oncogenic
transformation by MIF
/
cells could be overcome by
concomitant interference with p53- and E2F-responsive
transcriptional control. Our results demonstrate that MIF plays a role
in an E2F/p53 pathway that operates downstream of Rb regulation and
implicate MIF as a mediator of normal and malignant cell growth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activity (13), whereas Hudson
et al. (14) demonstrated that MIF functionally inactivates
the p53 tumor suppressor. The p53 gene has become the focus of intense
investigation since it became clear that p53 mutations are the most
common genetic alteration found in human tumors (15). However, in a
proportion of tumors in which p53 is functionally inactivated, the gene
remains intact, suggesting altered activity of p53-specific regulators
(16, 17). Coincidentally, MIF overexpression has been reported in various tumors (18-20). Immunostaining of brain tumors revealed predominantly nuclear localization (20), raising the possibility that
MIF overexpression may contribute to tumor proliferation via p53 inhibition.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-scintillation counting (Packard
Instrument Co.). The data are reported as the average cpm ± S.D.
(n = eight wells/condition). Two-tailed
Student's t test was used to determine the statistical significance.
N and E1A
CR1,
which contain deletions of amino acids 2-36 and 68-85, respectively
(a gift from Dr. Scott Lowe, Cold Spring Harbor Laboratory); a
dominant-negative human E2F1374 mutant (a gift from Dr.
Steven F. Dowdy, Washington University School of Medicine, St. Louis,
MO); and a human H-rasV12 mutant (a gift from
Dr. Dafna Bar-Sagi, State University of New York, Stony Brook).
Retroviral stocks were produced as previously described (21).
For viral infections, 105 MEFs plated onto a 6-cm dish were
incubated overnight with an appropriate amount of the corresponding
retroviral stock. Multiple infections were performed sequentially, with
a 12-24-h interval between each infection. When required, infected
fibroblasts were selected for 2 days in medium containing 2 µg/ml
puromycin. Typically, the efficiency of infection of primary MEFs
ranged from 60 to 70%, whereas the efficiency of infection of
immortalized cells was >95% as determined using retroviral constructs
containing the green fluorescent protein marker. Cells were analyzed
for the corresponding protein levels 2-4 days post-infection.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Fibroblasts--
To
examine the role of MIF in cell growth, MEFs were derived from
MIF-knockout and wild-type mice and assayed in several experimental settings. Under normal growth conditions, MIF
/
fibroblasts proliferated slightly more slowly than wild-type MEFs and
became contact-inhibited at cell densities that were 20-25% lower
than those of the wild-type controls (Fig.
1A). Immunoblot analysis
showed that these differences were not associated with alterations in
the expression levels of p53 (Fig. 1B). Moreover, following
infection of the cells with retroviruses encoding viral or cellular
oncogenes, such as SV40 large T-antigen (LT), adenoviral E1A,
and c-myc, the induction of p53 in both wild-type and
MIF
/
fibroblasts was similar (Fig. 1B).
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Fig. 1.
Growth properties of
MIF /
fibroblasts. A, growth curves of wild-type (wt)
and MIF
/
(knockout (ko)) fibroblasts before
and after infection with retroviruses expressing dominant-negative
p53H175. B, p53 expression in wild-type and
MIF
/
fibroblasts after infection with a control empty
retroviral vector (cntr) or with retroviruses encoding LT,
dominant-negative p53H175 (dnp53),
myc, or E1A. The film is shown after a short (upper
panel) or a long (lower panel) exposure.
Odd-numbered lanes correspond to wild-type cells, and
even-numbered lanes correspond to MIF
/
fibroblasts. C, growth curves of wild-type and
MIF
/
fibroblasts infected with LT-expressing
retroviruses.
/
fibroblasts (Fig. 1A). The delayed growth rate of
MIF
/
cells was not accounted for by differences in
mutant p53 expression (Fig. 1B).
/
fibroblasts transduced with LT-expressing
retroviruses. As expected, introduction of LT into the wild-type or
MIF
/
fibroblasts produced immortalized cell lines.
However, subsequent examination revealed that LT-expressing
MIF
/
fibroblasts were clearly growth-retarded compared
with the corresponding wild-type controls (Fig. 1C).
Immunoblot analyses showed no variations in the levels of LT expression
(Fig. 2A); LT-induced p53
stabilization (Fig. 1B); or LT binding to Rb, p107, and p130
(data not shown). Although LT has functions outside p53 neutralization,
these results suggest that MIF affects cell growth, in part,
through a p53-independent mechanism.
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Fig. 2.
MIF /
fibroblasts resist Ras-mediated transformation. A,
immunoblot analysis of wild-type and MIF
/
fibroblasts
transduced with LT, dominant-negative p53H175
(dnp53), myc, or E1A, followed by infection with
H-rasV12-expressing retroviruses. Uninfected
wild-type and MIF
/
fibroblasts are shown as controls
(cntr). The expression of ERK1 and ERK2 (MAPK) is shown as a
control for loading. Odd-numbered lanes correspond to
wild-type cells, and even-numbered lanes correspond to
MIF
/
fibroblasts. B, wild-type
(wt) and MIF
/
(knockout (ko))
fibroblasts sequentially infected with retroviruses expressing
p53H175 and H-rasV12. Representative
photographs were taken at the same magnification (×20) after 5 days in
culture. C, focus formation by wild-type (dish 1)
and MIF
/
(dish 2) fibroblasts coexpressing
p53H175 and H-rasV12 or by
MIF
/
fibroblasts that were infected with an empty
retroviral vector (dish 3) or a MIF-expressing retroviral
vector (dish 4), followed by infection with retroviruses
encoding p53H175 and H-rasV12.
Representative plates were stained with Giemsa and photographed.
D, upper panels, immunoblot analysis of
uninfected (control (C)) and p53H175 and
H-rasV12-transduced (R) wild-type and
MIF
/
fibroblasts. Human p53-specific antibody DO-1 was
used to examine the levels of p53H175. Lower
panels, expression levels of inducible transcription factors in
nuclear extracts prepared from uninfected or p53H175 and
H-rasV12-transduced wild-type and
MIF
/
fibroblasts. Cells were examined 4 days
post-infection.
/
Fibroblasts Resist Ras-mediated
Transformation--
Because the deficiency in tumor suppressor genes,
such as p53, facilitates oncogenic transformation, we next determined
whether MEFs expressing p53H175 are susceptible to
transformation. Introduction of the activated ras
(H-rasV12) allele into control wild-type
fibroblasts expressing p53H175 led to their clear
morphological transformation (Fig. 2B) and the loss of
contact inhibition, one of the hallmarks of the tumorigenic state.
Subsequently, these cells produced 50 ± 10 transformed colonies/plate in several independent experiments (Fig. 2C).
In marked contrast, expression of p53H175 and activated
ras in MIF
/
fibroblasts resulted in enlarged
cellular morphology (Fig. 2B) and increased
senescence-associated
-galactosidase expression (data not shown).
Accordingly, these cells remained contact-inhibited and produced only
1.8 ± 1.5 foci/plate, despite containing equivalent levels of the
mutant Ras protein (Fig. 2A) or growth regulatory molecules,
such as ARF and p16 (Fig. 2D). Transactivation mediated by
p53 was inhibited in cells expressing p53H175 and
ras, as evidenced by decreased Apaf-1 and p21 expression (Fig. 2D). However, MIF
/
fibroblasts
accumulated higher levels of the transcription factor E2F1, whereas
higher levels of c-Jun and c-Myc were found in the transformed
wild-type cells (Fig. 2D). It has been noted that a modest
reduction in c-Myc expression dramatically reduces susceptibility to
Ras transformation (25).
/
cells was
independent of MIF loss, we reintroduced the MIF gene into
MIF
/
fibroblasts. Restoration of MIF expression in
populations expressing p53H175 and
H-rasV12 led to efficient focus formation (Fig.
2C). However, we were unable to rescue the transformed
phenotype of MIF
/
fibroblasts using various
concentrations of recombinant murine MIF purified from bacteria or
wild-type MEF-conditioned medium (data not shown). Likewise,
MIF
/
fibroblasts expressing both p53H175
and H-rasV12 failed to produce transformed
colonies when plated on a monolayer of wild-type MEFs. These results
suggest that the effects of MIF on cell growth and transformation are
independent of its proposed role as a secreted cytokine. Furthermore,
our experiments imply that the functional inactivation of p53 is
insufficient to render MIF-deficient cells susceptible to
ras-mediated transformation. Alternatively, a complete
inactivation of p53 requires MIF involvement, even in the presence of
excess interfering mutant p53. To address these possibilities, we
performed the following experiments.
/
Fibroblasts--
To rule out the possibility that
MIF
/
fibroblasts are unable to become oncogenically
transformed, we examined cells expressing LT, whose activity is
associated with the disruption of the p53 and Rb tumor suppressor
pathways (24). Following the introduction of oncogenic ras
into wild-type and MIF
/
fibroblasts expressing LT, both
cell types were readily transformed and invariably produced numerous
foci (Fig. 3A), thus
demonstrating that inactivation of the p53 and Rb tumor suppressors
renders these cells susceptible to transformation. However, cell cycle analysis revealed that the transformed MIF
/
fibroblasts
accumulated in the G2/M phase (Fig. 3B).
Accordingly, when assayed for anchorage-independent growth in soft
agar, LT- and ras-transformed MIF
/
fibroblasts developed colonies with a 2-3-day delay compared with the
wild-type cells (data not shown).
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Fig. 3.
Viral oncogenes cooperate in the
transformation of
MIF /
fibroblasts. A, focus formation by wild-type
(wt) and MIF
/
(knockout (ko))
fibroblasts coexpressing H-rasV12 and LT,
H-rasV12 and E1A, or
H-rasV12 and the E1A
N or E1A
CR1 deletion
mutant. B, flow cytometric analysis of DNA content in
wild-type and MIF
/
fibroblasts expressing LT and
H-rasV12. C, immunoblot analysis of
wild-type and MIF
/
fibroblasts coexpressing
H-rasV12 and E1A or
H-rasV12 and the E1A
N or E1A
CR1 deletion
mutant.
/
fibroblasts coexpressing the E1A and Ras
oncoproteins also showed a 40% lower focus-forming ability
compared with the wild-type cells (Fig. 3A). This correlated
with the observed 20-30% slower proliferation by the transformed
MIF
/
cultures (data not shown). Because fibroblasts
that coexpress E1A and oncogenic ras are prone to
p53-mediated apoptosis in response to various insults, such as contact
inhibition, we next examined cells expressing E1A deletion mutants
E1A
N and E1A
CR1 (Fig. 3C). In previous work, these E1A
mutants showed a differential capacity to affect the functions of
p300/CBP coactivators and displayed a complete and partial defect,
respectively, in apoptosis induction (26-28). Remarkably,
E1A
N-expressing wild-type and MIF
/
fibroblasts
showed similar growth properties and focus-forming abilities when
tested in transformation assays (Fig. 3A). By contrast, E1A
CR1-expressing MIF
/
fibroblasts consistently
produced fewer transformed colonies than the corresponding wild-type
cells (Fig. 3A), thus indicating that p53 is more responsive
to oncogenic activation in MIF
/
cells.
/
Fibroblasts--
To explore the association
between the p53-dependent and p53-independent growth
inhibitory responses resulting from MIF deficiency, we utilized the
c-myc proto-oncogene. Unlike E1A, myc
promotes cell growth bypassing the normal Rb control (29-31). Under
certain conditions, however, overexpression of myc can cause
p53-dependent proliferative arrest or augment
p53-dependent apoptosis (32, 33). Expression of
myc in MIF
/
fibroblasts consistently
produced cell populations that proliferated at lower rates compared
with the corresponding wild-type controls (Fig.
4A) Morphologically,
MIF
/
populations contained many highly condensed cells
(Fig. 4B) and were blocked in the G1 phase
compared with myc-transduced wild-type cells (data not
shown). Although wild-type MEFs generally tolerated higher levels of
the exogenous Myc protein than MIF
/
cells, the
expression levels of the key functional targets of Myc regulation, such
as cyclins D1, cyclin E, CDK2, and p53, were similar in both cell types
(data not shown). Of note, equivalent levels of these proteins were
also found in wild-type and MIF
/
fibroblasts
immortalized by LT and E1A (data not shown). However, combined
expression of myc and p53H175 led to a greater
increase in proliferation by MIF
/
cells compared with
wild-type cells (Fig. 4A). These results are consistent with
the reported ability of MIF to bypass p53-mediated growth arrest
without altering p53 protein expression (14).
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Fig. 4.
c-myc induces a growth
inhibitory response in
MIF /
fibroblasts. A, growth curves of myc-expressing
wild-type (wt) and MIF
/
(knockout
(ko)) fibroblasts before and after infection with
retroviruses encoding p53H175. B, representative
photographs of myc-expressing wild-type and
MIF
/
fibroblasts (magnification ×40).
/
fibroblasts coexpressing
myc and H-rasV12 paralleled those of
MIF
/
cells transduced with myc alone. Thus,
the expression of myc and ras consistently
resulted in fewer transformed foci produced by MIF
/
cells compared with the respective wild-type cells (Fig.
5A). This correlated with
slower proliferation by myc- and ras-expressing MIF
/
fibroblasts than by the wild-type controls (Fig.
5B). In addition, myc- and
ras-transduced MIF
/
cultures contained a
larger percentage of apoptotic and necrotic cells (Fig. 5C).
Moreover, the proportion of live myc- and
ras-expressing MIF
/
fibroblasts rapidly
decreased when shifted to low serum conditions (Fig. 5C).
Immunoblot analysis of myc- and ras-transformed
wild-type and MIF
/
cells showed similar expression
levels for several growth regulatory molecules, including p53 and ARF
(Fig. 5D). However, the levels of pro-apoptotic molecules
(Apaf-1, caspase-3, and caspase-7) were higher in MIF
/
MEFs coexpressing the exogenous Myc and H-RasV12 proteins
(Fig. 5D). Accordingly, sequential introduction of
p53H175, myc, and ras into
MIF
/
MEFs resulted in a 5-fold increase in focus
formation, whereas the corresponding wild-type cells produced a 2-fold
increase (Fig. 5A). Noteworthy, transformation of wild-type
MEFs by coexpressing myc and ras correlated with
a significant up-regulation of MIF protein levels (Fig.
5D).
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Fig. 5.
MIF /
fibroblasts resist transformation by c-myc and
ras. A, focus formation by
myc- and ras-transformed wild-type
(wt) and MIF
/
(knockout (ko))
fibroblasts following ectopic expression of dominant-negative
p53H175 (dnp53) or E2F1374
(dnE2F1). B, proliferation of wild-type and
MIF
/
MEFs expressing myc or
myc plus ras as assessed by
[3H]thymidine incorporation. The data are representative
of three experiments. C, induction of apoptosis in wild-type
or MIF
/
MEFs expressing myc and
ras. Confluent cells were incubated in either
serum-containing or serum-free medium for 2.5 days prior to apoptosis
analyses. All cells (floating and attached) were harvested, stained
using annexin and/or propidium iodide, and examined by flow cytometry.
Live cells were annexin-negative and propidium iodide-negative
cells. D, immunoblot analysis of uninfected wild-type and
MIF
/
fibroblasts (lanes 1 and 3,
respectively) and myc- and ras-transformed
wild-type and MIF
/
fibroblasts (lanes 2 and
4, respectively). E, Western analysis of
myc- and ras-transduced wild-type and
MIF
/
fibroblasts following infection with a control
empty retroviral vector (V) or an
E2F1374-expressing vector.
/
Fibroblasts--
Because the
pro-apoptotic activity of c-Myc has been linked to E2F1 expression (32,
34), we examined the possibility that the differences observed in
transformation assays were related to E2F1 function. For this purpose,
cells transduced with myc and ras were infected
with a control green fluorescent protein-expressing retroviral vector
or a vector encoding E2F1374, a competitive inhibitor of
endogenous E2F activity (Fig. 5E). This E2F1 mutant contains
the DNA-binding domain, but lacks the transactivation and Rb-binding
regions and has been shown to block E2F-responsive gene
activation (35-37). The effect of green fluorescent protein expression
on the transforming capacity of the MEFs was insignificant (data not
shown). A mild inhibitory effect of E2F1374 expression on
focus formation was observed in myc and
ras wild-type cells (Fig. 5A), whereas
introduction of E2F1374 into myc and
ras MIF
/
fibroblasts increased the
efficiency of focus formation. These results suggest that
myc induces distinct proliferative and growth inhibitory
responses in MIF
/
MEFs. Although the intrinsic
growth-promoting myc activity cooperates with ras
in transformation, the growth inhibitory response observed in
MIF
/
cells is likely the result of inappropriate
induction of E2F activity and can be partially blocked by interfering
with p53 function.
/
Fibroblasts--
The Rb/E2F regulatory pathway controls the activity
of numerous genes essential for DNA replication and cell cycle
progression (37-39). Continuously growing MIF
/
cells
showed normal expression levels of several E2F-responsive genes, such
as cyclin A, cyclin E, cdc6, B-myb, and
p107 (data not shown). To assess the effect of MIF deficiency on
cell cycle regulation of E2F target genes, MEFs were synchronized by
serum deprivation for 72 h and then serum-stimulated to re-enter
the cell cycle. Subsequent examination of protein patterns showed no
differences between the wild-type and MIF
/
fibroblasts
in serum-induced accumulation of E2F-regulated gene products, such as
p107, cyclin A, B-Myb, and proliferating cell nuclear antigen (Fig.
6A) (data not shown). However,
the induction of cyclin E and cdc6 was delayed by an
average of 4 h as MIF
/
cells progressed toward S
phase (Fig. 6A).
View larger version (39K):
[in a new window]
Fig. 6.
Altered induction of E2F-responsive genes in
MIF /
fibroblasts. A, time course of cyclin A, cyclin E, and
CDC6 induction in wild-type (wt) and
MIF
/
(knockout (ko)) fibroblasts that were
serum-starved for 72 h and then serum-induced to re-enter the cell
cycle. MAPK and phosphorylated MAPK (MAPKP) are shown as
controls. B, induction of E2F1 expression examined in
nuclear extracts prepared from wild-type and MIF
/
fibroblasts after infection with a control empty retroviral vector
(cntr) or with retroviruses encoding dominant-negative
p53H175 (dnp53), LT, E1A, or c-Myc.
Odd-numbered lanes correspond to wild-type cells, and
even-numbered lanes correspond to MIF
/
fibroblasts.
/
MEFs could result from either inadequate
E2F-mediated gene activation or continued gene repression. Because
E2f1 is an E2F-regulated gene (40), we examined E2F1
expression levels in wild-type and MIF
/
fibroblasts
immortalized by various oncogenes. Notably, E2F1 expression was
up-regulated in MIF
/
cells containing
p53H175 (Fig. 6B, lane 4). By
contrast, c-Myc expression was similar between the corresponding
wild-type and MIF
/
fibroblasts (data not shown).
/
fibroblasts resulted in rapid cell death. The activity of this mutant
has been associated with sequestration of Rb and constitutive expression of E2F target genes (43). By contrast, the
E2F1E132 mutant stimulated growth in wild-type fibroblasts
(data not shown).
/
cultures
expressing the interfering E2F1374 mutant. Examination of
protein patterns in E2F1374-transduced wild-type
and MIF
/
fibroblasts showed largely unaltered
expression levels for several E2F-responsive genes, including cyclin A,
cyclin E, cdk2, and B-myb (Fig.
7A) (data not shown). By
contrast, the levels of p107 were down-regulated in both
E2F1374-expressing cell populations, as was the expression
of E2F target genes that play roles in DNA replication, such as
cdc6, mcm2, and mcm5 (Fig.
7A). The growth phenotype of E2F1374-transduced
wild-type and MIF
/
fibroblasts likely results from
preferential disruption of E2F1-, E2F2-, and E2F3-dependent
transactivation. Thus, the deletion of the E2f3 gene or the
combined loss of E2f1-3 results in the suppression of E2F
target genes involved in DNA replication and thus affects the ability
of cells to progress through mitosis (40, 44). By contrast, the loss of
E2f4 or the combined loss of E2f4 and
E2f5 has a modest effect on fibroblast cell proliferation (45, 46). Remarkably, sequential introduction of the
p53H175 and H-rasV12 mutants into
E2F1374-expressing wild-type and MIF
/
MEFs
resulted in clear morphological transformation of both cell types (data
not shown). In addition, both cell populations coexpressing E2F1374, p53H175, and
H-rasV12 produced transformed foci with a
similar efficiency (Fig. 7B), thus indicating that
interference with the E2F DNA-binding activity contributes to
overcoming the effect of MIF deficiency on ras-mediated transformation. Of note, a decrease in focus formation by
E2F1374-expressing wild-type fibroblasts is consistent with
the role of E2F3 in transformed cell growth (40).
View larger version (20K):
[in a new window]
Fig. 7.
Interference with E2F DNA-binding activity
blocks the effect of MIF deficiency on ras-mediated
transformation. A, immunoblot analysis of wild-type
(wt) and MIF /
(knockout (ko))
fibroblasts infected with a control empty retroviral vector
(V) or an E2F1374-expressing vector.
B, focus formation by wild-type and MIF
/
fibroblasts coexpressing p53H175 and
H-rasV12 or p53H175,
E2F1374, and H-rasV12.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, or p53 transcriptional activity (12-14). On the other hand,
two groups have used different strategies to generate MIF-knockout mice
(9, 10), but no growth-associated deficiencies have been reported in
these mutant animals. We found minor deviations in the growth
characteristics of MIF
/
MEFs under normal culture
conditions. However, major growth-related differences were observed
between the wild-type and MIF
/
cell populations
following the introduction of immortalizing oncogenes. Moreover, the
growth retardation observed with immortalized MIF
/
MEFs
correlated with their reduced susceptibility to oncogenic transformation.
/
cultures consistently resisted transformation under similar conditions. It appears that one major difference in the biology of
MIF
/
cells is the robustness of the senescence program,
which, in the absence of the functional p53 protein, retains the
capacity to restrain the inappropriate proliferation induced by
constitutively active Ras. The tumorigenic conversion of
MIF
/
fibroblasts required an additional cooperating
event compared with wild-type cells. Thus, the combined expression of
p53H175 and ras or of myc and
ras was insufficient to render MIF
/
cells
effectively tumorigenic, whereas the concomitant expression of
p53H175, myc, and ras resulted in
clear oncogenic transformation. Our results identify E2F as part of the
restraining mechanism that is activated in MIF
/
cells
in response to inappropriate proliferation. The consequences of E2F
induction in MIF
/
MEFs vary depending on the p53
status, inducing G1 phase arrest or apoptosis in
p53-positive cells or G2/M phase retardation when p53
function is compromised.
/
cultures disrupted normal
E2F-dependent gene activation and impaired cell growth.
However, in two experimental settings involving the expression of
either myc or p53H175,
E2F1374-transduced MIF
/
fibroblasts also
showed increased susceptibility to ras-mediated transformation. These results suggest that E2F-mediated inhibition of
MIF
/
cell growth involves its transcriptional activity.
Thus, we found that the cell cycle-regulated induction of cyclin E and
cdc6 was delayed by an average of 4 h in
MIF
/
fibroblasts compared with the wild-type cells. In
addition, the growth inhibition and apoptosis induced by E2F in
MIF
/
cells likely involves p73 (56), Apaf-1 (57), and
caspase proenzymes (58).
mutant mice and to extend their life span (59,
60). On the other hand, E2f1+/
and
E2f1
/
mutant mice also show increased tumor
susceptibility (61-63). Moreover, tumor formation in
E2f1+/
animals occurs without the loss of the
wild-type E2f1 allele, indicating that a mere reduction in
E2f1 dosage may be sufficient to either induce or sustain
tumorigenesis in vivo (61, 62). Although the
tumor-suppressive properties of E2F1 are not understood completely,
they are believed to result from the ability of E2F1 to induce
apoptosis, which can be both p53-dependent (55) and p53-independent (35). Notably, E2F1 plays a role in signaling apoptosis
not only as a consequence of the deregulation of the Rb pathway or Myc
activation, but also in response to DNA damage through a mechanism
involving the ATM pathway (64-70). It has been widely suggested that
ARF participates in the p53-dependent apoptosis induced by
E2F1. However, recent studies indicated that ARF might be dispensable
for E2F1-induced apoptosis (71-74) and that ARF-independent pathways
downstream of aberrantly induced E2F are largely responsible for p53
activation and subsequent apoptosis. Moreover, the disruption of
E2F-mediated transcription regulation in cells derived from FVB mice
causes an increase in the expression of E2F target genes, including ARF
(75). In this respect, it will be important to elucidate the pathway
that leads to the suppression of tumor cell growth and apoptotic
response in MIF
/
cells.
/
fibroblasts. Accordingly, the growth retardation was still present in
MIF
/
cells expressing viral oncoproteins, which bind Rb
and disrupt the Rb-E2F complexes (24, 76). Combined, our results
suggest that MIF plays a role in an E2F- and p53-dependent
pathway that operates downstream of Rb regulation. Based on recent
studies, the common biochemical mechanism by which p53 and E2F1 exert
an effect on target loci includes their association with
chromatin-modifying complexes through the involvement of overlapping
sets of transcription cofactors, such as p300, CBP, and TRRAP (77-80).
One possibility is that MIF might modulate the formation of p53 and/or
E2F1 complexes with the cofactors and their subsequent recruitment to
chromosomal sites. This would explain, in part, the ability of MIF to
modify p53 function without altering p53 protein levels (14).
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Picower Foundation (to C. N. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Pathology, State University of New York,
Stony Brook, NY 11794.
§ Present address: German CLL Study Group, Gene Centre, Munich University, Munich 81377, Germany.
¶ Present address: North Shore-Long Island Jewish Research Inst., Manhasset, NY 11030.
Present address: Dept. of Medicine, University of Louisville,
Louisville, KY 40202.
** To whom correspondence should be addressed: North Shore-Long Island Jewish Research Inst., 350 Community Dr., Manhasset, NY 11030. Tel.: 516-562-9471; Fax: 516-365-5090; E-mail: cmetz@nshs.edu.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M211985200
2 Available at www.sanger.ac.uk/HGP/Chr22/Mouse/.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MIF, macrophage migration inhibitory factor; MEFs, mouse embryonic fibroblasts; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MAPK, mitogen-activated protein kinase; LT, SV40 large T-antigen; ARF, alternate reading frame; CBP, cAMP-responsive element-binding protein.
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