From the Department of Neurological Surgery and the
§ Cancer Research Institute and Department of Cellular and
Molecular Pharmacology, University of California, San Francisco
Comprehensive Cancer Center, San Francisco, California 94115
Received for publication, December 20, 2000, and in revised form, March 12, 2001
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ABSTRACT |
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Normal human fibroblasts have been shown to
undergo a p16Ink4a-associated senescence-like growth
arrest in response to sustained activation of the Ras/Raf/MEK/ERK
pathway. We noted a similar p16Ink4a-associated,
senescence-like arrest in normal human astrocytes in response to
expression of a conditional form of Raf-1. While HPV16 E7-mediated
functional inactivation of the p16Ink4a/pRb pathway in
astrocytes blocked the p16Ink4a-associated growth arrest in
response to activation of Raf-1, it also revealed a second
p21Cip1-associated, senescence-associated,
Cell proliferation is dependent on the appropriate transmission of
growth signals from the cell membrane to the cell nucleus. Key in
regulating this process is the Ras family of proteins. Ras and
Ras-related proteins are membrane-bound, receptor-associated GTPases
(1, 2). These proteins are activated by ligand-bound growth factor
receptors (1, 2) or other engaged progrowth molecules (3) and in turn
activate a series of downstream effector proteins including Raf,
phosphatidylinositol 3-kinase, and RalGDS (4). While activation of
RalGDS and phosphatidylinositol 3-kinase undoubtedly play a role in Ras
function, activation of the Raf pathway appears to play a critical role
in cellular proliferation (5-7). Raf is recruited to the cellular
membrane by GTP-bound activated Ras, which results in Raf activation by
a variety of means, including phosphorylation (8). Activated Raf then
phosphorylates mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase
(MEK),1 which in turn
phosphorylates and activates p42/p44 mitogen-activated protein kinases
(ERK1 and ERK2) (9). ERK1/2 activation and nuclear translocation in
turn leads to phosphorylation of a variety of substrates, including
transcription factors, which ultimately contribute to proliferation (2,
10, 11).
The importance of the Ras/Raf/MEK/ERK pathway in growth is further
supported by its alteration in many human tumors. Approximately 50% of
human colon carcinomas contain mutant, constitutively active Ras (12),
an alteration that is believed to contribute to the proliferative
signaling necessary for tumor growth. Tumors that typically lack Ras
mutations such as brain tumors (gliomas) also have alterations in the
Ras/Raf/MEK/ERK pathway. Approximately 30% of high grade human gliomas
have an amplified, rearranged, and constitutively activated epidermal
growth factor receptor (13, 14), a receptor known to signal through the
Ras pathway (15). Additionally, Ras-GTP levels are elevated in a high
percentage of high grade human gliomas (16), presumably as a result of signaling from a number of different growth factor receptors. Disregulation of the Ras/Raf/MEK/ERK pathway is therefore a common means by which tumors maintain and amplify mitogenic stimuli and thereby sustain proliferation.
While Ras/Raf/MEK/ERK pathway activation can promote cellular
proliferation, it can also paradoxically provoke cell cycle arrest.
Sustained activation of oncogenic Ras in normal human fibroblasts (NHF)
leads not to proliferation but to permanent arrest in association with
elevated levels of p53 and of the cyclin-dependent kinase
inhibitors p16Ink4a and p21Cip1 (17). This
irreversible arrest is also associated with expression of
senescence-associated While normal human cells are programmed to undergo a senescence-like
growth arrest in response to excessive stimulation of the
Ras/Raf/MEK/ERK pathway, tumor cells could gain a growth advantage by
inactivating this tumor-suppressive response. Functional inactivation of the pRb/p16Ink4a pathway is a common feature of many if
not most human tumors, including human gliomas (13, 19). Given the role
of p16Ink4a in mediating Ras/Raf-induced senescence, it
seems likely that most human tumors lack important growth arrest
programs linked to excessive Ras/Raf/MEK/ERK pathway activation. The
cellular response to Ras/Raf/MEK/ERK activation in the absence of a
functional p16Ink4a/pRb pathway, however, remains poorly
defined. In NHF rendered unresponsive to p16Ink4a-mediated
cell cycle arrest by expression of cyclin D1 and a mutant Cdk4,
expression of oncogenic Ras resulted in growth arrest and alterations
in cell morphology similar to those noted in parental cells (20). It is
unclear, however, if the arrest pathway activated in the absence of
p16Ink4a was the senescence-like arrest pathway activated
in p16Ink4a-proficient cells and, if so, what proteins were
involved. Similarly, Raf activation in pRb-deficient small cell lung
carcinoma cells resulted in cell cycle arrest, although again it was
unclear if this arrest was associated with markers of senescence or
involved either p16Ink4a or p21Cip1 (21). To
more clearly define Raf-induced growth arrest pathways and, in
particular, potential p16Ink4a-independent alternate growth
arrest pathways, we characterized the response of normal human glial
cells to activation of a conditional form of Raf-1 and assessed whether
this response was similar in pRb-deficient glial cells and in glial
tumors lacking p16Ink4a and/or pRb. The results of these
studies show that rather than having a single
p16Ink4a-dependent senescence-associated arrest
pathway, astrocytes have a second distinct, SA- Cell Culture--
Normal human astrocytes (NHA), derived from
fetal tissue, were purchased from Clonetics (Walkersville, MD). NHA
were grown in phenol red-free astrocyte medium (Clonetics) containing
5% charcoal dextran-treated, estrogen-free fetal bovine serum (Omega Scientific, Inc., Tarzana, CA).
U251, SF126, U87, and U87 + E6 glioma cell lines were grown in phenol
red-free Dulbecco's modified Eagle's medium containing 20 mM HEPES, penicillin/streptomycin, and 10% charcoal
dextran-treated, estrogen-free fetal bovine serum.
Retrovirus Production and Infection--
To create cells that
express conditional Raf-1, Phoenix A cells were transfected using
LipofectAMINE (Life Technologies, Inc.) with EGFP
24 and 48 h after transfection of Phoenix A cells, the medium
containing the packaged virus was placed on NHA. Infected NHA were
selected for resistance to blasticidin S (25 µg/ml for 4-5 days)
(ICN, Costa Mesa, CA) or puromycin (1 µg/ml for 4-5 days) (Sigma-Aldrich), and resistant cells were pooled and expanded for
further studies. To ensure successful infection, relevant expanded
populations were analyzed by fluorescence-activated cell sorting to
verify expression of EGFP
To create pRb-deficient cells with conditional activation of
Raf-1, NHA first were infected with HPV16 E7 retroviral DNA packaged in
Phoenix A cells, as described above. The pLNCX retroviral vector allows
for constitutive expression of the HPV E7 protein (pRb inactivation)
and neomycin resistance. Cells expressing E7 were selected for
resistance to G418 (0.5 mg/ml for 6-7 days) (ICN). Infected cells that
survived drug selection were then infected with the EGFP
U251, SF126, U87, and U87 + E6 glioma cell lines were
infected with EGFP Raf Activation and Inactivation--
EGFP
For studies done to assess the reversibility of the Raf-induced growth
arrest, cells were incubated for 4 days with 1 µM
E2. Cells then were washed extensively with PBS, grown in
control medium for an additional 4 days, and analyzed.
[3H]Thymidine Incorporation--
The extent of
proliferation was assessed by monitoring incorporation of
[methyl-3H]thymidine (PerkinElmer Life
Sciences). Cells were seeded in at least quadruplicate in 96-well
plates, at a density of 16,000 cells/ml (NHA, NHA/E6, NHA/E7, and SF126
cells) or 12,000 cells/ml (U251 and U87 cells). Cells were grown for 4 days and then incubated with 20 µCi/well of
[methyl-3H]thymidine for 16 h.
Incorporation was measured with a Tomtec Micro 96-cell plate harvester
and a Betaplate 1205 liquid scintillation counter.
Analysis of Senescence-associated Western Blot Analysis--
For Western blot analysis, cells were
lysed in 0.5% SDS lysis buffer (100 mM NaCl, 50 mM Tris-Cl, pH 8.1, 5 mM EDTA, pH 8.0, 0.2%
NaN3, 0.5% SDS). Equal amounts of cellular lysates were
electrophoresed on 7.5-15% SDS-polyacrylamide gels and transferred
onto Immobilon-P polyvinylidene difluoride membrane (Millipore Corp.).
Western blots were probed with primary antibodies against the estrogen receptor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
phospho-p44/42 mitogen-activated protein kinase (Cell Signaling
Technology, Beverly, MA), p16Ink4a (Oncogene Research
Products, Boston, MA), p21Cip1 (Oncogene Research
Products), p53 (Santa Cruz Biotechnology), and pRb (Santa Cruz
Biotechnology). Anti-mouse and anti-rabbit horseradish
peroxidase-conjugated secondary antibodies were used (Santa Cruz
Biotechnology), and Western blots were developed with an enhanced
chemiluminescence kit (ECL; Amersham Pharmacia Biotech). Equal loading
was determined by staining the membrane with Ponceau S (Sigma-Aldrich).
Quantitative analysis was done on an AlphaImager 2200, using AlphaEase
Software (Alpha Innotech Corp., San Leandro, CA). Autoradiographs are
representative of 2-4 experiments.
Raf Activation Results in a Phenotype Resembling Senescence in
NHA--
4HT- or E2-mediated activation of Raf in
proliferating NHA retrovirally infected with EGFP
Activation of EGFP
Senescent cells have been shown to have increased expression of
p16Ink4a, p21Cip1, and p53 (20, 25, 26), and
human fibroblast studies have demonstrated that elevation of
p16Ink4a expression is associated with the maintenance of
senescence (18, 26). Therefore, we assessed the effect of Raf
activation on the expression of these cell cycle regulatory proteins in
NHA. As shown in Fig. 1d, incubation of uninfected NHA with
E2 had no effect on expression of p16Ink4a,
p21Cip1, or p53. Similarly, incubation of NHA/Raf301:ER
with 4HT did not affect p16Ink4a expression (data not
shown). EGFP
To address whether the Raf-induced growth arrest in NHA/RafER cells was
permanent and irreversible, NHA/RafER cells were incubated with
E2 for 4 days, washed extensively with PBS to remove
E2 from the culture, and grown in control medium for an
additional 4 days. E2 was used to activate EGFP SA-
To better understand the difference in Raf-induced arrest in
NHA/E7/RafER cells compared with NHA/RafER cells, Western blot analysis
was done to assess changes in protein expression following EGFP Activation of Raf Results in Growth Arrest in High Grade
Gliomas--
Although NHA have two pathways leading to growth arrest
following sustained Raf expression, glial tumors often have genetic defects that might influence their responses to Raf activation. For
example, the vast majority of high grade (grade IV) gliomas have
defects in the p16Ink4a/pRb pathway (13). To assess whether
glioma cells retained cell cycle arrest pathways in response to Raf
activation, high grade glioma cells were infected with EGFP
To further distinguish the Raf-activated,
p21Cip1-associated arrest noted in glioma cells from the
p16Ink4a-associated, senescence-like arrest seen in NHA,
further studies were done in U251/RafER and SF126/RafER cells to
determine the permanence of the Raf-induced arrest. When U251/RafER
cells were incubated for 4 days with E2 and then incubated
for 4 more days in control medium, cells resumed proliferating, as
determined by [3H]thymidine incorporation (Fig.
3a). Additionally, the cells reverted back to their original
morphology when the E2 was removed from culture, unlike the
NHA/RafER, which maintained their altered morphology following
E2 removal. Finally, when EGFP The data presented in this paper demonstrate the presence of two
distinct inducible growth arrest pathways in astrocytic cells. These
two pathways are qualitatively different in nature, with the choice of
which is used dependent on the presence of a functional p16Ink4a/pRb pathway. In the case of NHA, which have an
intact p16Ink4a/pRb pathway, Raf activation results in an
irreversible, senescence-like growth arrest associated with increased
p16Ink4a expression. However, when this pathway is
disrupted, as is the case in the NHA/E7/RafER cells and in the high
grade glioma cell lines, Raf activation leads to reversible growth
arrest associated with increased p21Cip1 expression.
The finding that normal human cells contain two distinct growth arrest
pathways in response to Raf activation is both novel and consistent
with previous published work. The ability of cells to undergo arrest in
response to exogenous expression of either p16Ink4a or
p21Cip1 is widely appreciated (18, 31-33), and Ras or Raf
activation in NHF leads to increased expression of both of these
proteins (17, 18). In NHF, however, Raf-induced senescence-like growth arrest has been shown to occur in the absence of induced levels of
p21Cip1 (18), a finding reinforced by our observation that
in NHA, p16Ink4a levels, but not levels of
p21Cip1, increase in response to EGFP A second important finding of the present work is that even tumors
which have dismantled the p16Ink4a-dependent
senescence-like growth arrest pathway retain the secondary, p21Cip1-associated growth arrest mechanism. Numerous tumor
cell types have been shown to respond to Raf induction by undergoing
growth arrest, although in these studies either the tumor cells used were p16Ink4a/pRb-proficient (35, 36) or the arrest did not
involve p21Cip1 (21). We also have noted that activation of
EGFP Having established the presence of a secondary
p21Cip1-associated, Raf-stimulated arrest pathway in NHA,
the question arises as to how Raf induces p21Cip1
expression and how p21Cip1 induces growth arrest in the
absence of pRb/p16Ink4a. With regard to the latter
question, p21Cip1 has been shown to inhibit the action of
E2F in human cervical and bladder carcinoma cell lines that lack
functional pRb (38), suggesting that the p21Cip1-associated
arrest noted in glioma cell lines and, in particular, in the
pRb/p16Ink4a-deficient SF126 line may similarly involve
E2F. Alternatively, p21Cip1 may regulate the activities of
cyclin E-Cdk2 and/or cyclin A-Cdk2 complexes (19). With regard to the
former question, p53-independent induction of p21Cip1 has
been associated with a variety of transcription factors including Sp1,
Sp3, STAT proteins, and E2Fs (39-41). Raf-induced increases in any of
these transcription factors could therefore lead to p53-independent
increases in p21Cip1 levels. These possibilities and, in
particular, the interplay between Raf, E2F, and p21Cip1,
remain to be examined.
Finally, the identification of two distinct growth arrest pathways in
astrocytes and astrocytic tumors may be important both to understanding
the genesis of tumors and to effectively stopping their growth.
Approximately half of grade III anaplastic astrocytomas have
inactivation of at least one component of the p16Ink4a/pRb
pathway, and homozygous deletion of p16Ink4a is seen in up
to 70% of grade IV glioblastoma multiforme (13). Thus, most
glial tumors, by dismantling the p16Ink4a/pRb pathway, have
eliminated the possibility of Ras/Raf-mediated p16Ink4a-associated permanent growth arrest. Inactivation
of p21Cip1, however, has rarely been reported in gliomas or
in other human tumors (13, 19, 42). These observations suggest
that tumor development may select for cells with Ras/Raf/MEK/ERK
pathway activation high enough to promote growth yet low enough to
avoid triggering p21Cip1-dependent growth
arrest. Indeed, this appears to be the case in experimental human
gliomas created by overexpression of mutant Ras, which, despite having
very high levels of activated Ras, have only modest levels of ERK1/2
activation.2 More
importantly, these results support the idea that intact arrest pathways
lie dormant in tumors (43), including gliomas. Selective activation of
these pathways in tumor cells would represent an ideal, noncytotoxic
way to prevent the growth and the ultimate fatal consequences of
tumors. The definition of at least two distinct growth arrest pathways
in cells is a first step toward this goal.
-galactosidase-independent growth arrest pathway. Importantly, the
p21Cip1-associated pathway was present not only in normal
astrocytes but also in p53-, p14ARF-, and
p16Ink4a/pRb-deficient high grade glioma cells that lacked
the p16Ink4a-dependent arrest mechanism. These
results suggest that normal human cells have redundant arrest pathways,
which can be activated by Raf-1, and that even tumors that have
dismantled p16Ink4a-dependent growth arrest
pathways are potentially regulated by a second
p21Cip1-dependent growth arrest pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (SA-
-gal) activity, suggesting that in response to high levels of activated Ras, normal human cells undergo a senescence-like growth arrest. The ability of
oncogenic Ras to induce a senescence-like growth arrest in NHF appears
to be closely linked to activation of Raf but not to other downstream
targets of Ras, such as phosphatidylinositol 3-kinase or RalGDS, since
only mutant forms of Ras that selectively activate the Raf pathway
induced growth arrest comparable with that induced by constitutively
expressed oncogenic Ras (17). This point is further supported by the
finding that sustained activation of a conditional form of Raf-1, or
overexpression of activated MEK1, in NHF leads to senescence-like
arrest comparable with that induced by Ras (17, 18). Although the
mechanism by which Ras or Raf induces a senescence-like arrest in NHF
remains to be fully defined, NHF expressing HPV16 E6 and therefore
lacking functional p53 (and p53-dependent induction of
p21Cip1) still undergo a p16Ink4a-associated,
senescence-like arrest in response to Raf activation (18). This
senescence-like, Raf-induced arrest could also be blocked by the MEK
inhibitor PD098059 (18), further suggesting a link between Raf
activation, p16Ink4a induction, and senescence-like arrest.
As a whole, these results suggest that the Ras/Raf/MEK/ERK pathway, in
addition to playing a key role in cellular proliferation, can also
trigger a senescence-like, p16Ink4a-associated growth
arrest that prevents proliferation of normal cells.
-gal-independent,
p21Cip1-associated arrest pathway activated in response to
Raf activation. This second pathway is present not only in normal
astrocytes but also in glial tumor cells that have lost the function of
several key cell cycle regulatory proteins including
p16Ink4a, p53, pRb, and p14ARF. These results
suggest that normal cells (in this case astrocytes) have both primary
and secondary mechanisms that can be used to prevent proliferation in
the face of sustained Raf/MEK/ERK signaling and that even highly
malignant tumors retain at least one pathway in a functional yet
dormant state.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Raf-1:ER retroviral
DNA. The pWZLblast3 EGFP
Raf-1:ER retroviral vector encodes a protein
that is a chimera of the catalytic domain of human Raf-1, the hormone
binding domain of the human estrogen receptor, and an enhanced version
of green fluorescent protein (EGFP). The vector also encodes
blasticidin resistance. A similar retroviral vector for the expression
of GFP
Raf-1:ER has been described previously (22). Infected cells
show little Raf kinase activity until the chimeric protein is activated
by the addition of 17
-estradiol (E2) or the
estrogen analog 4-hydroxytamoxifen (4HT) (23). As a control, cells were
infected with pBabepuro3
Raf301:ER retroviral DNA. The
pBabepuro3
Raf301:ER retroviral vector encodes a protein that is a
chimera of a kinase-inactive version of the catalytic domain of Raf-1
and the hormone-binding domain of the estrogen receptor. The vector
also encodes for puromycin resistance.
Raf-1:ER. Pooled populations of infected
NHA were used for all experiments, since the limited life span of
astrocytes in culture does not allow for development of clonal populations.
Raf-1:ER
retroviral DNA, as described. Cells were selected for resistance to
blasticidin/G418 and were pooled and expanded for further studies.
Raf-1:ER retroviral DNA as described and selected for resistance to blasticidin. U251 also were infected with
pBabepuro3
Raf301:ER retroviral DNA as described and selected for
resistance to puromycin. Following blasticidin selection, cells
infected with EGFP
Raf-1:ER were sorted using a Becton-Dickinson
FACSVantage SE Cell Sorter, and the cells expressing the highest level
of EGFP (upper 5% of expressers) were collected and expanded. All
experiments were performed using this population of high expressing
cells. These cells were periodically reanalyzed by
fluorescence-activated cell sorting to ensure maintenance of EGFP
expression level.
Raf-1:ER was
activated by incubating cells for 4 days with either 1 µM
E2 (Sigma-Aldrich) or 100 nM 4HT (Research
Biochemicals International, Natick, MA). 1 mM stocks of
E2 and 4HT were made in ethanol. For all studies, both
control and E2- or 4HT-containing media were changed
every 48 h.
-Galactosidase
Activity--
SA-
-gal activity was determined by the method of
Dimri et al. (24). Cells were fixed in 2% formaldehyde,
0.2% glutaraldehyde and incubated with a 1 mg/ml X-gal solution (40 mM citric acid/sodium phosphate buffer (pH 6), 5 mM potassium ferrocyanide, 5 mM ferricyanide, 150 mM NaCl, 2 mM MgCl2, 1 mg/ml
X-gal in dimethylformamide) for 16-24 h. Cells were then rinsed with
PBS and methanol and air-dried. The percentage of SA-
-gal-positive
cells was assessed by counting the number of blue cells in a × 10 microscopic field and dividing that number by the total number of cells
in the field. The percentage of positively stained cells in each plate
was the average of four fields per plate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Raf-1:ER
(NHA/RafER) led to elevated expression of the chimeric Raf:ER protein.
Western blot analysis showed that E2 incubation led to a
sustained high level of EGFP
Raf-1:ER expression in NHA/RafER cells
(Fig. 1a), consistent with
increased stability of the Raf-1:ER fusion protein, as reported
previously (23). EGFP
Raf-1:ER induction was noted within 24 h
of E2 addition and was sustained for 4 days (Fig.
1a). E2 incubation also led to an increase in
phosphorylated ERK1/2 in NHA/RafER cells, but not NHA or NHA containing
the kinase-inactive version of Raf-1 (NHA/Raf301:ER), as determined by
Western blot analysis using a phosphospecific p42/p44 antibody (Fig.
1a and data not shown). This increase in phosphorylated
ERK1/2 is consistent with p42/p44 ERK1/2 pathway stimulation following
Raf activation. The level of phosphorylated ERK1/2 remained high
throughout the experiment. As shown in Fig. 1a, the addition
of 4HT resulted in induction of EGFP
Raf-1:ER and phosphorylated
ERK1/2 similar to that seen with E2.
View larger version (20K):
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Fig. 1.
Analysis of Raf-induced senescence in
NHA. NHA and NHA/RafER were incubated for 24-96 h with
E2 or 4HT to activate Raf and then were harvested and
analyzed as described under "Experimental Procedures."
a, Western blot analysis of changes in expression of
chimeric RafER and phosphorylated ERK1/2 following
E2-mediated Raf activation. 4HT incubation was for 96 h. 96* values were derived from cells washed extensively to remove
E2 and then grown 4 days in control medium before analysis.
b, inhibition of proliferation following Raf activation was
assessed by [methyl-3H]thymidine
incorporation. All values are the mean ± S.D. of at least three
experiments. E2* values were derived from cells washed
extensively to remove E2 and then grown 4 days in control
medium before analysis. c, expression of the senescence
marker SA- -gal following Raf activation was determined by incubation
with a 1 mg/ml X-gal solution. All values are the mean ± S.D. of
at least three experiments. E2* values were derived from
cells washed extensively to remove E2 and then grown 4 days
in control medium before analysis. D, Western blot analysis
of changes in protein expression following E2-mediated Raf
activation. 96* values were derived as described for
a.
Raf-1:ER also led to changes in cell proliferation
and morphology. Beginning ~48 h following EGFP
Raf-1:ER activation,
cells became smaller and refractile but remained adherent and viable
(determined by trypan blue exclusion, data not shown). [3H]thymidine incorporation was reduced by 90% after 4 days of EGFP
Raf-1:ER activation (Fig. 1b). Incorporation
of [3H]thymidine by uninfected NHA and by NHA/Raf301:ER
was not affected by the addition of E2 to the medium (Fig.
1b). Additionally, 4 days after EGFP
Raf-1:ER activation
by the addition of either 4HT or E2, 45-50% of cells
stained positively for SA-
-gal, a common marker of senescent cells,
while control NHA/RafER cells and parental NHA had fewer than 10%
SA-
-gal positive cells (Fig. 1c).
Raf-1:ER activation in NHA/RafER cells similarly did not
significantly increase levels of p21Cip1 expression or p53
expression 4 days following Raf activation (54 ± 23 and 114 ± 31% of control values, respectively, by quantitative Western blot
analysis; Fig. 1d). EGFP
Raf-1:ER activation in NHA/RafER cells did, however, result in increased expression of the
cyclin-dependent kinase inhibitor p16Ink4a, at
72 h after Raf activation (Fig. 1d).
Raf-1:ER
in these experiments, since it is more easily removed from the cells
than 4HT. As shown in Fig. 1a, hormone removal resulted in
decreases of EGFP
Raf-1:ER levels and phosphorylated ERK1/2 levels to
those seen in control cells. Following E2 removal, however,
cells maintained an altered morphology, and thymidine incorporation
remained reduced by 90% (Fig. 1b). Additionally, there was
no change in the percentage of cells that stained positively for
SA-
-gal (Fig. 1c). Finally, consistent with the
senescence phenotype, p16Ink4a expression remained elevated
(Fig. 1d). Irreversible growth arrest, maintenance of
SA-
-gal expression, and sustained elevation of p16Ink4a
expression suggest that activation of Raf and the ERK1/2 pathway is
able to induce a p16Ink4a-associated senescence phenotype
in NHA.
-gal Activity, but Not Growth Arrest, of NHA following Raf
Activation Is Dependent on Functional pRb--
Because of the
association between increased p16Ink4a expression and
Raf-induced senescence in NHA, studies were initiated to determine if a
functional p16Ink4a/pRb pathway was required for
Raf-induced growth arrest in NHA. NHA constitutively expressing E7 and
conditional EGFP
Raf-1:ER (NHA/E7/RafER) were derived by retroviral
infection. Expression of the HPV16 E7 protein resulted in decreased
protein levels of pRb, as shown in the Western blot in Fig.
2a, lower
panel. While 4HT had no effect on protein expression in
parental NHA/E7 cells (data not shown), 4HT-mediated activation of
EGFP
Raf-1:ER (in NHA/E7/RafER cells) led to increased expression of
both chimeric EGFP
Raf-1:ER protein and phosphorylated ERK1/2 (Fig.
2a). EGFP
Raf-1:ER activation also resulted in a 60%
inhibition of [3H]thymidine incorporation, as compared
with control cells (Fig. 2b). EGFP
Raf-1:ER activation did
not, however, alter cell morphology, and after 4 days of 4HT exposure
fewer than 5% of cells stained positively for SA-
-gal.
View larger version (21K):
[in a new window]
Fig. 2.
Analysis of Raf-induced growth arrest in
NHA/E7/RafER. NHA/E7 and NHA/E7/RafER were incubated for 4 days
with 4HT to activate Raf and then were harvested and analyzed as
described under "Experimental Procedures." a, Western
blot analysis of changes in expression of chimeric RafER and
phosphorylated ERK1/2 following E2-mediated Raf activation.
b, inhibition of proliferation following Raf activation was
assessed by [methyl-3H]thymidine
incorporation. All values are the mean ± S.D. of at least three
experiments. c, changes in protein expression following Raf
activation were assessed by Western blot.
Raf-1:ER activation. Expression of p16Ink4a was
elevated in NHA/E7/RafER cells as compared with NHA/RafER cells (Fig.
2a, lower panel), which is consistent
with the destabilization of pRb by the E7 protein (27, 28). Unlike what
was noted in NHA/RafER cells with a functional p16Ink4a/pRb
pathway, however, there was no further increase in p16Ink4a
levels, relative to control cells (average p16Ink4a levels
were 65 ± 9% of control values) in the pRb-deficient
NHA/E7/RafER cells following Raf activation (Fig. 2c). There
was, however, an increase in p21Cip1 expression in
NHA/E7/RafER cells following EGFP
Raf-1:ER activation. This
p21Cip1 induction was apparent within 48 h (250 ± 43% of control values), persisted at least 4 days (250 ± 16%
of control values), and appeared to be independent of p53, the levels
of which remained unchanged throughout the experiment (Fig.
2c). These results indicate that activated Raf is able to
induce growth arrest in NHA in the absence of a fully functional
p16Ink4a/pRb pathway. This arrest, however, is associated
with increased p21Cip1 expression, is not associated with
increased SA-
-gal, and appears to be functionally different from
senescence-like arrest seen in NHA.
Raf-1:ER,
incubated with 4HT or E2 to activate EGFP
Raf-1:ER, and
monitored for cellular alterations and activation of cell signaling
pathways. While [3H]thymidine incorporation by U251 cells
containing the kinase-inactive form of Raf-1 (U251/Raf301:ER) was not
affected by incubation with 4HT (Fig.
3a),
[3H]thymidine incorporation was reduced by 80% in
U251/RafER cells, by 90% in SF126/RafER cells, and by 85% in
U87/RafER cells following 4 days of incubation with E2
(Fig. 3a). In contrast to the NHA/RafER cells, however,
which became smaller and refractile and SA-
-gal-positive following
EGFP
Raf-1:ER activation, the tumor cells became elongated with long
processes, and SA-
-gal activity did not increase following EGFP
Raf-1:ER activation. The response of these cells was therefore more similar to that of pRb-deficient NHA/E7/RafER cells than that of
NHA. Consistent with this idea, all cell lines showed elevated
p21Cip1 expression, ranging from 2-fold increase in
U87/RafER cells to 8-fold in U251/RafER and SF126/RafER cells, which
began 24-48 h after 4HT exposure and persisted throughout the
experiment (Fig. 3, b-e). As in the NHA/E7/RafER cells,
p21Cip1 induction appeared to be independent of p53, since
1) U251 and SF126 cells lack functional p53 (29, 30); 2) U87 cells
containing functional p53 (30) showed no increase in p53 protein levels following 4 days of 4HT exposure despite showing p21Cip1
induction (Fig. 3d); and 3) U87 + E6 cells, which lack
functional p53, displayed p21Cip1 induction and growth
arrest similar to that noted in U87 cells (Fig. 3e).
p16Ink4a did not have a role in growth arrest of these
glioma cell lines, since all lines had homozygous deletion of
p16Ink4a (21, 30). These results suggest that high grade
gliomas, which have lost the p16Ink4a-associated senescence
pathway, retain the p21Cip1-associated growth arrest
pathway present in NHA.
View larger version (36K):
[in a new window]
Fig. 3.
Analysis of Raf-induced growth arrest in high
grade gliomas. U251/RafER, SF126/RafER, U87/RafER, and U87 + E6/RafER were incubated for 4 days with 4HT to activate Raf and then
were harvested and analyzed as described under "Experimental
Procedures." a, inhibition of proliferation following Raf
activation in U251/Raf301:ER, U251/RafER, SF126/RafER, and U87/RafER
was assessed by [methyl-3H]thymidine
incorporation. All values are the mean ± S.D. of at least three
experiments. E2* values were derived from cells washed
extensively to remove E2 and then grown 4 days in control
medium before analysis. b-e, changes in protein expression
following Raf activation in U251/RafER cells (b),
SF126/RafER cells (c), U87/RafER cells (d), and
U87/E6/RafER cells (e) were assessed by Western blot. 96*
values were derived from cells washed extensively to remove
E2 and then grown 4 days in control medium before
analysis.
Raf-1:ER was inactivated
in U251/RafER and SF126/RafER cells by the removal of E2
from the culture, p21Cip1 expression levels dropped back to
the level seen in control cells (Fig. 3, b and
c). Therefore, EGFP
Raf-1:ER activation in the glioma cell
lines resulted in growth arrest that lasted only as long as Raf was
activated. This arrest also was associated with increased expression of
p21Cip1, which returned to normal levels with the
inactivation of EGFP
Raf- 1:ER. These data suggest that the
Raf-induced, p21Cip1-associated growth arrest in these
glioma cell lines is reversible and is dissimilar to the senescence
seen in NHA in response to Raf activation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Raf-1:ER
activation. The novel finding of the present study is that
p21Cip1, whose potential function in Ras- and Raf-induced
arrest in NHF was not closely examined, is associated not with
senescence-like growth arrest in NHA but rather with a second distinct
arrest pathway that is not associated with markers of senescence and whose presence can be noted only in the absence of the primary p16Ink4a-associated arrest pathway. The two growth arrest
pathways defined in NHA appear to be mutually exclusive
(i.e. functional p16Ink4a/pRb appears to be
unnecessary for p21Cip1-associated arrest, and vice versa).
In E7-expressing astrocytes, however, levels of pRb were low but not
absent, and these cells also contained high levels of
p16Ink4a. The possibility exists, therefore, that the
presumed p21Cip1-associated response might in fact be
simply a muted version of the already described
p16Ink4a-associated arrest. Additionally, since high levels
of p16Ink4a have been suggested to displace
p21Cip1 from cyclin D-Cdk4 complexes and to
stimulate formation of growth-inhibitory p21Cip1-cyclin
E-Cdk2 complexes (34), the possibility also exists that the presumed
p21Cip1-associated response might in fact be directly
dependent on high levels of p16Ink4a. The qualitative
difference in response to EGFP
Raf-1:ER activation in E7-expressing
cells (p21Cip1-associated arrest in the absence of
SA-
-gal expression or morphologic alterations) versus
that in NHA (p16Ink4a-associated senescence-like arrest),
however, argues against a single p16Ink4a-associated
pathway. The presence of a p21Cip1-associated arrest
pathway in p16Ink4a null tumor cell lines and the transient
as opposed to permanent nature of this arrest further support the
contention that two distinct arrest pathways exist in cells of glial origin.
Raf-1:ER induces a SA-
-gal-associated arrest in
p16Ink4a-proficient tumor cells derived from low grade
human gliomas (data not shown). It therefore seems likely that
p16Ink4a/pRb-proficient tumors retain senescence-like
arrest pathways similar to those noted in normal human cells following
activation of Raf. The arrest noted in the glioma cell lines used in
the present study, however, was clearly different from the
p16Ink4a-dependent arrest noted in NHA, since
it was reversible, not associated with SA-
-gal expression, and
activated even in tumor cells (U87, U251, and SF126) devoid of
p16Ink4a. Significantly, the p21Cip1-associated
arrest was also noted in tumor cells lacking functional p53 and
p14ARF (U251, SF126). Recent studies have shown that Ras or
Raf activation can induce expression of both p14ARF and the
p53 inhibitor Mdm2 and that the balance of these actions may affect p53
levels and downstream p53-dependent effects (37). The
presence of a Raf-induced, p21Cip1-associated growth arrest
pathway in p14ARF and p53-deficient cells therefore
suggests that the linkage between Raf induction and increased
p21Cip1 expression is independent of not only
p16Ink4a but also of effects of the Ras/Raf/MEK/ERK pathway
on the p14ARF/Mdm2/p53 pathway.
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FOOTNOTES |
---|
* 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.
¶ To whom all correspondence should be addressed: University of California, San Francisco Comprehensive Cancer Center, 2340 Sutter St., Box 0875, San Francisco, CA 94115. Tel.: 415-502-7132; Fax: 415-502-6779; E-mail: rpieper@cc.ucsf.edu.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M011514200
2 C. P. Fanton, M. McMahon, and R. O. Pieper, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
ERK1/2, extracellular signal-regulated kinase 1/2;
NHF, normal human fibroblasts;
SA--gal, senescence-associated
-galactosidase;
NHA, normal human astrocytes;
EGFP, enhanced green
fluorescence protein;
E2, 17
-estradiol;
4HT, 4-hydroxytamoxifen;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside.
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