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J Biol Chem, Vol. 274, Issue 34, 24263-24269, August 20, 1999
From the Cancer Research Institute, University of California,
School of Medicine, San Francisco, California 94143-0128
The phosphoinositide 3-OH kinase (PI3K)-PKB/Akt
signaling pathway has been shown to mediate both Ras- and
cytokine-induced protection from apoptosis. In addition, apoptosis
induced by the p53 tumor suppressor protein can be inhibited by Ras-
and cytokine-mediated signaling pathways. It was therefore of interest
to determine if the PI3K-PKB/Akt signaling pathway was capable of
conferring protection from apoptosis induced by p53. We demonstrate in
this report that constitutively active PI3K and PKB/Akt are capable of
significantly delaying the onset of p53-mediated apoptosis. This was
manifested as a delay in the kinetics of DNA degradation and cell death
as well as a profound attenuation in the accumulation of cells with a
sub-G1 DNA content. Moreover, we found that this effect is mediated in the absence of changes in expression of Bcl-2,
Bcl-Xl, and the pro-apoptotic protein Bax. Our results provide the
first direct and unambiguous link between p53-mediated apoptosis and
the PI3K-PKB/Akt signaling pathway.
The serine/threonine protein kinase PKB/Akt was originally
identified as the cellular counterpart of the v-Akt transforming protein present in AKT8, a retrovirus that causes T cell lymphomas in
mice (1). v-Akt was generated by a fusion event that juxtaposes the
retroviral glycosaminoglycan protein and the entire coding region of
PKB/Akt (2, 3). The fusion protein, designated glycosaminoglycan-PKB,
is constitutively active due to a myristoylation signal present within
the amino terminus of the glycosaminoglycan protein that targets
PKB/Akt to the plasma membrane.
In quiescent or serum-starved cells, PKB/Akt resides within the cytosol
in a catalytically inactive state. Upon stimulation of cells with
growth factors and cytokines, PKB/Akt is recruited to the plasma
membrane and catalytically activated by phosphorylation at threonine
308 and serine 473 (4-8). Phosphorylation of PKB/Akt at threonine 308 is catalyzed by the ubiquitously expressed and constitutively active
protein kinase PDK-1 (5, 7). The kinase responsible for phosphorylation
of PKB/Akt at serine 473 has not been definitively established,
although possible candidates have been proposed (9). Recruitment of
both PKB/Akt and PDK-1 to the plasma membrane is mediated by second
messenger phosphorylated phosphoinositides generated by the
phosphorylation of inositol lipids by phosphoinositide 3-OH kinase
(PI3K).1 Platelet-derived
growth factor- and IGF-mediated activation of PKB/Akt is inhibited by
two pharmacological inhibitors of PI3K, LY294002 and wortmannin (7, 10,
11). Also, platelet-derived growth factor receptor mutants incapable of
activating PI3K are likewise unable to activate PKB/Akt (12). Moreover,
PDK-1 potentiates the platelet-derived growth factor-mediated
activation of PKB/Akt, and this effect is abrogated by wortmannin (13).
Taken together, these results indicate that PI3K can function as an
upstream activator of PKB/Akt and can regulate the ability of PDK-1 to
modulate PKB/Akt activity
PI3K is a heterodimeric lipid kinase consisting of a p85 regulatory
subunit and a p110 catalytic subunit and is capable of triggering a
plethora of biological responses (14-16). PI3K is activated by the
interaction of the p85 regulatory subunit with phosphorylated tyrosine
residues on activated growth factor receptors (17). The binding of PI3K
to upstream signaling molecules leads to the recruitment of p85-p110
heterodimeric complexes to the plasma membrane and the subsequent
activation of the p110 catalytic subunit. The activated p110 catalytic
subunit phosphorylates inositol lipids at the 3-position of the
inositol ring, thereby generating the phospholipid second messenger
molecules required for the transposition of PKB/Akt and PDK-1 to the
plasma membrane.
Cytokines, growth factors, and certain oncogenes have been shown to be
effective inhibitors of apoptosis, and in many situations, this
anti-apoptotic effect is mediated by the PI3K-induced activation of
PKB/Akt (18-23). IGF-1 is a well documented activator of the PI3K-PKB/Akt signaling pathway (4, 23, 24). IGF-1 inhibits UV-induced
apoptosis in fibroblasts and prevents apoptosis in neuronal cells in
response to growth factor withdrawal (21, 23). In both cases,
IGF-1-mediated protection from apoptosis is abrogated either by
pharmacological inhibitors of PI3K or by dominant-negative PKB/Akt
constructs. Moreover, constitutively active PI3K or PKB/Akt mimics the
anti-apoptotic function of IGF-1. Ras activates the PI3K-PKB/Akt
signaling pathway by interacting directly with the p110 catalytic
subunit of PI3K (16). Ras-induced activation of the PI3K-PKB/Akt
signaling pathway confers protection from apoptosis in fibroblasts in
response to oncogenic Myc and protects epithelial cells from apoptosis
induced by anoikis (22, 25, 26). In this respect, PI3K-PKB/Akt-mediated
survival contributes to the ability of Ras to function as an oncogene.
The p53 tumor suppressor protein is a transcription factor capable of
inducing either growth arrest or apoptosis (27-29). In response to DNA
damage, p53 induces a G1-specific cell cycle arrest by
transcriptionally up-regulating the expression of the cyclin/Cdk inhibitor, p21/WAF-1-Cip1. This allows cells time to repair damaged DNA
before progressing into S phase (30, 31). However, in response to
oncogenic activation and/or growth factor withdrawal, p53 can induce
apoptosis. p53-mediated apoptosis in certain cell types requires a
transcriptionally functional p53. In this respect, the p53-inducible
proteins Bax and IGF-binding protein-3 have been shown to be capable of
inducing apoptosis. The inhibition of p53-mediated apoptosis in
vivo potentiates the rate at which a tumor progresses to the stage
of malignancy, and this is thought to be a major reason why p53 is so
often mutated in human cancers.
The cytokine IL-3 is a potent inhibitor of p53-mediated apoptosis in
erythroleukemia cells (32). In addition, the IL-3-mediated activation
of JAK kinase is sufficient to protect myeloid cells from p53-mediated
apoptosis in response to In light of these findings, it was of interest to determine if the
PI3K-PKB/Akt signaling pathway was capable of conferring protection
from apoptosis induced by p53. To this end, we employed a well
characterized cell culture system in which apoptosis is exclusively
p53-dependent (35-38). Using this cell culture system, we
demonstrate that both constitutively active PI3K and PKB/Akt compromise
the onset of p53-mediated apoptosis. Moreover, we find that this effect
is mediated in the absence of changes in expression of Bcl-2, Bcl-Xl,
and the pro-apoptotic protein Bax. These results provide the first
unambiguous and compelling evidence that the PI3K-PKB/Akt survival
pathway can protect from apoptosis induced by the p53 tumor suppressor protein.
Cell Lines and Tissue Culture--
The p53A and 4B cell lines
have been described (35, 37, 38). Briefly, both cell lines were
established from primary epithelial BRK cells transfected with genes
encoding adenovirus E1A and murine tsp53(Val-135), which is
predominantly in the mutant confirmation at the restrictive temperature
of 38.5 °C and predominantly in the wild-type confirmation at the
permissive temperature of 32 °C. The p53A cell line proliferates at
the restrictive temperature of 38.5 °C, but undergoes p53-mediated,
transcriptionally dependent apoptosis at the permissive temperature of
32 °C. The 4B cell line also constitutively expresses an ectopically
introduced human BCL-2 gene and proliferates at the
restrictive temperature of 38.5 °C. The 4B cell line is rescued from
p53-mediated apoptosis at the permissive temperature of 32 °C and
instead undergoes p53-mediated growth arrest predominantly in the
G2/M phase of the cell cycle.
The LXSN, Akt-1, and 110-1 cell lines were generated as follows. p53A
cells were seeded at 2.5 × 105 cells/35-mm dish. Twenty-four hours later, 1 ml of supernatant from the GP+E ecotropic packaging cell
line expressing empty retrovirus vector (LXSN), oncogenic v-Akt
(LXSN-v-Akt) (80), or the constitutively active p110 subunit of PI3K
(LXSN-p110caaxMyc) was added to the cells in the presence of
8 µg/ml Polybrene (Sigma), and the cells were allowed to incubate at
38.5 °C for 6 h. The supernatants were then aspirated, and the
cells were replenished with fresh growth medium and allowed to incubate
at 38.5 °C for 24 h. Cells were treated with G418 (Life
Technologies, Inc.) at a concentration of 0.5 mg/ml and maintained in
this selection medium until a proliferating pool of cells was obtained.
All BRK-derived cell lines were maintained at 38.5 °C in Dulbecco's
modified Eagle's medium (high glucose) + 5% fetal bovine serum.
Western Blotting and Antibodies--
Twenty-five micrograms of
whole cell extracts prepared from cell lines incubated at 38.5 and
32 °C were subjected to SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membranes (Millipore Corp.,
Bedford, MA) using standard procedures. Immunoblotting was performed
with the following antibodies: polyclonal anti-Bax (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), polyclonal anti-Bcl-Xl
(Calbiochem-Novabiochem), monoclonal anti-Bcl-2 (Transduction Laboratories, Lexington, KY), monoclonal anti-actin (Amersham Pharmacia
Biotech), polyclonal
anti-p110,2 and polyclonal
anti-PKB/Akt (Upstate Biotechnology, Inc.).
Plasmid Transfections--
Where indicated, cells growing in log
phase at 38.5 °C were transfected with 0.5 µg of Glu-Glu-tagged
PKB/Akt using Effectene reagent (QIAGEN Inc.) according to the
manufacturer's protocol. Twenty-four hours later, the Glu-Glu-tagged
PKB/Akt construct was immunoprecipitated and used in a PKB/Akt in
vitro kinase assay as described below.
Immunoprecipitations and PKB/Akt in Vitro Kinase
Assay--
Cells were lysed in ice-cold lysis buffer (0.5% Nonidet
P-40 in 1× phosphate-buffered saline (pH, 7.2), 10 mM
EDTA, 1 mM Na3VO4, 50 mM NaF, 1 µM leupeptin, 0.1 µM
aprotinin, and 0.5 mM benzamidine). After clearing the
lysate by centrifugation (14,000 rpm for 10 min at 4 °C), 200 µg
of total protein were immunoprecipitated with either 2 µl of
polyclonal antiserum generated against full-length recombinant PKB/Akt
(1prd1) or antibody raised against full-length Glu-Glu-tagged PKB/Akt
expressed in Sf9 cells. Immunoprecipitations were at 4 °C for
1 h. Immunocomplexes were then washed four times in cold lysis
buffer and subjected to an in vitro kinase assay as
described using crosstide as a substrate (4, 5). A 16% Tricine gel
(Novex) was used to separate the substrate peptide from free
[32P]ATP. Activity was then analyzed using a Storm
PhosphorImager (Molecular Dynamics, Inc.).
Viability and DNA Fragmentation Analysis--
Cells were plated
out at 106 cells/10-cm plate at 38.5 °C. Forty hours
post-plating, plates were shifted to 32 °C, and cell viability was
determined at 0, 12, 24, 48, and 72 h by trypan blue exclusion.
The 4B cell line was used as a positive control for viability at
32 °C (37). The viable number of cells at each time point is
represented as a percentage of viable cells relative to that at time 0. Low molecular weight DNA was prepared and analyzed in parallel with the
viability assay using procedures described previously (81). Samples
were normalized by loading low molecular weight DNA prepared from an
equal number of viable cells.
FACS Analysis--
Cells were propagated at 38.5 °C or were
shifted to 32 °C and cultured for 0, 12, 24, 48, and 72 h as
indicated. Cells were then harvested, fixed, and stained with propidium
iodide using methods previously described (82). Fluorescence
intensities were determined by quantitative flow cytometry, and
profiles were generated on a FACScan profile analyzer (Becton Dickinson).
Constitutively Active PI3K and PKB/Akt Delay the Onset of
p53-mediated Apoptosis--
The p53A cell line was generated from
primary BRK cells transformed by adenovirus E1A and murine
tsp53(Val-135) (35, 37, 38). p53A proliferates at the restrictive
temperature of 38.5 °C, but succumbs to p53-mediated,
transcriptionally dependent apoptosis at the permissive temperature of
32 °C. Therefore, apoptosis induced in the p53A cell line upon
incubation at 32 °C is entirely p53-dependent.
Derivatives of the p53A cell line that express either constitutively
active PI3K (110-1) or PKB/Akt (Akt-1) were generated from pooled
populations of G418-resistant p53A cells infected with the
LXSN-p110caaxMyc and LXSN-v-Akt retroviruses, respectively.
The LXSN cell line was generated in parallel from pooled populations of
p53A cells infected with the LXSN retrovirus alone and therefore serves
as an appropriate negative control. All three cell lines were
maintained at 38.5 °C. Western blot analysis indicated that the
Akt-1 and 110-1 cell lines expressed the v-Akt and
p110caaxMyc proteins, respectively, whereas neither protein
was expressed in the LXSN control cell line (Fig.
1). Moreover, PKB/Akt activity was
significantly higher in the Akt-1 and 110-1 cell lines compared with
the LXSN control cell line, thus demonstrating that the
p110caaxMyc and v-Akt proteins are catalytically intact
(Fig. 1).
To determine if constitutively active PI3K and PKB/Akt can abrogate
p53-mediated apoptosis, the Akt-1 and 110-1 cell lines were plated out
at 38.5 °C and 24 h later were incubated at 32 °C for 12, 24, 48, and 72 h. The 4B cell line has been described and is a
derivative of the p53A cell line, which constitutively expresses an
ectopically introduced BCL-2 proto-oncogene (37). The 4B
cell line is rescued from p53-mediated apoptosis at 32 °C and
instead undergoes p53-mediated growth arrest. The 4B cell line was
therefore used as a positive control for the inhibition of p53-mediated
apoptosis at 32 °C.
The p53A parental and LXSN control cell lines underwent a significant
decrease in viability by 12 h at 32 °C (Fig.
2A). The viability of both
lines progressively decreased at a comparable rate at subsequent time
points, and by 72 h at 32 °C, both the p53A and LXSN cell lines
were essentially nonviable. The onset of p53-mediated cell death in the
Akt-1 and 110-1 cell lines was significantly delayed by comparison in
that a net decrease in cell viability was not evident in either line
until after 24 h at 32 °C (Fig. 2A). Interestingly,
the decrease in viability observed in the Akt-1 and 110-1 cell lines
between 24 and 48 h at 32 °C was dramatic and occurred with
accelerated kinetics compared with the p53A and LXSN control lines. The
viability of the 4B cell line remained relatively constant at all time
points, as previously reported (37). The delayed onset of cell death
observed in the Akt-1 and 110-1 cell lines could be due to either an
inhibition of apoptosis or a balance between cell growth and cell
death. Therefore, additional experiments were performed to distinguish between these two possibilities.
The degradation of genomic DNA into small molecular weight
oligonucleosome-sized fragments is a characteristic of cells that succumb to apoptotic cell death (39). Moreover, this hallmark sign of
apoptosis is observed in the p53A cell line subsequent to incubation at
32 °C (35, 37, 38). In contrast, the 4B cell line, which is rescued
from p53-mediated apoptosis at the permissive temperature, displays no
sign of low molecular weight DNA at either 38.5 or 32 °C (37). The
absence of small molecular weight DNA is thus synonymous with
protection from p53-mediated apoptosis. It was therefore of interest to
determine if the delay in cell death observed in the Akt-1 and 110-1 cell lines was paralleled by the delayed kinetics in DNA degradation.
To this end, small molecular weight DNA from an equal number of viable
cells was prepared from each cell line in parallel with the viability
assay and analyzed for the presence of apoptotic DNA fragmentation.
DNA fragmentation in the characteristic nucleosomal pattern was evident
in both the p53A and LXSN cell lines by 12 h at the permissive
temperature and increased dramatically at later time points (Fig.
2B). These results reflect the swift and progressive decrease in cell viability observed in both lines at 32 °C. In contrast, the 4B cell line, which is rescued from p53-mediated apoptosis at 32 °C, was devoid of DNA degradation at all time points. With respect to the Akt-1 and 110-1 cell lines, DNA degradation was undetectable at the 0-, 12-, and 24-h time points when cell viability was maintained (Fig. 2B). These results indicate
that the preservation of cell viability observed in the Akt-1 and 110-1 cell lines at early time points subsequent to incubation at 32 °C is
due to the inhibition of p53-mediated apoptosis.
The percentage of apoptotic cells within a given population can be
quantitated by labeling cells with propidium iodide, subjecting them to
FACS analysis, and calculating the percentage of cells with a
sub-G1 DNA content. p53-mediated apoptosis in BRK cell lines transformed by E1A and tsp53(Val-135) is paralleled by a rapid
and dramatic increase in cells with a sub-G1 DNA content (35). Therefore, to quantitate the delayed kinetics of apoptosis observed in the Akt-1 and 110-1 cell lines, both lines were labeled with propidium iodide at 38.5 and 32 °C and subjected to FACS analysis. The population of cells with a sub-G1 DNA content
was then calculated and represented as a percentage of the total cell population.
The 4B cell line was virtually devoid of cells with a
sub-G1 DNA content at both 38.5 and 32 °C (Fig.
3). Moreover, the 4B cell line gradually
underwent a p53-mediated G2/M cell cycle arrest by 72 h at 32 °C, as previously observed (34). The population of cells
with a sub-G1 DNA content present in both the p53A and LXSN
cell lines had increased dramatically by 12 h at 32 °C and continued to increase at subsequent time points (Fig. 3). These results
correspond to the rapid kinetics of DNA degradation and cell death
observed in the p53A and LXSN cell lines upon incubation at 32 °C
(Fig. 2A). In contrast, neither the Akt-1 nor the 110-1 cell
line showed any significant increase in the percentage of cells with a
sub-G1 DNA content for up to 24 h at 32 °C. These results correspond to the delayed kinetics in p53-mediated DNA degradation and cell death observed in both lines. In addition, these
results further confirm the fact that both constitutively active PI3K
and PKB/Akt compromise the onset of apoptosis induced by p53, and, in
turn, demonstrate that this is a quantitatively significant event.
The Delayed Onset of p53-mediated Apoptosis Induced by
Constitutively Active PKB/Akt Is Not Due to Changes in the Levels of
Bax, Bcl-2, or Bcl-Xl--
In BRK cell lines transformed by E1A and
tsp53(Val-135), Bax is transcriptionally up-regulated by p53 at
32 °C, and this is sufficient to induce apoptosis (40). In addition,
in certain cell types, the survival function of the PI3K-PKB/Akt
signaling pathway is due, in part, to the PKB/Akt-mediated
up-regulation of Bcl-2 (20, 41). Bcl-2 inhibits apoptosis induced by a
variety of stimuli and can function as an oncogene by inhibiting
apoptosis induced by p53 (37, 42). The anti-apoptotic protein Bcl-Xl is
a homologue of Bcl-2 and is also capable of inhibiting p53-mediated apoptosis (43). In light of these findings, it was of interest to
determine if the delayed onset of p53-mediated apoptosis induced by
constitutively active PKB/Akt was due to attenuation of p53-mediated Bax induction or to increased expression of Bcl-2 or Bcl-Xl. To this
end, whole cell extracts were prepared from the Akt-1 cell line at
38.5 °C and at 12 and 24 h at 32 °C when all signs of p53-mediated apoptosis in the Akt-1 line were abrogated. Extracts were
then subjected to Western blot analysis for detection of Bax, Bcl-2,
and Bcl-Xl.
Bax protein levels in the LXSN cell line had increased 2-3-fold by
12 h at 32 °C (Fig. 4). This is
consistent with the fact that bax is a p53-inducible gene in
BRK cell lines transformed by E1A and tsp53(Val-135) (40, 44, 45). At
the 24-h time point, Bax protein levels in the LXSN cell line had
decreased to basal levels, possibly as a result of protein degradation. The p53-mediated up-regulation of Bax was intact in the Akt-1 cell
line, as Bax protein levels had increased significantly by 12 h at
32 °C (Fig. 4). The levels of Bax expressed in the LXSN and Akt-1
cell lines at the 12-h time point were comparable, despite the fact
that the Akt-1 cell line was completely protected from p53-mediated
apoptosis. Moreover, Bax protein levels in the Akt-1 cell line
continued to increase up to 24 h at 32 °C, although cell
viability was still completely maintained (Fig. 4). Bcl-2 protein
levels were detectable in the LXSN cell line and remained constant at
all time points (Fig. 4). Bcl-2 was also detectable at both
temperatures in the Akt-1 line, but the level of expression appeared to
decrease somewhat upon incubation at 32 °C. The Bcl-Xl protein was
detectable in both the LXSN and Akt-1 cell lines and was expressed at a
similar level at corresponding time points (Fig. 4). Thus, the delayed
kinetics of apoptosis observed in the Akt-1 cell line cannot be
explained by a corresponding delay in the p53-mediated up-regulation of
Bax or by an increased expression of Bcl-2 or Bcl-Xl. However, it is
possible that PI3K and PKB/Akt may regulate the function of these
proteins through some other mechanism that has yet to be
identified.
We demonstrate in this report that both PI3K and PKB/Akt are
capable of compromising the onset of apoptosis induced exclusively by
the tumor suppressor protein p53. This was manifested as a significant
delay in the kinetics of DNA degradation and cell death as well as a
profound attenuation in the accumulation of cells with a
sub-G1 DNA content. The protection from p53-mediated apoptosis conferred by PI3K and PKB/Akt was not permanent, as both the
Akt-1 and 110-1 cell lines ultimately succumbed to cell death at the
permissive temperature. One possible explanation for this observation
centers around the mechanism by which p53-mediated, transcriptionally
dependent apoptosis is induced.
p53-mediated apoptosis in BRK cell lines transformed by E1A and
tsp53(Val-135) is transcriptionally dependent and is triggered by a
class of enzymes known as caspases (44, 46). Caspases are a family of
aspartate-specific proteases that induce apoptosis by cleaving and
inactivating cellular substrates, which play an essential role in
maintaining cell viability (47). Apoptosis in mammalian cells results
from the activation of caspases in a cascade-like fashion, with
initiator caspases lying at the apex of the cascade and effector
caspases lying farther downstream (48, 49). Caspase-9 is an initiator
caspase that becomes activated by the release of cytochrome
c from mitochondria in response to many apoptotic stimuli
(reviewed in Ref. 50). Recent evidence indicates that PKB/Akt can
phosphorylate and inactivate caspase-9 and thereby abrogate
caspase-9-mediated apoptosis (51). Thus, in certain cell types, PKB/Akt
may abrogate apoptosis through the direct inhibition of caspases.
Constitutively active PKB/Akt was capable of abrogating the early
stages of p53-mediated apoptosis, but was incapable of conferring
protection at later time points (Fig. 2). Therefore, it is tempting to
speculate that the early stages of p53-mediated, transcriptionally
dependent apoptosis are triggered by initiator caspases such as
caspase-9 that are inhibitable by PKB/Akt. A role for capsase-9 in
p53-mediated apoptosis per se is implicated by the
observation that dominant-negative caspase-9 constructs can inhibit
apoptosis induced by E1A and Bax (52, 53). Moreover, a recent study
indicates that the inactivation of caspase-9 can substitute for p53
loss in permitting the oncogenic transformation of primary mouse embryo
fibroblasts by c-Myc (54). Experiments to determine if caspase-9 is
activated at the permissive temperature in BRK cell lines transformed
by E1A and tsp53(Val-135) are in progress.
The pro-apoptotic protein Bad antagonizes the anti-apoptotic function
of Bcl-2 and Bcl-Xl by forming inactivating Bad-Bcl-2 and Bad-Bcl-Xl
heterodimers (55). Bad has recently been shown to be a target of
PKB/Akt-mediated phosphorylation, and the phosphorylation of Bad by
PKB/Akt prevents Bad from heterodimerizing with Bcl-2 and Bcl-Xl
(55-57). When uncomplexed with Bad, Bcl-2 and Bcl-Xl are capable of
abrogating Bax-mediated apoptosis through the formation of
Bcl-2/Bcl-Xl-Bax heterodimers (42, 55). Bax is a transcriptional target
of p53 in BRK cell lines transformed by E1A and tsp53(Val-135), and its
expression is sufficient to induce apoptosis (40). Therefore, by
phosphorylating Bad and potentiating the interaction of Bcl-2 and
Bcl-Xl with Bax, PKB/Akt could conceivably protect from apoptosis induced by p53. However, Bad expression was completely undetectable in
the Akt-1 cell line at both 38.5 and 32 °C (data not shown). Therefore, the delayed onset of p53-mediated apoptosis observed in the
Akt-1 line cannot be explained by the PKB/Akt-mediated phosphorylation
of Bad. This result is not unexpected, as Bad has a very restricted
pattern of tissue expression (58). Moreover, the IL-4-mediated survival
of myeloid cells is paralleled by the activation of PKB/Akt in the
absence of Bad phosphorylation (59). Thus, PKB/Akt does not need to
phosphorylate Bad to protect from apoptosis induced by either IL-4
deprivation or p53. The possibility that PKB/Akt compromises the onset
of p53-dependent apoptosis by phosphorylating Bad-like
proteins, however, cannot be discounted at this time.
We have demonstrated that PI3K and PKB/Akt can promote cell survival by
compromising the kinetics of apoptosis induced by p53. It would
therefore be of interest to determine if PI3K-PKB/Akt-mediated survival
can restrict the efficacy of anticancer interventions that function by
triggering p53-induced apoptosis. Indeed, p53 has been shown to mediate
apoptosis in response to The abrogation of p53-mediated apoptosis through inactivating mutations
represents an important driving force in tumor development (68-70).
However, the inactivation of p53 is typically not involved in tumor
initiation, as it is frequently observed to be a late-onset event in
human cancers (71). Thus, there may exist other oncogenic mechanisms
that function to modulate the impact of p53-mediated apoptosis at the
early stages of cancer development. The transformation of colorectal
epithelium to carcinomas is associated with a progressive inhibition of
apoptosis, and p53 inactivation occurs near the transition from benign
to malignant growth (72). In contrast, Ras mutations occur most often
during the early adenomatous stage of the disease (73). Thus, it is
conceivable that Ras-mediated activation of the PI3K-PKB/Akt survival
pathway may function to limit the extent of apoptosis induced by p53
during the early premalignant stages of colon cancer. This, in turn,
would facilitate the progression of tumor growth until p53-mediated
apoptosis is completely abrogated by inactivating mutations. Ras is
capable of suppressing p53-mediated apoptosis in BRK cell lines
transformed by E1A and tsp53(Val-135), and the PI3K-PKB/Akt survival
pathway is one of many signaling pathways activated downstream of Ras (34, 74-79). It would be interesting to determine how these other signaling pathways influence PI3K-PKB/Akt-mediated protection from
apoptosis induced by p53, as this might help to explain how Ras
functions as an oncogene in vivo.
We thank Dr. Eileen White for the p53A and 4B
cell lines; Drs. David Stokoe, Pablo Rodriguez-Viciana, and Wayne
Zundel for retroviruses, plasmids, and helpful technical advice; Dr.
Lena Claesson-Welsh for the polyclonal anti-p110 antibody; and Dr. Art
Alberts for critical reading of the manuscript.
*
This work was supported in part by the Daiichi Cancer
Research Program.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.
2
R. Hooshmand-Rad, et al., manuscript
in preparation.
The abbreviations used are:
PI3K, phosphoinositide 3-OH kinase;
IGF, insulin-like growth factor;
IL, interleukin;
BRK, baby rat kidney;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
FACS, fluorescence-activated cell sorter.
Phosphoinositide 3-OH Kinase (PI3K) and PKB/Akt Delay the Onset
of p53-mediated, Transcriptionally Dependent Apoptosis*
and
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-radiation (33). IL-3 is also a well
established activator of the PI3K-PKB/Akt survival pathway (19).
Moreover, p53-mediated apoptosis in baby rat kidney (BRK) cell lines
transformed by E1A and tsp53(Val-135) (where ts is
temperature-sensitive) is suppressed by oncogenic Ras (34). These
findings indicate that p53-mediated apoptosis is inhibitable under
conditions in which the PI3K-PKB/Akt survival pathway is activated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of the Akt-1 and 110-1 cell
lines. A: upper panel, equal quantities of
whole cell lysates from the LXSN control and Akt-1 cell lines were
subjected to Western blot analysis for expression of v-Akt protein
using anti-Akt antibody (C-12); lower panel, shown is
PKB/Akt kinase activity in the LXSN and Akt-1 cell lines. Nonidet P-40
lysates from the LXSN and Akt-1 cell lines were immunoprecipitated with
anti-Akt antibody (1prd1), and the immunoprecipitates were subjected to
an in vitro kinase assay using crosstide as a substrate.
B: upper panel, equal quantities of whole cell
lysates from the LXSN and 110-1 cell lines were subjected to Western
blot analysis for expression of p110caaxMyc using anti-p110
antibody; lower panel, LXSN and 110-1 cell lines were
transfected at 38.5 °C with 0.5 µg of Glu-Glu-tagged PKB/Akt.
Twenty-four hours later, Nonidet P-40 lysates from transfected cells
were immunoprecipitated with an antibody raised against full-length
Glu-Glu-tagged PKB/Akt expressed in Sf9 cells. The
immunoprecipitates were then subjected to an in vitro kinase
assay using crosstide as a substrate.
View larger version (23K):
[in a new window]
Fig. 2.
Constitutively active PI3K and PKB/Akt
compromise the onset of p53-mediated cell death and DNA
fragmentation. A, the viable cell number was determined
for the p53A, LXSN, Akt-1, and 110-1 cell lines and the 4B control cell
line for 72 h at 32 °C by trypan blue exclusion and is
expressed as the percentage of the original viability at the time of
shift to 32 °C. B, low molecular weight DNA was prepared
and analyzed in parallel with the viability assay in A using
procedures described previously (38). DNA molecular weight
(MW) markers (50-10,000 base pairs) were from Bionexus,
Inc.
View larger version (25K):
[in a new window]
Fig. 3.
Akt-1 and 110-1 cell lines display a delay in
the appearance of cells with a sub-G1 DNA content upon
incubation at 32 °C. The p53A and 4B cell lines were used as
positive and negative controls, respectively, for the appearance of
cells with a sub-G1 DNA content upon incubation at 32 °C
(35, 37, 38). The x axis represents relative
fluorescence intensity, which is proportional to DNA content. The
y axis represents forward light scatter, which is
proportional to cell number. The sub-G1 DNA content is
indicative of apoptotic cell death. The percentage of cells with a
sub-G1 DNA content is represented numerically in each box.
The positions of sub-G1, G1, S, and
G2/M DNA contents are indicated.
View larger version (31K):
[in a new window]
Fig. 4.
Protection from p53-mediated apoptosis in the
Akt-1 cell line cannot be explained by changes in the levels of
expression of Bax, Bcl-2, or Bcl-Xl. Whole cell extracts were
prepared from the LXSN control and Akt-1 cell lines at 0, 12, and
24 h at 32 °C in parallel with the viability assay described in
the legend to Fig. 2A. Twenty-five micrograms of whole cell
extract from each cell line were subjected to SDS-polyacrylamide gel
electrophoresis and transferred to polyvinylidene difluoride membrane
using standard procedures. Immunoblotting was performed with polyclonal
antibodies specific for Bax and Bcl-Xl and monoclonal antibodies
specific for Bcl-2 and actin. The percent apoptosis at each time point
represents data taken from the viability assay depicted in Fig.
2A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-radiation and certain chemotherapeutic
reagents (60-64). This concern may be especially relevant in the
treatment of malignancies such as ovarian cancer, in which the
PI3K-PKB/Akt survival pathway has been shown to be aberrantly activated
(65-67).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 415-502-1720;
Fax: 415-502-3179; E-mail: sabbatini@cc.ucsf.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 D. N. Everly Jr., S. Kusano, and N. Raab-Traub Accumulation of Cytoplasmic {beta}-Catenin and Nuclear Glycogen Synthase Kinase 3{beta} in Epstein-Barr Virus-Infected Cells J. Virol., November 1, 2004; 78(21): 11648 - 11655. [Abstract] [Full Text] [PDF]
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 O. David, J. Jett, H. LeBeau, G. Dy, J. Hughes, M. Friedman, and A. R. Brody Phospho-Akt Overexpression in Non-Small Cell Lung Cancer Confers Significant Stage-Independent Survival Disadvantage Clin. Cancer Res., October 15, 2004; 10(20): 6865 - 6871. [Abstract] [Full Text] [PDF]
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J. Feng, R. Tamaskovic, Z. Yang, D. P. Brazil, A. Merlo, D. Hess, and B. A. Hemmings Stabilization of Mdm2 via Decreased Ubiquitination Is Mediated by Protein Kinase B/Akt-dependent Phosphorylation J. Biol. Chem., August 20, 2004; 279(34): 35510 - 35517. [Abstract] [Full Text] [PDF]
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H. A. Ferguson, P. M. Marietta, and C. L. Van Den Berg UV-induced Apoptosis Is Mediated Independent of Caspase-9 in MCF-7 Cells: A MODEL FOR CYTOCHROME c RESISTANCE J. Biol. Chem., November 14, 2003; 278(46): 45793 - 45800. [Abstract] [Full Text] [PDF]
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J. D. Su, L. D. Mayo, D. B. Donner, and D. L. Durden PTEN and Phosphatidylinositol 3'-Kinase Inhibitors Up-Regulate p53 and Block Tumor-induced Angiogenesis: Evidence for an Effect on the Tumor and Endothelial Compartment Cancer Res., July 1, 2003; 63(13): 3585 - 3592. [Abstract] [Full Text] [PDF]
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 Y. Lin, L. Brown, D. W. Hedley, D. L. Barber, and S. Benchimol The death-promoting activity of p53 can be inhibited by distinct signaling pathways Blood, December 1, 2002; 100(12): 3990 - 4000. [Abstract] [Full Text] [PDF]
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P. Watcharasit, G. N. Bijur, J. W. Zmijewski, L. Song, A. Zmijewska, X. Chen, G. V. W. Johnson, and R. S. Jope Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage PNAS, June 11, 2002; 99(12): 7951 - 7955. [Abstract] [Full Text] [PDF]
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Y. Ogawara, S. Kishishita, T. Obata, Y. Isazawa, T. Suzuki, K. Tanaka, N. Masuyama, and Y. Gotoh Akt Enhances Mdm2-mediated Ubiquitination and Degradation of p53 J. Biol. Chem., June 7, 2002; 277(24): 21843 - 21850. [Abstract] [Full Text] [PDF]
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 B. Singh, P. G. Reddy, A. Goberdhan, C. Walsh, S. Dao, I. Ngai, T. C. Chou, P. O-charoenrat, A. J. Levine, P. H. Rao, et al. p53 regulates cell survival by inhibiting PIK3CA in squamous cell carcinomas Genes & Dev., April 15, 2002; 16(8): 984 - 993. [Abstract] [Full Text] [PDF]
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M. W. Mayo, L. V. Madrid, S. D. Westerheide, D. R. Jones, X.-J. Yuan, A. S. Baldwin Jr., and Y. E. Whang PTEN Blocks Tumor Necrosis Factor-induced NF-kappa B-dependent Transcription by Inhibiting the Transactivation Potential of the p65 Subunit J. Biol. Chem., March 22, 2002; 277(13): 11116 - 11125. [Abstract] [Full Text] [PDF]
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 J. C. Wolfman, T. Palmby, C. J. Der, and A. Wolfman Cellular N-Ras Promotes Cell Survival by Downregulation of Jun N-Terminal Protein Kinase and p38 Mol. Cell. Biol., March 1, 2002; 22(5): 1589 - 1606. [Abstract] [Full Text] [PDF]
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L. D. Mayo, J. E. Dixon, D. L. Durden, N. K. Tonks, and D. B. Donner PTEN Protects p53 from Mdm2 and Sensitizes Cancer Cells to Chemotherapy J. Biol. Chem., February 8, 2002; 277(7): 5484 - 5489. [Abstract] [Full Text] [PDF]
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B. Zhang, D. F. Spandau, and A. Roman E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis J. Virol., January 1, 2002; 76(1): 220 - 231. [Abstract] [Full Text] [PDF]
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L. D. Mayo and D. B. Donner A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus PNAS, August 10, 2001; (2001) 181181198. [Abstract] [Full Text] [PDF]
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E. Y. Chen, N. M. Mazure, J. A. Cooper, and A. J. Giaccia Hypoxia Activates a Platelet-derived Growth Factor Receptor/Phosphatidylinositol 3-Kinase/Akt Pathway That Results in Glycogen Synthase Kinase-3 Inactivation Cancer Res., March 1, 2001; 61(6): 2429 - 2433. [Abstract] [Full Text]
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 H. Zhou, X.-M. Li, J. Meinkoth, and R. N. Pittman Akt Regulates Cell Survival and Apoptosis at a Postmitochondrial Level J. Cell Biol., October 23, 2000; 151(3): 483 - 494. [Abstract] [Full Text] [PDF]
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 I. J. Gonzalez-Robayna, A. E. Falender, S. Ochsner, G. L. Firestone, and J. S. Richards Follicle-Stimulating Hormone (FSH) Stimulates Phosphorylation and Activation of Protein Kinase B (PKB/Akt) and Serum and Glucocorticoid-Induced Kinase (Sgk): Evidence for A Kinase-Independent Signaling by FSH in Granulosa Cells Mol. Endocrinol., August 1, 2000; 14(8): 1283 - 1300. [Abstract] [Full Text]
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 W. Qian and K. G. Wiman Polyoma Virus Middle T and Small t Antigens Cooperate to Antagonize p53-induced Cell Cycle Arrest and Apoptosis Cell Growth Differ., January 1, 2000; 11(1): 31 - 39. [Abstract] [Full Text]
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R. E. Bachelder, M. J. Ribick, A. Marchetti, R. Falcioni, S. Soddu, K. R. Davis, and A. M. Mercurio p53 Inhibits {alpha}6{beta}4 Integrin Survival Signaling by Promoting the Caspase 3-dependent Cleavage of AKT/PKB J. Cell Biol., November 29, 1999; 147(5): 1063 - 1072. [Abstract] [Full Text] [PDF]
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J.-i. Okano, I. Gaslightwala, M. J. Birnbaum, A. K. Rustgi, and H. Nakagawa Akt/Protein Kinase B Isoforms Are Differentially Regulated by Epidermal Growth Factor Stimulation J. Biol. Chem., September 29, 2000; 275(40): 30934 - 30942. [Abstract] [Full Text] [PDF]
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A.-L. Mathieu, S. Gonin, Y. Leverrier, B. Blanquier, J. Thomas, C. Dantin, G. Martin, G. Baverel, and J. Marvel Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Protects against Interleukin-3 Starvation but Not DNA Damage-induced Apoptosis J. Biol. Chem., March 30, 2001; 276(14): 10935 - 10942. [Abstract] [Full Text] [PDF]
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J. M. Nelson and D. W. Fry Akt, MAPK (Erk1/2), and p38 Act in Concert to Promote Apoptosis in Response to ErbB Receptor Family Inhibition J. Biol. Chem., April 27, 2001; 276(18): 14842 - 14847. [Abstract] [Full Text] [PDF]
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J.-M. Lee, J. M. Hanson, W. A. Chu, and J. A. Johnson Phosphatidylinositol 3-Kinase, Not Extracellular Signal-regulated Kinase, Regulates Activation of the Antioxidant-Responsive Element in IMR-32 Human Neuroblastoma Cells J. Biol. Chem., June 1, 2001; 276(23): 20011 - 20016. [Abstract] [Full Text] [PDF]
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J. A. Gustin, T. Maehama, J. E. Dixon, and D. B. Donner The PTEN Tumor Suppressor Protein Inhibits Tumor Necrosis Factor-induced Nuclear Factor kappa B Activity J. Biol. Chem., July 13, 2001; 276(29): 27740 - 27744. [Abstract] [Full Text] [PDF]
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A. J. Barber, M. Nakamura, E. B. Wolpert, C. E. N. Reiter, G. M. Seigel, D. A. Antonetti, and T. W. Gardner Insulin Rescues Retinal Neurons from Apoptosis by a Phosphatidylinositol 3-Kinase/Akt-mediated Mechanism That Reduces the Activation of Caspase-3 J. Biol. Chem., August 24, 2001; 276(35): 32814 - 32821. [Abstract] [Full Text] [PDF]
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L. D. Mayo and D. B. Donner A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus PNAS, September 25, 2001; 98(20): 11598 - 11603. [Abstract] [Full Text] [PDF]
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