From the Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6160
Received for publication, November 21, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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A comparison of Akt- and
Bcl-xL-dependent cell survival was
undertaken using interleukin-3-dependent FL5.12 cells.
Expression of constitutively active Akt allows cells to survive for
prolonged periods following growth factor withdrawal. This survival
correlates with the expression level of activated Akt and is comparable
in magnitude to the protection provided by the anti-apoptotic gene Bcl-xL. Although both genes prevent cell
death, Akt-protected cells can be distinguished from
Bcl-xL-protected cells on the basis of increased
glucose transporter expression, glycolytic activity, mitochondrial
potential, and cell size. In addition, Akt-expressing cells require
high levels of extracellular nutrients to support cell survival. In
contrast, Bcl-xL-expressing cells deprived of interleukin-3
survive in a more vegetative state, in which the cells are smaller,
have lower mitochondrial potential, reduced glycolytic activity, and
are less dependent on extracellular nutrients. Thus, Akt and
Bcl-xL suppress mitochondrion-initiated apoptosis by
distinct mechanisms. Akt-mediated survival is dependent on promoting
glycolysis and maintaining a physiologic mitochondrial potential. In
contrast, Bcl-xL maintains mitochondrial integrity in the
face of a reduced mitochondrial membrane potential, which develops as a
result of the low glycolytic rate in growth factor-deprived cells.
There is increasing evidence that tissue homeostasis in
multicellular organisms is controlled by the availability of growth factors (1). Within a given tissue, high levels of relevant growth
factors promote increased cellular mass, metabolism, and proliferation.
In contrast, when the availability of growth factor becomes limiting,
cellular atrophy and an increased rate of apoptosis are observed. Many
growth factors affect cellular responses through receptor-mediated
recruitment and activation of the phosphoinositide 3-kinase
(PI3K)1 and the
serine/threonine kinase Akt (2, 3). Once activated, Akt can
phosphorylate substrates involved in controlling a variety of cellular
processes, including cellular metabolism and survival (4). The PI3K/Akt
pathway is an important regulator of cellular homeostasis in
vivo, as activating mutations of this pathway are correlated with
multiple types of cancer. Akt itself was first identified as
a viral oncogene, and deficiency in PTEN, an inositol phosphatase that
opposes the activity of PI3K, is frequently found in numerous types of
advanced cancers (5-7).
Akt activation by growth factor receptors prevents apoptosis by
blocking the release of cytochrome c (8). A number of
molecular targets for the inhibition of apoptosis by Akt have been
proposed. Akt phosphorylates and inactivates pro-apoptotic proteins,
including Bad and Forkhead family transcription factors (9, 10). In addition, Akt has been reported to stimulate the expression of anti-apoptotic Bcl-2 proteins, such as Bcl-xL and Mcl-1,
through the activation of NF- Members of the Bcl-2 family are attractive Akt targets, as they have
been shown to be potent regulators of apoptosis following growth factor
withdrawal. Transgenic overexpression of anti-apoptotic family members
prevents the induction of programmed cell death and leads to an
accumulation of cells, whereas transgenic overexpression of
pro-apoptotic family members can result in decreased cell numbers within an organ (13, 14). Anti-apoptotic Bcl-2 family proteins, such as
Bcl-xL, are localized to the outer mitochondrial membrane and function to maintain mitochondrial homeostasis upon growth factor
withdrawal. Bcl-xL and Bcl-2 have been reported to promote mitochondrial homeostasis by promoting continued transport of metabolites across the outer membrane, despite decreases in cellular metabolism (15, 16). In the absence of growth factor,
Bcl-xL facilitates cell survival by preserving cellular ATP
production following the decrease in glycolysis that accompanies growth
factor withdrawal.
Since both Akt and Bcl-xL can regulate cell survival and
cellular metabolism, we have compared the bioenergetic properties of
growth factor-deprived FL5.12 cells expressing either Akt or Bcl-xL. FL5.12 cells are nontransformed pro-B cells that
depend on IL-3 for survival and proliferation. The results demonstrate that cells expressing Akt or Bcl-xL maintain distinct
metabolic states in the absence of growth factor. Akt expression
sustains sufficient glucose uptake and glycolysis to maintain a
physiologic mitochondrial membrane potential in growth factor withdrawn
cells. In contrast, Bcl-xL promotes the survival of growth
factor-deprived cells by maintaining mitochondrial integrity and
function, despite a decrease in mitochondrial potential that results
from a decline in glucose-derived substrates. These differences in
cellular metabolism suggest that there are fundamental differences in
the mechanisms by which Akt and Bcl-xL promote cell survival.
Cell Culture--
FL5.12 cells were cultured as described
previously (17). Myristoylated Akt (mAkt)
gene expression was induced for 18 h with 1 µg/ml of doxycycline
(Sigma) treatment. Cell volume measurements were made using a Coulter
Z2 instrument (Beckman Coulter). Where indicated, wortmannin
(Calbiochem) and LY294002 (Sigma) were added to cultures at final
concentrations of 100 nM and 10 µM,
respectively. Glucose- and glutamine-free medium was made using the
RPMI 1640 Select Amine Kit (Life Technologies, Inc.) and 10% dialyzed
fetal bovine serum (Life Technologies, Inc.). Glucose and glutamine were supplemented to final concentrations as indicated.
Plasmid Constructs and Retroviral
Transductions--
pUHD172-1Neo expressing rtTA was generously
provided by J. Leiden, Abbott. Akt constructs were generously provided
by N. Hay, University of Illinois, Chicago. mAkt was hemagglutinin
(HA)-tagged at the C terminus, and both mAkt-HA and K179M Akt were
cloned into pRevTRE (pRT, CLONTECH).
Bcl-xL was ligated into pBabeMN-IRESGFP (Bcl-xL1, generously provided by G. Nolan, Stanford
University) or pRT (Bcl-xL2). Retroviral expression vectors
were virally transduced using the Phoenix packaging cell line
(generously provided by G. Nolan) as described previously (18).
Briefly, target cells were combined with the retrovirus and 4 µg/ml
hexadimethrine bromide (Sigma), spun at 2500 rpm for 1.5 h, and
selected with 3 mg/ml hygromycin (CLONTECH). Cell
populations were cloned by limiting dilution. Three independently
isolated clones expressing the epitope-tagged mAkt in an inducible
manner (mAkt1, mAkt2, and mAkt3) were obtained and subjected to further study.
Protein Expression--
Lysates from cell lines were
standardized for protein content and separated by SDS-polyacrylamide
gel electrophoresis (Invitrogen). Blots were probed with either rabbit
anti-Bcl-xL (13.6) (19), rabbit anti-Akt (New England
Biolabs), or mouse anti-tubulin (Santa Cruz Biotechnology) and
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Northern Blot--
Total RNA was prepared using TRIZOL (Life
Technologies, Inc.) from cells cultured with or without IL-3 for
14 h. 10 µg of RNA were separated on a 1% formaldehyde agarose
gel and probed with rat Glut1 cDNA (generously provided by M. Birnbaum, University of Pennsylvania). Loading was assessed by
visualizing gels stained with ethidium bromide.
Flow Cytometry--
Cell viability assays were performed using
propidium iodide as described (17). For mitochondrial potential
determination, live cells were enriched by centrifugation over Ficoll,
rested for 1 h, and incubated for 30 min at 37 °C with 200 nM tetramethylrhodamine ethyl ester (Molecular Probes).
Analysis was performed in a FacsCalibur flow cytometer (Becton Dickinson).
Glycolysis and ATP Assays--
After 6 days without IL-3, 1 × 106 Ficoll-enriched live cells were resuspended in
pre-warmed CO2-buffered Krebs solution (115 mM
NaCl, 2 mM KCl, 25 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2,
0.25% bovine serum albumin, pH 7.4) lacking glucose for 30 min at
37 °C. Cells were then washed and resuspended in 500 µl of Krebs buffer containing 10 mM glucose supplemented with 20 µCi/ml [5-3H]glucose (PerkinElmer Life Sciences) for
1 h at 37 °C, and the reaction was stopped by adding equal
volume 0.2 N HCl. 3H2O was
separated from [3H]glucose by evaporative diffusion of
3H2O in a closed chamber, as described (20).
Total ATP levels were determined from 1 × 106 live
cells, enriched by Ficoll, using the ATP bioluminescence assay kit HS
II (Roche Molecular Biochemicals) (15).
Akt is involved in transmitting intracellular signals from growth
factor receptors (21). We sought to determine whether Akt is involved
in IL-3-dependent signal transduction in FL5.12 cells.
FL5.12 cells were deprived of growth factor for 12 h, and recombinant IL-3 was reconstituted for the indicated time intervals. Addition of IL-3 resulted in rapid and transient phosphorylation of
Akt, which was maximal 15 min after IL-3 addition (Fig.
1A). Activation of Akt was
dependent on PI3K, since the PI3K inhibitors wortmannin or LY294002
prevented the induction of Akt phosphorylation (Fig. 1A and
data not shown). Thus, Akt is a component of the proximal signal
transduction machinery of the IL-3 receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (11, 12). However, it is not clear if
these targets entirely account for the effects of Akt on cell survival.
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.
The PI3K/Akt pathway transmits a growth
signal from the IL-3 receptor. A,
FL5.12-Bcl-xL cells were withdrawn from IL-3 for 18 h.
IL-3 was reconstituted in the absence (NO TX) or the
presence (WORT) of wortmannin, as indicated. Phosphorylated
and total Akt levels were assessed in immunoblots (top and
bottom panels, respectively). Lysates from cells
expressing constitutively active myristoylated Akt were loaded as a
positive control (mAkt). B, cellular volume was
determined in cells that had been withdrawn from IL-3 (open
diamonds) or in cells in which IL-3 had been reconstituted in the
absence (closed diamonds) or the presence (closed
squares) of LY294002 (LY). Cell volume is plotted as
the percentage of initial cellular volume of cells growing in
IL-3.
When FL5.12 cells are withdrawn from growth factor, they undergo progressive atrophy until they initiate programmed cell death. Bcl-xL protects from cell death in response to growth factor withdrawal, but it does not prevent cellular atrophy (22). Since the PI3K/Akt pathway is an important regulator of cell size in Drosophila (23), we investigated whether the PI3K/Akt pathway is required for cell growth following IL-3 readdition. Reconstitution of IL-3 in cultures that had been withdrawn from growth factor resulted in rapid recovery of cell size (Fig. 1B). Addition of the PI3K inhibitor LY294002 simultaneously with IL-3 resulted in a delay in the recovery of cell size, indicating that the PI3K pathway regulated cell growth in response to stimulation with IL-3. These data indicate that the PI3K/Akt pathway is important in transmitting cell growth signals in mammalian cells, as has been observed in Drosophila cells (23, 24).
In addition to promoting cell growth, the PI3K/Akt pathway also
transmits cell survival signals from growth factor receptors (2, 25).
To assess the role of Akt in promoting cell survival in FL5.12 cells,
we established cell lines expressing constitutively active, mAkt (26).
Addition of the Src myristoylation sequence to the N terminus of Akt
targets it to the plasma membrane, conferring constitutive activity to
the kinase (27). FL5.12 cells were infected with a retrovirus encoding
an epitope-tagged mAkt under the control of a tetracycline response
element. Three independent clones were isolated which expressed
doxycycline-induced mAkt at a level comparable to the level of total
Akt expressed in parental FL5.12 cells (Fig.
2). In the absence of induction, all
three clones expressed low levels of mAkt. Withdrawal of IL-3 from
wild-type FL5.12 cells resulted in loss of viability within 48 h,
as determined by the ability of cells to exclude propidium iodide (Fig.
2, A and B). All three mAkt clones maintained
viability over 6 days, whereas cells expressing kinase-deficient Akt
died with kinetics similar to wild-type cells (Fig. 2A). As
has been shown previously, cells expressing high and low levels of
Bcl-xL (Bcl-xL1 and 2, respectively) maintained
viability in the absence of growth factor in a
dose-dependent manner (28). After 6 days of growth factor withdrawal, the remaining viable cells could be recovered
quantitatively from mAkt- and Bcl-xL-expressing populations
as assayed by IL-3 readdition and cloning by limiting dilution (data
not shown). Thus, increased viability over 6 days represented true cell
survival over the entire time course. Constitutively active Akt
promotes cell survival in a dose-dependent manner, as
doxycycline addition increased mAkt expression and cell survival
concomitantly (Fig. 2, B and C).
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Akt signal transduction has been implicated in the control of cell
cycle progression by stimulating increased translation of cyclin D
(29). To determine whether Akt-dependent survival following
growth factor withdrawal involved continued cell proliferation, we
assessed the rates of DNA synthesis in cells growing in IL-3 and in
cells that had been withdrawn from IL-3. Incorporation of the
nucleotide analog bromodeoxyuridine (BrdUrd) was similar for all clones
growing in IL-3 (Fig. 3). Following
growth factor withdrawal, cells expressing either mAkt or
Bcl-xL lacked significant levels of DNA synthesis, while
maintaining significant viability (Fig. 3). In contrast, vector control
cells also lack the ability to incorporate BrdUrd, but this reflects
the fact that all these cells have died as indicated by their
sub-diploid DNA content and inability to exclude propidium iodide (Fig.
2 and data not shown). These data suggest that
Akt-dependent survival in FL5.12 cells does not require
cell cycle progression.
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FL5.12 cells undergo progressive atrophy when deprived of growth factor
(22). Since Akt transmits cell growth signals from the IL-3 receptor,
expression of constitutively active Akt could prevent cellular atrophy
upon growth factor withdrawal. Cell size was assessed in cells that had
been withdrawn from IL-3 by measuring forward scatter of equal numbers
of live cells in a flow cytometer. Cells expressing mAkt maintained a
larger cell size in the absence of growth factor, compared with cells
expressing Bcl-xL (Fig. 4).
Cell size correlated with mAkt expression, as cells induced with
doxycycline were larger than uninduced cells. Although cells expressing
mAkt remained larger than Bcl-xL cells, they still atrophied after growth factor withdrawal (data not shown). This indicates that mAkt can transmit a cell growth signal in the absence of
IL-3, but it does not completely substitute for the cell growth and
proliferative signals transmitted from the IL-3 receptor. Thus,
activated Akt both promotes cell survival and prevents cellular atrophy
in the absence of growth factor.
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Cellular atrophy is correlated with decreases in cellular metabolism
and macromolecular synthesis (30-32). Since Akt signaling attenuates
cellular atrophy, the metabolic status of mAkt and Bcl-xL
cells was compared in the absence of growth factor. Total cellular ATP
levels were assessed in cells that had been withdrawn from IL-3. Cells
expressing mAkt contained more ATP than cells expressing
Bcl-xL (Fig. 5), suggesting
that cells expressing mAkt remained metabolically more active than
cells expressing Bcl-xL. Furthermore, the prevention of
cellular atrophy by Akt is correlated with increased metabolism in
FL5.12 cells.
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In insulin receptor signal transduction, Akt regulates several aspects
of glucose uptake, metabolism, and storage (33-35). The elevated
amounts of ATP in mAkt cells may be due in part to the ability of mAkt
to maintain glucose metabolism in the absence of growth factor. Akt can
increase glucose metabolism in response to insulin by inducing the
expression of the glucose transporters in insulin-responsive cell types
(35). In contrast to insulin-responsive tissues, which primarily
express Glut4, the major glucose transporter in lymphocytes is
Glut1.2 Analysis of Glut1
mRNA in cells growing in the presence of IL-3 revealed no
difference in mRNA levels between vector control cells and cells
expressing mAkt or Bcl-xL (Fig.
6A). Following IL-3 withdrawal, Glut1 mRNA was undetectable in vector control and Bcl-xL cells but was still detectable in cells expressing
mAkt (Fig. 6A). In addition to promoting increased Glut1
expression, mAkt sustained an overall increase in glycolysis in the
absence of IL-3 (Fig. 6B). Following growth factor
withdrawal, cells expressing mAkt mediated significantly increased
glycolytic rates compared with cells expressing Bcl-xL
(Fig. 6B). However, constitutively active Akt did not
completely substitute for growth factor receptor signaling, since the
rates of glycolysis in Akt-protected cells were lower than in cells
growing in IL-3.
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Mitochondrial potential is indicative of mitochondrial activity, and
perturbations in inner membrane potential following growth factor
withdrawal have been associated with the commitment to cell death (15).
Therefore, mitochondrial membrane potential was assessed using the
potentiometric dye tetramethylrhodamine ethyl ester (TMRE). Following
IL-3 withdrawal, mitochondrial membrane potential was significantly
reduced in cells expressing Bcl-xL (Fig.
7A). In contrast, there was no
reduction in the mitochondrial potential of cells expressing mAkt
following IL-3 withdrawal. TMRE fluorescence was reflective of
mitochondrial potentials in these cells, since addition of agents that
collapse the mitochondrial potential resulted in a decrease in TMRE
staining (Fig. 7B). Thus, the difference in staining with
TMRE alone was due to the difference in mitochondrial potential in
cells expressing mAkt or Bcl-xL. These data indicate that
cells expressing mAkt contain sufficient electron transport substrates
to maintain mitochondrial potential, despite the absence of growth
factors.
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To survive growth factor withdrawal, cells must maintain cellular
metabolism at a level sufficient to sustain viability. The data
indicate that Akt-expressing cells are metabolically more active than
Bcl-xL-expressing cells in the absence of growth factor, as
determined by total cellular ATP, glycolytic rates, and mitochondrial potentials. One difference between Akt and Bcl-xL is the
ability of Akt to regulate glucose metabolism. Glucose and glutamine
are the major carbon sources for lymphocytes in culture medium (36, 37), and limiting concentrations of these nutrients resulted in
decreased cellular glycolysis (data not shown). To determine whether
the increased rate of glycolysis was necessary for
Akt-dependent survival, we tested cell survival under
conditions of nutrient limitation. As the concentrations of glucose and
glutamine were decreased in the culture medium, a progressive
impairment in the ability of mAkt to promote growth factor-independent
survival was evident (Fig. 8). In
contrast, Bcl-xL cells were unaffected by decreasing
concentrations of carbon sources. Cells expressing mAkt also decreased
in cell size prior to undergoing apoptosis as nutrients became more
limiting (data not shown). These data suggest that Bcl-xL
promotes cell survival by regulating a cell intrinsic effect on
bioenergetics, rather than by regulating glucose uptake or utilization.
In contrast, Akt is reliant on the availability of exogenous nutrients
to promote cell survival.
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DISCUSSION |
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The data indicate that Akt can function to promote both cell growth and cell survival downstream of growth factor receptors. Akt is rapidly phosphorylated following stimulation with IL-3, identifying the kinase as a component of the proximal IL-3 receptor signaling pathway. Inhibition of the PI3K/Akt pathway prevents the rapid induction of cell growth upon re-stimulation of FL5.12 cells with IL-3. Constitutively active Akt both promotes cell survival and attenuates cellular atrophy in the absence of growth factor. Thus, Akt regulates both cell survival and cell size in mammalian cells.
Increases in Akt activity have been shown to result in increases in cyclin D expression, which results in cell cycle progression (29). However, constitutively active Akt does not support IL-3-independent proliferation in FL5.12 cells. Cells expressing Akt are slower to exit the cell cycle than cells expressing Bcl-xL following growth factor withdrawal (data not shown). Nonetheless, cells expressing constitutively active Akt do exit from the cell cycle while maintaining cellular viability. This indicates that mAkt is not sufficient to transform FL5.12 cells and that cellular proliferation is not necessary for Akt-dependent cell survival.
The dual role of Akt in controlling both cell growth and cell survival has been suggested by genetic studies of Akt in Drosophila. Ablation of Akt in embryos results in increases in apoptosis throughout the embryo (38). Overexpression of Akt in eye and wing imaginal discs results in increases in cell size, without increases in cell number (23). Importantly, overexpression of Akt does not increase cellular proliferation and does not overcome cell cycle arrest in the zone of nonproliferating cells in the wing imaginal disc (23). This indicates that Akt can promote increases in cell size without altering cell proliferation in Drosophila, in agreement with the findings in FL5.12 cells.
How can Akt simultaneously control both cell growth and cell survival? The answer may be linked to the control of cellular metabolism. Cellular growth requires the uptake and utilization of energy-rich metabolites. Similarly, to survive growth factor withdrawal, cells must establish a mechanism to sustain their metabolism. It is possible that Akt influences both cell growth and cell survival by sustaining increased cellular bioenergetics. In the absence of growth factor, constitutively active Akt promotes increases in cellular ATP levels, glycolytic rates, and mitochondrial potential, indicating that Akt mediates a global increase in cellular metabolism. This global increase may attenuate the alterations in cellular metabolism that are associated with growth factor withdrawal-induced programmed cell death.
Akt can control cellular metabolism on a number of levels. Here we
report that IL-3 signal transduction is required to maintain Glut1
expression in FL5.12 cells. However, Akt activation is sufficient to
induce Glut1 expression in these cells, even in the absence of IL-3. In
addition to stimulating glucose uptake, Akt also controls glucose
utilization within cells. Constitutively active Akt is sufficient to
increase the overall rate of glycolysis in cells surviving growth
factor withdrawal. Akt may increase glucose utilization by
phosphorylating GSK-3 or PFK-2 (33, 34). The finding that GSK-3
overexpression results in apoptosis lends support to the possibility
that Akt control of glucose metabolism contributes to its ability to
promote cell survival (39).
The ability of Akt to maintain the glycolytic rate of a cell is sufficient to explain how Akt overexpression maintains the mitochondrial membrane potential. A higher glycolytic rate will result in greater substrate availability for mitochondrial electron transport. Consistent with this hypothesis, the ability of Akt to maintain the mitochondrial potential was found to be dependent on glucose. In contrast, Bcl-2 family proteins have been reported to maintain mitochondrial integrity following growth factor withdrawal by facilitating mitochondrial exchange of metabolites and ATP/ADP (15). This allows mitochondria to sustain coupled respiration in the face of a fall in membrane potential that occurs as a result of the decrease in glycolytic substrates.
Biochemical analysis of cells surviving growth factor withdrawal in an Akt-dependent manner indicates that these cells are metabolically more active than their Bcl-xL counterparts. To sustain high levels of metabolism and prevent atrophy, cells expressing Akt are reliant on external sources of energy. Akt cannot promote cell survival in the context of insufficient external energy sources. In contrast, cells that express Bcl-xL can maintain viability even in the context of limited external nutrient sources. Since Akt is a component of growth factor receptor signal transduction, constitutively active Akt may promote survival by transmitting partial cell growth signals that sustain cellular metabolism, whereas Bcl-xL promotes survival by allowing cells to adapt to a reduced metabolic state.
The data suggest that Akt does not promote cell survival solely
by inactivating pro-apoptotic factors. If Akt functioned exclusively as
an anti-apoptotic protein, cells surviving growth factor withdrawal would adopt a low energy phenotype, as do cells expressing
Bcl-xL. The difference in the metabolic requirements of
cells surviving due to Akt or Bcl-xL
indicates cellular contexts in which each gene might be functional.
Bcl-xL can mediate survival under limiting concentrations
of nutrients. Thus, newly transformed cells that have not yet
established an effective blood supply would be better served by
expressing Bcl-xL than by expressing Akt. Vascularized
tumors, in contrast, can survive the absence of specific growth factors
by expressing Akt. These cells would maintain greater levels of
metabolism, which might contribute to their oncogenic potential. The
requirement for a rich source of nutrients may partially explain why
mutations in PTEN, which result in elevations in Akt
activity, are correlated with late stage, aggressive tumors (6, 7). In
contrast, overexpression of Bcl-2 proteins correlates with low grade
tumors with low mitotic indices (40).
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ACKNOWLEDGEMENTS |
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We thank N. Hay (University of Illinois, Chicago) for Akt constructs and F. Matschinsky (University of Pennsylvania) for help with the glycolysis assay. In addition, we thank members of the Thompson laboratory for technical help and reviewing the manuscript, especially Matthew Vander Heiden.
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FOOTNOTES |
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* 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.
Both authors contributed equally to this work.
§ Supported by grants from the Irvington Institute for Immunological Research.
¶ Supported by the Helen Hay Whitney Foundation.
To whom correspondence should be addressed: Dept. of Cancer
Biology, University of Pennsylvania, 421 Curie Blvd., Philadelphia, PA
19104-6160. Tel.: 215-746-5514; Fax: 215-746-5511; E-mail: drt@mail. med.upenn.edu.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M010551200
2 J. C. Rathmell, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PI3K, phosphoinositide 3-kinase; mAkt, myristoylated Akt; TMRE, tetramethylrhodamine ethyl ester; HA, hemagglutinin; IL, interleukin; BrdUrd, bromodeoxyuridine.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Raff, M. C. (1992) Nature 356, 397-400[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Dudek, H.,
Datta, S. R.,
Franke, T. F.,
Birnbaum, M. J.,
Yao, R.,
Cooper, G. M.,
Segal, R. A.,
Kaplan, D. R.,
and Greenberg, M. E.
(1997)
Science
275,
661-665 |
3. | Yao, R., and Cooper, G. M. (1995) Science 267, 2003-2006[Medline] [Order article via Infotrieve] |
4. | Kandel, E. S., and Hay, N. (1999) Exp. Cell Res. 253, 210-229[CrossRef][Medline] [Order article via Infotrieve] |
5. | Bellacosa, A., Testa, J. R., Staal, S. P., and Tsichlis, P. N. (1991) Science 254, 274-277[Medline] [Order article via Infotrieve] |
6. | Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D. H., and Tavtigian, S. V. (1997) Nat. Genet. 15, 356-362[Medline] [Order article via Infotrieve] |
7. |
Li, J.,
Yen, C.,
Liaw, D.,
Podsypanina, K.,
Bose, S.,
Wang, S. I.,
Puc, J.,
Miliaresis, C.,
Rodgers, L.,
McCombie, R.,
Bigner, S. H.,
Giovanella, B. C.,
Ittmann, M.,
Tycko, B.,
Hibshoosh, H.,
Wigler, M. H.,
and Parsons, R.
(1997)
Science
275,
1943-1947 |
8. |
Kennedy, S. G.,
Kandel, E. S.,
Cross, T. K.,
and Hay, N.
(1999)
Mol. Cell. Biol.
19,
5800-5810 |
9. | Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve] |
10. | Kops, G. J., and Burgering, B. M. (1999) J. Mol. Med. 77, 656-665[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Jones, R. G.,
Parsons, M.,
Bonnard, M.,
Chan, V. S.,
Yeh, W. C.,
Woodgett, J. R.,
and Ohashi, P. S.
(2000)
J. Exp. Med.
191,
1721-1734 |
12. |
Zong, W. X.,
Edelstein, L. C.,
Chen, C.,
Bash, J.,
and Gelinas, C.
(1999)
Genes Dev.
13,
382-387 |
13. | Chao, D. T., Linette, G. P., Boise, L. H., White, L. S., Thompson, C. B., and Korsmeyer, S. J. (1995) J. Exp. Med. 182, 821-828[Abstract] |
14. | Grillot, D. A., Merino, R., Pena, J. C., Fanslow, W. C., Finkelman, F. D., Thompson, C. B., and Nunez, G. (1996) J. Exp. Med. 183, 381-391[Abstract] |
15. | Vander Heiden, M. G., Chandel, N. S., Schumacker, P. T., and Thompson, C. B. (1999) Mol. Cell 3, 159-167[Medline] [Order article via Infotrieve] |
16. |
Vander Heiden, M. G.,
Chandel, N. S.,
Li, X. X.,
Schumacker, P. T.,
Colombini, M.,
and Thompson, C. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4666-4671 |
17. | Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., Schumacker, P. T., and Thompson, C. B. (1997) Cell 91, 627-637[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Pear, W. S.,
Nolan, G. P.,
Scott, M. L.,
and Baltimore, D.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8392-8396 |
19. |
Chang, B. S.,
Kelekar, A.,
Harris, M. H.,
Harlan, J. E.,
Fesik, S. W.,
and Thompson, C. B.
(1999)
Mol. Cell. Biol.
19,
6673-6681 |
20. | Ashcroft, S. J., Weerasinghe, L. C., Bassett, J. M., and Randle, P. J. (1972) Biochem. J. 126, 525-532[Medline] [Order article via Infotrieve] |
21. | Conlon, I., and Raff, M. (1999) Cell 96, 235-244[Medline] [Order article via Infotrieve] |
22. | Rathmell, J. C., Vander Heiden, M. G., Harris, M. H., Frauwirth, K. A., and Thompson, C. B. (2000) Mol. Cell 6, 683-692[Medline] [Order article via Infotrieve] |
23. | Verdu, J., Buratovich, M. A., Wilder, E. L., and Birnbaum, M. J. (1999) Nat. Cell Biol. 1, 500-506[CrossRef][Medline] [Order article via Infotrieve] |
24. | Weinkove, D., Neufeld, T. P., Twardzik, T., Waterfield, M. D., and Leevers, S. J. (1999) Curr. Biol. 9, 1019-1029[CrossRef][Medline] [Order article via Infotrieve] |
25. | Kulik, G., Klippel, A., and Weber, M. J. (1997) Mol. Cell. Biol. 17, 1595-1606[Abstract] |
26. | Kennedy, S. G., Wagner, A. J., Conzen, S. D., Jordan, J., Bellacosa, A., Tsichlis, P. N., and Hay, N. (1997) Genes Dev. 11, 701-713[Abstract] |
27. |
Kohn, A. D.,
Takeuchi, F.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
21920-21926 |
28. | Boise, L. H., Gonzalez-Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., Mao, X., Nunez, G., and Thompson, C. B. (1993) Cell 74, 597-608[Medline] [Order article via Infotrieve] |
29. |
Muise-Helmericks, R. C.,
Grimes, H. L.,
Bellacosa, A.,
Malstrom, S. E.,
Tsichlis, P. N.,
and Rosen, N.
(1998)
J. Biol. Chem.
273,
29864-29872 |
30. |
Franklin, J. L.,
and Johnson, E. M.
(1998)
J. Cell Biol.
142,
1313-1324 |
31. | Deshmukh, M., Vasilakos, J., Deckwerth, T. L., Lampe, P. A., Shivers, B. D., and Johnson, E. M., Jr. (1996) J. Cell Biol. 135, 1341-1354[Abstract] |
32. | Deckwerth, T. L., and Johnson, E. M., Jr. (1993) J. Cell Biol. 123, 1207-1222[Abstract] |
33. |
Deprez, J.,
Vertommen, D.,
Alessi, D. R.,
Hue, L.,
and Rider, M. H.
(1997)
J. Biol. Chem.
272,
17269-17275 |
34. | Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995) Nature 378, 785-789[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Barthel, A.,
Okino, S. T.,
Liao, J.,
Nakatani, K.,
Li, J.,
Whitlock, J. P., Jr.,
and Roth, R. A.
(1999)
J. Biol. Chem.
274,
20281-20286 |
36. | Brand, K., Leibold, W., Luppa, P., Schoerner, C., and Schulz, A. (1986) Immunobiology 173, 23-34[Medline] [Order article via Infotrieve] |
37. | Brand, K., Fekl, W., von Hintzenstern, J., Langer, K., Luppa, P., and Schoerner, C. (1989) Metabolism 38, 29-33[Medline] [Order article via Infotrieve] |
38. | Staveley, B. E., Ruel, L., Jin, J., Stambolic, V., Mastronardi, F. G., Heitzler, P., Woodgett, J. R., and Manoukian, A. S. (1998) Curr. Biol. 8, 599-602[Medline] [Order article via Infotrieve] |
39. |
Pap, M.,
and Cooper, G. M.
(1998)
J. Biol. Chem.
273,
19929-19932 |
40. | Zhang, G. J., Kimijima, I., Abe, R., Watanabe, T., Kanno, M., Hara, K., and Tsuchiya, A. (1998) Anticancer Res. 18, 1989-1998[Medline] [Order article via Infotrieve] |