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NEOPLASIA
DNA damage–induced cell-cycle arrest of hematopoietic cells is overridden
by activation of the PI-3 kinase/Akt signaling pathway
Matthew K. Henry, Jeffrey T. Lynch, Alex K. Eapen, and Frederick W. Quelle
Exposure of hematopoietic cells to DNAdamaging agents induces cell-cycle arrest at G1 and G2/M checkpoints. Previously, it was shown that DNA damage–
induced growth arrest of hematopoietic
cells can be overridden by treatment with
cytokine growth factors, such as erythropoietin (EPO) or interleukin-3 (IL-3). Here,
the cytokine-activated signaling pathways required to override G1 and G2/M
checkpoints induced by ␥-irradiation
(␥-IR) are characterized. Using factor-
dependent myeloid cells stably expressing EPO receptor (EPO-R) mutants, it is
shown that removal of a minimal domain
required for PI-3K signaling abrogated
the ability of EPO to override ␥-IR–
induced cell-cycle arrest. Similarly, the
ability of cytokines to override ␥-IR–
induced arrest was abolished by an inhibitor of PI-3K (LY294002) or by overexpression of dominant-negative Akt. Moreover,
the ability of EPO to override these checkpoints in cells expressing defective EPO-R
mutants could be restored by overexpression of a constitutively active Akt. Thus,
activation of a PI-3K/Akt signaling pathway is required for cytokine-dependent
suppression of DNA-damage induced
checkpoints. Together, these findings
suggest a novel role for PI-3K/Akt pathways in the modulation of growth arrest
responses to DNA damage in hematopoietic cells. (Blood. 2001;98:834-841)
© 2001 by The American Society of Hematology
Introduction
Exposure of mammalian cells to DNA-damaging agents triggers a
variety of responses that include apoptotic cell death and cell-cycle
arrest.1 Failure of DNA-damaged cells to commit to either of these
pathways, before the restoration of DNA integrity, may contribute
to tumorigenic development by allowing the accumulation of new
mutations.2,3 Cellular determinants that predict whether cells will
arrest or undergo apoptosis in the presence of genotoxic stress are
not completely defined. Protection from DNA damage–induced
apoptosis may be conferred by the expression of antiapoptotic
proteins, such as Bcl-2 and Bcl-XL.4 Various hematopoietic cytokines induce the expression of antiapoptotic proteins, and this
coincides with their ability to protect against DNA damage–
induced cell death.5-7 In addition to the inhibition of apoptosis,
growth factors acting through the hematopoietic cytokine receptor
superfamily have also been shown to override the cell-cycle arrest
response to DNA damage in hematopoietic cell lines.7 However,
the signaling pathways responsible for this ability to override DNA
damage–induced cell-cycle checkpoints have not been defined.
The classical pathway coupling DNA damage with cell-cycle
arrest involves up-regulation of p53 and its transcriptional targets.
In response to DNA damage, p53 induces the expression of growth
inhibitory genes, such as p21Cip1 and GADD45.8 However, the
existence of p53-independent mechanisms resulting in cell-cycle
arrest has been demonstrated in lymphoid cells derived from
p53⫺/⫺ mice.9 Similarly, hematopoietic cell lines lacking p53
protein arrest in the G1 and G2/M phases after treatment with
ionizing irradiation.7 Thus, it remains unclear what mechanisms
regulate the cell-cycle arrest response to DNA damage in hematopoietic cells. Further, it is not obvious how signaling pathways
regulated by growth-promoting cytokines influence these G1 and
G2/M cell-cycle checkpoints.
Members of the hematopoietic cytokine receptor superfamily
include the receptors for erythropoietin (EPO) and interleukin-3
(IL-3).10 These receptors regulate a number of cellular functions in
hematopoietic cell lineages, including growth and development.
Receptors of this family function by activating one or more
members of the Jak family of tyrosine kinases. For example, the
receptor for EPO (EPO-R) associates with and activates Jak-2
through a cytoplasmic domain proximal to the membrane.11,12 This
membrane-proximal domain of EPO-R is sufficient to mediate
EPO-dependent proliferation and up-regulation of antiapoptotic
genes in factor-dependent myeloid cell lines. The precise signaling
pathways that mediate proliferative and antiapoptotic responses to
EPO are undefined. However, it has been clearly demonstrated that
EPO-R truncation mutants, lacking as many as 106 amino acids of
the C-terminal domain (eg, EPO-R[H]), retain all activities required to inhibit cell death and to promote growth of factordependent myeloid cell lines under normal culture conditions.13-16
Notably, the EPO-R(H) truncation mutant lacks the domain responsible for EPO-dependent activation of Ras/Erk- and PI-3 kinase
(PI-3K)–dependent signaling pathways. This domain contains a
recruitment site for the p85 subunit of PI-3K at tyrosine 479 of
EPO-R.17,18 Activated PI-3K phosphorylates inositol lipids resulting in the recruitment of pleckstrin homology (PH) domaincontaining proteins to the membrane.19 In response to cytokines
such as EPO and IL-3, the activation of PI-3K leads to activation of
the PH domain-containing serine–threonine kinase, Akt.20-22 Alternatively, EPO activation of the Ras/Erk pathway occurs through the
From the Department of Pharmacology and the Immunology Graduate
Program, The University of Iowa College of Medicine, Iowa City.
Reprints: Frederick W. Quelle, Department of Pharmacology, 2-210 Bowen
Science Bldg, The University of Iowa College of Medicine, Iowa City, IA 52242;
e-mail: [email protected].
Submitted September 26, 2000; accepted April 3, 2001.
Supported by a Howard Hughes Medical Institute Biomedical Research
Support Program grant and by Public Health Service grant CA-79889 from the
National Cancer Institute.
834
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
recruitment of adaptor proteins, including Shc, CrkL, and Ship1, to
unidentified sites in the C-terminal tail of EPO-R.23-25 The ability of
EPO-R(H) to support proliferation suggests that EPO activation of
Ras/Erk and PI-3K/Akt pathways is dispensable to EPO-dependent
cell growth under normal culture conditions.
We have previously shown that murine myeloid cell lines stably
expressing wild-type EPO-R are resistant to both ␥-IR–induced
apoptosis and cell-cycle arrest when cultured in EPO or IL-3.7 In
these cells, the apoptotic response to DNA damage could also be
bypassed by the ablation of p53 protein or by the forced expression
of Bcl-2 or Bcl-XL. However, all cells lacking this apoptotic
response still arrested in both G1 and G2/M after ␥-IR in the
absence of cytokine treatment. Similarly, cells expressing EPOR(H) remained viable but arrested in G1 and G2/M after treatment
with ␥-IR in the presence of EPO. Thus, activities associated with
the C-terminal domain of EPO-R are dispensable to cell growth
under normal culture conditions, but they are required for EPO to
override DNA damage–induced cell-cycle checkpoints in G1 and
G2/M. In the present study, we have shown that the ability of EPO
and IL-3 to override DNA damage–induced growth arrest is
dependent on their ability to activate PI-3K/Akt signaling
pathways. These studies demonstrate that PI-3K/Akt signaling,
which is normally dispensable to cytokine-dependent proliferation, is required for promoting cell-cycle progression in the face
of DNA damage.
Materials and methods
Complemetary DNA constructs
The truncated EPO-R complementary DNA (cDNA), EPO-R(d465), was
prepared by polymerase chain reaction (PCR) mutagenesis in which a stop
codon was introduced after the codon for tyrosine 463 of the murine EPO-R
cDNA.26 The truncated receptor, EPO-R(H), has been previously described.15 A point mutation converting Y479 of EPO-R to phenylalanine
(EPO-R[Y479F]) was prepared by PCR mutagenesis.27 A cDNA encoding
Bcl-XL28 was obtained by reverse transcription–PCR amplification from
RNA obtained from murine myeloid DA3 cells. cDNAs encoding HAtagged wild-type Akt, constitutively active Akt (myrAkt-HA), and dominantnegative Akt (HA-Akt[K179M]) have been described.29 All cDNAs were
cloned into the pXM expression vector for stable expression in 32D cells.
Cell lines and culture conditions
Cells expressing wild-type EPO-R or various mutant EPO-Rs were
generated by electroporation of 10 ␮g pXM expression vector containing
EPO-R cDNA or mutant EPO-R cDNAs into 32D cells. Stable clones of
32D cells expressing either EPO-R(wt), EPO-R(H), EPO-R(d465), or
EPO-R(Y479F) were selected for their ability to grow in 5 U/mL EPO.
Cells stably expressing Bcl-XL, wild-type HA-Akt, myrAkt-HA, or HAAkt(K179M) were obtained by cotransfecting 10 ␮g pXM expression
vector plus 1 ␮g pSV2-Neo, followed by selection with 1 mg/mL G418.
G418-resistant clones were screened by Western blot analysis for the
expression of the appropriate constructs. All cell lines were maintained in
RPMI 1640 plus 10% fetal bovine serum (FBS) and recombinant murine
IL-3 (70 pg/mL). For stimulation experiments, cells were washed free of
cytokine and were cultured in RPMI plus 10% FBS for 6 hours. In
experiments involving inhibition of PI-3K, 10 ␮M LY294002 (Calbiochem,
San Diego, CA) or dimethyl sulfoxide (DMSO; 0.2%) was added at the
beginning of the 6-hour incubation. Subsequently, cells were treated with
EPO or IL-3 or were left unstimulated for 8 minutes before lysis.
Western blot analysis
Cells were lysed in 0.5% NP-40, 10% glycerol, 50 mM Tris (pH 8.0), 0.1
mM EDTA, 150 mM NaCl, 0.1 mM Na3VO4, 50 mM NaF, 1 mM
PI-3K/Akt OVERRIDES CELL-CYCLE CHECKPOINTS
835
dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride, and 1 ␮M
microcystin. Lysates were cleared of insoluble material by centrifugation at
4°C. Whole cell lysates (5 ⫻ 105 cell equivalents per lane) were resolved by
SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane, and membranes were probed with appropriate antisera or antibodies
and were visualized by ECL (Amersham, Piscataway, NJ). Expression of
HA-tagged Akt was detected using an anti-HA antibody (Roche Molecular
Biochemicals, Indianapolis, IN) or an anti–human Akt1/PKB PH domain
antiserum (Upstate Biotechnology, Lake Placid, NY). Erk phosphorylation
was determined using an anti–phospho-Erk (P-Erk) antibody (Santa Cruz,
Santa Cruz, CA). Stat-5 phosphorylation was determined using an anti–
phospho-Stat-5A/B antibody (Upstate Biotechnology). Additionally, membranes were probed with an anti-Erk2 antiserum (Santa Cruz) to control for
equal loading in each lane.
Cell-cycle analysis
Cells were collected, washed in phosphate-buffered saline, and resuspended
(5 ⫻ 105 cells/mL) in RPMI 1640 medium containing 10% FBS and either
IL-3 (1.4 ng/mL or 0.07 ng/mL), EPO (5 U/mL), or no cytokine. For
experiments involving the inhibition of PI-3K, cells were cultured in 10 ␮M
LY294002 or 0.2% DMSO immediately after resuspension. All cultures
were incubated for 2 hours at 37°C, 5% CO2. Subsequently, parallel cultures
were either exposed to 4 Gy ␥-IR from a cesium (Cs) 137 source or left
untreated. Cells were returned to a 37°C, 5% CO2 incubator for 24 hours
and were then assayed for viability and DNA content. Cell viability was
determined by trypan blue exclusion. For cell-cycle analysis, cells were
collected by centrifugation and resuspended at 1 ⫻ 106 cells/mL in
propidium iodide (PI) staining buffer (0.1% sodium citrate, 0.1% Triton-X
100, and 50 ␮g/mL PI) and were treated with 1 ␮g/mL RNase at room
temperature for 30 minutes. Cell-cycle histograms were generated after
analysis of PI-stained cells by fluorescence-activated cell sorting (FACS)
with a Becton Dickinson FACScan. For each culture, at least 1 ⫻ 104 events
were recorded. Histograms generated by FACS were analyzed by ModFit
Cell Cycle Analysis Software (Verity, Topsham, ME) to determine the
percentage of cells, in each phase (G1, S, and G2/M).
Akt kinase assay
Akt kinase assays were performed in vitro using an Akt Kinase Assay Kit
(catalog no. 9840; New England Biolabs, Beverly, MA). Akt was immunoprecipitated from whole cell lysates of stimulated 32D cells (2 ⫻ 106 cell
equivalents) with immobilized anti-Akt antibodies. The immunoprecipitated Akt was subjected to a kinase reaction containing 25 mM Tris (pH
7.5), 5 mM ␤–glycerol-phosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM
MgCl2, 200 ␮M adenosine triphosphate, and 1 ␮g GSK-3␣ fusion protein.
Subsequently, the GSK-3␣ fusion protein was resolved by SDS-PAGE and
transferred to PVDF membranes, and the membranes were probed with an
anti-phospho–GSK antibody and visualized using enhanced chemiluminescence (ECL; Amersham).
Results
Cytokine-treatment of 32D cells overrides a DNA
damage–induced cell-cycle arrest
Factor-dependent 32D cells expressing wild-type EPO-R (32D–
EPO-R[wt]) exhibit an asynchronous cell-cycle profile when
cultured in EPO (Figure 1). When withdrawn from cytokine growth
factors (⫺GF), these cells arrested largely in the G1 phase (77%)
and much less in G2/M (5%). ␥-IR of 32D–EPO-R(wt) cells
resulted in complete cell death in the absence of cytokine, whereas
irradiated cells treated with EPO remained viable and asynchronously growing. To confirm that cytokine treatment specifically
overrides a DNA damage–induced growth arrest, 32D–EPO-R(wt)
cells were stably transfected with a Bcl-XL expression construct to
prevent DNA damage–induced cell death in the absence of
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836
HENRY et al
Figure 1. DNA damage specifically induces a G2/M-phase arrest in the absence
of cytokine treatment. 32D cells expressing EPO-R(wt) or cells expressing both
EPO-R(wt) and Bcl-XL (32D–EPO-R/BclXL) were cultured in medium lacking cytokine
growth factors (⫺GF) or with 5 U/mL EPO. Subsequently, cultures were exposed to 4
Gy ␥-IR (⫹␥) or were left untreated (⫺␥). Thirty-three hours after irradiation, cells
were stained with PI and analyzed by FACS.
cytokine treatment. Similar to parental cells, 32D–EPO-R/Bcl-XL
cells proliferate asynchronously when cultured in EPO and arrest
predominantly in G1 phase (77%) rather than in G2/M (13%) in the
absence of cytokine (Figure 1). By contrast, irradiated 32D–EPO-R/
Bcl-XL cells arrested in both G1 (54%) and G2/M (34%) phases in
the absence of cytokine. Therefore, 32D cells arrest in G2/M
specifically in response to DNA damage, not simply as a result of
cytokine withdrawal. Moreover, this G2/M arrest is overridden in
response to signals activated by EPO-R.
BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
phosphorylation was retained. Interestingly, no EPO-induced Erk
phosphorylation could be detected in 32D–EPO-R(d465) cells,
whereas 32D–EPO-R(Y479F) cells were fully competent for this
activity. The lack of EPO-induced activation of both Erk and Akt in
32D–EPO-R(d465) cells was similar to that observed in cells
expressing the more severely truncated EPO-R(H) receptor. As
controls, IL-3 induced the activation of Akt, Erk, and Stat-5 in all
cell lines assayed, whereas none of these activities were observed
in cells cultured in the absence of cytokine. These data indicate a
dual role for the 17 C-terminal amino acids of EPO-R in the
regulation of PI-3K and Ras/Erk pathways, and they confirm a
specific requirement for tyrosine 479 in the regulation of PI-3K.
32D cell expressing the various EPO-R mutants were then
tested for their ability to override DNA damage–induced cell-cycle
arrest in response to EPO treatment. Each line was cultured in the
presence of EPO and was exposed to 4 Gy ␥-IR or was left
unirradiated. Twenty-four hours after irradiation, the cell-cycle
status of each culture was determined by flow cytometry. As a
control for the activity of a wild-type cytokine receptor in each cell
line, cells cultured in IL-3 were also examined. Similar to the
experiment shown in Figure 1, all cell lines cultured in the absence
of cytokine were nonviable after ␥-IR, whereas unirradiated
cultures in the absence of cytokine were largely viable yet arrested
in G1 (data not shown). 32D–EPO-R(wt) cells maintained an
asynchronous cell-cycle profile after exposure to ␥-IR when
cultured in EPO or IL-3 (Figure 3A). A slight decrease in S phase
and an increase in the G2/M population (less than 2-fold; Figure
3B) after ␥-IR are indicative of a partial effect on cell-cycle
progression, even in the presence of cytokine. In contrast, 32D–EPOR(H), 32D–EPO-R(d465), and 32D–EPO-R(Y479F) cells were
largely arrested in G1 and G2/M phases after exposure to ␥-IR in
the presence of EPO, as indicated by the near absence of S phase
(Figure 3A) and a substantial increase in G2/M (4.5- to 8-fold)
(Figure 3B). All cells cultured in IL-3 remained asynchronous, with
only a modest increase in G2/M after ␥-IR.
p85 recruitment site of EPO-R is required for EPO to override
␥-IR–induced cell-cycle arrest
Previous studies have shown that activated receptors for EPO and
IL-3 are coupled to a variety of signaling pathways, including the
activation of Ras/Erk, PI-3K/Akt, and Stat-5. Truncation of the 106
C-terminal amino acids from EPO-R (EPO-R[H]) removes domains required for PI-3K/Akt and Ras/Erk activation and also
abrogates the ability of EPO to override DNA damage–induced
cell-cycle checkpoints.7 To further characterize the region of
EPO-R that is necessary for preventing the ␥-IR–induced cell-cycle
arrest, a series of C-terminal truncated receptors was constructed.
EPO-R(d465) lacks the 17 C-terminal amino acids of EPO-R,
which includes a single tyrosine at position 479 (Figure 2A). In
addition, a point mutant was prepared in which the reported PI-3K
recruitment site at tyrosine 47917 was converted to phenylalanine
(EPO-R[Y479F]). Each receptor construct was stably expressed in
32D cells.
Before assessing their roles in overriding cell-cycle checkpoints, the signaling activities of each form of EPO-R were
assessed through their ability to activate Akt and to phosphorylate
Erk and Stat-5 in response to EPO (Figure 2B). In cells expressing
EPO-R(wt), treatment with EPO resulted in activation of Akt, Erk,
and Stat-5, as expected. By comparison, 32D–EPO-R(H), 32D–
EPO-R(d465), and 32D–EPO-R(Y479F) contained little or no
EPO-stimulated Akt activity, yet the ability to induce Stat-5
Figure 2. Signaling activities of wild-type and mutant cytokine receptors.
(A) Structures of wild-type and mutant receptors are diagrammed. Positions of the
transmembrane domain (TM) and phosphorylatable tyrosine (Y) are indicated.
(B) 32D cells stably expressing wild-type (wt) or mutated EPO-R were left unstimulated (⫺) or were stimulated with 1.4 ng/mL IL-3 or 5 U/mL EPO for 8 minutes. Akt
catalytic activity was assayed from cell lysates in an in vitro kinase assay (see
“Materials and methods”). Alternatively, whole cell lysates were Western blotted with
anti–phospho-Erk (␣ P-Erk) or anti–phospho-Stat-5A/B (␣ P-STAT5) antibodies. As a
control for loading, lysates were probed with anti-Erk2 (␣ Erk) antiserum.
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
PI-3K/Akt OVERRIDES CELL-CYCLE CHECKPOINTS
837
cells caused arrest in G1 and G2/M despite treatment with EPO or
IL-3 (Figure 4A-B). For all experiments, cells remained at least
94% viable regardless of treatment conditions (data not shown).
To confirm the specific inhibitory activity of LY294002 in 32D
cells, we compared the ability of IL-3 or EPO to activate Akt, Erk,
and Stat-5 in the presence or absence of the inhibitor. As shown in
Figure 4C, treatment of 32D–EPO-R(wt) cells with 10 ␮M
LY294002 did not completely abolish Akt activity but did suppress
both IL-3 and EPO-induced Akt activation by more than 2-fold.
LY294002 treatment had no detectable effect on cytokinedependent Erk or Stat-5 phosphorylation.
Expression of a dominant-negative Akt prevents cytokines
from overriding ␥-irradiation–induced growth arrest
Figure 3. The p85 recruitment site of EPO-R is required for EPO-dependent
suppression of ␥-IR–induced cell-cycle arrest. (A) 32D cells expressing wild-type
(wt) or mutated EPO-R were cultured in medium that contained either 1.4 ng/mL IL-3
or 5 U/mL EPO. Subsequently, cultures were exposed to 4 Gy ␥-IR (⫹␥) or were left
untreated (⫺␥). Twenty-four hours after irradiation, cells were stained with PI and
analyzed by FACS. Before PI staining, the viability of the cells was determined by
trypan blue exclusion and was 94% or greater in all experiments. Percentage of
S-phase cells is indicated for each culture. (B) Percentage of G2/M cells in
␥-IR–treated cultures versus nonirradiated cultures shown in panel A was determined, and the fold increase in G2/M was calculated (G2⫹␥/G2⫺␥). Values
presented represent the average of 3 independent experiments. Similar results were
obtained with at least 3 independent clones of each cell type. f, EPO; 䡺, IL-3.
PI-3K activity is necessary for cytokines to override
␥-irradiation–induced growth arrest
The inability of EPO to override DNA damage–induced growth
arrest resulting from the mutation of tyrosine 479 implies a
requirement for the activation of PI-3K. To confirm this requirement, the effects of ␥-IR on cell-cycle status were assessed for
32D–EPO-R(wt) cells cultured in IL-3 or EPO and the presence or
absence of the PI-3K inhibitor, LY294002 (Figure 4A). In the
absence of irradiation, 32D–EPO-R(wt) cells continued to proliferate when cultured in EPO or IL-3 plus LY294002, though there was
a reduction of S-phase cells compared to DMSO (vehicle)-treated
controls. By contrast, ␥-IR of LY294002-treated 32D–EPO-R(wt)
Activation of Akt is a well-characterized consequence of PI-3K
activation. To assess the requirement for Akt activity in overriding
the DNA damage–induced arrest, 32D cells were prepared that
stably overexpressed wild-type Akt (32D-Akt) or a dominantnegative Akt (32D-Akt[KM]). As shown in Figure 5A, both Akt
and Akt(KM) were expressed at higher levels in multiple clones
than endogenous Akt. 32D-Akt or 32D-Akt(KM) proliferated
normally when cultured in IL-3 (Figure 5B), as previously
reported.22 Similar to parental 32D cells, IL-3 was able to prevent
␥-IR–induced growth arrest in 32D-Akt. Conversely, the dominantnegative form of Akt blocked the ability of IL-3 to override these
checkpoints because 32D-Akt(KM) cells cultured in IL-3 were
dramatically arrested in G1 and G2/M after ␥-IR.
It was noted in the previous experiments that higher concentrations of IL-3 could overcome the dominant-negative activity of
Akt(KM). Therefore, the dose dependence of IL-3 suppression of
␥-IR–induced arrest was compared for 32D-Akt(KM) versus
32D-Akt cells. As shown in Figure 6, IL-3 was able to override
␥-IR–induced cell-cycle arrest of 32D-Akt(KM) cells in a dosedependent manner. However, significantly higher concentrations of
IL-3 were required to override ␥-IR–induced growth arrest in
32D-Akt(KM) than in 32D-Akt cells. Specifically, when cultured
in low concentrations (less than 100 pg/mL) of IL-3, 32D-Akt(KM)
cells experienced a substantial reduction in S phase after ␥-IR
(3.4-fold at 17.5 pg/mL IL-3). IL-3 concentrations less than 17.5
pg/mL were insufficient to maintain viability of 32D-Akt(KM) cell
cultures after ␥-IR. In contrast, 32D-Akt(KM) cells treated with
IL-3 concentrations greater than 150 pg/mL resulted in less than a
2-fold reduction in S phase after ␥-IR. By comparison, the slight
reductions in S phase (1.5-1.8 fold) after ␥-IR of 32D-Akt cells was
not altered by increasing IL-3 concentrations. A similar dose
dependence was observed for IL-3 suppression of ␥-IR–induced
increases in the G2/M population for 32D-Akt(KM) versus 32DAkt cells (Figure 6B). Notably, treatment with 10 ␮M LY294002
completely abolished the ability of 280 pg/mL IL-3 to override
␥-IR–induced arrest in 32D-Akt(KM) cells (3.75-fold decrease in S
phase and 8.3-fold increase in G2/M after irradiation). Thus, the
ability of higher concentrations of IL-3 to override ␥-IR–induced
cell-cycle arrest of 32D-Akt(KM) cells requires PI-3K activity and
is not indicative of aberrant activation of an alternative PI-3K–
independent pathway.
To confirm that the increased IL-3 concentrations required to
override ␥-IR–induced checkpoints in 32D-Akt(KM) cells correlated with activation of endogenous Akt, Akt kinase activity was
measured in cells treated over a range of IL-3 concentrations. As
shown in Figure 6C, 32D-Akt(KM) cells contained no detectable
Akt activity when treated with less than 140 pg/mL IL-3, but they
contained increasing levels of Akt activity with higher doses of
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838
BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
HENRY et al
Figure 4. Inhibition of PI-3K–dependent signaling pathways prevents IL-3 and EPO from overriding ␥-IR–induced growth arrest. (A) 32D–EPO-R(wt) cells were
cultured in medium that contained either 1.4 ng/mL IL-3 or 5 U/mL EPO. Cultures were then supplemented with either DMSO (0.2%) or LY294002 (10 ␮M). Subsequently,
cultures were exposed to 4 Gy ␥-IR (⫹␥) or were left untreated (⫺␥). Twenty-four hours after irradiation, cells were stained with PI and analyzed by FACS. Before PI staining,
viability of the cells was determined by trypan blue exclusion and was 94% or greater in all experiments. Percentage of S-phase cells is indicated for each culture.
(B) Percentage of G2/M cells in ␥-IR–treated cultures versus nonirradiated cultures shown in panel A was determined, and the fold increase in G2/M was calculated
(G2⫹␥/G2⫺␥). Values presented represent the average of 3 independent experiments. 䡺, DMSO; o, LY294002. (C) 32D–EPO-R(wt) cells treated with either DMSO (0.2%) or
LY294002 (10 ␮M) were left unstimulated (⫺) or were stimulated with 1.4 ng/mL IL-3 or 5 U/mL EPO for 8 minutes. Akt catalytic activity was assayed from cell lysates in an in
vitro kinase assay (see “Materials and methods). Alternatively, whole cell lysates were Western blotted with anti–phospho-Erk (␣ P-Erk) or anti–phospho-Stat-5A/B (␣
P-STAT5) antibodies. As a control for loading, lysates were probed with anti-Erk2 (␣ Erk) antiserum.
IL-3. By comparison, 32D-Akt cells contained significant Akt
activity when treated with as little as 35 pg/mL IL-3, and they
reached maximum activity with 140 pg/mL IL-3. As a control,
levels of IL-3–induced Erk and Stat-5 phosphorylation were found
to reach maximum levels with approximately 140 pg/mL IL-3 in
both 32D-Akt and 32D-Akt(KM) cells.
Expression of constitutively active Akt restores the ability of
EPO-R mutants to override DNA damage checkpoints
To determine whether PI-3 kinase activation by the C-terminal
domain of EPO-R is sufficient to override DNA damage–induced
checkpoints, 32D–EPO-R(d465) cells were stably transfected with
a constitutively active form of Akt (myrAkt). As shown in Figure
7A, expression of HA-tagged myrAkt was detectable in clonal cell
lines (32D-d465/myrAkt). However, levels of myrAkt expression
were substantially lower than those achieved in clones expressing
wild-type Akt (32D-d465/Akt). Nonetheless, 32D-d465/myrAkt
cells contained constitutive levels of Akt activity in the absence of
cytokine or after treatment with EPO (Figure 7B). IL-3 treatment
modestly enhanced Akt kinase activity in 32D-d465/myrAkt cells.
32D-d465/Akt cells contained little or no Akt activity in the
absence of cytokine or after EPO treatment. In all cell lines, EPO
induced Stat-5 phosphorylation but failed to activate Erk. As with
parental 32D–EPO-R(d465) cells, EPO treatment of 32D-d465/Akt
cells did not prevent ␥-IR–induced growth arrest (Figure 7C).
However, expression of myrAkt restored the ability of EPO to
override these checkpoints because 32D-d465/myrAkt cells cultured in EPO remained asynchronous after ␥-IR treatment
(Figure 7C-D).
Discussion
Cellular responses to DNA-damaging agents include apoptotic cell
death and cell-cycle arrest at specific checkpoints.1 In hematopoietic cells, both these responses can be overridden by growthpromoting cytokines such as EPO or IL-3.5-7 We have previously
shown that the abilities of EPO to override DNA damage–induced
apoptosis and growth arrest are regulated by distinct signaling
pathways.7 EPO-R truncation mutants, lacking as many as 106
C-terminal amino acids (ie, EPO-R[H]), retain the ability to
suppress the apoptotic response induced by ␥-IR. The membraneproximal domain retained by EPO-R(H) associates with Jak-2 and
is sufficient to mediate EPO-dependent up-regulation of antiapoptotic genes, including Bcl-2 and Bcl-XL. This receptor domain is
necessary and sufficient to promote the proliferation of hematopoietic cells under normal culture conditions. However, EPOdependent activation of EPO-R(H) and Jak-2 is not sufficient to
override ␥-IR–induced G1 and G2 phase growth arrest. Therefore,
the C-terminal domain of EPO-R is dispensable for EPO-induced
cell growth under nonstressing conditions but is required to
override ␥-IR–induced cell-cycle arrest.
In the present study, we have further defined regions within the
C-terminal domain of EPO-R that are required for EPO to override
the ␥-IR–induced cell-cycle arrest. Notably, this activity is shown
to require a single C-terminal tyrosine (Y479) that is also required
for effective PI-3K activation. Conversion of Y479 to phenylalanine (EPO-R[Y479F]) disrupted EPO-induced Akt activation and
abrogated the ability of EPO to override the ␥-IR–induced cellcycle arrest. The lack of Akt activation by EPO-R(Y479F) is
consistent with the previous observation that Y479 is a primary
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
Figure 5. Overexpression of a dominant-negative Akt (Akt[KM]) impairs the
ability of IL-3 to override ␥-IR–induced cell-cycle arrest. (A) Whole cell lysates
from 32D-Neo (lane 1) or 2 clones of 32D-Akt (lanes 2 and 3) or 32D-Akt(KM) (lanes 3
and 4) cells were Western blotted with anti-HA antibodies (␣HA) or anti-Akt/PKB PH
domain antiserum (␣Akt). (B) 32D-Neo, 32D-Akt, and 32D-Akt(KM) cells were
cultured in medium that contained 70 pg/mL IL-3. Subsequently, cultures were
exposed to 4 Gy ␥-IR (⫹␥) or were left untreated (⫺␥). Twenty-four hours after
irradiation, cells were stained with PI and analyzed by flow cytometry. Before PI
staining, cell viability was determined by trypan blue exclusion and was 94% or
greater in all experiments. Percentage of S-phase cells is indicated for each culture.
recruitment site for the p85 subunit of PI-3K.17,18 Although it
remains possible that other signaling molecules may also be
recruited to phosphorylated Y479 of EPO-R, the addition of the
PI-3K inhibitor, LY294002, to cells expressing wild-type EPO-R
PI-3K/Akt OVERRIDES CELL-CYCLE CHECKPOINTS
839
similarly abolished the ability of EPO to override ␥-IR–induced
growth arrest. These data indicate a specific requirement for PI-3K
in overriding these DNA damage–induced checkpoints. Moreover,
LY294002 also inhibited the ability of IL-3 to override ␥-IR–
induced growth arrest. Thus, it appears that the activation of PI-3K
may be a general requirement for cytokines to override the growth
arrest induced by DNA damage.
PI-3K regulates a large number of pleckstrin homology (PH)domain containing effector proteins, including Akt and Pdk1,
through the phosphorylation of inositol lipids at the plasma
membrane.19 In the present study, we show that the expression of
dominant-negative Akt (Akt[KM]) inhibits the ability of IL-3 to
override ␥-IR–induced cell-cycle arrest. In contrast, the expression
of wild-type Akt at comparable levels to dominant-negative Akt
had no effect on the ability of IL-3 to override ␥-IR–induced arrest.
This demonstrates that the simple overexpression of a PH domaincontaining protein (eg, Akt) is insufficient to block a PI-3K–
activated pathway needed to override the ␥-IR–induced growth
arrest. Thus, we conclude that dominant-negative Akt does not
inhibit the recruitment of other PH-domain–containing effector
proteins to the plasma membrane, nor does it inhibit their subsequent activation. Instead, the ability of cytokines to override
damage–induced checkpoints is dependent on the catalytic activity
of Akt. Moreover, treatment of Akt(KM) cells with increased
concentrations of IL-3 overcame the dominant-negative Akt inhibition of endogenous Akt activity while overriding DNA damage–
induced growth arrest. Therefore, the activation of Akt is required
for cytokines to override the DNA damage–induced cell-cycle
arrest. Furthermore, the inability of the EPO-R(d465) truncation
mutant to override these checkpoints can be alleviated by the
expression of active Akt, suggesting that restoration of this activity
alone is sufficient to override the checkpoint.
In the present study, we found that the EPO-R–(Y479F)
mutation had no effect on EPO-induced Erk phosphorylation.
Conversely, a previous report found that EPO-induced Erk activation was disrupted by an apparently identical Y479F point mutation.18 Although these studies were performed in different cell
lines, the basis for these differing results remains unclear. Ultimately, it was concluded that Erk activation by EPO was dependent
on the activation of PI-3K.18 However, it is noteworthy that we also
failed to detect any reduction of EPO- or IL-3–induced Erk
phosphorylation in cells treated with the PI-3K inhibitor, LY294002.
Instead, we found that the truncation of as few as 17 C-terminal
amino acids, including Y479, from EPO-R (EPO-R[d465]) abrogated EPO-dependent phosphorylation of Erk1 and Erk2. The lack
Figure 6. DNA damage–induced growth arrest of IL-3–treated cells is correlated with a lack of IL-3 induced Akt activity. (A, B) 32D-Akt (E) or 32D-Akt(KM) (F) cells
were cultured over a range of IL-3 concentrations. Percentage of S-phase cells in ␥-IR–treated cultures versus nonirradiated cultures was determined, and the fold reduction in
S phase was calculated (S⫺␥/S⫹␥) (A). Alternatively, the percentage of G2/M cells in the ␥-IR–treated versus the nonirradiated cultures was determined, and the fold increase
in G2/M was calculated (G2⫹␥ /G2⫺␥) (B). (C) 32D-Akt or 32D-Akt(KM) cells were left unstimulated (⫺) or were stimulated with the indicated concentration of IL-3 for 8
minutes. Akt catalytic activity was assayed from cell lysates in an in vitro kinase assay (see “Materials and methods”). Alternatively, whole cell lysates were Western blotted with
anti–phospho-Stat-5 (␣ P-STAT5) or anti–phospho-Erk (␣ P-Erk) antibodies. As a control for loading, lysates were probed with anti-Erk2 (␣ Erk) antiserum.
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840
HENRY et al
BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
Figure 7. Expression of constitutively active Akt in 32D–
EPO-R(d465) restores the ability of EPO to override
␥-IR–induced growth arrest. (A) Whole cell lysates of
32D–EPO-R(d465) clones stably expressing a Neo control
plasmid, wild-type Akt, or myrAkt were Western blotted with
anti-HA (␣HA) antibodies. (B) Cells were left unstimulated (⫺)
or were treated with IL-3 (1.4 ng/mL) or EPO (5 U/mL) for 8
minutes. Akt catalytic activity was assayed from cell lysates in
an in vitro kinase assay (see “Materials and methods”).
Alternatively, whole cell lysates were Western blotted with
anti–phospho-Stat-5 (␣ P-STAT5) or anti–phospho-Erk (␣
P-Erk) antibodies. As a control for loading, lysates were
probed with anti-Erk2 antiserum (␣ Erk). (C) Cells were
cultured in medium containing either 1.4 ng/mL IL-3 or 5 U/mL
EPO. Subsequently, cultures were exposed to 4 Gy ␥-IR (⫹␥)
or were left untreated (⫺␥). Twenty-four hours after irradiation, cells were stained with PI and analyzed by FACS. Before
PI staining, cell viability was determined by trypan blue
exclusion and was 80% or greater in all experiments. Percentage of S-phase cells is indicated for each culture. (D)
Percentage of G2/M cells in ␥-IR–treated cultures versus
nonirradiated cultures shown in panel C was determined, and
the fold increase in G2/M was calculated (G2⫹␥/G2⫺␥).
Values presented represent the average of 3 independent
experiments. Similar results were also obtained from polyclonal populations of each cell type. 䡺, IL-3; f, EPO.
of Erk phosphorylation resulting from the d465 truncation could
stem from loss of a recruitment site within this 17–amino acid
domain or a general perturbation of the C-terminal region of
EPO-R. In this regard, Klingmuller et al18 found that mutation of
the other 7 tyrosines of EPO-R (except Y479) also resulted in the
significant loss of EPO-induced Shc phosphorylation, Grb2 association, and Erk activation. Thus, it appears likely that Erk activation
by EPO-R may not be associated simply with a single receptor
domain. Regardless of the mechanism by which Erk is activated,
the observation that the expression of active Akt restores the ability
of EPO-R(d465) to override DNA damage–induced growth arrest
suggests that activation of the Ras/Erk pathway is not required to
override these checkpoints.
In hematopoietic cell lines, such as 32D, ␥-IR induces both G1
and G2/M phase arrest. Our previous work demonstrated that
cytokines override these cell-cycle checkpoints and promote growth
in the face of DNA damage. Here we argue that PI-3K/Akt
activation is essential for this activity. However, the targets of this
signaling pathway in G1 and G2/M remain to be identified. In many
cell types, DNA damage–induced cell-cycle arrest in G1 is thought
to be dependent on p53-mediated up-regulation of the cyclin/cdk
inhibitor p21Cip1. However, we have previously shown that ␥-IR–
induced cell-cycle arrest of hematopoietic cell lines occurs in the
absence of p53.7 We have not ruled out the possibility that recently
identified relatives of p53, p63, and p73 could contribute to DNA
damage–induced G1 arrest through the up-regulation of p21Cip1 or
GADD45 protein levels.30,31 Other pathways mediating DNA
damage–induced cell-cycle arrest have been described, such as the
Chk1-mediated phosphorylation and inactivation of Cdc25.32,33
Although this pathway has clearly been shown to contribute to
G2/M phase arrest, there is no evidence for its involvement in a
DNA damage–induced G1 checkpoint. We are investigating
whether these or other DNA damage–induced responses are
responsible for the cell-cycle arrest observed in hematopoietic
cell lines. It is clear, however, that whatever mediates this arrest
can be regulated by cytokine-dependent signaling pathways
involving PI-3K and Akt.
Evidence suggests a general role for PI-3K signaling pathway in regulating cell-cycle progression. Notably, PI-3K activity can be sufficient to induce G1 transit in fibroblasts34 and is
required for IL-2–dependent activation of E2F in T cells.35 Thus,
downstream targets of PI-3K/Akt–dependent pathways that
regulate normal cell-cycle progression may also participate in
overriding ␥-IR–induced checkpoints. PI-3K activity has been
shown to contribute to induced expression of D-type cyclins36
and to increase cyclin D1 stability through Akt-dependent
phosphorylation of GSK-3␤.37 Alternatively, PI-3K effects on
G1-phase progression may be mediated through the downregulated expression of the Cdk inhibitor, p27kip1, because
various inhibitors of PI-3K pathways have been shown to cause
enhanced expression of p27kip1 protein.35,38,39 Although PI-3K
activation does not appear to be required for EPO- or IL-3–
dependent proliferation of nonirradiated cells, lack of PI-3K
activity in DNA-damaged cells could impair some or all of these
events, resulting in growth arrest.
The results of the present study demonstrate that cytokine
receptors use multiple signaling pathways to regulate cell growth.
Under normal culture conditions, EPO-induced proliferation is
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
PI-3K/Akt OVERRIDES CELL-CYCLE CHECKPOINTS
primarily dependent on signaling pathways activated through the
membrane-proximal domain of EPO-R and is not dependent on the
activation of PI-3K. However, the present study demonstrates
that cytokine-dependent activation of PI-3K/Akt signaling pathways is specifically required to override the effects of DNA
damage on cell-cycle progression. The targets of this pathway
are likely to be cell-cycle regulators governing G1/S- and
G2/M-phase transit. Identification of these targets should provide a better understanding of how DNA damage–induced
cell-cycle arrest is regulated in hematopoietic cells, and it may
provide insight into how these checkpoints might be bypassed
during leukemogenic development.
841
Acknowledgments
We thank Dr Naheed Ahmed (Fox Chase Cancer Center, Philadelphia, PA) for providing Akt cDNA constructs, Ann Friedman for
the construction of truncated EPO-R cDNA, Dr James W. Osborne
(Department of Radiation Biology, University of Iowa) for assistance in use of the 137Cs source, Justin Fishbaugh and Gene Hess
for assistance in the Flow Cytometry Facility (University of Iowa),
core facilities of The Diabetes and Endocrinology Research Center
at the University of Iowa, and Dr Dawn Quelle for helpful
discussion and review of this manuscript.
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2001 98: 834-841
doi:10.1182/blood.V98.3.834
DNA damage−induced cell-cycle arrest of hematopoietic cells is overridden by
activation of the PI-3 kinase/Akt signaling pathway
Matthew K. Henry, Jeffrey T. Lynch, Alex K. Eapen and Frederick W. Quelle
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