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NEOPLASIA
From the Department of Pharmacology and the Immunology
Graduate Program, The University of Iowa College of Medicine, Iowa
City.
Exposure of hematopoietic cells to DNA-damaging 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 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 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 factor-dependent 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) domain-containing 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 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 Complemetary DNA constructs
Cell lines and culture conditions
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 dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride, and 1 µM 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 µM 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 µg/mL PI) and were treated with 1 µg/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 µM adenosine triphosphate, and 1 µg 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).
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 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.
p85 recruitment site of EPO-R is required for EPO to override
-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 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
PI-3K activity is necessary for cytokines to override
-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) 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 µM 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 cytokine-dependent Erk or Stat-5 phosphorylation. Expression of a dominant-negative Akt prevents cytokines from
overriding -IR-induced
growth arrest in 32D-Akt. Conversely, the dominant-negative 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
To confirm that the increased IL-3 concentrations required to override
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).
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
growth-promoting 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 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
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 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 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, 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 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 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.
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.
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.
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.
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: frederick-quelle{at}uiowa.edu.
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J.-H. Yuan, Y. Feng, R. H. Fisher, S. Maloid, D. L. Longo, and D. K. Ferris Polo-Like Kinase 1 Inactivation Following Mitotic DNA Damaging Treatments Is Independent of Ataxia Telangiectasia Mutated Kinase Mol. Cancer Res., July 1, 2004; 2(7): 417 - 426. [Abstract] [Full Text] [PDF]
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J. E. Lancet and J. E. Karp Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy Blood, December 1, 2003; 102(12): 3880 - 3889. [Abstract] [Full Text] [PDF]
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A. D. Friedman, D. Nimbalkar, and F. W. Quelle Erythropoietin Receptors Associate with a Ubiquitin Ligase, p33RUL, and Require Its Activity for Erythropoietin-induced Proliferation J. Biol. Chem., July 11, 2003; 278(29): 26851 - 26861. [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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Top
Abstract
Introduction
/
, EPO; 
, IL-3.
,
LY294002. (C) 32D-EPO-R(wt) cells treated with either DMSO (0.2%) or
LY294002 (10 µM) were left unstimulated (
) or 32D-Akt(KM) (
) cells were cultured over a
range of IL-3 concentrations. Percentage of S-phase cells in
