1 Inhibition of the PI-3 kinase pathway induces a senescent-like arrest mediated by p27Kip1 ...

44 Pages·2000·5.24 MB·English

JBC Papers in Press. Published on April 28, 2000 as Manuscript M000759200
Inhibition of the PI-3 kinase pathway induces a senescent-like arrest mediated
by p27Kip1
Manuel Collado1, 6, 7, René H. Medema4, 7, Isabel García-Cao2,
Marlène L.N. Dubuisson1, Marta Barradas2, Janet Glassford1, Carmen Rivas3,
Boudewijn M. T. Burgering5, Manuel Serrano2, and Eric W-F Lam1
1 Ludwig Institute for Cancer Research and Section of Virology and Cell Biology,
Imperial College School of Medicine at St Mary’s Campus, London, United Kingdom,
2Department of Immunology and Oncology, Centro Nacional de Biotecnologia,
Campus UAM, Cantoblanco, Madrid, Spain,
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3Department of Haematology, Royal Postgraduate Medical School, Imperial College School o
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of Medicine at Hammersmith’s Campus, London, United Kingdom loa
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4 Jordan Laboratory G03-647, Departments of Haematology, University fro
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Medical Center Utrecht, 3584 CX Utrecht, The Netherlands ttp
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5Laboratory of Physiological Chemistry, University Medical Center w
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Utrecht, The Netherlands c
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6 Present address: Department of Biochemistry, NYU Medical Center, 550 First b/
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Avenue, New York City, NY10016 USA es
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7Contributed equally to the study and should be considered joint first authors No
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Running title: p27Kip1 mediates senescent-like arrest induced by PI-3 kinase b
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inhibitors , 2
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Corresponding Author:
Eric W-F Lam, Ludwig Institute for Cancer Research and Section of Virology and
Cell Biology, Imperial College School of Medicine at St Mary’s Campus, Norfolk
Place W2 1PG London, United Kingdom.
Telephone: [phone]. Fax: [phone].
Keywords:p27/ PI-3 kinase pathway/LY294002/senescence/MEF/p130/CDK2
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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Abstract
A senescent-like growth arrest is induced in mouse primary embryo
fibroblasts by inhibitors of PI-3 kinase. We observed that the senescent-like
growth arrest is correlated with an increase in p27Kip1 but down-regulation of
other CKI including p15INK4b, p16INK4a, p19 NK4d and p21Cip1 as well as other
negative cell cycle regulators such as p53, p19ARF, implying that this senescence
related growth arrest is independent of the activity of p53, p19ARF, p16INK4a and
p21Cip1, which are associated with replicative senescence. The p27Kip1 binds to the
cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We
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demonstrated that ectopic expression of p27Kip1 can induce permanent cell-cycle lo
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arrest and a senescent-like phenotype in wild-type mouse embryo fibroblasts. fro
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We also obtained results suggesting that the kinase inhibitors LY294002 and ://w
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Wortmannin arrest cell growth and induce a senescent-like phenotype, at least .jb
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partially, through inhibition of PI3-K and PKB/Akt, activation of the forkhead b/
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protein AFX, and up-regulation of p27Kip1expression. In summary, these t o
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observations taken together suggest that p27Kip1 could be an important mediator ve
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of the permanent cell cycle arrest induced by PI-3 kinase inhibitors. Our data 8
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suggest that repression of CDK2 activity by p27Kip1 is required for the P-I3 kinase
induced senescence, yet mouse embryo fibroblasts derived from p27Kip1-/- mice
entered cell cycle arrest after treatment with LY294002. We show that this is due
to a compensatory mechanism by which p130 functionally substitutes for the loss
of p27Kip1. This is the first description that p130 may have a role in inhibiting CDK
activity during senescence.
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Introduction
Normal somatic cells undergo a limited number of divisions when
cultured in vitro before entering an irreversible state of cell cycle arrest known as
replicative senescence (1). This process has been demonstrated to occur also in
vivo (2), and is believed to play a major role in safeguarding against tumour
formation by suppressing the emergence of immortal cells (3). The biological
significance of replicative senescence has been highlighted by observations
showing that the in vitro lifespan of cells is related to the age of the donor as well
as the general life expectancy of the species (4). The molecular basis underlying D
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this physiological process and how it is overcome in tumour cells is at present ad
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not very well understood. Nevertheless, replicative senescence is associated with m
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specific physiological and morhological changes (3), including a reduction in ://w
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proliferative capacity which is refractory to mitogenic stimulation, telomere c.o
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shortening, adoption of a flat and enlarged cell shape and in human cells and the y g
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appearance of senescence related -galactosidase activity (2). The molecular n
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mechanism that regulates the replicative senescence is not well understood, but b
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the accompanied growth arrest is associated specifically with the up-regulation of , 2
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negative regulators of cell cycle progression, including the tumour suppressor
p53 and the cyclin-dependent kinase inhibitors (CKIs), p21Cip1 and p16INK4a (5-8).
Overexpression of either p53, p16 NK4A, or p21Cip1 has been shown to be able to
cause premature senescence related cell cycle arrest in low passage fibroblasts
(9,10). The CKIs p21Ciip1 and p16INK4a can arrest cell cycle progression through
inhibiting the activity of cyclin-dependent kinases (CDKs) directly (11-13), while
p53 presumably may act indirectly by inducing the transcription of p21CIP1 (14). It
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has also been shown that pRB is present in its hypophosphorylated forms in
senescent cells (15). Thus, it is conceivable that the increased expression of p21Cip1
and p16INK4A detected in senescent cells can arrest cell cycle progression through
inhibition of the G1 cyclin-dependent CDK activity and thus, preventing the
phosphorylation of pRB. The hypophosphorylated pRB will in turn repress
transcription factors, including E2F which regulate the expression of genes
essential for cell cycle progression (16-18). Indeed, E2F-regulated genes including
cyclin A and E, CDK2, CDC2, dihydrofolate reductase(DHFR), and E2F1 have been
demonstrated to be down-regulated in a variety of senescent cells (19-21). These
genes that have been implicated in the senescence program are somatically D
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mutated in a variety of cancers, and such mutations to these genes may ad
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contribute to development of malignant clones (22). m
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The INK4a/ARF locus encodes two potent tumour-suppressor proteins, ://w
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p16IINK4a and p19ARF, that regulate the anti-proliferative and tumour suppressor c.o
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functions of pRB and p53 proteins (23,24), respectively. Recent evidence has y g
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shown that expression of p19ARF alone is sufficient to induce cell cycle arrest (25), t on
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and the ability of p19ARF to induce cell cycle arrest depends on the presence of m
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functional p53 and is achieved through stabilisation of p53; p19ARF sequesters the , 2
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oncogene MDM2, thus preventing the MDM2 induced degradation of p53 (26-
29).
Though oncogenic Ras can transform immortal rodent cells to a
tumourigenic state, introduction of oncogenic Ras into primary fibroblasts can
trigger premature senescence through the activation of the tumour suppressor
p16INK4A, p19ARF, and p53 (30,31). The ability of the oncogenic Ras to transform
immortal rodent cell lines involves its capacity to interact and activate a range of
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downstream effectors, including Raf-1, phosphoinositide 3-kinases, and Ral.GDS
(32-34). These signalling molecules, in turn, activate their respective downstream
targets and signalling pathways. Although the involvement of these signalling
pathways in mediating the senescence process is unclear, constitutive activation
of molecules along the Raf-1/MAPK signalling cascade, including Raf-1, MEKs,
and MAPKs, have been demonstrated to be able to induce premature senescence
through activating p16INK4A and p53 (35,36).
Phosphoinositide 3-kinases (PI 3-Ks) are a group of lipid kinases that
catalyse the specific phosphorylation of the inositol ring of phosphoinositides at
position 3 (37), and are involved in a variety of cellular responses, including cell D
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growth, survival, metabolism, differentiation, cytoskeletal organisation, and ad
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membrane trafficking (38). Several nematode genes, including age-1, daf-2, akt-1 m
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and –2, and daf-16, shown to affect the life-span of Caenorhabditis elegans have ://w
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been identified to encode homologues of molecules making up the PI 3-kinase c.o
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signal transduction pathway. For instance, AGE-1 is a nematode homologue of y g
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the p110 subunit of PI 3-kinase; DAF-2 encodes a member of the insulin/IGF 1 n
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receptor family, which generally signals through PI 3-kinase; AKT-1 and AKT-2 m
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are nematode homologues of mammalian Akt/PKB, which commonly acts , 2
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downstream of PI 3-kinase; DAF-18 encodes the PTEN which is an antagonist of
PI 3-kinase activity, by removing the 3-phosphate from 3-phosphoinositides;
DAF- 16, a member of the forkhead/winged-helix family of transcriptional
regulators (39). These findings from C elegans strongly suggested that the PI 3-
kinase signal transduction pathway has a role in mediating senescence signals.
Apart from an isolated study showing that inhibitors of PI 3-kinases can shorten
the lifespan of human diploid fibroblast cell line WI-38 (40), the role of PI 3-
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kinases in mediating senescence in mammalian cells has not been investigated. In
the present study, we explore the role of PI 3-kinases in regulating cell
proliferation and senescence as well as the mechanisms involved.
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Experimental Procedures
MEFs isolation and cell culture
Wild-type mice and mice with deletion of either INK4a/ARF (41), p21Cip1 (42),
p27Kip1 (43), or p53 (44) genes were maintained at the animal facilities of the
Imperial College, (London) and CNB (Madrid). Primary mouse embryo
fibroblasts (MEFs) were isolated from day 13.5 embryos derived form the
corresponding colonies of wild-type or gene ‘knock-out’ mice as described
previously (45). Briefly, each embryo was dispersed and trypsinised for 20 min at
37°C and the resulting cells were grown for 1 day in a 10 cm diameter tissue
culture plate. After which, the cells were replated onto a 15 cm dish and allowed D
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to grow for 2 days. These cells, designated passage number 0 cells, were stored ad
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in liquid nitrogen for later use. MEFs derived from ARF -/- mices were kindly m
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provided by Dr. Charles Sherr. The MEFs were cultured and passaged as ://w
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described previously (46). Briefly, 106 cells were replated every three days on 10 c.o
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cm plates. MEFs were cultured in Dulbecco's modified Eagle's Medium (DMEM) y g
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supplemented with 10% foetal calf serum and 100 g/ml of n
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penicillin/streptomycin. For kinase inhibitor treatment, fibroblasts were grown b
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to 60% confluence and the tissue culture medium changed before addition of , 2
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inhibitors. Cells were treated for 24-48 h with PD98059 (50 µM; Calbiochem) or
LY294002 (25 µM; Calbiochem) or Wortmannin (10 nM; Calbiochem) unless
specified otherwise.
Western blot analysis and antibodies
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Western blot cell extracts were prepared by lysing cells with 3 times
packed cell volume of lysis buffer (20mM HEPES pH7.9, 150mM NaCl, 1mM
MgCl , 5mM EDTA (pH.8.0), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
2
SDS, 50mM NaF, 5mM sodium orthovanadate) on ice for 20 min. Protein yield
was quantified by Bio-Rad Dc protein assay kit (Bio-Rad). Samples corresponding
to 50µg of lysates were separated by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membranes and recognised by appropriate
antibodies. The antibodies against p15 (M-20), p16INK4a (M-156), p18INKc (M-168),
p19 INK4d (M-167), p21Kip1 (M-19), p57Kip2 (M-20), p107 (C-18), CDK2 (M-2), p27Kip1
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(C-19), and cyclin E (M-20) were purchased from Santa Cruz Biotechnology. o
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Anti-p19ARFARF (R562) antibody was purchased from ABCam. Anti-p130 (anti- ad
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pRB2) and anti- p27Kip1 (K25020) monoclonal antibodies were acquired from m
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Transduction Laboratories. The antibodies were detected using horseradish ://w
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peroxidase-linked goat anti-mouse or anti-rabbit IgG (Dako) and visualised by c.o
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the enhanced chemiluminescent (ECL) detection system (Amersham Pharmacia g
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Biotech, UK). n
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Immunoprecipitation, CDK2 kinase assays and immunodepletion , 2
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For immunoprecipitation and CDK2 kinase assays, cells collected were washed
with PBS and lysed in lysis buffer containing 20mM Tris-HCl at pH7.9, 150mM
NaCl, 1mM EGTA, 1mM EDTA (pH.8.0), 1% Triton X-100, 2.5mM sodium
pyrophosphate, 1mM sodium orthovanadate, 1mM PMSF. Protein lysates
(100µg) were then incubated with 5µg of anti-CDK2 (M2) for 2 h at 40C.
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Afterwards, 20 l of protein A-Sepharose beads (Pharmacia) (50%) in lysis buffer
were added and the mixture were incubated for a further 2 h at 4 h. the anti-
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CDK2 immunoprecipitates were then washed substantially and resuspended in
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20 l of kinase buffer (20 mM Tris-HCl [pH 8.0], 10 mM MgCl , 1 mM EDTA, 1
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µ µ γ
mM DTT), supplemented with 2.5 g of histone H1 (Sigma) and 10 Ci of [ -
32P]ATP (3000 Ci/mmol; Amersham). Reaction mixtures were incubated for 15
min at room temperature and the phosphorylatyed histone H1 resolved on 10%
SDS-PAGE gels. The gels were then dried and exposed to X-ray films. For
immunodepletion experiments, two extra immunoprecipitations were
performed with the anti- p27Kip1 antibody (K25020).
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Retroviral infections o
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Phoenix cells (5 x 106) were plated in a 10 cm dish, incubated for 24 h, and then de
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transfected by calcium-phosphate precipitation (47) with 20 µg of the p27Kip1 hm
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expressing retroviral plasmid, pLPC-h p27Kip1, or the empty control vector, pLPC ://ww
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(16 h at 37°C) (30). After 48 h, the virus containing-medium was filtered (0.45 µm c.o
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filter, Millipore) and supplemented with with 4 µg/ml polybrene. Recipient cells g
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were plated the night before the infection at 8 x 105 cells per 10 cm dish. N
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Retrovirus-containing supernatants were added to the target cells and plates be
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were centrifuged for 1h at 1500 rpm and incubated at 37°C overnight. Infected 0
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cells were selected 16 h later by incubating the cell population with medium
containing 2 µg/ml of puromycin. For AFX and p27Kip1 transduction, MEFs were
infected with 20 µg of retroviral plasmid, pBabe-p27Kip1, or pBabe-AFX (48) or the
empty control vector pBabe-puro for 16 h at 37°C.
Cell cycle analysis
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Cell cycle analysis was performed by combined propidium iodide (PI) and
bromodeoxyuridine (BrdU) staining. Subconfluent cells with or without drug
treatment were labelled for 30 min with 10µM bromodeoxyuridine (BrdU;
Sigma, UK). Cells were trysinised, collected by contrifugation, and resuspended
in PBS, before fixing in 80% ethanol. The fixed cells were incubated first with 2M
HCl, then with 0.5% Triton X-100 for 30 min at room temperature, and then
with FITC–conjugated anti-BrdU antibodies at 1:3 dilution for 30 min, with PBS
washes between each treatments. The cells were incubated with 5µg/ml
propidium iodide, 0.1 mg/ml RNAse A, 0.1% NP-40, and 0.1% trisodium citrate
for 30 min prior to analysis using a Becton Dickinson FACSort analyser. The cell D
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cycle profile was analysed using the Cell Quest software. ad
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Growth Curves ://w
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Cell proliferation was monitored by [3H]-thymidine incorporation assays. MEFs c.o
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were seeded into 96-well plates at 2x103 cells per well and cultured in DMEM, and g
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10% FCS. [3H]-thymidine was added for the final 20 h of the indicated times. n N
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Cells were collected and [H3]-thymidine incorporation into DNA was quantified be
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by scintillation counting. Each point was determined in triplicate, and each 01
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growth curve was performed at least twice.
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