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The Prostate 32:266–271 (1997)
Transient Tyrosine Phosphorylation of p34cdc2 Is
an Early Event in Radiation-Induced Apoptosis of
Prostate Cancer Cells
Natasha Kyprianou,1,3* Arvinder Bains,1 and Juong G. Rhee2
1
Division of Urology, University of Maryland School of Medicine, Baltimore, Maryland
2
Department of Radiation Oncology, University of Maryland School of Medicine,
Baltimore, Maryland
3
Department of Molecular Biology and Biochemistry, University of Maryland School of
Medicine, Baltimore, Maryland
BACKGROUND. Previous studies have demonstrated that androgen-independent human
prostate cancer cells undergo radiation-induced apoptosis. The present study investigated the
early events that trigger the apoptotic response of prostate cancer cells after exposure to
ionizing irradiation.
METHODS. Human prostate cancer cells (PC-3) were exposed to single doses of ionizing
irradiation, and the immediate protein phosphorylation events were temporally correlated
with induction of apoptosis. Apoptosis among the irradiated cell populations was evaluated
using the fluorescein-terminal transferase assay.
RESULTS. The kinetics of phosphorylation of a Mr 34,000 substrate followed a transient
course: an initial increase was observed after 10 min postirradiation, reaching maximum
levels by 60 min, and the protein subsequently underwent rapid dephosphorylation. Subsequent analysis revealed that the substrate for this tyrosine phosphorylation is the serine/
threonine p34cdc2 protein kinase, a cell cycle regulatory protein that controls cell entry into
mitosis. This enhanced phosphorylation temporally preceded the radiation-induced apoptotic
DNA fragmentation as detected by the terminal transferase technique. Arresting the cells in
G0/G1 phase by pretreatment with suramin totally abrogated radiation-induced phosphorylation of p34cdc2 protein at the tyrosine residue, indicating that this posttranslational modification occurs in cell populations that escape G2 arrest and undergo apoptosis in response to
radiation.
CONCLUSIONS. These results suggest that a rapid and transient phosphorylation of a
protein that controls mitotic progression precedes and potentially triggers radiation-induced
apoptosis in prostate cancer cells. Prostrate 32:266–271, 1997. © 1997 Wiley-Liss, Inc.
KEY WORDS:
prostate cancer; suramin; radiation-induced apoptosis; tyrosine phosphorylation
INTRODUCTION
The cell nucleus has been identified as the primary
target for the lethal effects of ionizing irradiation, with
radiation-induced DNA damage ultimately leading to
cell death [1]. Following exposure to ionizing irradiation, mammalian cells progress into and are arrested
in the G1-S or G2 phase of the cell cycle, an arrest
required for repair of DNA damage before entry into
mitosis [2]. This implicates the existence of a surveillance mechanism operating in mammalian cells, bio© 1997 Wiley-Liss, Inc.
logically analogous to the RAD 9 protein identified in
Sacharomyces cerevisiae [3], which prevents a mitotic
catastrophe by inducing G2 arrest following detection
Abbreviations: PBS, phosphate-buffered saline; BSA, bovine serum
albumin; PMSF, phenyl methyl sulphonyl fluoride.
*Correspondence to: Dr. Natasha Kyprianou, Division of Urology,
Department of Surgery, University of Maryland School of Medicine,
22 South Greene Street, Baltimore, MD 21201.
Received 16 April 1996; Accepted 13 December 1996
Transient p34cdc2 Phosphorylation Signals Apoptosis in Response to Radiation
of DNA damage [4]. Its mammalian biological equivalent, p34cdc2, is a cell cycle-regulated serine/threonine
protein kinase that controls cell entry into mitosis [5].
The enzymatic function of p34cdc2 is highly regulated
by a complex series of phosphorylation/dephosphorylation reactions on Ser, Thr, and Tyr residues,
and by its association with cyclins [6]. When phosphorylated on a tyrosine residue, p34cdc2 kinase is inactive until dephosphorylation results in its activation
at the G2/M transition. Ionizing irradiation has been
demonstrated to induce transient inactivation of
p34cdc2 kinase by rapid tyrosine phosphorylation as a
mechanism of G2 arrest in several cellular settings
[7–9]. Transient activation/inactivation of this critical
cellular radioresponsive element of the signal transduction pathway can potentially determine the cell’s
ability to survive a radiation challenge, by initiating a
series of biochemical events leading to cell cycle delay,
altered DNA repair mechanisms, or death (in response
to radiation), by triggering its genetic program of apoptosis.
Apoptosis is the molecular mechanism of physiologically relevant cell death, that plays a major regulatory role in the programmed deletion of cells during
embryonic development, growth, and maintenance of
mammalian tissue homeostasis [10]. During apoptosis, target cells commit suicide in response to specific
signals, such as hormonal depletion, DNA-targeted
chemotherapeutic drugs, and ionizing irradiation
[11,12]. This mode of cell death is a geneticallydictated pathway that involves the sequential activation of distinct biochemical events, which eventually
lead to the disintegration of the dying cells into numerous apoptotic bodies [11]. A characteristic early
commitment step in the apoptotic pathway is the fragmentation of genomic DNA into nucleosomal oligomers [12]. Signals generated at the end of the pathway
activate adjacent cells and infiltrating macrophages to
phagocytize the dying cell and its disintegrating
nucleus [11].
Previous studies have implicated apoptosis as the
molecular mechanism underlying the lethal effects of
radiation in human prostatic tumor cells [13]. Although a potential functional link, between certain key
components of the cell cycle regulatory pathway and
activation of programmed cell death in response to
ionizing irradiation, has been investigated in other cellular settings [11,14,15], studies of the potential role of
p34cdc2 in radiation-induced apoptosis of human cancer cells have been scarce. The present findings identified the rapid and transient tyrosine phosphorylation
of p34cdc2, a protein that controls mitotic progression,
as an early event that precedes and possibly mandates
radiation-induced apoptotic cell death of prostate cancer cells.
267
MATERIALS AND METHODS
Cell Culture
Androgen-independent prostate cancer cells (PC-3)
were grown as monolayer cultures in RPMI-1640 medium (GIBCO, Gaithersburg, MD), containing 10% fetal calf serum (Hyclone, Logan, UT), supplemented
with 1 mM L-glutamate, 100 U/ml potassium penicillin G, and 100 U/ml streptomycin sulfate. Cells were
maintained in exponential growth in monolayer cultures with regular medium changes. Subconfluent cultures of PC-3 cells were incubated with suramin (FBA
Pharmaceuticals, New York, NY; 300 mg/ml) for 3
days prior to radiation. Clonogenic survival of prostate cancer cells after radiation exposure (with or without suramin pretreatment) was determined postirradiation by a standard colony formation assay. Cells
were irradiated, and 24 hr postirradiation, cells were
seeded in six-well plates (1,000 cells/well), and after 7
days colonies greater than 30 cells were counted.
Radiation Treatment
Irradiation was performed at room temperature using a Seifert 250 kv/15 mA irradiator unit (Seifert,
Bonn, Germany) with a 0.5-mm Ca/1.00-mm A1
added open-field filter. Exponentially growing cells
were irradiated with a fixed dose rate of 172.2 cGy/
min (average) as determined by dositometry.
Detection of Apoptotic DNA Fragmentation
In Situ
Prostate cancer cells undergoing apoptosis following radiation treatment were identified by immunofluorescence using the ApoTag Fluorescein Kit from
Oncor (Gaithersburg, MD), according to the manufacturer’s instructions, after 1, 3, 6, 12, and 24 hr postirradiation. This technique provides a unique visualization of the apoptotic cells by end-labeling of fragmented DNA using the terminal transferase and
dixogenin-11-deoxyuridine triphosphate reaction [16].
The cells were visualized by epifluorescence using a
fluorescence microscope (Axiovert-10, Zeiss model;
Zeiss, Inc., Thornwood, NY) with standard fluorescein
excitation and emission filters, and the percentage of
apoptotic cells was determined by counting a total
field of 200–400 cells, under ×400 magnification.
Immunoblot Analysis of p34cdc2
Protein Phosphorylation
Following exposure to ionizing irradiation, cells
were washed twice with PBS (ice-cold) and lysed in
268
Kyprianou et al.
buffer A (10 mM Tris (pH 7.4), 1 mM EGTA, 1 mM
EDTA, 50 mM NaCl, 5 mM 6-glycerol phosphate, 1%
Triton X-100, 0.1% NP-40, 1 mM sodium vanadate, 1
mM dithithreitol, 1 mM PMSF, and 10 mg/ml leupeptin). Samples were centrifuged at 14,000 rpm (15 min,
4°C) to remove insoluble material. Protein concentration in the supernatant fractions was determined by
Coomassie blue staining, using BSA as a standard.
Soluble fractions (approximately 100 mg of protein)
were electrophoretically separated by sodium-dodecyl
sulphate polyacrylamide gel electrophoresis (PAGE)
and then transferred onto nitrocellulose membranes.
The residual binding sites were blocked by incubating
the filters with 5% BSA, in PBS, for 1 hr at room temperature. The filters were then incubated with the antimouse anti-P-Tyr monoclonal antibody (4G10; UBI,
Lake Placid, NY), according to the manufacturer’s instructions, washed, and reprobed with a mouse antip34cdc2 monoclonal antibody which is unreactive with
other cyclin-dependent kinases (SC-54, Santa Cruz
Biotechnology, Santa Cruz, CA). After washing twice
with Tween-20 (0.1% v/v in PBS), the blots were incubated with anti-mouse IgG peroxidase conjugate
(Sigma Chemical Co., St. Louis, MO). The antigen/
antibody complexes were visualized by chemiluminescence (ECL detection system, Amersham Corp.,
Arlington Heights, IL).
RESULTS
When a population of cells is exposed to ionizing
irradiation, the probability of cell death is a function of
the radiation dose and is commonly assessed by the
loss of clonogenic capacity. Exposure of human prostate cancer cells to single doses of ionizing irradiation
results in loss of clonogenic survival in a dosedependent manner. Figure 1 shows the log of the percentage surviving fraction of irradiated cells vs. radiation dose. A high dose of radiation (2,000 cGy) leads to
complete loss of clonogenic ability. Suramin pretreatment (3 days prior to irradiation) renders these prostate cancer cells radioresistant, consistent with suramin’s ability to arrest proliferating cells in the G0/G1
phase of the cell cycle [17], thus preventing them from
entering G2 phase, and ultimately from undergoing
radiation-induced growth arrest or apoptosis.
We previously demonstrated by agarose gel electrophoresis that DNA fragmentation into the characteristic ladder of oligonucleosomal fragments preceded radiation-induced toxicity in human prostate
cancer cells [13]. This apoptotic DNA fragmentation
was partially abrogated by suramin pretreatment. In
the present study, apoptotic DNA fragmentation
among the irradiated PC-3 cell populations was detected in situ using the ApoTag fluorescein procedure,
Fig. 1. Radiation survival curves for the PC-3 prostate cancer
cells treated with radiation only, and those pretreated with suramin prior to irradiation. Points represent the mean value of three
separate experiments. Bars, SE.
that identifies apoptosis at the single-cell level (Fig.
2B). As shown in Figure 2A, there was a gradual increase in the number of apoptotic cells with increasing
periods of time postirradiation, which was entirely
blocked when cells were treated with suramin prior to
irradiation. Since radiation-induced DNA damage
most frequently occurs when cells are in the G2 phase
of the cell cycle [2], suramin pretreatment of an asynchronous population of radiosensitive cells (possibly
by preventing cell entry into G2) abrogates the apoptotic effects of radiation, possibly through various radiation protection mechanisms activated intracellularly [15].
Protein phosphorylation is an early signal in the
generation of positive growth signals [6]. In this study
we investigated whether transient protein phosphorylation changes are potentially involved in signaling
apoptotic cell death in human prostate cancer cells
following exposure to ionizing irradiation. Exponentially growing cultures of PC-3 cells were irradiated
(600 cGy) and were subsequently analyzed for proteins with increased levels of phosphotyrosine. Figure
3 (top) reveals an immunoblot analysis of protein
phosphorylation status using a specific anti-phosphotyrosine antibody. A significantly increased reactivity
with a protein of Mr 34,000 was initially observed as
early as 10 min after irradiation and then declined
after 60 min. Reprobing the filters with an antibody
against the p34cdc2 kinase revealed a signal that was
entirely superimposed to that obtained for the protein
(Mr 34,000) with the predominant tyrosine phosphorylation (Fig. 3, bottom). The observation that there was
no significant change in p34cdc2 protein levels in the
control and irradiated cells implicates p34cdc2 as a substrate for increased tyrosine phosphorylation following exposure to ionizing radiation. A similar phosphorylation pattern was obtained with increasing
Transient p34cdc2 Phosphorylation Signals Apoptosis in Response to Radiation
269
Fig. 2. Induction of apoptosis in prostate cancer cells following exposure to ionizing irradiation. A: PC-3 cells were exposed to a single
radiation dose (600 cGy), with or without pretreatment with suramin (300 µg/ml, for 3 days). Cells exhibiting DNA fragmentation (as
detected by ApoTag fluorescence staining) were determined among a population of 300–400 cells at 1, 3, 6, 12, and 24 hr postirradiation.
Values represent the number of apoptotic cells expressed as percentage of the total number of cells counted. B: A representative field of
irradiated PC-3 cells (at 24 hr postirradiation), indicating single apoptotic cells as identified by the fluoroscein ApoTag technique. ×400.
Suramin pretreatment completely abrogated the radiation-induced tyrosine phosphorylation of p34cdc2,
irrespective of the time or the radiation dose (Fig.
4A,B). The observation that both processes, tyrosine
phosphorylation of p34cdc2 and apoptosis activation in
response to radiation, are inhibited by suramin pretreatment, suggests that they share a functional relationship. Since suramin causes a G1 arrest [17], this
rapid phosphorylation change probably occurs in cells
that had entered the G2 phase of the cell cycle. Flow
cytometric analysis is currently being performed to
confirm that the observed phosphorylation changes
occur in G2-phase cells.
DISCUSSION
Fig. 3. Ionizing irradiation induces transient tyrosine phosphorylation of a Mr 34,000 protein substrate. PC-3 prostate cancer
cells were exposed to ionizing irradiation (600 cGy), cell lysates
were prepared at indicated times, and soluble proteins (100 µg)
were sequentially subjected to immunoblot analysis with (top) the
anti-phosphotyrosine and (bottom) p34cdc2 antibodies, as shown.
Arrows indicate position of Mr 34,000 signals.
doses of ionizing irradiation, i.e., 200, 400, 600, 800,
1,000, and 1,200 cGy (data not shown). The findings,
that the signals obtained with the anti-p34cdc2 antibody were consistently superimposable over those detected with the anti-phosphotyrosine, imply that
p34cdc2 protein undergoes a rapid and transient phosphorylation on the tyrosine residue. Furthermore, this
process precedes radiation-induced apoptotic cell
death (Fig. 2).
The present study demonstrates that treatment of
prostate cancer cells with ionizing irradiation leads to
a rapid transient tyrosine phosphorylation of p34cdc2
protein, in accordance with similar observations in
HL-60 cells [8]. The transient kinetics of the translational modification of this cell cycle regulatory protein
implicate its occurrence as a signaling event for the
apoptotic pathway in response to radiation. The concept gains support from evidence suggesting that transient tyrosine phosphorylation of cyclin-dependent
protein kinases dictates the cytotoxic activity of chemotherapeutic drugs [18,19]. These results however,
indirectly challenge previous studies reporting that
transient activation of protein kinases through tyrosine dephosphorylation (occurring at an inappropriate
time during the cell cycle) can trigger apoptosis
[14,20]. This apparent discrepancy could be due to differences in the kinetics of regulation of p34cdc2 phosphorylation-dephosphorylation relative to the cell
270
Kyprianou et al.
Fig. 4. A: Time course of tyrosine phosphorylation in PC-3 cells incubated with suramin for 3 days (300 µg/ml) prior to exposure to
ionizing irradiation. Immunoblot analysis was performed on cell lysates (prepared at various times postirradiation as shown), using the
anti-phosphotyrosine (top) and p34cdc2 (bottom) antibodies. Control lane comprises an asynchronous population of untreated (nonirradiated) PC-3 prostate cancer cells. B: Effect of suramin pretreatment on tyrosine phosphorylation of p34cdc2 following exposure of PC-3
cells to increasing doses of radiation. Cell lysates were prepared after 15 min of radiation treatment. Control lane, nonirradiated
suramin-treated cells.
synchronization status of different cell types responding to diverse stimuli. One has also to consider that
tyrosine dephosphorylation alone might not be sufficient for the functional activation of p34cdc2, and that
threonine dephosphorylation might be an additional
requirement, as previously reported for other mammalian cells [6]. At present, it is unknown whether, in
addition to tyrosine phosphorylation, a transient
threonine phosphorylation is also induced following
exposure of prostate cancer cells to ionizing irradiation.
Studies are currently in progress to investigate
whether this radiation-induced transient phosphorylation of p34cdc2 protein temporally correlates with a
decrease in the kinase activity of irradiated prostate
cancer cells. The present results, taken together with
previous evidence implicating p34cdc2 tyrosine phosphorylation in triggering other early characteristic
events in the radiation-induced apoptotic pathway
[21], will enhance our understanding of the role of
p34cdc2 phosphorylation, not only as a DNA damagedetection system controlling mitotic progression, but
also in transducing specific key signals to activate the
preprogrammed apoptotic mechanism of cell death in
tumor cells.
ACKNOWLEDGMENTS
The authors acknowledge the support of the American Foundation of Urologic Disease and of the Depart-
ment of Surgery at the University of Maryland School
of Medicine in funding this study.
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