¡CANCERRESEARCH 50. 3761-3766, June 15, 1990]
Inhibition of p34cdc2Kinase Activity by Etoposide or Irradiation as a Mechanism
of G2 Arrest in Chinese Hamster Ovary Cells1
Richard B. Lock2 and Warren E. Ross3
The J. Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40292
ABSTRACT
The mammalian homologue of the yeast cdc2 gene product, p34"*''2,is
a cell cycle-regulated
protein essential
for mitosis. We have used poly-
clonal antisera raised against a peptide corresponding to the carboxyl
terminus of the sequence of human cdc2 to study p34dc2 in Chinese
hamster ovary (CHO) cells. Major bands are immunoprecipitated at a
molecular weight of 34,000, although not in the presence of competing
antigenic peptide. p34"" is coimmunoprecipitated
with proteins of mo
lecular weights of 52,000 and 57,000.
Immunoprecipitates
express his-
tone HI kinase activity which varies throughout the cell cycle, maximal
activity being observed in G2-M. The activity of the p34cic2 kinase varies
according
according
ous CHO
etoposide,
to its association with the M, 52,000 and 57,000 proteins and
to their phosphorylation state. Treatment of either asynchron
cells or an enriched G2 population with the antitumor agent,
results in rapid inhibition of immunoprecipitated p34rfc2 kinase
activity, which is not due to a direct effect of drug upon the enzyme.
p34cdc2kinase activity recovers as cells arrest in G2 and a second etoposide
treatment further inhibits p34cdc2kinase activity and prolongs G2 arrest.
Exposure of asynchronous CHO cells to 7-irradiation also inhibits p34cdc2
kinase activity within 1 h. Again this activity recovers as cells accumulate
in G2. These results suggest that DNA damage in CHO cells elicits a
response which results in inhibition of p34cdc2 kinase activity and, con
sequently, G2 arrest.
INTRODUCTION
G2 arrest is a response exhibited by proliferating eukaryotic
cells exposed to a variety of DNA damaging agents including
UV light (1), DNA alkylators (2), A'-irradiation (3), radiomimetic agents such as bleomycin (4), and topoisomerase II inhib
itors (5, 6). It is presumed that G2 arrest allows cells to repair
DNA lesions prior to mitosis. Caffeine exposure during G2
induces mitosis before DNA repair is complete, resulting in
enhanced cell killing (7). The biochemical mechanisms under
lying G2 arrest are not well understood. It has been proposed
that G2 arrest occurs as a result of the failure of essential genes
to be transcribed, leading to an inability to divide (8, 9), al
though there is no direct evidence to support this hypothesis.
Indeed, it seems equally likely that G2 arrest results from a
broad-based cellular response to DNA damage which alters the
normal processes which regulate the transition of cells from G2
to mitosis.
The antitumor epipodophyllotoxin, etoposide,4 appears to
exert cytotoxicity via an interaction with the nuclear enzyme
DNA topoisomerase II. Treatment of mammalian cells with
this agent results in the rapid formation of DNA single- and
double-strand breaks (10). These breaks involve the interaction
Received 10/18/89; revised 2/14/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by funds from the NIH (USPHS Grant CA-24586).
2To whom requests for reprints should be addressed.
3 Present address: The College of Medicine, University of Florida, Gainesville,
FL 32610.
4 The abbreviations and trivial name used are: etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-0-ethylidene-/3-D-glucopyranoside);
CHO, Chinese hamster
ovary; WT, wild-type; PBS, phosphate-buffered saline; FCS, fetal calf serum;
DMSO, dimethyl sulfoxide; PMSF, phenylmethylsulfonyl fluoride; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol.
of drug, DNA, and topoisomerase II to form a stable interme
diate, termed the "cleavable complex" (11), in which one subunit of the topoisomerase II homodimer is covalently attached
to the 5'-end of the broken DNA strand via a tyrosine residue
(12). A linear correlation exists between etoposide-induced
cytotoxicity and DNA double-strand breaks (13), although,
upon removal of drug, rapid resealing of DNA lesions occurs
(10). This indicates that cleavable complex formation induces
a chain of events which results in cell death. Processes distal to
cleavable complex formation are poorly understood, but have
been proposed to involve illegitimate DNA recombination
events (14) or an "SOS"-like response analogous to bacterial
systems (15). As noted, one response which has been demon
strated for epipodophyllotoxins and all other topoisomerase II
inhibitors is cell cycle arrest in the G2 phase (5, 6).
The mechanisms which control cell cycle progression have
been the subject of intensive research. Recently mammalian
proteins have been identified which share homology with yeast
cell division cycle control gene products, suggesting that ele
ments of the mitotic control are conserved in all eukaryotes
(16). Indeed, a homologue of the cdc2 gene product from
Schizosaccharomyces pombe is a component of maturation pro
moting factor (17), a cytoplasmic extract from metaphasearrested Xenopus oocytes that was originally characterized by
its ability to induce meiosis in G2-arrested oocytes (18). The
human homologue of the S. pombe cdc2 gene product, p34cdc2,
is a protein kinase maximally active at the G2-M transition
(19). Indeed, it copurifies with histone HI kinase, predicting
an intracellular function for this latter enzyme during mitosis
(17). Enzymatic activity correlates with increased association
of p34cdc2with a M, 62,000 protein (p62, a human cyclin) and
varies according to the phosphorylation state of p34cdl:2(19).
The p34cdc2kinase interacts directly with p 13, the human hom
ologue of the S. pombe sud gene product (20), and with M,
50,000 to 60,000 proteins, cyclins A and B, in an active complex
in the clam (21). Circumstantial evidence would imply that
p34cdc2 plays a central role in the protein phosphorylation
cascade which results in nuclear envelope disassembly, chro
mosome condensation, and construction of the mitotic spindle,
by directly phosphorylating relevant proteins or stimulating
other regulatory and effector enzymes (for review see Ref. 22).
Microinjection of anti-p34cdc2 antibodies into serum-stimulated
rat fibroblasts has yielded direct evidence that p34cdc2is required
in order for cell division to occur (23). Exit from mitosis is
accompanied by inactivation of the p34cdc2/cyclin complex,
p34cdc2phosphorylation, and cyclin proteolysis (21, 24).
The lack of understanding of G2 arrest and cell death proc
esses in mammalian cells led us to analyze the role that p34cdc2
may play in these important biological responses. We have used
antisera prepared against the carboxyl terminus of human
p34cdc2protein to verify that p34cdc2is a cell cycle-regulated
protein in CHO cells, and that maximal activity is attained in
G2-M. In addition, we demonstrate that treatment of CHO cells
with either etoposide or 7-irradiation, at doses which result in
G2 arrest, causes rapid inhibition of p34cdc2activity.
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INHIBITION OF pit"*2 KINASE ACTIVITY BY DNA-DAMAGING
MATERIALS
AND METHODS
Cell Culture, Synchronization, and Labeling. WT and etoposideresistant (VpmR-5) CHO cell lines were maintained as monolayer
cultures at 37°Cin <. minimal essential medium supplemented with 5%
PCS in a 5% CO2 atmosphere. The selection and characterization of
the resistant VpmR-5 subline have been described (25, 26). Drug
treatments were carried out using 25 /¿Metoposide for l h at 37°C
(1.5% survival compared with nondrug-treated controls by colonyforming assay) unless stated otherwise. A 10 IÕIM
working solution of
etoposide was prepared immediately prior to use from a 100 mM stock
dissolved in DMSO which had been stored at -20°C.Control cultures
received the equivalent solvent exposure (0.04% for 25 fiM etoposide).
Following drug treatment, cells were rinsed twice with drug-free me
dium (37°C)and incubated in drug-free medium at 37°C.Cells were
irradiated using a 60Co source at a dose rate of 196 cGy/min.
Synchronization of CHO cells was achieved using a double thymidine
block (27). Exponentially growing cells were incubated at 37°Cfor 12
h in medium containing 2 mM thymidine, followed by a 6-h incubation
in thymidine-free medium. Synchronization of cells at the G,-S bound
ary was then facilitated by a further 12-h incubation in thymidinecontaining medium (2 mM).
For labeling of proteins, 1 to 2 x IO6 log-phase CHO cells were
incubated for 2 h with 50 ^Ci/ini of Tran[35S]label (ICN) in methioninefree medium, or with 100 /iCi/ml of [32P]orthophosphate (ICN) in
phosphate-free medium.
Flow Cytometry. Approximately IO6cells were harvested by trypsinization. After neutralization of the trypsin with medium containing 5%
FCS, the cells were centrifuged (1000 rpm for 5 min), washed twice in
ice-cold PBS, and finally resuspended in 0.3 ml of PBS. Cells were
fixed by the gradual addition of 0.9 ml of ice-cold ethanol. After
fixation, cells were washed twice with PBS and finally resuspended in
0.4 ml of PBS. Fifty ß\of RNase A (5 mg/ml in PBS) and 50 jul of
propidium iodide solution (50 /¿g/mlin 50 mM sodium citrate, pH 7.6)
were then added. Cell cycle distribution was estimated using an Ortho
Cytofluorograph Us with an Ortho 2151 data handling system with
doublet discrimination. The cell cycle distribution often thousand cells
was analyzed and quantitated with reference to asynchronously dividing
control samples.
p34cdc2Kinase Studies. CHO cell extracts were prepared by lysis of
monolayer cells in 50 mM Tris-Cl, pH 7.4. 250 mM sodium chloride,
0.1% Nonidet P-40, 5 mM EDTA, 50 mM sodium fluoride containing
the protease inhibitors antipain, aprotinin, chymostatin, leupeptin, and
pepstatin A, each at a concentration of 20 /ig/ml, and 2 mM PMSF.
After incubation on ice for 30 min, the samples were centrifuged at
13,000 x g for 5 min at 4°C,and the supernatant was used for the
subsequent assays. Aliquots were removed for protein determination
(28) or quantification of TCA-precipitable radioactive material.
For immunoprecipitation reactions, equal amounts of protein (100
jig for kinase assays) or TCA-precipitable radioactivity (IO7 cpm for
"S- and 32P-labeling studies) were normalized by dilution with the
above lysis buffer containing 1 mM PMSF. Immunoprecipitation was
accomplished by the addition of 3 n\ of affinity-purified polyclonal
antisera, raised against a synthetic peptiede corresponding to the carboxyl terminus of the human cdc2 protein (19); this was kindly provided
by Dr. Giulio Draetta, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY. Titration of the antisera revealed its optimal dilution to
be 1:100 (data not shown). In some cases 100 nmol of competing
antigenic peptide were included in the reactions. Following incubation
for l h at 4°C,3 mg of Protein A-Sepharose (Pharmacia) were added
and the mixture was incubated on a rotator for 20 min at 4°C.Following
centrifugation (13,000 x g for 10s) pellets were washed 3 times with 1
ml of lysis buffer containing 1 mM PMSF at 4°C,and to the final pellet
were added 60 n\ of 2x SDS sample buffer [100 mM Tris-Cl (pH 6.8)4% SDS-20 mM DTT-20% (v/v) glycerol-0.05% bromophenol blue],
and the samples were boiled for 3 min and electrophoresed on a 7.5 to
15% linear gradient SDS-polyacrylamide gel. For 32P-labeled proteins,
gels were fixed with 7% acetic acid, dried, and autoradiographed. "Slabeled proteins were detected using fluorography by impregnating the
gel after fixation (7% acetic acid) with l M sodium salicylate, pH 6.8.
Kinase assays were carried out using immunoprecipitates from un-
AGENTS
labeled cell lysates as previously described (16) in a reaction volume of
25 p\ containing 50 mM Tris-Cl, pH 7.4, 10 mM magnesium chloride,
1 mM DTT, l ¿IM
ATP, 50 ng/ml of histone HI (Boehringer Mann
heim), and 5 nC\ of [-y-32P]ATP(specific activity, 3000 Ci/mmol). The
radioactivity in each band was quantitated by excising the band and
incubating it in 2 ml of 30% H2O2 overnight at room temperature.
After the addition of 6 ml of ScintiVerse II (Fischer), radioactivity was
determined by liquid scintillation counting.
Alkaline phosphatase treatments of immunoprecipitated proteins
were carried out as previously described (19).
For Western blot analysis, proteins from cell lysates were electro
phoresed as described above, transferred to nitrocellulose filters (29),
and probed with anti-p34cdc2antisera. Bands were visualized using an
alkaline phosphatase detection system (Bio-Rad Laboratories).
RESULTS
p34cdc2Kinase Activity in CHO Cells. Immunoprecipitation
of [15S]methionine-labeled CHO cell lysates, prepared from
asynchronously dividing cells, with anti-cdc2 peptide serum
followed by SDS-polyacrylamide gel electrophoresis resolved
major bands with a molecular weight of approximately 34,000
(Fig. \A, Lane /), which were not precipitated in the presence
of competing antigenic peptide (Lane 2). In addition, proteins
with molecular weights of approximately 52,000, 57,000, and
64,000 were identified in immunoprecipitations carried out in
the absence of competing antigenic peptide (compare Lanes 1
and 2 in Fig. IA). HeLa p34cdc2protein élûtes
at molecular
weights of both 34,000 and >200,000 when subjected to gel
filtration chromatography (19). The higher molecular weight
complex includes a M, 62,000 protein, which has more recently
been proposed to be a cyclin (21). Neither the M, 52,000 nor
the 57,000 proteins were immunoprecipitated from CHO cells,
which had been lysed in 50 mM Tris-Cl (pH 6.8)-0.5% SDS-1
97.4-
57kD-I
52kD-I
66.2>•
»•
57kD
42.7-
Histone HI
31.0-
21.514.4-
1234
123
A
B
Fig. I. A, p34cdc2immunoprecipitated from [35Sjmethionine-labeled cell lysates
in the absence (Lanes I and 3) or presence (Lane 2) of competing antigenic
peptide. In Lane 3, the immunoprecipitate was treated with 6.5 units of purified
alkaline phosphatase for 15 min at 37°Cprior to electrophoresis. Lane 4 repre
sents an immunoprecipitate from [3!P]orthophosphate-labeled cells. Numbers on
the left correspond to molecular weight markers which were as follows: phosphorylase ft (M, 97,400); bovine serum albumin (M, 66,200); ovalbumin (M, 42,700);
carbonic anhydrase (M, 31.000); soybean trypsin inhibitor (M, 21,500); and
lysozyme (MT14.400). B, kinase activity of CHO immunoprecipitates. p34cdc2was
immunoprecipitated from 100 ^g of total cellular protein and incubated with [732P]ATP in the presence (Lanes I and 3) or absence (Lane 2) of exogenously
added histone HI. In Lane 3 the immunoprecipitation was carried out in the
presence of competing antigenic peptide.
3762
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INHIBITION OF p34**2 KINASE ACTIVITY BY DNA-DAMAGING
AGENTS
HIM DTT and boiled for 5 min (data not shown). This lysis
procedure disrupts protein-protein complexes, suggesting that
the M, 52,000 and 57,000 proteins exist in a high-molecularweight complex with p34cdc2.Incubation of anti-cdc2 peptide
immunoprecipitates with alkaline phosphatase resulted in the
disappearance of the slower migrating bands with a molecular
weight at around 34,000 (Fig. \A, Lane 3). This treatment also
resulted in less of the immunoprecipitated proteins being de
tected in the M, 40,000 to 90,000 range which, unlike the
proteins in the M, 34,000 range, did not include a shift in
mobility and may represent dilution of 35S-labeled protein.
Immunoprecipitation of 32P-orthophosphate-labeled cell lysates
with anti-peptide serum revealed a M, 34,000 doublet (Fig. \A,
Lane 4), which corresponded to the higher molecular weight
forms from 35S-methionine-Iabeled lysates (compare Lanes 1
and 4).
Incubation of immunoprecipitates prepared from lysates of
asynchronously dividing CHO cells with exogenously added
histone HI and [-y-"P]ATP resulted in phosphorylation of the
histone (Fig. ÃŒB,
Lane 1). In the absence of exogenously added
substrate a M, 57,000 protein was phosphorylated (Fig. IB,
Lane 2). No kinase activity was observed when the antecedent
immunoprecipitation was carried out in the presence of com
peting antigenic peptide (Fig. IB, Lane 3).
Thus, we have demonstrated that a protein with a molecular
weight of approximately 34,000 (p34cdc2),which exists in phos
phorylated and unphosphorylated forms, is specifically immu
noprecipitated from CHO cells by affinity-purified human cdc2
antipeptide sera.
p34cdc2Kinase Activity throughout the CHO Cell Cycle. Using
CHO cells which were synchronized at the G,-S boundary by a
double thymidine block, we have evaluated the regulation of
immunoprecipitated p34cdc2kinase activity throughout the cell
cycle (Fig. JA). In these experiments, where p34cdc2 kinase
activity was immunoprecipitated from equal amounts of total
cellular protein, exogenously added histone HI was used as a
substrate for phosphorylation. The kinase activity of immuno
precipitated p34cdc2 increased as cells progressed through S
phase (Fig. 2B, Lanes 1 to 3) and reached a peak in G2-M
(Lanes 4 to 6). This activity decreased as cells divided and
entered Gì(Fig. 2B, Lanes 7 and 8). It is unlikely that these
changes were due to differences in cellular content of the p34cdc2
protein, as Draetta and Beach (19) demonstrated that p34cdc2
protein levels do not alter throughout the HeLa cell cycle.
Furthermore, by Western blot analyses, we could detect no
significant changes in the cellular content of p34cdc2throughout
the CHO cell cycle (data not shown). Fig. 2C demonstrates
that, as CHO cells progressed through S (Lanes 1 and 2) and
into G2-M (Lanes 3 and 4), there was an increased association
of p34cdc2with the M, 52,000 and M, 57,000 proteins in [35S]methionine-labeled lysates, which decreased as cells entered G,
(Lane 5). In addition, the data indicate that p34edc2and the M,
52,000 and 57,000 proteins existed in a higher phosphorylation
state in G2-M cells compared with Gìcells (compare Lanes 10
and 11 in Fig. 2C). Thus, we have demonstrated that maximal
immunoprecipitated p34cdc2kinase activity in CHO cells occurs
during G2-M and that, during this period, p34cdc2is more highly
phosphorylated and associated with M, 52,000 and 57,000
proteins.
Etoposide Treatment and p34cdc2Kinase Activity. Treatment
of
of
in
of
CHO cells with etoposide (25 MMfor 1 h) at 4 h postrelease
a double thymidine block, when over 50% of the cells were
G2-M (Fig. 2A; Fig. 3, Lane 1), caused significant inhibition
p34cdc2kinase activity (compare Lanes 2 and 8 in Fig. 3).
S«
-- c
o ~
34567
Hours Post Release
Histone HI
B
10
11
12
66.2-
42.7-
31.021.5-
Fig. 2. Variation of p34cdc2activity throughout the cell cycle. A, CHO cell
cycle distribution following release of the thymidine block, analyzed by flow
cytometry. The distribution of asynchronous control cells was 54% Gì,18% S,
and 23% G2-M. B, p34cdclkinase activity estimated from immunoprecipitates of
cells which were harvested at hourly intervals between 1 and 8 h (Lanes I to 8)
after release of the thymidine block. Lane 9 represents the kinase activity from
asynchronously dividing cells. C, SDS-polyacrylamide gel electrophoresis of
immunoprecipitates from cells which had been labeled for 2 h with [3!S]methionine (Lanes 1 to 6), or [32P]orthophosphate (Lanes 7 to 12) at 0 (Lanes I and 7),
2 h (Lanes 2 and 8), 4 h (Lanes 3 and 9), 6 h (Lanes 4 and 10), and 8 h (Lanes 5
and //) following release of the thymidine block. Lanes 6 and 12 show ¡inmuno
precipitations from asynchronously dividing cells which also had been labeled for
2 h. The molecular weight standards are the same as those used in Fig. 1/4.
CONTROL
ETOPOSIDE
9
10 11
12 13
14 15
Histone HI
Fig. 3. Inhibition of p34coc2kinase activity in G2 cells by etoposide treatment.
Synchronous cells were treated for 1 h with solvent control (Lanes 2 to 7) or 25
fiM etoposide (Lanes S to 15) at 4 h postrelease of the thymidine block (Lane 1).
At this point approximately 55% of cells were in G2-M (Fig. 2A). Cells were
harvested and processed for p34cdc2kinase assay at 0 (Lanes 2 and S), 0.5 h (Lanes
3 and 9), 1 h (Lanes 4 and 10), 1.5 h (Lanes 5 and //), 2 h (Lanes 6 and 12), 3 h
(Lanes 7 and 13), 6 h (Lane 14) and 8 h (Lane 15) posttreatment. Lanes 6 and 7
represent normal turnover of p34cdc2kinase activity as cells progressed through
mitosis (Fig. 2, .1 and /' ).
This activity did not recover until at least 8 h post-etoposide
treatment (Lane 15). Lanes 6 and 7 in Fig. 3 represent normal
turnover of p34cdc2activity as control cells progressed into G,.
Asynchronously dividing CHO cells which were exposed to
etoposide (25 /¿M
for 1 h) gradually accumulated in the G2-M
phase of the cell cycle (approximately 80% at 12 h posttreat3763
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INHIBITION OF
KINASE ACTIVITY BY DNA-DAMAGING AGENTS
ment; Fig. 4A), consistent with a G2 blockade. Exposure to
etoposide also caused a rapid inhibition of immunoprecipitated
p34cdc2activity (compare Lanes 1 and 2 in Fig. 4B). Upon
further incubation in drug-free medium, this activity gradually
recovered in concert with accumulation of cells in G2 (Fig. 4, A
and B). By 10 h posttreatment p34cdc2 kinase activity was
300
200-
equivalent to that of asynchronously dividing controls (compare
Lanes 1 and 7 in Fig. 4B). However, this activity was less than
that of synchronized G2-M cells at 5 and 6 h postrelease of a
double thymidine block (compare Lane 7 with Lanes 8 and 9 in
Fig. 4B).
Since the recovery of p34cdc2kinase activity occurred as cells
II
accumulated in G2, it was of
second drug treatment would
were retreated with etoposide
original drug treatment. This
B
interest to determine
again delay mitosis.
(25 UM for l h) 10
retreatment inhibited
whether a
CHO cells
h after the
p34cdc2ki
18
24
30
36
12
18
24
30
36
100
O o
'c Z.
60
40
20
O
6
Post Treatment Incubation (h)
Fig. 5. Inhibition of p34cdc2kinase activity and prolongation of G¡arrest by
retreatment with etoposide. Asynchronously dividing cells were treated with
etoposide (25 MM,1 h), rinsed twice with drug-free medium (37"C), and reincubated in drug-free medium (O). At 10 h posttreatment cells were retreated with
etoposide in an identical manner as the first treatment and incubated in drug-free
medium (•).. I. immunoprecipitated p34cdc2kinase activity. Radioactive bands
were excised and quantitated by liquid scintillation counting. In B, cells were
harvested, and their phase distribution was estimated by flow cytometry.
at etoposide concentrations as low as 2.5 n\t (Fig. 6, Lanes 1
to 8). On the other hand, significant inhibition of p34cdc2activity
occurred only in VpmR-5 cells at 50 UM etoposide (Fig. 6,
Lanes 9 to 16). Densitometric scans indicated that the etoposide
80
12
80-
nase activity (Fig. 5/4), although to a lesser extent than the
original treatment. Cell cycle progression was further delayed
by 10 to 12 h by this second drug exposure (Fig. 5B). In both
cases (single etoposide treatment and retreatment with the drug)
etoposide treatment caused inhibition of p34cdc2kinase activity,
followed by recovery of its activity. Recovery of p34cdc2activity
accompanied the exit of cells from G2 (Fig. 5).
In order to examine the possibility that etoposide had a direct
effect on p34cdc2kinase activity, one which was not mediated
by its putative intracellular target topoisomerase II, etoposideresistant (VpmR-5) cells were treated with etoposide, and im
munoprecipitated p34cdc2kinase activity was assayed (Fig. 6).
The VpmR-5 cell line is 20- to 30-fold resistant to the cytotoxic
and DNA-damaging effects of etoposide compared with WT
cells (26). Purified topoisomerase II from the resistant cell line
displays characteristics consistent with a mutant form of the
enzyme (30). p34cdc2kinase activity was inhibited in WT cells
100-
VpmR-5
WT
4
5
10 11 12
13
14
15
16
Fig. 6. Inhibition of p34cdc2 kinase activity by etoposide in WT but not
etoposide-resistant CHO cells. WT (Lanes 1 to 8) or etoposide-resistant VpmR5 (Lanes 9 to 16) cells were treated with 0.08% DMSO (solvent control. Lanes I
and 9), 0.5 MM(Lanes 2 and 10), 1.0 MM(Lanes 3 and //), 2.5 MM(Lanes 4 and
12), 5.0 MM(Lanes 5 and 13), 10 MM(Lanes 6 and 14), 25 MM(Lanes 7 and 15),
and 50 MM(Lanes 8 and 16) etoposide for 1 h. Cells were harvested at 2 h
postetoposide treatment, and immunoprecipitated p.14"'1- kinase activity was
estimated. Densitometric scans indicated that intrinsic P34"102kinase activity of
WT (Lane 1) and VpmR-5 (Lane 9) cells differed by less than 10%.
G2/M
*•c
—«H
.S 2
°C40
concentrations required to induce equivalent inhibition of
p34cdc2kinase in WT and VpmR-5 cells differed by a factor of
8
10
12
Post Treatment Incubation (h)
B
Histone HI
Fig. 4. Etoposide treatment of asynchronously dividing CHO cells induces G2
arrest and inhibition of p34cdc2kinase activity. A, flow cytometric analysis of cell
cycle distribution following treatment with 25 MMetoposide for 1 h. ( r;M (•),S
(*), and Gì
(O)- B, kinase activity of immunoprecipitated p34e*:2.The treatments
were as follows: solvent control (Lane 1); 25 MMetoposide for 1 h (Lane 2): 2 h,
4 h, 6 h, 8 h, and 10 h postetoposide (Lanes 3 to 7, respectively); Lanes 8 and 9
represent p34cocl kinase activity immunoprecipitated from synchronized cells at
5 and 6 h postrelease of a double thymidine block, respectively (see Fig. 2).
ten. This is consistent with the degree of etoposide resistance
exhibited by VpmR-5 cells. Thus, for etoposide-treated cells,
DNA damage via cleavable complex formation appears a pre
requisite for the cellular processes resulting in inhibition of
p34cdc2kinase activity and G2 arrest.
p34cdc2Kinase Activity following 7-Irradiation of CHO Cells.
The response of p34cdc2kinase to etoposide-induced cleavable
complex formation suggests the possibility that other DNAdamaging agents which cause G2 arrest would have a similar
effect. The following experiment supports this speculation.
Exposure of asynchronously dividing CHO cells to -y-irradia
tion (800 cGy, 2.2% survival compared with uniiradiated con
trols) caused an accumulation of cells in G2 (approximately
50% at 12 h posttreatment, data not shown). Immunoprecipi
tated p34cdc2kinase activity was significantly inhibited within 1
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INHIBITION OF
KINASE ACTIVITY BY DNA-DAMAGING
AGENTS
mechanism may involve turnover of cyclin-like proteins or
discreet differences in phosphorylation of p34tdc2,based on the
known pathways of p34cdc2regulation within the normal mam
Fig. 7. Inhibition of p34c*2 kinase activity in CHO cells following 7-irradiation. Asynchronously dividing cells were irradiated with 800 cGy and harvested
at 0 (Lane I), l h (Lane 2), 2 h (Lane 3), 6 h (Lane 4), and 12 h (Lane 5)
postirradiation. Lane 6 represents a mock-irradiated culture harvested at 12 h
postirradiation. Cells were harvested, and p34cdc2kinase activity was estimated.
h of irradiation (compare Lanes I and 2 in Fig. 7) and recovered
as cells accumulated in G2 (Lanes 4 and 5). No significant
differences were detected in the p34cdc2kinase activity of mockirradiated
shown).
cultures over an identical time course (data not
DISCUSSION
Antiserum raised against a peptide corresponding to the
carboxyl terminus of human cdc2 immunoprecipitates a M,
34,000 protein from CHO cell lysates. These immunoprecipi
tates express protein kinase activity in vitro in a cell cycleregulated fashion. Kinase activity peaks in the G2-M phase, at
which point the M, 34,000 protein exhibits maximal phosphorylation and coimmunoprecipitation with M, 52,000 and 57,000
proteins. Taken together, these data indicate that the M, 34,000
protein is the CHO homologue of the yeast cdc2 and human
p34cdc2proteins (16, 19).
Exposure of CHO cells to the topoisomerase II inhibitor,
etoposide, at a concentration which induces G2 arrest, results
in a substantial and rapid inhibition of immunoprecipitated
p34cdc2 kinase activity. This may be in response to cleavable
malian cell cycle (21, 24). However, any inhibitory mechanism
proposed would have to be sufficiently short-lived in order to
allow for the observation that p34cdc2activity recovers as cells
accumulate in G2 (Fig. 4).
The gradual recovery of p34cdc2activity in etoposide-treated
or 7-irradiated CHO cells (Figs. 4 and 7) leads to the intriguing
possibility that, in response to DNA damage, p34cdc2activity is
reset to an "early G,-like state." Interestingly, the time neces
sary for maximum recovery of p34coc2activity (between 10 and
18 h; Fig. 5) approximates the CHO population doubling time
(around 15 h). Following recovery of p34cdc2 activity, G2-arrested cells are driven through mitosis, as shown by a decrease
in G2-phase cells (Fig. 5B) and an increase in cell number (see
Ref. 31). Retreatment of G2-arrested cells with etoposide leads
to a further delay in G2 by approximately 12 h, concomitant
with inhibition and recovery of p34cdc2activity (Fig. 5). There
fore, in the experiments described in this study, G2 arrest may
be explained by a rapid inhibition of an enzyme required for
mitosis. Following recovery of p34cdc2activity the enzyme may
carry out the role required for mitosis.
Upon sustaining DNA damage, eukaryotic cells initiate a
chain of events which results in G2 arrest (4). This may allow
repair of DNA lesions necessary for a successful mitosis. By
artificially driving nitrogen mustard-treated baby hamster kid
ney cells through mitosis with caffeine, the drug's cytotoxicity
is potentiated (7), indicating the protective role of G2 arrest. It
is prudent to observe that the mechanism by which caffeine
interrupts G2 arrest is unknown. In their study, Lau and Pardee
complex formation rather than a direct effect of drug on the (7) observed that, in cells induced to circumvent G2 arrest,
mitosis resulted in nuclear fragmentation. This phenomenon is
kinase, as treatment of a cell line which is etoposide resistant,
by virtue of an altered topoisomerase II (30), does not result in also observed in etoposide-treated CHO cells which have over
inhibition of kinase activity. It is unlikely that VpmR-5 cells come the G2 blockade (see Ref. 31), indicating that agents
contain an altered p34cdc2 kinase in addition to an altered
which cause G2 arrest via different types of DNA damage may
topoisomerase II, although the experiments described herein
induce similar pathways of cell death.
do not exclude this possibility. Irradiation of CHO cells also
Until now, there has been little indication of the cellular
causes rapid inhibition of p34cdc2activity, indicating that this events which may account for G2 arrest in mammalian cells.
response may be general to DNA-damaging agents. Currently,
Studies using radiation-sensitive yeast mutants indicate that the
we are evaluating the effects of a broad range of antitumor
RAD9 gene product is necessary for G2 arrest and protection
agents on p34cdc2 kinase activity in CHO cells in order to from the cytotoxic effects of X-irradiation (1). The nature of
determine whether this response is limited to those drugs which this gene product is unknown, and an analogous mutation has
induce G2 arrest, or whether it can be induced by any agent
yet to be isolated in mammalian systems. Rao (32) proposed
which interrupts cell cycle progression.
that extensive chromosomal damage might account for irre
These observations raise interesting questions regarding the versible G2 arrest, and that specific cell cycle-regulated proteins
role played by p34cdc2in the G2 arrest response of eukaryotic
necessary for mitosis were absent from G2-arrested cells. The
cells. From this study it seems likely that inactivation of p34cdc2 latter explanation is also favored by Eastman's group when
kinase, a protein necessary for mitosis in mammalian cells (23), studying cisplatin-induced G2 arrest (9). Specific transcripts or
may participate in the processes leading to G2 arrest. In our proteins absent in G2-arrested cells have yet to be identified.
study, p34cdc2activity is approximately 24-fold greater in G2-M
The data reported here suggest a different possibility, however.
than in G,, in agreement with other studies (19). Inhibition of Specifically we hypothesize that the rapid loss of p34cdc2kinase
p34cdc2 kinase activity in asynchronously dividing cultures,
activity is one component of a general response of cells to DNA
therefore, is due mainly to those cells which are in the G2-M damage, perhaps in some way analogous to the bacterial "SOS"
compartment. At this juncture, however, it is not possible to response (15).
determine whether identical responses to DNA damage are
An association between DNA damage, inhibition of p34cdc2
induced throughout the cell cycle. The precise mechanism of kinase activity, and G2 arrest has been established. Much of the
this observed inhibition of p34cdc2activity is undefined. Cellular
p34cdc2 content by Western blot, p34cdc2 synthesis, and the significance of this work is that it provides a pathway to examine
overall phosphorylation state estimated by [35S]methionine and the nature of the cellular response to cleavable complex for
mation. The mechanism of p34cdc2kinase inhibition, the direct
[32P]orthophosphate incorporation studies, respectively, remain
consequences of this inhibition on G2 arrest, and the role of
unchanged following etoposide treatment.5 The inactivation
cell cycle-regulated proteins in the processes of cell death are
5 Unpublished data.
now open to experimental characterization. In the accompa3765
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INHIBITION
OF pi***2 KINASE ACTIVITY BY DNA-DAMAGING
nying paper (31) we consider the potential relationship between
G2 arrest and cell death.
15.
16.
ACKNOWLEDGMENTS
We gratefully acknowledge the helpful discussions with Dr. Giulio
Iinietta and Dr. Dan Sullivan and the Macintosh Computer for giving
us the power to be our best!
17.
18.
19.
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3766
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Inhibition of p34cdc2 Kinase Activity by Etoposide or Irradiation
as a Mechanism of G 2 Arrest in Chinese Hamster Ovary Cells
Richard B. Lock and Warren E. Ross
Cancer Res 1990;50:3761-3766.
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