From the Vanderbilt University Departments of Cell Biology and Pathology and the Vanderbilt University Cancer Center, Nashville, Tennessee 37232
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ABSTRACT |
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An increasing body of evidence suggests that
mitogen-induced activation of the RAF/ERK signaling pathway is
functionally separate from the stress-induced activation of the
SEK/JNK/p38 signaling pathway. In general, stress stimuli strongly
activate the p38s and the JNKs while only weakly activating
ERK1 and ERK2. However, a number of independent
groups have now shown that the RAF/ERK signaling pathway is strongly
activated by ionizing radiation. In this work, we examine this paradox.
We show that both mitogen-activated protein (MAP) kinase kinase 1 (MEK1) and MAP kinase kinase 2 (MEK2) are activated by ionizing
radiation. Blockage of this activation through the use of dominant
negative MEK2 increases sensitivity of the cell to ionizing radiation
and decreases the ability of a cell to recover from the
G2/M cell cycle checkpoint arrest. Blocking MEK2 activation
does not affect double-strand DNA break repair, however. Although MEK1
is activated to a lesser extent by ionizing radiation, expression of a
dominant negative MEK1 does not affect radiation sensitivity of the
cell, the G2/M checkpoint of the cell, or double-strand
break repair. Because ionizing radiation leads to a different cell
cycle arrest (G2/M arrest) than that typically seen with
other stress stimuli, and because we have shown that MEK2 can affect
G2/M checkpoint kinetics, these results provide an
explanation for the observation that the MEKs can be strongly activated
by ionizing radiation and only weakly activated by other stressful stimuli.
Double-strand DNA breaks
(dsbs)1 are
recognized as key components of the initiation of multi-step
carcinogenesis. A number of cancer predisposition genes and oncogenes
exert their effects through the cell signaling pathways initiated by
dsbs. For example, the cancer predisposition genes ATM and DNA-PK are
kinases that are activated by dsbs. Cells with mutations in these two
genes show a decreased ability to repair dsbs, and it is thought that this inability leads to the genomic instability that is characteristic of cancers containing ATM or DNA-PK mutations (Refs. 1-3 and reviewed
in Refs. 4 and 5). Whereas ATM and DNA-PK are required for the
signaling initiated by dsbs, the cancer predisposition gene
BRCA2 is required for the actual repair of dsbs. Cells that lack BRCA2 show decreased ability to survive ionizing
radiation (6-8), and this inability to repair dsbs is central to
BRCA2-initiated carcinogenesis (8-10). Several oncogenes
also have similar effects on dsb signaling and dsb repair. The
c-abl proto-oncogene has been shown to be activated upon the
formation of dsbs via an interaction with DNA-PK (12-15), and the
retroviral oncogene, FBR v-fos, has been shown to inhibit
the response of the cell to ionizing radiation (16). All of these
findings implicate dsb formation in the initiation of carcinogenesis.
Much of the work implicating dsb formation in the initiation of
carcinogenesis has focused on the signaling pathways induced by dsbs
(e.g. ATM/DNA-PK) and on the repair of those dsbs
(e.g. BRCA2). Less is known about the signaling
induced by the agent causing the dsb. The most commonly used agent to
induce dsbs is ionizing radiation. Whereas dsbs are the most prominent
effect of ionizing radiation exposure, ionizing radiation also causes lipid peroxidation, glutathione depletion, and protein oxidation (reviewed in Refs. 17 and 18). Thus, ionizing radiation activates the
stress response pathway of the cell in a manner that is not necessarily
dependent on the formation of dsbs (19).
Ionizing radiation will activate JNK (20), p38, SEK (21), and NF- The activation of the MAP kinase pathway by the stress stimulus of
ionizing radiation could be due to the fact that dsbs lead to different
cell cycle checkpoint control than what is normally seen for
DNA-damaging agents (reviewed in Refs. 42 and 43). Repair of dsbs
follows two pathways, nonhomologous recombinational repair (occurring
in G1) and homologous recombination repair (44-47). Homologous recombinational repair occurs in G2, the time
point when homologous, undamaged double-strand DNA is present to serve as a template for correct repair. Because the damaged DNA strand has a
template for repair, homologous recombinational repair leads to fewer
mutations than nonhomologous repair (44, 47, 48). Therefore, the
G2/M checkpoint is essential for the proper repair of DNA
damaged by ionizing radiation. Recently, the MAP kinase pathway has
been implicated in G2/M cell cycle regulation. In Xenopus oocytes, MAP kinase activity has been shown to be
necessary for progression through G2 (49-51). The MEK1 and
MEK2 activator, c-mos, has also been shown to be necessary
for progression through G2 (52), and in mouse oocytes, MAP
kinase becomes activated at metaphase and localizes to the
microtubule-organizing centers (53). Because the MAP kinase pathway is
required for G2/M progression in a number of systems and
because ionizing radiation leads both to activation of the MAP kinase
pathway and to G2/M arrest, it is possible that ionizing
radiation activates the MAP kinase cascade to exert an effect on
G2/M checkpoint control.
To address these questions, we focused on MEK1 and MEK2, two components
of the MAP kinase cascade. These two proteins are phosphorylated
in vitro by RAF (54-55) and can both phosphorylate ERK1 and ERK2 (30). MEK1 and MEK2 are
approximately 80% homologous and are very similar in size (MEK1 = 45 kDa; MEK2 = 46 kDa). They differ in the first 30 amino
acids of the N terminus and in a proline-rich region that is only found
in MEK1 (56, 57). In this work, we find that both MEK1 and MEK2 are
specifically activated by ionizing radiation in a variety of cell
lines. We show that cells that express dominant negative MEK2
show radiation hypersensitivity that is not seen in cells
which equivalently express dominant negative forms of MEK1. Expression
of dominant negative MEK2 leads to a slightly delayed G2/M
arrest upon ionizing radiation exposure and a substantial inability to
progress through the G2/M arrest upon recovery from that
radiation exposure. Finally, we show that the effect of MEK2 on
radiation sensitivity can be reversed by forcing G2
progression through the use pharmacological agents. These data imply
that ionizing radiation activates the MAP kinase cascade in a specific
manner to maintain G2/M checkpoint fidelity.
Cell Culture and Transfection--
HeLa cells, NIH 3T3-L1 cells,
and BxPC-3 cells were obtained from the American Type Tissue Collection
(ATCC) and grown in Dulbecco's modified Eagle's media supplemented
with 10% fetal bovine serum (Sigma), 2 mM
L-glutamine (Sigma), and 1× antimycotic/antibiotic (Sigma). HBL100 cells were obtained from the ATCC and grown in McCoy's
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum. Serum starvation was performed in Dulbecco's modified Eagle's
media supplemented with 0.5% fetal bovine serum, 2 mM
L-glutamine, and 1× antimycotic/antibiotic for 24 h
followed by incubation in 0.1% fetal bovine serum for 24 h.
Metabolic labeling was performed by incubating the cells in
methionine-free media (Life Technologies, Inc.) for 30 min, followed by
the addition of methionine-free media supplemented with 50 µCi of
[35S]methionine for 3 h. Stably transfected cell
lines were generated by transfecting HeLa cells at 80% confluence with
20 µg of indicated construct (pREP4 (Invitrogen), pREP4-K97A MEK1,
pREP4-S222A MEK1, pREP4-K101A MEK2, pREP4-wt MEK2, pREP4-S222E MEK1)
using the calcium phosphate precipitation. Cells were split 1:3 the
following day. Two days after transfection, selection was begun in 500 µg/ml hygromycin B (Life Technologies, Inc.). Three weeks later,
approximately 1000 colonies were pooled and shown to express the
desired protein by Western blotting. All cell lines were used within 2 weeks of establishment or within 3 weeks following liquid nitrogen thawing.
Plasmids--
The plasmids used in this work were the gifts of
Edwin Krebs, University of Washington, Seattle (K97A MEK1, S222A MEK1);
Natalie Ahn, University of Colorado, Boulder (pCML-MEK2); and Kun-Liang Guan, University of Michigan, Ann Arbor (pGEX-MEK2). The K97A MEK and
S222A MEK1 plasmids were supplied in the vector pCDNA 3.1 (Invitrogen). To subclone these genes into the pREP4 vector (Invitrogen), pCDNA-K97A MEK1, pCDNA-S222A MEK1, and pREP4 were digested with XhoI and HindIII (Life
Technologies, Inc.). The K97A MEK1 fragment and the S222A MEK1 fragment
was then ligated into pREP4. To generate the dominant negative K101A
MEK2 construct, pCML-MEK2 was digested with BamHI (Life
Technologies, Inc.) and ligated into pGEM4Z (Promega). Pgem4Z-MEK2 was
then digested with PstI (Life Technologies, Inc.) to
generate a 400-bp fragment containing the region to be mutated. This
400-bp PstI fragment was then ligated into pGEM7Z (Promega).
This pGEM7Z-400-bp MEK2 construct was then digested with
BalI and NaeI (Life Technologies, Inc.), and an oligonucleotide containing the sequence
5'-CCAGGGCGCTGATCCACCTC GAGATCAAGCC-3' was
ligated into the digested MEK2. This oligonucleotide sequence contains
a 2-base pair replacement (in bold) designed to mutate Lys-101 to
alanine and a single base pair replacement (in italics) designed to
insert an XhoI site without changing the amino acid
sequence. The altered 400-bp fragment was then sequenced to ensure that
the desired mutation was present and in frame. The pGEM7Z-400-bp K101A
MEK2 construct was then digested with PstI, and the mutated
400-bp fragment was reinserted into pGEM4Z-MEK2. This plasmid was then
sequenced to ensure that the mutation was present and in frame. In
addition, in vitro translation of the protein gave a 46-kDa
fragment that could be immunoprecipitated with an antibody to MEK2. The
pGEM4Z-K101A MEK2 fragment was then digested with BamHI and
inserted into the pREP4 vector. All pREP4 plasmids used for
transfections were purified by ultracentrifugation using CsCl gradients
prior to transfection.
Immunoprecipitations and Kinase Assays--
Immunoprecipitations
and kinase assays were performed using a method described previously
with slight modifications (58). Cells were washed twice in
phosphate-buffered saline (PBS) and harvested by scraping. Cells were
pelleted by centrifugation at 2000 rpm and rewashed with PBS followed
by another 2000 rpm centrifugation. Cells were then lysed in Lysis
Buffer (20 mM Tris (pH 7.5), 0.27 M sucrose, 1 mM sodium orthovanadate (Sigma), 10 mM sodium
Western Blotting--
Cells were washed twice with PBS and then
harvested by scraping. Cells were centrifuged at 2000 rpm, rinsed again
with PBS, and centrifuged at 2000 rpm. The pellet was then resuspended
in Lysis Buffer and allowed to sit on ice for 10 min. The suspension was then sonicated. The suspension was centrifuged at 14,000 rpm for 10 min, and the pellet was discarded. Western blotting was then performed
as described previously (59). Briefly, protein concentration was
standardized using the Bio-Rad Protein Assay. Equal amounts of lysate
were subjected to polyacrylamide gel electrophoresis (10% gel).
Lysates were transferred to nitrocellulose filters (Amersham Pharmacia
Biotech) for 1 h at 40 mV. To ensure equal loading of protein and
equal transfer efficiency, the membrane was stained with Ponceau S
prior to blocking. The membrane was blocked using 5% nonfat dry milk,
0.3% Tween 20 in Tris-buffered saline. After blocking and subsequent
washing, the blot was exposed to a 1:1000 dilution of the given
antibody overnight at 4 °C. The blot was then washed extensively
with Tris-buffered saline, 0.3% Tween 20 before being exposed to a
horseradish peroxidase-conjugated secondary antibody (Santa Cruz
Biotechnology) at a dilution of 1:2500 at room temperature for 1 h. Bands were visualized using the ECL detection system (Amersham
Pharmacia Biotech) using the manufacturer's instructions. Antibodies
used were anti-phosphorylated MEK1/2 (New England Biolabs),
anti-nonphosphorylated MEK1/2 (New England Biolabs),
anti-N-terminal MEK1 (Santa Cruz Biotechnology), and anti-N-terminal
MEK2 (Santa Cruz Biotechnology).
Irradiation and Cell Survival Assays--
Cells were irradiated
using a 137Cs source at a dose rate of 2.35 Gy/min. Colony
forming assays were performed as described previously (16). Briefly,
cells were plated at 500 cells/dish and irradiated at a given dose.
After irradiation, cells were returned to the 37 °C incubator. They
were refed fresh media every 2 days. After 14 days, the colonies were
fixed in ethanol and stained with crystal violet. Colonies were counted
manually. Relative survival was determined by comparing colony number
of cells irradiated at a given dose to the colony number of
unirradiated cells plated at the same density and cultured for the same
amount of time.
For cellular survival following treatment with vanadate, approximately
2000 cells of each cell line (vector-only cells, K97A MEK1 cells, and
K101A MEK2 cells) were plated 12 h prior to the commencement of
the experiment. These cells were then treated with 50 µM
vanadate (Sigma) for 8 h prior to irradiation. 16 h after
irradiation with 5 Gy, cells were washed extensively and refed with
normal media. All cells lines showed a strong G2 arrest by
flow cytometry analysis. Three weeks later, cell survival was quantitated as described above (survival was compared with cells treated with vanadate in the same manner, but not irradiated). For
cellular survival following treatment with caffeine, approximately 2000 cells of each cell line studied were plated as above. The cells were
irradiated with 5 Gy and then treated with 2 mM caffeine (Calbiochem) 24 h after irradiation. 32 h after irradiation,
flow cytometry showed recovery from G2 arrest in K101A MEK2
cells (relative to irradiated/no caffeine cells) and no difference
between irradiated vector-only cells and K97A MEK1 cells either treated
or untreated with caffeine (32 h after irradiation, vector-only cells
and K97A MEK1 cells show normal cell cycle distribution; therefore,
treatment with caffeine at 24 h has no effect on the cell cycle
profile). Survival was measured as described above.
DNA Repair Assays--
Equal cell numbers were embedded into
agarose plugs as described previously (8). The plugs were then
exposed to the indicated doses of ionizing radiation at 4 °C. To
allow the cells to repair damaged DNA, the cell plugs were then covered
with 10% fetal bovine serum/Dulbecco's modified Eagle's media in a
37 °C incubator for the indicated period. The plugs were then
digested overnight in a solution containing 10 mM Tris (pH
7.4), 20 mM NaCl, 50 mM EDTA, and 1 mg/ml
proteinase K (Life Technologies, Inc.) at 50 °C. The plugs were then
embedded in a 0.7% agarose gel and subjected to pulsed field gel
electrophoresis at 3 V/cm, 45-s pulse time at 14 °C for 48 h
(CHEF II system, Bio-Rad). Southern blotting was then performed using
random prime labeled HeLa cell genomic DNA as a probe. Quantitation of
repair was performed using the Packard Electronic Autoradiography
Instant Imager.
Flow Cytometry--
Cells were plated to 40% confluency on
100-mm plates. 18 h later, the cells were exposed to 5 Gy ionizing
radiation (except for the unexposed plate). At the indicated time
point, the cells were trypsinized, centrifuged, and washed 2× with
PBS. 1 × 106 cells were then suspended in 1 ml of PBS
supplemented with 5 µg/ml propidium iodide. 1 ml of Vindelov's
solution (10 mM Tris (pH 8.0), 10 mM NaCl, 0.7 units/ml RNase, 50 µg/ml propidium iodide, 0.1% Nonidet P-40) was
then added, and the cells were allowed to incubate in the dark at
4 °C overnight. Flow cytometry was performed using a Becton
Dickinson FACS Caliber using the Vanderbilt University Flow Cytometry
Core. Data acquisition software is CellQuest Version 3.1. Excitation
was with 488 nm air-cooled argon-ion laser at 15 milliwatts. Emission
was collected at 585/42 nm band pass filter at a flow rate of 12 ± 3 µl/min. 10000 events were measured per experiment.
MEK1 and MEK2 Are Activated by Ionizing Radiation--
The MAP
kinase kinases (MEKs) are a central component of the growth-response
signaling pathway (reviewed in Refs. 29 and 30). Whereas a great deal
of work has centered on their role in transmitting mitogenic signals,
there is an increasing body of evidence that the MEKs could play a role
in the response of the cell to the stress of ionizing radiation
(37-39). Activation of MEK1 and MEK2 involves phosphorylation upon
conserved serine residues (Ser-218 and Ser-222 on MEK1,
Ser-222 and Ser-226 on MEK2; see Refs. 55 and 60). To test the
activation of MEK1 and MEK2 upon ionizing radiation exposure, an
antibody that specifically recognizes phosphorylated MEK1 and MEK2 was
obtained. HeLa cells were serum-starved and exposed to increasing doses
of ionizing radiation. As a control, one plate of cells was
mock-irradiated (0 Gy). 10 min after exposure, lysates were generated,
and Western blotting was performed using the antibody directed against
either phosphorylated MEK1 or phosphorylated MEK2. Under these
conditions, we were unable to distinguish between the similarly sized
MEK1 and MEK2. However, the Western blot shown in the upper
panel of Fig. 1 shows that in HeLa
cells, the 45-kDa MEK1 and/or the 46-kDa MEK2 are phosphorylated in
response to physiological doses of ionizing radiation (upper
blot, Fig. 1). To determine whether this activation of MEK1 and
MEK2 is generalizable over cell lines, the same experiment was
performed in a human non-transformed breast cell line (HBL-100), in a
human pancreatic adenocarcinoma cell line (BxPC-3), and in a mouse
pre-adipocyte cell line (NIH 3T3-L1, the phosphorylated sites
recognized by the antibody are conserved in mouse MEK1 and MEK2 and
human MEK1 and MEK2; see Refs. 56 and 57). In all of these
cell lines either MEK1 or MEK2 were phosphorylated in response to
ionizing radiation and showed various dose responses (Fig. 1,
bottom three blots). The * in Fig. 1 refers to a 32-kDa
cross-reacting band that is seen in all cell lines and that is
unresponsive to serum starvation or ionizing radiation. This
cross-reactive band can be used to standardize for equal protein
loading (Fig. 1). In addition, Western blots using antibody against
total MEK1 or total MEK2 indicate that ionizing radiation does not lead
to increased total MEK protein in the time course of this experiment
(data not shown). Thus, these experiments show that phosphorylation of
the MEKs in response to ionizing radiation is a general effect of
ionizing radiation exposure.
To show that MEK1 and MEK2 are not only phosphorylated in
response to ionizing radiation, but are activated as well, IP kinase assays were performed. Antibodies directed against the non-conserved regions of MEK1 and MEK2 (N terminus) were used to limit
cross-reactivity between the two proteins, and catalytically inactive
ERK1 (K71A ERK1) was used as a substrate. Because
MEK is downstream of Ras (30), as a positive control, activity of the
MEKs from HeLa cells transformed with v-ras were compared
with activity of the MEKs from HeLa cells transfected with empty
vector. Fig. 2A shows that
MEK1 and MEK2 have much higher activity in cells transformed with
v-ras, so these antibodies perform appropriately in IP
kinase assays. HeLa cells were then serum-starved and exposed to
various doses of ionizing radiation. As a positive control for
increased kinase activity, cells were restimulated with 20% serum
(Fig. 2B, far left lane). MEK1 shows a slight
increase in activity at both 2.5 and 10 Gy, whereas MEK2 is strongly
activated by 10 Gy of ionizing radiation (Fig. 2B). The same
experiment was then performed using an IP-depletion strategy aimed at
eliminating the remaining MEK1 and MEK2. Cells were metabolically
labeled with [35S]methionine, and immunodepletion was
performed using antibodies directed against either c-fos,
MEK1, or MEK2. Lysates were cleared by adding protein
A/G-agarose and, after 1 h incubation, retaining the supernatant.
These depleted lysates were then immunoprecipitated with either MEK1
and MEK2. The antibody to MEK1 immunoprecipitates a 45-kDa protein when
depleted either of c-fos or MEK2, whereas this antibody
precipitates decreased 45-kDa protein when depleted of MEK1 (Fig.
2C, left three lanes). The antibody directed against MEK2
immunoprecipitates a 46-kDa protein when depleted of either c-fos or MEK1 but not when depleted of MEK2 (Fig. 2C,
right three lanes). Thus, the antibodies employed in Fig. 2 do not
show a high degree of cross-reactivity. Immunodepletion was then
coupled with the IP kinase assay to show activity of MEK1 and MEK2.
After serum starvation and depletion of either MEK1 or MEK2, IP kinase assays were performed. Fig. 2D shows that both MEK1 and MEK2
show increased kinase activity upon exposure to 10 Gy of ionizing
radiation and that MEK2 shows increased activity relative to MEK1 upon
ionizing radiation exposure.
Overexpression of Dominant Negative MEK2, but Not Dominant Negative
MEK1, Increases Sensitivity of the Cell to Ionizing Radiation--
To
determine whether the activation of MEK1 and MEK2 is significant for
the response of the cell to ionizing radiation, dominant negative forms
of these two proteins were used. Two dominant negative forms of MEK1
were obtained (60). The first, K97A MEK1, replaces a lysine in the
ATP-binding domain, with an alanine, such that the kinase cannot bind
ATP to transfer the phosphate. The second, S222A MEK1, replaces a
serine, which is essential for activation, with an alanine, such that
full activation cannot be achieved (generous gifts of Edwin Krebs,
University of Washington, Seattle). These two dominant negative
constructs have been previously shown to slow cell growth and inhibit
activation by EGF and serum stimulation (60). In addition, as a control
for overexpression of MEK1, a constitutively active form of MEK1 (S222E
MEK1) was also obtained (gift of Edwin Krebs, University of Washington,
Seattle). We subcloned these three constructs into pREP4 (Invitrogen).
This vector replicates episomally in HeLa cells and contains the
hygromycin resistance gene driven by the cytomegalovirus promoter. The
constructs were transfected into HeLa cells, selected for 3 weeks in
hygromycin, and approximately 1000 clones were pooled. Because activity
of dominant negative proteins is dependent on their expression relative to wild-type protein, we wanted to show expression of the dominant negative constructs in these cells relative to wild-type
MEK1 expression. Lysates were generated and Western blots
were performed (with equivalent lysate protein concentration) using an
antibody against MEK1. This antibody will recognize both endogenous
wild-type protein and overexpressed dominant negative protein. Fig.
3A shows that relative to
cells stably transfected with vector only, both K97A MEK1 cells and
S222A MEK1 cells have greatly overexpressed dominant negative MEK1
(Fig. 3A). In addition, the constitutively active S222E MEK1
is also expressed to a high degree (Fig. 3A). To show that
these constructs affect the activity of MEK1 in response to ionizing
radiation, IP kinase assays were performed using exposed and unexposed
cells. The cells containing the stably transfected empty vector show
the 1.6-1.8-fold activation of MEK1 upon exposure to ionizing
radiation, whereas the cells containing the dominant negative
constructs showed no activation upon exposure to ionizing radiation
(Fig. 3B). In addition, the S222E MEK1 cell line shows elevated basal activity which can be increased approximately 1.4-fold upon ionizing radiation exposure (Fig. 3B).
To test the K97A MEK1 and the S222A MEK1 dominant negative effects of
constructs on cell survival in response to ionizing radiation, colony
forming assays were performed. This assay measures the ability of a
cell to survive ionizing radiation exposure and to proliferate
following ionizing radiation exposure (16). Approximately 500 cells
were plated per ionizing radiation dose. The plates were exposed to the
indicated dose of ionizing radiation, and colony formation was scored 2 weeks later. The S222A MEK1 cell line had slightly decreased survival
at both 4 and 6 Gy of ionizing radiation, whereas the K97A MEK1 cell
line only showed slightly decreased survival at 6 Gy of ionizing
radiation (Fig. 3C). Neither of these cell lines showed
significant difference from the S222E MEK1 cell line (Fig.
3C), so the dominant negative MEK1 constructs have little
effect on HeLa cell survival in response to ionizing radiation.
Because MEK2 is activated in IP kinase assays to a greater extent then
MEK1, it is possible that a dominant negative form of MEK2 would have a
larger effect on the ability of HeLa cells to survive ionizing
radiation exposure. To test the effect of a dominant negative MEK2, we
mutated wild-type MEK2 (57, 61) (generous gift of Natalie Ahn,
University of Colorado, Boulder) to K101A MEK2. This mutation was
designed to be analogous to the K97A MEK1 mutation, as this region of
MEK2 is highly conserved with MEK1 (56, 57). By analogy, the
K101A MEK2 mutation should render MEK2 unable to bind ATP to transfer
the phosphate to its substrate. Both the dominant negative K101A MEK2
and wild-type MEK2 were subcloned into the pREP4 vector and transfected
into HeLa cells. After 3 weeks of hygromycin selection, approximately 1000 colonies were pooled. Western blots were performed (equivalent protein concentrations of lysates) using an antibody that recognizes both endogenous, wild-type MEK2 and transfected, dominant negative MEK2. Fig. 4A shows that K101A
MEK2 and wt MEK2 are highly expressed relative to cells stably
transfected with vector alone (Fig. 4A). To show that
expression of the dominant negative MEK2 leads to decreased MEK2
activation in response to ionizing radiation, IP kinase assays were
performed on these cells using kinase-inactive ERK1 as
substrate. The cells containing only the empty vector show a 4-6-fold
up-regulation of MEK2 activity in response to ionizing radiation,
whereas the K101A MEK2 cells show no activation in response to ionizing
radiation (Fig. 4B). In addition, expression of
wild-type MEK2 shows higher basal MEK2 activity that can be increased only slightly in response to ionizing radiation (Fig. 4B).
Colony forming assays were then performed to determine the influence of
MEK2 on the cell's survival response to ionizing radiation. 500 cells
were plated 12 h prior to radiation exposure. Cells were exposed
to the given dose, and colony formation was scored 2 weeks later. At
low doses of ionizing radiation, the K101A MEK2 cells do not show
decreased cell survival, but at higher doses of radiation (>3 Gy),
these cells are significantly more radiosensitive (Fig. 4C).
Since cells containing wild-type MEK2 show no differences in survival
relative to the vector-only cells, the decreased survival of cells
expressing dominant negative MEK2 is not simply an overexpression phenomenon. Thus, whereas both MEK1 and MEK2 are activated in response
to ionizing radiation, only expression of dominant negative MEK2 leads
to radiosensitivity.
Dominant Negative MEK2 Has No Effect on Double-strand DNA Repair
but Leads to Defective G2/M Checkpoint Control--
Two
explanations are possible for the effect of MEK2 on radiation survival.
First, MEK2 may influence DNA repair. Ionizing radiation causes
double-strand DNA breaks, and a number of proteins that cause radiation
hypersensitivity do so by not allowing efficient double-strand break
repair (62). In addition, the MEK activator, c-mos, has been
shown to influence cellular genomic stability (63). For these reasons,
the effect of MEK2 on double-strand break repair was tested
using the K101A MEK2, K97A MEK2, and vector-only cell lines.
Equal numbers of cells were embedded in agarose plugs and exposed to 10 Gy of ionizing radiation. These cells were then allowed to repair the
damaged DNA for 30, 60, or 120 min or not allowed to repair (0 min) the
damage. After the plugs were digested overnight with proteinase K, they
were embedded in a 0.7% agarose gel and subjected to pulse-field
electrophoresis. Southern blotting was then performed, and percent
repair was quantitated. A representative experiment is shown in Fig.
5A. Under these
electrophoretic conditions, the damaged, unrepaired DNA migrates as a
discrete band, whereas the undamaged or repaired DNA barely migrates
out of the agarose plug. The results of three independent experiments
are quantitated in Fig. 5. There are no significant differences in the
abilities of these three cell lines to repair double-strand DNA breaks
(Fig. 5). This result is expected because the K101A MEK2 survival curve (Fig. 4C) shows a shoulder at low doses of ionizing
radiation, implying the ability to repair sublethal DNA damage (3).
Since the MAP kinase pathway has been shown to be necessary for
progression through G2/M (50, 52), a second explanation for
the effect of dominant negative MEK2 on ionizing radiation sensitivity
might be a dysregulated G2/M cell cycle checkpoint. In HeLa
cells, the G2/M checkpoint shows the greatest arrest due to
ionizing radiation damage (48). To determine the effect of MEK2 on the
G2/M checkpoint, the dominant negative K101A MEK2 cell line
was used. As controls, the vector-only cell line, the K97A MEK1 cell
line and the S222A MEK1 cell line were also used. Asynchronously
growing cells were exposed to 5 Gy ionizing radiation; the difference
in survival between the K101A MEK2 cell line and the vector-only cell
line is maximal at this dose. At the indicated time points after
exposure, cells were harvested and stained with propidium iodine, and
flow cytometry was performed. The vector-only cells and the K97A MEK1
cells show G2 arrest 12 h after exposure with recovery
24 h after exposure (Fig. 6,
vector-only cells are shown in upper panels and K97A MEK1
cells are shown in middle panels). In contrast, the K101A
MEK2 cells show slower G2/M arrest. Full arrest eventually
occurs after approximately a 5-7-h delay (Fig. 6). However, the major
cell cycle dysfunction in the K101A MEK2 cell line is an inability to
recover from G2/M arrest. At both the 24- and 36-h time
points, significant numbers of K101A MEK2 cells are still arrested in
G2 (Fig. 6). At these time points, both the vector-only
cells and the K97A MEK2 cells have recovered and show relatively normal
cell cycle profiles (Fig. 6). The S222A MEK1 cell line showed similar
profiles to the K97A MEK1 cell line (data not shown). Since effective
G2/M arrest and recovery from that arrest are essential for
the ability of the cell to respond effectively to ionizing radiation,
and since inhibiting MEK2 activity with a dominant negative construct
leads to a slightly delayed G2/M arrest and to a grossly
delayed recovery from that arrest, it is likely that activation of MEK2
in response to ionizing radiation influences the G2/M
checkpoint control of the cell.
To determine whether an inability to arrest in G2 or an
inability to recover from G2 arrest is responsible for the
radiosensitivity of the K101A MEK2 cell line, pharmacological
manipulation was performed. Vanadate has been shown to inhibit
dephosphorylation of CDC2, thereby leading to an arrest in
G2 (64). K101A MEK2 cells were exposed to vanadate for
8 h before ionizing radiation exposure. 16 h after radiation
exposure, cells were washed extensively, refed with normal growth
media, and colony forming assays were performed. Fig.
7A shows that treatment of
K101A MEK2 cells with ionizing radiation and vanadate leads to an
increased G2 arrest when compared with K101A MEK2 cells
treated with ionizing radiation alone (Fig. 7A, middle
panels). This forced G2 arrest does recover radioresistance in the K101A MEK2 cell line, as vanadate-treated K101A
MEK2 cells show similar radiosensitivity to untreated K101A MEK2 cells
(Fig. 7B).
To study recovery from the G2/M arrest, caffeine was used.
Caffeine is typically regarded as an agent that sensitizes cells to
ionizing radiation (65, 66). Treatment of cells with caffeine prior to
irradiation abolishes the G2 arrest and leads to radiation sensitivity (65, 66). We used caffeine in a slightly different manner.
By exposing K101A MEK2 cells to caffeine 24 h after ionizing radiation exposure, we were able to use caffeine to force the K101A
MEK2 cells to recover from the G2/M arrest in a timely
manner. Fig. 7A shows that irradiated, non-caffeine-treated
K101A MEK2 cells have significant numbers of cells arrested in
G2 32 h after ionizing radiation exposure. When these
cells are treated with caffeine 24 h after irradiation, the cells
show a normal cell cycle distribution 8 h later (Fig. 7A,
right panels). Thus, caffeine can be used to force recovery from
the G2/M arrest in otherwise terminally arrested K101A MEK2
cells. This forced G2/M arrest recovery reverses the
radiosensitivity of K101A MEK2 cells upon ionizing radiation exposure
(Fig. 7B), whereas treatment of vector-only cells or K97A
MEK1 cells at these time points with either caffeine or vanadate has no
appreciable effect on cell survival (Fig. 7B). Therefore,
the radiosensitivity of cells that express dominant negative MEK2 is
most likely due to an inability to recover from G2 arrest
and not due to an inability to arrest in G2 in a timely manner.
The RAF/ERK signaling pathway is generally regarded to be strongly
responsive to mitogenic signals and only weakly responsive to stressful
signals (11, 32, 36). The only stressful stimulus that strongly
activates the MAP kinase pathway is ionizing radiation (37-41).
Whereas a number of cell stressors and DNA-damaging agents cause a
G1/S arrest, physiologic doses of ionizing radiation cause a G2/M arrest (42). In this work, we have studied the
possibility that ionizing radiation activates the RAF/ERK signaling
pathway to influence G2/M checkpoint kinetics. We have
shown that both MEK1 and MEK2 are activated in response to ionizing
radiation (Figs. 1 and 2). This activation has functional significance
as dominant negative MEK2 is essential for cell survival in response to
ionizing radiation (Fig. 4). We have also shown that expression of
dominant negative MEK2 leads to a delayed induction of the G2/M cell cycle checkpoint and a vastly decreased ability
to recover from that checkpoint once it is established (Fig. 6).
Finally, we show using pharmacological means that it is the faulty
recovery from the G2/M arrest and not the delayed
G2/M arrest that is responsible for the radiosensitivity in
cells that express dominant negative MEK2 (Fig. 7).
Previous work on activation of the MAP kinase pathway by ionizing
radiation focused on the activation of the components of the pathway
and not on the cellular effects of that activation. We have carried
that work a step further by showing activation of MEK1 and MEK2 at
doses (2.5 Gy) that are likely to be physiologically relevant. In
addition, we have focused on the effect that activation of these
kinases has on cell survival and cell cycle checkpoint control.
Although there is a pharmacological agent (PD98059) that inhibits
activation of MEK1 (IC50 = 5 µM) and MEK2
(IC50 = 50 µM) (54, 67), the doses required
to completely inhibit MEK2 activity (100 µM) were toxic
to HeLa cells. For this reason, dominant negative MEK1 and MEK2
constructs were employed. The dominant negative constructs allowed us
to selectively block MEK1 and MEK2 activity, respectively. Our results
show that the inhibition of MEK2 activation causes cells to become both
radiosensitive and checkpoint-defective. These defects in cells
expressing dominant negative MEK2 suggest that MEK activation by
ionizing radiation is a specific component of the stress response of
the cell and not a nonspecific effect of cellular stress as previously
suggested (41).
The importance of the MAP kinase pathway in all phases of cell cycle
regulation is being increasingly recognized. The MEK inhibitor,
PD98059, has been shown to reverse the nerve growth factor-induced
G1 arrest in fibroblasts (68). In addition, expression of a
constitutively active form of MEK1 induces differentiation in PC12
cells (69) and in megakaryocytes (70). These findings indicate that in
some cell lines, MEK activity leads to G0/G1 arrest. In other instances, however, such as upon growth factor stimulation and upon cellular transformation by v-ras and
v-raf, MEK activity is necessary for progression through
G1 (67, 71). Inhibition of MEK activity in pre-adipocytes
also enhances adipocyte differentiation (72). Constitutively active
MEK1 will also transform some cell lines (71). These findings imply
that MEK activity helps the cell progress through G1.
Therefore, the data surrounding the activity of MEK in G1
cell cycle regulation is largely contradictory. The effect of MEK on
G1 cell cycle regulation is largely dependent on the
circumstance and cell line tested.
Recently, the MAP kinase pathway has also been implicated in
G2 cell cycle regulation. In Xenopus oocytes,
MAP kinase activity has been shown to be necessary for progression
through G2, possibly by inhibiting an inhibitor of cyclin
B/CDC2 kinase activity (49-51). c-mos (an activator of MEK1
and MEK2) has been shown to be essential for progression through
G2 (52). In addition, there is mounting circumstantial
evidence that the MAP kinase pathway plays an essential role in
meiosis. In Caenorhabditis elegans, activation of the MAP
kinase pathway is necessary for meiotic cell cycle progression (73),
and in mouse oocytes, MAP kinase becomes activated at metaphase and
becomes localized to the microtubule-organizing centers in meiotic
maturation (53). Because the repair of double-strand DNA breaks seen
during physiological crossing-over during meiosis is similar to the
repair of ionizing radiation-induced double-strand DNA breaks (74),
activation of the MAP kinase cascade may lead to similar effects in
meiosis and DNA repair. To this point, however, the MEKs have not been
implicated in the maintenance of stress-induced cell cycle checkpoints
or in progression through those checkpoints. In this article, we have
shown that cells that express dominant negative MEK2 have a defective
G2/M arrest. Although these cells eventually arrest at
levels seen in controls, these cells take slightly longer
(approximately 5-7 h longer in asynchronous cells) to fully enter
G2 arrest. In addition, these cells take remarkably longer
to recover from G2 arrest (Fig. 6). Because our cell
survival assays require the cells to recover from the insulting agent
and to proliferate after that recovery, we are not measuring only cell
death but instead measuring both cell death and proliferation capacity
following recovery. Thus, a dysfunctional recovery from G2
arrest would manifest in our assay by showing increased cell death and
decreased proliferation capacity, leading to decreased colony
formation. We show through the use of caffeine to force recovery from
G2 arrest that the delay in recovery from radiation-induced G2 arrest mediates the radiosensitivity of the K101A MEK2
cells. Activation of MEK2 by ionizing radiation likely influences the progression through the G2 checkpoint. Because ionizing
radiation causes a G2/M arrest, this could provide an
explanation for the finding that the RAF/ERK pathway is activated by
ionizing radiation but not activated by other stress stimuli.
INTRODUCTION
Top
Abstract
Introduction
References
B
(22), and it will induce the immediate early genes, c-jun,
c-fos, and egr-1 (23-27). Although it is not
surprising that ionizing radiation activates the stress response
pathway, it is unexpected that the typically growth-responsive MAP
kinase cascade (reviewed in Refs. 28-30) is also activated by ionizing radiation. Although exceptions exist (31), an increasing body of
evidence suggests that the stress response pathway is functionally separate from the growth-responsive MAP kinase pathway (11, 32-36).
The stress response pathway (SEK, JNK, and p38) is poorly activated by
mitogens, whereas the MAP kinase pathway is generally poorly activated
by stressful stimuli (UV radiation, osmotic stress, etc.) (11, 32-36).
However, c-ras, c-RAF, ERK1, and
ERK2 have all been shown by independent groups to be
activated by ionizing radiation (37-41). It is unknown how activation
of the MAP kinase pathway affects cellular survival in response to
ionizing radiation, but the fact that activation has been seen so
consistently suggests that activation of the MAP kinase cascade could
be important for response of the cell to ionizing radiation.
EXPERIMENTAL PROCEDURES
-glycerophosphate (Sigma), 0.5 µM okadaic acid (Life
Technologies, Inc.), 50 mM NaF (Sigma), 5 mM
sodium pyrophosphate (Sigma), 1% Triton X-100, 0.1%
-mercaptoethanol, 1 mM benzamidine (Sigma), 0.2 mM phenylmethylsulfonyl fluoride (Sigma), 5 µg/ml
leupeptin (Sigma), and 2 µg/ml aprotinin (Sigma)) for 10 min at
4 °C. The suspension was then sonicated briefly and centrifuged at
14,000 rpm at 4 °C for 10 min. The supernatant was then extracted.
Protein concentration was standardized using the Bio-Rad protein assay.
100 µg of total protein was then incubated with 10 µl of the given
antibody (anti-N-terminal MEK1 or anti-N-terminal MEK2, and total
volume was adjusted to 700 µl using Lysis Buffer) for 2 h at
4 °C. Protein A/G-Sepharose (Santa Cruz Biotechnology) was then
added for 1 h. The suspension was then centrifuged, and the
precipitate was washed five times with Lysis Buffer (the 2nd and 3rd
lysis buffer contained 0.5 M NaCl). For the metabolic
labeling experiment, 2× SDS-PAGE buffer was added. The suspension was
boiled and electrophoresed on a 10% SDS-PAGE gel. The gel was then
dried and subjected to autoradiography. For the kinase assays, the
precipitate was then washed an additional three times in Kinase Buffer
(50 mM Tris (pH 7.5), 0.03% Brij 35 (Sigma), 0.1%
-mercaptoethanol, 0.5 µM okadaic acid (Life Technologies, Inc.), 0.27 mM sodium orthovanadate, and 10 mM magnesium chloride). After the washes, Kinase Buffer was
added to the immunoprecipitate such that the total volume was 50 µl.
25 µl of this solution was added to a microcentrifuge tube containing
2.5 mg of GST-K71A ERK1 (Upstate Biotechology, Inc.) and
incubated at 30 °C for 5 min. The kinase reaction was then initiated
by the addition of 10 µl of 0.5 mM ATP supplemented with
1 µl of [
-32P]ATP (NEN Life Science Products, 3000 µCi/ml) and allowed to incubate for 10 min at 30 °C. The reaction
was terminated by the addition of 2× SDS-PAGE Sample Buffer followed
by boiling. 10 µl of the kinase reaction was then electrophoresed on
a 7% polyacrylamide gel, dried, and subjected to autoradiography.
Counts/min phosphate transferred per µg of substrate were quantitated
using the Packard Electronic Autoradiography Instant Imager and
comparing activity of the sample with the activity of the unstimulated sample.
RESULTS
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Fig. 1.
MEK1 and MEK2 are phosphorylated in response
to ionizing radiation. Cells were serum-starved for 2 days and
then exposed to the different doses of ionizing radiation. 10 min after
exposure, the cells were lysed, and equal amounts of lysates were
electrophoresed. Western blotting was performed using an antibody
that recognizes active, phosphorylated MEK1 (Ser-218 and Ser-222
phosphorylation) or MEK2 (Ser-222 and Ser-226
phosphorylation). The cell lines used to show MEK phosphorylation in
response to ionizing radiation were (top to
bottom) as follows: HeLa cells, a human cervical cancer cell
line; HBL-100 cells, a human non-transformed breast epithelial cell
line; BxPC-3 cells, a human pancreatic adenocarcinoma cell line and NIH
3T3-L1 cells, a mouse pre-adipocyte cell line. MEK1 runs as a 45-kDa
band and MEK2 runs as a 46-kDa band and are indistinguishable in this
assay. The * refers to a 32-kDa band that cross-reacts with the
antibody in all cell lines and is unresponsive to both serum starvation
and to ionizing radiation. This band can be used to normalize for equal
protein loading. In addition, Western blots against total MEK protein
indicated that in the time course of this experiment, MEK protein
levels were not up-regulated by ionizing radiation exposure (data not
shown).
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Fig. 2.
MEK1 and MEK2 are activated by ionizing
radiation. A, as a positive control to show that the
antibodies obtained could be used to perform MEK1 and MEK2 IP kinase
assays, IP kinase assays were performed on cells stably transfected
with v-ras or cells stably transfected with empty vector.
Cells were serum-starved, and equal amounts of lysate were
immunoprecipitated with an antibody that recognizes only MEK1 or only
MEK2. The substrate K71A ERK1 (kinase-dead) was then added
to the immunoprecipitate in the presence of [ -32P]ATP.
The kinase reaction was halted by the addition of SDS-PAGE buffer
followed by boiling. The reaction was then electrophoresed and
autoradiographed. B, IP kinase assays were performed on
serum-starved HeLa cells after exposure to serum, 2.5 Gy ionizing
radiation, and 10 Gy ionizing radiation. Equal amounts of protein were
immunoprecipitated with antibody directed against MEK1 or MEK2.
Phosphorylation of the K71A ERK1 substrate is shown in the
autoradiograph. C, to test specificity of the antibodies and
their suitability for IP depletion/IP kinase assays, cells were
metabolically labeled with [35S]methionine. Lysates were
pooled and divided into three tubes. For control immunodepletions, to
one tube antibody to c-fos was added. To another, antibody
to MEK1 was added, and to the last, antibody to MEK2 was added. After
overnight incubation, protein A/G-agarose was added for an hour. After
centrifugation, each supernatant was divided in two, and
immunoprecipitations were performed using antibody to either MEK1 or
MEK2. The 35S-labeled MEK1 or MEK2 is indicated in the
autoradiograph of the electrophoresed immunoprecipitation product.
D, immunodepletion was followed by IP kinase assays. HeLa
cells were serum-starved. Equal amounts of lysates were immunodepleted
of either MEK1 or MEK2, and then IP kinase assays were performed using
K71A ERK1 as a substrate. Again, although both MEK1 and MEK2
are activated by ionizing radiation, MEK2 shows a greater increase in
activation relative to unstimulated cells. Each IP kinase assay
presented in Fig. 2 was performed a minimum of three times with similar
results each time.
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Fig. 3.
Cells that are stably transfected with
dominant negative MEK1 show only modestly increased sensitivity to
ionizing radiation. A, cell lines that were stably
transfected with K97A MEK1, S222A MEK1, or S222E MEK1 were
generated from pools of approximately 1000 clones. As a control, stably
transfected cells containing only empty vector were also made. To show
expression, lysates from the four cell lines (vector, K97A MEK1, S222A
MEK1, and S222E MEK1) were generated, and Western blots were performed
using an antibody that recognizes the C terminus of MEK1 (Santa Cruz
Biotechnology) to probe the blot. The K97A MEK1, the S222A
MEK1, and the S222E MEK1 cell lines all overexpress
transfected MEK1 as shown by this blot. B, to test whether
endogenous MEK1 becomes activated in response to ionizing radiation in
the K97A MEK1 and S222A MEK1, two separate plates of each cell line
(including vector-only cells) were serum-starved. IP kinase assays were
then performed after one plate of cells was exposed to 15 Gy radiation,
whereas the other plate of cells was not exposed. Phosphorylation of
kinase-dead (K71A ERK2) was then quantitated. Each data
point was performed in triplicate. Counts/min of phosphate transferred
per mg of substrate is shown in a graph. C,
survival of the cells stably transfected with the given MEK1 construct
was measured using the colony forming assay. Approximately 500 cells
were plated for an indicated time point. Each plate was then exposed to
the given dose of ionizing radiation. Cells were refed every 2 days for
2 weeks. Colonies were then stained by crystal violet and counted
manually. Relative survival refers to the survival relative to an
unirradiated plate of cells from the same cell line plated on the same
day at the same density. Each experiment was performed in triplicate.
Relative survivals and S.E. are graphed. All cell lines were used
within 3 weeks of generation or within 2 weeks of thawing.
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Fig. 4.
Cells that express dominant negative MEK2 are
hypersensitive to ionizing radiation. A, a
site-directed MEK2 mutant (K101A MEK2) designed to mimic the K97A MEK1
dominant negative was made. Both the wild-type MEK2 gene and
the K101A MEK2 gene were transfected into HeLa cells
independently. After 3 weeks of hygromycin selection (pREP4 vector),
approximately 1000 colonies from each transfection were pooled. Again,
a separate control cell line containing only the vector was also made.
Lysates from the three cell lines (K101A MEK2 cells, vector-only cells,
and wild-type MEK2 cells) were generated, and Western blotting was
performed using an antibody that recognizes the N terminus of MEK2.
Both the wild-type MEK2 cells and the K101A MEK2 cells overexpress MEK2
as shown by this blot. B, to test whether the stably
transfected K101A MEK2 blocked MEK2 activation in response to ionizing
radiation, IP kinase assays were performed. Two serum-starved plates of
cells were used. One was exposed to 10 Gy ionizing radiation and the
other was not exposed. Phosphorylation of kinase-dead (K71A
ERK2) was then quantitated. Each data point was performed in
triplicate. Counts/min phosphate transferred per mg of substrate is
shown in a graph. C, survival of the cells stably
expressing the given MEK2 construct was measured using the colony
forming assay. Again, relative survival refers to the survival relative
to an unirradiated plate of cells from the same cell line plated on the
same day at the same density. Each experiment was performed in
triplicate. Relative survivals and S.E.s are graphed. All cell lines
were used within 3 weeks of generation or within 2 weeks of
thawing.
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Fig. 5.
Cells that express dominant negative MEK1 or
dominant negative MEK2 do not show a defect in double-strand DNA break
repair. A, the ability of each cell line to repair
double-strand DNA breaks was measured by embedding the cells from the
three cell lines (K101A MEK2, vector- only, or K97A MEK1) in agarose
plugs and exposing the plugs to 10 Gy ionizing radiation. The cells
were allowed to repair the damage for the given amount of time before
the plugs were digested with proteinase K and subjected to pulse-field
electrophoresis. Southern blotting was then performed using
random-prime labeled human DNA as a probe, and the data were then
quantitated. A representative experiment is shown. B, the
graph shows the quantitation of three DNA repair
experiments. Percent repair was determined by comparing the amount of
damaged DNA at a certain time point to the amount of damaged DNA at 0 min of repair (after standardization to equal amounts of DNA). Percent
repair and S.E.s are graphed.
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Fig. 6.
Cells expressing dominant negative MEK2 show
delayed induction of the G2 arrest and decreased ability to
recover from that G2 arrest. Asynchronously growing
cells from the respective cell lines (vector only, top; K97A
MEK1, middle; K101A MEK2, bottom) were exposed to
5 Gy ionizing radiation. Propidium iodine staining was performed at the
indicated time points. Flow cytometry was then performed. This
experiment was performed five times with similar results all five
times. A representative experiment is shown. Similar results were
obtained using the S222A MEK1 cells line (not shown).
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Fig. 7.
Delayed recovery from the G2
arrest leads to radiosensitivity in the K101A MEK2 cell line.
A, flow cytometry was performed to monitor G2
arrest after irradiation and treatment of the cells with the various
pharmacological agents. Far left profile, cell cycle profile
of asynchronously growing K101A MEK2 cells. Middle profile,
the timing of the experiment is shown in the upper panel.
Below this is cell cycle profile of K101A MEK2 cells irradiated with 5 Gy and harvested 16 h later. Full arrest has not occurred at this
time point. The bottom panel shows the cell cycle profile of
K101A MEK2 cells pretreated with vanadate followed by irradiation with
5 Gy. Cells treated with vanadate plus irradiation in this manner show
a significantly increased G2 arrest relative to K101A MEK2
cells treated with only irradiation. Far right panels, the
upper panel shows the time course of the experiment. Below
this panel is a cell cycle profile of K101A MEK2 cells irradiated with
5 Gy and harvested 32 h later. These cells show a significant
number still arrested in G2. The bottom panel
shows the cell cycle profile of K101A MEK2 cells irradiated with 5 Gy
and treated with caffeine 24 h following irradiation. Eight hours
following caffeine treatment (32 h following irradiation), cells were
harvested. Caffeine treatment leads to a recovery from the
G2 arrest and allows a cell cycle profile which is now
indistinguishable from asynchronously growing cells. B,
survival of cells treated with 5 Gy ionizing radiation and either
caffeine or vanadate in the time course described above. Neither
caffeine nor vanadate lead to a change in cell survival in the
vector-only cells or the K97A MEK1 cells. Vanadate treatment (forced
G2/M arrest) had no effect on K101A MEK2 cell survival,
whereas caffeine treatment (forced recovery from G2/M
arrest) leads to significantly increased survival.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Edwin Krebs (University of Washington, Seattle), Natalie Ahn (University of Colorado, Boulder), and Kun-Liang Guan (University of Michigan, Ann Arbor) for their generous gifts of plasmids. We thank Marylin Thompson and Michael Freeman for critical comments on the manuscript and Philip Browning, Steve Hann, David Miller, and P. Anthony Weil for helpful discussions concerning the data.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Public Service Grant R01CA51735.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Medical Scientist
Training Program Grant 5T32GM07347 from the National Institutes of Health.
§ To whom correspondence should be addressed: 2220 Pierce Ave. South, Rm. 659 MRB II, Vanderbilt University Cancer Center, Nashville, TN 37232. Tel.: 615-936-3114; Fax: 615-936-1790; E-mail: jeff.holt{at}mcmail.vanderbilt.edu.
The abbreviations used are: dsb, double-strand DNA break; ATM, ataxia telangectasia gene; DNA-PK, DNA-dependent protein kinase; BRCA2, breast cancer 2 gene; MEK, MAP kinase kinase; SEK, stress-activated protein kinase kinase; MAP kinase, mitogen-activated protein kinase; JNK, Jun kinase; Gy, gray; bp, base pair; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; IP, immunoprecipitation.
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REFERENCES |
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