From the Cellular and Molecular Biochemistry Research Laboratory (151), Department of Laboratory Medicine and Pathology and University of Minnesota Cancer Center, University of Minnesota and the Department of Veterans Affairs Medical Center, Minneapolis, Minnesota 55417 and the § Transgenic Oncogenesis Group, Laboratory of Cell Regulation and Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, June 5, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Protein kinase CK2 (CK2) has long been implicated
in the regulation of cell growth and proliferation. Its activity is
generally elevated in rapidly proliferating tissues, and nuclear matrix (NM) is an important subnuclear locale of its functional signaling. In
the prostate, nuclear CK2 is rapidly lost commensurate with induction
of receptor-mediated apoptosis after growth stimulus withdrawal. By
contrast, chemical-induced apoptosis in prostate cancer and other cells
(by etoposide and diethylstilbestrol) evokes an enhancement in CK2
associated with the NM that appears to be because of translocation of
CK2 from the cytoplasmic to the nuclear compartment. This shuttling of
CK2 to the NM may reflect a protective response to chemical-mediated
apoptosis. Supporting evidence for this was obtained by employing cells
that were transiently transfected with various expression plasmids of
CK2 (thereby expressing additional CK2) prior to treatment with
etoposide or diethylstilbestrol. Cells transfected with the CK2 Protein kinase CK2
(CK2)1 has been extensively
studied in recent years for its potential role in multiple functional
activities including the regulation of cell growth and proliferation.
It is a ubiquitous protein Ser/Thr kinase, localized in the cell cytoplasm and nucleus, existing as a heterotetramer consisting of In previous work, we have demonstrated that CK2 is dynamically
regulated with respect to its nuclear localization, such that chromatin
and NM appear to be its preferential sites of association within the
nucleus. The association of CK2 in these compartments is profoundly
responsive to the status of growth stimuli (1, 9, 11-13). To that end,
we have employed androgen action in the prostate epithelial cells that
is mediated via the androgen-receptor system as an experimental model.
It is well known that withdrawal of androgenic growth signal via
castration of adult rats induces rapid apoptosis in the epithelial
cells of the gland and that this process is reversed on androgen
administration to castrated rats (14, 15). In the same paradigm,
androgen withdrawal evoked a rapid loss of nuclear CK2 that was most
apparent in the NM fraction. The treatment of castrated rats with
androgen, and consequent induction of gene activity and cell growth in
the prostatic epithelial cells, concurred with a rapid translocation of
the CK2 from cytoplasm to the nuclear compartment such that the
enhancement in the NM-associated CK2 was the most prominent (1, 2, 9,
11-13).
The above studies also point to a hitherto unappreciated functional
role of CK2, namely that growth stimulus-mediated changes in nuclear
CK2 were associated not only with early growth responses but also with
changes in apoptotic activity in the cells, depending on the
availability of physiological growth factor signals such as androgens
or other mitogenic growth factors (1-3, 16). Thus, under conditions of
androgenic stimulation in the prostate the growth-related activities
would predominate, whereas under conditions of growth stimulus
withdrawal the apoptosis would predominate. This would imply that the
presence or absence of CK2 in the nucleus may exert a significant
effect on apoptotic activity. Apoptosis, or programmed cell death, has
been increasingly appreciated to play a significant role in the
maintenance of tissue homeostasis (for examples see Refs. 17-20). The
process of apoptosis can be initiated by either the receptor-mediated
or chemical-mediated pathway. The former relates to withdrawal of
growth stimuli (e.g. on androgen deprivation in
the prostate), whereas the latter type of apoptosis is induced by
physical agents such as In the present paper we report that etoposide and diethylstilbestrol
(DES), employed as models for chemical-induced apoptosis, demonstrate a
distinctly different response of CK2 in the NM when compared with the
previous observations on receptor-mediated induction of apoptosis.
Unlike the latter, which evoked a rapid loss of CK2 from the NM in the
prostate epithelial cells preceding apoptosis, the chemical-induced
apoptosis mediated by etoposide or DES treatment of cells
resulted in a dramatic initial increase in the CK2 association with the
NM. A possible explanation of these results is that the presence of CK2
in the nuclear compartment may exert a protective role against
apoptosis, in addition to its previously suggested role in activities
related to stimulation of growth. A direct support for this notion was
obtained from the observation that overexpression of CK2 by transient
transfection of cells with expression plasmids of catalytic subunit
CK2 Materials
Chemicals--
Synthetic dodecapeptide substrate (RRRADDSDDDDD)
(25) was purchased from Peptide Technologies (Gaithersburg, MD).
Etoposide and DES were purchased from Sigma. Cell proliferation
reagent WST-1 was supplied by Roche Molecular Biochemicals. The
expression plasmids pCI-CK2 Cells--
Human prostate adenocarcinoma cell lines LNCaP and
PC-3 were purchased from American Type Culture Collection (Manassas,
VA). They were maintained in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 2 mM L-glutamine,
25 mM HEPES, 10% defined FBS (Hyclone Laboratories, Logan,
UT) in an atmosphere containing 5% CO2. Culture medium for
LNCaP cells also included 10 Methods
Cell Proliferation Assay--
Cell viability and proliferation
was determined by employing the cell proliferation reagent WST-1, a
tetrazolium salt that is cleaved by mitochondrial dehydrogenases in
viable cells. Briefly, 100 µl of cell suspension (containing 0.5 to
2 × 104 cells) was plated in each well of 96-well
plates. Cells were cultured for 24 h to allow reattachment.
Following the treatment of cells with etoposide or DES, 10 µl of
WST-1 was added to each well, and incubation was carried out at
37 °C for 30 min. An automated plate reader was employed to measure
A450.
Cell Transfection with Expression Plasmids of CK2--
The
transient transfection with expression plasmids pCI-CK2 Preparation of Nuclear Matrix--
Subfractionation of cells to
isolate NM was carried out as described previously (16, 26, 27). All
the procedures were performed at 4 °C except where indicated
otherwise. Cells were scraped from the flasks after various treatments,
washed twice with cold 0.9% saline solution, and then suspended in
CSK Buffer (10 mM PIPES, pH 6.8, 100 mM
NaCl, 0.3 M sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 4 mM vanadyl
ribonucleoside complex, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin) for preparation of the cytosolic
and NM fractions as described previously (16, 26, 27). The final NM
fraction was suspended in a buffer consisting of 0.2 M
NaCl, 5 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 50 mM Tris-HCl, pH 7.9. Protein content of these preparations was assayed as described
previously (28).
Protein Kinase CK2 Assays--
Synthetic dodecapeptide was used
as the substrate to assess CK2 activity associated with various cell
fractions (cytosol and NM) as described previously (16, 26, 27).
Briefly, the reaction buffer consisted of 30 mM Tris-HCl,
pH 7.4, 5.0 mM MgCl2, 150 mM NaCl,
1.0 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 40 mM Western Blot Analysis--
Samples were denatured by heating at
95 °C for 5 min in a sample buffer consisting of 10 mM
sodium phosphate buffer, pH 7.0, 4 M urea, 2.5% SDS, 1%
2-mercaptoethanol and were subjected to SDS/urea/10% polyacrylamide
gel electrophoresis as described previously (16, 26). The separated
proteins were transferred from the gel to a nitrocellulose sheet. After
blocking the sheet with a medium consisting of 10 mM
Tris-HCl, pH 7.4, 0.9% NaCl, 3% dry milk, the blot was successively
incubated with mouse anti-human CK2 RNA Analysis--
Cells subjected to various treatments were
collected for total RNA isolation using TRIZOL reagent according to the
manufacturer's guidelines. Different amounts (0.2, 1, and 5 µg) of
total RNA from each sample were used for hybridization with CK2 General--
Each experiment was repeated at least three times
and included multiple replicates for each condition. The S.E. was
determined for all assays, and where appropriate, statistical analyses
were carried out to establish the significance of the results using ANOVA.
To investigate the dynamics of CK2 in response to chemical-induced
apoptosis, we have employed LNCaP, PC-3, and ALVA-41, three prostate
cancer cell lines of diverse biological properties. Further, to confirm
the general nature of these responses, we have also included three
diverse nonprostate cell lines, which are Shionogi mouse mammary
carcinoma, CA-9-22 (a squamous cell carcinoma of the head and neck),
and CHO (a noncarcinoma cell line). For induction of chemical-mediated
apoptosis we have utilized two well established agents, namely
etoposide (22) and DES (29); these drugs have also been employed as
cancer chemotherapeutic agents.
Effect of Etoposide on Prostate Cancer Cells--
The results
represented in Fig. 1 show that etoposide
is a potent inducer of cell death in a time- and
dose-dependent manner in the three diverse prostate cancer
cell lines LNCaP, PC-3, and ALVA-41. For example, the viability was
most markedly reduced at concentrations of etoposide between 50 and 100 µM in the culture medium and was apparent within 20 h of
treatment with the drug. The nature of the cell death under these
conditions was confirmed by DNA ladder analysis, and Fig.
2 shows a representative result of the
effect of 48-h treatment of ALVA-41 cells with varying concentrations
of etoposide (Fig. 2A) and the effect of 30 µM etoposide over a period of 1 to 96 h (Fig. 2B). These
results confirmed that etoposide-induced cell death was via apoptosis; similar results were observed with the other two prostate cancer cell
lines (result not shown).
Effect of Etoposide on Cytosolic and NM-associated CK2 in Prostate
Cancer Cells--
Fig. 2C shows that ALVA-41 cells treated
with 30 µM etoposide over time evoked a marked change in
NM-associated CK2 activity compared with the corresponding controls.
This effect was time-dependent and was maximally apparent
at about 48 h following the etoposide treatment of cells.
Western blot analysis was undertaken to determine whether the change in
the NM-associated CK2 in response to etoposide treatment was explained
by translocation of CK2 from the cytoplasmic compartment, as observed
previously when prostatic cells were subjected to changes in growth
stimuli (1, 9, 11, 16, 24). Fig. 2D shows a representative
result employing ALVA-41 cells treated with varying concentrations of
etoposide for a period of 48 h. The results suggested that
etoposide treatment produced a dose-dependent change in the
immunoreactive CK2
Fig. 3 shows the effect of varying
concentrations of etoposide on ALVA-41, LNCaP, and PC-3 cells with
respect to CK2 activity in the cytosolic and NM fractions. A small
change was observed in the cytosolic compartment of each cell line;
however, a remarkable dose-dependent change was apparent in
the NM-associated CK2 in each cell line although the sensitivity of the
three cell lines to etoposide varied somewhat. Thus, whereas the
maximal effect was noted at 48 h of treatment with etoposide at a
concentration of 30 µM for ALVA-41 cells, the
concentration of etoposide required for similar effects on LNCaP and
PC-3 cells was 100 µM. This correlated with the
sensitivity of these various cell lines to induction of apoptosis by
etoposide shown in Fig. 1.
Response of Various Other Cell Lines to Etoposide--
The effects
of etoposide on growth of Shionogi, CA-9-22, and CHO cell lines were
also studied (Fig. 4A). The
results indicated that cell growth was affected in a manner similar to
that observed for prostate cancer cell lines (Fig. 1). Measurement of
NM-associated CK2 activity in these cell lines showed an enhancement in
CK2 in response to etoposide analogous to that observed for the
prostate cancer cell lines (Fig. 4B compared with Fig.
3).
Other Considerations of Etoposide Action on CK2--
In control
experiments we established that treatment of ALVA-41 cells with
etoposide at 10, 30, and 100 µM did not evoke a significant change in the expression of CK2 Effect of Diethylstilbestrol on Prostate Cancer Cells--
The
effect of DES, another agent for induction of chemical-mediated
apoptosis (29), on growth of ALVA-41, PC-3, and LNCaP cell lines was
examined. The results represented in Fig.
5A show that DES affects the
growth of ALVA-41 cells in a dose- and time-dependent manner similar to that observed for the effects of etoposide (Fig. 1A). Further, PC-3 and LNCaP cell lines also respond
similarly to DES in a dose-dependent manner (Fig.
5B compared with Fig. 1B). The nature of the cell
death induced in these cell lines by DES as being due to apoptosis was
confirmed by DNA ladder analysis (Fig. 5C).
Effect of Transient Overexpression of CK2 on Chemical-mediated
Apoptosis--
The above results, taken together with our previous
observations on receptor-mediated apoptosis in prostate cells and the associated changes in CK2 (1, 9, 11, 16, 24), hinted that CK2 may exert
a protective effect against apoptosis. To test this more directly, we
transfected PC-3 cells (as a representative prostate cancer cell line)
with expression plasmids of CK2
We also confirmed the above observation by employing ALVA-41 cell line
treated with DES. ALVA-41 cells were transiently transfected with
various expression plasmids of CK2 as described above for PC-3 cells.
The profile of CK2 expression in these cells was similar to that
observed for PC-3 cells under the same conditions (result not shown).
These cells were treated with 30 µM DES for 48 h, which resulted in a loss of about 53% of cell viability in control and
pCI-transfected cells (Fig. 7). On the
other hand, cells transfected with pCI-CK2 Protein kinase CK2 has long been known as a multifunctional
protein Ser/Thr kinase with possible roles in the cytoplasmic and
nuclear compartments (1-9, 30). In particular, a considerable amount
of evidence has accumulated implicating CK2 in normal and abnormal cell
growth and proliferation (1-9, 30). Along these lines, our studies on
the functional dynamics of CK2 in relation to growth control have
employed the temporal androgenic response of prostatic epithelial cells
as a model. In the in vivo model of rat ventral prostate,
androgen deprivation results in receptor-mediated apoptosis, which is
reversed on androgen administration. Cell culture models employing
various prostate cancer cells responsive to androgens and/or growth
factors have also demonstrated similar growth responses in
vitro (1, 16, 31). By employing the in vivo, as well as
in vitro androgenic regulation of prostate cells, our
previous studies have established that CK2 undergoes dynamic
modulations in the nuclear compartment in response to altered status of
growth signals, and within the nucleus there appears to be distinct
loci of the functional association of CK2. For example, removal of the
growth signal that controls rat prostatic growth
(i.e. by androgen deprivation in the animal)
evokes a rapid differential loss of CK2 from the NM, whereas
introduction of the growth stimulus for the prostatic epithelial cells
(by administration of androgen to the animal) results in a rapid
translocation of CK2 from the cytoplasmic compartment to the nuclear
compartment where it demonstrates a differential association with the
NM (1, 9, 11-13). These responses of CK2 are among the earliest events that occur with altered growth signals. Analogous studies carried out
on prostate cancer cells in culture have also demonstrated that NM is a
common downstream site of CK2 signaling that is profoundly modulated by
altered androgenic or growth factor stimulus in the corresponding
responsive cells (1, 16). In other studies, transcriptionally active
nucleosomes that are organized on NM were found to harbor a higher
level of CK2 compared with that in the transcriptionally inactive
nucleosomes (2, 10). Further, it is noteworthy that transient
overexpression of CK2 in prostate cells by transfection with expression
vectors was found to yield a higher degree of differential enhancement
in the NM as compared with that in the cytoplasm (24). Thus, based on
several lines of evidence it appears that NM is a key site of CK2
signaling in the nucleus (1, 9).
In the aforementioned rat prostate epithelial cell model, removal of
the growth stimulus resulted in an early loss of CK2 associated with
the cessation of cell growth and initiation of receptor-mediated
apoptosis. It may be recalled that receptor-mediated apoptosis in this
case does not involve events such as an initial DNA damage or
up-regulation of p53 (14, 15) and is a direct consequence of the
removal of the growth stimulus, androgen, or the growth factors, as the
case may be. More importantly for the present discussion, the
receptor-mediated dynamic loss of CK2 from the NM is initiated prior to
a significant appearance of apoptosis in rat prostate epithelial cells
(1, 9, 11). Accordingly, it would be pertinent to discuss the present
results on CK2 dynamics in chemical-mediated apoptosis in the context of the previous observations on CK2 dynamics in receptor-mediated apoptosis.
To reiterate, CK2 has been proposed to have a major functional role in
growth-related activities, especially in stimulation of cell growth
(1-4, 6-8). However, based on our previous observations taken
together with the present results, an adjunct functional role of CK2
under various growth conditions also merits consideration. We suggest
that an early and rapid loss of NM-associated CK2 in the
receptor-mediated induction of apoptosis may additionally be
interpreted in terms of the removal of a protective role of CK2 against
apoptosis under these conditions. Analogous considerations on the role
of CK2 should apply to the chemical-mediated apoptosis induced by the
commonly employed agents such as etoposide and DES. Unlike the
receptor-mediated apoptosis, the chemical-mediated apoptosis initially
involves damaging effects at upstream sites prior to the execution
phase of apoptosis mediated by caspases (21-23, 29, 32). With respect
to the present results on the dynamics of CK2 in response to etoposide
and DES, an increase in the NM-associated CK2 was observed as an early
response to this mode of induction of apoptosis. Clearly, this is
opposite of the previously observed response of CK2 to
receptor-mediated apoptosis where a rapid loss of CK2 from the nuclear
compartment was demonstrated. However, based on the present results we
propose that these two apparently disparate responses of NM-associated CK2 to the receptor-mediated apoptosis compared with chemical-mediated apoptosis can be interpreted to reflect a common role of CK2 as a
protective agent against apoptosis. If the presence of CK2 would be
expected to block receptor-mediated apoptosis, early loss of CK2 from
the nucleus following androgen deprivation (1, 9, 14, 15) would be
commensurate with the progression of apoptosis. On the other hand, in
chemical-mediated induction of apoptosis, a rapid recruitment of CK2 to
the NM may be interpreted to serve a primary protective response under
these conditions. As discussed subsequently, our results also provide a
more direct support for this notion.
A key test of the role of CK2 in protection against apoptosis, as
proposed above, would be to demonstrate that cells overexpressing CK2
would exhibit a resistance to apoptosis. Thus, our results provided a
direct evidence that etoposide and DES induction of apoptosis was
modulated by the overexpression of the CK2 catalytic subunit or
holoenzyme. The fact that overexpression of the regulatory subunit of
CK2 ( The results discussed above suggest an important role of dynamic CK2
translocation to the NM in response to the chemical-mediated cell
injury. It may be noted that we observed a somewhat disparate increase
in the NM-associated CK2 activity compared with the immunoreactive protein in etoposide-treated cells. This may be because of the technical problem of precisely equating the immunoreactive protein with
the enzyme activity. However, it may also reflect involvement of
additional factors such as the availability of the previously sequestered or unavailable CK2 and/or some activation of the enzyme activity through unknown mechanisms. It has been noted that a significant part of CK2 in the cell nucleus is present in a sequestered form, presumably bound to DNA and other elements in the nucleus (35).
Considering this, the percent change in CK2 by translocation measured
by immunoblotting would be masked somewhat by that originally present
in the nucleus in a sequestered form (but not active). Additionally, a
factor in the case of apoptosis involving DNA damage would also be the
release of agents such as histones and polyamines that may produce an
activation or enhancement of the CK2 activity as described previously
(36-41). However, such an enhancement in CK2 activity would have to
occur prior to the isolation of NM and persist in the final preparation.
In summary, we have shown that two different chemical agents that are
known to induce apoptosis evoke a qualitatively similar response of CK2
translocation to the NM in diverse type of cells. The distinct nature
of the dynamic response of NM-associated CK2 to chemical-mediated or
receptor-mediated apoptosis is compatible with its possible
role in the process of apoptosis. This is supported by the observation
that overexpression of CK2 catalytic subunit or the holoenzyme
significantly blocks the chemical-mediated apoptosis. The lack of such
an effect of the overexpression of the or
CK2
showed significant resistance to chemical-mediated
apoptosis commensurate with the corresponding elevation in
CK2 in the NM. Transfection with CK2
did not demonstrate this
effect. These results suggest, for the first time, that besides the
commonly appreciated function of CK2 in cell growth, it may also have a
role in protecting cells against apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
', and
subunits (~ 42, 38, and 28 kDa, respectively) with the
possible
2
2,
'
2, or
'
'
2 configuration. A number of putative substrates
for CK2 have been identified in both the cytoplasm and the nucleus.
Many of these are based on in vitro phosphorylation studies
although a number of them have also been shown to be substrates for CK2
in vivo. Among the nuclear substrates are proteins involved
in growth including RNA polymerases, topoisomerase II, protein B23,
nucleolin, SV40 large T antigen, certain proto-oncogene products, and
growth factors, as well as certain nonhistone proteins, which might
include transcription factors, etc. (for examples see Refs.
1-10).
-radiation or by chemicals such as
etoposide. Some of the apoptosis-inducing chemical agents are known to
induce DNA damage as an initial step, which is a distinguishing feature
from receptor-mediated apoptosis (21-23). Our previous work employing
receptor-mediated apoptosis in the prostate epithelial cells has shown
a distinct response of NM-associated CK2 such that a rapid loss of CK2
from the NM compartment temporally precedes the appearance of apoptosis
(1, 9, 11, 14, 15, 24). However, no such studies have been reported on
the response of the CK2 signal in relation to chemical-mediated apoptosis in cancer cells. Considering the distinct nature of the
receptor-mediated and chemical-mediated apoptosis, we decided to
examine the nature of CK2 dynamics in the NM under the latter conditions.
or bicistronic holoenzyme CK2
resulted in a significant
protection against chemical-induced apoptosis. The specificity of this
action appears to reside in the catalytic subunit
of CK2, because
transfection with the regulatory subunit CK2
did not afford such a
protection.2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, pCI-CK2
, and bicistronic
pCI-CK2
were prepared as detailed previously (26). Anti-CK2
antibody was purchased from Transduction Laboratories (Lexington, KY).
TRIZOL reagent was from Life Technologies, Inc., Grand Island, NY. All
other reagents were of the highest purity available.
10 M
5
-dihydrotestosterone. Human prostate carcinoma cell line ALVA-41
was obtained from Dr. Richard C. Ostenson (University of Washington,
Seattle, WA). These cells were maintained in RPMI 1640 supplemented
with 2 mM L-glutamine, 25 mM HEPES,
6% defined FBS in an atmosphere containing 5% CO2. A cell
line from squamous carcinoma of head and neck CA-9-22 was a gift from
Dr. T. Kuroki, University of Tokyo, Tokyo, Japan. These cells were
also maintained in the same medium used for ALVA-41 cells. Shionogi
mouse mammary cancer cells were kindly supplied by Dr. John Isaacs of
Johns Hopkins University, Baltimore, MD. They were maintained in RPMI 1640 supplemented with 2 mM L-glutamine, 25 mM HEPES, 10
8 M
5
-dihydrotestosterone, 10% charcoal/dextran-stripped defined FBS in
an atmosphere containing 5% CO2. The CHO cell line was purchased from CLONTECH, Palo Alto, CA. They were
maintained in Ham's F-12 medium supplemented with 2 mM
L-glutamine, 6% defined FBS in an atmosphere containing
5% CO2.
, pCI-CK2
,
and pCI-CK2
was carried out in PC-3 cells as described previously
(26). The period of transfection was 24 h, yielding a transfection
efficiency of about 50-60%. These cells were treated with etoposide
for varying periods of time as described in the text and figure
legends. Control cells carrying only the pCI vector were treated in
parallel in a similar manner. Similar approaches were followed for
transfection of ALVA-41 cells.
-glycerophosphate,
0.2 mM synthetic dodecapeptide substrate, 0.05 mM [
-32P]ATP (specific radioactivity,
3 × 106 dpm/nmol of ATP). The reaction was started by
the addition of the enzyme source, generally as 20 µl of sample
(equivalent to 2-10 µg of protein) and was carried out for 30 min at
37 °C. Control experiments were carried out to ensure that linear
rates of reaction were obtained with respect to time and amount of the
enzyme source.
IgM and goat anti-mouse
IgM-alkaline phosphatase-conjugated antibody. Immobilized alkaline
phosphatase was visualized using 5-bromo-4-chloro-indolyl phosphate and
nitro blue tetrazolium as described previously (16, 26).
and
CK2
cDNA probes as described previously (26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
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Fig. 1.
Effect of etoposide on the growth of various
prostate cancer cells in culture. A, effect of varying
concentrations of etoposide on viability of ALVA-41 cells over time.
Control (no added etoposide), ; 10 µM etoposide,
;
30 µM etoposide,
; and 100 µM etoposide,
. The time of treatment with etoposide was as shown. B,
effect of treatment for 48 h with etoposide at varied
concentrations on the viability of LNCaP (
) and PC-3 (
) cells.
Procedures for cell culture and measurement of cell viability were as
described under "Methods." The results are expressed as
A450 ± S.E.
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Fig. 2.
Analysis of genomic DNA, CK2 activity, and
immunoreactive CK2 in ALVA-41 cells treated
with etoposide. A, DNA was isolated after a 48-h
treatment with varying concentrations of etoposide. Lane a,
no etoposide; lane b, 10 µM etoposide;
lane c, 30 µM etoposide; and lane
d, 100 µM etoposide. B, DNA was isolated
from cells treated with 30 µM etoposide for various
periods of time. Lane a, 0 h; lane b, 1 h; lane c, 8 h; lane d, 24 h;
lane e, 48 h; and lane f, 96 h.
Treatment of cells, isolation of genomic DNA, and agarose gel
electrophoresis were as described under "Methods." The
amount of DNA in each lane is 10 µg. Arrows indicate the
markers for size of the DNA fragments. C, ALVA-41 cells were
treated with 30 µM etoposide for the periods of time
shown, after which the CK2 activity was measured in the isolated
fractions of cytosol (
) and NM (
) as described under
"Methods." CK2 activity associated with NM of control
ALVA-41 cells was 33.9 ± 3.1 nmol of 32P/mg of
protein/h. CK2 activity in the cytosol of control ALVA-41 cells was
21.6 ± 2.0 nmol of 32P/mg of protein/h. CK2 activity
in fractions from etoposide-treated cells is expressed as a percentage
of that in the corresponding fractions from untreated controls ± S.E. D, ALVA-41 cells were treated with etoposide at varying
concentrations for a period of 48 h, after which cytosolic and NM
fractions were isolated. Cytosolic fractions (20 µg of protein each)
and NM fractions (50 µg of protein each) were fractionated by gel
electrophoresis and subjected to immunoblot analysis by employing
anti-CK2
antibodies. Lane a, controls (no etoposide
added); lane b, 10 µM etoposide; lane
c, 30 µM etoposide; and lane d, 100 µM etoposide. The relative densitometric values for
cytosolic fractions were 1.00, 0.96, 0.25, and 0.19 for lanes
a-d, respectively. The relative densitometric values for the NM
fractions were 1.00, 1.28, 1.81, and 2.69 for lanes a-d,
respectively.
in the cytosolic and NM fractions such that a
decrease was observed in the former compartment with a corresponding
significant increase in the latter. Interestingly, a comparison of the
relative change in the immunoreactive CK2 with that of the CK2 activity
in the NM of cells treated with the highest concentration of etoposide
suggested that the change in CK2 activity was somewhat greater than
that in the immunoreactive CK2 (Fig. 2, C compared with
D). This implies additional means that might influence the
NM-associated CK2 activity under these conditions, as considered
subsequently under "Discussion."
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Fig. 3.
Effect of varying concentrations of etoposide
on CK2 activity in the cytosol and NM fractions of ALVA-41, LNCaP, and
PC-3 cells. Cells were treated for 48 h with etoposide at the
concentrations shown, after which CK2 activity was determined in the
cytosolic and NM fractions isolated from various cells. CK2 activity
associated with the NM fractions from control ALVA-41, LNCaP, and PC-3
cells was 33.9 ± 3.1, 9.6 ± 0.7, and 15.2 ± 1.5 nmol
of 32P/mg of protein/h, respectively. CK2 activity in the
cytosol fractions of control ALVA-41, LNCaP, and PC-3 cells was
21.6 ± 2.0, 17.9 ± 1.2, and 35.8 ± 2.2 nmol of
32P/mg of protein/h, respectively. The CK2 activity is
expressed as a percentage of that in the fractions from
etoposide-treated cells compared with that in the corresponding
fractions from control untreated cells ± S.E.
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Fig. 4.
Response of various nonprostate cell lines to
etoposide. Shionogi mouse mammary carcinoma, human squamous cell
carcinoma of head and neck CA-9-22, and CHO cell lines were treated
with etoposide for 48 h, after which cell viability and
NM-associated CK2 activity were determined as described under
"Methods." A, growth response of the cells after
treatment with 10 or 30 µM etoposide, expressed as
A450 ± S.E. B, the CK2 activity
associated with the NM fraction following treatment with 30 µM etoposide, expressed as a percentage of untreated
control ± S.E. Control values of the CK2 activity in the NM
fraction from Shionogi, CA-9-22, and CHO cells were 45.3 ± 3.6, 28.4 ± 2.6, and 27.8 ± 0.7 nmol of 32P/mg of
protein/h, respectively.
or CK2
mRNA over a time course of 0 to 48 h, as examined by slot blot analysis using CK2-specific cDNA probes. This suggests that the
above-described effects were not due to changes at the transcriptional
level. Likewise, addition of etoposide at these concentrations to
isolated nuclei or nuclear matrix or purified CK2 had no influence on
the enzyme activity thus ruling out a direct effect of this agent on
the enzyme activity itself (results not shown).
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Fig. 5.
Effect of diethylstilbestrol on growth and
genomic DNA of prostate cancer cells. A, the growth
response of ALVA-41 cells over time to various concentrations of DES.
Control (no added DES), ; DES present at 10,
; 30,
; and 100 µM,
. B, the growth response of LNCaP (
)
and PC-3 (
) cells to varied concentrations of DES at 48 h of
treatment. Results, based on measurement of
A450, are expressed as a percentage of untreated
control ± S.E. C, cells were treated with 30 µM DES for 48 h, after which DNA ladder analysis was
carried out as for Fig. 2. Lanes a, c, and
e, control ALVA-41, LNCaP, and PC-3 cells, respectively.
Lanes b, d, and f, ALVA-41, LNCaP, and
PC-3 cells, respectively, treated with DES. Markers for size of the DNA
fragments are shown in the ordinate.
(the catalytic subunit), CK2
(the
regulatory subunit), and bicistronic CK2
for 24 h prior to
treatment with etoposide. Measurement of CK2 activity in the cytosolic
and NM fractions from transiently transfected cells was carried out to
confirm the CK2 overexpression. The relative overexpression of CK2
activity in the cytosolic and NM fractions in response to transient
transfection with various plasmids of CK2 was analogous to our previous
observations in that despite a modest change in the cytosolic CK2
activity there was a much greater increase in the NM-associated CK2
activity (26). A representative result of the CK2 expression in PC-3 cells in response to transient transfection with bicistronic
pCI-CK2
followed by etoposide treatment is shown in Fig.
6A. Transfected cells treated
with 30 µM etoposide demonstrated modest change in CK2
activity in the cytosolic fraction, but there was a significantly greater enhancement of CK2 activity in the NM, compared with that in
the corresponding controls. Fig. 6B shows the corresponding effect of transient transfection with CK2 plasmids on the
etoposide-mediated apoptosis of PC-3 cells. Treatment of control or
pCI-transfected cells with 30 µM etoposide for 48 h
resulted in about 51% loss in cell viability. This was partially
reversed in cells transfected with pCI-CK2
, and the effect was even
more pronounced in cells transfected with the bicistronic
pCI-CK2
, such that in the latter case the cell viability as
compared with the control cells was about 71%. Under the same
conditions, transfection of cells with pCI-CK2
was without effect
suggesting that the presence of the catalytic subunit of CK2 is the
primary mediator of this protective effect. Considering that the
efficiency of transient transfection was no more than about 60%, these
results suggest that the extent of protection against apoptosis by
CK2
is likely to be of a much higher magnitude than that observed
in Fig. 6B.
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Fig. 6.
Protection against etoposide-mediated
apoptosis in PC-3 cells transiently transfected with various expression
plasmids of CK2. PC-3 cells were transfected with the various
expression plasmids for CK2 subunits (pCI-CK2 , pCI-CK2
, or
pCI-CK2
) for 24 h, after which they were subjected to
treatment with 30 µM etoposide for a period of 48 h.
Cell viability was determined, and cytosolic and NM fractions were
isolated for CK2 activity analysis. A, CK2 activity in
fractions from treated cells is expressed as a percentage of that in
the untreated controls ± S.E. B, cell viability in
etoposide-treated cell cultures was determined and is expressed as
A450 ± S.E. ANOVA was used to test the
significance of the differences between the CK2-overexpressing cells
and the cells treated only with etoposide. The p
values of 2.9 × 10
4, 0.24, and 1.2 × 10
4 were obtained for cells overexpressing CK2
,
CK2
, and CK2
, respectively.
and the bicistronic
pCI-CK2
resulted in a significant protection (about 72% of the
control) against DES-induced apoptosis. No protection against
chemical-mediated apoptosis induced by DES was observed when cells were
transfected with the pCI-CK2
expression plasmid (Fig. 7).
View larger version (39K):
[in a new window]
Fig. 7.
Protection against
diethylstilbestrol-mediated apoptosis in ALVA-41 cells transiently
transfected with various expression plasmids of CK2. Cells were
transfected with various expression plasmids for CK2 subunits for a
period of 24 h, as for Fig. 6. They were then treated with 30 µM DES for 48 h, and cell viability was determined.
Results are expressed as A450 ± S.E. ANOVA was
used to test the significance of the differences between the
CK2-overexpressing cells and the cells treated only with DES. The
p values of 6.3 × 10 6, 0.17, and
2.9 × 10
5 were obtained for cells overexpressing
CK2
, CK2
, and CK2
, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit) did not have a protective effect against the
chemical-mediated apoptosis suggested the specificity of the
subunit of CK2 in mediating this response. It may be noted that there
is no evidence that the level of transient overexpression of CK2
achieved in these experiments significantly alters the growth rate of
the transfected cells. The mechanism by which CK2
provides such a
protection is not clear, but phosphorylation of certain
nuclear-associated substrates of CK2 may have a potential role (1, 2,
4, 6-8, 33, 34). In this context, noteworthy is the observation that
CK2-mediated phosphorylation of B23 plays a significant role in its
stability in the nuclear matrix (24). Specific alterations in the NM
protein composition during receptor-mediated apoptosis in rat embryo
cells have been reported. Of note were the time-dependent
changes in NM composition, which included appearance of new proteins,
as well as increase in several other intrinsic proteins that were
present in the NM prior to the induction of apoptosis (34). Future work
will determine the details of the nature of the processes involved in
the blocking of apoptosis by CK2.
subunit of CK2 suggests a
specificity of the role of the
subunit in protection against
apoptosis. Thus, our results document for the first time a potential
role of CK2 in protection of cells against apoptosis, in addition to
the previously recognized role of CK2 in cell growth. These
observations could have significant pathobiological implications as
they may explain a mechanism of the resistance of cancer cells to
apoptosis-inducing therapeutic approaches.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Research Grant CA-15062 awarded by the NCI, DHHS, National Institutes of Health and by the Medical Research Fund of the Department of Veterans Affairs.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.
Present address: Duke University Medical Center, Durham, NC.
¶ To whom correspondence should be addressed: Cellular and Molecular Biochemistry Research Laboratory (151), Veterans Affairs Medical Center, One Veterans Dr., Minneapolis, MN 55417. Tel.: 612-725-2000 (ext. 2594); Fax: 612-725-2093; E-mail: ahmedk@tc.umn.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M004862200
2 An abstract based on aspects of this work was presented at the 18th International Congress of Biochemistry and Molecular Biology. An abstract on parts of this work has also been accepted by the American Society for Cell Biology for presentation at its annual meeting, San Francisco, 2000.
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ABBREVIATIONS |
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The abbreviations used are: CK2 or protein kinase CK2, formerly casein kinase 2 or II; NM, nuclear matrix; DES, diethylstilbestrol; FBS, fetal bovine serum; PIPES, 1,4-piperazinediethanesulfonic acid; CHO, Chinese hamster ovary; ANOVA, analysis of variance.
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