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Articles |
Lund University, Department of Laboratory Medicine, Molecular Medicine, Malmö University Hospital, 205 02 Malmö, Sweden
Abstract
To investigate a putative involvement of protein kinase C (PKC) isoforms
in supporting neuroblastoma cell proliferation, SK-N-BE(2)
neuroblastoma cells were transfected with expression vectors coding for
the C2 and V5 regions from different PKC isoforms. These structures
have been suggested to inhibit the activity of their corresponding PKC
isoform. The PKC fragments were fused to enhanced green fluorescent
protein to facilitate the detection of transfected cells. Expression of
the C2 domain from a classical PKC isoform (PKC
), but not of C2
domains from novel PKC
or PKC
, suppressed the number of
neuroblastoma cells positive for cyclin A and bromodeoxyuridine
incorporation. This indicates a role for a classical isoform in
regulating proliferation of these cells. Among the V5 fragments from
PKC, PKCßI, and PKCßII, the PKCßI V5 had the most suppressive
effect on proliferation markers, and this fragment also displaced
PKCßI from the nucleus. Furthermore, a PKCß-specific inhibitor,
LY379196, suppressed the phorbol ester- and serum-supported growth of
neuroblastoma cells. There was a marked enhancement by LY379196 of the
growth-suppressive and/or cytotoxic effects of paclitaxel and
vincristine. These results indicate that PKCßI has a positive effect
on the growth and proliferation of neuroblastoma cells and
demonstrate that inhibition of PKCß may be used to enhance the effect
of microtubule-interacting anticancer agents on neuroblastoma cells.
Introduction
PKC3
comprises a family of at least 10 related serine/threonine protein
kinases. Based on structural properties and cofactor requirements, the
family is subgrouped into classical (PKC, PKCßI, PKCßII, and
PKC
), novel (PKC, PKC, PKC
, and PKC
), and atypical
(PKC
and PKC
) PKCs (1, 2, 3)
. The enzymes of the PKC
family have been implicated in the regulation of growth and
differentiation in a number of cell types, and several studies suggest
that different isoforms may have unique or even opposite effects on
cell growth. For instance, in NIH3T3 cells, overexpression of PKC
leads to an increased growth rate, whereas PKC has the opposite
effects (4)
, and in R6 embryo fibroblasts, PCKßI
overexpression has a positive effect on cell growth, whereas PKC has
a negative effect on cell growth (5)
. In vascular smooth
muscle cells, the two closely related isoforms PKCßI and PKCßII
have opposite effects, with PKCßI leading to a decreased doubling
time and PKCßII leading to an increased doubling time
(6)
.
In SH-SY5Y neuroblastoma cells, treatment in the presence of serum with
a phorbol ester concentration that does not down-regulate PKC leads to
neuronal differentiation (7
, 8)
. We have recently shown
that PKC seems to be critical for the induction of neurites that
accompanies neuronal differentiation (9)
. On the other
hand, for phorbol ester-mediated increased expression of
differentiation marker genes such as neuropeptide Y, a classical PKC
isoform is likely to be of importance (10)
. PKC also seems
to be able to support the growth of neuroblastoma cells. The growth of
several neuroblastoma cell lines can be supported by phorbol ester in
the absence of serum, and inclusion of PKC inhibitors in
serum-containing medium leads to a suppressed growth of these cells
(10)
. The identity of the PKC isoform that mediates
this effect is currently unknown.
One problem in determining which PKC isoform mediates a certain biological effect is the relative lack of isoform-specific inhibitors. However, in recent years, a picture has emerged suggesting that there may be isoform-specific docking proteins in the cell and that the interaction with these proteins may be essential for the activity of the isoform. RACKs constitute a group of proteins that bind activated PKC, and this interaction is mediated to a large extent by the C2 domain in the PKC molecule (11) . In line with these results, expression of isolated C2 domains has been shown to block the translocation and/or activation of PKC isoforms in a specific manner (12, 13, 14) .
The COOH-terminal V5 region, which comprises approximately 50 amino
acids, has also been shown in several studies to mediate
isoform-specific interactions or functions. This region is the only
difference between the PKCßI and PKCßII isoforms and is thus likely
to determine their unique functions. The V5 region from PKCßII was
demonstrated to be the site for the specific interaction of this
isoform with F-actin (15)
and also to determine the
specific function of PKCßII in erythroleukemia K256 cells
(16)
. The corresponding region was shown to be involved in
the isoform-specific interaction of PKC with the PDZ domain of PICK1
(17)
and in the interaction of PKC with a PDZ domain of
INAD (18)
. The latter findings are particularly
interesting because PDZ domain-containing proteins may act as scaffolds
assembling components of a transduction cascade within one
complex (19)
. There are also results indicating a
role for the V5 region in the binding of PKCßII to its RACK
(20)
.
The aim of this study was to elucidate which PKC isoform is important for neuroblastoma cell proliferation. This was done primarily by using an approach in which the above-mentioned domains of different PKC isoforms were introduced into the cells to inhibit endogenous PKC isoforms. The effects of this expression on proliferation markers were then analyzed.
Results
The C2 domain has been suggested to act as a PKC isoform-specific
inhibitor when introduced into cells (11)
. cDNAs coding
for C2 domains from the PKC isoforms that are consistently expressed in
neuroblastoma cells (PKC, PKCß, PKC, and PKC) were amplified
with PCR and introduced into the pEGFP-N1 expression vector, which
generated a PKC C2-EGFP fusion protein when transfected into mammalian
cells. PKC, PKC, and PKC C2-EGFP fusions were readily
expressed when introduced into SK-N-BE(2)
cells (Fig. 1)
, whereas the corresponding fusion construct for PKCß generated
essentially no fusion protein (data not shown).
|
, PKC, and PKC C2
constructs was evaluated for a possible influence on neuroblastoma cell
proliferation using SK-N-BE(2)
cells. Because the transfection
efficiency of these cells is generally between 2% and 5%
(9)
, the expression of EGFP was used to identify
transfected cells. After transfection, the cells were incubated with
medium containing 10% FCS for 40 h, and then the percentage of
transfected cells staining positive for two proliferation markers,
incorporation of BrdUrd and cyclin A expression, was assessed (Fig. 2A)
. This experiment revealed that the C2 domain from PKC
suppressed the number of SK-N-BE(2)
cells positive for the two
proliferation parameters. The number of BrdUrd-positive cells was
suppressed by 29%, and the number of cyclin A-positive cells was
suppressed by 35% by expression of the PKC C2 domain. These effects
were statistically significant. The effects of C2 domains from PKC
and PKC were minor (8–15% inhibition) and not significant,
indicating that the inhibition is specific for the PKC C2 domain.
The fact that the results were similar for both BrdUrd incorporation
and cyclin A staining indicates that either marker can be used to
analyze proliferation of neuroblastoma cells.
|
. As was the case for cells
grown in the presence of serum, only the C2 domain of PKC caused a
potent statistically significant suppression (27%) of the number of
cells positive for BrdUrd incorporation. Under these conditions, there
were only minor nonsignificant effects of the PKC (16% inhibition)
and PKC (6% inhibition) C2 domains.
The selective suppression of neuroblastoma cell proliferation markers
by the C2 domain from PKC suggests that this isoform may be of
importance in regulating the proliferation of these cells. However, C2
domains of classical isoforms have a large degree of homology, and
this, together with the fact that it was not possible to test the C2
domain of PKCß, precludes the drawing of definite conclusions.
We have previously demonstrated that mRNA for classical PKC isoforms
PKC, PKCßI, and PKCßII but not PKC is present in SK-N-BE(2)
neuroblastoma cells (10)
. We also demonstrated the
presence of PKC and PKCßII protein, but due to antibody
cross-reactivity, we could not determine whether PKCßI protein is
present in neuroblastoma cells. To clarify this issue, several
antibodies were examined for cross-reactivity with PKC and PKCßII.
To obtain extracts with large amounts of the different isoforms,
expression vectors coding for PKC, PKCßI, and PKCßII were
constructed and transfected into CHO cells. This revealed that a
monoclonal anti-PKCßI antibody from Santa Cruz Biotechnology
displayed no cross-reactivity with PKC or PKCßII and also showed
that PKCßI protein is expressed in SK-N-BE(2)
cells (Fig. 3, A and B)
. Hence, of the classical isoforms, these
cells express PKC, PKCßI, and PKCßII, and any of these may have
been influenced by the PKC C2 domain.
|
, PKCßI and PKCßII) fused to the COOH
terminus of EGFP were constructed, and the generation of proteins of
anticipated size was shown with Western blot after transfecting the
constructs into SK-N-BE(2)
cells (Fig. 1)
. The PKCV5-EGFP fusion
proteins migrated as doublets, perhaps reflecting different states of
phosphorylation of the two characterized phosphorylation sites in this
region (21)
. SK-N-BE(2)
cells were transfected with the
vectors, and the number of transfected cells expressing cyclin A was
analyzed (Fig. 3C)
. The V5 region from PKCßI significantly
suppressed the number of cyclin A-positive cells with 47%,
whereas the V5 regions from PKC and PKCßII had only minor effects
on this parameter. This result highlights PKCßI as the PKC isoform of
importance for neuroblastoma cell proliferation.
If the V5 domains act as inhibitors of PKC activity, it is likely that
they localize to the same intracellular sites as the endogenous PKC
isoform that they inhibit. The EGFP-PKCV5 fusion proteins all displayed
a similar intracellular distribution (Fig. 4, A–C)
. There was a prominent localization to the nucleus of
all fusion proteins, but in most cells, a presence in the cytosol could
also be seen. Analysis of the intracellular distribution of the
corresponding endogenous PKC isoforms (Fig. 4, D–F)
revealed that PKC and PKCßII immunoreactivity was essentially
localized to the cytosol, with no staining in the nucleus. In contrast,
PKCßI was found primarily in the nucleus. To investigate whether the
most effective V5 fragment, PKCßIV5, influences the nuclear
localization of PKCßI, cells were transfected with expression vector
coding for the EGFP-PKCßIV5 fusion protein. Endogenous PKCß was
visualized with an antibody that reacts with the PKCß regulatory
domain and thus detects both PKCß isoforms (Fig. 4, G–I)
.
The staining with this antibody was generally weaker than that with the
other PKCß antibodies, revealing PKCß immunoreactivity only in the
nucleus and no reactivity corresponding to the PKCßII localization to
the cytosol. In many cells overexpressing EGFP-PKCßIV5, there
was a marked reduction in nuclear staining with the PKCß antibody.
Because PKCßI is the isoform found in the nucleus, it suggests that
the PKCßIV5 fragment indeed displaces PKCßI, implying that this
isoform is inhibited by the PKCßIV5 fragment.
|
. The expression
vectors coding for PKCßI and PKCßII were transfected into
SK-N-BE(2)
cells, and 40 h later, the incorporation of BrdUrd in
transfected cells was assessed (Fig. 5)
. Overexpression of neither PKCß isoform had any marked effect on the
percentage of cells staining positive for BrdUrd, which may indicate
that the amount of PKCßI molecules is not rate-limiting for
neuroblastoma cell proliferation.
|
. The incorporation of
[3
H]thymidine by cells that had been grown in
serum-free medium or in medium supplemented with 16
nM TPA was suppressed by LY379196 in a
concentration-dependent manner. Culture in the presence of 100
nM LY379196 led to a 28% suppression for cells
grown in serum-free medium. There was a tendency toward a similar
effect on TPA- and serum-supported cells, but the effect was not as
pronounced. The concentration of LY379196 needed for this effect is in
accordance with an effect on PKCß isoforms because the
IC50 of this compound is 50
nM for PKCßI and 30 nM
for
PKCßII.4
|
. SK-N-BE(2)
neuroblastoma cells were cultured in either regular growth medium
(i.e., in the presence of 10% serum) or SHTE medium
containing 16 nM TPA. For this parameter, there
was a concentration-dependent effect of LY379196 for both culture
conditions. Inclusion of 100 nM LY379196 in the
medium caused a decrease in the amount of viable cells of 16% for
cells cultured with serum and 30% for cells cultured with TPA.
By itself, the PKCß inhibitor did not completely block proliferation,
and there was still a significant growth of neuroblastoma cells in the
presence of LY379196. This raises the possibility that it is necessary
to influence other mechanisms of importance for growth to detect a
substantial effect of the PKCß inhibitor. LY379196 was therefore
combined with increasing concentrations of the anticancer agents
etoposide, paclitaxel, or vincristine. The effect of the drug
combinations on the number of viable SK-N-BE(2)
cells after culture for
3 days in regular growth medium was analyzed with a MTT assay. For
etoposide, no substantial enhancement of the
cytotoxic/growth-suppressive effect was observed when 100
nM LY379196 was included in the medium (data not shown). On
the other hand, when paclitaxel or vincristine was combined with 100
nM LY379196, the potency of these drugs for reduction of
viable cell number was enhanced (Fig. 7)
. For paclitaxel, the concentration that induced half maximal effect
was reduced from 540 nM (range, 500–580 nM,
two experiments) to 130 nM (range, 110–140 nM)
in the presence of LY379196. The corresponding effect on vincristine
was a reduction from 18 nM (range, 18–19 nM,
two experiments) to 8 nM (range, 5–13 nM).
|
The aim of this study was to elucidate which PKC isoform is
involved in regulating the proliferation of neuroblastoma cells and to
investigate whether inhibition of this isoform could be used to
attenuate the growth of these cells. To selectively block individual
PKC isoforms, we used the fact that the introduction of
RACK-interacting C2 domains has been shown to block activation and
translocation of specific PKC isoforms (12, 13, 14)
. C2
domains from different PKC isoforms were expressed in neuroblastoma
cells, and the number of transfected cells positive for proliferation
markers was estimated. This demonstrated that only a C2 domain from a
classical isoform, PKC, caused a substantial decrease in the amount
of cells displaying markers representing proliferation activity.
Significantly, the same pattern was observed for both BrdUrd
incorporation and expression of cyclin A, clearly indicating that it is
an effect on the amount of cells going through the cell cycle. It also
demonstrates that either of these markers may be used to estimate the
proliferation activity in these cells.
These results could suggest that PKC has a positive effect on the
proliferation of neuroblastoma cells. However, the C2 domains of PKC
and PKCß are highly homologous, and because the PKCß C2 construct
could not be expressed, the effect of the PKC C2 domain is not
sufficient to conclude that PKC is the isoform involved in
proliferation. Neuroblastoma cells express PKC and PKCßII
(10)
and PKCßI (Fig. 3B)
, and it is possible
that any of these is the target for inhibition by the PKC C2
domain.
In an attempt to distinguish the activities of these isoforms, we used
the COOH-terminal V5 region, which comprises approximately 50 amino
acids, and hypothesized that this fragment would function as an
inhibitor of the respective isoform. The basis for the hypothesis is
the fact that the V5 region is the only difference between the two
PKCß isoforms and is thus likely to be involved in the specific
effects of these isoforms. The V5 region has also been demonstrated to
be involved in protein-protein interactions and PKC functions
(15
, 17
, 18)
. When the effects of the expression of V5
domains were analyzed in this study, it was evident that the V5 region
from PKCßI, as opposed to V5 fragments from PKC or PKCßII,
caused a substantial suppression of the number of cyclin A-positive
cells. This highlights PKCßI as a PKC isoform of relevance for
neuroblastoma cell proliferation and adds to those studies that have
suggested that introduction of the V5 fragment may be a fruitful
approach for intervening with the function of certain isoforms.
However, it is not clear whether the V5 regions will function as
isoform-specific inhibitors in all cellular settings. The
isoform-selective effects of the C2 domains have been demonstrated by
their capacity to specifically block the translocation of the
corresponding isoform to membranes or other intracellular sites. The
limited transfection efficiency of neuroblastoma cells only allows
analysis of subcellular localization at the single cell level. The
localization pattern of PKC and PKCßII, which was rather diffuse
in the cytosol, has not allowed us to determine whether the
V5-EGFP fusion protein influences the localization or the activity of
these isoforms. Despite the lack of effects of the PKC and PKCßII
V5 fragments on the proliferation parameters, the possibility that the
activities of these isoforms are also of importance for neuroblastoma
cell proliferation cannot be excluded. All PKCV5-EGFP fusion
proteins displayed a high density in the nucleus, but a substantial
amount of fluorescence could also be noted in the cytosol. Endogenous
PKCßI was also shown to be present primarily in the nucleus, in
contrast to PKC and PKCßII, which were expressed in the cytosol.
The PKCß immunoreactivity in the nucleus, most likely representing
PKCßI, was frequently displaced in cells expressing PKCßIV5, which
indicates that the effect of PKCßIV5 on proliferation is indeed due
to interference with PKCßI function.
The hypothesis that a PKCß isoform is of importance for optimal
proliferation activity of neuroblastoma cells is further supported by
the effects of the PKCß inhibitor LY379196. Although the suppression
of proliferation and growth by this compound was not as great as
the effects of the PKCßIV5 region, the effect of LY379196 was
obtained at concentrations that inhibit only the PKCß isoforms. The
IC50 of LY379196 for PKCßI has been shown to be
50 nM, and the corresponding values for PKC, PKC, and
PKC are 600 nM, 700 nM, and 5
µM.4
Because concentrations higher
than 100 nM may partially inhibit other PKC isoforms,
higher concentrations were not used to exclude partial inhibition of
other PKC isoforms. The maximal concentration used in our experiments
(100 nM) was only two times the IC50
for PKCßI (50 nM) that would lead to a 60–70%
inhibition of PKCßI, assuming Michaelis-Menten kinetics. The
potentially remaining 30–40% PKCßI activity may explain the
relatively smaller effect of LY379196 compared with the PKCßIV5
fragment.
The discrepancy between the two methods of inhibition could also be due to the fact that LY379196 will inhibit both PKCß isoforms, whereas the PKCßIV5 fragment may block only PKCßI. There are indeed reports indicating that PKCßI will augment and PKCßII will suppress proliferation in the same cell (6) . It is also possible that the BrdUrd assay with 30 min of labeling is more sensitive than the thymidine incorporation assay, in which there is a 3-h labeling period.
PKC has been shown to be involved in the regulation of
proliferation in several cell systems, but the isoform of importance
may vary depending on cell type. Overexpression of PKC was shown to
enhance smooth muscle cell proliferation (23)
, whereas
decreased PKC levels led to increased proliferation of CaCo-2 cells
(24)
. Furthermore, overexpression of PKC in 3Y1 cells
was shown to suppress the activity of the transcription factor E2F that
is positively linked to cell cycle progression (25)
. A
specific effect of PKCßI on proliferation and growth has been shown
for rat fibroblasts (5)
and vascular smooth muscle cells
(6)
. For both these cell types, overexpression of other
classical isoforms, PKC in fibroblasts and PKCßII in smooth muscle
cells, resulted in a suppression of growth and proliferation. A PKCß
isoform was shown to be positively involved in endothelial cell growth
(26)
, and PKCßII has been demonstrated to enhance the
proliferation of colonic epithelium (27)
. In fibroblasts,
PKC was shown to enhance the growth rate, whereas PKC suppressed
the growth rate (4)
. Taken together, these data
demonstrate that there are cell type-specific effects of different PKC
isoforms on proliferation. This may reflect that PKC has no direct
general effect on the cell division machinery but rather influences
proliferation by modifying cell-specific pathways controlling the cell
division. The present study highlights PKCßI as a PKC isoform with
positive effects on neuroblastoma cell proliferation and growth.
When the PKCß inhibitor LY379196 was combined with paclitaxel or vincristine, a substantial enhancement of their effect was observed. This effect was obtained using 100 nM LY379196, a concentration specific for PKCß but not sufficient to obtain maximal inhibition of PKCßI. These data are in line with the finding that PKCß inhibition sensitized Lewis lung carcinoma cells to paclitaxel (28) and suggest that PKCß inhibition could also be used to increase the sensitivity of neuroblastoma cells to microtubule-interacting anticancer drugs.
In conclusion, this study provides indications that PKCßI is of importance for optimal neuroblastoma cell proliferation and growth. It suggests that introduction of a PKCßIV5 fragment into the cells is one way to intervene with this effect. It also shows that inhibition of a PKCß isoform, together with treatment with microtubule-interacting anticancer agents, may be a fruitful approach to attenuate the growth of neuroblastoma cells.
Materials and Methods
Plasmids.
Expression vectors coding for C2 domains of PKC, PKCßI/ßII,
PKC, and PKC and V5 domains from PKC, PKCßI, and PKCßII
fused to EGFP were generated by inserting cDNA coding for the C2
domains in the pEGFP-N1 vector (Clontech) and cDNA coding for the V5
domains in the pEGFP-C1 vector (Clontech). The cDNAs were generated
with PCR using cDNA from SH-SY5Y cells (PKC, PKCßII, PKC, and
PKC constructs) and from IMR-32 neuroblastoma cells (PKCßI
constructs) as template. Full-length cDNA for PKC, PKCßI, and
PKCßII was amplified and subcloned in the pEGFP-N1 vector, and then
the NheI/SalI fragment containing full-length PKC
cDNA was transferred to the CMS-EGFP vector (Clontech). The generation
of PKC cDNA has been described previously (9)
.
Restriction enzyme cleavage sites were introduced in the primers listed
in Table 1
. The PCR reactions were done using Pfu polymerase, and the resulting
cDNA products were sequenced to ensure that no mutations were
introduced in the PCR reaction.
|
Western Blot Analysis.
SK-N-BE(2)
or CHO cells were transfected with different expression
vectors, washed with PBS, and lysed in buffer [10 mM Tris
(pH 7.2), 160 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 1 mM EGTA, 1 mM EDTA,
and Complete protease inhibitor mixture (Roche)]. Lysates were
centrifuged for 10 min at 15,000 x g, and 20 µg (CHO
cell lysates) or 60 µg [SK-N-BE(2)
cell lysates] of protein were
electrophoretically separated on a SDS polyacrylamide gel and then
transferred to a Hybond-C extra nitrocellulose filter (Amersham). EGFP
immunoreactivity was analyzed with antibodies directed against green
fluorescent protein (Clontech) and detected with a horseradish
peroxidase-labeled secondary antibody (Amersham) together with the
SuperSignal system (Pierce). PKC and PKCß isoforms were detected
with isoform-specific antibodies (Santa Cruz Biotechnology). The
chemiluminescence was detected with a charge-coupled device camera
(Fujifilm).
BrdUrd Incorporation.
Cells were incubated with 10 µM BrdUrd for 30 min,
fixed in 4% paraformaldehyde in PBS for 4 min, treated with 0.2
M HCl and 0.7% Triton X-100 in PBS for 10 min, washed
several times with PBS, and incubated with 8 M urea for 30
min. Because the acid treatment abrogates the fluorescence of EGFP,
both EGFP and BrdUrd were visualized with immunofluorescence. The cells
were incubated for 30 min with primary antibodies [mouse anti-BrdUrd
(Boehringer Mannheim; 1:10) and rabbit anti-GFP (Clontech; 1:200) in
PBS], followed by a 30-min incubation with secondary antibodies,
TRITC-conjugated donkey antimouse IgG and FITC-conjugated donkey
antirabbit IgG (Jackson Laboratories) diluted 1:100 in PBS. The
coverslips were mounted on microscope slides using a PVA-DABCO solution
[9.6% polyvinyl alcohol, 24% glycerol, and 2.5%
1,4-diazabicyclo[2.2.2]octane in 67 mM Tris-HCl (pH
8.0)]. For experiments in Fig. 5
, cells were permeabilized in the
absence of HCl and then incubated directly with the mouse anti-BrdUrd
antibody together with DNase I (0.1 unit/µl) and exonuclease III (4
units/ml). Under these conditions, the fluorescence of EGFP could be
used to identify transfected cells. Using fluorescence microscopy, the
percentage of EGFP-expressing cells that were BrdUrd positive was
counted.
Immunofluorescence.
Cells were fixed with 4% paraformaldehyde in PBS for 4 min and treated
for 30 min with 5% donkey serum and 0.3% Triton X-100 in TBS. The
cells were then incubated for 60 min with primary antibody rabbit
anti-cyclin A diluted 1:150 in TBS, rabbit anti-PKC (1:400),
-PKCßI (1:400), -PKCßII (1:100; all from Santa Cruz Biotechnology),
or with mouse anti-PKCß (1:50; Transduction Laboratories). After
extensive washing, cells were incubated with TRITC-conjugated donkey
antirabbit or antimouse IgG (Jackson Laboratories) for 60 min. The
coverslips were mounted, and for cyclin A experiments, transfected
cells were scored for cyclin A positivity.
[3H]Thymidine Incorporation.
SK-N-BE(2)
cells were seeded at a density of 8000 cells/well in 96-well
culture plates. The indicated compounds, TPA (Sigma) and LY379196
(kindly provided by Dr. D. K. Ways, Eli Lilly, Indianapolis,
IN), had been added in 50 µl of medium before the addition of
cells in the same volume. Two days after seeding, 20 µCi of
[3
H]thymidine were added to each well, and
after incubation for 3 h, incorporated
[3
H]thymidine was recovered with a cell
harvester (Skatron). The radioactivity was measured with scintillation
counting.
Analysis of Cell Growth/Viability.
SK-N-BE(2)
neuroblastoma cells were seeded at a density of 3000
cells/well in 96-well culture plates. The indicated compounds had been
added in 50 µl of medium before the addition of cells in the same
volume. After 3 days, the amount of viable cells was analyzed measuring
the conversion of the tetrazolium salt MTT to formazan according to the
supplier’s protocol (Promega). Paclitaxel (Sigma) was solubilized in
DMSO, and vincristine sulfate (Sigma) was solubilized in water.
For paclitaxel, control cells were incubated with 0.4% DMSO. For
calculations of the concentration that elicited a half-maximal effect,
a nonlinear curve fit was performed with the equation y = A2 + (A1- A2)/(1 +
x/x0) where x0
corresponds to the half-maximal concentration.
Acknowledgments
Dr. D. K. Ways is gratefully acknowledged for providing LY379196 and for fruitful suggestions and comments.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by the Swedish Cancer Society; the
Swedish Medical Research Council; the Child Cancer Foundation of
Sweden; the Swedish Society for Medical Research; the Crafoord
Foundation; Magnus Bergvall Foundation; Gunnar, Arvid, and Elisabeth
Nilsson Foundation; Ollie and Elof Ericsson Foundation; John and
Augusta Persson Foundation; and Malmö University Hospital
Research Funds. 
2 To whom requests for reprints should be
addressed, at Lund University, Molecular Medicine, Entrance 78,
3rd Floor, Malmö University Hospital, 205 02
Malmö, Sweden. Phone: 46-40-337404; Fax: 46-40-337322; E-mail: Christer.Larsson{at}molmed.mas.lu.se
3 The abbreviations used are: PKC, protein kinase
C; EGFP, enhanced green fluorescent protein; RACK, receptor for
activated C-kinase; TPA,
12-O-tetradecanoylphorbol-13-acetate; BrdUrd,
bromodeoxyuridine; CHO, Chinese hamster ovary; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
TRITC, tetramethylrhodamine isothiocyanate.
4 D. K. Ways, personal communication.
Received for publication 5/10/00. Revision received 9/ 6/00. Accepted for publication 9/27/00.
References
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 H. Stensman, A. Raghunath, and C. Larsson Autophosphorylation Suppresses Whereas Kinase Inhibition Augments the Translocation of Protein Kinase C{alpha} in Response to Diacylglycerol J. Biol. Chem., September 24, 2004; 279(39): 40576 - 40583. [Abstract] [Full Text] [PDF]
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 Q. J. Wang, G. Lu, W. A. Schlapkohl, A. Goerke, C. Larsson, H. Mischak, P. M. Blumberg, and J. F. Mushinski The V5 Domain of Protein Kinase C Plays a Critical Role in Determining the Isoform-Specific Localization, Translocation, and Biological Function of Protein Kinase C-{delta} and -{varepsilon} Mol. Cancer Res., February 1, 2004; 2(2): 129 - 140. [Abstract] [Full Text] [PDF]
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in
A and B) and detection of cyclin A with
immunofluorescence (
in A) were used to estimate
proliferating activity of transfected cells. Data are expressed as the
percentage of transfected cells positive for either BrdUrd or cyclin A
and are the mean ± SE of four (A) or three
(B) separate experiments. Controls (-) are cells
expressing EGFP only. Approximately 200 transfected cells identified by
the presence of EGFP (A) or an average of around 100
transfected cells (B) were scored on each coverslip.
Levels of significance: P < 0.05 (*) and
P < 0.01 (**) compared with control cells
(Student’s t test).
) or with 10%
serum (
) or presence (•) of 100
nM LY379196. The viable cell number was analyzed with a MTT
assay, and data (mean ± SE; n = 6) are
expressed as a percentage of the values obtained in the absence of
drugs.