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INTRODUCTION |
Oncogenically transformed cells display many properties that are
not observed in normal cells. One such transformed phenotype is the
change in cell morphology. For example, the introduction of
oncogenically activated Ras into fibroblasts induces a round-up or
spindle-shaped morphology, which in turn disrupts the regulatory mechanisms controlling cell-cell contact and cell-substratum adhesivity and, thus, is believed to contribute to malignant (metastatic) phenotypes of cancer cells (reviewed in Ref. 1). Intracellular calcium
is known to play an important role in establishment of cellular
morphology. Early studies showed the calcium sensitivity of actin and
actin-binding proteins (reviewed in Ref. 2), major cytoskeletal
components (reviewed in Refs. 3 and 4). Moreover, calcium ions regulate
the formation of actin bundles and networks (5, 6). It has also been
shown that ectopic expression of calmodulin, a major intracellular
Ca2+-binding protein (reviewed in Ref. 7) or administration
of calmodulin-antagonists disrupts Ca2+ homeostasis,
leading to changes in cell morphology and cytoskeleton organization
(8). Microinjection of villin, a Ca2+-regulated F-actin
bundling and nucleating protein (reviewed in Refs. 9 and 10), into NIH
3T3 cells results in disruption of the stress fiber networks, leading
to morphological changes (11). These findings indicate that calcium
signaling is important for maintaining proper cell morphology.
In fibroblasts, two major voltage-dependent
Ca2+ channels are present: L (long-lasting, large
conductance)-type and T (transient)-type channels (12, 13). L- and
T-type channels are distinguished by differences in their
electrophysiological and pharmacological properties (reviewed in Refs.
14-19). The L-type channel requires strong depolarization for
activation, shows high sensitivity to dihydropyridines, and inactivates
slowly. In contrast, the T-type is a rapidly inactivating channel
activated by weak depolarizations and is resistant to organic blockers
of L-type channels but relatively sensitive to mibefradil. Because of
the low fluctuation of membrane potential in fibroblasts, T-type
channels are thought to impart a greater influence on fibroblast
physiology than L-type (13, 20, 21). In Swiss 3T3 fibroblasts
transformed by various oncogenes (i.e., v-ras,
v-fms, or polyoma virus middle T oncogenes), T-type channel
currents have been shown to be specifically suppressed (12).
Ras is a plasma membrane-localized GDP/GTP-binding protein that is
active in the GTP-bound state and functions as an extracellular mitogenic signal transducer. Activating mutations of Ras results in
constitutive signaling to downstream elements, leading to cellular transformation (reviewed in Refs. 22 and 23). To date, three major
effector pathways of Ras have been characterized in detail: the
mitogen-activated protein kinase
(MAPK)1 pathway, the
phosphatidylinositol 3-kinase pathway, and the Ral/GDS pathway (most
recently reviewed in Refs. 24 and 25). In the MAPK pathway, activated
Ras promotes the movement of Raf to the plasma membrane where it
becomes a functional kinase (26-31). Raf then phosphorylates MAPK
kinases (MEK) (32-36), which in turn activates MAPK through
phosphorylation (37). In the phosphatidylinositol 3-kinase pathway, Ras
binds and activates phosphatidylinositol 3-kinase (38, 39), which
results in activation of a variety of effector molecules including
Akt/PKB, Vav, SOS, and GRP1 (40-42). In the Ral/GDS pathway, Ras binds
and activates Ral/GDS (43-45), which in turn activates effectors such
as CDC42 and Rac1 (46, 47).
Using various Ha-Ras effector loop mutations (39, 48-50), we first
attempted to identify which of the downstream effector pathway(s) of
Ras are responsible for inhibiting the T-type Ca2+ channel
current. We found that Ras mutants lacking the ability to activate the
MAPK pathway failed to inhibit T-type Ca2+ channel current,
whereas Ras mutants that activate only the MAPK pathway efficiently
inhibit T-type channel activity. Moreover, the introduction of the
constitutively active MEK results in inhibition of the T-type
Ca2+ channel current. By use of the T-type channel blocker,
mibefradil, we further found that the suppression of the T-type
Ca2+ channel is essential for the induction and/or
maintenance of spindle-shape transformation morphology in fibroblasts.
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EXPERIMENTAL PROCEDURES |
Cells, Plasmids, and Transfection--
Swiss 3T3 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum (FBS), penicillin (100 units/ml), and
streptomycin (100 µg/ml) (this medium will be referred to as complete
medium hereafter) at 37 °C in 10% CO2. Plasmids were
transfected by the calcium phosphate method previously described (51).
Approximately 1 × 106 cells were plated in 100-mm
dishes 24 h before transfection. The Swiss 3T3 cells were
transfected with the following Ras mutant plasmids: 12V, 12V35S,
12V37G, 12V40C with pKOneo plasmid encoding a neomycin-resistance gene
in a 20:1 molar ratio. The ras mutant genes were tagged with
the hemagglutinin (HA) epitope, and expression was controlled by the
cytomegalovirus promoter. Swiss 3T3 cells were also transfected with a
plasmid encoding a gain-of-function MAPK kinase (MEK) mutant gene
(
N3-S218E-S222D (52)). Transfected cells were selected in medium
supplemented with G418 (400 µg/ml). In ras
mutant-transfected cells, G418-resistant colonies were pooled. For
cells transfected with the MEK mutant, G418 resistant colonies were subcloned.
Electrophysiological Analysis--
Ba2+ currents
through endogenous voltage-dependent Ca2+
channels expressed in cells stably transfected with 12V, 12V35S,
12V37G, 12V40C, and MEK were recorded using the whole cell
configuration of the patch-clamp technique (53). All recordings were
done at room temperature. Cells were incubated in a bath solution
containing (20 mM BaCl2, 135 mM
tetraethylammonium chloride, 10 mM HEPES (pH 7.4)). The
patch electrodes were pulled and fire polished, after which they had an
electrical resistance of ~3 megaohms when filled with an internal
solution containing (140 mM L-aspartate, 5 mM EGTA, 5 mM pyruvate Na+, 5 mM oxaloacetate, 5 mM creatine, 10 mM HEPES/CsOH (pH 7.4)). Currents were recorded using an
Axopatch 200A patch-clamp amplifier and analyzed using the pClamp
(version 6.02, Axon instruments, Inc.) software. Whole cell capacitive
transients were compensated on-line, and linear leakage currents were
cancelled using a P/4 subpulse protocol. The data were filtered at 2 kHz and digitized at 10 kHz via a Lambaster interface (Axon instruments
Inc., Burlingame, CA). L-type currents were elicited by ten 10-mV
depolarizing test pulses between
20 to +70 mV from a holding
potential of
30 mV, where T-type was completely inactivated. T-type
currents were evoked by using ten 10-mV step pulses from
40 to +50
mV, with a holding potential of
80 mV in the presence of 1 µM nifedipine. Identity of L- and T-type currents were
confirmed by their sensitivity to nifedipine and mibefradil, respectively.
Immunoblot Analysis--
To prepare cell lysates, cells were
washed twice with PBS and lysed in SDS-Nonidet P-40 lysis buffer (1%
SDS, 1% Nonidet P-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 4 mM Pefabloc SC (Roche Molecular Biochemicals), 2 µg/ml leupeptin, 2 µg/ml aprotinin). The lysates were sonicated, boiled for 5 min, and then centrifuged at 4 °C for
10 min at 20,000 × g. The lysates were further
denatured for 5 min at 95 °C in sample buffer (2% SDS, 10%
glycerol, 60 mM Tris (pH 6.8), 5%
-mercaptoethanol,
0.01% bromphenol blue). The proteins were fractionated by
SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P
membranes (Millipore). The membranes were blocked with TBST (20 mM Tris HCl (pH 7.6), 137 mM NaCl, and 0.2%
Tween 20) with 5% (w/v) dry milk) at room temperature (20-22 °C).
After multiple washes with TBST, the membranes were incubated with
primary polyclonal antibodies: anti-HA (clone Y-11, Santa Cruz),
anti-MEK-1 (clone 12-B, Santa Cruz), or anti-phosphorylated MAPK (New
England Biolab). The blots were then washed thoroughly with TBST and
then incubated with horseradish peroxidase-conjugated goat anti-rabbit
IgG for 1 h at room temperature. The antibody-antigen complex was
detected by the enhanced chemiluminescence procedure (ECL, Amersham
Pharmacia Biotech).
Morphological Transformation Assay--
Because of the
p53-dependent toxicity associated with overexpression of
Ras, Swiss 3T3 cells stably transfected with human p53 mutant (Val at
amino acid residue 143 was changed to Ala) were transiently transfected
with ras mutant plasmids (25 µg/100-mm plate). Cells were
then incubated in media supplemented with 1% FBS and appropriate
Ca2+ channel antagonists. Five to 7 days after
transfection, cells were fixed, and either Giemsa-stained or processed
for immunostaining as described below.
Indirect Immunofluorescence--
Transiently transfected cells
grown on slides with 1% FBS and respective Ca2+ channel
antagonist were washed with PBS and then fixed with 10% formalin. The
cells were then washed with PBS and permeabilized with 1% Nonidet P-40
in PBS for 5 min at room temperature. The cells were incubated with
blocking solution (10% normal goat serum in PBS) for 1 h and
probed with an anti-HA monoclonal antibody (Roche Molecular
Biochemicals) for 1 h. The antibody-antigen complexes were
detected with rhodamine-conjugated goat anti-mouse IgG antibody. After
30 min of incubation at room temperature, the samples were washed three
times with PBS. Cells were also stained with
4',6-diamidino-2-phenylindole (DAPI) DNA dye for visualization of nuclei.
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RESULTS |
Electrophysiological Dissection of Ras Downstream Effectors in
T-type Ca2+ Channel Inhibition--
To elucidate which
downstream Ras signaling pathway(s) is involved in suppression of the
T-type Ca2+ channel, Swiss 3T3 cells were transfected with
four different Ha-ras mutants (12V, 12V35S, 12V37G, 12V40C)
(39, 48-50). The 12V35S (Gly-12
Val,Thr-35
Ser) mutant retains
the Raf-1 binding site and thus can activate the Raf-1/MEK/MAPK
pathway. The 12V37G (Gly-12
Val,Glu-37
Gly) and 12V40C (Gly-12
Val,Tyr-40
Cys) mutants were identified by their ability to
signal downstream effectors via a Raf-independent pathway. The
downstream effector activated by the 12V37G mutant is the Ral/GDS
pathway and that activated by the 12V40C mutant is the
phosphatidylinositol 3-kinase pathway (48). Swiss 3T3 cells transfected
with these mutants tagged with influenza virus HA epitope were pooled
and selected, and expression of the Ras mutants were examined by
immunoblot analysis using an anti-HA monoclonal antibody (Fig.
1). All pooled cells expressed similar
levels of transfected Ras proteins.

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Fig. 1.
Immunoblot analysis of Ha-Ras
(H-Ras) effector mutant expression in pooled
Ha-ras-transfectants. Cell lysates prepared from
control Swiss 3T3 cells (lane 1) and cells transfected with
12V (lane 2), 12V35S (lane 3), 12V37G (lane
4), and 12V40C (lane 5) ras effector mutants
were resolved in 12% SDS-polyacrylamide gel electrophoresis. The
expression of transfected Ras mutant proteins were detected with
anti-HA polyclonal antibody (clone Y-11, Santa Cruz).
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Calcium channel currents were measured in the mutant Ras-expressing
cells by the whole-cell patch-clamp technique using Ba2+ as
the external charge carrier. Electrophysiological dissection of
Ca2+ channels required that we take advantage of the
different voltage dependence of channel gating between the L- and
T-type channels. L-type currents were recorded using a holding
potential of
30 mV, a voltage in which T-type channels are
inactivated and 10-mV step pulses from
20 mV to +70 mV.
Representative Ba2+ current (IBa)
traces of the control cells as well as 12V, 12V35S, 12V37G, and 12V40C
Ras mutant-expressing cell lines are shown in Fig.
2 (columns A and
B), and the corresponding current-voltage relationships of
peak IBa density are shown in column
C. L-type currents were detected in all the cell lines tested. The
average L-type current density for control Swiss 3T3 cells was
3.21 ± 0.58 pA/pF (n = 9) and 2.82 ± 0.37 pA/pF (n = 12) for 12V, 2.33 ± 0.51 pA/pF
(n = 8) for 12V40C, 1.68 ± 0.34 pA/pF
(n = 12) for 12V35S, and 2.20 ± 0.37 pA/pF
(n = 9) for 12V37G (Fig.
3). The L-type channel current peaked at
+30 mV, and there was no significant difference in the voltage
dependence among the controls and mutant Ras expressors.

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Fig. 2.
Whole cell currents recorded from the control
Swiss 3T3 cells and cells expressing H-ras effector
domain mutants. A, representative L-type
Ca2+ channel currents. Currents were elicited by a 500-ms
depolarizing pulse to +30 mV from a holding potential of 30 mV.
B, representative T-type Ca2+ channel currents.
Currents were evoked by a depolarizing pulse to +10 mV from a holding
potential of 80 mV in the presence of 1 µM nifedipine.
C, current density-voltage relationships for L-type
(closed circle) and T-type (open circle)
Ca2+ channels.
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Fig. 3.
Current densities of L- and T-type
Ca2+ channels of the control Swiss 3T3 cells and Ha-Ras
effector domain mutant-expressing cells. The current density of
each cell line was calculated from the results shown in Fig. 2 and
presented as mean ±S.E. The statistical significance was analyzed
using the Student's t test. The decrease of T-type
Ca2+ currents in 12V and 12V35S Ras mutant-expressing cells
is statistically significant (p < 0.05) as indicated
by asterisks.
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To isolate T-type current, Ba2+ currents were elicited from
a holding potential of
80 mV in the presence of the L-type channel blocker, nifedipine (1 µM). The control cells elicited a
T-type peak IBa with a current density of
0.62 ± 0.10 pA/pF (n = 5). The 12V37G mutant line
had a current density of similar amplitude (0.55 ± 0.14 pA/pF
(n = 5)), whereas the 12V40C line displayed a slightly
higher T-type current density (0.87 ± 0.12 pA/pF
(n = 8)). The 12V mutant had a T-type current density
of 0.008 ± 0.003 pA/pF (n = 7) that was
significantly less than that of the control (p < 0.001). Similar to 12V, diminished T-type current was observed in the
12V35S expressor, which had a T-type current density of peak
IBa of 0.005 ± 0.003 pA/pF
(n = 12), which is also significantly less than that of
the control (p < 0.001). The threshold current for the
L-type channel activation in both the mutants and control cells was
approximately
10 mV (Fig. 2, column C). The T-type
currents in the control, 12V37G, and 12V40C cells were activated
approximately at a threshold of
30 mV and peaked at +10 mV with no
significant difference in their voltage dependence (Fig. 2,
column C). The absence of T-type currents in both 12V- and
12V35S- expressing cells suggests that the activation of the MAPK
pathway may be responsible for suppression of T-type Ca2+
channel activity.
Mibefradil is a T-type-selective Ca2+ channel antagonist
(54, 55). As such, we sought to determine the sensitivity of endogenous T-type Ca2+ channels to mibefradil in Swiss 3T3 cells. The
treatment of Swiss 3T3 cells with 1 µM mibefradil caused
approximately 50% reduction in T-type Ca2+ channel
currents (Fig. 4). In the presence of 3 µM mibefradil, T-type Ca2+ channel currents
were completely abolished (Fig. 4), whereas there was no change in
L-type channel currents (data not shown). Similar results have been
previously reported for the recently cloned
1H T-type
Ca2+ channel from heart (56).

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Fig. 4.
Dose-dependent inhibition of
T-type Ca2+ channels in Swiss 3T3 cells by mibefradil.
Mibefradil was administered to Swiss 3T3 cells by its sequential
addition to the bath solution from 0, 1, and 3 µM in the
presence of 1 µM nifedipine. Upon each dose change of
mibefradil, cells were voltage-clamped with a holding potential of 80
mV, and a 150-ms step pulse to +10 mV was applied.
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Activation of the MAPK Pathway Is Responsible for Inhibition of
T-type Ca2+ Channels--
To test whether the activation
of the MAPK pathway is responsible for inhibition of the T-type
Ca2+ channel, Swiss 3T3 cells were stably transfected with
a gain-of-function MEK mutant (
N3-S218E-S222D (52)). The expression
of this MEK mutant results in constitutive activation of MAPK (52). The G418-resistant colonies were subcloned (3T3/MEK1 and -2), and subjected
to immunoblot analysis for expression of MEK (Fig.
5A). Both 3T3/MEK1 and -2 expressed higher levels of MEK than the control Swiss 3T3 cells that
were transfected with a vector plasmid. The 3T3/MEK1 and -2 cell lines
were further tested for activation of MAPK by immunoblot analysis using
anti-phosphorylated (activated) MAPK antibody (Fig. 5B). As
expected, activated MAPK was present in both MEK-transfected cell
lines.

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Fig. 5.
Generation of Swiss 3T3 cells stably
transfected with a gain-of-function MEK mutant. The G418-resistant
colonies from cells transfected with either vector plasmid or a MEK
mutant plasmid ( N3-S218E-S222D) were subcloned. The control Swiss
3T3 cells and two MEK-transfected cell lines (3T3/MEK1 and 3T3/MEK2)
were analyzed by immunoblot analysis using anti-MEK1 polyclonal
antibody (clone 12-B, Santa Cruz) (A) and
anti-phosphorylated MAPK (MAPK-P) polyclonal antibody (New
England Biolabs) (B). NS, non-specific
band.
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The 3T3/MEK1 and 2 cell lines were tested electrophysiologically.
Similar to 12V and 12V35S-expressing cell lines, the 3T3/MEK1 cells
exhibited an L-type current density of 3.3 ± 0.39 pA/pF (n = 11) and a T-type density of 0.005 ± 0.003 pA/pF (n = 8). The latter was significantly less
(p < 0.001) than control cells (Fig.
6D). We obtained similar
recordings in the 3T3/MEK2 cells (data not shown). To further show that
activation of the MAPK pathway is responsible for inhibition of T-type
channels in ras-transformed cells, Swiss 3T3 cells
expressing the 12V Ras mutant were pretreated with the MEK-specific
inhibitor, PD98059 (57, 58) for 24 h. We found that the T-type
Ca2+ channel currents recovered to the levels similar to
that of control Swiss 3T3 cells (Fig. 6, column B) with a
current density of 0.67 ± 0.2 pA/pF (n = 6) after
PD98059 treatment (Fig. 6D). Thus, we conclude that
activation of the MAPK pathway is responsible for the T-type
Ca2+ channel depression in ras-transformed
cells.

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Fig. 6.
Activation of the MEK-MAPK pathway is
essential for inhibition of T-type Ca2+ channels in
ras-transformed cells. a, whole cell
currents from Swiss 3T3 cells expressing gain-of-function mutant MEK
(3T3/MEK1) as well as ras-transformed Swiss 3T3 cells
treated with 75 µM MEK-specific inhibitor (PD98059) were
recorded for L-type Ca2+ channels as described in the
legend to Fig. 2A. B, whole cell currents from
the 3T3/MEK1 line as well as ras-transformed Swiss 3T3 cells
treated with PD98059 were recorded for T-type Ca2+ channels
as described in the legend for Fig. 2B. C,
current-voltage relations for peak density of L-type (closed
circle) and T-type (open circle) Ca2+
channels. D, current densities of L- and T-type
Ca2+ channels from the 3T3/MEK1 line and PD98059-treated
ras-transformed Swiss 3T3 cells were elucidated from the
results shown in A and B and presented as mean
±S.E. The statistical significance was analyzed using a Student's
t test. The decrease of T-type Ca2+ currents in
3T3/MEK1 is statistically significant (p < 0.05) as
indicated by an asterisk.
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Inhibition of T-type Ca2+ Channels Induces
Morphological Transformation--
Activation of the MAPK pathway is
common in cells transformed by many oncogene products and frequently
observed in cancer cells (most recently reviewed in Ref. 59).
Accordingly, we examined whether inhibition of the T-type
Ca2+ channel by the MAPK pathway would have a role in
cellular transformation. It has been shown that Swiss 3T3 cells are
transformed efficiently with oncogenically activated Ras (Ras 12V) when
co-transfected with mutant p53 (60, 61). Moreover, overexpression of
oncogenically activated Ras induces cell cycle arrest and cell death in
a p53-dependent manner (62-64). We therefore decided to
use the Swiss 3T3 cell line stably transfected with the dominant
negative mutant p53 (V143A), 3T3/p53(V143A). We found that the
expression of mutant p53 did not affect the overall Ca2+
channel activity because the 3T3/p53(V143A) cells exhibited both L- and
T-type Ca2+ channel currents similar to those of control
Swiss 3T3 cells (data not shown). We then transiently transfected
3T3/p53(V143A) cells with the following Ras effector mutants: 12V,
12V37G, 12V40C, and 12V37G + 12V40C. The transfected cells were
incubated under low growth conditions (1% serum) for ~7 days. As
expected, 12V-transfected cells showed a spindle morphology typical of
transformed cells (Fig. 7, panel
a). In contrast, none of the cells transfected with 12V37G,
12V40C, or 12V37G + 12V40C showed such morphological changes (Fig. 7,
panels e, i, and m). The transfected
cells in parallel were treated with 1 µM nifedipine.
Nifedipine treatment (inhibition of L-type Ca2+ channels)
did not affect cell morphology; 12V-transfected cells showed spindle
morphology (panel b), whereas cells transfected with 12V37G,
12V40C, or 12V37G + 12V40C retained a flat morphology (panels
f, j, and n). Thus, the L-type
Ca2+ channel does not play a role in induction and/or
maintenance of transformed morphology. In addition, the 12V-transfected
cells showed a spindle-like morphology in the presence of 3 µM mibefradil (selective inhibition of T-type channels
(panel c). However, when cells transfected with 12V37G,
12V40C, and 12V37G + 12V40C were treated with 3 µM
mibefradil, the cells became morphologically transformed, similar to
the 12V-transfected expressing cells (panels g,
k, and o). Consistent with the finding that
inhibition of L-type channels does not affect cell morphology,
co-treatment of cells with 3 µM mibefradil and 1 µM nifedipine gave similar results with mibefradil
treatment alone (panels d, h, l, and
p).

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Fig. 7.
Photomicrographs of 3T3/p53(V143A) cells
transiently transfected with ras mutants. The
3T3/p53(V143A) cells were transfected with 12V, 12V37G, 12V40C, and
12V37G + 12V40C. Two days after transfection, cells were split into
four culture dishes and fed with media containing 1% FBS. Plates from
each transfection were treated with 1 µM nifedipine
(panels b, f, j, and n), 3 µM mibefradil (panels c, g,
k, and o), and nifedipine (1 µM) + mibefradil (3 µM) (panels d, h,
l, and p). Five days after cell splitting, cells
were fixed, Giemsa-stained, and examined under a microscope.
Magnification, 400×.
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To confirm whether the morphologically transformed cells express the
transfected Ras mutants, 3T3/p53(V143A) cells transfected with 12V,
12V35S, 12V37G, and 12V40C were fixed and subjected to immunostaining
using anti-HA antibody. The cells expressing transfected 12V or 12V35S
Ras mutants are spindle-shaped (Fig. 8,
panels a and c), whereas cells expressing
transfected 12V37G and 12V40C mutants show a flat morphology
(panels e and g). By treatment of 12V37G- and
12V40C-transfected cells with 3 µM mibefradil, cells
acquired a spindle like morphology (panels f and
h). These results demonstrate that inhibition of the T-type
channel, but not the L-type channel, is essential for induction and/or
maintenance of morphological transformation.

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Fig. 8.
Immunofluorescence analysis of cells
transiently transfected with ras mutants. Swiss
3T3 cells were transfected with 12V, 12V35S, 12V37G, and 12V40C. Two
days after transfection, cells were split into two culture dishes and
fed with media containing 1% FBS. One plate from each transfection was
treated with 3 µM mibefradil (panels b,
d, f, and h and b',
d', f', and h'), and another plate was
untreated (panels a, c, e, and
g and a', c', e', and
g'). Five days after splitting, cells were fixed and
processed for immunostaining with anti-HA antibody (panels
a-h). Cells were also counter-stained with
4',6-diamidino-2-phenylindole (DAPI) (panels
a'-h'). Magnification, 400×.
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Requirement of Other Ras Effectors in Addition to the MAPK Pathway
for Morphological Transformation--
Under a serum-starved condition,
normal cells become arrested at resting state (G0). It has
been shown that expression of 12V, 12V35S, 12V37G, and 12V40C Ras
mutants drives cells to enter mitotic cycling in a serum-independent
manner (50). The findings, in which 12V37G- or 12V40C-transfected cells
become morphologically transformed under serum-starved condition in the
presence of the T-type Ca2+ channel inhibitor, raises two
possibilities: 1) cell cycling may be required for morphological
transformation by inhibition of T-type channels, and 2) other
downstream effector(s) of Ras may be required for morphological
transformation in addition to inhibition of T-type Ca2+
channels. To test this, we treated normal Swiss 3T3 cells with 3 µM mibefradil under optimal growth conditions (in the
presence of 10% FBS) (Fig. 9). We found
that the mibefradil-treated cells retained a flat morphology similar to
untreated cells, suggesting that mere cell cycling does not allow for
morphological transformation even if the T-type channel is blocked
(Fig. 9, panel b). Because Ras targets multiple effectors,
in addition to the inhibition of the T-type channels by the MAPK
pathway, one or more of those downstream effectors of Ras other than
the pathways examined in this study appear to be required for
induction/maintenance of morphological transformation.

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Fig. 9.
Inhibition of T-type Ca2+
channels does not induce morphology change in exponentially growing
Swiss 3T3 cells. Swiss 3T3 cells growing in complete media were
treated with mibefradil (3 µM) for 48 h, then fixed
and Giemsa-stained. The treatment of Swiss 3T3 cells with 3 µM mibefradil resulted in almost complete inhibition of
T-type channels (Fig. 4). There was no morphological change in the
mibefradil-treated cells (panel b) in comparison with
untreated control cells (panel a), suggesting that the
inhibition of T-type channels alone does not induce changes of cell
morphology. Magnification, 400×.
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Suppression of T-type Ca2+ Channel Activity by
Activation of the MAPK Pathway Is Essential for Morphological
Transformation--
By use of MEK-specific inhibitor PD98059, we
directly examined whether the suppression of the T-type
Ca2+ channel by activation of the MAPK pathway is important
for maintenance of a transformed morphology. Swiss 3T3 cells stably
transfected with oncogenically activated Ras (12V Ras) were treated
with 75 µM PD98059 for 24 h and examined under a
light microscope. PD98059 treatment (thus inactivation of the MAPK
pathway) resulted in reversion of the transformed morphology to a flat
morphology similar to normal Swiss 3T3 cells (Fig.
10, panel b). Together with
the findings that PD98059 treatment recovers the T-type
Ca2+ channel activity (Fig. 6, column b), our
observations demonstrate that inhibition of T-type Ca2+
channels by activation of the MAPK pathway is critical for maintaining morphological transformation.

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Fig. 10.
Inhibition of the MEK-MAPK pathway by
PD98059 reverts cell morphology of ras
(12V)-transformed cells. Swiss 3T3 cells transformed with
ras (12V) were treated with the MEK inhibitor, PD98059 (75 µM) for 24 h. Cells were then fixed, Giemsa-stained,
and examined under a light microscope. Magnification, 400×.
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DISCUSSION |
Calcium plays an important role in a variety of fundamental
cellular events, including muscle contraction, hormone secretion, gene
expression, cell cycle control, and cell morphology (65). The levels of
intracellular Ca2+ are differentially regulated depending
on the cell type. In fibroblasts, L- and T-type Ca2+
channels are present. Fluctuations or oscillations of membrane potential is generally low enough to activate T-type but not L-type channels. Thus, the T-type channel likely plays a predominant role in
regulating intracellular concentration of Ca2+ in
fibroblasts (13). It has been shown that the T-type Ca2+
channel current, but not the L-type, are inhibited in cells transformed by various oncogenes including ras (12). In this study, we
show that activation of the MAPK pathway is responsible for the
inhibition of T-type channels in ras-transformed cells.
Because MAPK activation is a converging point of the transforming
functions of various oncogene products (reviewed in Refs. 59 and 66),
specific inhibition of T-type channels in cells transformed by various oncogenes, shown by Chen et al. (12), is likely mediated by the activation of the MAPK pathway.
One of the major transformation-associated phenotypes induced by
activation of the MAPK pathway is change of cell shapes (from flat to
round-up or spindle-shaped morphology). For example,
ras-transformed cells show a marked morphological reversion
from spindle-shaped to flattened upon exposure to the MEK-specific
inhibitor. Such morphological changes are believed to play a critical
role in malignant transformation. By use of the T-type channel blocker, mibefradil, we investigated the role of T-type Ca2+ channel
inhibition in cellular transformation. We found that loss of T-type
Ca2+ channel activity is essential for induction and/or
maintenance of morphological transformation. However, abrogation of
T-type Ca2+ channel activity alone does not appear to be
sufficient for inducing morphological changes, because treatment of
either serum-starved or actively proliferating nontransformed cells
with the T-type channel blocker fails to induce morphological
transformation. Thus, in addition to inhibition of the T-type channels,
activation of another downstream effector(s) of Ras, which is activated
by all the Ras effector domain mutants examined in this study, appears to be required for induction of morphological transformation.
The actin cytoskeleton is known to play a major role in regulation of
cell morphology. It has recently been shown that Rho and Rac proteins,
both of which are targeted by activated Ras, are involved in actin
organization. Rho is required for regulation of the assembly of actin
stress fibers and focal adhesions (67), and Rac1 is necessary for the
formation of membrane ruffles (68). Our present findings of the
involvement of the MAPK pathway in blocking T-type Ca2+
channel activity, which results in the induction of a spindle-shape morphology, demonstrates that morphological transformation by Ras is
achieved by an integration of events imposed by multiple downstream
pathways of Ras.
It remains to be elucidated how activation of the MAPK pathway inhibits
T-type Ca2+ channels. The several regulatory mechanisms of
L-type channel activity have been proposed. For example, direct
phosphorylation of the pore-forming subunit of the L-type channel by
protein kinase C, protein kinase A, as well as calmodulin kinase II
have been reported (69-71) and is predicted to modulate the activity
of L-type channels (72, 73). Accessory subunits also regulate the
activity of L-type channels via an allosteric mechanism as well as by
chaperoning the pore-forming subunit (Ref. 74, also reviewed in Ref.
75). However, little is known about regulatory mechanisms of T-type channels, and in fact, cDNA of the T-type channel has just recently been cloned (56, 76). There are several potential mechanisms that may
explain how activation of the MAPK pathway inhibits T-type channels.
MAPK is known to regulate the transcription of a variety of genes
through direct phosphorylation of major transcription factors,
including ELK-1, Sap1, bZIP, ATFa, ATF2, Net/Erp/Sap2, AP-1, c-Jun,
Fos, Fra1, Fra2 and c-Myc (for reviews, see Refs. 59 and 66). Thus,
MAPK may inhibit T-type Ca2+ channels in Swiss 3T3 cells by
modulating the expression of the T-type channel itself or accessory
proteins (at present, it is not known whether the T-type channel is a
multisubunit complex or a single subunit). Alternatively, MAPK may
directly inactivate T-type channels through phosphorylation. Indeed,
there are two MAPK phosphorylation consensus sites in the C-terminal
region of the T-type channel. These questions are currently under
investigation in our laboratory. Because alteration of cell morphology
is a critical event for cells to acquire a metastatic phenotype, the protocols that are designed to re-establish the proper T-type Ca2+ channel activity may prove to be effective in cancer chemotherapy.