From the
Thapsigargin (TG), a specific inhibitor of intracellular
Ca
The Sarco-Endoplasmic Reticulum Ca
Determination of
SERCA protein in microsomes was obtained via SDS-polyacrylamide gel
electrophoresis gels (7.5% polyacrylamide), run as per the method of
Laemmli
(14) and blotted onto nitrocellulose paper. The filters
were blocked with 3% gelatin in Tris-buffered saline solution for 1 h
at room temperature, and then probed with either the SERCA1
CaF3-5C3 antibody or a mouse SERCA2 antibody that recognizes both
isoforms of SERCA2 (IID8 F6)
(15) . Bound antibody was detected
with a secondary antibody (goat anti-mouse IgG conjugated to
horseradish peroxidase) and a commercially available ECL kit (Amersham
Corp.). Densitometry was carried out using an LKB Ultroscan XL enhanced
laser densitometer.
Owing to uncertainties in the estimation of the affinity of fura-2
for Ca
It is
noteworthy that the uptake studies in permeabilized cells were carried
out in the presence of FCCP, which inhibits uptake of Ca
Western blot analysis reveals that
SERCA1 expression gradually decreases upon removal of TG, so that 12
days after TG removal the overall expression of SERCA1 is reduced to
one-third of that seen when cells are continuously maintained on TG
(Fig. 6, ). Expression of the endogenous SERCA2 also
decreases in DC-3F/CaTG as these cells are kept off TG (Fig. 6).
It has been clearly
demonstrated that, due to its extremely high affinity, the amount of TG
required to inhibit isolated wild-type ATPase is stoichiometrically
equivalent to the target protein, even when the ATPase is present at
subnanomolar concentrations
(2) . For this reason, the higher TG
concentration required to obtain inhibition of microsomes obtained from
the DC-3F/CaTG cells (Fig. 5B) may be related to the
higher ATPase content of these microsomes, in addition to the presence
of TG-resistant ATPase within these cells. On the other hand, the
disproportionately high concentrations of TG (Fig. 5B)
required to inhibit the very low amount of ATPase (Figs. 5A and 6) recovered from the (nontransfected) DC-3F/TG cells must be
attributed to a distinct, TG-resistant SERCA ATPase, which may
correspond to an endogenous isoform selected during the induction of TG
resistance. Therefore, the TG-resistant cells developed in our
experiments contain two ATPase populations: a population which is
inactivated by stoichiometric titration with TG (prevalent in the
transfected DC-3F/CaTG cells) and a population which is resistant to TG
and derives in DC-3F/CaTG from exogenous and/or endogenous ATPase, and
in DC-3F/TG from an endogenous isoform.
The overexpression of the TG
sensitive SERCA, in addition to the TG-resistant SERCA, indicates that
the experimental protocol used here favors the selection of cells with
high expression of SERCA ATPases. Resistance to TG then develops in
parallel with (a) selection of cells expressing high level of
SERCA ATPases and (b) selection of a TG-resistant SERCA ATPase
isoform. It is apparent that overexpression of SERCA1 and SERCA2
ATPases does not completely account for the TG resistance acquired by
the DC-3F/CaTG and DC-3F/TG cells. Ultimately, selection of a
TG-resistant SERCA ATPase is required to provide sufficient function to
sustain cytosolic Ca
We thank Leonardo Vieira for participation as a summer
student.
transport ATPases (SERCA), inhibits cell
proliferation when added to culture media in the nanomolar
concentration range. However, long term exposure to gradually
increasing concentrations of TG induces resistance to TG inhibition in
both the parental Chinese hamster lung fibroblast DC-3F and a subline
derived from it via transfection and stable expression of a full-length
cDNA encoding avian SERCA1 ATPase (DC-3F/Ca cells). TG resistance
develops in parallel with selection of cells expressing higher levels
of the endogenous SERCA2 as well as of the exogenous transfected SERCA1
ATPase, whose Ca
transport function can be studied
in situ by imaging techniques and following isolation in
microsomal fractions. Microsomes isolated from resistant cells contain
two functionally distinct populations of ATPases: a population that is
inhibited by stoichiometric titration with TG, and a population
displaying resistance to inhibition even when TG exceeds the enzyme
stoichiometry. It is apparent that resistance to TG develops in
parallel with (a) selection of cells expressing high levels of
SERCA ATPases, and (b) selection of an ATPase that is
resistant to TG.
(SERCA)
(
)
ATPases are key enzymes in the
regulation of cytosolic Ca
through the establishment
and maintenance of intracellular Ca
stores. Specific
inhibitors of the SERCA pumps have been identified, of which the
sesquiterpene lactone thapsigargin (TG) is the most potent and specific
(1-5). Exposure of cell cultures to TG results in several
effects, including alterations in intracellular Ca
homeostasis and inhibition of cell
proliferation
(6, 7) . However, resistance to TG
inhibition of cell proliferation can be developed
(8) and is
associated with TG-resistant ATP-dependent calcium transport into
intracellular stores, as shown in situ(9) . To
facilitate characterization of the TG-resistant enzyme, we have used a
cell line transfected and selected for stable expression of exogenous
SERCA1 ATPase (DC-3F/Ca cells), in addition to a parent line (DC-3F
cells) sustaining only expression of endogenous ATPase. We describe
here the functional properties of the SERCA ATPases affected by the
development of resistance to TG, both in situ and following
isolation.
Cell Lines
DC-3F is a cell line that was
originally established from Chinese hamster lung (CHL)
fibroblasts
(10) . As described previously, DC-3F/Ca was derived
from DC-3F by transfection of the dual promoter expression vector
pHFCaA3 containing a mutant methotrexate (MTX)-resistant
dihydrofolate reductase (DHFR) cDNA (under the control of a SV4O
promoter) and an avian SERCA1-encoding cDNA (under the control of a
-actin promoter), followed by selection in MTX
(11) .
TG-resistant cell lines (DC-3F/TG and DC-3F/CaTG) were developed via
stepwise selection of DC-3F or DC-3F/Ca cells in gradually increasing
concentrations of TG (LC Laboratories) over the course of several
months, with the final maintenance concentration being 2 µM TG for each line
(8) . All these cell lines were maintained
in
-minimum essential medium media (Sigma) supplemented with 7%
fetal calf serum (Life Technologies, Inc.) plus appropriate
concentrations of drug where indicated. Thus, the cells were maintained
in the following: DC-3F/Ca in 25 µg/ml MTX, DC-3F/TG in 2
µM TG, DC-3F/CaTG in 25 µg/ml MTX plus 2 µM TG. Parental DC-3F cells were cultured in the absence of any drug.
In some of the experiments with DC-3F/CaTG cells where TG was removed
from the culture media for various lengths of time, the cells were
still kept on 25 µg/ml MTX.
Cell Growth Assays
Cells were plated at
10,000-15,000 cells/plate in 35-mm plates and incubated overnight
at 37 °C. TG was added the next day, and the cells were incubated
for an additional 72 h at 37 °C, then trypsinized and counted.
Microsome Preparation
The procedure was described
by Sumbilla et al.(12) .
Immunodetection Studies
Immunofluorescent
detection of transfected SERCA1 in the cells was carried out as
described by Karin et al.(13) .
High Resolution Ca
The
procedures for subcellular imaging of Ca Imaging
stores in
permeabilized cells were as described by Short et
al.(16) , who demonstrated that under appropriate
permeabilization conditions, fura-2 remains trapped within
intracellular organelles where it reports the local
[Ca
]. Ca
uptake into
organelles was initiated by adding ICM plus 3 mM ATP and 1 or
10 µM free Ca
. TG was added to the
appropriate solution as indicated, from a 1000-fold concentrated stock
solution dissolved in Me
SO. Changes produced by
Ca
binding to fura-2 within the organelles were
determined by fluorescence microscopy, using dual excitation
fluorescence at 380 and 358 nm, nearest neighbor deblurring of images
at 2-µm focal planes for each wavelength and conversion to the
pseudo-color images using image processing procedures described by
Short et al.(16) .
Ca
Ca Transport
Studies
transport was assayed by incubating
5 µg/ml DC-3F/Ca or DC-3F/CaTG microsomes, or 10 µg/ml DC-3F/TG
microsomes, in a reaction mixture containing 20 mM MOPS, pH
7.0, 80 mM KCl, 5 mM MgCl
, 0.2 mM CaCl
supplemented with
Ca
(specific activity of 3000 dpm/nmol), 5 mM K
oxalate, and 0.2 mM EGTA at 37 °C. For the
Ca
affinity experiments, free Ca
concentrations were calculated from total Ca
and EGTA according to Fabiato and Fabiato
(17) . The
reaction was initiated by the addition of 5 mM ATP. At
sequential times (up to 60-90 min), 1-ml samples were filtered
through a 0.45-µm Millipore filter and quenched with 20 ml of
chilled lanthanum chloride/MOPS, pH 7.0, solution. Ca
uptake was determined by quantitating the radioactivity on the
filters via liquid scintillation counting and was expressed per mg of
microsomal protein.
RESULTS
Characterization of the TG-resistant Cell Lines
Fig. 1
demonstrates that the DC-3F/TG and the DC-3F/CaTG cell
lines, developed by exposure to increasing concentrations of TG, are
highly resistant to inhibition by TG. That is, DC-3F/TG and the
DC-3F/CaTG cells are nearly 3 orders of magnitude more resistant to TG
than parental DC-3F and DC-3F/Ca cells.
Figure 1:
Relative resistance of cells to TG.
Cell viability (expressed as percent of control growth) after treatment
with various concentrations of TG was determined via cell growth assays
as detailed under ``Experimental Procedures.'' , DC-3F;
, DC-3F/Ca;
, DC-3F/CaTG; ▾,
DC-3F/TG.
It can be also shown by
immunofluorescence staining that resistance to TG develops in parallel
with selection of cells expressing high levels of SERCA ATPase. In
fact, it is shown in Fig. 2(compare A and C)
that in the DC-3F/Ca population used in these studies, more than 50% of
the cells have lost expression of the transfected SERCA1 when compared
to the original DC-3F/Ca cell line previously described
(11) . On
the other hand, it is clear that 100% of the cells express SERCA1
ATPase following selection of DC-3F/Ca in TG (compare B and
D). Furthermore, the overall expression of SERCA1 within each
cell in the TG-resistant population appears to be increased, as judged
by fluorescence intensity. Western blotting of microsomal fractions
reveals that SERCA expression is greatly increased following the
acquisition of resistance to TG, with expression of the exogenous
SERCA1 being 20-fold higher and that of the endogenous SERCA2 being
2-fold higher in DC-3F/CaTG when compared to DC-3F/Ca cells
(Fig. 3, ).
Figure 2:
Immunofluorescent visualization of SERCA1
in DC-3F/Ca (panels A and C) and DC-3F/CaTG
(i.e. TG-resistant) cells (B and D).
PanelsA and B show phase contrast images of
a field of cells. PanelsC and D show the
corresponding immunofluorescence images. Cells were fixed and stained
with a primary antibody to SERCA1, then a secondary FITC-conjugated
goat anti-mouse IgG, as described under ``Experimental
Procedures.''
Figure 3:
Western blotting. Microsomal fractions
were obtained from DC-3F/Ca and DC-3F/CaTG and analyzed with SERCA
isoform-specific antibodies. Top row, blot probed with an
antibody (CaF3-5C3) directed against the exogenously transfected
and expressed avian SERCA1. Lane 1, DC-3F/Ca (5 µg of
microsomal protein); lanes 2-4, DC-3F/CaTG (0.5, 1.0,
and 5.0 µg of microsomal protein, respectively). Bottom
row, a separate blot was probed with an antibody (IID8 F6) that
recognizes the endogenous SERCA2 (IID8 F6 cannot distinguish between
SERCA2a and SERCA2b). Lane 1, DC-3F/Ca (5 µg); lane2, DC-3F/CaTG (5 µg). Relative SERCA1 content is
20-fold greater and SERCA2 content 2-fold greater in DC-3F/CaTG than in
DC-3F/Ca.
Evaluation of Ca
Direct in situ imaging of Ca Storing Capacity in
Situ
within the organelles of DC-3F/Ca and DC-3F/CaTG cells was
undertaken following the loading of organelles with fura-2 and
permeabilization of the plasma membrane with saponin. In these studies,
the ratio of fura-2 fluorescence at excitation wavelengths of 380 and
358 nm provided a relative estimate of the Ca
concentration within the organelles, represented by pseudo-color
images of the perme-abilized cells in Fig. 4. Fig. 4B (compared to Fig. 4A) shows that in permeabilized
DC-3F/Ca cells, exposure to ICM plus 1 µM free
Ca
and 3 mM ATP resulted in uptake of
Ca
into the organelles. Ca
uptake
was dependent on the presence of both Ca
and ATP (not
shown) and was completely blocked by 2 µM TG
(Fig. 4, C and D). No additional uptake was
observed in the presence of ICM containing ATP plus 10 µM Ca
(not shown).
Figure 4:
Pseudo-color Ca images of permeabilized DC-3F cells. Within each pair of images,
leftpanel shows the organellar Ca
image just before, and rightpanel after
exposure of the cells to ICM containing 1 µM Ca
and 3 mM ATP. Each pair of images
shows a different cell. The Ca
calibration is given
as the calculated [Ca
] normalized to the
K of the indicator (see text). The 10-µm bar reflects
relative cell size. A and B, DC-3F/Ca cells. C and D, DC-3F/Ca cells in the presence
of 2 µM TG. E and F,
DC-3F/CaTG cells. G and H, DC-3F/CaTG
cells in the presence of 4 µM TG. I and J, parental DC-3F cells. Note that DC-3F/Ca cells take
up Ca
to levels about 2-fold higher than DC-3F/CaTG
cells; the parental line takes up relatively little
Ca
.
Fig. 4
(E and
F) shows that the organelles of DC-3F/CaTG cells are able to
sustain ATP-dependent Ca uptake, which is not
inhibited by 4 µM TG (Fig. 4, G and
H). Ca
uptake within the DC-3F/CaTG cells,
however, was lower than in DC-3F/Ca cells by about one-half, even
though the immunofluorescence results in Fig. 2suggest that
DC-3F/CaTG cells have significantly higher SERCA1 expression than the
DC-3F/Ca cells. The reduced Ca
uptake in the
DC-3F/CaTG cells may in part be a consequence of partial inhibition of
the SERCA ATPases by the TG originally present in the culture medium.
It should be pointed out that Ca
uptake within the
DC-3F/CaTG cells is still much higher than that obtained with the
parental line DC-3F (Fig. 4, compare H with J).
within the organelles, we have expressed the
calibration of fluorescence ratio as [Ca
]
normalized to the K
of the indicator. In
spite of this approximation, the images shown in Fig. 4, which
are examples derived from observations made in the course of 11
independent experiments, give a clear indication of the occurrence of
TG sensitive and TG-resistant Ca
uptake by ER stores
in situ. A more quantitative evaluation was obtained by
measuring directly the ATP-dependent Ca
uptake by
microsomal vesicles isolated from the cells (see below).
by mitochondria. Therefore, the contribution of mitochondrial
Ca
in our imaging studies was negligible.
Ca
These studies were best performed with microsomes
isolated from DC-3F/Ca cells due to the relatively high expression of
SERCA1 and, hence, the relatively high rates of Ca Transport in Isolated
Microsomes
transport in such cells. The activities of microsomes prepared
from DC-3F/Ca and DC-3F/CaTG (TG-resistant) cells are shown in
Fig. 5A. The average initial rates of Ca
transport are 21.2 ± 2.3 nmol of Ca
uptake/mg of protein/min for DC-3F/Ca microsomes and 14.2
± 2.6 nmol of Ca
uptake/mg of protein/min for
the DC-3F/CaTG microsomes (). Therefore, the microsomal
membranes of the TG-resistant cells retain approximately half of their
overall capacity for Ca
transport as compared with
controls (i.e. DC-3F/Ca cells), which have not been exposed to
TG. Since the SERCA1 content is approximately 20-fold greater and
SERCA2 content is 2-fold greater in the microsomes derived from the
TG-resistant cells ( Fig. 3and ), the percentage of
functional SERCA ATPase in the DC-3F/CaTG cells is apparently less than
5%, due to the inhibition produced by the TG present in the culture
media until cell harvest. Nevertheless, this residual activity is much
higher than that recovered from (nontransfected) DC-3F or DC-3F/TG
cells (Fig. 5A).
Figure 5:
Ca transport activity of
microsomes. A, microsomal vesicles were obtained from DC-3F/Ca
(
), DC-3F/CaTG (
), DC-3F (
), and DC-3F/TG cells
(▾). The DC-3F/CaTG and DC-3F/TG cells were maintained on TG
until the day of microsomal harvest, but the reaction medium for
Ca
uptake contained no TG. B, Ca
uptake rates were determined in the presence of various
concentrations of TG added to the reaction medium for Ca
uptake. Rates of Ca
transport (expressed as
fractional values of the maximal rate obtained in the absence of TG)
are plotted as a function of TG concentration for microsomes obtained
from DC-3F/Ca (
), DC-3F/CaTG (
), and DC-3F/TG cells
(▾).
When Ca uptake by
isolated microsomes is measured as a function of the concentration of
TG added to the reaction mixture for Ca
uptake, 50%
inhibition of the maximal rates of uptake requires 100 nM TG
for the microsomes obtained from DC-3F/CaTG cells, as compared to 0.1
nM TG for the microsomes obtained from DC-3F/Ca cells
(Fig. 5B). Most importantly, the low
(Fig. 5A) activity recovered from (nontransfected)
DC-3F/TG cells requires TG concentrations in the micromolar range for inhibition (Fig. 5B). In fact, this low
level of highly resistant ATPase may be present even in the DC-3F/CaTG
microsomes as a small percentage of the total ATPase, whose inhibition
spans a wide range of TG concentrations. Note that in Fig. 5B the activities are normalized; in fact, in DC-3F/TG it is only a
small fraction of the DC-3F/CaTG activity (Fig. 5A).
Removal of TG from Culture Media following Acquisition of
Resistance by DC-3F/CaTG Cells
Since stepwise selection in TG
results in the overexpression of TG-resistant SERCA pumps in the
DC-3F/CaTG cell line, we explored the consequences of removing TG
selection on the functional properties of SERCA in these resistant
lines. Thus, in some experiments, TG was removed from the culture media
and the microsomes subsequently harvested for analysis after 2, 6, or
12 days following the TG removal.
Figure 6:
Analysis of DC-3F/CaTG cells taken off TG.
Western blotting of microsomal fractions obtained from DC-3F/CaTG at
various times following removal of TG from the culture medium. SERCA1:
lane 1, DC-3F (5 µg); lane 2, DC-3F/Ca (10
µg); lane 3, microsomes from DC-3F/CaTG cells maintained
on TG until day of harvest (2 µg); lane 4, microsomes from
DC-3F/CaTG harvested 2 days following removal of TG from culture medium
(2 µg); lane 5, 6 days following TG removal (2 µg);
lane 6, 12 days following TG removal (2 µg). SERCA2:
lane 1, DC-3F (40 µg); lane2, DC-3F/TG
(40 µg); lane3, DC-3F/Ca (40 µg); lane
4, microsomes from DC-3F/CaTG cells maintained on TG until day of
harvest (40 µg); lane5, microsomes from
DC-3F/CaTG cells harvested 2 days following removal of TG from the
culture medium (40 µg); lane6, 6 days (40
µg); lane7, 12 days (40
µg).
When the microsomes obtained serially from DC-3F/CaTG cells
following withdrawal of TG were tested for their Ca transport function, we found a 4-fold increase in the overall
Ca
transport activity after only 2 days following TG
withdrawal, as compared to DC-3F/CaTG cells that had been continuously
maintained on TG (). This overall increase in activity
tapered off in several days following withdrawal of TG ().
Considering the overall expression of SERCA protein, and assuming no
changes in ATPase turnover, our experiments indicate that the
percentage of functional enzyme was reduced to 5% as a consequence of
continuous exposure to TG, and then increased gradually by the 12th day
following removal of TG. Furthermore, SERCA-dependent Ca
transport became progressively more sensitive to TG inhibition as
microsomes were harvested following withdrawal of TG from the cell
culture media (, Fig. 7). It is noteworthy that even
though a minimal change in overall SERCA1 and SERCA2 expression
occurred within the first 2 days following withdrawal of TG
(Fig. 6), the ED
value for TG inhibition of
Ca
transport by isolated microsomes decreased by more
than 16-fold (Fig. 7, ). This suggests that a small
fraction of the SERCA pump is TG-insensitive and operative primarily in
the TG-resistant cells, while operation of a TG-sensitive enzyme
becomes more prominent following withdrawal of TG.
Figure 7:
Rates of Ca transport
(expressed as fractional values of maximal rate of transport obtained
in the absence of TG) are plotted as a function of TG concentration.
, microsomes from DC-3F/CaTG cells maintained on TG until day of
harvest;
, microsomes from DC-3F/CaTG harvested 2 days
following removal of TG from the culture medium; +, 6 days;
, 12 days;
, DC-3F/Ca, i.e. no exposure to TG in
the culture medium. Experimental points are the average of three
measurements for two different microsomal
preparations.
Ca
As the SERCA enzymes require
Ca Transport as a Function of
Ca
Concentration
for activation of their function, characterization
of these ATPases in the TG-resistant line DC-3F/CaTG was extended to
determine the Ca
concentration dependence of their
activation. Fig. 8compares the rates of Ca
transport as a function of Ca
concentration for
microsomes obtained from DC-3F/CaTG and from the same cell line
harvested 12 days after withdrawal of TG. These experiments show hardly
any difference in the Ca
concentration range required
for activation of microsomes obtained from cells exposed to TG until
harvest or cells harvested 12 days following removal of TG. Therefore,
it is apparent that TG resistance does not impair the ability of the
SERCA pumps to function within the Ca
concentration
range required for homeostasis.
Figure 8:
Ca dependence of
Ca
transport by various microsomes. Microsomes
obtained from DC-3F/CaTG maintained on TG until day of harvest (
)
or harvested 12 days following removal of TG from the culture medium
(
). The experimental points are averages from three experiments.
In each experiment the velocity was obtained from six time points taken
over a 40-min incubation.
DISCUSSION
Interference with various cell functions and signaling
pathways as a consequence of specific inhibition of SERCA ATPases by TG
has been reported in several studies (for recent review, see Ref. 18).
An important effect of TG is the inhibition of cell proliferation in
parallel with depletion of intracellular Ca stores
(6) . It is of interest that DC-3F cells develop
resistance to TG and continue to proliferate following gradual exposure
to increasing concentrations of the inhibitor
(8) . Although a
2-3-fold overexpression of the multidrug resistance transporter
p-glycoprotein occurs in association with the development of resistance
to TG, this modest overexpression of the drug transporter does not
account for the very large (more than 2 orders of magnitude)
increase in resistance of DC-3F cells to TG
(8) . Therefore, with
the experiments described in this report we attempted to clarify
whether SERCA ATPases are directly involved in the mechanism of TG
resistance. A most important finding is that microsomes obtained from
cells exposed for a long time to inhibitory concentrations of TG retain
residual Ca
transport activity. This demonstrates
that, under our experimental conditions, resistance to TG is due to
factors related directly to the ATPase.
housekeeping and proliferation
of TG-resistant cells. The TG-resistant ATPase could derive from a
``latent'' inherently resistant endogenous SERCA isoform
which may be present in at least a few of the DC-3F cells. The latter
suggestion is consistent with the known presence of TG-resistant SERCA
ATPase isoforms in platelets
(19) and in association with the
inositol 1,4,5-trisphophate-insensitive Ca
stores
(20) .
Table: Characteristics of DC-3F/CaTG microsomes
isolated from cells harvested after removal of TG from the culture
media
; TG, thapsigargin; CHL, Chinese hamster
lung; MTX, methotrexate; MOPS, 4-morpholinepropanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.