From the
CD20 is a transmembrane protein that functions as a
Ca
Mammalian nontransformed fibroblasts, such as Balb/c 3T3 cells,
become quiescent when they are cultured in serum-free medium for a
sufficient time. Multiple growth factors are required for such
quiescent cells to reenter the cell cycle and to initiate DNA
replication
(1) . Although there is a great deal of information
available about the molecular events regulating G
G
CD20 is a 35-kDa integral membrane
protein expressed in B lymphocytes (13). It is unique in that
monoclonal antibodies (mAbs) against CD20 modulate proliferation and
differentiation of B lymphocytes; some antibodies inhibited DNA
synthesis
(14) , whereas others stimulated it (15). These
observations raise the possibility that CD20 is involved in the
regulation of B lymphocyte growth. The cDNA encoding CD20 was cloned,
and the primary structure of CD20, which contains four predicted
membrane-spanning domains with both C and N termini located in the
cytoplasm
(16) , suggests that it may function as an ion channel.
Indeed, when CD20 molecules were expressed in non-lymphoid cells, they
generated transmembrane Ca
For immunostaining, cells
were grown on coverslips, fixed for 10 min in 10% formalin/PBS, washed
with PBS, blocked with PBS containing 3% BSA, and incubated with
anti-CD20 overnight at 4 °C. After washing with PBS, the coverslips
were incubated with horseradish peroxidase-conjugated rabbit anti-mouse
IgG antibody (Amersham Japan) at room temperature for 1 h, and, after a
final wash with PBS, immunostaining was carried out using
diaminobenzidine.
Electrophoresis in the presence of 0.1% (w/v) SDS was
carried out using 10% (w/v) polyacrylamide slab gels as described by
Laemmli
(22) , after which gel was fixed using 30% (v/v)
methanol, 10% (v/v) acetic acid, and 10% (w/v) trichloroacetic acid for
20 min at room temperature. The gels were autoradiographed using XAR-5
X-Omat film (Eastman Kodak Co.) and an intensifying screen. The
relative molecular weights of the proteins were determined using
rainbow-colored protein weight markers (Amersham Japan).
In order to
determine the time course of entry into the S phase, the time course of
nuclear labeling with BrdUrd was measured. When control cells rendered
primed competent were incubated with 1 nM IGF-I, they
traversed the cell cycle, and nuclear labeling took place after 12 h.
As shown in Fig. 5A, nuclear labeling of control cells
was first detected at 14 h and increased exponentially thereafter.
These results are identical to those observed with untransfected cells
(12) and cells transfected with vector alone (data not shown).
In contrast, CD20-expressing cells began to enter the S phase after
about 8 h. Thus, nuclear labeling was detected at 10 h and increased
exponentially thereafter. At time points later than 10 h, the numbers
of labeled nuclei were significantly greater than those in control
cells (p < 0.05). The slopes of the two lines were
identical.
We have shown that IGF-I stimulates
[
Insulin-like growth factor-I exerts its
progression activity by acting on the Ca
Transfection of Balb/c 3T3 cells with pMEP4-CD20 followed by
treatment with ZnCl
When the mRNA for CD20 was induced by
the addition of Zn
Cells expressing CD20 protein have some interesting
properties in terms of growth factor responsiveness. First,
approximately 30% of the CD20-expressing cells traversed the cell cycle
toward the S phase in response to IGF-I alone. In sharp contrast, DNA
synthesis by control cells was never initiated in response to IGF-I
unless they had been pretreated with PDGF and EGF
(Fig. 5B). These results suggest that at least some of
CD20-expressing cells were competent in terms of responsiveness to
IGF-I. Therefore, the effects of PDGF and EGF were reproduced partially
by CD20 expression. Pledger et al.(24) showed that the
competence-inducing activity of PDGF required RNA synthesis and that
PDGF rendered quiescent cells competent by stimulating transcription of
a certain gene(s). Such effects of PDGF can be mimicked partly by CD20
expression. It is an interesting possibility that PDGF and/or EGF
induce the production of a protein that functionally resembles CD20,
possibly the IGF-sensitive cation channel
(25) , by quiescent
Balb/c 3T3 cells.
Second, DNA synthesis by CD20-expressing cells was
initiated in response to IGF-I 4 h earlier than that by control cells
(Fig. 5, A and B). Therefore, G
As discussed above, IGF-I activates a
Ca
Recent studies have shown that CD20 is
phosphorylated in B lymphocytes and that the phosphorylation state
correlates with the rate of cell proliferation
(23) . When CD20
is cross-linked with mAbs, a certain protein-tyrosine kinase associates
with CD20
(29) . It is possible that a related protein kinase(s)
also associates with CD20 expressed in Balb/c 3T3 cells. Although we
incubated CD20-expressing cells without an antibody against CD20,
assuming that the association of such a kinase would be negligible, we
cannot rule out the possibility that such a kinase is at least partly
responsible for the properties of CD20-expressing cells. Nevertheless,
CD20-mediated alterations are dependent on extracellular Ca
In conclusion, expression of
the CD20 Ca
CD20-transfected cells were incubated with ZnCl
We thank Dr. Thomas Tedder of the Dana-Farber Cancer
Institute for providing us with CD20 cDNA and are grateful to Romi
Nobusawa and Kiyomi Ohgi for secretarial assistance and Dr. Norio
Kawamura for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-permeable cation channel (Bubien, J. K., Zhou, L.
J., Bell, P. D., Frizzel, R. A., and Tedder, T. F.(1993) J. Cell
Biol. 121, 1121-1132) and is involved in growth regulation
of B lymphocytes. In order to further investigate the role of calcium
entry in cell cycle progression, we introduced the cDNA encoding a
Ca
-permeable cation channel, CD20, into Balb/c 3T3
cells. Balb/c 3T3 cells transfected with a vector containing cDNA
encoding CD20 expressed the CD20 protein, which was detected by
assaying the binding of a monoclonal antibody against CD20.
Calcium-permeable cation channel activity was detected in
CD20-expressing cells by whole cell patch clamp recording and
microfluorometric determination of the cytoplasmic Ca
concentration using fura-2. The expression of CD20 induced
significant alterations in the responses of the cells to insulin-like
growth factor-I (IGF-I). IGF-I induced DNA synthesis by control cells
only when they had been pretreated with both platelet-derived growth
factor (PDGF) and epidermal growth factor (EGF). In contrast, DNA
synthesis by 30% of the quiescent CD20-expressing cells was initiated
in response to IGF-I in the absence of priming with PDGF and EGF. When
control quiescent cells were primed with PDGF and EGF, the addition of
IGF-I led to the initiation of DNA synthesis after 14 h or more,
whereas it induced DNA synthesis by CD20-expressing cells primed with
PDGF and EGF 4 h earlier. The IGF-induced DNA synthesis was dependent
on extracellular Ca
, and expression of CD20 reduced
the concentration of extracellular Ca
required for
it. Furthermore, DNA synthesis by approximately 25% of the
CD20-expressing cells was initiated after priming with PDGF and EGF,
even in the absence of the progression factor IGF-I. These results
indicate that CD20 expressed in Balb/c 3T3 cells functions as a
constitutively active Ca
-permeable cation channel and
that expression of CD20 accelerates G
progression in a
Ca
-dependent manner.
-G
transition (see Ref. 2 for review) and transition of
G
-S boundaries (see Ref. 3 for review), the molecular
events controlling mid-G
progression remain relatively
uncharacterized.
progression is the rate-limiting step
in the entire cell cycle
(1) . Pledger and colleagues
(4) showed that platelet-derived growth factor
(PDGF)
(
)
renders quiescent Balb/c 3T3 cells
``competent'' in terms of responding to growth factors in
plasma. Competent cells enter the cell cycle and progress to the S
phase in the presence of progression factors in plasma
(5) . The
active components in plasma are epidermal growth factor (EGF) and
insulin-like growth factor-I (IGF-I)
(6) , and it is the latter
that promotes progression through the G
phase
(6, 7) . We extended these data by showing
that EGF renders competent cells responsive to IGF-I
(8) and
that such EGF-primed competent cells, designated ``primed
competent cells,'' progress toward the S phase when incubated with
IGF-I alone. It should be emphasized that neither quiescent nor
PDGF-treated competent cells are able to initiate DNA synthesis in the
presence of IGF-I
alone
(4, 6, 7, 8, 9) . In this
respect, primed competent cells are responsive to IGF-I in terms of
cell growth. It is well known that Ca
is required for
cell proliferation
(10) and is indispensable for cells to
progress to the S phase. Our previous results indicated that IGF-I
activated a Ca
-permeable cation channel in primed
competent cells (8). Blockade of the IGF-sensitive channel attenuated
IGF-induced DNA synthesis and stimulation of Ca
entry
into primed competent cells by adding BAYK8644 increased DNA synthesis
to some extent
(11) . Moreover, reduction of Ca
entry terminated G
progression induced by
IGF-I
(12) . On the basis of these observations, we postulated
that continuous stimulation of Ca
entry may be a
critical intracellular message of the progression activity of
IGF-I
(8, 12) .
conductance
(17) .
Therefore, CD20 appears to function as a Ca
-permeable
cation channel, which may be involved in growth regulation. In this
respect, CD20 and the IGF-sensitive channel have some properties in
common
(8, 17) . Both are Ca
-permeable
channels and activate processes that are independent of the membrane
potential and, furthermore, may be involved in cell growth. It is
therefore of great interest to establish whether expression of CD20 in
fibroblasts alters the cell cycle progression induced by IGF-I. In the
present study, we cultured CD20-expressing Balb/c 3T3 cells and
assessed their G
progression in response to IGF-I.
Materials
Recombinant human IGF-I was supplied
by Fujisawa Pharmaceutical Co. (Osaka, Japan); recombinant PDGF-BB was
purchased from PeproTech (Rocky Hill, NJ). EGF was from Collaborative
Research (Lexington, MA), and the cell proliferation detection kit was
purchased from Amersham Japan (Tokyo, Japan).
Cell Culture
Balb/c 3T3 cells (clone A31) provided
by the RIKEN cell bank (Tsukuba, Japan) were cultured in
Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal calf serum (FCS; Life Technologies, Inc.) under humidified
conditions of 95% air and 5% CO at 37 °C. Quiescent
cells were obtained by incubating confluent cells in DMEM containing
0.5% platelet-poor plasma for 24 h, and IGF-responsive primed competent
cells were obtained by incubating quiescent cells sequentially with 1
nM PDGF and 10 nM EGF as described
previously
(8, 9) .
Amplification of CD20 cDNA from Raji Cell
Poly(A)
The CD20 cDNA was
amplified from Raji cell (B lymphoblastoid cell line)
poly(A)RNA
RNA using reverse transcription-polymerase
chain reaction. Briefly, poly(A)
RNA was isolated from
Raji cells using oligo(dT)-cellulose (Pharmacia Biotech Inc.), and the
RNA was reverse transcribed using Superscript Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Inc.). The synthesized
cDNA was used for subsequent polymerase chain reaction as follows: 94
°C for 1 min, 50 °C for 2 min, 74 °C for 2 min, 20 cycles.
The sense and antisense primers used were 5`-AGGAGTCTCGAGAGCAAAATG-3`
and 5`-CAGTCGACAGAAGAATCAC-3`, respectively. The polymerase chain
reaction product was purified by 1% agarose gel electrophoresis and
ligated into a TA Cloning pCRII vector (Invitrogen, San Diego, CA).
Transfection of CD20 cDNA into Balb/c 3T3
Cells
The human CD20 cDNA clone was subcloned into the
XhoI site of the Epstein-Barr virus vector pMEP4, which
possesses an inducible human metallothionein IIa gene enhancer/promoter
(Invitrogen). The CD20 expression vector (CD20-pMEP4) construct was
purified by cesium chloride gradient centrifugation, and Balb/c 3T3
cells were transfected with the CD20-pMEP4 vector by electroporation
using a Gene Pulsar (Bio-Rad). The cells were washed once with
Ca- and Mg
-free phosphate-buffered
saline (PBS), harvested with 0.05% trypsin, 0.02% EDTA, and then
resuspended in PBS at a concentration of 2
10
cells/ml. A 0.8-ml aliquot of this suspension with 100 µg of
CD20-pMEP4 was transferred to an electroporation cuvette (Bio-Rad) and
placed on ice for 5 min, after which electroporation at a setting of
300 V and 500 microfarads was carried out. The cells were allowed to
recover on ice for 10 min and then were divided into 100-mm culture
dishes containing culture medium. 12 h after electroporation, the
culture medium was removed, and the cells were cultured in fresh DMEM
containing 10% FCS. 48 h after electroporation, the cells were replaced
at a lower concentration, and transfected cells were selected using 100
µg/ml hygromycin B (Wako Pure Chemicals, Osaka, Japan). After
10-14 days, independent colonies were picked up, grown in 35-mm
dishes, and screened for a high level of expression of human CD20 by
determining the binding of a mAb against CD20 as described below.
Binding of
A
monospecific mAb against CD20 (CBL 456; Cymbus Bioscience Ltd.,
Southampton, UK) was used for Western blotting and immunostaining. For
the binding assay, the IgG was iodinated by the chloramine T
method
(18) . In brief, 5 ml of 250 mCi of NaI-Labeled
Monoclonal Antibody against CD20 and Immunostaining
I
dissolved in 0.2 M phosphate buffer (pH 7.4) and 5 ml of
chloramine T solution (2 mg/ml in 0.2 M phosphate buffer) were
added to the IgG (3 mg in 30 ml of 0.1 M phosphate buffer),
and the mixture was incubated at room temperature for 1 min. The
reaction was terminated by adding sodium metabisulfite solution, and
the labeled IgG was collected by chromatography on a 1
6-cm
Bio-Gel P-60 column equilibrated with PBS containing 1% bovine serum
albumin (BSA). The specific activity of the
I-labeled IgG
was approximately 80 mCi/mg. The confluent cells grown in 6-well plates
were washed twice with binding buffer (DMEM containing 0.2% BSA) and
incubated for 1 h in this buffer containing
I-labeled IgG
(2
10
cpm/0.4 ml) at 32 °C. Then, the cells
were washed three times with the binding buffer and lysed by adding 0.5
ml of 0.5 N NaOH, and the radioactivity associated with the
cell lysate was counted. Nonspecific binding was determined in the
presence of excess unlabeled antibody.
Northern Blotting Analysis
The cells were
harvested, and the total RNA was isolated using Isogene and quantitated
spectrophotometrically. 20 µg of total RNA was electrophoresed on a
1.2% agarose gel containing 2.2 M formaldehyde, 20 mM
MOPS (pH 7.0), 8 mM sodium acetate, and 1 mM EDTA and
transferred to a nylon membrane (Hybond-N+; Amersham Japan) using
a capillary blotting technique with 10 sodium citrate buffer.
Hybridization was performed with a probe of the CD20 cDNA fragment,
provided by Dr. T. Tedder of the Dana-Farber Cancer Institute (Boston,
MA) labeled with fluorescein-CTP by the random priming procedure
according to the manufacturer's instructions (Amersham Japan).
Positive signals were detected using the alkaline
phosphatase-conjugated anti-fluorescein antibody and Limiphos AP
detection reagent (Amersham Japan).
Measurement of DNA Synthesis
Synthesis of DNA was
assessed in two ways. For the measurement of
[H]thymidine incorporation into trichloroacetic
acid-precipitable material, cells were cultured in 24-well plates and
rendered primed competent. The primed competent cells were incubated
for 24 h in DMEM containing IGF-I and 0.5 µCi/ml
[
H]thymidine, and
[
H]thymidine incorporation was measured as
described previously
(8) . In order to measure the labeling
index, bromodeoxyuridine (BrdUrd; Amersham Japan) was added instead of
[
H]thymidine and was incubated for the times
indicated in Fig. 5. The cells were fixed with PBS containing 10%
formalin, and immunocytochemical detection of the BrdUrd incorporated
into the nuclei was performed using a detection kit (Amersham Japan)
according to the manufacturer's instructions. Statistical
analysis was done by Student's t test.
Figure 5:
Time
course of nuclear labeling in response to IGF-I in primed competent
cells and in quiescent cells. A, quiescent CD20-transfected
cells treated with () or without (
) ZnCl
were
rendered primed competent. Cells were then incubated for the indicated
periods with 1 nM IGF-I, and nuclear BrdUrd labeling was
measured. Values are the means ± S.E. for four experiments.
B, quiescent CD20-transfected cells treated with (
) or
without (
)) ZnCl
were incubated for the indicated
times with 1 nM IGF-I. Nuclear BrdUrd labeling was then
measured. Values are the means ± S.E. for four
experiments.
Measurement of Cytosolic Free Calcium
Concentration
The cytosolic free Ca concentration was monitored using fura-2, as described
previously
(19) . Briefly, cells cultured on coverglasses were
incubated with 2 µM fura-2 acetoxymethyl ester (Dojin
Laboratories, Kumamoto, Japan) for 20 min at room temperature
(20-25 °C), and each coverglass was then placed on a
flow-through chamber mounted on the stage of a TMD microscope (Nikon,
Tokyo, Japan). The perifusion medium comprised 135 mM NaCl,
4.5 mM KCl, 1.25 mM CaCl
, 1.2 mM
MgCl
, and 20 mM Hepes/NaOH (pH 7.4). Dual
wavelength microfluorometry of the fura-2 fluorescence was carried out
using an image-intensifying charge-coupled device camera (Hamamatsu
Photonics, Hamamatsu, Japan) equipped with a fluorimeter (Nikon). The
fluorescence excited at both 340 and 380 nm was measured at 510 nm. The
emission signals excited at 340 and 380 nm and the ratio of these
signals (340/380 ratio) were recorded by a computer system (Hamamatsu
Photonics). In some experiments, the cytoplasmic free Ca
concentration was calibrated as described elsewhere
(20) .
Whole Cell Patch Clamp Analysis
Whole cell
currents were recorded by the method described by Hamil et al.(21) using a computer-based amplifier system, a List EPC 9 patch
clamp amplifier (List, Darmstadt, Germany) controlled by E9 screen
software (HEKA, Lambrecht, Germany). The whole cell configuration was
achieved by a sharp aspiration immediately after seal formation to
rupture the plasma membrane within the seal. In this configuration, the
soluble cytosolic contents are dialyzed by the pipette solution.
Voltage clamp recording that had been compensated by capacity and leak
current subtraction was started after the series resistance fell below
20 megaohms, taking the voltage error into consideration. All these
experiments were carried out at 26-30 °C. The pipette
solution comprised 120 mMN-methyl-D-glucamine glutamate, 5 mM Hepes
(pH 7.0), 1 mM EGTA, and 1 µM free
Ca. The bath solution comprised 150 mMN-methyl-D-glucamine glutamate, 5 mM Hepes
(pH 7.4), 1 mM EGTA, and 1 mM free
Ca
. Under these conditions, the principal
membrane-permeant ion was Ca
, and the only ionic
gradient was Ca
. The free Ca
concentration was determined by the calcium-EGTA
buffer
(8) .
Phosphorylation of CD20 Expressed in Balb/c 3T3
Fibroblasts
Quiescent cells were washed 3 times with phosphate-
and Ca-free DMEM, incubated for 1 h in phosphate-free
DMEM, and labeled metabolically by culturing in DMEM containing
[
P]orthophosphate (100 mCi/ml) for 3 h. After
labeling, the cells were washed with PBS, incubated in
Ca
-free DMEM for 15 min in the presence or absence of
2 mM Ca
, washed 3 times with PBS, 0.1%
sodium azide, and lysed in 0.5 ml of 20 mM sodium phosphate
butter (pH 7.4) containing 1% (v/v) Triton X-100, 0.68 M
sucrose, 0.15 M NaCl, 5 mM EDTA, 50 mM NaF,
5 mM sodium pyrophosphate, 2 mM sodium vanadate, 1
mg/ml BSA, 20 mg/ml soybean trypsin inhibitor, 1 mg/ml leupeptin, 2
mg/ml pepstatin A, 2 mg/ml iodoacetamide, and 50 mg/ml
phenylmethylsulfonyl fluoride. Immunoprecipitation was carried out
using an anti-CD20 mAb and protein G-Sepharose (50%, v/v) as follows.
The cell lysates were centrifuged at 12,000
g for 15
min at 4 °C to remove the detergent-insoluble materials and nuclei.
Then each lysate was transferred to a tube containing 30 ml of
prewashed protein G-Sepharose and 2 mg of anti-CD20 mAb, and the
mixture was incubated for 12 h at 4 °C with constant rotation. The
resulting immunoprecipitates were washed twice with 50 mM
Tris-HCl (pH 7.4) containing 0.5% (v/v) Triton X-100, 0.2% (w/v) sodium
deoxycholate, 10 mM EDTA, 10 mM EGTA, 10 mM
NaF, 0.5 M NaCl, and 1 mg/ml BSA and then twice with PBS. The
precipitated proteins were eluted from the Sepharose gel by incubation
in 30 ml of Laemmli sample butter
(22) in a boiling water bath
for 3 min.
Expression of CD20 after cDNA Transfection
Human
cDNA encoding the entire translated region of the CD20 gene was
subcloned into an Epstein-Barr virus vector, pMEP4, and transfected
into Balb/c 3T3 cells. Hygromycin-resistant colonies were isolated, and
the expression levels of the positive clones were analyzed by Northern
blotting. A stable oligoclonal cell line that expressed CD20 was
isolated. Fig. 1shows the Northern blotting analysis results of
these CD20-transfected cells. These cells were incubated in DMEM
containing 10% FCS with or without 80 µM ZnCl for 24 h. CD20-transfected cells treated with ZnCl
expressed CD20 mRNA, whereas untreated cells did not. We used two
methods to show that the CD20-transfected cells but not the control
cells expressed CD20 protein. Fig. 2shows the time course of the
binding of a mAb against CD20 to the cells. The CD20-transfected cells
cultured with DMEM containing 80 µM ZnCl
expressed immunoreactive CD20 protein (CD20-expressing cells),
whereas those cultured without ZnCl
(control cells) did
not. The levels of immunoreactive CD20 remained elevated for at least
24 h after the removal of ZnCl
from the CD20-transfected
cells (Fig. 2). Immunohistochemically, over 80% (206 of 250
cells) of the CD20-transfected cells treated with 80 µM
ZnCl
expressed immunoreactive CD20 protein, which was
absent in the cells that were not treated thus.
Figure 1:
Northern blotting of mRNA for
CD20. CD20-transfected cells were incubated for 24 h in DMEM containing
10% FCS in the presence or absence of 80 µM ZnCl. mRNA was extracted, and Northern blotting was
performed.
Figure 2:
Binding of antibody against CD20 in
CD20-expressing cells. CD20-transfected cells were incubated for the
indicated periods with () or without (
) 80 µM ZnCl
, and binding of
I-labeled antibody
was measured as described under ``Experimental Procedures.''
In some experiments, binding was measured in cells 24 h after the
removal of ZnCl
.
In order to
determine whether the expressed CD20 protein acts as a
Ca-permeable channel, we monitored the changes in the
cytosolic free Ca
concentration
([Ca
]
) in response to
elevation of the extracellular Ca
concentration.
Fig. 3A shows the changes in
[Ca
]
in the
CD20-expressing cells monitored by measuring the fluorescence of a
Ca
indicator, fura-2. Increasing the extracellular
Ca
concentration from almost zero to 1.25 mM
resulted in a [Ca
]
rise in 27.8% (89 of 320) of the CD20-expressing cells but no
change in the other 72.2%. However, when the extracellular
Ca
concentration was raised from almost zero to 10
mM, the [Ca
]
increased in approximately 75% (241 of 320) of the
CD20-expressing cells. A typical response is shown in
Fig. 3B. Therefore, approximately 75% of the
CD20-expressing cells tested responded to changes in extracellular
Ca
concentration, and 28% were considered to be good
responders (). The
[Ca
]
of control and
untransfected cells did not change significantly under these conditions
(data not shown). The resting
[Ca
]
values in the
responders and non-responders measured in 1.25 mM
Ca
-containing medium were 186 ± 70 and 104
± 54 nM (mean ± S.E., n = 20),
respectively. The resting
[Ca
]
of the control
cells was identical to that of the non-responders. Next, we examined
Ca
conductance by the whole cell patch clamp
technique. Fig. 4shows the leak-subtracted steady-state
current-voltage relationship for the CD20-expressing cells. Expression
of CD20 induced an increase in Ca
conductance. In 25%
of the cells (27 of 108), the inward Ca
current in
response to a voltage jump to -100 mV was greater than 30 pA, as
shown in Fig. 4, and, in approximately 50% (52 of 108) of the
cells, a smaller inward current (less than 20 pA in response to
-100 mV) was observed. The inward Ca
current
was negligible in the control cells (data not shown). These results
demonstrate that constitutive expression of CD20 enhanced the plasma
membrane Ca
permeability of approximately 75% of the
CD20-expressing cells and confirm the previous observations of Bubien
et al.(17) . Furthermore, Ca
entry
into approximately 25% of the CD20-expressing cells was greater than
that into the rest. These results are summarized in .
Figure 3:
Effect of elevation of extracellular
calcium on cytoplasmic free calcium concentration. A,
CD20-transfected cells treated with ZnCl were loaded with
fura-2. Extracellular Ca
concentration was elevated
from almost zero to 1.25 mM as indicated. Cytoplasmic
Ca
concentration was monitored by measuring the
340/380 ratio of fura-2 fluorescence. A representative response is
shown. Solid and open circles represent good
responder and non-responder, respectively. B, fura-2-loaded
CD20-expressing cells were incubated, and extracellular Ca
concentration was changed stepwise from nominally zero to 10
mM as indicated. A representative response is shown (see Table
I for the number of cells responded to changes in extracellular
Ca
).
Figure 4:
Calcium current in CD20-expressing cells.
CD20-transfected cells were treated with ZnCl for 24 h.
Whole cell mode patch clamp recording was performed as described under
``Experimental Procedures,'' and the current-voltage
relationship for Ca
current is presented.
,
cells in which calcium current in response to -100 mV was greater
than 30 pA;
, cells in which calcium current in response to
-100 mV was less than 20 pA. Values are the means ± S.E.
for 30 determinations (see Table I for the number of cells in each
group).
Effect of Expression of CD20 on DNA Synthesis
In
order to examine the effect of CD20 expression on the growth of Balb/c
3T3 cells, CD20-transfected cells were cultured in DMEM containing 10%
FCS to confluence and then starved, and CD20 protein expression was
induced by incubation in DMEM containing 0.2% platelet-poor plasma and
80 µM ZnCl for 24 h. The resulting starved
CD20-expressing cells were used in this experiment. Control quiescent
cells were obtained by incubating confluent CD20-transfected cells in
DMEM containing 0.2% platelet-poor plasma for 24 h. When the
CD20-expressing and control cells were treated with 0.2% platelet-poor
plasma for 24 h, no nuclear labeling was detected (see below).
Pretreatment with ZnCl
for 24 h did not affect DNA
synthesis by untransfected cells (data not shown).
H]thymidine incorporation by primed competent
cells specifically but has no such effect on quiescent
cells
(9) . However, some CD20-expressing cells were able to
respond to IGF-I, which stimulated DNA synthesis without PDGF and EGF
pretreatment. Fig. 5B shows the time course of nuclear
labeling mediated by IGF-I in quiescent cells. Approximately 30% of the
CD20-expressing cells entered the S phase in response to IGF-I, whereas
few of the control cells did so. As described above, CD20-expressing
cells began to enter the S phase after 8 h, and the number of labeled
cells increased markedly thereafter. In contrast, no nuclear labeling
was observed in control cells that had not been pretreated with PDGF
and EGF. Note that, in a previous study, no nuclear labeling of
quiescent untransfected cells was observed when they were incubated
with IGF-I
(12) .
messenger
system
(8) . In order to determine whether CD20 expression
induced changes in the Ca
requirement for
IGF-I-mediated DNA synthesis, we examined the effects of changes in the
extracellular Ca
concentration on IGF-I-stimulated
[
H]thymidine incorporation by primed competent
cells. Reduction of the extracellular Ca
concentration resulted in a decrease in IGF-I-induced
[
H]thymidine incorporation by both
CD20-expressing and control cells (Fig. 6). It is noteworthy that
CD20-expressing cells required lower Ca
concentration
for IGF-I-mediated [
H]thymidine incorporation
than control cells, and the concentration-response curve for the
CD20-expressing cells was shifted to the left of that for the controls.
Furthermore, after priming with PDGF and EGF, some of the
CD20-expressing cells were able to progress toward the S phase, even in
the absence of IGF-I, and 25% of the cells were labeled with BrdUrd in
24 h. The labeling was blocked by the addition of mAb against CD20.
Figure 6:
Calcium sensitivity of IGF-I-induced DNA
synthesis in CD20-expressing and control cells. Quiescent CD20-treated
cells treated with () or without (
) ZnCl
were
rendered primed competent. Cells were then incubated in DMEM containing
1 nM IGF-I and various amounts of Ca
.
[
H]Thymidine incorporation was measured as
described under ``Experimental Procedures.'' Values are the
means ± S.E. for four experiments.
In lymphocytes, CD20 is phosphorylated, and various kinases,
including calmodulin-dependent protein kinase
(23) , are involved
in its phosphorylation. In the present study, we examined whether CD20
was phosphorylated in Balb/c 3T3 cells. As shown in Fig. 7, CD20
was phosphorylated under basal conditions, and this phosphorylation was
reduced by the removal of extracellular Ca from the
incubation medium.
Figure 7:
Phosphorylation of CD20 expressed in
Balb/c 3T3 cells. Balb/c 3T3 cells prelabeled with
[P]orthophosphate were incubated for 15 min in
DMEM with (lane 2) or without (lane 1) 2 mM
Ca
. Cells were lysed, and CD20 was immunoprecipitated
as described under ``Experimental Procedures.'' After
separation by SDS-polyacrylamide gel electrophoresis, an autoradiogram
was taken.
induced CD20 protein expression.
Immunological and functional analyses confirmed that CD20 protein was
present in CD20-transfected cells but not in control cells. We tried to
achieve constitutive expression of high levels of CD20 cDNA in a
variety of fibroblast cell lines but were unable to establish stable
cell lines that did so. This may be due to a toxic effect of long term
expression of high levels of Ca
-permeable channels.
Therefore, we used an expression system that enabled stable
transfectant cells to be recovered under conditions in which CD20 was
not expressed until dictated by the experimental design. Thus,
potential hazards associated with chronic expression during the weeks
of selection could be avoided.
, the CD20 protein, detected by
antibody binding, appeared several hours later, and the amount produced
increased up until 24 h. More importantly, CD20 expression remained
elevated for at least 24 h after the removal of Zn
from the medium (Fig. 2). This enabled us to assess the
effects of growth factors on CD20-expressing cells with the minimal
contribution of Zn
to cell growth. In agreement with
the results of Bubien et al.(17) , CD20 expressed in
Balb/c 3T3 cells functioned as a voltage-independent
Ca
-permeable cation channel. As shown in
Fig. 3
, CD20 expressed in Balb/c 3T3 cells was constitutively
operational; in other words, Ca
can permeate the CD20
channels in the absence of any stimulator, such as an antibody against
CD20. Despite the fact that a significant amount of CD20 was expressed
in the plasma membranes of approximately 80% of the cells, which was
detected by anti-CD20 antibody binding, the plasma membrane
Ca
permeability properties of the CD20-positive cells
were not uniform. As shown in , approximately 28% of these
cells responded well to a fairly modest rise in the extracellular
Ca
concentration, and the transmembrane
Ca
currents were higher in 25% of the CD20-expressing
cells than the rest. Our interpretation of these data is that
25-30% of the CD20-expressing cells possessed more functional
CD20 protein units than the rest. In any event, our system enables the
effects of IGF-I on cell cycle progression under conditions in which
Ca
entry is constitutively facilitated to be
assessed.
progression was accelerated in the CD20-expressing cells. It has
been established that the G
-S transition is regulated by
expression of G
cyclins and cyclin-dependent
kinases
(3) . With regard to Ca
-dependent
regulation, calcium and calmodulin have been shown to regulate the
expression of cyclins and cyclin-dependent kinases and the subsequent
phosphorylation state of RB protein
(26, 27) . Our
observations that increasing Ca
calcium entry
shortened the G
phase are in agreement with these findings.
Third, expression of CD20 reduced the concentration of extracellular
Ca
required for IGF-I-mediated G
progression. Given that CD20 is a Ca
-permeable
channel
(17) and that IGF-I promotes G
progression
by a mechanism dependent on Ca
entry
(8) , it
is conceivable that this is a property of CD20-expressing cells.
Fourth, some CD20-expressing cells pretreated with PDGF and EGF
progressed through the G
phase in the absence of the
progression factor IGF-I, which implies that CD20 can bypass, at least
partly, the action of IGF-I. At present, we have no direct evidence
that DNA synthesis by cells that are good responders can be initiated
in the absence of IGF-I, but it seems probable, because pharmacological
stimulation of Ca
entry by BAYK8644 led to the
initiation of DNA synthesis by primed competent Balb/c 3T3
cells
(11) .
-permeable cation channel
(8) . Previously,
we demonstrated that the addition of BAYK8644, a pharmacological
activator of voltage-dependent Ca
channels
(28) , stimulated DNA synthesis by primed
competent, but not quiescent, cells to some extent
(11) . Taken
together, the findings discussed above suggest that augmentation of
Ca
entry into primed competent cells via three types
of Ca
-permeable channels, CD20, voltage-dependent,
and IGF-sensitive cation channels, eventually leads to the initiation
of DNA synthesis. Yet, IGF-I is the most effective at promoting G
progression, presumably because it activates other signaling
cascades as well as activating these cation channels. In any event, our
results lend further support to the concept that Ca
entry is an intracellular message for the promotion of G
progression.
and therefore may be related to the function of CD20 as a
Ca
-permeable channel.
-permeable channel in Balb/c 3T3 cells
accelerates G
progression. Furthermore, our results suggest
that Ca
entry into cells is important for G
progression.
Table:
Responsiveness of CD20-expressing cells
for 24 h
to induce CD20. [Ca
]
response was determined as described in the legend for Fig. 3.
Moderate responder is the cell that responded to the elevation of
extracellular Ca
from almost zero to 10 mM but not to 1.25 mM. Good responder is the cell that
responded to the elevation of extracellular Ca
from
nominally zero to 1.25 mM. Ca
current was
determined as described in the legend for Fig. 4. The good and moderate
responders were the cells in which inward current in response to a
hyperpolarization pulse of -100 mV was greater than 30 pA and
less than 20 pA, respectively.
], concentration of
cytosolic free Ca
; MOPS, 4-morpholinepropanesulfonic
acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.