©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Calcium-permeable Cation Channel CD20 Accelerates Progression through the G Phase in Balb/c 3T3 Cells(*)

Makoto Kanzaki, Hiroshi Shibata, Hideo Mogami, and Itaru Kojima (§)

From the (1) Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD20 is a transmembrane protein that functions as a Ca-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.


INTRODUCTION

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 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.

G 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) .

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 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.


EXPERIMENTAL PROCEDURES

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)RNA

The CD20 cDNA was amplified from Raji cell (B lymphoblastoid cell line) poly(A) 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 ofI-Labeled Monoclonal Antibody against CD20 and Immunostaining

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 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.

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.

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.

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).


RESULTS

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).

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 [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) .

Insulin-like growth factor-I exerts its progression activity by acting on the Ca 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.




DISCUSSION

Transfection of Balb/c 3T3 cells with pMEP4-CD20 followed by treatment with ZnCl 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.

When the mRNA for CD20 was induced by the addition of Zn, 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.

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 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) .

As discussed above, IGF-I activates a Ca-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.

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 and therefore may be related to the function of CD20 as a Ca-permeable channel.

In conclusion, expression of the CD20 Ca-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

CD20-transfected cells were incubated with ZnCl 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.



FOOTNOTES

*
The present study was supported in part by a grant from the Growth Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Inst. for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan. Tel.: 81-272-20-8835; Fax: 81-272-20-8893.

The abbreviations used are: PDGF, platelet-derived growth factor; EGF, epidermal growth factor; IGF-I, insulin-like growth factor-I; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; BrdUrd, bromodeoxyuridine; [Ca], concentration of cytosolic free Ca; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

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.


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