Expression of CD38 Increases Intracellular Calcium Concentration and Reduces Doubling Time in HeLa and 3T3 Cells*

Elena ZocchiDagger §, Antonio Daga, Cesare Usaipar , Luisa FrancoDagger , Lucrezia GuidaDagger , Santina BruzzoneDagger , Aurora Costa**, Carla Marchettipar , and Antonio De FloraDagger

From the Dagger  Institute of Biochemistry, University of Genova, Viale Benedetto XV No. 1, 16132 Genova,  Immunobiology, Advanced Biotechnology Centre (CBA), Largo Rosanna Benzi No. 1, 16132 Genova, par  Institute of Cybernetics and Biophysics, National Research Council, Via de Marini 6, 16149 Genova, and ** Experimental Oncology C, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian No. 1, 20133 Milano, Italy

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38- HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38+ HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

CD38 is a type II transmembrane glycoprotein predominantly expressed on lymphocytes (1) but also present in a number of different cell types, including erythrocytes (2), hematopoietic progenitor cells (1), beta -pancreatic cells (3), and cerebral (4) and cerebellar (5) neurons. Immunologically, CD38 can be defined as an "orphan receptor" since its binding by specific monoclonal antibodies directed against ectocellular epitopes elicits cellular responses in lymphocytes, including proliferation, activation, and rescue from apoptosis (6, 7). The signal transduction pathways implicated in these events are under study, but phosphorylation/dephosphorylation reactions of target kinases have been already demonstrated (6, 8, 9). Biochemically, CD38 is a bifunctional ectoenzyme that catalyzes the synthesis of cADPR1 from NAD+ and also its hydrolysis to ADPR (2, 10). Cyclic ADPR is a potent Ca2+ mobilizer from intracellular stores, in invertebrate as well as in mammalian cells (11), and its presence has been described in most mammalian tissues (12).

The widespread tissue distribution of the CD38/cADPR system suggests its involvement in the control of pivotal Ca2+-controlled functions like contraction, secretion, cell proliferation/differentiation, and apoptosis. However, the topological paradox of the ectocellular production of an intracellular Ca2+ mobilizer has raised questions on both the immunological and the biochemical functions of the CD38/cADPR system (13, 14). Cell death has been advocated as a means for local increase in extracellular NAD+ concentrations, sufficient to elicit the production of cADPR by the ectoenzyme CD38; in this respect, nanomolar concentrations of NAD+ have been detected in plasma (15) and cerebellar interstitial fluid (5) suggesting the theoretical possibility of an extracellular production of cADPR by CD38 "in vivo." In few selected cell systems, i.e. murine B-lymphocytes (10), rat cerebellar granule cells (5), and rat osteoclasts (16), extracellular, exogenously added cADPR was demonstrated to elicit functional responses in intact cells, but most reported effects of cADPR on cellular functions require permeabilization of target cells to ensure binding of the nucleotide to its intracellular receptor(s).

Internalization of membrane-bound CD38, as observed in human Namalwa B-lymphocytes upon incubation with NAD+ or thiol reagents, is followed by an increase of intracellular cyclase activity (insensitive to protein synthesis inhibitors) and of intracellular cADPR concentration ([cADPR]i) (17), suggesting that cytosolic NAD+ is available to intravesicular CD38. This observation prompted us to investigate whether modulation of the ectocellular expression of CD38 could affect both the [cADPR]i and the free cytosolic ionized calcium concentration ([Ca2+]i).

The experimental models developed for this study were human HeLa and murine 3T3 cells transfected or retrovirally infected with human full-length CD38. Neither wild type cell line expresses CD38. Several biochemical parameters were investigated during the progressive selection for high CD38 expression and on established CD38+ cell lines. The results obtained indicate that expression of CD38 is associated with decrease of [NAD+ + NADH]i, appearance of intracellular cADPR, elevation of basal [Ca2+]i, and reduction of cell doubling time. Cell cycle analysis of CD38+ HeLa cells demonstrates a significant shortening of the S phase. These findings provide new insight into the physiological significance of the modulation of CD38 expression observed during hematopoietic stem cell and lymphocyte proliferation/differentiation processes (1).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Fura2-AM was purchased from Fluka, Milano, Italy. [3H]Thymidine (80 Ci/mmol) and [3H]NAD+ (30 Ci/mmol) were obtained from NEN Life Science Products, Milano, Italy. Cyclic ADPR and [3H]cADPR were prepared enzymatically from NAD+ and [3H]NAD+, respectively, with Aplysia californica cyclase (courtesy of Prof. H. C. Lee, Minneapolis, MN) and HPLC-purified. Anti-CD38 monoclonal antibody (mAb), IB4, was kindly provided by Prof. F. Malavasi, Torino, Italy, and anti-bromodeoxyuridine mAb was from Becton Dickinson, Mountain View, CA. Goat anti-mouse Ig-coated magnetic beads (4.4 µm diameter) were from Unipath, Milano, Italy. 8-Amino-cADPR was generously provided by Prof. H. C. Lee. All other chemicals were purchased from Sigma, Milano, Italy.

Cell Lines and Culture Conditions-- HeLa and NIH 3T3 cells (3T3), obtained from ATCC (Rockville, MD), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and glutamine (2 mM) in a humidified 5% CO2 atmosphere at 37 °C. Transfected and infected cells were routinely maintained under geneticin (1 mg/ml) selection in the above medium.

Construction of Plasmids-- The complete coding sequence of the human full-length CD38 cDNA (1) was recovered as XbaI fragment and cloned in the same restriction site of the pRC-CMV expression plasmid. The same full-length cDNA recovered as XhoI fragment was cloned in the XhoI restriction site of LXSN retroviral vector to generate the LCD38SN sense and antisense vectors. From a number of mutant CD38 forms obtained in our laboratory by site-directed mutagenesis, we selected the K129N/L131H mutant, which expressed very low NAD+-glycohydrolase activity (3% of wild type CD38), to be used as a control in this study. The synthetic oligonucleotide used for site-directed mutagenesis was as follows: 5'-ATAAATGATCACGCCCATCAGTTC-3'. Mutant CD38 was cloned into the XbaI site of pRC-CMV (CMV-CD38mut).

Retroviral Production-- To generate the amphotropic producer cell lines, the GP+E-86 ecotropic packaging cell line (18) was transfected with 10 µg of LXSN or LCD38SN sense and antisense vectors using a standard Ca2+ phosphate method (19). Two days post-transfection the supernatant was collected and used to infect the GP+env AM-12 amphotropic packaging cell line (20) in the presence of 8 µg/ml Polybrene (Sigma) for 16 h, followed by three subsequent cross-infections using the ping-pong technique. The amphotropic packaging cell line was then plated at low density and selected with geneticin (Life Technologies, Inc., Paisley, Scotland) at 1.2 mg/ml (active form) for 10 days. After geneticin selection, individual clones were collected, and their titer was assessed by infecting HeLa cells with a serial dilution of the different viral stocks. The absence of replication competent retrovirus was assessed by the absence of serially transmitted geneticin resistance on HeLa and 3T3 cells (21).

Transfection and Infection of HeLa and 3T3 Cells with Sense and Antisense CD38-- CMV-CD38 and CMV-CD38mut, both sense and antisense, were transfected into HeLa and 3T3 cells using a standard Ca2+ phosphate procedure (19). The lower NAD+-glycohydrolase (NADase) specific activity of mutant, as compared with wild type CD38, was demonstrated by SDS-polyacrylamide gel electrophoresis and Western blot analysis of Triton X-100-solubilized pellets of transfected 3T3 cells; samples with similar protein content showed a 46-kDa band of comparable intensity (as detected by ECL), whereas samples with similar NADase activity showed a much higher band intensity in the mutant lane as compared with wild type CD38 (not shown). For cell line infection, HeLa and 3T3 cell lines were plated on 6-well plates, and the filtered retroviral supernatant of a high titer, cloned packaging cell line was added overnight in the presence of Polybrene (8 µg/ml).

Two days after gene transfer, geneticin (1 mg/ml) was added to the culture medium, and transduced cells were maintained under antibiotic selection throughout the study. Several successive magnetic bead selections for high expression of CD38 were performed on transduced cells during the first 2 weeks following gene transfer and were thereafter repeated monthly using anti-CD38 mAb and goat anti-mouse Ig-coated magnetic beads. Expression of CD38 was monitored routinely by immunofluorescence (>95% of cells were positive) and by assay of CD38 ectoenzyme activities on intact cells (see below): NADase (production of ADPR from NAD+), cyclase (production of non-hydrolyzable cGDPR from the NAD+ analog NGD+), and hydrolase (production of ADPR from cADPR). Detachment of adherent cells with a brief trypsin treatment was found not to decrease either CD38 immunofluorescence or any of its enzyme activities as compared with detachment with PBS/EDTA. Thus, trypsin was used throughout this study for removing adherent cells.

Assay of CD38 Enzyme Activities-- NADase, cyclase, and hydrolase activities were assayed on NAD+, NGD+, and cADPR, respectively, by incubating 2 × 105 cells in a total volume of 400 µl of PBS/glucose (10 mM) with 0.2 mM NAD+, NGD+, or cADPR. At different times, 60-µl aliquots of the incubations were removed and briefly centrifuged, and 50-µl supernatants were analyzed by HPLC (22). Protein determination was performed on an aliquot of the incubations by the method of Bradford (23).

Determination of Intracellular Concentrations of (NAD+ + NADH) and cADPR-- The total pyridine dinucleotide content of cells was determined by a modification of a sensitive enzymatic cycling method (24), as described (25), on alkaline extracts from 106 cells. Values were occasionally confirmed by HPLC analysis of neutralized perchloric acid extracts (3 × 106 cells). The intracellular cADPR concentration was determined with a specific radioimmunoassay, as described in detail in Ref. 26, on neutralized perchloric acid extracts (2 × 107 cells). The cADPR-specific chicken antibody used was obtained following immunization of the animals with mono-succinylated cADPR and partially purified from eggs as described (26). The antibody was specific for cADPR; the half-maximal inhibitory concentration, IC50, for cold cADPR was approximately 2 nM, whereas other nucleotides (ADPR, NAD+, NMN, and ATP) produced no displacement at similar concentrations. However, the millimolar range of the intracellular concentrations of some nucleotides could interfere with the radioimmunoassay (26). Thus, prior to radioimmunoassay, cell extracts were incubated for 4 h at 37 °C with the following enzymes: NADase (from Neurospora crassa), 0.25 units/ml; nucleotide pyrophosphatase (from Crotalus atrox), 1.75 units/ml; alkaline phosphatase (from bovine intestine), 50 units/ml; and apyrase (from potato) 5 units/ml, as described (26). Although cADPR is resistant to these enzymes, other nucleotides potentially interfering with the assay were completely removed. Finally, each extract was divided into 2 aliquots, one of which was incubated in the presence of 0.2 µg of purified CD38 for 1 h at 37 °C to hydrolyze cADPR. Both aliquots were then heated at 100 °C for 15 min to inactivate CD38, and the concentration of cADPR in the cell extract was calculated from the difference between the radioactivity values of the CD38-treated (control) and untreated sample.

Estimation of Cell Doubling Time-- For each experiment, cell doubling time was determined according to three different methods as follows: [3H]thymidine (TdR) incorporation, direct cell count, and protein determination. For [3H]TdR incorporation cells were seeded in triplicate at 104/well in 24-well plates, and 1 µCi of [3H]TdR was added to each well. At different times (typically 14, 19, 24, 36, 40, and 48 h) cells were removed with trypsin, and the trichloroacetic acid-insoluble, incorporated radioactivity was harvested on glass fiber filters (Whatman, Maidenstone, UK). For direct cell count and protein determination 2 × 104 cells were seeded, in duplicate, in 25-cm2 flasks. At different times (typically 24, 48, 60, 72, 84, and 96 h) cells were removed and counted, and the cell pellets were washed twice with PBS in a microcentrifuge, and total protein content was determined by the method of Bradford (23). The cell doubling times obtained with the different methods, i.e. the time required to double either the trichloroacetic acid-insoluble incorporated [3H]TdR radioactivity, the cell number, or the total protein content, were always closely comparable. The cell doubling time was calculated as the mean of the above three values.

Fluorimetric Determination of the [Ca2+]i-- Adherent (on 20-mm coverslips) cells were incubated with 6 µM Fura2-acetoxymethyl ester (Fura2-AM) in standard saline containing 5 mM glucose for 45 min at 37 °C and then washed several times with standard saline at room temperature. This protocol resulted in optimal loading of the dye, which lasted for several hours. The specimen was then mounted on the stage of an inverted microscope (Zeiss IM35, Germany) and continuously perfused with solutions fed by gravity, through solenoid microvalves and removed by a hydraulic vacuum pump. Cells were viewed through a 100 × Nikon Fluor objective. The field contained from 1 to 10 cell bodies. Cells were illuminated by a 75-watt Xenon lamp (PTI, Brunswick, NJ) equipped with a rotating wheel with six interference filters, four filters centered at 340 nm and two centered at 380 nm. A computer-driven spectrophotometer system (Cairn Research, Sittingbourne, UK) controlled both rotor speed and emitted light acquisition through a photomultiplier (ThornEMI, Ruinslip, UK). The emissions relative to each series of filters (E340 and E380, respectively) were mediated, and the ratio R = E340/E380 was calculated after subtraction of background fluorescence. The rotor speed was set at 32 rps, and emission ratios were averaged on-line over 16 revolutions to maximize the signal-to-noise ratio. The final frequency of recording was 1 point/0.5 s.

Calculation of Cytosolic Ca2+ Concentration-- The intracellular free calcium concentration was calculated according to Equation 1 (27).
[<UP>Ca</UP><SUP>2+</SUP>]<SUB>i</SUB>=&bgr;K<SUB>D</SUB>(R−R<SUB><UP>min</UP></SUB>)/(R<SUB><UP>max</UP></SUB>−R) (Eq. 1)
where R is E340/E380; Rmin is E340/E380 in zero calcium; Rmax is E340/E380 in calcium-saturated solution; beta  is E380 in zero calcium/E380 in calcium-saturated solution; and KD = 140 nM (the dissociation constant of the dye at room temperature). To obtain the parameter values, after each experiment cells were incubated in 10 µM 4-bromo-calcium ionophore A23187 for 20-40 min in a zero-calcium bath (0 calcium + 1 mM EGTA) and then perfused with the saturated Ca2+ solution. At the end of this procedure, 5 mM MnCl2 was added to the bath to quench the fluorescence of the dye and determine the background values.

Solutions-- The standard physiological saline contained the following (in mM): NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, Hepes 5, pH 7.4. The volume of the recording chamber was 200 µl, and the bath solution flowed at the rate of 100 µl/s, assuring a complete exchange of the solution in few seconds.

Cell Membrane Permeabilization-- Cytoplasmic membrane permeabilization was achieved by incubating cells with 25 µM digitonin in Ca2+-free standard solution. The experiments were performed in Ca2+-free external solution to observe modifications of intracellular ionic calcium originated from internal compartments only.

Atomic Absorption Determination of Total Cell Calcium-- This was obtained as described (28) on La3+ trichloroacetic acid extracts of 107 cells.

Determination of Ca2+ Exchange-- For determination of calcium influx, 5 × 105 cells per time point were incubated at 37 °C in standard saline solution (see above) with 10 mM glucose containing 5 µCi/ml 45Ca2+. At various times, individual incubation mixtures were centrifuged; the cell pellets were rapidly washed in chilled standard solution without 45Ca, and the incorporated radioactivity was determined.

In efflux experiments, cells were equilibrium-loaded with 45Ca2+ by culture for 72 h in complete medium supplemented with 45Ca2+ (5 µCi/ml). Thereafter, cells were detached with trypsin, rapidly washed twice in chilled standard solution, and resuspended at 4 × 106/ml in the same buffer containing EGTA (2 mM). The radioactivity released in the supernatant, after incubation at 37 °C, was determined at various times after brief centrifugation of the cells.

Reversible Permeabilization of Cells with Streptolysin O (SLO) and Entrapment of cADPR or ADPR-- HeLa cells, washed twice with PBS, were reversibly permeabilized with SLO, essentially as described (29), except that cells were treated with SLO in suspension (400 units/ml/106 cells) for 20 min at room temperature. Permeabilization was confirmed on aliquots of cells by detection of fluorescent lucifer yellow (molecular weight, 572) uptake. Permeabilized cells were divided into 2 aliquots, either cADPR or ADPR (100 µM final concentration) was added, and cells were further incubated for 10 min at room temperature. Thereafter, cells were resealed by addition of 1 volume of complete medium, incubated for 10 min at 37 °C, and washed twice in complete medium, and resealing was checked by lucifer yellow permeability; routinely more than 97% of resealed cells were again impermeant to the fluorescent dye. Resealed cells were then seeded in duplicate in 25-cm2 flasks, as described above, for determination of total protein content and cell number during the following 5 days' culture. Aliquots of cells were seeded on glass coverslips for measurement of [Ca2+]i and cultured for up to 5 days in sterile Petri dishes. To monitor the metabolic fate of encapsulated cADPR, the same protocol was used for entrapment of [3H]cADPR (250 cpm/pmol, 100 µM final concentration) into wild type HeLa cells. Immediately after resealing and at various times of culture, 2-4 × 106 cells were trichloroacetic acid-extracted, and the radioactivity incorporated into nucleotides was analyzed by HPLC (22).

Cell Cycle Analysis-- Exponentially growing CD38+/- transfected HeLa cells (seeded at 106/75-cm2 flasks 12 h before) were labeled for 30 min with 30 µM 5'-bromodeoxyuridine (BrdUrd). Cells were then washed with PBS and either immediately fixed (time 0) or further cultured in complete medium for 4 h. BrdUrd-labeled cells were detached with trypsin, washed twice with ice-cold PBS containing 2 mM EDTA, and fixed in chilled 70% ethanol. For detection of BrdUrd incorporation into DNA, double-stranded DNA was denatured with 3 N HCl for 20 min at room temperature, and DNA denaturation was stopped with 0.1 M sodium tetraborate, pH 8.5. The pellet was incubated with 1 ml of 0.1% Tween 20 in PBS (PBS/Tween) containing 0.5% normal goat serum (Dakopatts, Denmark) for 10 min at room temperature. The incorporated BrdUrd was then visualized by incubating cells with the anti-BrdUrd mAb diluted 1:10 in PBS/Tween for 30 min and then with a fluorescein isothiocyanate-conjugated goat anti-mouse IgG mAb diluted 1:50 in PBS/Tween for 30 min at room temperature in the dark. The cells were then stained for their DNA content with a propidium iodide solution containing 5 µg/ml propidium iodide and 25 µg/ml RNase in PBS for 1 h at 4 °C in the dark and analyzed in a FACScan flow cytometer (Becton Dickinson). Simultaneous measurements of red (DNA content) and green (BrdUrd content) fluorescence were done on 30,000 cells for each sample.

BrdUrd-positive (BrdUrd+) subpopulations were identified on each sample by biparametric BrdUrd/DNA analysis, and cell cycle phase percentages of BrdUrd+ cells were calculated. The DNA synthesis time (tS) was calculated from the measurement of relative movement (RM) (30) which assumes that the rate of cell progression through the S phase is constant and that at the time of BrdUrd pulse labeling the mean DNA content of BrdUrd-labeled S phase cells is in the middle of the interval between the G1 and G2M peaks. The RM was calculated using Equation 2.
<UP>RM</UP>=(F<UP>S</UP>−F<UP>G</UP><SUB>1</SUB>)/(F<UP>G</UP><SUB>2</SUB>−F<UP>G</UP><SUB>1</SUB>) (Eq. 2)
where F is the mean red fluorescence of the corresponding phase of the cell cycle.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Establishment of Transfected/Infected 3T3 and HeLa Cell Lines Expressing Human Wild Type and Mutant CD38-- Following transfection or infection of 3T3 and HeLa cells with sense (CD38+) and antisense (CD38-) CD38, establishment of geneticin-resistant cell lines required about 2 weeks. No growth of geneticin-resistant clones in wild type (not transfected/infected), HeLa, or 3T3 cells was observed during this period; no wild type cells survived longer than 4-6 days in geneticin (1 mg/ml). After 15-20 days, CD38 surface expression of geneticin-resistant, transfected cells was low as compared with infected cells (0.17 and 0.31 nmol/min/mg GDP-ribosyl cyclase activity as compared with 4.31 and 4.38 for 3T3 and HeLa cells, respectively, Fig. 1). Magnetic beading was performed once on infected and 4 times on transfected cells to enrich for high CD38 expression. Both CD38+ and CD38- cells were subjected to immunoselection, although no attachment of magnetic beads to CD38- cells was ever observed. At this point, all CD38+ cell lines were approximately 100% positive for CD38 expression as detected by immunofluorescence (not shown). Although surface cyclase activity of both transfected and infected HeLa cells remained stable after the initial increase following immunoselection, CD38 expression on 3T3 cells, transfected as well as infected, decreased rapidly (1-2 weeks) and sharply (>50%) in the first weeks following gene transfer, despite repeated magnetic beadings (see Fig. 1). On the other hand, cyclase expression of 3T3 cells transfected with mutant CD38 was stable at approximately 3 nmol/min/mg, and this level of expression was maintained for 10 months with regular immunoselection. Stable expression of the cyclase activities listed in Table I on transduced cells has been maintained for 14 (transfected) and 8 (infected) months by constant culture in geneticin and regular monthly immunoselection. The ratio between NADase, GDP-ribosyl cyclase, and cADPR hydrolase activities (approximately 100:10:1) expressed on 3T3 cells transduced with wild type CD38 (33 nmol of ADPR/min/mg, 2.8 nmol of cGDPR/min/mg, and 0.4 nmol of ADPR/min/mg, respectively) was the same as that observed on human purified CD38 (5). On the other hand, this ratio was significantly different in 3T3 cells expressing mutant CD38; although cyclase activity on NGD+ was comparable to that of wild type CD38, NADase and hydrolase activities were approximately 3 and 7% of wild type (1.1 nmol of ADPR/min/mg, 2.38 nmol of cGDPR/min/mg, and 0.03 nmol of ADPR/min/mg). Thus, the mutant CD38 was selectively deficient in its enzyme activities on the physiological substrates NAD+ and cADPR.


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Fig. 1.   Establishment of a stable and high CD38 expression in transduced 3T3 and HeLa cells. Transfections or infections were performed at time 0, and ectocellular cyclase activities, measured on NGD+, expressed on infected (A) or transfected (B) cells are shown (mean of >= 2 determinations). Arrowheads indicate magnetic immunoselections (see "Experimental Procedures"). 3T3 mut are cells transfected with mutant CD38.

                              
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Table I
Cyclase activity, intracellular NAD+, NADH, and cADPR concentrations, cell doubling time, and cytosolic free Ca2+ concentrations of CD38+ HeLa and 3T3 cells
Determinations of cyclase activity on NGD+, total cell pyridine dinucleotide content ([NAD+ + NADH]), intracellular cADPR concentration ([cADPR]), cell doubling time, and cytosolic free calcium ([Ca2+]i) were performed as described under "Experimental Procedures" on 3T3 and HeLa cell lines transfected (t) or retrovirally infected (i) with human full-length CD38 or a mutant CD38 (mut) and compared to antisense-transduced (CD38) controls. Mean results ± S.D. of >= 4 determinations are shown. ND, not detectable.

Correlation between CD38 Expression, [NAD+ + NADH]i, [Ca2+]i, and Cell Doubling Time-- During the first three magnetic bead selections of freshly transfected CD38+ 3T3 cells (Fig. 1B), we observed a progressive decrease in intracellular NAD+ + NADH content (98, 80, and 73% of control, antisense-transfected cells), an increase in [cADPR]i (from undetectable levels to 0.29 and 0.6 pmol/mg), and a decrease in cell doubling time (102, 85, and 75% of control) that paralleled the increase in cyclase activity. These determinations were repeated when a stable expression of CD38 was obtained, 8-14 months later (Fig. 1), and results are shown in Table I. 3T3 and HeLa cells transduced with sense CD38 showed appearance of [cADPR]i, a decrease in [NAD++NADH]i, increased basal cytoplasmic free calcium concentration ([Ca2+]i), and a reduction in cell doubling time as compared with antisense-transduced cells. These parameters correlated well with the amount of surface cyclase activity expressed; CD38+ HeLa cells showed a higher cyclase activity than CD38+ 3T3 cells and also higher [cADPR]i and [Ca2+]i, lower [NAD++NADH]i, and shorter doubling time than their respective CD38- controls, as compared with CD38+ 3T3 cells. Transfection or infection per se did not modify [NAD+ + NADH]i or [Ca2+]i, since CD38- 3T3 and HeLa cells did not differ significantly from either mock-transduced cells (transduced with vector alone) or wild type cells (not shown). 3T3 cells transfected with a mutant, enzymatically defective CD38 (3% of enzyme activity on NAD+ but similar cyclase activity on NGD+, as compared with wild type) did not contain detectable amounts of cADPR, and the [NAD+ + NADH]i, [Ca2+]i, and cell doubling times were similar to those of CD38- cells (Table I).

Effect of cADPR on [Ca2+]i in Permeabilized 3T3 and HeLa Cells-- To confirm a direct Ca2+ releasing activity of cADPR in 3T3 and HeLa cells, two types of experiments were devised. cADPR was either exogenously added to permeabilized wild type Fura2-loaded cells or intact Fura2-loaded CD38+ cells were preincubated with NAD+ and subsequently permeabilized. Addition of 100 µM cADPR to intact cells was without effect, whereas addition to permeabilized cells elicited an immediate Ca2+ release from intracellular stores (Fig. 2A); maximal release was obtained with cADPR concentrations ranging from 2 to 100 µM. This effect was subject to desensitization and was inhibited by preincubation with the cADPR antagonist 8-amino-cADPR (Fig. 2B). ADPR (100 µM) was without effect. A calcium release was also observed after permeabilization of CD38+ cells that had been previously incubated with 1 mM NAD+ for 20 min. HPLC analysis of the incubation supernatant demonstrated the presence of 1 µM cADPR, produced by the intact cells. This value is in the range of the [cADPR]i of CD38+ 3T3 and HeLa cells, i.e. 0.5 and 5 µM, respectively, as calculated from the values reported in Table I and a cell volume of 2 µl/mg protein, estimated as reported by Gershengorn et al. (31). Thus, ectocellular cADPR did not have any effect on [Ca2+]i of intact 3T3 and HeLa cells, whereas exogenously added or ectocellularly produced cADPR increased [Ca2+]i in permeabilized cells. Furthermore, cADPR concentrations in the micromolar range, as detected in CD38+ 3T3 and HeLa cells, were effective in releasing calcium from intracellular stores in these cell types.


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Fig. 2.   Effect of cADPR on permeabilized 3T3 cells. Fura2-loaded, wild type 3T3 cells were permeabilized with 25 µM digitonin. cADPR (100 µM) induced a remarkable calcium release in the absence of extracellular calcium (A). Pretreatment of the permeabilized cells with 8-amino-cADPR (100 µM) completely abolished the response to the subsequent addition of 100 µM cADPR (B). Traces shown are representative of 4 consistent experiments. Similar results were obtained with wild type HeLa cells.

Cell Calcium Compartments in CD38+ and CD38- Infected 3T3 and HeLa Cells-- Prompted by the demonstration of an increased basal free Ca2+ concentration in CD38+ cells, as compared with controls, we investigated the size of the major Ca2+ compartments of infected CD38+ and CD38- HeLa and 3T3 cells. Results are shown in Table II. Total cell calcium, determined by atomic absorption spectroscopy, was similar for CD38+ and CD38- infected cells. This was confirmed also by experiments with 45Ca2+; loading of cells for 72 h with the tracer ion resulted in comparable radioactivity incorporated per mg of protein in CD38+ and CD38- cells (not shown). On the other hand, both the total ionized Ca2+ releasable from thapsigargin-sensitive stores and the [Ca2+]i were significantly perturbed in CD38+ cells as compared with controls (Table II and Fig. 3). The size of the thapsigargin-sensitive Ca2+ stores was markedly reduced (43 and 52% of controls in CD38+ HeLa and 3T3 cells, respectively), whereas the [Ca2+]i was elevated (247 and 174% of controls in CD38+ HeLa and 3T3 cells, respectively). No significant differences in the rate or amount of 45Ca2+ influx or efflux between CD38+ and CD38- cells were observed (Fig. 4). These results indicate that the mechanism underlying the increased basal [Ca2+]i in CD38+ cells is a sustained calcium release from internal stores.

                              
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Table II
Calcium content of HeLa and 3T3 cells infected with sense and antisense CD38
Measurements of calcium content were performed on sense- (CD38+) and antisense- (CD38-) infected HeLa and 3T3 cells. Total cell calcium was determined by atomic absorption spectroscopy (mean ± SD of 3 experiments). Ionized calcium released from stores by thapsigargin (1 µM) and free cytosolic calcium were measured fluorimetrically on Fura 2-loaded cells (mean ± SD of 5 experiments: in each experiment responses were recorded from groups of 3-10 cells).


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Fig. 3.   Thapsigargin-releasable Ca2+ in CD38 sense and antisense infected 3T3 cells. Infected 3T3 cells were challenged with 1 µM thapsigargin (Thapsi) (arrow) in calcium-free standard saline (see "Experimental Procedures"). Traces shown are representative of results obtained in 5 consistent experiments. Similar results were obtained with infected HeLa cells.


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Fig. 4.   Calcium exchange in infected 3T3 cells. Calcium influx (A) and efflux (B) in sense (CD38+) and antisense (CD38-) infected 3T3 cells were determined as described under "Experimental Procedures." Results shown are mean ± S.D. of three separate experiments. Comparable results were obtained with infected HeLa cells.

Reversible Permeabilization of HeLa Cells and Entrapment of cADPR or ADPR, Determination of [Ca2+]i, and Cell Growth Rate-- To demonstrate a direct, causal effect of cADPRi on cytosolic calcium concentration and cell growth rate, either cADPR or ADPR was encapsulated into wild type HeLa cells. A conservative, reversible cell permeabilization technique was essential for long term (48-72 h) survival of treated cells and evaluation of [Ca2+]i and cell growth. Permeabilization with SLO proved to be effective (60-70% of cells were permeabilized by incubation with 400 units/ml/106 cells for 20 min) and reversible (>95% resealing by addition of fetal calf serum). Permeabilization of more than 80% of the cells could be achieved by increasing SLO concentration but resulted in poor viability of the resealed cells. Wild type HeLa cells were loaded with either cADPR or ADPR and [Ca2+]i and cell growth rates were compared during 5 days of culture. Results of a representative experiment are shown in Fig. 5. Immediately after resealing, the [Ca2+]i of cADPR-loaded cells was twice that of ADPR-loaded controls (53 and 26 nM, respectively). Although decreasing with time (an effect which might be related to dilution of encapsulated cADPR due to cell division), significantly higher [Ca2+]i, 25% compared with controls, was still measured in cADPR-loaded cells 3 days after resealing. cADPR-loaded cells also showed a higher growth rate than ADPR-loaded controls, as determined by assay of total protein content and direct cell count of the cultures. This effect was most evident 5 days after resealing, when the protein content of cADPR-loaded cell cultures was almost twice the amount measured in controls (Fig. 5). Direct cell count after 5 days of culture confirmed doubling of cell number in cADPR-loaded cultures compared with controls.


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Fig. 5.   Growth rate and intracellular free calcium concentration of cADPR- or ADPR-loaded wild type HeLa cells. Wild type HeLa cells were reversibly permeabilized with SLO (68% lucifer yellow permeable cells) and 100 µM cADPR or ADPR (control) were encapsulated at time 0. Total protein (black-square) and [Ca2+]i (black-triangle) were determined as described under "Experimental Procedures." Data obtained on cADPR-encapsulating cells are expressed as relative increase over control values, recorded in ADPR-loaded cells. Results of a representative experiment are shown. Each point represents the mean of 2 (protein) or 6 (calcium) determinations. The doubling of cell number after 5 days of culture was confirmed by direct cell count.

The long lasting effect of encapsulated cADPR on [Ca2+ ]i of HeLa cells suggested a long life span of this molecule inside CD38- cells. Stability of cADPR in wild type HeLa cells was first investigated by incubation of cADPR (100 µM) with cell lysates (107 cells/ml in PBS containing 1% Triton X-100) at 37 °C for 5 days. Degradation of cADPR was constant at a rate of approximately 0.4% per h, similar to the rate observed for control cADPR, incubated in PBS containing 1% Triton (0.6% per h). Although the only degradation product of cADPR in PBS/Triton was ADPR, in the cell lysate the main degradation product was IMP, probably due to the activity of cell dinucleotide pyrophosphatases and deaminases on ADPR. This confirms our previous observation that ADPR is readily converted into both the adenylic and the inosinic nucleotide pools in erythrocyte hemolysates (32) as well as in lysates from lymphocytic cell lines.2 To monitor the metabolic fate of intracellular cADPR, we analyzed by HPLC the radioactivity incorporated into nucleotides at various times of culture of [3H]cADPR-loaded HeLa cells. [3H]cADPR was encapsulated into living HeLa cells at a starting concentration of 1.2 ± 0.4 µM (mean ± S.D. of three different experiments). The rate of degradation of the cyclic nucleotide was low and similar to that observed in cell lysates or in the presence of detergent (see above); the percentage of total radioactivity associated with the cADPR peak decreased by approximately 10% per day over 5 days of culture of the resealed cells. Most of the radioactivity incorporated into nucleotides other than cADPR co-eluted with ATP (76 ± 8%) and ADP (15 ± 5%, mean ± S.D. of three different experiments).

Cell Cycle Analysis-- Fig. 6 shows the results of BrdUrd-labeling experiments with CD38 sense- and antisense (control)-transfected HeLa cells. Immediately after pulse labeling with BrdUrd (time 0) the cells with significant green fluorescence (S phase cells) lay between two populations with no significant green fluorescence, G1 on the left and G2 on the right. All of the S phase cells were BrdUrd+. Cell distribution in the various cycle phases and the BrdUrd level were the same in control and CD38+ cells as demonstrated also by the DNA histograms.


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Fig. 6.   Cell cycle analysis of CD38 sense- and antisense-transfected HeLa cells. Exponentially growing HeLa cells were labeled for 30 min with 30 µM 5'-bromodeoxyuridine. At time 0 (immediately after pulse labeling) and after 4 h of culture without BrdUrd, cells were fixed and stained with anti-BrdUrd mAb and propidium iodide. A, control, antisense-transfected HeLa cells; B, CD38+ transfected HeLa cells. Top panels are biparametric BrdUrd/DNA cytograms; center panels are DNA histograms relative to the BrdUrd+ cells, and lower panels are DNA histograms of the total cell populations (BrdUrd+ + BrdUrd-). Traces are representative of three consistent experiments.

Four hours later, the BrdUrd+ populations were progressively moving through S phase into G2M, although with a different relative movement (RM) for control and CD38+ cells. As shown by bivariate BrdUrd/DNA analysis and BrdUrd+ cell cycle distribution, CD38+ cells were faster than controls in traversing the S phase. Duration of S phase was 8.4 and 12 h for CD38+ and control cells, respectively.

DNA histograms of sense- and antisense-transfected cells showed a similar distribution of total (BrdUrd+ + BrdUrd-) cells in the various cycle phases, both at time 0 and after 4 h of culture, confirming that both cell lines were exponentially growing during the experiment.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Transfection of a gene into cells that do not normally express it is a useful model to gain insight into the function(s) of the transfected gene product. Transient expression of wild type and mutant CD38 in mammalian cells has been obtained and used by some investigators to study the enzymatic properties of this glycoprotein (10, 33, 34). To our knowledge, transfection of CD38- cells with human wild type, full-length CD38 to study the metabolic consequences of its expression has not been reported until now.

We transfected and retrovirally infected human HeLa and murine 3T3 cells with human CD38, both wild type and a mutant, enzymatically defective form, to explore the intracellular modifications in [cADPR]i and [Ca2+]i possibly induced by CD38 expression and to correlate them with the levels of ectoenzyme activity. Retroviral infection proved to be a more effective method than transfection for CD38 gene transfer; surface cyclase activity on freshly infected, geneticin-resistant 3T3 and HeLa cells was significantly higher as compared with freshly transfected cells (Fig. 1). Low expression of CD38 on transfected cells, despite geneticin resistance, was not due to changes in methylation patterns of the transferred gene since culture of CD38+ 3T3 cells for up to 7 days in 5-10 µM 5-azacytidine failed to significantly increase surface cyclase activity or immunofluorescence positivity (not shown). Repeated magnetic selections with anti-CD38 mAb and anti-mouse Ig-coated magnetic beads enriched both transfected and infected cell populations for high CD38 expressing cells. Cloning of transduced cells, to select for high CD38 expression, was deliberately avoided in order not to unwillingly co-select for rapidly growing subclones. A sharp and rapid decrease in cyclase activity occurred in 3T3 cells following the first round of immunoselections, despite the fact that cells remained >90% positive to beading and >97% positive at immunofluorescence. Thus, the decline in cyclase activity was not due to loss of expression but to selection for lower expressing cells. Since the [Ca2+]i is known to control proliferation rate as well as apoptosis in a large number of cell types (35), it is possible that the latter event negatively selected for the cells with the highest CD38 expression and [Ca2+]i.

The cyclase activities expressed on the established, transduced CD38+ HeLa and 3T3 cell lines are in the range of physiological values for CD38+ cells as follows: Jurkat (1.0 ± 0.2 nmol/min/mg), Molt (1.25 ± 0.2), resting and activated Namalwa (3.3 ± 0.4 and 8.21 ± 0.6, respectively), and resting and activated peripheral blood lymphocytes (3.0 ± 0.2 and 11.5 ± 0.7).3 Both HeLa and 3T3 cells are responsive to the calcium releasing activity of cADPR; in permeabilized cells cADPR released Ca2+ from intracellular stores in both cell types (Fig. 2). The effect of cADPR was specific (inhibited by preincubation with 8-amino-cADPR) and subject to desensitization. This is the first demonstration that 3T3 cells are responsive to cADPR; although HeLa cells have been already described to express type II ryanodine receptor (36), 3T3 cells are reported not to express any known type of ryanodine receptor (36). In addition we found that repeated Ca2+ loading of permeabilized 3T3 cells eventually resulted in Ca2+ release from intracellular stores,4 this strongly suggesting a Ca2+-induced Ca2+ release mechanism. This fact and responsiveness of permeabilized 3T3 cells to cADPR seem to imply the occurrence in these cells of an unidentified type of cADPR receptor. In this respect, a recent report provides evidence for cADPR-induced, ryanodine receptor-independent calcium release from dog cardiac sarcoplasmic reticulum (37).

Due to the physiological levels of the cyclase activity expressed on transduced HeLa and 3T3 cells and to responsiveness of both cell types to cADPR in terms of Ca2+ release, these cell lines proved to be an appropriate model to investigate the metabolic consequences of CD38 expression. Table I shows that expression of cyclase activity correlated with appearance of [cADPR]i, which was undetectable in wild type and antisense-transduced cells, and with decrease in [NAD+ + NADH]i in CD38+ cells compared with control (antisense-transduced) CD38- cells. The fact that the [ATP]i was similar in CD38+ and CD38- cells (not shown) suggests that the decrease in pyridine dinucleotide content is not due to defective synthesis. Rather, CD38 substrate consumption and product accumulation point to an intracellular enzymatic activity of transfected CD38. This conclusion is further supported by the observation that expression in 3T3 cells of a mutant type of CD38, displaying only 3% of wild type ADP-ribosyl cyclase activity, failed to determine any modifications of [NAD+ + NADH]i, as compared with CD38- cells, whereas intracellular cADPR was undetectable (Table I). Therefore, expression of CD38 results in intracellular cADPR production. This is also confirmed by the fact that Namalwa cells, which show ectocellular cyclase levels similar to the CD38+ 3T3 cells of this study, also have comparable [cADPR]i (17). The mechanism whereby NAD+ gains access to the apparently secluded catalytic domain of CD38 in (exo/endocytotic) vesicles remains undetermined. Intracellular production of cADPR determined an elevation of the basal [Ca2+]i, which was highest in cells expressing the highest value of surface cyclase activity (Table I). This demonstrates that [cADPR]i in the transduced cells is in the range of functionally significant values and that cADPRi does have accessibility to its receptors on intracellular calcium stores. Finally, cell doubling time was demonstrated to be shorter in CD38+ cells, as compared with controls, and again higher cyclase activity correlated with higher [Ca2+]i and shorter cell doubling time. 3T3 cells expressing mutant CD38 showed a [Ca2+]i and a doubling time comparable to controls.

In an effort to better characterize the perturbations elicited by [cADPR]i on the individual cell calcium compartments, total cell calcium and thapsigargin-releasable Ca2+ were measured in CD38 sense (CD38+) and antisense (CD38-) infected 3T3 and HeLa cells. Thapsigargin is a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca2+-ATPases that causes the emptying of the ER calcium stores (38). Although no difference in total cell calcium content was apparent between CD38+ and CD38- HeLa or 3T3 cells, a marked decrease in the ER Ca2+ stores was observed in CD38+ cells (Table II and Fig. 3). Concomitantly, CD38+ cells showed a consistent elevation of cytosolic free calcium concentration over the levels measured in the corresponding CD38- cells. Store depletion and elevation of cytosolic free Ca2+ concentration in CD38+ cells caused the percentage of cytosolic Ca2+ to increase from 7 to 43% of ER deposits in CD38+ HeLa cells and from 5 to 18% in 3T3 cells (Table II). Since expression of CD38 did not cause significant perturbations of Ca2+ fluxes at the plasma membrane level (Fig. 4), these differences reflect an ongoing stimulation of Ca2+ release from ER stores in CD38+ cells.

The experiments of reversible permeabilization of wild type HeLa cells showed that encapsulation of cADPR in these cells results in the same functional consequences afforded by expression of CD38 (Fig. 5). Accordingly, a causal relationship was established between [cADPR]i, increased [Ca2+]i, and increased proliferation rate. Due to lack of CD38 expression in wild type HeLa cells, exogenously added cADPR is expected to be stable for a relatively long time; this molecule is known to be resistant to dinucleotide pyrophosphatase(s) (26) and is cleaved only by the hydrolase activity of CD38. Indeed, substantial stability of cADPR both in HeLa cell lysates and inside living wild type HeLa cells could be demonstrated. At the same time, as shown in Fig. 5, a long lasting (20-120 h) elevation of the [Ca2+]i occurred in cells loaded with cADPR over controls (ADPR-loaded); the slow decrease of [Ca2+]i after the initial doubling over control values (recorded approximately 2 h after resealing) suggests progressive dilution of the encapsulated cADPR due to cell division. Furthermore, cADPR-containing cells showed an increased proliferation rate, as indicated by the double amount of total protein and cell number measured in cultures from cADPR-loaded cells as compared with ADPR-loaded controls 5 days after resealing (Fig. 5).

Cell cycle analysis of BrdUrd-labeled, exponentially growing CD38 sense- and antisense-transfected HeLa cells (Fig. 6) demonstrated a significant reduction of the S phase duration in CD38+ cells as compared with controls (8.4 and 12 h, respectively). Recently, oscillations of ADP-ribosyl cyclase activity during cell cycle have been reported in the unicellular protist Euglena gracilis, and participation of cADPR in the Ca2+-mediated regulation of cell cycle progression in G2/M transition phase has been proposed (39). The present observations demonstrate that cADPR may play a role in the regulation of cell cycle progression also in mammalian cells.

In conclusion, this study provides evidence that, despite the eventual ectocellular localization of plasma membrane-bound CD38, both cADPR metabolism and its functional consequences on Ca2+i levels do occur intracellularly. Expression of CD38 in HeLa and 3T3 cells is associated with changes of intracellular calcium homeostasis (increased cytosolic free calcium concentration and depletion of calcium stores) that quantitatively parallel the intracellular cADPR concentration and the ectocellular expression of cyclase activity. Accessibility of the NAD+/NADH pool to intracellular CD38 and of cADPRi to its receptors is functionally demonstrated, and a causal relationship is established between increased [cADPR]i, elevated [Ca2+]i, and higher proliferation rate. These results may allow a deeper understanding of the functional significance of the discontinuous pattern of CD38 expression observed during lymphocyte and hematopoietic stem cell differentiation (1).

    ACKNOWLEDGEMENTS

We are indebted to Profs. L. Alberghina and G. Corte for helpful suggestions.

    FOOTNOTES

* This study was supported in part by the Italian Ministry of University and Scientific and Technological Research (MURST), by the Associazione Italiana per la Ricerca sul Cancro (AIRC), and by the CNR Target Project on Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Institute of Biochemistry, University of Genova Viale Benedetto XV No. 1, 16132 Genova, Italy. Tel.: 39-10-3538151; Fax: 39-10-354415; E-mail: ezocchi{at}unige.it.

1 The abbreviations used are: cADPR, cyclic ADP-ribose; ADPR, adenosine diphosphate ribose; NGD+, nicotinamide guanine dinucleotide; cGDPR, cyclic GDP-ribose; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; SLO, streptolysin O; [Ca2+]i, ionized cytosolic calcium; BrdUrd, bromodeoxyuridine; mAb, monoclonal antibody; TdR, thymidine; RM, relative movement; ER, endoplasmic reticulum.

2 L. Guida, unpublished data.

3 S. Bruzzone, unpublished data.

4 L. Franco, unpublished data.

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Abstract
Introduction
Procedures
Results
Discussion
References

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