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Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors

MARINA PODESTÀ^*,1, ELENA ZOCCHI^,1, ANNA PITTO^*, CESARE USAI, LUISA FRANCO^,2, SANTINA BRUZZONE, LUCREZIA GUIDA, ANDREA BACIGALUPO^*, DAVID T. SCADDEN^§, TIMOTHY F. WALSETH^**, ANTONIO DE FLORA³ and ANTONIO DAGA^,1

^* Department of Hematology, S. Martino Hospital, Genova, Italy;
Department of Experimental Medicine, Section of Biochemistry, University of Genova, Italy;
Institute of Cybernetics and Biophysics, National Research Council, Genova, Italy;
^§ Department of Experimental Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA;
^** Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, USA; and
IST, National Institute for Cancer Research, Genova, Italy

³Correspondence: Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV/1, 16132 Genova, Italy. E-mail: toninodf{at}unige.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD⁺ (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 µM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3-deaza-cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.—Podestà, M., Zocchi, E., Pitto, A., Usai, C., Franco, L., Bruzzone, S., Guida, L., Bacigalupo, A., Scadden, D. T., Walseth, T. F., De Flora, A., Daga, A. Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors.

Key Words: bone marrow cells • intracellular calcium homeostasis • cytokine-like activity of cyclic ADP-ribose • expansion of hemopoietic progenitors

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CYCLIC ADP-RIBOSE (CADPR) is a powerful regulator of calcium mobilization from intracellular stores. In sea urchin eggs, where it was first described (1) , it is synthesized from NAD⁺ by a soluble and a membrane-bound ADP-ribosyl cyclase (2) . Several observations indicate involvement of cADPR in the calcium-controlled events taking place during the process of egg fertilization, completion of the oocyte meiotic cycle, and the first stages of cell division in invertebrates (3 , 4) . Moreover, oscillations of ADP-ribosyl cyclase activity and of cADPR concentration during cell cycle progression have also been reported in the unicellular protist Euglena gracilis (5) . These observations point to an ancient evolutionary role of cADPR as a calcium-releasing signal involved in the control of cell proliferation.

In vertebrates, cADPR has been identified in most tissues (6) ; two ADP-ribosyl cyclases, CD38 and BST-1, have been described so far: CD38 is a type II transmembrane glycoprotein (7 , 8) , whereas BST-1 is a glycosyl-phosphatidylinositol-anchored protein (9) . Both are bifunctional ectoenzymes, catalyzing the synthesis and hydrolysis of cADPR at their ectocellular domain. The widespread tissue distribution of cADPR in mammals suggests a role for this nucleotide in calcium-controlled, tissue-specific cell functions that include secretion, contraction, cell proliferation, and apoptosis (10) . Indeed, extracellularly added cADPR has been demonstrated to release calcium from ryanodine-sensitive intracellular stores in a wide range of permeabilized cell types (11) and to elicit tissue-specific functional responses in permeabilized ß-pancreatic cells (12) , smooth muscle myocytes (13) , and oocytes (14) . Observations reporting effects of extracellular cADPR on intact cells are less abundant; however, cADPR has been shown to increase cytokine-induced B lymphocyte proliferation (15) and depolarization-induced elevation of the intracellular free calcium concentration ([Ca²⁺]_i) in cerebellar neurons (16) .

Recently, de novo expression of CD38 in CD38^- cells was demonstrated to induce a shortening of the cell cycle, an effect that was shown to be causally related to the intracellular production of cADPR and to the consequent increase of the [Ca²⁺]_i (17) . This observation suggests that cADPR may also play a role in the regulation of cell cycle progression in mammalian cells.

Both mammalian cyclases are expressed in the bone marrow (BM): CD38 on hemopoietic cells (7 , 18) and BST-1 on stromal cells (9) ; the presence of NAD⁺ in plasma and interstitial fluids (16) could enable production of extracellular cADPR in the BM microenvironment. Taken together, these observations suggest a possible role of the CD38/cADPR system in the regulation of hemopoiesis. Thus, we investigated the effect of extracellular cADPR on the proliferation and on the [Ca²⁺]_i of human hemopoietic progenitors originating from different sources: cord blood, bone marrow, or peripheral blood. Results obtained support the conclusion that cADPR is indeed a hitherto unrecognized signal molecule in hemopoiesis, controlling [Ca²⁺]_i and proliferation in hemopoietic progenitors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nucleotides
cADPR and [³H]-cADPR were prepared enzymatically from NAD⁺ and [³H]-NAD⁺, respectively, with Aplysia californica cyclase (courtesy of Prof. H. C. Lee, Minneapolis, Minn.) and high-performance liquid chromatography (HPLC) purified. Recombinant CD38 was kindly provided by Prof. H. C. Lee. 8-N₃-NAD⁺ was synthesized as described (19) and reduced to 8-NH₂-NAD⁺ with DTT; 8-NH₂-cADPR was then obtained by incubation with Aplysia cyclase and HPLC purified; 3-deaza-cADPR was synthesized as described (20) . ADP-ribose (ADPR) was obtained from Sigma (Milan, Italy).

Samples
Cord blood (CB) samples were collected from umbilical cords into tubes containing preservative-free heparin after normal full-term deliveries. Maternal informed consent was obtained in all cases.

Normal bone marrow cells were obtained from donors providing marrow for allogeneic transplantation. Leukapheresis samples were obtained from normal donors providing peripheral blood (PB) progenitor cells for allogeneic transplant after a short course of granulocyte colony-stimulating factor. All donors gave informed consent.

Mononuclear cells (MNC) from CB, BM, and PB were separated by density gradient centrifugation on Ficoll-Hypaque (Sigma). After separation, cells were washed twice in Iscove’s modified Dulbecco’s medium (IMDM) containing 5% fetal calf serum (FCS) and resuspended in IMDM containing 10% FCS, penicillin 100 U/ml, streptomycin 100 µg/ml, and glutamine 2 mM (complete medium). The cADPR-hydrolase activity of freshly isolated MNC was assayed as described (17) .

CD34⁺ cell enrichment and CD34⁺38^low separation
Immunoaffinity purification of CD34⁺ cells was used to enrich MNC in colony-forming cells (CFC) and early progenitors (21) . CD34⁺ cell enrichment was performed by positive selection using the miniMACS immunomagnetic separation system (Miltenyi Biotec GmBH, Germany). Briefly, PB- or CB-derived MNC were suspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 5 mM EDTA, and CD34⁺ cells were separated using anti-CD34 antibody (clone: QBEND/10; isotype: mouse IgG1) and colloidal superparamagnetic beads. After labeling, the cell suspension was passed through a column held within a magnetic field causing CD34⁺ cells to be retained in the column. Purified CD34⁺ cells were then collected after removal of the column from the magnet and washing with buffer.

Enrichment of the MNC in the earliest HP was obtained by negative selection of the CD34⁺38^low cells (22) using the StemSep Separation System (StemCell Technologies Inc.). Cells were labeled for magnetic depletion by incubation with a mixture of tetrameric antibody complexes (anti-CD38, CD66b, CD19, CD2, CD45RA, CD16, CD24, CD3, CD36, CD14, CD56, glycophorin A) and magnetic dextran iron particles. The cell suspension was then passed through a high-gradient magnetic column of stainless steel mesh. The magnetically labeled cells bind to the column whereas the unlabeled cells, containing the most immature HP (CD 34⁺38^low), pass through; these cells do not have antibodies bound to their surface and are suitable for further functional studies.

Liquid cultures
Incubations were performed in a total volume of 1 ml complete medium (see above) with 10⁶ MNC/ml for 24 h in a humidified, 5% CO₂ atmosphere at 37°C. cADPR or ADPR (100 µM final concentration) was added once at the beginning of the incubation period. When the incubation time exceeded 24 h (2, 5, 9, 15 days), cADPR or ADPR was added twice a week and different tubes were used for each time point.

3-deaza-cADPR, a nonhydrolyzable cADPR analog (20) , was used under the same culture conditions described above at a final concentration of 1 to 1000 nM and was added only at the beginning of the incubation.

Semisolid colony growth assay
After liquid culture, an appropriate number of MNC or CD34⁺-enriched cells were plated in methylcellulose medium supplemented with a mixture of hemopoietic growth factors (Methocult; Stem Cell Technologies, Vancouver, B.C., Canada). After 14 days of incubation in a humidified, 5% CO₂ atmosphere at 37°C, colonies were scored using an inverted microscope, applying standard criteria for their identification.

Thereafter, an appropriate number of the recovered cells (usually 1/30–1/10 from the first generation and 1/10–1/2 from subsequent generations) was replated in complete methylcellulose medium for assessment of the replating efficiency (23) . The total number of CFC grown in each generation (14 days) was calculated by dividing the number of scored colonies by the fraction of the total cell number recovered from the previous generation(s) that was plated. In each experiment, the sum of the colonies grown throughout the generations from cADPR-primed HP was then divided by the sum obtained in the respective control to calculate the expansion factor.

Cytosine arabinoside susceptibility of CB MNC
Cytosine arabinoside (c-ARA) was used to determine the proportion of cycling CFC, as described (24) . MNC (10⁶/ml) were incubated at 37°C in a humidified 5% CO₂ atmosphere for 24 h in complete medium (containing 20% FCS) with or without 1 µM c-ARA and subsequently washed twice in IMDM containing 5% FCS. Incubation with or without cADPR (100 µM) was performed before or after c-ARA treatment, under the liquid culture conditions described above, for 24 h. Thereafter, cells were washed, counted, and plated in semisolid medium for assay of CFC at a concentration of 10⁴ MNC/plate.

Fluorimetric determination of the [Ca²⁺]_i
CB-derived MNC (10⁷/ml) were incubated with 6 µM Fura 2 acetoxymethyl ester (FURA 2 AM) in complete culture medium for 45 min at 37°C, washed with standard saline (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose and 5 mM HEPES, pH 7.4), and resuspended in the same solution at 0.5 x 10⁶/ml. Measurements were performed at room temperature in a 2 ml cuvette under continuous stirring. Parameter values for the calculation of the [Ca²⁺]_i were obtained as described in detail (17) . The frequency of recording was 1 point/s.

Due to the small number of CD34⁺/CD38^± cells that can be recovered from CB samples, calcium measurements on these subpopulations were performed on cells settled on glass coverslips (3–4 cells per field), using the same experimental setting described previously (17) , except that measurements were performed under conditions of stopped flow. Briefly, 20 µl of FURA 2 AM-loaded cells (10⁶/ml) were seeded on 20 mm glass coverslips. After 15 min, the specimen was mounted in a 200 µl chamber on the stage of an inverted microscope (Zeiss IM35, Germany) and the chamber was slowly filled with 200 µl of standard saline taking care not to disturb the settled cells. All measurements were performed in Ca²⁺-free external solutions.

Determination of intracellular cADPR concentration
The intracellular cADPR concentration was determined by HPLC analysis on neutralized trichloroacetic acid (TCA) cell extracts. MNC were incubated at 1.5 x 10⁷/ml in complete medium in the presence of 1 mM cADPR at 37°C. After 10 min, 1 h, or 24 h, 3 x 10⁷ cells were washed four times in PBS at 25°C; the cell pellet was resuspended in 300 µl of water, sonicated, and TCA extracted. After removal of excess TCA with diethyleter, 2 x 10³ cpm of internal standard [³H]cADPR was added to the cell extract, which was subjected to the first HPLC analysis on an anion exchange PL-1000 SAX column (HP, Milan, Italy) (25) . An aliquot of each 0.5 min fraction was counted in a ß-counter and the fractions containing the radioactive cADPR standard were pooled, lyophilized, and redissolved in 0.5 ml of 20 mM TrisHCl, pH 6.5. Recombinant CD38 (4 µg/ml) was added to one 0.25 ml aliquot and both samples were incubated at 37°C for 12 h After TCA extraction, both incubations were analyzed on a reverse phase Hypersil C18 column (HP) (26) and the radioactivity of the 0.3 min fractions was determined. The identity of the cADPR peak in the cell extract was confirmed by coelution with the radioactive standard, by comparison of the absorbance spectrum with a computer-stored standard and by an absence of the corresponding peak in the CD38-hydrolyzed sample. The concentration of intracellular cADPR per 10⁸ cells was calculated from the area of its HPLC peak and taking into account the percentage of nucleotide recovery obtained with the radioactive standard.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Selection of the protocol for cADPR treatment
To investigate a possible effect of cADPR on the proliferation and on the [Ca²⁺]_i of hemopoietic progenitors, CB-derived MNC (10⁶ cells in 1 ml complete medium) were exposed to increasing concentrations of the cyclic nucleotide (1, 10, 100, and 1000 µM) for 24 h in liquid culture. Controls were untreated cells as well as cells incubated with ADPR, i.e., the product of the cADPR-hydrolase activity expressed on the surface of CB MNC. This hydrolase activity (0.014±0.006 nmol/min/10⁶ cells) would allow extracellularly added cADPR (1, 10, 100, and 1000 nanomol) to last ~1 h, 12 h, 5 days, and 50 days, respectively, under the culture conditions described above. After incubation with cADPR, cells were plated in semisolid medium for assay of CFC number. No effect of ADPR on CFC growth was observed, as compared to untreated cells, at any of the concentrations tested. Conversely, cADPR increased CFC output in a concentration-dependent way, with 100 and 1000 µM inducing a significantly (P<0.01) higher clonogenic growth compared to controls (1400 and 2800 vs. 700 CFC/ml, respectively; n=3). The lowest cADPR concentrations tested, 1 and 10 µM, were ineffective. The fact that they would be hydrolyzed within 12 h culture of the cells suggested that 12–24 h exposure to cADPR was necessary to stimulate cell growth.

To test the effect of the length of cADPR exposure on CFC growth, we prolonged the liquid culture up to 15 days; cADPR or ADPR was added every three days to maintain the nucleotide concentration approximately constant. At each time point (1, 2, 5, 9, and 15 days) cells were counted and plated in semisolid medium. cADPR induced a variable increment of MNC but a remarkable expansion of CFC at all time points tested (Fig. 1 ), with the most significant difference between cADPR-treated and control progenitors being recorded after 15 days (P<0.002). However, the net CFC output decreased significantly over time, in control as well as in cADPR-treated cultures. Moreover, repeated additions to the liquid cultures could by themselves affect cell growth/survival.

View larger version (16K): [in this window] [in a new window]   Figure 1. CFC output of CB MNC after different times of cADPR incubation. Cells were cultured in complete medium with or without 100 µM cADPR or ADPR for the times indicated and subsequently plated in semisolid medium. Results are expressed as CFC number/ml of liquid culture suspension. Each point is the median value of 7–9 experiments. ) cADPR; ) ADPR; •) untreated.

Finally, in the absence of growth factors cADPR did not produce any colony growth when added directly to the semisolid medium.

These preliminary observations allowed us to devise the protocol for cADPR treatment; the lowest cADPR concentration (100 µM) sufficient to survive degradation by the hydrolase activity of 10⁶ MNC/ml, without the need for repeated additions, and the shortest incubation time necessary to stimulate cell growth (24 h) were the culture conditions subsequently used.

Stimulatory effect of cADPR on the growth of CFC
In all experiments (n=25), extracellular 100 µM cADPR significantly enhanced CFC output from CB MNC: the median was 5000 CFC/ml (range 720–17,340) compared with ADPR-treated cells, median 2141 (range 600–13,120; P<0.01), or with untreated cells, median 2900 (range 450–12,670; P<0.01) (Table 1 ). The wide range in the absolute numbers reflects the variability of the proliferative capacity of CFC from different CB samples (27) ; however, in each experiment cADPR induced a higher CFC output compared to controls (Table 1) . On the other hand, the number of cells recovered after liquid culture was not significantly higher in cADPR-treated cells compared to controls (cADPR- vs. ADPR- and cADPR- vs. un-treated cells, P=0.4). To correlate the functional effect to cADPR itself, we also assayed the nonhydrolyzable cADPR analog 3-deaza-cADPR on CFC output. Different concentrations (1, 10, 100, and 1000 nM) were tested under the same culture conditions described above. 3-deaza-cADPR induced a significant increase of the CFC output compared to control, untreated cells at each concentration tested (P<0.04); thus, 1 nM of the analog produced 95% of the effect induced by 1 µM, with medians of 2050 and 2200 CFC/ml, respectively, compared to a median of 1150 obtained with the control (n=5).

The experimental protocol described above (10⁶ MNC/ml, 100 µM cADPR for 24 h) was also tested on bone marrow-derived and on peripheral blood-derived clonogenic progenitors. Higher CFC numbers grew from BM (n=3) and from PB (n=2) after cADPR treatment as compared to the respective controls, incubated with ADPR; medians were 2660 vs. 1500 for BM-derived and 2750 vs. 1460 for PB-derived CFC/ml. These results demonstrate that the effect of cADPR is not restricted to ontogenetically immature (CB-derived) progenitor cells.

Finally, the effect of cADPR treatment on colony size and type was analyzed. Colonies arising from cADPR-primed MNC were significantly larger than ADPR-treated or untreated controls (Table 2 ), indicating an increased proliferation rate within the single colony. The increase of CFC output was statistically significant for all colony types, i.e., colony-forming units granulocyte macrophage (CFU-GM) (²=28.5; <0.005), burst-forming units erythroid (BFU-E) (²=27.5; <0.005), and multipotential colony-forming units (CFU-GEMM) (² = 7.1; <0.01), indicating a stimulatory effect of cADPR on all hemopoietic lineages.

Increased [Ca²⁺]_i in CB MNC incubated with cADPR or 3-deaza-cADPR
The same cADPR and 3-deaza-cADPR concentrations used in the liquid culture experiments described above were tested for their effect on the [Ca²⁺]_i of CB MNC. The [Ca²⁺]_i in freshly isolated CB MNC was found to be 20.3 ± 1.8 nM (n=20). Closely comparable values were obtained with cells in suspension or on glass coverslips (see Materials and Methods). Upon incubation with 100 µM cADPR, or 10 nM 3-deaza-cADPR the [Ca²⁺]_i increased in a time-dependent manner (Fig. 2 ) up to 82.6 ± 3.4 nM and 96.4 ± 5.0 nM for cADPR and 3-deaza-cADPR, respectively, after 24 h (n=7). To simulate a constant supply of micromolar cyclic nucleotide, repeated additions of 1–10 µM cADPR were performed during 24 h liquid culture; this treatment determined a final [Ca²⁺]_i increase in the MNC similar to that observed after a single addition of 100 µM cADPR (not shown). Upon removal of cADPR by repeated washings of the cells, the [Ca²⁺]_i decreased progressively to 160% and 110% of basal values after 24 and 48 h, respectively, probably due to the CD38-catalyzed hydrolysis of cADPR. On the other hand, calcium levels remained unchanged in 3-deaza-cADPR-treated cells for at least 48 h after removal of the nucleotide (Fig. 2) , probably reflecting the insensitivity of the cADPR analog to the intracellular hydrolase activity of CD38 (20) .

View larger version (11K): [in this window] [in a new window]   Figure 2. [Ca2+]i of CB MNC incubated with cADPR or 3-deaza-cADPR. [Ca2+]i was determined on FURA 2-loaded cells at different times during incubation with 100 µM cADPR () or 10 nM 3-deaza-cADPR () and 24 and 48 h after removal of the cyclic nucleotide (arrow). Each point is the mean ± SD of at least 7 determinations obtained from independent experiments.

The time course of the [Ca²⁺]_i increase in 3-deaza-cADPR-incubated cells (Fig. 2) can be described by the following three-parameter, single exponential regression curve:

where [Ca²⁺]_i (t=0) is the intracellular free calcium concentration before incubation, _max [Ca²⁺]_i is the maximal calcium change and is the time constant of the process, i.e., the time required to reach 65% of the maximal value. The experimental data are best fitted by the parameters [Ca²⁺]_i (t=0) = 21.1 ± 0.6 nM, _max [Ca²⁺]_i = 74.0 ± 0.7 nM, and = 6.5 h, with R = 0.999 (P<0.0001). This result suggests that the increase of [Ca²⁺]_i is the result of a single rate-limiting event, described by the curve itself, rather than the sum of multiple steps having different kinetics. Moreover, a time constant of the order of hours strongly suggests a slow influx of the cADPR analog into the cells rather than binding to a hypothetical membrane receptor, followed by signal-transducing events, eventually leading to calcium release from internal stores.

To elucidate whether slow cADPR influx across the cell membrane was the rate-limiting event, we followed the calcium increase in real time upon addition of cADPR to intact or to permeabilized MNC cells. Whereas addition of 100 µM cADPR to digitonin-permeabilized cells elicited immediate and considerable calcium release (Fig. 3 , inset), intact cells responded with a slow and progressive increase of [Ca²⁺]_i detectable over a 30 min time span (Fig. 3) . The same rate and extent of calcium increase were observed upon addition of 10 nM 3-deaza-cADPR (Fig. 3) . The fact that these calcium increases were obtained in zero-calcium external solutions rules out any influx of extracellular Ca²⁺.

View larger version (18K): [in this window] [in a new window]   Figure 3. Time course of [Ca2+]i increase in CB MNC exposed to cADPR or 3-deaza-cADPR: effect of cell permeabilization. The [Ca2+]i was continuously monitored for 30 min after addition (time zero) of 100 µM cADPR () or 10 nM 3-deaza-cADPR () to FURA 2-loaded CB MNC suspended in zero calcium external solution. ) Untreated cells. Traces shown are representative of 3 consistent experiments. Each point is the mean of the values (=200) recorded in 2 min. Inset: trace from a representative experiment showing the effect on [Ca2+]i of the addition of 100 µM cADPR to digitonin-permeabilized (17) CB MNC.

Determination of intracellular cADPR concentration
To achieve direct evidence for cADPR influx across the cell membrane, we investigated the presence of the cyclic nucleotide in TCA extracts of MNC cultured in the presence of 1 mM cADPR for different periods. No cADPR was detectable in extracts of cells incubated with cADPR for 10 min, indicating that the repeated washings of the cells were adequate to remove all extracellular and/or surface-bound nucleotide. After 1 h incubation of the cells with cADPR, however, the cyclic nucleotide became detectable in cell extracts, and after 24 h its intracellular concentration was 0.55 ± 0.16 nanomol/10⁸ cells. These values are in the range of reported intracellular cADPR concentrations in CD38⁺ human lymphoid and myeloid cell lines (28 , 29) .

Inhibitory effect of cADPR antagonists on CFC growth and on [Ca²⁺]_i
In an attempt to causally correlate the increase of [Ca²⁺]_i with the growth-promoting effect induced by cADPR, CB MNC were incubated with a specific cADPR antagonist, 8-NH₂-cADPR, prior to exposure to cADPR. Pretreatment of the cells with 8-NH₂-cADPR (10 µM) for 24 h, although not affecting cell viability during the liquid culture, completely inhibited colony growth in the subsequent 2 wk, eventually leading to cell death in the semisolid medium; the median was zero CFC/ml (no growth was ever observed) compared to a median of 3400 (range 2000–3800) for untreated controls (n=3). Prolonging the liquid culture for further 24 h with the addition of cADPR (100 µM) did not restore colony growth. However, the presence of a 10- or 100-fold excess of cADPR (100 µM or 1 mM) together with 10 µM 8-NH₂-cADPR in the liquid cultures for 24 h reduced or abolished, respectively, the inhibitory effect of the cADPR antagonist on colony growth. Mean values calculated from closely comparable results were 2800, zero, and 1200 CFC/ml for control, 8-NH₂-cADPR, and 8-NH₂-cADPR plus 10-fold excess cADPR (n=3) and 2600, zero, and 2500 CFC/ml for control, 8-NH₂-cADPR, and 8-NH₂-cADPR plus 100-fold excess cADPR (n=3), respectively. Similar results, in terms of cADPR-induced recovery from inhibition of colony growth, were obtained with another cADPR antagonist, 8-N₃-cADPR.

Incubation of the cells with 10 µM 8-NH₂-cADPR for 24 h resulted in a significant decrease of the basal [Ca²⁺]_i from 20 ± 2 to 14 ± 1 nM (n=3) and in a consistent increase of the amount of Ca²⁺ releasable by the calcium ionophore A23187, compared to untreated control cells, indicating an increased repletion state of microsomal stores in 8-NH₂-cADPR-treated cells.

Moreover, 8-NH₂-cADPR completely inhibited the calcium-releasing activity of cADPR on CB MNC. Specifically, preincubation of the intact cells for 2 h in the presence of 100 µM 8-NH₂-cADPR completely inhibited calcium increase induced by the subsequent addition of 100 µM cADPR for 4 h; the [Ca²⁺]_i values measured after exposure to cADPR were 20 nM in the in 8-NH₂-cADPR-preincubated and 58 nM in the untreated cells (n=2).

Stimulatory effect of cADPR on growth and on [Ca²⁺]_i of CD34⁺-enriched MNC
To minimize a possible influence of accessory cells in mediating the stimulation of CFC growth by cADPR, the effect of the cyclic nucleotide was tested on CD34⁺-enriched fractions (10⁶ cells/ml) derived from either PB (n=2) or CB (n=2). The CD34⁺ subpopulation, representing 0.1–0.5% of the total nucleated cells derived from PB or CB, contains the earliest and committed precursors for all hemopoietic lineages (21) . A stimulatory effect of cADPR (100 µM for 24 h) was observed on the growth of CFC from these HP-enriched subpopulations: cADPR-treated (median 121,000 CFC/10⁶ MNC, range 1800–151,000) vs. ADPR-treated (median 18,000, range 360–70,000, P<0.01) and untreated cells (median 16,000, range 800–23,000, P<0.01) (n=6). In parallel, the effect of cADPR on the [Ca²⁺]_i of CD34⁺ MNC from CB was investigated; the kinetics of calcium increase after addition of 100 µM cADPR to cells settled on glass coverslips, as monitored in real time on 3–4 cells per field, closely paralleled those of total MNC shown in Fig. 3 . Incubation with 100 µM cADPR for 24 h resulted in an increase of the [Ca²⁺]_i from 20 nM to 81 nM and to 93 nM in CD34⁺/38⁺ and CD34⁺/38^low subpopulations, respectively (mean of two experiments, giving closely comparable results). Moreover, the [Ca²⁺]_i remained unchanged in CD34⁺/38^low MNC for at least 24 h after removal of external cADPR through repeated washings, in agreement with the lack of hydrolase activity in this MNC subpopulation.

Increased replating efficiency of cADPR-primed hemopoietic progenitors
To assess whether a short exposure to cADPR (100 µM for 24 h) might have long-term effects on hemopoietic progenitors, we investigated the replating efficiency of cADPR-primed CB MNC. Indeed, cADPR-primed progenitors were found to be able to grow for multiple generations (up to four) when replated, whereas control, untreated cells never produced any colony beyond the second generation. The expansion factor (see Materials and Methods) for cADPR-primed CFC compared to control was calculated to be between 10 and 700 (Table 3 ).

Moreover, second generation colonies arising from cADPR-primed (100 µM for 24 h) MNC were found to contain mostly elements with the morphology of undifferentiated cells: indented nucleus with sharp chromatin and high nuclear/cytoplasmic ratio. On the contrary, the majority of the cells from control second-generation colonies were terminally differentiated, mature macrophages with foaming cytoplasms and pyknotic nuclei (Fig. 4 ).

View larger version (110K): [in this window] [in a new window]   Figure 4. Secondary cord blood cultures: cytological characteristics of cADPR-primed and control cells. Second-generation colonies grown from cADPR-primed (100 µM for 24 h) or control (untreated) CB MNC were pooled and an aliquot of the cells was stained with May-Grünwald Giemsa. The rest of the cells were plated again in semisolid medium. Although no further colony growth was observed in the control, cADPR-primed cells produced third- and fourth-generation colonies in the subsequent 5 wk. A) Cells from cADPR-primed colonies, showing two differentiated macrophages surrounded by a number of smaller, undifferentiated cells; B) cells from untreated, control colonies. Original magnification: 600x.

Cytosine arabinoside-susceptibility of the cADPR-sensitive CFC
To characterize the cycling status of the cADPR target cells in the CFC assay (24) , we tested whether the cADPR-sensitive cells were c-ARA sensitive. When c-ARA-pretreated CB MNC (1 µM for 24 h) were incubated with cADPR (100 µM for 24 h), the colony number was not significantly different from controls incubated without cADPR: the corresponding medians were 18 vs. 21 per 10⁴ plated MNC, P=0.7 (n=8). Conversely, the colony number after cADPR incubation alone was significantly higher compared with controls (39 vs. 23/10⁴ MNC; P<0.02) and with colonies exposed to c-ARA, followed by cADPR (39 vs. 18/10⁴ MNC; P<0.004). Similar results (i.e., complete inhibition of the growth-stimulating effect of cADPR by c-ARA) were obtained when cADPR-treatment preceded c-ARA incubation (not shown). Thus, in the liquid culture conditions used for the CFC assays (100 µM cADPR for 24 h), the cADPR-sensitive cell is cycling, i.e., c-ARA sensitive.

On the contrary, c-ARA pretreatment of CB MNC (1 µM for 24 h) did not affect the increased replating efficiency produced by cADPR priming and recorded over several weeks (not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Results reported here demonstrate that extracellularly added cADPR stimulates the proliferation of human hemopoietic progenitors and induces an expansion of CFC via its intracellular calcium-releasing activity. cADPR seems to act on cells at different differentiation stages and possibly in different cell cycle phases. Indeed, a 24 h priming of MNC (obtained from CB, PB, or BM) induces an increase in CFC output and colony size, as scored in the ensuing 2 wk (Tables 1 and 2) , an effect completely abolished by c-ARA pretreatment of the cells. Thus, this assay highlights the stimulatory effect of cADPR on the proliferative potential of a cycling CFC. However, the same protocol of cADPR treatment (24 h with 100 µM cADPR) also induces a marked increase of the replating efficiency of CFC, generating a 10- to 700-fold expansion of the total CFC number compared to control. Since this effect is not inhibited by c-ARA pretreatment of the cells, this assay demonstrates a stimulatory effect of cADPR on a noncycling hemopoietic progenitor. Prolonging the cADPR incubation time for up to 15 days in liquid culture increases the statistical significance of its effect compared to control cultures (Fig. 1) ; in principle, this increased CFC output could result from 1) an increased CFC viability (e.g., by protection from apoptosis), an effect not detectable in the 24 h priming protocol, 2) expansion of CFC, or 3) recruitment into cell cycle of immature progenitors, unable to form colonies at the beginning of the culture (30) .

The stimulatory effect of cADPR on HP growth is not mediated by accessory cells (e.g., through an increased production of endogenous cytokines) because it was observed on total MNC as well as on the CD34⁺ subpopulation (markedly enriched in both the committed and early hemopoietic progenitors). That cADPR itself is responsible for these effects is indicated by the lack of activity of ADPR and by the stimulatory effect of a nonhydrolyzable cADPR analog and agonist, 3-deaza-cADPR. Notably, 3-deaza-cADPR stimulates CFC growth at nanomolar concentrations, thus holding promise for future pharmacological applications.

Stimulation of cell growth is paralleled by an increase of [Ca²⁺]_i, similar in extent and kinetics for cADPR and 3-deaza-cADPR (Fig. 2) . The kinetics of calcium increase are consistent with a slow influx of both cyclic nucleotides into the cells, as also supported by the different time course of the calcium increase induced by cADPR on permeabilized vs. intact MNC (Fig. 3) .

The pleiotropic effects of cADPR described above could result from the different duration of the calcium increase elicited on CD38^± subpopulations. In fact, the increase of [Ca²⁺]_i in MNC extends beyond the washout of cADPR, up to 24–36 h in CD38⁺ MNC (Fig. 2) and much longer, possibly several days, in CD38^- MNC (see Results). The long life span of the cADPR molecule in CD38^- cells (17) is due to resistance of the cyclic nucleotide to cell nucleotidases and pyrophosphatases (31) . Thus, long-term effects (possibly activation of gene transcription) could be elicited in resting CD34⁺/38^low progenitors by a [Ca²⁺]_i increase lasting several days. These effects could account for the remarkable observation that cADPR priming for 24 h affects the growth of CFC for up to four generations, over a span of more than 8 wk. Moreover, the immature features displayed by cells growing after several replatings after cADPR priming contrast with the differentiated morphology of control cells (Fig. 4) . Thus, cADPR priming may induce an expansion of immature progenitors, which undergo proliferation and differentiation in subsequent generations. This horizontal expansion of CFC induced by a 24 h incubation of MNC with cADPR is possibly due to an asymmetric cell division, with each progenitor giving rise to one colony and to one cell capable of forming a colony in a subsequent generation (32) .

A causal relationship between the [Ca²⁺]_i increase and the stimulation of cell proliferation was established by using the cADPR antagonists 8-NH₂- and 8-N₃-cADPR (10–20 µM). Treatment of MNC with either cADPR antagonist for 24 h induces a significant reduction of the basal [Ca²⁺]_i, an increased repletion state of ionophore-sensitive calcium stores, and an inhibition of colony growth. These results suggest that the antagonists may interfere with a physiological calcium ‘leakage’ from internal stores, possibly controlled by endogenous cADPR, responsible for maintaining basal [Ca²⁺]_i values and HP viability. A similar role in maintenance of intracellular calcium homeostasis by providing a Ca²⁺ leak channel has been proposed for ryanodine receptors (RyR) in muscle cells (33) . Indeed, mutant cardiac myocytes lacking RyR-2 and double-mutant skeletal muscle cells lacking RyR-1 and RyR-3 show similar ultrastructural abnormalities caused by overloading of intracellular calcium stores (34) . Reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme analysis of the RyR PCR product indicate that RyR-1 is the major isoform expressed in CB MNC (E. Zocchi and L. Sturla, unpublished results).

Surface expression of CD38 on MNC should enable the extracellular production of cADPR in the presence of NAD⁺. However, addition of NAD⁺ to the liquid culture (100 µM for 24 h) did not consistently stimulate CFC growth (not shown). This failure could be due to the low cyclase activity expressed on the surface of MNC, which is overcome by a 5- to 10-fold higher hydrolase activity of bifunctional CD38. Indeed, addition of monofunctional ADP-ribosyl cyclase from Aplysia californica to NAD⁺-supplemented liquid cultures increased CFC output similarly to cADPR-treated cultures (not shown).

Taken together, these observations suggest that cyclase expression on HP, though insufficient to elicit production of extracellular cADPR concentrations capable of stimulating CFC proliferation in vitro, could be competent for the production of low levels of intracellular cADPR, responsible for maintenance of basal [Ca²⁺]_i and cell viability. Recently, it has been demonstrated that intracellular generation of cADPR in CD38⁺ cells is made possible by two transport mechanisms circumventing the intravesicular compartmentation of the CD38 active site: a pyridine dinucleotide transport system, mediating access of cytoplasmic NAD⁺ to CD38 (35) , and the cADPR-transporting activity of CD38 itself, actively pumping cADPR across the vesicle’s membrane into the cytosol (36) . Both mechanisms are present and active in several constitutively and transduced CD38⁺ cell lines (35 , 36) . The endogenous, basal [cADPR]_i of CB MNC was below the threshold of detectability of our HPLC analyses (i.e., < 70 pmol/10⁸ cells); however, the presence and functional significance of intracellular cADPR are inferred by the effects of its specific antagonist, 8-NH₂-cADPR, on the basal [Ca²⁺]_i and on colony growth (see Results). Thus, results obtained with extracellular cADPR and its antagonists seem to suggest that the [Ca²⁺]_i of hemopoietic cells may control cell viability and proliferation, with low (<20 nM) levels inducing cell death and higher concentrations (>20 nM) supporting cell growth.

Recently, CD38-deficient mice have been shown to exhibit marked deficiencies in the humoral immune response but normal peripheral blood cell counts and hemopoietic organ cellularities (37) . Since no in vitro assay for CFC was described, the cyclase activity displayed in vivo by BST-1 in the BM microenvironment could have compensated for the absence of endogenous cyclase activity on HP. These observations may suggest distinctive roles for the cyclases described so far, CD38 (a catalytically active cADPR transporter) and BST-1 (a GPI-linked ectoenzyme unsuitable for the transport of cADPR across membranes), in the physiology of lymphocyte activation and hemopoietic progenitor differentiation, respectively. Ectocellular generation of cADPR by BST-1 on the surface of stromal cells could be supported by NAD⁺ efflux (35) from these cells (E. Zocchi and M. Podestà, unpublished results) and the extended cell contacts existing in the BM microenvironment between the cADPR-generating stromal feeder and the cADPR-sensitive HP may play a critical role in preventing diffusion of the cyclic nucleotide. These cell-to-cell interactions are expected to ensure a constant and targeted supply of growth-promoting cADPR to HP, whose effect may exceed that of a single in vitro pulse of cADPR. Accordingly, the effects of cADPR on HP growth should be maximized in vivo, thus enhancing the physiological significance of the present results.

Finally, influx of cADPR, its agonist, 3-deaza-cADPR, and antagonist, 8-NH₂-cADPR, across the membrane of intact MNC opens the way to a possible pharmacological utilization of these calcium modulators.

	ACKNOWLEDGMENTS

 
We are indebted to Prof. H. C. Lee for critical reviewing of the manuscript. This study was supported in part by the Associazione Italiana per la Ricerca sul Cancro (AIRC, 1998) to A.D.F., by the Italian Ministry of University and Scientific and Technological Research (MURST-PRIN 1998) to A.D.F., by the 5% Project on Biotechnology (MURST-CNR) to A.D.F., by the CNR Target Project on Biotechnology to E.Z., and by NIH grant DA11806 to T.F.W. The precious collaboration of the maternity ward’s staff in Galliera Hospital, Genova, Italy, is gratefully acknowledged.

	FOOTNOTES

 
¹ These authors contributed equally to the work.

² Current address: Biocrystallography Center, National Research Council, Naples, Italy.

Received for publication May 27, 1999. Revised for publication October 25, 1999.

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