[Carbonyl-¹⁴C] NAD⁺ (specific activity 53 Ci/mol) was purchased from Amersham International. Chemicals of analytical grade were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and AG-X4 resin from Bio-Rad Laboratories (Hercules, CA, USA). Amerlite CEA-60 assay kit was purchased from Kodak Clinical Diagnostics (Rochester, NY, USA). EATC were obtained from the Research Institute for Experimental Medicine of Istanbul University where the cells had been maintained through successive passages in BALB/c mice over a long period of time. Mouse CD38-specific goat polyclonal IgG, CD-38 (M-19), which was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), was used in the experiments.

Blood samples were obtained from apparently healthy individuals (controls) and from one of the Tumor Marker Laboratories of the Istanbul Faculty of Medicine. Erythrocytes were purified from anticoagulated blood samples by centrifugation for 20 min at 3,000 × g through a Histopaque gradient (Histopaque-1077; Sigma, St. Louis, MO, USA) and resuspended in phosphate-buffered saline (PBS). Erythrocyte numbers were determined after spectroscopic measurements were obtained using calibration curves where 10⁶ erythrocytes corresponded to 0.6 A₄₀₀. Erythrocyte ghosts were prepared as previously described (9). Erythrocytes were briefly suspended in H₂O and lysed by repeated drawing and pressing of the suspension through the outlet of a micropipette. The lysate was centrifuged for 10 min at 10,000 × g and the pellet was washed repeatedly by resuspension and centrifugation in PBS.

Lymphocytes were isolated by layering the blood on a Histopaque gradient and undergoing centrifugation for 30 min at 400 × g. Interfaced cells were harvested, resuspended in PBS and washed twice in PBS by centrifugation for 10 min at 3000 × g in order to separate lymphocytes from thrombocytes (10). The lymphocyte pellet was finally resuspended in PBS. For membrane isolation, the lymphocytes were lysed by 3 freeze/thaw cycles and the supernatants, obtained by centrifugation of lysates for 10 min at 3000 × g, were used as the lymphocyte membrane fraction (11). The total cell counts were determined on aliquots using a hemocytometer. Cell viability was determined by trypan blue exclusion by mixing one drop of trypan blue with an aliquot of cell suspension and examining the cells under a microscope for dye exclusion (12).

EATC (n=8×10⁵) was administered in 0.2 ml PBS to BALB/c mice by intraperitoneal injections. For each group, four mice were used. Alternatively, 50 μl of ascites fluid or serum from mice with developed EAT, serum from cancer patients or EATC supernatant were administered intraperitoneally to new groups of mice. Where indicated, these injections were repeated on day 4. Control groups of mice received PBS, irradiated EATC (50 Gy total dose) or control serum. As an alternative to irradiated EATC, EATC incubated with 1 mM cycloheximide were used, and similar results were obtained. If not otherwise indicated, blood samples were obtained from such mice on every fourth day and subjected to assays as described below.

Peripheric mouse lymphocytes isolated as described above by centrifugation in Histopaque gradients were cultured in a humidified atmosphere of 5% CO₂ in 2 ml Dulbecco’s modified Eagle’s medium (DMEM) in 24-well plates at 37°C for 7 days. The lymphocytes were supplemented with 10% fetal calf serum (FCS), serum from normal BALB/c mice, serum from BALB/c mice with developed EAT (Day 8), serum from normal individuals, serum from cancer patients with high CEA values, ascites fluid or EATC supernatant, obtained as described below.

EATC were propagated in RPMI-1640, supplemented with 10% FCS, then with 1% FCS and finally with serum-free medium with no supplements. EATC culture was maintained in this manner over several passages and for several weeks. The resulting cell culture medium (EATC supernatant) was then assayed either directly or after concentration, using IVSS vivaspin 2 centrifugal concentrator (Sartorius Stedim Biotech, Aubagne, France).

NAD⁺ glycohydrolase activity in serum samples was determined by separation of [carbonyl-¹⁴C] nicotinamide released from [carbonyl-¹⁴C] NAD⁺ on Bio-Rad AG1×4 anion exchange resin (13). Reaction mixtures (20 μl) containing 12 μl serum, 10 mM NaCl, 500 μM ZnCl₂, 50 μM CaCl_2, 20 mM Tris-HCl, pH 9.0 and 5 μM [carbonyl-¹⁴C] NAD⁺ were incubated for 30 min at 37°C (7). Reactions were stopped with 1 ml 0.1% SDS. The samples were then applied to the Bio-Rad AG1×4 column and [carbonyl-¹⁴C] nicotinamide was eluted with H₂O. Unhydrolised [carbonyl-¹⁴C] NAD⁺ was retained on the column and then eluted with 0.5 M NaCl. Radioactivity was determined by counting aliquots from the eluate in a liquid scintillation counter (Packard Tri-Carb 1000 TK, Meriden, CT, USA); counting efficiency for ¹⁴C was 90%. Ecto-NAD⁺ glycohydrolase activity was assayed in 20 μl reaction mixtures containing 5×10⁶ erythrocytes (or 5×10⁵ lymphocytes) in PBS. After incubation for 30 min at 37°C and subsequent centrifugation of cells, the supernatants were subjected to analysis as above (14).

SDS-PAGE and Western blotting using the CD38-specific goat polyclonal IgG antibody (M-19) were performed as previously described (15,16). The erythrocyte ghost or lymphocyte membrane fractions were solubilised in 1 M Tris-HCl, pH 6.8, 2% glycerin, 10% SDS, 5% 2-mercaptoethanol, 0.1% bromphenol blue, and subjected to SDS-PAGE. The separated proteins were then transferred electrophoretically onto nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany). The blots were blocked by 1 h incubation with 0.5% BSA in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween-20) followed by successive 1 h incubations with CD38-specific goat polyclonal IgG (M-19). Detection of immunocomplexes was achieved using alkaline phosphatase-conjugated bovine anti-goat antibody (Sigma) (1:1000 in TBST) and BCIP/NBP as a substrate (7).

The procedure described by the producing company was followed for the determination of CEA.

Mouse peripheric lymphocytes (n=10,000), which were prepared as described above were resuspended in 100 μl PBS and incubated with anti-mouse CD38 monoclonal antibody, fluorescein (FITC) conjugated (Cedarlane), at a concentration of 1 μg/ml for 30 min at 4°C. Lymphocytes were then washed three times in PBS and analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA), equipped with a 5-W argon ion laser. FITC excitation occured at 488 nm, and forward and side scatter properties of lymphocytes were used to establish sorting gates. Data acquisition and analysis were carried out using CellQuest Pro software (Becton-Dickinson).

Each experiment was repeated at least four times and in each experiment each value was the mean of 4 mice. Statistical analysis was performed according to the Kruskal-Wallis ANOVA test and Kruskal-Wallis multiple comparison z-value test.

The investigation was carried out in line with the ethical principles and approval of the Ethics Committee of the Istanbul Faculty of Medicine.

The objective of this study was to reproduce, in an appropriate animal model, the increases observed in serum and erythrocyte NAD⁺ glycohydrolase in blood samples from cancer patients. To that effect, EATC were administered to BALB/c mice by intraperitoneal injections and enzyme activities were then followed up concomitantly to ascites tumor development. In general, a complete image of a developed ascites tumor was observed in these animals in the course of 10–12 days after the injection of EATC. A leukocytosis of 18,000–20,000 (cells/μl) appeared to precede the onset of ascites symptoms. It was already detected on day 4 after the application of EATC and remained at this level until the termination of the experiment.

The development of ascites tumors was accompanied by a considerable enhancement in CD38 expression, as revealed by a comparison of anti-CD38 reactive proteins in erythrocyte ghost and lymphocyte membrane fractions in mice with developed ascites tumors versus the controls (Fig. 1A). In line with recent findings of studies pertaining to cancer cases (7), an anti-CD38 reactive protein band of 45 kDa was detected in the Western blot analyses of erythrocyte ghost proteins from mice to which live EATC had been administered. In these mice, the 45 kDa band became visible 8 days after the EATC injection and reached a maximum intensity by day 12 after the injection. Western blot analyses of lymphocyte membrane proteins revealed similar results. No anti-CD38 reactive protein band was found in the controls. Western blot analysis findings were supported by the increases observed in erythrocyte and lymphocyte NAD⁺ glycohydrolase (Fig. 1B). Elevations in these activities were observed 8 days after the application of EATC and reached maximum values after 12 days. These values were maintained until day 20 when the experiment was terminated. The increases in erythro- cyte and lymphocyte NAD⁺ glycohydrolase activities were on average not less than approximately 3-fold higher than the controls. However, in this case, the increases caused by tumor development were 2- to 3-fold higher than the controls.

The results suggest the existence of certain serum factors as mediators of the stimulatory effect on CD38 expression and related enzymatic activities. Thus, the experiments were repeated with serum and ascites fluid samples from mice with fully developed tumors as well as serum samples from cancer patients. The administration of these samples was carried out twice with a 4-day interval was almost as effective as that of live Ehrlich cells in inducing CD38-expression. As shown in Fig. 2A, the injection of serum and ascites samples from mice with developed EATC, as well as serum samples from cancer patients with high (>100 ng/ml) CEA values again gave rise to the appearance of the anti-CD38 reactive band of 45 kDa in Western blot analyses of erythrocyte ghost and lymphocyte membrane proteins. The administration of serum samples from control mice and normal individuals failed to exhihibit a similar inducing effect. The serum samples from mice with tumors also gave rise to increases in NAD⁺ glycohydrolase (Fig. 2B). Ascites fluid had a similar and even a higher stimulatory impact. The highest stimulation was achieved with intraperitoneal injections of serum samples from cancer patients. The elevated activity declined gradually in erythrocytes from day 12 to reach control levels by day 20, but activity persisted in the lymphocytes until day 20.

Of note is that the injection of EATC culture medium propagated either with or without FCS supplement, i.e., EATC supernatant with or without FCS supplement, also resulted in the expression of CD38. In this case, the immunoreactive band, although sharp and distinct, was not as prominent as the bands obtained after injection of serum or ascites fluid samples. However, since this was achieved with trace amounts of protein present in serum-free culture medium, the finding indicated a high level of inducing activity (Fig. 3A). The increase in NAD⁺ glycohydrolase activity promoted by EATC supernatants was also less than that obtained with serum samples or ascites fluid, but significant in comparison to the controls (Fig. 3B). Moreover, the inducing effect on CD38 expression may be shown by flow cytometry (Fig. 3C-J).

Finally, CD38 expression was investigated in a peripheric lymphocyte culture system. The findings obtained in whole mice were reproduced in this system by the addition of serum samples from mice with developed EAT, patients with high CEA values, ascites fluid and, in particular, EATC (Fig. 4).