Figure 4

CD38 enzymatic functions in primary myeloma cells and the myeloma cell line. Nucleotide consumption was evaluated using MM cells (2 × 10⁶ cell/mL) incubated in HBSS containing NAD⁺ or NGD⁺ (100 µmol/L), in the presence of a STOP Solution (see Materials and Methods). After centrifugation, supernatants were processed for HPLC. (A) NAD⁺/cyclase/hydrolase activity of CD38 determined in the LP-1 myeloma cell line. Supernatants revealed the presence of non-consumed NAD⁺ (R_t = 3.0). The products derived were cADPR (R_t = 4.1), ADPR (R_t = 5.6) and NAM (R_t = 7.0). (B) CD38 cyclase activity was measured using NGD⁺ as substrate. The breakdown of exogenous NGD⁺ (R_t = 3.2) in the supernatants of LP-1 myeloma cells revealed the presence of guanosine diphosphate ribose (GDPR) (Rt = 4.2), cGDPR (R_t = 5.8) and NAM (R_t = 7.0) products. (C) Primary myeloma (CD138⁺/CD38⁺) cells produce cADPR, ADPR and NAM from NAD⁺ (a CD38-dependent step) and AMP (a CD203a-dependent step), confirming that the CD38/CD203a enzymatic tandem is functionally active. The absence or low expression of CD73 precludes the formation of detectable ADO. (D) LP-1 myeloma cells (n = 5) were pre-in-cubated with kuromanin (100 µmol/L, 30 min) before assaying the conversion of NAD⁺ into ADPR or NGD⁺ to cGDPR. Results are expressed as percentage of inhibition of the enzymatic activity of CD38. Kuromanin reduced (range 25% to 48%) the conversion of NGD⁺ to cGDPR, with no effects on NAD⁺ hydrolytic activity. Values are the mean ± SD of triplicate determinations. All data are representative of at least three experiments.