Formation of the N′-methylnicotinamide adenine dinucleotide derivative of NAD in intact rat pituitary tumor GH 3 and human promyelocytic leukemia HL-60 cells

Abstract

The NAD analog N′-methylnicotinamide adenine dinucleotide (N′AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH 3 cells during treatment of the cultured cells with the nicotinamide derivative N′-methylnicotinamide (N′CH 3NAm). N′AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH 3 cells during treatment with the nicotinamide derivative. N′AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N′AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N′CH 3NAm. Second, the putative N′AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N′AD formation is prevented by addition of nicotinamide along with N′CH 3NAm to compete with binding of N′CH 3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N′AD formation was evaluated. The appearance of N′AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N′CH 3NAm. Therefore, we propose that N′AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells.