Low nicotinamide mononucleotide adenylyltransferase activity in a tiazofurin-resistant cell line: effects on NAD metabolism and DNA repair

Poly(ADP-ribose) polymerase (PADPRP), which uses NAD to synthesize ADP-ribose polymers, is activated by DNA strand breaks and mediates cellular responses to DNA damage. The consequences of low cellular NAD levels in a cell line deficient in nicotinamide mononucleotide adenylyltransferase (NMNAT), an enzyme essential for NAD biosynthesis, were investigated by assessing NAD metabolism and DNA repair after treatment with alkylating agents. A tiazofurin-resistant L1210 cell line (TZR) was isolated. NAD levels were approximately 5933 and 3375 pmol mg(-1) protein for parental (wild type, WT) and TZR cells respectively, and NMNAT levels were reduced by > 95%. TZR cells were more sensitive to temozolomide (TM) and 1-methyl-3-nitro-1-nitroso-guanidine (MNNG), particularly at concentrations that caused > 50% NAD depletion. TM and MNNG treatment decreased NAD levels in both cell lines, but took longer to return to control levels in TZR cells. For example, MNNG (5 mu M), depleted NAD levels at 6 h to approximately 4512 (WT) and 1442 (TZR) pmol mg(-1) protein; however, NAD levels had returned to control levels by 8 h in WT cells, but were not restored by 16 h in TZR cells. Both cell lines were equisensitive to the growth-inhibitory effects of NU1025 per se (IC50 370 mu M). Co-exposure of the cell lines to TM (100 mu M) with increasing concentrations of NU1025 led to a synergistic enhancement of cytotoxicity, with IC50 values for NU1025 decreasing to 17 +/- 4 mu M (TZR) and 37 +/- 6 mu M (WT). A similar enhanced sensitivity to NU1025 (approximately 2.7-fold) was obtained when TZR cells were co-exposed to MNNG + NU1025. TM-induced DNA strand breaks were increased by co-incubation with NU1025, and again the TZR cell line showed increased sensitivity to NU1025. There were no significant changes in NMNAT activity in response to MNNG treatment over 24 h, either in the presence or in the absence of NU1025. These data demonstrate that modest decreases in cellular NAD levels can sensitize cells to alkylating agents and PADPRP inhibitors.