Abstract
changes in nicotinamide adenine dinucleotide (nAD +) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD + levels also affect DNA repair capacity as nAD + is a substrate for pARp-enzymes (mono/poly-ADp-ribosylation) and sirtuins (deacetylation). The ecto-5′-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular nAD + levels by processing NAD + and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular nAD + content and nAD +-dependent DNA repair capacity. Reduced intracellular NAD + levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD + reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through nAD + or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular nAD + bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD +. A positive correlation between CD73 expression and intracellular NAD + content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD + when supplemented with nAD + or NMN. The tumor microenvironment has the potential to provide critical metabolites to promote tumor cell growth and immune modulation 1 as well as support cellular metabolism via metabolic coupling 2 or metabolic plasticity 3,4. A key energy metabolite in the tumor microenvironment is the hydride exchanger nicotinamide adenine dinu-cleotide (NAD + /NADH) which plays an important role in redox reactions for a number of energy-related and regulatory processes 5-8. Mammalian cells, in order to sustain intracellular NAD + homeostasis, can preferentially utilize a de novo synthesis pathway from L-tryptophan (Trp) or the Preiss-Handler pathway from nicotinic acid (NA), or employ the more effective salvage pathway 9 , which initiates from nicotinamide (NAM), or the nicotina-mide riboside (NR) kinase pathway. It is suggested that a source of NAD + and related NAD + metabolites arises from cell lysis at sites of inflammation or tumor cell necrosis 10 , providing substrates for NAD +-consuming gly-cohydrolase ectoenzymes such as CD38 in concert with connexin 43 11 or NAD +-consuming pyrophosphatases such as NPP5 12. NAD + is also an essential substrate for signaling and protein modification factors that impact cell death, stress responses and genome stability via mono-or poly-ADP-ribosylation (PARP family proteins) 13 , chromatin status via deacetylation (sirtuins) 14 and overall functional capacity of mitochondria 15. Importantly, nuclear/mitochon-drial crosstalk is mediated in part by NAD + and NAD + precursors to facilitate genome stability and the cellular response to genotoxic and cytostatic insults 16,17. The last few years have opened a new chapter in NAD + biology since a decrease in the cellular NAD + level has been associated with aging and a variety of pathological syndromes including obesity, neurodegenerative diseases,