Abstract
Cellular senescence is a mechanism by which cells permanently withdraw from the cell cycle in response to stresses including telomere shortening, DNA damage, or oncogenic signaling. Senescent cells contribute to both age-related degenerative pathologies and hyperplastic pathologies including cancer. In culture, normal human epithelial cells enter senescence after a limited number of population doublings, known as replicative senescence. Here, to investigate how metabolic pathways regulate replicative senescence, we used liquid chromatography-mass spectrometry (LC-MS)-based metabolomics to analyze senescent primary human mammary epithelial cells (HMEC). We did not observe significant changes in glucose uptake or lactate secretion in senescent HMEC. However, analysis of intracellular metabolite pool sizes demonstrated that senescent cells exhibited depletion of metabolites from nucleotide synthesis pathways. Furthermore, stable isotope tracing with 13C-labeled glucose and glutamine revealed a dramatic blockage of flux into nucleotide synthesis pathways in senescent HMEC. To test whether cellular immortalization would reverse these observations, we expressed telomerase in HMEC. In addition to preventing senescence, telomerase expression maintained metabolic flux from glucose into nucleotide synthesis pathways. Finally, we tested whether inhibition of nucleotide synthesis in proliferating HMEC was sufficient to induce senescence. Treatment of proliferating HMEC with an inhibitor of ribonucleotide reductase regulatory subunit (RRM2), a rate limiting enzyme in dNTP synthesis, induced premature senescence with concomitantly decreased metabolic flux from glucose into nucleotide synthesis. Taken together, our results suggest that inhibition of nucleotide synthesis plays a causative role in establishment of replicative senescence in HMEC.
