Figure 4.

hTERT immortalization restores nucleotide pools and fluxes. A, expression of hTERT in proliferating HMECs prevented occurrence of senescence growth arrest. Shown is a representative growth curve demonstrating that hTERT-expressing HMECs continued to grow logarithmically in culture, whereas HMECs expressing luciferase ceased to proliferate at PD ∼33. Filled triangles and squares indicate the PD at which cells were extracted for metabolomics. The x axis represents days since completion of drug selection. hTERT and luciferase expression were confirmed by Western blotting. Actin was used as an equal loading control. B, phase-contrast images of luciferase- and hTERT-expressing HMECs at PD 35 and 85, respectively. Luciferase-expressing HMECs acquired enlarged, flattened, and irregular morphology typical of senescent cells. hTERT-expressing HMECs maintained their epithelial cell morphology. Scale bar, 100 μm. C, luciferase-expressing HMECs showed increased activity of SA-β-gal measured by fluorescence signal of C₁₂FDG at SA-β-gal measurements are shown at day ∼60 following drug selection. SA-β-gal activity was calculated as follows: mean of samples labeled with C₁₂FDG − mean of samples without C₁₂FDG. **, p < 0.0001 by Student's t test. D, measurement of DNA synthesis by EdU incorporation showed decreased DNA synthesis in senescent, luciferase-expressing cells at day ∼45 following drug selection. **, p < 0.00004 by Student's t test. E, immunoblot for p21, PAI-1, and actin with lysates from luciferase- and hTERT-expressing HMECs at day ∼45 following drug selection. F, hTERT-expressing HMECs maintain purine and pyrimidine pools. Luciferase- and hTERT-expressing cells were profiled by LC-MS metabolomics at days ∼15 and ∼45 following drug selection. Luciferase-expressing cells were senescent at day ∼45, but all other samples were still proliferating. Data from proliferating (PD 9) and senescent primary HMECs (PD 37) is shown for comparison. * and **, p < 0.04 and 0.008, respectively. G, hTERT-expressing HMECs maintain glucose flux to purines and pyrimidines as measured by fractional contribution of U-¹³C–labeled glucose for selected purines and pyrimidines, as well as the nucleotide synthesis precursor PRPP. The number of days following drug selection for hTERT- and luciferase-expressing HMECs was the same as in F. Data from proliferating and senescent primary HMECs are shown for comparison. * and **, p value less than 0.04 and 0.002, respectively. H, metabolic pathway map depicting the average log₂ -fold change of [U-¹³C]glucose fractional contribution for senescent, luciferase-expressing (day ∼45) compared with proliferating, hTERT-expressing cells (day ∼45) on the indicated color scale for metabolites in glycolysis, pentose phosphate pathway, nucleotide synthesis, and the TCA cycle. Metabolites that were not measured are shown as small circles with gray color. Isomers that were not resolved with LC-MS are shown as diamonds. I, immunoblot for RRM2 and actin with lysates from senescent, luciferase-expressing and proliferating, hTERT-expressing HMECs. Error bars, S.D.