Fig. 8.

AEC2 isolated from normoxia-maintained terc−/− F2, F3, and F4 lung exhibit altered expression of markers for DNA damage, oxidative stress, and apoptosis. A: expression of markers for apoptosis and stress in AEC2 isolated from normoxia-maintained terc−/− lung. AEC2 freshly isolated from normoxia-maintained WT and terc−/− lung were analyzed for protein expression by Western blotting. Data presented are representative of expression in AEC2 isolated from 3–5 separate animals from each generation (WT F4, terc−/− F2, F3, and F4). Equality of loading was determined by probing for β-actin expression. The levels of expression of apoptotic markers caspase-3 (uncleaved and cleaved), caspase-6, apoptotic agonist Bax, and stress marker heat shock protein (HSP)-25 were then analyzed. B: TUNEL FACS analysis of AEC2 isolated from normoxia-maintained WT and terc−/− lung. Fresh, uncultured AEC2 isolated from WT F4 and terc−/− F2, F3, and F4 lung were fixed and analyzed by FACS to ascertain level of TUNEL. A representative experiment from 3 repetitions is shown. C: quantitation of TUNEL-positive AEC2 isolated from normoxia-maintained WT and terc−/− lung. Data from 3 separate experiments were analyzed, and the mean numbers of TUNEL-positive AEC2 were compared. In WT F4 animals maintained in normoxia, mean % of TUNEL-positive cells was 2.82% (SE ±1.38). Under the same conditions, mean % of TUNEL-positive AEC2 in terc−/− F2 fresh isolates increased to 11.1% (SE ±2.15). In terc−/− F3 isolates, % was 16.91% (SE ±0.57), while % in terc−/− F4 AEC2 was 19.9% (SE ±4.04). By Spearman nonparametric rank correlation analysis of the difference between WT F4 and terc−/− samples, *P = 0.001. For differences among terc−/− samples as a group, **P = 0.011. D: correlation of telomere length to the level of TUNEL present in AEC2 isolated from normoxia-maintained WT and terc−/− lung. Mean % of AEC2 that were TUNEL positive from each generation were correlated with mean telomere length. Regression analysis gave an R² value of 0.9631.