Abstract 2095
Effects of Oxygen on Lung Oxidant/Antioxidant Balance Poster Symposium, Sunday, 5/2
Hyperoxic exposure is associated with decreased proliferation and increased p21WAF1 expression in pulmonary epithelial cells and fibroblasts through a p53-dependent process. Recently, we found that hyperoxia-exposed human bronchial smooth muscle cells (BSMC) also undergo non-lethal, growth arrest in conjunction with increased expression of p21WAF1 and p53. Although p21WAF1 is known to inhibit cell cycle progression, evidence implicating p21WAF1 as the cause of O2-mediated growth arrest is lacking. For this reason, we examined O2-mediated changes in proliferation and p21WAF1 levels in BSMC and in SKLMS cells, a human smooth muscle cell line with a mutation in the DNA binding region of p53. Methods. Actively proliferating BSMC and SKLMS were incubated for 72 hrs in either room air (RA) or 95%O2 plus 5%CO2 and removed for analysis every 24 hrs for 72 hrs. Proliferation was assessed with cell counts and DNA synthesis by [3H]thymidine incorporation. Cell death was determined by dye exclusion while p53 and p21WAF1 protein levels were determined by ELISA. Trends were analyzed by ANOVA and Newman-Keuls multiple comparison testing with p<0.05 being significant. Results. 95%O2 exposure lead to a time-dependent decrease in proliferation and DNA synthesis in both BSMC and SKLMS (p<0.0001). At 72 hrs, BSMC and SKLMS exposed to 95%O2 incorporated just 7.5% and 12%, respectively, of the thymidine incorporated by RA cells (p<0.01). Although the viability of BSMC was unaffected by O2 concentration, the viability of SKLMS dropped from 97% to 83% within 72 hrs of exposure to 95%O2 exposure also lead to increased p53 levels in both cell types with differences noted at 24 and 72 hrs in BSMC and at 72 hrs in SKLMS (p<0.01). p21WAF1 levels, on the other hand, were greater in 95%O2-exposed BSMC (p<0.01) but not in 95%O2-exposed SKLMS cells when compared to RA counterparts. Conclusions. These findings provide preliminary evidence that p21WAF1 is not essential for O2-induced growth arrest. Furthermore, the loss of viability in cells with low p21WAF1 levels may signify that p21WAF1 serves to rescue cells from apoptosis. Studies assessing the degree that apoptosis contributes to the cell death of SKLMS cells are in progress.
