Delayed recovery from hyperoxia-induced growth arrest by human airway smooth muscle cells. • 1826

In animal models, hyperoxia causes extensive lung injury characterized by varying degrees of airway smooth muscle hyperplasia and hypertrophy which resolve upon oxygen removal. In vitro, however, oxygen frequently decreases cellular proliferation. Since the ability of cells in culture to recover from oxygen-induced growth inhibition is unknown, we studied proliferative indices of human bronchial smooth muscle cells exposed to 1-5 days of O₂ followed by a 72 hour recovery period in room air. METHODS: Cells were plated 2.5×10⁴ cells/well and placed in either 21% O₂(CON), 70% O₂, or 95% O₂ plus 5%CO₂. Data was collected at 24, 72, 120, and 72-hours post-O₂ exposure. Proliferation was assessed via ₃H-thymidine incorporation (³H-thym, dpm/well). Nuclear protein content was measured using a standard BCA colorimetric assay(protein, μg/well) following trichloroacetic acid extraction of nuclear protein. Plates were run in replicates of 12 and significance of relationships tested by repeated measures ANOVA. RESULTS:Table ³H-thymidine counts were similar between 70% and 95% O₂ groups, however, O₂ was associated with a decrease in counts over time and compared to CON (p<0.001, ANOVA). Nuclear protein increased over time in all groups although CON displayed a greater increase than either O₂ group (p<0.001, ANOVA). CONCLUSIONS: Human bronchial smooth muscle cells exposed to high dose O₂ exhibit decreased ³H-thymidine incorporation and nuclear protein content indicating growth arrest. Furthermore, these proliferative changes persist at least 72 hours beyond oxygen withdrawal, implying that growth arrested cells are incapble of re-entering the cycle cell during this period. We speculate that oxygen alters cyclindependent protein kinases which trap cells within the G_o phase of the cell cycle.

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