Am. J. Respir. Cell Mol. Biol., Volume 21, Number 3, September, 1999 395-402

Oxygen Induces S-Phase Growth Arrest and Increases p53 and p21^WAF1/CIP1 Expression in Human Bronchial Smooth-Muscle Cells

Jeffrey S. Shenberger and Patricia S. Dixon

Departments of Pediatrics and Clinical Investigations, Wilford Hall United States Air Force Medical Center, Lackland Air Force Base, Texas

Hyperoxia increases free radical production, leading to DNA damage. Recent studies indicate that oxygen augments the expression of p53 and p21^WAF1/CIP1, and increases apoptotic labeling of airway epithelial cells. Similar changes in regulatory gene products have not been reported in other pulmonary cells, nor have these changes been investigated in conjunction with alterations in cell-cycle distribution. The present study was conducted to determine whether oxygen alters the expression of p53 and p21^WAF1/CIP1 in human bronchial smooth-muscle cells (BSMC). BSMC placed in room air (RA), 40% O₂, or 95% O₂ were examined for 3 d to determine cell number, thymidine incorporation, cell-cycle distribution, and lactate dehydrogenase release. Apoptosis was assessed through the terminal deoxynucleotidyl transferase-deoxyuridine triphosphate end-nick labeling (TUNEL) technique, and p53 and p21^WAF1/CIP1 protein levels were determined through enzyme-linked immunosorbent assay. Exposure of BSMC to 95% O₂ decreased proliferation and DNA synthesis within 24 h, and was accompanied by an increase in S-phase cells (72 h; RA: 12.9 ± 4.6%, versus 95% O₂: 34.6 ± 7.0%; P < 0.01). By comparison, exposure to 40% O₂ resulted in decreased proliferation at 48 h without significant alterations in cell-cycle distribution. Both p53 and p21^WAF1/CIP1 levels were increased by 95% O₂, with maximal differences noted at 24 and 48 h, respectively. All atmospheres showed < 8% cell death and few TUNEL-positive cells. Our results indicate that oxygen-mediated alterations in BSMC proliferation are time- and concentration-dependent. Furthermore, high oxygen levels induce S-phase arrest and increased expression of p53 and p21^WAF1/CIP1. Activation of these genes may prevent replication without inducing apoptosis to allow for the repair of oxidative damage.

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