The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia.
This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G(1) when exposed to 95% oxygen. Instead, cells arrested in S and G(2). Stable expression of p53 restored induction of p21 and G(1) arrest without affecting mRNA expression of the other Cip or INK4 G(1) kinase inhibitors. To confirm the role of p21 in G(1) arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G(1) arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G(1) during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G(1) arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.
Helt
Christopher E
CE
Department of Enviromental Medicine, School of Medicine and Dentistry, The University of Rochester, Rochester, NY 14642, USA. michael_oreilly@urmc.rochester.edu
Staversky
Rhonda J
RJ
Lee
Yi-Jang
YJ
Bambara
Robert A
RA
Keng
Peter C
PC
O'Reilly
Michael A
MA
D000230:Adenocarcinoma; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics*; D016213:Cyclins / Q000737:chemistry / Q000235:genetics*; D016193:G1 Phase / Q000502:physiology; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D018496:Hyperoxia / Q000503:physiopathology*; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008175:Lung Neoplasms; D010957:Plasmids; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D016196:S Phase / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D018844:Cyclin-Dependent Kinases