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PUMA Overexpression Induces Reactive Oxygen Species Generation and Proteasome-Mediated Stathmin Degradation in Colorectal Cancer Cells

¹ Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine and ² The EPR Center and Toxicology Program, University of New Mexico Health Sciences Center, Albuquerque, New Mexico; ³ Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico; and ⁴ Department of Pathology, Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania

Requests for reprints: Chien-an A. Hu, Department of Biochemistry and Molecular Biology, MSC08 4670 1, University of New Mexico, Albuquerque, NM 87131-001. Phone: 505-272-8816; Fax: 505-272-6587; E-mail: ahu{at}salud.unm.edu.

Increased amounts of reactive oxygen species (ROS) induce apoptosis in mammalian cells. PUMA (P53 up-regulated modulator of apoptosis), a mitochondrial proapoptotic BH3-only protein, induces rapid apoptosis through a Bax- and mitochondria-dependent pathway. However, the molecular basis of PUMA-induced apoptosis is largely not understood. Using a combination of biophysical and biochemical methods and PUMA-inducible colorectal cells, DLD-1.PUMA, we showed that (a) PUMA-induced apoptosis is dose and time dependent; (b) PUMA-induced apoptosis is directly associated with ROS generation; (c) diphenyleneiodonium chloride, a ROS blocker, or BAX-inhibiting peptide, a suppressor of BAX translocation, decreased ROS generation and apoptosis in DLD-1.PUMA cells; (d) overexpression of PUMA induced up-regulation (>1.34-fold) of peroxiredoxin 1 and down-regulation (by 25%) of stathmin through proteasome-mediated degradation; and (e) hydrogen peroxide down-regulated stathmin and disrupted the cellular microtubule network. Our findings indicate that PUMA induces apoptosis, in part, through the BAX-dependent generation of superoxide and hydrogen peroxide. ROS overproduction and oxidative stress induce proteome-wise alterations, such as stathmin degradation and disorganization of the cell microtubule network, in apoptotic cells.

Key Words: PUMA • Apoptosis • ROS • EPR • Quantitative Proteomics

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