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

Abstract

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. (Cancer Res 2005; 65(5): 1647-54)

Apolipoprotein L6, Induced in Atherosclerotic Lesions, Promotes Apoptosis and Blocks Beclin 1-dependent Autophagy in Atherosclerotic Cells

“…ApoL6 Adenovirus (AD.ApoL6)-Construction of adenovirus (AD) harboring full-length ApoL6 cDNA (AD.ApoL6) and infection of cells by viruses were performed as previously described (37,38). In one group, pan caspase inhibitor (Z-VAD-FMK, 50 M; R&D Systems) was added 2 h prior and during viral infection for 48 h. Cell numbers and apoptosis were measured over time by cell counting, analysis of cell morphology, and assay of caspase activation as previously described (14,37,38).…”

“…In one group, pan caspase inhibitor (Z-VAD-FMK, 50 M; R&D Systems) was added 2 h prior and during viral infection for 48 h. Cell numbers and apoptosis were measured over time by cell counting, analysis of cell morphology, and assay of caspase activation as previously described (14,37,38).…”

“…Transient co-transfection of LDCs was carried out using a 1:1 mixture of pApoL6-EGFP and pmcherry-LC3 (39) with Lipofectamine 2000 reagent (Invitrogen). 24 h after co-transfection, cells were incubated in HBSS, an isotonic buffer lacking nutrients, for 2 h to induce autophagy, and then subjected to confocal microscopy analysis as previously described (38).…”

“…ROS Detection and Autophagy Flux Determination by Fluorescence Microscopy-LDCs were grown on glass coverslips, treated as indicated, fixed in 4% (v/v) PBS-buffered paraformaldehyde solution and subjected to fluorescent microscopic analysis as described (14,38). Two methods were used to assay ROS generation in cells using the fluorescence dye DHE (dihydroethidium; Sigma) (38,40).…”

“…Two methods were used to assay ROS generation in cells using the fluorescence dye DHE (dihydroethidium; Sigma) (38,40). In brief, LDCs were plated on a glass slip in 6-well plates in medium, infected with indicated virus (AD or AD.ApoL6), with or without 1 M DPI (diphenylene iodonium; Sigma).…”