Tumor Necrosis Factor α Induces Spermidine/Spermine N1-Acetyltransferase through Nuclear Factor κBin Non-small Cell Lung Cancer Cells

Abstract

Tumor necrosis factor ␣ (TNF␣) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNF␣ are due to its ability to activate multiple signal transduction pathways, including nuclear factor B (NFB), which plays critical roles in cell proliferation and survival. TNF␣ displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNF␣ can lead to the induction of NFB signaling with a concomitant increase in spermidine/spermine N 1 -acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a ratelimiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IB␣ protein led to the suppression of SSAT induction by TNF␣ in these cells, thereby substantiating a role of NFB in the induction of SSAT by TNF␣. SSAT promoter deletion constructs led to the identification of three potential NFB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFB response elements play an important role in the regulation of SSAT in response to TNF␣. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFB signaling pathway in non-small cell lung cancer cells.Polyamines are aliphatic cations present in all cells, whose levels are intricately controlled by their transport and metabolic enzymes. Spermidine/spermine N 1 -acetyltransferase (SSAT) 2 is a rate-limiting step in polyamine catabolism, which catalyzes the transfer of the acetyl group from acetyl-CoA to the N 1 position of spermidine or spermine and has a predominant role in the regulation of intracellular polyamine concentrations in mammalian cells (1, 2). Decreases in polyamines have been shown to promote decreased growth or apoptosis (3-6), depending on the cell type and the particular stimulus, suggesting a complex interaction between polyamines, cell growth, and cell death. Therefore, although polyamines are required for cell growth and differentiation, SSAT is thought to prevent overaccumulation of the higher polyamines from becoming toxic to the cell and may play a role in reducing the growth rate by decreasing intracellular polyamines.Recently, considerable attention has been paid to SSAT as a target for cancer chemotherapy. SSAT activity is highly regulated and is induced rapidly in response to a number of stimuli, including polyamines, polyamine analogues, hormones, physiological stimuli, drugs, and toxic agents (1). It has been shown that the regulation of SSAT by the natural polyamines and the anti-tumor polyamine analogues is th...

Generation of Prostate Tumor–Initiating Cells Is Associated with Elevation of Reactive Oxygen Species and IL-6/STAT3 Signaling

“…The mechanisms involved were transcriptional activation of the IL-6 gene through an NF-kB-dependent pathway (39,40) or mitogen-activated protein kinase (MAPK) activity (41). On the other hand, IL-6 has been reported to induce ROS by activating the transcription and enzyme activity of spermine oxidase (SMO), which oxidizes spermine into spermidine, 3-aminopropanal, and H 2 O 2 (42). Spermine can also be first acetylated by spermidine/spermine N1-acetyltransferase (SAT) and then oxidized by N1-acetylpolyamine oxidase (APAO), producing H 2 O 2 as a byproduct (42).…”

Tumor Necrosis Factor-α Increases Reactive Oxygen Species by Inducing Spermine Oxidase in Human Lung Epithelial Cells: A Potential Mechanism for Inflammation-Induced Carcinogenesis

“…Intracellular H 2 O 2 was quantified using flow cytometry and the cell-permeable redox-sensitive dye CM-H 2 DCFDA as reported previously (14). Briefly, cells were treated for the indicated times with TNF-a, harvested with trypsin, and washed with 1Â PBS (Mediatech, Herndon, VA), and 1 Â 10 6 cells were treated with 10 Amol/L CM-H 2 DCFDA for 20 minutes at 37jC.…”

“…Total cellular RNA was extracted using Trizol reagent and RNA isolation according to the method developed by Chomczynski and Sacchi (13). To determine changes in levels of SMO/PAOh1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), quantitative PCR (qPCR) was done as reported previously (14). SMO/PAOh1 cDNA (287 bp) was amplified using the following primers: 5 ¶-GATCCCGGCGGACCAT-GTGATTGTG-3 ¶ ( forward) and 5 ¶-CTCAGGCGGGTAGAGGACATCAAA-3 ¶ (reverse) and, as a loading control, GAPDH cDNA (188 bp) was also amplified using the following primers: 5 ¶-GAAGGTGAAGGTCGGAGTC-3 ¶ ( forward) and 5 ¶-GAAGATGGTGATGGGATTTC-3 ¶ (reverse).…”

Paramecium bursaria Chlorella Virus 1 Encodes a Polyamine Acetyltransferase

“…In mammalian cells, polyamine acetylation is the first ratelimiting step in polyamine catabolism, and the human acetyltransferase, spermidine/spermine N 1 -acetyltransferase, is highly regulated due to the association of polyamine abundance with cellular proliferation (19,20,28). Through our newly determined crystal structure of vPAT and characterization of its catalytic activity as a polyamine acetyltransferase, we describe for the first time a virally encoded polyamine acetyltransferase with a catalytic efficiency (K m ϭ 21 M and k cat ϭ 131 min…”