Julia M Coppola | PubMed Health

PubMed2005

Phosphorylated FADD induces NF-kappaB, perturbs cell cycle, and is associated with poor outcome in lung adenocarcinomas.

In an effort to identify a clinical biomarker for lung cancer, we used cDNA microarray and 2D protein analyses to demonstrate that increased Fas-associated death domain (FADD) mRNA and protein were significantly associated with poor survival. Analyses of copy number and sequence of the FADD gene in 24 independent tumors ruled out the existence of an amplified and/or mutated FADD gene in aggressive lung cancers. Immunohistochemistry-based tissue microarray analysis showed that nuclear localization of FADD and elevation of the phosphorylated form of FADD (p-FADD) correlated with poor outcome (P = 0.003). Tumors with increased p-FADD expression showed elevated NF-kappaB (P = 0.004) activation, a frequent molecular alteration associated with tumorigenesis and metastasis in a variety of cancers. To provide a link between p-FADD and NF-kappaB, cell culture studies demonstrated that overexpression of p-FADD leads to an increase in NF-kappaB activity and a decrease in the number of cells in the G2 phase of the cell cycle, compared with cells expressing the nonphosphorylatable form of FADD or the vector control. Furthermore, cDNA microarray analyses of lung tumor samples showed that increased levels of FADD transcripts were significantly correlated with overexpression of cyclins D1 (P < 0.01) and B1 (P < 0.01), genes that are involved in the regulation of cell cycle progression and are inducible by NF-kappaB. These studies demonstrate that induction of NF-kappaB activity and its effects on cell-cycle progression may represent a molecular basis underlying the aggressive tumor behavior associated with elevated p-FADD expression in lung adenocarcinoma.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Imaging of proteolytic activity using a conditional cell surface receptor.

Programmed cell death (apoptosis) is a ubiquitous means utilized by multicellular organisms for elimination of unwanted cells during development and homeostasis. Dysregulated apoptosis is implicated in an array of clinical disorders including cancer, autoimmune diseases, neurodegenerative disorders, and ischemia. During programmed cell death, a series of proteases, known as caspases, with different specificities play crucial roles in the apoptotic process. Caspase-3, a group II cysteine aspartate protease, recognizes and cleaves substrates harboring the amino acid sequence aspartic acid-glutamic acid-valine-aspartic acid (DEVD), and it plays an important role in the terminal phase of apoptosis. Here we report the development of a novel imaging platform for sensing the activation of cellular proteases. A recombinant chimeric protein was constructed, composed of a cell-surface-targeted single-chain antibody (sFv) fused to a Golgi retention signal. The DEVD tetrapeptide sequence was included between the single-chain antibody and the Golgi retention signal as a caspase-3 protease cleavage site. When expressed in cultured cells this fusion protein was localized to Golgi bodies and was not detected on the cell surface. Induction of apoptosis resulted in cleavage of the fusion protein releasing the single-chain antibody from the Golgi retention signal in a caspase-dependent manner. As a result, in cells undergoing apoptosis the single-chain antibody was visualized at the cell surface by immunofluorescence microscopy. The expression of sFv on the surface of cells in a protease-dependent manner provides a unique opportunity for real-time imaging through the use of targeted nanoparticles. This methodology may provide for a multimodal noninvasive real-time imaging of apoptosis and a new opportunity for high-throughput screening of cell-death-modulating therapeutic agents.

Animals↗