NF-κB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-κB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-κB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-κB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-κB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-κB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.
In adult vertebrates, most cells are not in the cell cycle at any one time. Physiological nonproliferation states encompass reversible quiescence and permanent postmitotic conditions such as terminal differentiation and replicative senescence. Although these states appear to be attained and maintained quite differently, they might share a core proliferation-restricting mechanism. Unexpectedly, we found that all sorts of nonproliferating cells can be mitotically reactivated by the sole suppression of histotype-specific cyclin-dependent kinase (cdk) inhibitors (CKIs) in the absence of exogenous mitogens. RNA interference–mediated suppression of appropriate CKIs efficiently triggered DNA synthesis and mitosis in established and primary terminally differentiated skeletal muscle cells (myotubes), quiescent human fibroblasts, and senescent human embryo kidney cells. In serum-starved fibroblasts and myotubes alike, cell cycle reactivation was critically mediated by the derepression of cyclin D–cdk4/6 complexes. Thus, both temporary and permanent growth arrest must be actively maintained by the constant expression of CKIs, whereas the cell cycle–driving cyclins are always present or can be readily elicited. In principle, our findings could find wide application in biotechnology and tissue repair whenever cell proliferation is limiting.
The flagellated protozoan Giardia duodenalis (syn. lamblia or intestinalis) has been chosen as a model parasite to further investigate the multifunctional 14-3-3s, a family of highly conserved eukaryotic proteins involved in many cellular processes, such as cell cycle, differentiation, apoptosis, and signal transduction pathways. We confirmed the presence of a single 14-3-3 homolog gene (g14-3-3) by an in silico screening of the complete genome of Giardia, and we demonstrated its constitutive transcription throughout the life stages of the parasite. We cloned and expressed the g14-3-3 in bacteria, and by protein-protein interaction assays we demonstrated that it is a functional 14-3-3. Using an anti-peptide antibody raised against a unique 18-amino acid sequence at the N terminus, we observed variations both in the intracellular localization and in the molecular size of the native g14-3-3 during the conversion of Giardia from trophozoites to the cyst stage. An affinity chromatography, based on the 14-3-3 binding to the polypeptide difopein, was set to purify the native g14-3-3. By matrix-assisted laser desorption ionization mass spectroscopy analysis, we showed that polyglycylation, an unusual post-translational modification described only for tubulin, occurred at the extreme C terminus of the native g14-3-3 on Glu 246 , Glu 247 , or both and that the Thr 214 , located in the loop between helices 8 and 9, is phosphorylated. We propose that the addition of the polyglycine chain can promote the binding of g14-3-3 to alternative ligands and that the differential rate of polyglycylation/deglycylation during the encystation process can act as a novel mechanism to regulate the intracellular localization of g14-3-3.
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