Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression colocalizes with LC3 in pancreas tissue undergoing pancreatitisinduced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells (1-3). This catabolic process is involved in the turnover of long lived proteins and other cellular macromolecules, and it might play a protective role in development, aging, cell death, and defense against intracellular pathogens (4 -8). By morphological studies, autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance (4, 9, 10). Early reports of autophagy in human disease include the ultrastructural autophagic features described in pancreas from human pancreatitis (11,12).Autophagy is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes, which eventually acquire lysosomal-like features (13,14). Autophagy is mediated by a set of evolutionarily conserved gene products (termed the Atg proteins) originally discovered in yeast (15). In mammalian cells, Beclin 1 (3, 16 -18) promotes autophagosome formation when it functions as part of a complex with the Class III phosphatidylinositol 3-kinase (PI3K) 6 mediating the localization of other autophagic proteins to the autophagosomal membrane (19)...
Using a bioinformatic approach, we identified a TP53INP1-related gene encoding a protein with 30% identity with tumor protein 53-induced nuclear protein 1 (TP53INP1), which was named TP53INP2. TP53INP1 and TP53INP2 sequences were found in several species ranging from Homo sapiens to Drosophila melanogaster, but orthologues were found neither in earlier eukaryotes nor in prokaryotes. To gain insight into the function of the TP53INP2 protein, we carried out a yeast two-hybrid screening that showed that TP53INP2 binds to the LC3-related proteins GABARAP and GABARAPlike2, and then we demonstrated by coimmunoprecipitation that TP53INP2 interacts with these proteins, as well as with LC3 and with the autophagosome transmembrane protein VMP1. TP53INP2 translocates from the nucleus to the autophagosome structures after activation of autophagy by rapamycin or starvation. Also, we showed that TP53INP2 expression is necessary for autophagosome development because its small interfering RNA-mediated knockdown strongly decreases sensitivity of mammalian cells to autophagy. Finally, we found that interactions between TP53INP2 and LC3 or the LC3-related proteins GABARAP and GABARAP-like2 require autophagy and are modulated by wortmannin as judged by bioluminescence resonance energy transfer assays. We suggest that TP53INP2 is a scaffold protein that recruits LC3 and/or LC3-related proteins to the autophagosome membrane by interacting with the transmembrane protein VMP1. It is concluded that TP53INP2 is a novel gene involved in the autophagy of mammalian cells.
Gemcitabine is the only available chemotherapeutic treatment of pancreatic cancers. It is, however, moderately effective, showing a tumor response rate of only 12%. The aim of this work was to identify new pathways involved in the resistance of pancreatic cancer cells to gemcitabine, in the hope of developing new adjuvant strategies to enhance its therapeutic efficacy. Comparison of gene expression patterns of five human pancreatic cancer cell lines showing different degrees of resistance to gemcitabine revealed specific overexpression of several genes in the most resistant. One of them encoded the antiapoptotic p8 protein.We found that (a) knocking down p8 expression in gemcitabine-resistant cells promoted cell death and increased caspase-3 activity; (b) forced overexpression of p8 in gemcitabine-sensitive cells increased their resistance to gemcitabine-induced apoptosis; and (c) gemcitabine down-regulated p8 mRNA expression. These results suggest that, in pancreatic cancer cells, a large part of gemcitabine-induced apoptosis results from the inhibition of the constitutive antiapoptotic activity of p8. Hence, targeting the p8-associated pathway could be a new adjuvant therapy improving the response of patients with pancreatic cancer to gemcitabine treatment.Pancreatic cancer is the fifth most common cause of death by cancer in the Western world (1). The anatomic localization of the pancreas and the lack of specificity of the symptoms result in a complex and delayed diagnosis. Therefore, at the time of detection, 85% of patients show metastatic infiltration in proximal lymph nodes, liver, or lungs. The current 5-year survival rate is f3% and only surgical resection (possible in 15% of the cases) can increase survival rate to 20%.
Our results show that 18F-FDG PET/CT complements adrenal washout CT in the evaluation of adrenal masses and should be recommended in the evaluation of large and/or indeterminate adrenal masses.
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