CD95 promotes metastatic spread via Sck in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) have been implicated in the initiation and maintenance of tumour growth as well as metastasis. Recent reports link stemness to epithelial-mesenchymal transition (EMT) in cancer. However, there is still little knowledge about the molecular markers of those events. In silico analysis of RNA profiles of 36 pancreatic ductal adenocarcinomas (PDAC) reveals an association of the expression of CD95 with EMT and stemness that was validated in CSCs isolated from PDAC surgical specimens. CD95 expression was also higher in metastatic pancreatic cells than in primary PDAC. Pharmacological inhibition of CD95 activity reduced PDAC growth and metastasis in CSC-derived xenografts and in a murine syngeneic model. On the mechanistic level, Sck was identified as a novel molecule indispensable for CD95's induction of cell cycle progression. This study uncovers CD95 as a marker of EMT and stemness in PDAC. It also addresses the molecular mechanism by which CD95 drives tumour growth and opens tantalizing therapeutic possibilities in PDAC.

Chemical modification and structural analysis of the progesterone membrane binding protein from porcine liver membranes.

In addition to the classical genomic steroid actions on modulation of transcription and protein synthesis, rapid, nongenomic effects have been described for various steroids. These effects on cellular signaling and function are supposed to be transmitted by membrane binding sites unrelated to the classical intracellular receptors. Recently, a high affinity progesterone membrane binding protein (mPR) has been characterized in porcine liver membranes. In the present study, amino acid residues that are essential for progesterone binding to porcine liver microsomal mPR have been identified by the use of protein modifying reagents. Among all reagents tested, agents with specificity for carboxyl groups, methionine and tryptophan such as N,N'-dicyclohexylcarbodiimide, chloramine T and N-bromosuccinimide induced a reduction in [3H]progesterone binding. To evaluate the presence of essential disulfide bridges, porcine liver microsomes were incubated with the disulfide reducing agent dithiothreitol (DTT) and [3H]progesterone binding was measured. This treatment also resulted in a reduction of binding activity with an IC50 of 20 mM for DTT. Western-blotting analysis in the presence or absence of the reducing agent suggested that mPR--in its binding state--consists of at least two identical subunits with an apparent molecular mass of 28 kDa which are linked by a disulfide bridge. In conclusion, in the present study evidence for an involvement of carboxyl-, tryptophan- and methionine residues in [3H]progesterone binding to porcine liver microsomes is given. In addition, it is shown that mPR can form disulfide-linked homodimers.

Staging of malignant pleural mesothelioma: comparison of CT and MR imaging.

OBJECTIVE: This article compares the accuracy of CT with that of MR imaging in staging of malignant pleural mesothelioma. SUBJECTS AND METHODS: Ninety-five patients were enrolled in a prospective staging protocol based on the International Mesothelioma Interest Group staging system. Sixty-five patients underwent CT and MR imaging and a surgical procedure (excluding percutaneous needle biopsy) to stage and resect the tumor. Receiver operating characteristic analyses were performed. CT and MR scans were interpreted independently by observers who were unaware of the results of the other imaging study; these imaging findings were compared with the results of surgery and pathologic examination. RESULTS: The areas under the receiver operating characteristic curves for eight of 10 features revealed by imaging showed no statistically significant differences between CT and MR imaging. However, MR imaging was superior to CT in revealing invasion of the diaphragm (A(z) = .55 for CT versus .82 for MR imaging) and in revealing invasion of endothoracic fascia or solitary resectable foci of chest wall invasion (A(z) = .46 for CT; A(z) = .69 for MR imaging). Several anatomic regions could not be evaluated because positive findings at surgery were rare. CONCLUSION: CT and MR imaging are of nearly equivalent diagnostic accuracy in staging malignant pleural mesothelioma. MR imaging is superior to CT in revealing solitary foci of chest wall invasion and endothoracic fascia involvement and in showing diaphragmatic muscle invasion; however, this advantage does not affect surgical treatment. For cost reasons, CT should be considered the standard diagnostic study before therapy.

Characterization of progesterone membrane binding sites from porcine liver probed with a novel azido-progesterone radioligand.

A new derivative of progesterone was synthesized for photoaffinity labelling of specific binding sites in porcine liver microsomes. Using progesterone-3-(O-carboxymethyl)-oximino-(3-125I-iodo-4-azidosa licylamidobutylamine) as a photoactivatable radioligand, selective covalent labelling of proteins was detected in porcine liver microsomes at apparent molecular weights of 90-100 kDa and 60-65 kDa by SDS-PAGE and subsequent radioautography. These proteins showed different ligand specifity for various steroids tested. On blue native polyacrylamide-gels three selectively labelled proteins were found corresponding to apparent molecular weights of approximately 310 kDa, approximately 215 kDa and approximately 75 kDa, respectively. Using two-dimensional electrophoresis for the analysis of these protein complexes, the 215 kDa-band could be correlated to the 90-100 kDa-band, while the 75 kDa-band may correspond to the 60-65 kDa-band at one-dimensional SDS-PAGE, respectively. One or more of these proteins may be involved in the rapid progesterone-induced increase of intracellular Ca2+ described previously in cultured hepatic cells.

Biotin-labelled and photoactivatable aldosterone and progesterone derivatives as ligands for affinity chromatography, fluorescence immunoassays and photoaffinity labelling.

New derivatives of progesterone and aldosterone were synthesized and functionally tested with commercially available antibodies. The covalent labelling of antibodies specific for aldosterone and progesterone was detected by SDS/PAGE analysis and subsequent autoradiography after using 3-(O-carboxymethyl)-oximino-(3-[125I]iodo-4-azidosalicylamidobu tylamine) derivatives of aldosterone and progesterone, respectively, as photoactivatable radioligands. Labelling was not observed in the presence of an excess of the unlabelled steroid. Aldosterone was labelled with biotin and used as a tracer in a time-resolved fluorescence immunoassay. The nonradioactive tracer is highly selective for its antibody-binding site, with almost no detectable cross-reactivity for other steroids. Biotin-labelled progesterone was immobilized by avidin-agarose and used for affinity chromatography. This yielded a more than 20-fold enrichment of an anti-progesterone polyclonal antibody. These results demonstrate that derivatives of steroids are particularly useful for the development of nonradioactive assays for the determination of natural steroids and may be also useful for the detection of specific binding sites in biological material such as plasma membranes.

Aldosterone-specific membrane receptors and rapid non-genomic actions of mineralocorticoids.

Functional studies in extrarenal, non-epithelial cells such as smooth muscle cells and more recently circulating human lymphocytes have provided increasing evidence that aldosterone produces not only classical genomic effects, but also rapid, non-genomic effects on transmembrane electrolyte movements. These involve activation of the sodium/proton exchanger of the cell membrane at very low, physiological concentrations of aldosterone with an acute onset within 1-2 min. A second messenger cascade involved is the inositol 1,4,5-trisphosphate/calcium pathway which responds over the same rapid time course. Such changes clearly cannot be explained by genomic mechanisms, which are responsible for later effects than the membrane related rapid responses. The mechanisms underlying these rapid effects of aldosterone on electrolytes have been extensively studied in human lymphocytes, which thus may represent valuable tools in the delineation of the receptor-effector mechanisms involved. The unique characteristics of this new pathway for steroid action include its rapid time course, 10,000-fold selectivity for aldosterone over cortisol and the ineffectiveness of spironolactones, classical mineralocorticoid antagonists, as antagonists of the response.