Qdot Nanocrystal Conjugates conjugated to bombesin or ANG II label the cognate G protein-coupled receptor in living cells

Quantum dots (Qdot Nanocrystal Conjugates; Quantum Dot, Hayward, CA) exhibit high fluorescence and low photobleaching compared with organic dyes, properties that should enhance their detection at low densities. In view of the properties of Qdots and the biological and pharmaceutical importance of G protein-coupled receptors (GPCRs), we attempted to use Qdots to label GPCRs in a variety of live cell types. An agonist consisting of biotinylated bombesin or ANG II was conjugated to Qdot Nanocrystal Conjugates coated with streptavidin through a biotin-streptavidin linkage (Qdot agonist). Herein we demonstrate that Qdot-bombesin conjugate can label the bombesin-preferring GPCR in living mouse Swiss 3T3 cells and in Rat-1 cells. Similarly, we used the Qdot-ANG II conjugate to label GPCR in intact rat intestinal epithelial cells (IEC)-18 and in a human pancreatic adenocarcinoma cell line of ductal origin, HPAF-II cells. We demonstrate that Qdot-ANG II is brighter and more photostable than agonist labeled with the organic dye Cy3. Our results demonstrate that Qdot technology can be adapted to monitor ligand binding to GPCRs. Combined with the narrow and symmetric emission profile of Qdot Nanocrystal Conjugates, this information suggests the potential for a new multiplex strategy to determine the effect of agonists and/or antagonists on agonist binding to several GPCRs simultaneously in living cells.

Identification of a Specific Vimentin isoform that Induces an Antibody Response in Pancreatic Cancer

Pancreatic cancer has a poor prognosis, in part due to lack of early detection. The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Proteins from a pancreatic adenocarcinoma cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for autoantibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis and 15 healthy subjects were analyzed. Autoantibodies were detected against a protein identified by mass spectrometry as vimentin, in sera from 16/36 patients with pancreatic cancer (44.4%). Only one of 18 chronic pancreatitis patients and none of the healthy controls exhibited reactivity against this vimentin isoform. Interestingly, none of several other isoforms of vimentin detectable in 2-D gels exhibited reactivity with patient sera. Vimentin protein expression levels were investigated by comparing the integrated intensity of spots visualized in 2-D PAGE gels of various cancers. Pancreatic tumor tissues showed greater than a 3-fold higher expression of total vimentin protein than did the lung, colon, and ovarian tumors that were analyzed. The specific antigenic isoform was found at 5–10 fold higher levels. The detection of autoantibodies to this specific isoform of vimentin may have utility for the early diagnosis of pancreatic cancer.