Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF.

BACKGROUND: Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. METHODS: Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. RESULTS: SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. CONCLUSIONS: The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.

Development of F-18-labeled fluoroerythronitroimidazole as a PET agent for imaging tumor hypoxia.

PURPOSE: To develop a hydrophilic ligand to image tumor hypoxia at positron emission tomography (PET). MATERIALS AND METHODS: Biodistribution of fluorine-18-labeled fluoroerythronitroimidazole (FETNIM) and F-18-labeled fluoromisonidazole (FMISO) was determined at PET and autoradiography in three mammary-tumor-bearing rats. The partition coefficient of FETNIM, FMISO, and misonidazole was determined. RESULTS: Biodistribution of F-18-labeled FETNIM at 1, 2, and 4 hours showed tumor-to-blood ratios of 2.29 +/- 0.599, 2.41 +/- 0.567 and 8.02 +/- 2.420, respectively, and tumor-to-muscle ratios of 0.66 +/- 0.267, 2.11 +/- 0.347, and 5.92 +/- 2.240, respectively. The tumor-to-blood count density ratio with F-18-labeled FETNIM at 4 hours after injection was significantly higher than with F-18-labeled FMISO. Autoradiographs indicated that both agents could help differentiate hypoxic versus necrotic region in the tumor. CONCLUSION: F-18-labeled FETNIM can help detect tumor hypoxia and is easier to prepare, less costly, and more hydrophilic than F-18-labeled FMISO.

Diagnostic and therapeutic potential of poly(benzyl L-glutamate).

Poly(benzyl L-glutamate) (PBLG) microcapsules, prepared by a solvent evaporation technique for intravenous injection, are evaluated for their potential use in diagnostic computed tomographic enhancement of liver images. The smaller microcapsules, < 3 microns, loaded with a radiopaque contrast material, ethyl iopanoate (IOPAE), produced prolonged opacification of the liver when delivered intravenously. In vivo tissue distribution studies of PBLG-131I-IOPAE (5 microCi/rat, iv) showed that liver had the highest uptake (percent of injected dose/g of tissue) among other organs 24 h postinjection. An in vitro estrogen receptor assay in pig uteri indicated that PBLG conjugated with estrone did not interfere with estrogen receptor affinity, suggesting the estrogen therapy potential of PBLG-estrone.