Stromal-epithelial measurements of prostate cancer in native Japanese and Japanese-American men.

We measured the histologic stromal and epithelial tissue components of the benign (normal) and malignant tissue compartments of Japanese-Americans (J-A) and native Japanese (NJ) men living in Japan. The patient cohort included 25 NJ men undergoing radical prostatectomy (RP) in Nagoya, Japan and 25 J-A (second or third generation US born). We conducted tissue image quantitation (in-house image software) of the stromal and epithelial compartments in malignant and adjacent normal tissue areas from a tissue microarray (TMA) selected from radical prostatectomy (RP) blocks. Stromal-epithelial (S-E) areas were determined using immunohistochemical stains for CAM-5.2 epithelial cytokeratin marker and the Masson trichrome stain to measure the stroma component. We observed differences in the volumes of normal and cancer epithelium and stroma within both the J-A and NJ study populations (P<0.01). Only the individual average cancer epithelium (CE) volume (JA=24.1 vs NJ=29.9) differed significantly between the NJ and J-A study populations (P=0.03). Consequently, the S-E ratio in NJ group was significantly different from that of J-A population (P=0.05). The decrease in S-E ratio noted in the malignant tissues of NJ prostate tissue may provide a biological marker for differentiation of the two groups and suggests a need for further investigations into the molecular basis for these histologic differences.

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.