References.......................................................................................14 Appendices.......................................................................................16 4 BODY Task 1 -To demonstrate proteins binding resveratrol in prostate cancer cells. Overall research activities and accomplishments. As indicated in SOW, the primary focus of Task 1 was to test the hypothesis that proteins capable of interacting and binding resveratrol, denoted RTPs, exist in both

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... androgen-dependent and -independent prostate cancer cells. Our experimental strategy for detecting RTPs, as proposed in Task 1, sought to determine whether [ 3 H]resveratrol forms a stable complex with RTPs, as monitored by (i) retention of complex and not the free ligand on nitrocellulose filters, and (ii) shift in molecular weight of complex on gel filtration columns. Pilot experiments to test the feasibility of these approaches used extracts prepared from LNCaP and PC-3 cells. Low though reproducible uptake of [ 3 H]resveratrol into LNCaP and PC-3 cell extracts were observed. In neither cell types, however, was uptake sufficiently robust to allow the demonstration of complexes based on retention on nitrocellulose filters. This negative research outcome may be attributable to scarcity of RTPs and/or low stability of [resveratrol.RTP] complexes. Task 1a aimed to develop a nitrocellulose filter-binding assay for detecting RTPs in androgen-dependent LNCaP cells. Rationale. Previous studies of interferon-inducible enzymes -2',5'-oligoadenylate dependent ribonuclease L (RNase L) and the double-stranded RNA-activated 2',5'-oligoadenylate synthetase (2-5AOS) -by the PI utilized assays based on binding of radioactive ligands to low abundance RNase L and 2-5AOS; formation of ligand:enzyme complex can be detected by retention on nitrocellulose membranes [5] [6] [7] [8] [9] [10] . We reasoned that the existence of RTPs might be similarly ascertained and established by testing whether a complex between [ 3 H]resveratrol and RTPs forms with sufficient stability as to show quantitative retention on nitrocellulose filters. Repeated attempts using LNCaP cell extracts yielded negative results. Uptake studies to quantify the cellular uptake of [ 3 H] resveratrol provided an explanation for the difficulties we encountered. As illustrated in Table 1 , little [ 3 H] resveratrol -typically only 300-750 cpm in 0.1 ml cell extracts containing 200-300 µg total protein -was taken up by LNCaP cells, suggesting that this experimental objective was unlikely to be successfully completed using this approach. Despite the noted technical challenges, it is worth mentioning that a time dependent increase in [ 3 H] resveratrol assumed to be in complex with proteins in LNCaP cell lysates and in amounts significantly above background radioactivity, was repeatedly observed, thus providing support for the notion that RTPs are present in CaP cells. is correlated with upregulation of quinone reductase 2 and p53 Abstract Resveratrol (trans-3,4 0 ,5-trihydroxystilbene) is a grape-derived polyphenol under intensive study for its potential in cancer prevention. In the case of cultured human melanoma cells, no one to our knowledge has investigated whether resveratrol exerts similar anti-proliferative activities in cells with different metastatic potential. Therefore, we examined the effects of this polyphenol on the growth of weakly metastatic Line IV clone 3 and on autologous, highly metastatic Line IV clone 1 cultured melanoma cells. Comparable inhibition of growth and colony formation resulted from treatment by resveratrol in both cell lines. Flow cytometric analysis revealed that resveratrol-treated clone 1 cells had a dose-dependent increase in S phase and a concomitant reduction in the G 1 phase. No detectable change in cell cycle phase distribution was found in similarly treated clone 3 cells. Western blots demonstrated a significant increase in the expression of the tumor suppressor gene p53, without a commensurate change in p21 and several other cell cycle regulatory proteins in both cell types. Chromatography of Line IV clone 3 and clone 1 cell extracts on resveratrol affinity columns revealed that the basal expression of dihydronicotinamide riboside quinone reductase 2 (NQO2) was higher in Line IV clone 1 than clone 3 cells. Levels of NQO2 but not its structural analog NQO1 were dose-dependently increased by resveratrol in both cell lines. We propose that induction of NQO2 may relate to the observed increased expression of p53 that, in turn, contributes to the observed suppression of cell growth in both melanoma cell lines.