A bioorthogonal organometallic reaction is a biocompatible transformation undergone by a synthetic material exclusively through the mediation of a non-biotic metal source; a selective process used to label biomolecules and activate probes in biological environs. Here we report the in vitro bioorthogonal generation of 5-fluorouracil from a biologically inert precursor by heterogeneous Pd0 catalysis. Although independently harmless, combined treatment of 5-fluoro-1-propargyl-uracil and Pd0-functionalized resins exhibits comparable antiproliferative properties to the unmodified drug in colorectal and pancreatic cancer cells. Live-cell imaging and immunoassay studies demonstrate that the cytotoxic activity of the prodrug/Pd0-resin combination is due to the in situ generation of 5-fluorouracil. Pd0-resins can be carefully implanted in the yolk sac of zebrafish embryos and display excellent biocompatibility and local catalytic activity. The in vitro efficacy shown by this masking/activation strategy underlines its potential to develop a bioorthogonally activated prodrug approach and supports further in vivo investigations.

Estrogens play a significant role in the development, growth, invasion and metastasis of ovarian tumors. The transcriptional program regulated by 17b-estradiol (E 2 ) in human ovarian cancer cell lines was analyzed using cDNA microarrays containing 1200 cancer-related genes. Twenty-eight transcripts had at least a threefold change in expression in E 2 -treated PEO1 ovarian carcinoma cells compared with controls. These differences were confirmed by real-time quantitative PCR and shown to be dependent upon the expression of functional estrogen receptor-a (ERa). Consistent with this, these gene expression changes were blocked by the anti-estrogen tamoxifen. The use of ERa-and ERb-specific ligands allowed molecular dissection of the E 2 response and showed that ERa activation was responsible for the observed changes in gene expression, whereas ERb played no significant role. Inhibition of de novo protein synthesis by cycloheximide was used to distinguish between primary and secondary target genes regulated by E 2 . Actinomycin D was used to show that changes in gene expression levels induced by E 2 were a result of changes in transcription and not due to changes in mRNA stability. The results presented here demonstrate that estrogen-driven growth of epithelial ovarian carcinoma is mediated by activation of ERa-mediated, and not ERbmediated, transcription.

The majority of ovarian cancer patients are treated with platinum-based chemotherapy, but the emergence of resistance to such chemotherapy severely limits its overall effectiveness. We have shown that development of resistance to this treatment can modify cell signaling responses in a model system wherein cisplatin treatment has altered cell responsiveness to ligands of the erbB receptor family. A cisplatinresistant ovarian carcinoma cell line PE01 CDDP was derived from the parent PE01 line by exposure to increasing concentrations of cisplatin, eventually obtaining a 20-fold level of resistance. Whereas PE01 cells were growth stimulated by the erbB receptor-activating ligands, such as transforming growth factor-A (TGFA), NRG1A, and NRG1B, the PE01 CDDP line was growth inhibited by TGFA and NRG1B but unaffected by NRG1A. TGFA increased apoptosis in PE01 CDDP cells but decreased apoptosis in PE01 cells. Differences in extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling were also found, which may be implicated in the altered cell response to ligands. Microarray analysis revealed 51 genes whose mRNA increased by at least 2-fold in PE01 CDDP cells relative to PE01 (including FRA1, ETV4, MCM2, AXL, MT3, TRAP1, and FANCG), whereas 36 genes (including IGFBP3, TRAM1, and KRT4 and KRT19) decreased by a similar amount. Differential display reverse transcriptase-PCR identified altered mRNA expression for TCP1, SLP1, proliferating cell nuclear antigen, and ZXDA. Small interfering RNA inhibition of FRA1, TCP1, and MCM2 expression was associated with reduced growth and FRA1 inhibition with enhanced cisplatin sensitivity. Altered expression of these genes by cytotoxic exposure may provide survival advantages to cells including deregulation of signaling pathways, which may be critical in the development of drug resistance. (Cancer Res 2005; 65(15): 6789-800)

No abstract

No abstract

No abstract

B Kuske and C Naughton contributed equally to this work. AbstractHormone-dependent estrogen receptor (ER)-positive breast cancer cells may adapt to low estrogen environments such as produced by aromatase inhibitors. In many instances, cells become insensitive to the effects of estrogen but may still retain dependence on ER. We have investigated the expression, function, and activation of ERa in two endocrine-resistant MCF-7 models to identify mechanisms that could contribute to resistance. While MCF-7/LCC1 cells are partially estrogen dependent, MCF-7/LCC9 cells are fully estrogen insensitive and fulvestrant and tamoxifen resistant. In both MCF-7/LCC1 and MCF-7/LCC9 cell lines, high expression of ERa was associated with enhanced binding to the trefoil factor 1 (TFF1) promoter in the absence of estrogen and increased transcription of TFF1 and progesterone receptor. In contrast to the observations derived from hypersensitive and supersensitive models, these cells were truly estrogen independent; nevertheless, removal of ERa by siRNA, or fulvestrant, a specific ER downregulator, inhibited growth indicating dependence on ERa. In the absence of estrogen, neither ERa Ser 118 nor Ser 167 were phosphorylated as frequently found in other ligandindependent cell line models. Addition of estrogen activated ERa Ser 118 in MCF-7 and LCC1 cells but not in LCC9 cells. We suggest that the estrogen-independent growth within these cell lines is accounted for by high levels of ERa expression driving transcription and full estrogen independence explained by lack of ERa activation through Ser 118 .

No abstract