Microsatellite DNA markers have been widely used as a tool for the detection of loss of heterozygosity and genomic instability in primary tumors. In a blinded study, urine samples from 25 patients with suspicious bladder lesions that had been identified cystoscopically were analyzed by this molecular method and by conventional cytology. Microsatellite changes matching those in the tumor were detected in the urine sediment of 19 of the 20 patients (95 percent) who were diagnosed with bladder cancer, whereas urine cytology detected cancer cells in 9 of 18 (50 percent) of the samples. These results suggest that microsatellite analysis, which in principle can be performed at about one-third the cost of cytology, may be a useful addition to current screening methods for detecting bladder cancer.

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)-embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT-embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis.

S U M M A R Y Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharidestimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-kappaB function. In the current study 17beta-estradiol-bound ERalpha interfered with cytokine-induced activation of a NF-kappaB reporter in HepG2 cells. The estrogen metabolite, 17alpha-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-kappaB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-kappaB interfered with the trans-activation properties of ERalpha. Ligand-bound ERalpha did not inhibit NF-kappaB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-kappaB transcriptional activity. Coexpression of the transcription coactivator CREB binding protein (CBP), but not steroid receptor coactivator 1a, reversed the ERalpha-mediated inhibition of NF-kappaB activity. Mammalian two-hybrid experiments also revealed that ligand-bound ERalpha can interact functionally with CBP-NF-kappaB complexes. We suggest that CBP targeting by ERalpha results in the inhibition of NF-kappaB and may occur through formation of transcriptionally inert multimeric complexes that are dependent upon the nature of the ERalpha ligand.

Estrogen replacement therapy increases plasma concentrations of high density lipoprotein and its major protein constituent, apolipoprotein AI (apoAI). Studies with animal model systems, however, suggest opposite effects. In HepG2 cells stably expressing estrogen receptor alpha (ERalpha), 17beta-estradiol (E2) potently inhibited apoAI mRNA steady state levels. ApoAI promoter deletion mapping experiments indicated that ERalpha plus E2 inhibited apoAI activity through the liver-specific enhancer. Although the ERalpha DNA binding domain was essential but not sufficient for apoAI enhancer inhibition, ERalpha binding to the apoAI enhancer could not be detected by electrophoretic mobility shift assays. Western blotting and cotransfection assays showed that ERalpha plus E2 did not influence the abundance or the activity of the hepatocyte-enriched factors HNF-3beta and HNF-4, two transcription factors essential for apoAI enhancer function. Expression of the ERalpha coactivator RIP140 dramatically repressed apoAI enhancer function in cotransfection experiments, suggesting that RIP140 may also function as a coactivator on the apoAI enhancer. Moreover, estrogen regulation of apoAI enhancer activity was dependent upon the balance between ERalpha and RIP140 levels. At low ratios of RIP140 to ERalpha, E2 repressed apoAI enhancer activity, whereas at high ratios this repression was reversed. Regulation of the apoAI gene by estrogen may thus vary in direction and magnitude depending not only on the presence of ERalpha and E2 but also upon the intracellular balance of ERalpha and coactivators utilized by ERalpha and the apoAI enhancer.