A detailed understanding of the assortment of genes that are expressed in breast tumor vessels is needed to facilitate the development of novel, molecularly targeted anti-angiogenic agents for breast cancer therapies. Rapid immunohistochemistry using factor VIII-related antibodies was performed on sections of frozen human luminal-A breast tumors (n ‫؍‬ 5) and normal breast (n ‫؍‬ 5), followed by laser capture microdissection of vascular cells. RNA was extracted and amplified, and fluorescently labeled cDNA was synthesized and hybridized to 44,000-element longoligonucleotide DNA microarrays. Statistical analysis of microarray was used to compare differences in gene expression between tumor and normal vascular cells, and Expression Analysis Systematic Explorer was used to determine enrichment of gene ontology categories. Protein expression of select genes was confirmed using immunohistochemistry. Of the 1176 genes that were differentially expressed between tumor and normal vascular cells, 55 had a greater than fourfold increase in expression level. The extracellular matrix gene ontology category was increased while the ribosome gene ontology category was decreased. Fibroblast activation protein, secreted frizzledrelated protein 2, Janus kinase 3, and neutral sphingomyelinase 2 proteins localized to breast tumor endothelium as assessed by immunohistochemistry, showing significantly greater staining compared with normal tissue. These tumor endothelial marker proteins also exhibited increased expression in breast tumor vessels compared with that in normal tissues. Therefore, these genetic markers may serve as potential targets for the development of angiogenesis inhibitors.

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Endometrial cancer, the most common gynecological malignancy in western countries, is characterized by a favorable prognosis. Nonetheless, deep myometrial invasion correlates with more undifferentiated tumors, lymph-vascular invasion, node involvement and decreased global survival. We have described previously the Ets family member ERM/ETV5 specifically upregulated in endometrial endometrioid carcinoma (EEC) associated with myometrial infiltration. To understand the role of this transcription factor during myometrial infiltration, we analyzed by two-dimension differential gel electrophoresis (2D-DIGE) technology those proteins whose expression was altered in endometrial cell lines stably overexpressing ERM/ETV5. Pathway analysis pointed to actin regulation and transforming growth factor beta and progesterone signaling as processes regulated by ERM/ETV5. In addition, we characterized the specific upregulation of the nuclear dehydrogenase/reductase Hep27 as well as its ERM/ETV5-dependent mitochondrial localization. Further functional studies demonstrated a protective role of Hep 27 against apoptosis induced by oxidative stress. Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumor behavior and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. Unraveling the molecular events in EEC associated with the initiation of tumor invasion would represent an obvious improvement in the pursuit of rational targets for the onset of metastasis. This knowledge would also be a valuable tool for the molecular stratification of patients since myometrial affectation determines an increase in the rate of recurrence after a first surgical treatment and a decrease in 5 year survival.

The histone variants and high-mobility-group (HMG) proteins of a transcribing fraction of chromatin, described in the preceeding paper of this journal, have been analysed qualitatively and quantitatively by a combination of one-dimensional and two-dimensional gel electrophoresis. The stoichiometry of the four core histones (all variants included) in this fraction is equimolar and is not detectably different from that in the nontranscribing fraction or in total chromatin. The molar ratio of histone H I to the core histones is markedly lower, by approximately 72 %, than that in the nontranscribiiig fraction. A minor histone variant identified as M1(an H2A variant) is detected only in the transcribing fraction, while variant H3.1 is found only in the nontranscribing fraction. Proteins A24, H M G l and H MG2 are essentially absent from the transcribing fraction ; HMG14 is found uniquely in this fraction, while HMG17 occurs at a relatively lower level.In preceding papers [l -31 we have described the separation and properties of a fraction of chromatin which, by a number of criteria, is greatly enriched in transcribing chromatin. Although it is clear [4] that transcribing chromatin possesses a nucleosome-like organisation, the possibility that nucleosomal structure is different in regions which are undergoing transcription, due to inclusion of high-mobilitygroup (HMG) proteins [5-71 or of specific histone variants [8,9], or to differences in tlic relative amounts of histone HI [lo-131, has frequently been discussed (reviewed in [4]). T o examine these possible differences, we have analysed quantitatively the histones and H M G proteins in the transcribing chromatin fraction from mouse cells. MATERIALS AND METHODS MaterialsChemicals and materials were as follows: Triton N-101, 2-mercaptoethanol and sodium dodecyl sulfate (Sigma); acrylamide, bisacrylamide and PhMeS02F (Fluka) ; pronase (Serva) ; [4,5-3H]lysine, [G-3H]tryptophan, I4C-labelled amino acid mixture and aquasol (NEN); PPO (Merck); X-Omat R X-ray film (Kodak). The sources of other chemicals arc given in the preceding paper [ 3 ] . Preparation unlf Fractionation qf ChromatinThe proteins of growing mouse P815 cells were labelled during at least three generations of growth using [3H]lysine (2-10 FCi/ml), [3H]tryptophan (6.6 pCi/ml) or a 14C-labelled amino acid mixture(0.25pCi/ml).To monitor chromatin fractionation, DNA was labelled at a low level using [14C]thyAbbreviutions. HMG, high-mobility-group; PhMeSOZF, phenylmethylsulfonyl fluoride. midine (4 pCi/l) when 'H-labelled amino acids were used. Growth conditions, chromatin preparation and fractionation were as described [2]. The appropriate chromatin fractions were pooled, precipitated with MgClz [3] and stored at -70 C. Extraction of Clwomatin ProteinsTotal Chromatin proteins were extracted with phenol [14], precipitated overnight at -20°C by addition of 6 vol. of acetone at -20 "C, washed three times with acetone, and resuspended in 0.8 M urea, 10 % 2-mercaptoethanol and stored at -70 "C until loading onto g...