Prostate cancer (PCa) remains the most frequently diagnosed male malignancy in Western countries and the second most common cause of male cancer death in the United States. The relatively elevated PCa incidence and mortality among African American men makes this cancer type a challenging health disparity disease. To increase the chance for successful trea tment, earlier detection and prediction of tumor aggress iveness will be important and need to be resolved. This study demonstrates that small membrane-bound vesicles shed from the tumor called exosomes contain ethnically and tumor-specific biomarkers, and could be exploited for their diagnostic and therapeutic potential.

Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the crosstalk relationship between H2B ubiquitination and H3 Lys-79 methylation.Acetylation, methylation, and ubiquitination are widely known modifications of lysine in histones (1). Histone acetylation normally plays a vital role in gene activation with an exception when acetylation is utilized for other functions such as protein-protein interactions (2, 3). The function of histone methylation depends on the site that is modified, i.e. H3 Lys-9, Lys-27 and H4 Lys-20 methylation are associated with heterochromatin where genes are predominantly in a silenced state, whereas H3 Lys-4 and Lys-79 methylation are linked to euchromatin where the majority of genes are in an active format (4). Methylation may also function oppositely, under two different conditions such that both H3 Lys-9 and Lys-36 methylation have a positive effect in the coding region and negative effect in the promotor region (5, 6). Histone H2B ubiquitination has been demonstrated in vitro to be required for histone H3 Lys-4 and Lys-79 di-and tri-methylation, indicating a cross-talk pathway exists between the two types of modifications in two histone subclasses in the process of regulating gene expression (7-9).Human leukemia cell line U937 typically contains reciprocal CALM-AF10 2 fusion genes resulting from the rearrangement of CALM and AF10 genes (10). These CALM-AF10 and/or AF10/CALM fusion proteins were identified in many leukemia patients with acute myeloid, acute lymphoblastic, and T cell acute lymphoblastic leukemia (11-13). The AF10 protein associates with the histone H3 methyltransferase hDot1L in the nucleus (14, 15). Literally, the fusion protein, CALM-AF10, might compete with the endogenous AF10 protein for hDot1L binding and bring the bound Dot1 out of the nucleus to the cytoplasm along with the shuttling vehicle of CALM. As a consequence, the net concentration of the hDot1L protein in the nucleus decreases, ...

The persistent activation of innate immune cells in chronic inflammation is gaining recognition as a contributing factor in a number of human diseases. A distinguishing feature of activated leukocytes at sites of inflammation is their production of reactive species such as hypochlorous acid (HOCl). Investigating the role of reactive molecules such as HOCl in inflammation and human disease requires appropriate biomarkers. The preferred biomarker for HOCl, and by extension its synthesizing enzyme myeloperoxidase, is 3-chlorotyrosine. 3-Chlorotyrosine is a chemically stable product formed when HOCl, or an HOCl-generated chloramine, reacts with the tyrosine side chain and is readily measured by sensitive mass spectrometry methods. However, Whiteman and Spencer ((2008) Biochem. Biophys. Res. Commun., 371, 50 - 53.) noted that 3-chlorotyrosine is degraded by HOCl, calling into question its use as a biomarker. The kinetic rate constants for the reaction of 3-chlorotyrosine with HOCl, histidine chloramine, or lysine chloramine to form 3,5-dichlorotyrosine are reported. The kinetics of tyrosine chlorination in the context of a peptide with a nearby lysine residue was also determined and further supports the role of chloramines in the chlorination of protein-bound tyrosine residues. The likelihood of free and protein-bound 3,5-dichlorotyrosine occurring in vivo, given the reported rate constants, is discussed.

The markers 3-nitrotyrosine and 3-chlorotyrosine are measured as surrogates for reactive nitrogen species and hypochlorous acid respectively, which are both elevated in inflamed human tissues. Previous studies reported a loss of 3-nitrotyrosine when exposed to hypochlorous acid, suggesting that observations of 3-nitrotyrosine underestimate the presence of reactive nitrogen species in diseased tissue (Whiteman and Halliwell, Biochemical and Biophysical Research Communications, 258, 168-172 (1999)). This report evaluates the significance of 3-nitrotyrosine loss by measuring the kinetics of the reaction between 3-nitrotyrosine and hypochlorous acid. The results demonstrate that 3-nitrotyrosine is chlorinated by hypochlorous acid or chloramines to form 3-chloro-5-nitrotyrosine. As 3-nitrotyrosine from in vivo samples is usually found within proteins rather than as free amino acid, we also examined the reaction of 3-nitrotyrosine modification in the context of peptides. The chlorination of 3-nitrotyrosine in peptides was observed to occur up to 700-fold faster than control reactions using equivalent amino acid mixtures. These results further advance our understanding of tyrosine chlorination and the use of 3-nitrotyrosine formed in vivo as a biomarker of reactive nitrogen species.