Kenneth B. Tomer

Testosterone and dihydrotestosterone tissue levels in recurrent prostate cancer

Purpose: Prostate cancer eventually recurs during androgen deprivation therapy despite castrate levels of serum androgens. Expression of androgen receptor and androgen receptor–regulated proteins suggests androgen receptor activation in recurrent prostate cancer. Many groups have pursued mechanisms of ligand-independent androgen receptor activation but we found high levels of testicular androgens in recurrent prostate cancer tissue using RIA. Experimental Designs: Prostate specimens from 36 men were procured preserving blood flow to prevent ischemia and cyropreserved immediately. Recurrent prostate cancer specimens from 18 men whose cancer recurred locally during androgen deprivation therapy and androgen-stimulated benign prostate specimens from 18 men receiving no hormonal treatments were studied. Tissue levels of testosterone and dihydrotestosterone were measured in each specimen using liquid chromatography/electrospray tandem mass spectrometry. Testosterone and dihydrotestosterone levels were compared with clinical variables and treatment received. Results: Testosterone levels were similar in recurrent prostate cancer (3.75 pmol/g tissue) and androgen-stimulated benign prostate (2.75 pmol/g tissue, Wilcoxon two-sided, P = 0.30). Dihydrotestosterone levels decreased 91% in recurrent prostate cancer (1.25 pmol/g tissue) compared with androgen-stimulated benign prostate (13.7 pmol/g tissue; Wilcoxon two-sided, P < 0.0001) although dihydrotestosterone levels in most specimens of recurrent prostate cancer were sufficient for androgen receptor activation. Testosterone or dihydrotestosterone levels were not related to metastatic status, antiandrogen treatment, or survival (Wilcoxon rank sum, all P > 0.2). Conclusions: Recurrent prostate cancer may develop the capacity to biosynthesize testicular androgens from adrenal androgens or cholesterol. This surprising finding suggests intracrine production of dihydrotestosterone and should be exploited for novel treatment of recurrent prostate cancer.

Fast atom bombardment and tandem mass spectrometry of phosphatidylserine and phosphatidylcholine

Fast atom bombardment (FAB) desorption of phosphatidylserine and various phosphatidylcholines produces a limited number of very informative negative ions. Especially significant is the formation of (M-H)− ions for phosphatidylserine, a compound which does not yield informative high mass ions by other ionization methods. Phosphatidylcholines of not yield (M-H)− ions but instead produce three characteristic high mass ions, (M-CH3+_−, [M-HN(CH3)3+]− and [M-HN(CH33+-C2H2]−. Both classes of lipids also yield anions attributed to the carboxylate components of these complex lipids. FAB desorption in combination with collisional activation allows for characterization of fragmentation and determination of structural features. Collisional activation of the carboxylate anion fragments from the complex lipids is especially informative. Structural characterization of the fatty acid chain can be achieved as the released saturated carboxylate anions undergo a highly specific 1,4-elimination of H2, which results in the losses of the elements of CH4, C2H6, C3H8...in a fashion entirely consistent with the chemistry of carboxylate anions desorbed from free fatty acids. These CnH2n+2 losses begin at the alkyl terminus and progress along the entire alkyl chain. Modified fatty acids undergo a similar fragmentation; however, the modification affects the series of CnH2n+2 losses in a manner which permits determining the type of modification and its location on the fatty acid chain.

Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5

ABSTRACT Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.

Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, β-casein and α-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.

FAB MS/MS for phosphatidylinostitol,-glycerol,-ethanolamine and other complex phospholipids

Fast atom bombardment (FAB) of phosphatidylinositol, phosphatidylethanolamine, cardiolipin, phosphatidic acid and phosphatidylglycerol produces a limited number of very informative negative ions. Especially significant is the formation of (M−H)− ions and ions that correspond to the carboxylate portions of these molecules. FAB desorption in combination with collisional activation allows for characterization of fragmentation and determination of structural features. Collisional activation of the carboxylate anion from complex lipids is especially informative. Structural characterization of the fatty acids can be achieved as the released saturated carboxylate anions undergo highly specific charge remote fragmentations that are entirely consistent with the chemistry of carboxylate anions desorbed from free fatty acids. This permits both identification of the modification and assignment of its location on the acid chain. FAB-desorbed alkyl acetyl glycerophosphocholines (platelet-activating factor) do not produce (M−H)− ions. However, significant high mass ions are formed, and these can be collisionally activated for structural characterization.

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.

CEBS—Chemical Effects in Biological Systems: a public data repository integrating study design and toxicity data with microarray and proteomics data

Abstract CEBS (Chemical Effects in Biological Systems) is an integrated public repository for toxicogenomics data, including the study design and timeline, clinical chemistry and histopathology findings and microarray and proteomics data. CEBS contains data derived from studies of chemicals and of genetic alterations, and is compatible with clinical and environmental studies. CEBS is designed to permit the user to query the data using the study conditions, the subject responses and then, having identified an appropriate set of subjects, to move to the microarray module of CEBS to carry out gene signature and pathway analysis. Scope of CEBS: CEBS currently holds 22 studies of rats, four studies of mice and one study of Caenorhabditis elegans. CEBS can also accommodate data from studies of human subjects. Toxicogenomics studies currently in CEBS comprise over 4000 microarray hybridizations, and 75 2D gel images annotated with protein identification performed by MALDI and MS/MS. CEBS contains raw microarray data collected in accordance with MIAME guidelines and provides tools for data selection, pre-processing and analysis resulting in annotated lists of genes of interest. Additionally, clinical chemistry and histopathology findings from over 1500 animals are included in CEBS. CEBS/BID: The BID (Biomedical Investigation Database) is another component of the CEBS system. BID is a relational database used to load and curate study data prior to export to CEBS, in addition to capturing and displaying novel data types such as PCR data, or additional fields of interest, including those defined by the HESI Toxicogenomics Committee (in preparation). BID has been shared with Health Canada and the US Environmental Protection Agency. CEBS is available at http://cebs.niehs.nih.gov. BID can be accessed via the user interface from https://dir-apps.niehs.nih.gov/arc/. Requests for a copy of BID and for depositing data into CEBS or BID are available at http://www.niehs.nih.gov/cebs-df/.

Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides

The solution chemistry conditions necessary for optimum analysis of peptides by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry and CZE electrospray ionization tandem mass spectrometry have been studied. To maximize the signal-to-noise ratio of the spectra it was found necessary to use acidic CZE buffers of low ionic strength. This not only increases the total ion current, but it also serves to fully protonate the peptides, minimizing the distribution of ion current across the ensemble of possible charge states.The use of acidic buffers protonates the peptides, which is advantageous for mass spectrometry and tandem mass spectrometry analysis, but is problematic with CZE when bare fused silica CZE columns are used. These conditions produce positively charged peptides, and negatively charged silanol moieties on the column wall, inducing adsorption of the positively charged peptides, thus causing zone broadening and a loss in separation efficiency. This problem was circumvented by the preparation of chemically modified CZE columns, which, when used with acidic CZE buffers, will have a positively charged inner column wall. The electrostatic repulsion between the positively charged peptides and the positively charged CZE column wall minimizes adsorption problems and facilitates high efficiency separations. Full-scan mass spectra were acquired from injections of as little as 160 fmols of test peptides, with CZE separation efficiencies of up to 250,000 theoretical plates.

Activation of the Androgen Receptor by Intratumoral Bioconversion of Androstanediol to Dihydrotestosterone in Prostate Cancer

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report, we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17β-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17β-HSD6 in prostate-derived cell lines and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor-bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis.