Blase Billack

Analysis of a set of missense, frameshift, and in-frame deletion variants of BRCA1

Germline mutations that inactivate BRCA1 are responsible for breast and ovarian cancer susceptibility. One possible outcome of genetic testing for BRCA1 is the finding of a genetic variant of uncertain significance for which there is no information regarding its cancer association. This outcome leads to problems in risk assessment, counseling and preventive care. The purpose of the present study was to functionally evaluate seven unclassified variants of BRCA1 including a genomic deletion that leads to the in-frame loss of exons 16/17 (Delta exons 16/17) in the mRNA, an insertion that leads to a frameshift and an extended carboxy-terminus (5673insC), and five missense variants (K1487R, S1613C, M1652I, Q1826H and V1833M). We analyzed the variants using a functional assay based on the transcription activation property of BRCA1 combined with supervised learning computational models. Functional analysis indicated that variants S1613C, Q1826H, and M1652I are likely to be neutral, whereas variants V1833M, Delta exons 16/17, and 5673insC are likely to represent deleterious variants. In agreement with the functional analysis, the results of the computational analysis also indicated that the latter three variants are likely to be deleterious. Taken together, a combined approach of functional and bioinformatics analysis, plus structural modeling, can be utilized to obtain valuable information pertaining to the effect of a rare variant on the structure and function of BRCA1. Such information can, in turn, aid in the classification of BRCA1 variants for which there is a lack of genetic information needed to provide reliable risk assessment.

In Vitro evaluation of the cytotoxic and anti-proliferative properties of resveratrol and several of its analogs

Resveratrol (RES), a component of red wine, possesses anti-inflammatory properties. The studies described in the present work were aimed at evaluating the potential for RES and related stilbene analogs (piceatannol, PIC; pterostilbene, TPS; trans-stilbene, TS; and trans-stilbene oxide, TSO) to exhibit toxicity towards RAW 264.7 mouse macrophages. The effect of TS, TSO, RES and TPS on RAW 264.7 macrophage viability was determined by two standard methods: (a) the MTT assay and (b) the trypan blue dye exclusion test. Whereas macrophages were more sensitive to PIC (LC50 trypan ∼ 1.3 μM) and to TPS (LC50 trypan ∼ 4.0 μM and LC50 MTT ∼ 8.3 μM) than to RES (LC50 trypan ∼ 8.9 μM and LC50 MTT ∼ 29.0 μM), they were relatively resistant to TSO (LC50 trypan ∼ 61.0 μM and LC50 MTT > 100 μM) and to TS (LC50 trypan ≥ 5.0 μM and LC50 MTT ≥ 5.0 μM). The ability of selected stilbenes (RES, TPS and PIC) to exhibit growth inhibitory effects was also examined. Although RES and TPS were observed to inhibit cell proliferation in macrophages (IC50 ≤ 25 μM), these cells were resistant to growth inhibition by PIC (IC50 ≥ 50 μM). The data obtained in the present analysis demonstrate that substituted stilbene compounds such as RES have the capacity to exhibit cytotoxic and anti-proliferative activities in macrophages.

BRCA1 Protein and Nucleolin Colocalize in Breast Carcinoma Tissue and Cancer Cell Lines

The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. Immunoelectron microscopic studies of estrogen-stimulated MCF7 cells demonstrated BRCA1 protein localization in the granular components of the nucleolus. Moreover, immunofluorescence of BRCA1 and nucleolin double-labeling showed colocalization in both nucleoli and nucleoplasmic foci in breast tumor cells and asynchronously growing MCF7 and HeLa cells. Multiparameter analysis of BRCA1 and nucleolin in relation to cell cycle position (DNA content) showed expression during G1-S and persistence of BRCA1 during G2/M. After gamma-irradiation of MCF7 cells, BRCA1 protein dispersed from nucleoli and nucleoplasmic foci to other nucleoplasmic sites, which did not colocalize with nucleolin. Small interfering RNA-mediated knockdown of BRCA1 protein resulted in decreased immunofluorescence staining, which was confirmed by Western blotting. The observed colocalization of BRCA1 and nucleolin raises new possibilities for the nucleoplasm-nucleolus pathways of these proteins and their functional significance.

Growth Inhibitory Action of Ebselen on Fluconazole-Resistant Candida albicans: Role of the Plasma Membrane H+-ATPase

PMA1 is a yeast gene that codes for the plasma membrane H(+)-ATPase, a protein commonly referred to as Pma1p. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a synthetic selenium-containing compound that has recently been shown to display antimicrobial activity owing to its ability to inhibit Pma1p. Ebselen is able to block the activity of Pma1p not only in opportunistic pathogens such as Cryptococcus neoformans and Candida albicans but also in nonpathogenic yeasts such as Saccharomyces cerevisiae. A series of in vitro studies aimed at evaluating the antifungal activity of ebselen were performed. At low concentrations (<10 microM), ebselen was fungistatic against three strains of S. cerevisiae (IC(50) approximately 3 microM) and one fluconazole-resistant strain of C. albicans (IC(50) approximately 6 microM), and at a high concentration (30 microM) it was fungicidal against C. albicans. Moreover, ebselen was found to inhibit medium acidification by the fluconazole-resistant strain of C. albicans in a concentration-dependent manner. In comparison to currently used antifungal agents represented by azole (itraconazole, ketoconazole, fluconazole) and polyene (amphotericin B) compounds, ebselen was at least 10-fold more potent than fluconazole but less active than the other compounds tested. The present results suggest that the growth inhibitory activity of ebselen toward fluconazole-resistant yeast cells is due, at least in part, to inhibition of Pma1p. Ebselen may also serve as a useful agent in the treatment of infections caused by fluconazole-resistant fungi.

Evaluation of the antifungal and plasma membrane H+-ATPase inhibitory action of ebselen and two ebselen analogs in S. cerevisiae cultures

The plasma membrane H+-ATPase pump (Pma1p) has been proposed as a viable target for antifungal drugs since this high capacity proton pump plays a critical role in the intracellular regulation of pH and in nutrient uptake of yeast and other fungi. In recent years, this and other laboratories have verified that the antifungal activity of 2-phenylbenzisoselenazol-3(2H)-one, an organoselenium compound commonly referred to as ebselen (1), stems, at least in part, from its inhibitory action on the fungal Pma1p. In the present study, the antifungal efficacy of 2-(3-pyridinyl)-benzisoselenazol-3(2H)-one (2) and 2-phenylbenzisoselenazol-3(2H)-one 1-oxide (3), two ebselen analogs, was evaluated using a strain of S. cerevisiae and compared against that of 1. In addition, the study also examined the inhibitory potential of these three compounds toward the Pma1p of S. cerevisiae. Based on mean IC50 values, the antifungal potency was found to decrease in the order 3 > 1 > 2. However, in terms of inhibitory action on Pma1p, the potency decreased in the order 1 > 3 > 2. The magnitude of these activities appears to be correlated with the corresponding log P values, with compound 2 being the most hydrophilic and the least active of the three.

A Naturally Occurring Allele of BRCA1 Coding for a Temperature-Sensitive Mutant Protein

Recent evidence suggests that the breast and ovarian cancer susceptibility gene product BRCA1 is involved in at least two fundamental cellular processes: transcriptional regulation and DNA repair. However, the mechanism of action of BRCA1 in either of these processes is still unknown. Here, we report the characterization of a disease-predisposing allele of BRCA1, identified in a family with several cases of ovarian cancer, coding for a protein that displays temperature-sensitive activity in transcriptional activation. The mutant protein differs from the wild type protein at a single amino acid, R1699W that occurs in a region at the N-terminal BRCT domain that is highly conserved among BRCA1 homologs. When the C-terminus of the mutant protein (aa 1560–1863) was fused to a heterologous GAL4 DNA-binding domain and expressed in yeast or mammalian cells, it was able to activate transcription of a reporter gene to levels observed for wild type BRCA1 at the permissive temperature (30?C) but exhibited significantly less transcription activity at the restrictive temperature (37?C or 39?C). Our results indicate that the transcriptional activity of the R1699W mutant can be modulated as a function of temperature and provide a novel experimental approach which can be utilized to dissect the molecular mechanism(s) of BRCA1 in processes related to transcription. Key Words: BRCA1, Yeast, Transcription, Temperature-sensitive mutants, BRCT domain, Ovarian cancer

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280 in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.

Ebselen protects brain, skin, lung and blood cells from mechlorethamine toxicity

Nitrogen mustards are vesicants capable of burning the skin, eyes and respiratory tract of exposed individuals. While generally less toxic than sulfur mustards, these compounds have the potential for use as chemical warfare agents. Presently, no antidote exists for treatment against nitrogen mustard toxicity. The purpose of this study was to investigate the in vitro toxicity of the nitrogen mustard mechlorethamine (HN2) in four cell models: CEM-SS human T cells, A431 human skin epithelial cells, rat hippocampal astrocytes and rat pleural mesothelial cells. Furthermore, the efficacy of the synthetic seleno-organic compound ebselen (Eb) (2-phenyl-1,2- benzisoselenazol-3(2H)-one) as a cytoprotective agent against such toxicity was evaluated. Significant increases in cell viability, as assessed using an MTT assay for viability, was demonstrated when 30μM Eb was used as a cotreatment with HN2 in all cell models tested at the following doses of HN2: A431 skin cells,10—40μM; rat astrocytes, 20 and 40μM; rat mesothelia, 10—40 μM; and human T cells 4—16 μM. Decreases in cell viability and toxicity to HN2 were confirmed using light and scanning electron microscopy. Membrane damage, observed with HN2 exposure, such as blebbing and loss of cell projections, was ameliorated with Eb cotreatment. Our results demonstrate a generalized protective effect observed with Eb cotreatment that suggests that this agent may have potential as an antidote for HN2 exposure and toxicity.

Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin Cells

Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin Cells The cytotoxicity of resveratrol and of piceatannol, a structural analog of resveratrol, was examined in cultured cells. Using a MTT-based assay, which measures the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) to a colored formazan product in living cells, resveratrol was found to inhibit the viability of transformed mouse macrophages, tumor-derived human T cells and human epidermoid carcinoma cells in a concentration-dependent manner, with the effect decreasing in the order: T cells (LC50 ~27 μmol L-1, 24 h; ~9 μmol L-1; 48h) > macrophages (LC50~29 μmol L-1, 24 h; 39 μmol L-1, 48 h) > skin cells (LC50 ~91 μmol L-1, 24 h; ~66 μmol L-1, 48 h). Paradoxically, a high concentration of resveratrol (50 μmol L-1) inhibited the proliferation of all three cell types, and a low concentration (5 μmol L-1) stimulated the proliferation of macrophages. The viability of macrophages was also decreased by piceatannol in a concentration-dependent manner. The stimulation of macrophages with zymosan lowered the cytotoxicity of both resveratrol and piceatannol. Scanning electron microscopy of cells treated with resveratrol revealed changes in cellular morphology that were consistent with toxicity. In macrophages and skin cells, resveratrol (50 μmol L-1) induced a time-dependent increase in reduced glutathione levels but did not alter the background levels of thiobarbituric acid-reactive substances. Taken together, the present data indicate that resveratrol is toxic to cultured macrophages, T cells and skin cells at concentrations ≥25 μmol L-1, and that the cytotoxicity occurs via a mechanism that does not involve oxidative stress. Furthermore, the degree of toxicity of both resveratrol and piceatannol towards macrophages depends on the activation status of these cells, with zymosan-activated cells appearing more resistant than nonstimulated cells. Evaluacija Citotoksičnosti Resveratrola I Piceatanola U Makrofazima, T-Stanicama I Stanicama Kože Citotoksičnost resveratrola i piceatanola, strukturnog analoga resveratrola, ispitivana je u uzgojenim stanicama. Primjenom MTT-testa koji mjeri pretvorbu 3-[4,5-dimetiltiazol-2-il]2,5-difenil-tetrazolijeva bromida (MTT) u obojeni formazan produkt u živim stanicama, nađeno je da resveratrol inhibira preživljavanje transformiranih makrofaga miša, ljudskih tumorskih T-stanica i humanih stanica epidermoidnog karcinoma u ovisnosti o koncentraciji, a učinak opada redom: T-stanice (LC50 ~27 μmol L-1, 24 h; ~9 μmol L-1; 48 h) > makrofazi (LC50 ~29 μmol L-1, 24 h; 39 μmol L-1, 48 h) > stanice kože (LC50 ~91 μmol L-1, 24 h; ~66 μmol L-1, 48 h). Paradoksalno, pri visokoj koncentraciji resveratrola (50 μmol L-1) inhibirana je proliferacija svih triju tipova stanica, a pri niskim koncentracijma (5 μmol L-1) stimulirana je proliferacija makrofaga. Preživljavanje makrofaga bilo je također smanjeno primjenom piceatanola ovisno o koncentraciji. Stimulacija makrofaga zimosanom smanjila je citotoksičnost i resveratrola i piceatanola. Skenirajuća elektronska mikroskopija stanica tretiranih resveratrolom pokazala je promjene u morfologiji stanica, što je bilo u skladu s toksičnosti. U makrofazima i stanicama kože resveratrol (50 μmol L-1) inducirao je porast smanjenja razina glutationa ovisan o vremenu, ali nije mijenjao osnovne razine reaktivnih spojeva tiobarbiturne kiseline. Gledajući skupno, prikazani rezultati indiciraju da je resveratrol toksičan za uzgojene makrofage, T-stanice i stanice kože pri koncentracijama ≥25 μmol L-1 i da se citotoksičnost zbiva mehanizmom koji ne uključuje oksidativni stres. Nadalje, stupanj toksičnosti resveratrola i piceatanola prema makrofagima ovisi o aktivacijskom statusu tih stanica, pri čemu su stanice aktivirane zimosanom rezistentnije od nestimuliranih stanica.

The role of surface receptor stimulation on the cytotoxicity of resveratrol to macrophages.

The present work has evaluated the role of lipopolysaccharide (LPS) stimulation and of TLR4 receptors on the cytotoxicity of resveratrol (RES), a polyphenolic stilbene component of red wine, in TLR4-bearing (i.e., RAW 264.7) and TLR4-deficient (i.e., 10ScNCr/23) macrophages. Based on the results of the MTT assay, DNA fragmentation analysis, and scanning electron microscopic examination, cell stimulation with LPS was found to maintain the viability of, attenuate DNA fragmentation in, and preserve normal morphology of TLR4-proficient macrophages exposed to RES. In contrast, LPS failed to spare TLR4-deficient macrophages from the deleterious effects of RES. Moreover, while LPS treatment conferred protection from RES toxicity in TLR4-bearing macrophages, this stilbene inhibited the production of nitric oxide by stimulated cells in a concentration-dependent fashion. It is therefore likely that the cytotoxic effects of RES towards non-stimulated macrophages and the propensity of RES to inhibit nitric oxide production by activated macrophages are both contributing, at least in part, to the anti-inflammatory activity of this natural product.