Emmanuelle Nicolas

Publications of Emmanuelle Nicolas

• Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time.

  Authors: Eric A Ariazi, Heather E Cunliffe, Joan S Lewis-Wambi, Michael J Slifker, Amanda L Willis, Pilar Ramos, Coya Tapia, Helen R Kim, Smitha Yerrum, Catherine G N Sharma, Emmanuelle Nicolas, Yoganand Balagurunathan, Eric A Ross, V Craig Jordan

  Proceedings of the National Academy of Sciences of the United States of America. 11/2011; 108(47):18879-86.

  In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with eitherIn laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

• APC +/- alters colonic fibroblast proteome in FAP.

  Authors: Bhavinkumar B Patel, Xin-Ming Li, Maketa P Dixon, Elena L Blagoi, Emmanuelle Nicolas, Steven H Seeholzer, David Cheng, Yin A He, Renata A Coudry, Sharon D Howard, Dawn M Riddle, Harry C Cooper, Bruce M Boman, Peggy Conrad, James A Crowell, Alfonso Bellacosa, Alfred Knudson, Anthony T Yeung, Levy Kopelovich

  Oncotarget. 03/2011; 2(3):197-208.

  Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffectedHere we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.

• Interferon impedes an early step of hepatitis delta virus infection.

  Authors: Ziying Han, Shoko Nogusa, Emmanuelle Nicolas, Siddharth Balachandran, John Taylor

  PloS one. 01/2011; 6(7):e22415.

  Hepatitis delta virus (HDV) infects hepatocytes, the major cell type of the liver. Infection of the liver may be either transient or chronic. The prognosis for patients with chronic HDV infection isHepatitis delta virus (HDV) infects hepatocytes, the major cell type of the liver. Infection of the liver may be either transient or chronic. The prognosis for patients with chronic HDV infection is poor, with a high risk of cirrhosis and hepatocellular carcinoma. The best antiviral therapy is weekly administration for at least one year of high doses of interferon alpha. This efficacy of interferon therapy has been puzzling in that HDV replication in transfected cell lines is reported as insensitive to administration of interferon alpha or gamma. Similarly, this study shows that even when an interferon response was induced by transfection of poly(IC) into a cell line, HDV RNA accumulation was only modestly inhibited. However, when the HDV replication was initiated by infection of primary human hepatocytes, simultaneous addition of interferons alpha or gamma at 600 units/ml, a concentration comparable to that achieved in treated patients, the subsequent HDV RNA accumulation was inhibited by at least 80%. These interferon treatments were shown to produce significant time-dependent increases of host response proteins such as for Stat-1, phosphoStat-1, Mx1/2/3 and PKR, and yet interferon pretreatment of hepatocytes did not confer an increased inhibition of HDV replication over interferon treatment at the time of (or after) infection. These and other data support the interpretation that interferon action against HDV replication can occur and is largely mediated at the level of entry into primary human hepatocytes. Thus in vivo, the success of long-term interferon therapy for chronic HDV, may likewise involve blocking HDV spread by interfering with the initiation of productive infection of naïve hepatocytes.

• Drosophila Boi limits Hedgehog levels to suppress follicle stem cell proliferation.

  Authors: Tiffiney R Hartman, Daniel Zinshteyn, Heather K Schofield, Emmanuelle Nicolas, Ami Okada, Alana M O'Reilly

  The Journal of cell biology. 11/2010; 191(5):943-52.

  Stem cells depend on signals from cells within their microenvironment, or niche, as well as factors secreted by distant cells to regulate their maintenance and function. Here we show that Boi, aStem cells depend on signals from cells within their microenvironment, or niche, as well as factors secreted by distant cells to regulate their maintenance and function. Here we show that Boi, a Hedgehog (Hh)-binding protein, is a novel suppressor of proliferation of follicle stem cells (FSCs) in the Drosophila ovary. Hh is expressed in apical cells, distant from the FSC niche, and diffuses to reach FSCs, where it promotes FSC proliferation. We show that Boi is expressed in apical cells and exerts its suppressive effect on FSC proliferation by binding to and sequestering Hh on the apical cell surface, thereby inhibiting Hh diffusion. Our studies demonstrate that cells distant from the local niche can regulate stem cell function through ligand sequestration, a mechanism that likely is conserved in other epithelial tissues.

• Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells.

  Authors: Callinice D Capo-Chichi, Jennifer L Smedberg, Malgorzata Rula, Emmanuelle Nicolas, Anthony T Yeung, Richard F Adamo, Andrey Frolov, Andrew K Godwin, Xiang-Xi Xu

  Stem cells international. 01/2010; 2010:602068.

  Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonicBackground. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (-/-) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (-/-) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (-/-) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.

• The evolutionarily conserved TSC/Rheb pathway activates Notch in tuberous sclerosis complex and Drosophila external sensory organ development.

  Authors: Magdalena Karbowniczek, Diana Zitserman, Damir Khabibullin, Tiffiney Hartman, Jane Yu, Tasha Morrison, Emmanuelle Nicolas, Rachel Squillace, Fabrice Roegiers, Elizabeth Petri Henske

  The Journal of clinical investigation. 01/2010; 120(1):93-102.

  Mutations in either of the genes encoding the tuberous sclerosis complex (TSC), TSC1 and TSC2, result in a multisystem tumor disorder characterized by lesions with unusual lineage expressionMutations in either of the genes encoding the tuberous sclerosis complex (TSC), TSC1 and TSC2, result in a multisystem tumor disorder characterized by lesions with unusual lineage expression patterns. How these unusual cell-fate determination patterns are generated is unclear. We therefore investigated the role of the TSC in the Drosophila external sensory organ (ESO), a classic model of asymmetric cell division. In normal development, the sensory organ precursor cell divides asymmetrically through differential regulation of Notch signaling to produce a pIIa and a pIIb cell. We report here that inactivation of Tsc1 and overexpression of the Ras homolog Rheb each resulted in duplication of the bristle and socket cells, progeny of the pIIa cell, and loss of the neuronal cell, a product of pIIb cell division. Live imaging of ESO development revealed this cell-fate switch occurred at the pIIa-pIIb 2-cell stage. In human angiomyolipomas, benign renal neoplasms often found in tuberous sclerosis patients, we found evidence of Notch receptor cleavage and Notch target gene activation. Further, an angiomyolipoma-derived cell line carrying biallelic TSC2 mutations exhibited TSC2- and Rheb-dependent Notch activation. Finally, inhibition of Notch signaling using a gamma-secretase inhibitor suppressed proliferation of Tsc2-null rat cells in a xenograft model. Together, these data indicate that the TSC and Rheb regulate Notch-dependent cell-fate decision in Drosophila and Notch activity in mammalian cells and that Notch dysregulation may underlie some of the distinctive clinical and pathologic features of TSC.

• Altered gene expression in morphologically normal epithelial cells from heterozygous carriers of BRCA1 or BRCA2 mutations.

  Authors: Alfonso Bellacosa, Andrew K Godwin, Suraj Peri, Karthik Devarajan, Elena Caretti, Lisa Vanderveer, Betsy Bove, Carolyn Slater, Yan Zhou, Mary Daly, Sharon Howard, Kerry S Campbell, Emmanuelle Nicolas, Anthony T Yeung, Margie L Clapper, James A Crowell, Henry T Lynch, Eric Ross, Levy Kopelovich, Alfred G Knudson

  Cancer prevention research (Philadelphia, Pa.). 01/2010; 3(1):48-61.

  We hypothesized that cells bearing a single inherited "hit" in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even preventWe hypothesized that cells bearing a single inherited "hit" in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of "one hit" represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention.

• Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells.

  Authors: Jane J Yu, Victoria A Robb, Tasha A Morrison, Eric A Ariazi, Magdalena Karbowniczek, Aristotelis Astrinidis, Chunrong Wang, Lisa Hernandez-Cuebas, Laura F Seeholzer, Emmanuelle Nicolas, Harvey Hensley, V Craig Jordan, Cheryl L Walker, Elizabeth P Henske

  Proceedings of the National Academy of Sciences of the United States of America. 03/2009;

  Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking femaleLymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-beta-estradiol (E(2)) causes a 3- to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E(2)-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E(2) inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E(2) enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEK-dependent pathways by E(2) leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.

• Properties of subviral particles of hepatitis B virus.

  Authors: Ning Chai, Ho Eun Chang, Emmanuelle Nicolas, Ziying Han, Michal Jarnik, John Taylor

  Journal of virology. 08/2008; 82(16):7812-7.

  In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composedIn the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.

• A Novel Cas Family Member, HEPL, Regulates FAK and Cell Spreading.

  Authors: Mahendra K Singh, Disha Dadke, Emmanuelle Nicolas, Ilya G Serebriiskii, Sinoula Apostolou, Adrian Canutescu, Brian L Egleston, Erica A Golemis

  Molecular biology of the cell. 05/2008; 19(4):1627-36.

  For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focalFor over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.

• Assembly of hepatitis B virus envelope proteins onto a lentivirus pseudotype that infects primary human hepatocytes.

  Authors: Ning Chai, Ho Eun Chang, Emmanuelle Nicolas, Severin Gudima, Jinhong Chang, John Taylor

  Journal of virology. 11/2007; 81(20):10897-904.

  This study demonstrates that the envelope proteins of hepatitis B virus (HBV) could be incorporated into the lipid membrane of lentivirus pseudotype particles. The assembly procedure was initiated byThis study demonstrates that the envelope proteins of hepatitis B virus (HBV) could be incorporated into the lipid membrane of lentivirus pseudotype particles. The assembly procedure was initiated by the transfection of 293T cells with three plasmids: (i) a human immunodeficiency virus (HIV) packaging construct, (ii) a transfer plasmid expressing a reporter gene, and (iii) a plasmid expressing large (L), middle (M), and small (S) HBV envelope proteins. After 2 days, hepatitis B surface antigen and the antigenic forms of L, M, and S were detected at the cell surface by flow cytometry. Also, virus particles that were able to infect cultured primary human hepatocytes (PHH) were released. Under optimal conditions, 50% of PHH could be infected. In addition, the susceptibility of PHH and the resistance of other cell types to the pseudotype particles were similar to those observed for HBV and hepatitis delta virus (HDV), which shares the same L, M, and S. Furthermore, the infection of PHH by the pseudotype was sensitive to known inhibitors of HBV and HDV entry. These findings of specific and efficient infection of hepatocytes could be applicable to liver-specific gene therapy and may help clarify the attachment and entry mechanism used by HBV and HDV.

• The Birt-Hogg-Dube and tuberous sclerosis complex homologs have opposing roles in amino acid homeostasis in Schizosaccharomyces pombe.

  Authors: Marjon van Slegtenhorst, Damir Khabibullin, Tiffiney R Hartman, Emmanuelle Nicolas, Warren D Kruger, Elizabeth Petri Henske

  The Journal of biological chemistry. 09/2007; 282(34):24583-90.

  Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cellBirt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Deltatsc1 and Deltatsc2, were up-regulated in Deltabhd, and levels of specific intracellular amino acids known to be low in Deltatsc1 and Deltatsc2 were elevated in Deltabhd. This "opposite" profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1(+) dramatically increased permease expression levels in Deltabhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Deltabhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.

• Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes.

  Authors: Severin Gudima, Yiping He, Anja Meier, Jinhong Chang, Rongji Chen, Michal Jarnik, Emmanuelle Nicolas, Volker Bruss, John Taylor

  Journal of virology. 05/2007; 81(7):3608-17.

  Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins ofEfficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.

• High-frequency epigenetic repression and silencing of retroviruses can be antagonized by histone deacetylase inhibitors and transcriptional activators, but uniform reactivation in cell clones is restricted by additional mechanisms.

  Authors: Richard A Katz, Emily Jack-Scott, Anna Narezkina, Ivan Palagin, Pamela Boimel, Joseph Kulkosky, Emmanuelle Nicolas, James G Greger, Anna Marie Skalka

  Journal of virology. 04/2007; 81(6):2592-604.

  Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here weIntegrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(-)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(-) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(-) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(-) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(-) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.

• Increased expression of the pro-protein convertase furin predicts decreased survival in ovarian cancer.

  Authors: Robert E Page, Andrés J P Klein-Szanto, Samuel Litwin, Emmanuelle Nicolas, Raid Al-Jumaily, Peter Alexander, Andrew K Godwin, Eric A Ross, Russell J Schilder, Daniel E Bassi

  Cellular oncology : the official journal of the International Society for Cellular Oncology. 02/2007; 29(4):289-99.

  BACKGROUND: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factorsBACKGROUND: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. METHODS: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). RESULTS: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). CONCLUSIONS: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

• Enzymatic mutation detection technologies.

  Authors: Anthony T Yeung, Deepali Hattangadi, Lauryn Blakesley, Emmanuelle Nicolas

  BioTechniques. 06/2005; 38(5):749-58.

  Mutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variabilityMutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Indeed, recent mutations that have not yet become polymorphisms are often deleterious and pertinent to the disease history of afflicted individuals. This review discusses the principles behind a variety of methods for the detection of mutations and factors that should be considered in future methods design. One enzymatic approach in particular using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust. Further developments of this and other methods will allow mutation detection to become an integral component of individualized medicine.

• The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment.

  Authors: Xiao He, Xi He, Vibhuti P Dave, Yi Zhang, Xiang Hua, Emmanuelle Nicolas, Weihong Xu, Bruce A Roe, Dietmar J Kappes

  Nature. 03/2005; 433(7028):826-33.

  Development of immature T-cell precursors (thymocytes) to either the CD4 helper or CD8 killer T-cell lineages correlates precisely with their T-cell receptor specificity for major histocompatibilityDevelopment of immature T-cell precursors (thymocytes) to either the CD4 helper or CD8 killer T-cell lineages correlates precisely with their T-cell receptor specificity for major histocompatibility complex class II or class I molecules, respectively, indicating that the process is carefully regulated. Although intensively studied owing to its importance in determining the composition of the mature T-cell compartment and as a general model of binary lineage decisions, the underlying molecular pathways remain obscure. We have previously reported a spontaneous mouse mutant (HD (helper deficient) mice) in which lineage commitment is specifically perturbed without affecting positive selection. Here we show that a point mutation in the zinc finger transcription factor Th-POK (T-helper-inducing POZ/Krüppel-like factor) is responsible for redirection of class-II-restricted thymocytes to the CD8 lineage in HD mice. Furthermore, we demonstrate that constitutive expression of this factor during thymic development leads to redirection of class-I-restricted thymocytes to the CD4 lineage, indicating that Th-POK is a master regulator of lineage commitment.

• Altered gene expression in phenotypically normal renal cells from carriers of tumor suppressor gene mutations.

  Authors: Radka Stoyanova, Margie L Clapper, Alfonso Bellacosa, Elizabeth P Henske, Joseph R Testa, Eric A Ross, Anthony T Yeung, Emmanuelle Nicolas, Nicolaos Tsichlis, Yue-Sheng Li, W Marston Linehan, Sharon Howard, Kerry S Campbell, Andrew K Godwin, Bruce M Boman, James A Crowell, Levy Kopelovich, Alfred G Knudson

  Cancer biology & therapy. 01/2005; 3(12):1313-21.

  BACKGROUND: The inherently complex signaling networks of tumors result from genetic and epigenetic alterations that occur during cancer initiation and progression. METHODS: In an attempt to identifyBACKGROUND: The inherently complex signaling networks of tumors result from genetic and epigenetic alterations that occur during cancer initiation and progression. METHODS: In an attempt to identify early molecular changes associated with dominantly inherited predisposition to "two-hit" renal tumors, the expression profiles of primary cultures of phenotypically normal renal epithelial cells from individuals bearing a germline mutation in either the von Hippel-Lindau (VHL) or the tuberous sclerosis complex (TSC) gene were compared to that of renal epithelial cells from control nonmutation carriers by microarray analysis. RESULTS: Reliability of the microarray data from pooled samples was confirmed by real-time RT-PCR. Principal Component Analysis revealed substantial differences in the gene expression profiles of the renal epithelial cells from VHL and TSC mutation carriers. In several instances, the microarray data confirm our present knowledge of the cellular pathways affected by biallelic VHL and TSC mutations. CONCLUSIONS: These findings demonstrate that heterozygosity for a mutant tumor suppressor gene may alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner. Detectable effects of "one-hit" represent early molecular changes in tumorigenesis that may serve as targets for chemopreventive intervention.

• miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

  Authors: Jinhong Chang, Emmanuelle Nicolas, Debora Marks, Chris Sander, Anthony Lerro, Marie-Annick Buendia, Chunxiao Xu, William S Mason, Thomas Moloshok, Roque Bort, Kenneth S Zaret, John M Taylor

  RNA biology. 08/2004; 1(2):106-13.

  These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specific noncoding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122 as well as theThese studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specific noncoding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122 as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalian species back to fish. Levels of miR-122 in the mouse liver increase to half maximal values around day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per average cell before birth. Lewis et al. (2003) predicted the cationic amino acid transporter (CAT-1 or SLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammalian tissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1 mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 target sites were predicted within the human CAT-1 mRNA, with six in the 3'-untranslated region. Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to strongly inhibit protein synthesis and reduce mRNA levels. In summary, these studies followed the accumulation during development of miR-122 from its mRNA precursor, hcr, through to identification of what may be a specific mRNA target, CAT-1.