O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1.

The human epigenetic cell-cycle regulator HCF-1 undergoes an unusual proteolytic maturation process resulting in stably associated HCF-1(N) and HCF-1(C) subunits that regulate different aspects of the cell cycle. Proteolysis occurs at six centrally located HCF-1(PRO)-repeat sequences and is important for activation of HCF-1(C)-subunit functions in M phase progression. We show here that the HCF-1(PRO) repeat is recognized by O-linked β-N-acetylglucosamine transferase (OGT), which both O-GlcNAcylates the HCF-1(N) subunit and directly cleaves the HCF-1(PRO) repeat. Replacement of the HCF-1(PRO) repeats by a heterologous proteolytic cleavage signal promotes HCF-1 proteolysis but fails to activate HCF-1(C)-subunit M phase functions. These results reveal an unexpected role of OGT in HCF-1 proteolytic maturation and an unforeseen nexus between OGT-directed O-GlcNAcylation and proteolytic maturation in HCF-1 cell-cycle regulation.

A novel mucosal orthotopic murine model of human papillomavirus-associated genital cancers.

Cervical cancer results from infection with high-risk type human papillomaviruses (HPV). Therapeutic vaccines aiming at controlling existing genital HPV infections and associated lesions are usually tested in mice with HPV-expressing tumor cells subcutaneously implanted into their flank. However, effective vaccine-induced regression of these ectopic tumors strongly contrasts with the poor clinical results of these vaccines produced in patients with HPV-associated genital neoplasia. To assess HPV therapeutic vaccines in a more relevant setting, we have, here, established an orthotopic mouse model where tumors in the genital mucosa (GM) develop after an intravaginal instillation of HPV16 E6/E7-expressing tumor cells transduced with a luciferase-encoding lentiviral vector for in vivo imaging of tumor growth. Tumor take was 80-90% after nonoxynol-9 induced damage of the epithelium. Tumors remained localized in the genital tract, and histological analysis showed that most tumors grew within the squamous epithelium of the vaginal wall. Those tumors induced (i) E7-specific CD8 T cells restricted to the GM and draining lymph nodes, in agreement with their mucosal location and (ii) high Foxp3+ CD4+ infiltrates, similarly to those found in natural non-regressing HPV lesions. This novel genital HPV-tumor model by requiring GM homing of vaccine-induced immune responses able to overcome local immuno-suppression may be more representative of the situation occurring in patients upon therapeutic vaccination.

In vivo localisation of radiolabelled antibodies to carcinoembryonic antigen in human colon carcinoma grafted into nude mice.

SEVERAL attempts have been made to show the specific localisation in vivo of anti-tumour antibodies. Most of these studies, however, either in experimental animals1,2 or in humans3 were performed with antibodies obtained by adsorption and elution from poorly characterised crude tumour fractions.

Comparative localization of glioma-reactive monoclonal antibodies in vivo in an athymic mouse human glioma xenograft model.

Radioiodinated murine monoclonal antibodies (Mabs) 81C6, Me 1-14, C12, D12, and E9, made against or reactive with human gliomas but not normal brain, and Mab UJ13A, a pan-neuroectodermal Mab reactive with normal human glial and neural cells, were evaluated in paired label studies in the D-54 MG subcutaneous human glioma xenograft model system in nude mice. Following intravenous injection in the tail vein of mice bearing 200-400 mm3 tumors, specific localization of Mabs to tumor over time (6 h-9 days) was evaluated by tissue counting; each Mab demonstrated a unique localization profile. The comparison of localization indices (LI), determined as a ratio of tissue level of Mab to control immunoglobulin with simultaneous correction for blood levels of each, showed Mabs 81C6 and Me 1-14 to steadily accumulate in glioma xenografts, maintaining LI from 5-20 at 7-9 days after Mab injection. Mab UJ13A peaked at day 1, maintaining this level through day 2, and declining thereafter. Mabs D12 and C12 peaked at days 3 and 4, respectively, and E9 maintained an LI of greater than 3 from days 3-9. Percent injected dose localized/g of tumor varied from a peak high of 16% (81C6) to a low of 5% (Me 1-14 and UJ13A). Immunoperoxidase histochemistry, performed with each Mab on a battery of primary human brain neoplasms, revealed that Mabs 81C6 and E9, which demonstrated the highest levels of percent injected dose localized/g of tumor over time, reacted with antigens expressed in the extracellular matrix. This finding suggests that extracellular matrix localization of antigen represents a biologically significant factor affecting localization and/or binding in the xenograft model used. The demonstration of significant localization, varied kinetics and patterns of localization of this localizing Mab panel warrants their continued investigation as potential imaging and therapeutic agents for human trials.

The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily, which act as transcription factors upon binding to specific DNA sequences called thyroid hormone (T3) response elements (TREs). Such elements are found in the upstream regulatory region of promoters as well as in intragenic sequences of T3-responsive genes. In this report, we demonstrate that SV40 late (SVL) promoter activity is strongly down-regulated by TR in the absence of ligand. Addition of T3 releases this repression, but does not further induce SVL promoter activity. Electrophoretic mobility shift analyses reveal a TR binding element that overlaps with the SV40 major late transcription initiation site. This element closely fits the consensus TRE, formed of two hexanucleotides organized in a tandem repeat separated by 4 nt, and is able to confer T3 responsiveness on a heterologous promoter. We further show that, although the presence of TR leads to quantitatively modified expression of an SVL-driven reporter gene, neither displacement of the site of transcription initiation nor modification of the splicing pattern of the primary transcripts occur.

Transplantation of a human mammary carcinoma cell line (BT 20) into nude mice.

Cell suspensions of a human mammary carcinoma cellline (BT 20), wh en injected subcutaneously into nude athymie mice (BALB/c NujNu), produced tumor nodules at the injection site. Subsequent seriai transM plantations also gave rise to neoplastic nodules after latency periods averaging 3 weeks. The nodules displayed morphologie and functional characteristics comparable to those of the original tumor cells. Metastases, however, were not observed in any of the tumor-bearing mice.

Identification and functional characterization of auxiliary enzymes of the β-oxidation in " Arabidopsis thaliana "

Abstract: The ß-oxidation is the universal pathway that allows living organisms to degrade fatty acids. leading to lipid homeostasis and carbon and energy recovery from the fatty acid molecules. This pathway is centred on four core enzymatic activities sufficient to degrade saturated fatty acids. Additional auxiliary enzymes of the ß-oxidation are necessary for the complete degradation of a larger array of molecules encompassing the unsaturated fatty acids. The main pathways of the ßoxidation of fatty acids have been investigated extensively and auxiliary enzymes are well-known in mammals and yeast. The comparison of the established ß-oxidation systems suggests that the activities that are required to proceed to the full degradation of unsaturated fatty acids are present regardless of the organism and rely on common active site templates. The precise identity of the plant enzymes was unknown. By homology searches in the genome of Arabidopsis thaliana, I identified genes. encoding for proteins that could be orthologous to the yeast or animal auxiliary enzymes Δ 3, Δ 2-enoyl-CoA isomerase, Δ 3,5, Δ 2,4 -dienoyl-CoA isomerase, and type 2 enoyl-CoA hydratase. I established that these genes are expressed in Arabidopsis and that their expression can be correlated to the expression of core ß-oxidation genes. Through the observation of chimeric fluorescent protein fusions, I demonstrated that the identified proteins are localized in the peroxisóme, the only organelle where the ß-oxidation occurs in plants. Enzymatic assays were performed with the partially purified enzymes to demonstrate that the identified enzymes can catalyze the same in vitro reactions as their non-plant orthologs. The activities in vivo of the plant enzymes were demonstrated by heterologous complementation of the corresponding yeast Saccharomyces cerevisiae mutants. The complementation was visualized using the artificial polyhydroxyalkanoate (PHA) production in yeast peroxisomes. The recombinant strains, expressing a Pseudomonas aeruginosa PHA synthase modified for a peroxisomal localization, produce this polymer that serves as a trap for the 3-hydroxyacyl-CoA intermediaries of the ßoxidation and that reflects qualitatively and quantitatively the array of molecules that are processed through the ß-oxidation. This complementation demonstrated the implication of the plant Δ 3, Δ 2-enoyl-CoA isomerases and Δ3,5, Δ2,4-dienoyl-CoA isomerase in the degradation of odd chain position unsaturated fatty acids. The presence of a monofunctional type 2 enoyl-CoA hydratase is a novel in eukaryotes. Downregulation of the corresponding gene expression in an Arabidopsis line, modified to produce PHA in the peroxisome, demonstrated thàt this enzyme participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2Eenoyl-CoA for further degradation through the core ß-oxidation cycle. Résumé: La ß-oxydation est une voie universelle de dégradation des acides gras qui permet aux organismes vivants d'assurer une homéostasie lipidique et de récupérer l'énergie et le carbone contenus dans les acides gras. Le coeur de cette voie est composé de quatre réactions enzymatiques suffisantes à la dégradation des acides gras saturés. La présence des enzymes auxiliaires de la ß-oxydation est nécessaire à la dégradation d'une gamme plus étendue de molécules comprenant les acides gras insaturés. Les voies principales de la ß-oxydation des acides gras ont été étudiées en détail et les enzymes auxiliaires sont déterminées chez les mammifères et la levure. La comparaison entre les systèmes de ß-oxydation connus suggère que les activités requises pour la dégradation complète des acides gras insaturés reposent sur la présence de site actifs similaires. L'identité précise des enzymes auxiliaires chez les plantes était inconnue. En cherchant par homologie dans le génome de la plante modèle Arabidopsis thaliana, j'ai identifié des gènes codant pour des protéines pouvant être orthologues aux enzymes auxiliaires Δ3 Δ2-enoyl-CoA isomérase, Δ 3,5 Δ 2,4-dienoyl-CoA isomérase et enoyl-CoA hydratase de type 2 d'origine fongique ou mammalienne. J'ai établi la corrélation de l'expression de ces gènes dans Arabidopsis avec celle de gènes des enzymes du coeur de la ß-oxydation. En observant des chimères de fusion avec des protéines fluorescentes, j'ai démontré que les protéines identifiées sont localisées dans le péroxysomes, le seul organelle où la ß-oxydation se déroule chez les plantes. Des essais enzymatiques ont été conduits avec ces enzymes partiellement purifiées pour démontrer que les enzymes identifiées sont capables de catalyser in vitro les mêmes réactions que leurs orthologues non végétaux. Les activités des enzymes végétales in vivo ont été .démontrées par complémentation hétérologue des mutants de délétion correspondants de levure Saccharomyces cerevisiae. La visualisation de la complémentation est rendue possible par la synthèse de polyhydroxyalcanoate (PHA) dans les péroxysomes de levure. Les souches recombinantes expriment la PHA synthase de Pseudomonas aeruginosa modifiée pour être localisée dans le péroxysome produisent ce polymère qui sert de piège pour les 3-hydroxyacylCoAs intermédiaires de la ß-oxydation et qui reflète qualitativement et quantitativement la gamme de molécules qui subit la ß-oxydation. Cette complémentation a permis de démontrer que les Δ3, Δ2-enoyl-CoA isomérases, et la Δ3.5, Δ2,4-dienoyl-CoA isomérase végétales sont impliquées dans la dégradation des acides gras insaturés en position impaire. L'enoyl-CoA hydratase de type 2 monofonctionelle est une enzyme nouvelle chez les eucaryotes. La sous-expression du gène correspondant dans une lignée d'Arabidopsis modifiée pour produite du PHA dans le péroxysome a permis de démontrer que cette enzyme participe in vivo à la dégradation des acides gras ayant une double liaison en conformation cis (Z) en position paire.

Probing the role of point mutations in the cyp51A gene from Aspergillus fumigatus in the model yeast Saccharomyces cerevisiae.

Azole-resistant strains of Aspergillus fumigatus have been detected and the underlying molecular mechanisms of resistance characterized. Point mutations in the cyp51A gene have been proved to be related to azole resistance in A. fumigatus clinical strains and with different resistance profiles depending on the amino acid change (G54E, G54V, G54R, G54W, M220V, M220K, M220T, M220I). The aim of this work was to express A. fumigatus cyp51A genes in the yeast Saccharomyces cerevisiae in order to better assess the contribution of each independent amino acid substitution to resistance. A tetracycline regulatable system allowing repression of the endogenous essential ERG11 gene was used. The expression of Aspergillus cyp51A alleles could efficiently restore the absence of ERG11 in S. cerevisiae. In general, S. cerevisiae clones expressing. A. fumigatus cyp51A alleles from azole-resistant isolates showed higher MICs to all azoles tested than those expressing alleles from susceptible isolates. The azole susceptibility profiles obtained in S. cerevisiae upon expression of specific cyp51A alleles recapitulated susceptibility profiles observed from their A. fumigatus origins. In conclusion this work supports the concept that characteristics of specific A. fumigatus cyp51A alleles could be investigated in the heterologous host S. cerevisiae.

Bystander activation of CD4+ T cells.

Bystander activation of T cells, i.e. the stimulation of unrelated (heterologous) T cells by cytokines during an Ag-specific T-cell response, has been best described for CD8(+) T cells. In the CD8(+) compartment, the release of IFN and IFN-inducers leads to the production of IL-15, which mediates the proliferation of CD8(+) T cells, notably memory-phenotype CD8(+) T cells. CD4(+) T cells also undergo bystander activation, however, the signals inducing this Ag-nonspecific stimulation of CD4(+) T cells are less well known. A study in this issue of the European Journal of Immunology sheds light on this aspect, suggesting that common gamma-chain cytokines including IL-2 might be involved in bystander activation of CD4(+) T cells.

The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element.

The estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.

Lack of original antigenic sin in recall CD8(+) T cell responses.

In the real world, mice and men are not immunologically naive, having been exposed to numerous antigenic challenges. Prior infections sometimes negatively impact the response to a subsequent infection. This can occur in serial infections with pathogens sharing cross-reactive Ags. At the T cell level it has been proposed that preformed memory T cells, which cross-react with low avidity to epitopes presented in subsequent infections, dampen the response of high-avidity T cells. We investigated this with a series of related MHC class-I restricted Ags expressed by bacterial and viral pathogens. In all cases, we find that high-avidity CD8(+) T cell precursors, either naive or memory, massively expand in secondary cross-reactive infections to dominate the response over low-avidity memory T cells. This holds true even when >10% of the CD8(+) T cell compartment consists of memory T cells that cross-react weakly with the rechallenge ligand. Occasionally, memory cells generated by low-avidity stimulation in a primary infection recognize a cross-reactive epitope with high avidity and contribute positively to the response to a second infection. Taken together, our data show that the phenomenon of original antigenic sin does not occur in all heterologous infections.

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.

Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

G Protein-Coupled Receptor heterodimerization in the brain

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

Cell viability and noninvasive in vivo MRI tracking of 3D cell encapsulating self-assembled microcontainers.

Several molecular therapies require the implantation of cells that secrete biotherapeutic molecules and imaging the location and microenvironment of the cellular implant to ascertain its function. We demonstrate noninvasive in vivo magnetic resonance imaging (MRI) of self-assembled microcontainers that are capable of cell encapsulation. Negative contrast was obtained to discern the microcontainer with MRI; positive contrast was obtained in the complete absence of background signal. MRI on a clinical scanner highlights the translational nature of this research. The microcontainers were loaded with cells that were dispersed in an extracellular matrix, and implanted both subcutaneously and in human tumor xenografts in SCID mice. MRI was performed on the implants, and microcontainers retrieved postimplantation showed cell viability both within and proximal to the implant. The microcontainers are characterized by their small size, three dimensionality, controlled porosity, ease of parallel fabrication, chemical and mechanical stability, and noninvasive traceability in vivo.