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S Nasi

Publications and source records attributed to S Nasi.

At least 19 recordsLinked to original sources
PubMed2004

Omomyc expression in skin prevents Myc-induced papillomatosis.

Obligate sensitization to apoptosis provides a safeguard mechanism against the oncogenic potential of Myc. Omomyc is a mutant bHLHZip domain that sequesters Myc in complexes that are unable to bind to the E box recognition element and activate transcription but remain competent for transcriptional repression. Omomyc has the peculiar properties of reverting Myc-induced transformation of tissue culture cells and enhancing Myc proapoptotic function. Thus, Omomyc has the potential to act as a potent suppressor of Myc-induced oncogenesis. To validate the therapeutic potential of Omomyc in vivo, we targeted its expression to the adult suprabasal epidermis of Inv-c-MycER (TAM) transgenic mice which express a switchable form of the Myc protein in suprabasal cells. Activation of Myc induces rapid epidermal hyperplasia and papillomatosis. We show that Omomyc inhibits such Myc-induced papillomatosis, potentiating Myc-dependent apoptosis in a tissue in which it is usually strongly suppressed. Furthermore, Omomyc expression restores the normal keratinocyte differentiation program and skin architecture, both of which are otherwise disrupted by Myc activation. These findings indicate that it is possible to selectively enhance the intrinsic apoptotic pathway mediated by Myc and so quell its oncogenic action.

Animals↗
PubMed2001

Making decisions through Myc.

c-Myc is a transcriptional regulator involved in carcinogenesis through its role in growth control and cell cycle progression. Here we attempt to relate its role in stimulating the G1-S transition to the ability to affect functioning of key cell cycle regulators, and we focus on how its property of modulating transcription of a wide range of target genes could explain its capacity to affect multiple pathways leading to proliferation, apoptosis, growth, and transformation.

Animals↗
PubMed1998

Design and properties of a Myc derivative that efficiently homodimerizes.

bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.

3T3 Cells↗
PubMed1997

Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.

3T3 Cells↗
PubMed1995

Analysis of the Myc and Max interaction specificity with lambda repressor-HLH domain fusions.

The basic helix-loop-helix domain (bHLH) is present in a large class of transcriptional regulators involved in developmental processes and oncogenesis. It determines DNA binding and specific homo- and heterodimeric protein associations, crucial for protein function. Myc and Max belong to a subset of HLH proteins, containing a leucine zipper (LZ) adjacent to the bHLH domain. They differ in dimerization and functional properties such as DNA binding and transcriptional activation, and their association is required for malignant transformation by Myc. To analyze the interaction specificity of Myc and Max bHLH-LZ domains, we developed a simple Escherichia coli genetic system, which uses the amino-terminal lambda phage cI repressor as a reporter for dimerization and allows an easy detection of dimeric interactions. By reciprocal exchanges of different Myc and Max subdomains (helix 1, helix 2 and leucine zipper), we showed that the recognition specificity of Max homodimers as well as of Myc/Max heterodimers is entirely determined by the helix 2-leucine zipper region, the major role being played by the leucine zipper. The Myc LZ was found to prevent homodimeric interactions, thus explaining Myc inability to homodimerize efficiently. Moreover, we showed that the system is valid as well for reproducing the interaction of HLH proteins not containing a leucine zipper and that the chimerical proteins maintain sequence-specific DNA binding.

Bacteriophage lambda↗
PubMed1993

Ras oncogene transformation of human B lymphoblasts is associated with lymphocyte activation and with a block of differentiation.

The p21ras small GTP binding proteins participate in signal transduction from cell surface receptors and affect neoplastic transformation and development in many different cell types. In the present study, we examined the relationship between ras transformation and differentiation of human B lymphocytes. We show that the constitutive expression of the T24 Ha-ras oncogene in EBV-immortalized B lymphoblasts was associated with the induction of the interleukin 2 receptor alpha subunit, with an impaired immunoglobulin gene expression, altered adhesion properties and increased survival in serum-free medium. Since induction of the IL-2 receptor alpha subunit is a hallmark of lymphocyte activation, we suggest that p21ras naturally triggers B cell activation. The ras-transformed lymphocytes displayed a fully functional IL-2r, as assessed by c-fos induction following treatment with IL-2; nevertheless, they were not growth stimulated by this lymphokine. The decreased expression of immunoglobulin genes indicates that the ras oncogene blocks terminal differentiation to plasma cells, possibly by inhibiting the activity of lymphocyte-specific transcription factors. Somewhat unexpectedly, the constitutive p21ras activity did not cause an increased DNA binding of transcription factors PEA1 (AP1), PEA3, Oct-2 or NF-kB.

B-Lymphocytes↗
PubMed1992

Regulatory elements in the promoter region of vgf, a nerve growth factor-inducible gene.

vgf, a gene coding for a protein secreted through the regulated pathway, is rapidly up-modulated by nerve growth factor in PC12 cells and is expressed in vivo only in cell subpopulations of neuronal and endocrine origin. Here we demonstrate the following: (i) the nerve growth factor-dependent induction of vgf mRNA occurs at the transcriptional level and requires ongoing protein synthesis, (ii) lack of vgf expression in the nonneuronal cell line HTC is in part mediated by the presence of a repressor, (iii) a 110-base-pair sequence in the vgf promoter region contains positive and negative regulatory elements that partially account for its regulated expression, and (iv) a 47-base-pair oligonucleotide within this sequence specifically binds nuclear proteins that differ between vgf-expressing and non-expressing cells.

Adrenal Gland Neoplasms↗
PubMed1990

Induction of the neoplastic phenotype of EBV-established B lymphocytes by the human Ha-ras oncogene.

We report on the use of human B lymphocytes immortalized by the Epstein-Barr virus (EBV) as targets for transformation by the c-Ha-ras oncogene of bladder carcinoma cells T24. Several stably transformed cell lines were obtained and their in vivo and in vitro growth properties as well as levels of expression of the ras gene were studied. The transformed phenotype in these cells was correlated to ras oncoprotein expression level; only the cell lines which overproduce p21 ras, by at least six-fold, were tumorigenic in nude mice. In this regard, our ras transformed cells behave as lymphoblastoid cells transformed by the c-myc oncogene, suggesting that c-myc and c-Ha-ras might act on the same regulatory level.

Animals↗
PubMed1983

Hidden receptors for nerve growth factor in PC12 cells.

The binding of nerve growth factor (NGF) to its receptors in PC12 cells was studied in two experimental conditions: (a) cell fixation with paraformaldehyde followed by permeabilization of the plasma membrane with methanol and (b) metabolic poisoning of living cells with sodium azide. Paraformaldehyde fixation of PC12 cells causes a 60-70% reduction of NGF binding capacity; the original binding capacity is restored following permeabilization with methanol. A kinetic analysis of NGF binding under these conditions reveals a single homogeneous population of receptors at variance with experiments performed in living cells where two kinetically distinct types of NGF receptors were demonstrated [Landreth, G. E. and Shooter, E. M. (1980) Proc. Natl Acad. Sci. USA, 77, 4751-4755; Schechter, A. L. and Bothwell, M. A. (1981) Cell, 24, 867-874]. Our results suggest that a proportion of the NGF receptors in PC12 cells is hidden, i.e. not available for binding to the ligand, and in a dynamic equilibrium with exposed receptors. The existence of hidden receptors is confirmed by treatment of PC12 cells with sodium azide, which causes a 50% reduction in NGF binding capacity and protection from trypsin digestion of the remaining pool of hidden receptors. The latter become exposed at the cell surface following removal of sodium azide. Our data provide an interpretation for the as yet unsatisfactorily explained data on NGF receptors.

Cell Line↗
PubMed1982

An HMBA-resistant and heme-synthesizing variant clone of Friend leukemia cells.

A clone of Friend leukemia (FL) cells has been isolated which, in the presence of hexamethylene bisacetamide (HMBA) as inducer, is capable of growing and producing heme for long periods of time. The differentiation program expressed by this clone is very similar to that observed in the 5-86 parent, that, however, is arrested in its growth. The expression of the differentiated state in this clone depends on the continuous presence of an inducer; if the inducer is removed from a culture containing a high percentage of B+ (benzidine-positive) cells, the cells rapidly (5-7 days) revert to an undifferentiated state and appear to lose their differentiated character consecutively rather than simultaneously. The clone described in this paper is unstable; when grown for more than two months in the presence of an inducer it shifts toward a population containing a low percentage of heme-synthesizing cells. This instability seems to be due to the accumulation of a faster growing and more undifferentiated cell population.

Acetamides↗
PubMed1982

Microtubules and microfilaments in fixed and permeabilized cells are selectively decorated by nerve growth factor.

A specific antibody against nerve growth factor (NGF) and indirect immunofluorescence microscopy have been used to follow the in vitro binding of NGF to cells made permeable to large molecules. All cells tested, both target (sensory neurons and PCI2 cells) and nontarget (3T3, BKH 2I, C6 glioma cells), revealed a decoration of cytoskeletal structures which on the basis of their form, reactivity with antibodies, and sensitivity to specific drugs may be identified as microtubules (MTs) and microfilaments (MFs). The decoration of either structure depends on the fixation and permeabilization conditions: MFs, in the form of stress fibers, are stained by NGF when the plasma membrane is permeabilized with methanol/acetone; MTs become intensely stained when the plasma membrane is solubilized with a nonionic detergent in the presence of a MT-stabilizing medium. The two procedures do not affect the staining of these structures with specific antibodies. Binding of 125I-labeled NGF to PCI2 cells was not competitively inhibited by a 100-fold excess of several positively charged proteins but it was markedly decreased in the presence of DNase I. 125I-Labeled NGF interacted with MTs and F-actin (fixed with paraformaldehyde) in a range of concentrations similar to that used for their cellular localization with NGF-anti-NGF. Our studies show that the specificity and affinity of NGF binding to MTs and MFs is in the range of that of antibodies against tubulin and actin. The possible relevance of these findings to the mechanism of action of NGF in target cells is discussed.

Actins↗
PubMed1978

Isolation and properties of DMSO resistant variant clones of Friend leukemia cells.

DMSO resistant clones have been isolated from the inducible Friend leukemia cell line 5-86 both from unmutagenized cultures and following EMS mutagenesis. All the clones can grow in the presence of 1.8% DMSO and are non-inducible or poorly inducible for hemoglobin synthesis by DMSO as well as by other known inducers of Friend leukemia (FL) cells differentiation like hemin, hypoxanthine, hexamethylene bisacetamide. The clones are also defective for the expression of other properties of differentiating Friend cells like agglutinability by plant lectins and expression of the surface protein glycophorin. Some of the clones show an impaired ability to form tumors in vivo. These resistant clones might be useful for a genetic analysis of the differentiation process of Friend leukemia cells.

Animals↗
PubMed1977

Surface changes in differentiating Friend erythroleukemic cells in culture.

The sensitivity to agglutination by several plant lectins has been studied during the induced erythroid differentiation of Friend erythroleukemic cells in culture. In addition, the number of lectin receptors on the cell has been measured. It is shown that early during the differentiation, there is an increase in agglutinability while the receptor density remains constant. In the later phase of the differentiation process, the cells lose their sensitivity to agglutination while the receptor number and density increases. These changes were not observed on nonerythroid mastocytoma culture cells. Two nondifferentiating variants of the FL cells were shown to have altered sensitivities to agglutination by ConA.

Agglutination↗
PubMed1976

Isolation of suppressor sensitive mutants in the Ai gene of phage lambda.

Previous experiments have shown that mutations in the Ai gene can suppress the growth defect of lambda N- phages. Many temperature resistant derivatives of phage lambdatsN9 have been isolated and among these 5 have been found which are Ai- and have an amber suppressible behaviour. These mutants can help in defining the role of the Ai gene in phage lambda development.

Coliphages↗
PubMed1976

Deletions induced by heat treatment of E. coli K12 lysogenic for lambda prophages.

We have investigated the production of prophage deletions in heat-induced lambda lysogens of E. coli K12. Our results are indicative of a direct action of the heat-induced prophage in producing deletions. The temperature of 40 degrees used for the experiments may be critical to prove this effect. The phage function involved in deletion formation is not known.

Chromosome Aberrations↗
PubMed1975

Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation.

A temperature-sensitive mutant having a lethal mutation in the gene for the beta subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) exhibits an apparent 2- to 3-fold decrease in the rates of both beta and beta' subunit synthesis at the non-permissive temperature, relative to total protein. In contrast, a temperature-sensitive mutant with a lethal mutation in the gene encoding beta' has a 5- to 6-fold increase in the rates of beta and beta' synthesis at 42 degrees. These beta and beta' mutants also exhibit rapid degradation of both subunits at the high temperature.

DNA-Directed RNA Polymerases↗