Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation.

Inhibitor of DNA-binding (Id) proteins prevent cell differentiation, promote growth and sustain tumour development. They do so by binding to E proteins and other transcription factors through the helix-loop-helix (HLH) domain, and inhibiting transcription. This makes HLH-mediated Id protein interactions an appealing therapeutic target. We have used the dominant interfering HLH dimerization mutant 13I to model the impact of Id inhibition in two human neuroblastoma cell lines: LA-N-5, similar to immature neuroblasts, and SH-EP, resembling more immature precursor cells. We have validated 13I as an Id inhibitor by showing that it selectively binds to Ids, impairs complex formation with RB, and relieves repression of E protein-activated transcription. Id inactivation by 13I enhances LA-N-5 neural features and causes SH-EP cells to acquire neuronal morphology, express neuronal proteins such as N-CAM and NF-160, proliferate more slowly, and become responsive to retinoic acid. Concomitantly, 13I augments the cell-cycle inhibitor p27(Kip1) and reduces the angiogenic factor vascular endothelial growth factor. These effects are Id specific, being counteracted by Id overexpression. Furthermore, 13I strongly impairs tumorigenic properties in agar colony formation and cell invasion assays. Targeting Id dimerization may therefore be effective for triggering differentiation and restraining neuroblastoma cell tumorigenicity.

Disfagia en paciente joven con historia de atopia / Dysphagia in young patient with an atopic background

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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.

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.

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.

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.

[Atypical cat-scratch disease: a case report of splenic granulomatosis]. / La malattia da graffio di gatto atipica: descrizione di un caso di granulomatosi splenica.

Generally cat-scratch disease is a benign inflammatory adenopathy. The Authors describe an atypical form of this disease, characterized by persistent fever and splenic granulomatosis requiring a diagnostic and therapeutic prolonged effort. They point out the important role of new immuno-fluorescent techniques to exactly identify the bacterium--Bartonella henselae--causing cat-scratch disease and suggest to include cat-scratch disease among the causes of unknown origin fever.

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.

[Dermatitis herpetiformis and latent celiac disease in two siblings]. / Dermatite erpetiforme e malattia celiaca silente in due fratelli.

The association between coeliac disease (CD) and dermatitis herpetiformis (DH) is well known. Moreover, this cutaneous disease may be the only sign of an otherwise asymptomatic CD. Subjects presenting with both CD and DH generally show an HLA pattern in which A1, B8, DR2, DR7, DQw2 are the most frequent antigens one can find. We report about 2 brothers presenting with DH, clinically asymptomatic, without antigliadin serum antibodies (AGA), but positive to the research of antiendomysial (EMA) ones. The biopsy performed by digestive endoscopy showed a complete atrophy of duodenal villi and the diagnosis of CD was confirmed according to the European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) criteria. The diet without gluten caused the DH to recovery and the duodenal villi microscopic aspect to normalize as well. Both the brothers had the same HLA pattern: A1, B8, DR3-DR2, DQw2. Our clinical study suggests that it is very important, especially for the general practitioner, to recognize a DH and in every child presenting with a dermatitis like that it will be mandatory to perform a laboratory research of both AGA and EMA.

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.

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.

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.

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.

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.

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.

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.

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.