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N C Reich

Publications and source records attributed to N C Reich.

32 records · Page 2Linked to original sources
PubMed1994

Interferon alpha (IFN alpha) signaling in cells expressing the variant form of the type I IFN receptor.

Two different Type I interferon receptors (IFN-R) have been described: the normal and the variant receptors. The alpha subunit of the Type I IFN-R has a molecular mass of 110 kDa in cells expressing normal and variant receptors. The beta subunit has a molecular mass of approximately 100 kDa in cells that express normal receptors and 55 kDa in cells expressing the variant form of the receptor. The IFN alpha-resistant U-937 cell line expresses variant receptors and fails to down-regulate and phosphorylate the alpha subunit on tyrosine residues. We report that two other myelomonocytic cell lines, YK-M2 and ML-2, also expressing the variant form of the receptor, fail to down-regulate and phosphorylate the alpha subunit on tyrosine residues. However, YK-M2 and ML-2 cells are sensitive to the antiproliferative and antiviral effects of IFN alpha 2, indicating that phosphorylation of the alpha subunit is not necessary to elicit an IFN alpha response and that expression of variant receptors is not a source of IFN alpha resistance. We also determined if other proteins involved in the IFN alpha signal transduction pathway had a different phosphorylation pattern. Treatment of cells expressing variant receptors induced tyrosine phosphorylation of the p135tyk2 tyrosine kinase, and the three interferon-stimulated gene factor 3 alpha (ISGF3 alpha) polypeptides (p113, p91, and p84), albeit at lower levels. These results indicate that cells expressing either form of the Type I IFN-R phosphorylate a similar set of proteins, with the exception of the alpha subunit.

Base Sequence↗
PubMed1993

Requirement of tyrosine phosphorylation for rapid activation of a DNA binding factor by IL-4.

Interleukin-4 (IL-4) is an immunoregulatory cytokine produced by activated T lymphocytes to promote the growth and differentiation of cells that participate in immune defense. This study demonstrates the rapid activation of a specific DNA binding factor by IL-4. The IL-4 nuclear-activated factor (IL-4 NAF) appeared within minutes of IL-4 stimulation and recognized a specific DNA sequence found in the promoters of IL-4-responsive genes. Activation of this putative transcription factor required tyrosine phosphorylation, and antibodies specific for phosphotyrosine recognize the IL-4 NAF-DNA complex. Thus, IL-4 appears to transduce a signal to the nucleus through tyrosine phosphorylation of a latent DNA binding factor.

Base Sequence↗
PubMed1993

Double-stranded RNA activates novel factors that bind to the interferon-stimulated response element.

Infection of cells with adenovirus or transfection of cells with double-stranded RNA (dsRNA) activates transcription of the alpha/beta interferon-stimulated genes (ISGs). Induction of ISG expression by adenovirus appears to be mediated through the same DNA target that is responsive to alpha/beta interferons, the interferon-stimulated response element (ISRE). Transcriptional induction by alpha/beta interferons has been shown previously to be mediated by the activation of a latent cytoplasmic transcription factor, ISGF3, that translocates to the nucleus and binds to the ISRE. However, ISG expression induced by adenovirus or dsRNA appears to be mediated by unique dsRNA-activated factors (DRAFs) that bind to the ISRE. The activation of these preexisting factors by dsRNA does not require new protein synthesis. Two DRAFs, DRAF1 and DRAF2, have been identified in our studies as ISRE-binding complexes in gel mobility shift assays. The ISRE-binding specificity of DRAF1 is similar to that of ISGF3; however, the ISRE-binding specificity of DRAF2 is distinct. Activation of DRAF1 and DRAF2 is independent of interferon action since it occurs in cells that are nonresponsive to interferon and in cells that lack the alpha/beta interferon locus. The activation pathway of DRAF1 and DRAF2 is blocked by the protein kinase inhibitors staurosporine and genistein. This is analogous to the interferon signal transduction pathway and suggests that phosphorylation, possibly tyrosine phosphorylation, is involved in activation of these factors.

Actins↗
PubMed1992

Tyrosine phosphorylation is required for activation of an alpha interferon-stimulated transcription factor.

The signal transduction pathway of alpha interferon utilizes tyrosine phosphorylation to transmit a signal generated at the cell surface to the transcriptional machinery in the nucleus. Activation of the interferon pathway initiates with the binding of alpha interferon to its cell surface receptor. The ligand-receptor complex signals the activation of a latent cytoplasmic transcription factor. The active form of the interferon-stimulated gene factor (ISGF3) is phosphorylated on tyrosine residues. ISGF3 subsequently translocates to the nucleus and binds to a DNA sequence, the interferon-stimulated response element, found within the promoter of inducible genes. ISGF3 is a multicomponent factor consisting of four proteins of 113 kDa, 91 kDa, 84 kDa, and 48 kDa. Three proteins consistent with sizes of 113 kDa, 91 kDa, and 84 kDa copurify with ISGF3 and are phosphorylated on tyrosine residues after stimulation by alpha interferon. Tyrosine phosphorylation is essential for activation of ISGF3. Genistein, a tyrosine kinase inhibitor, blocks the appearance of ISGF3 and blocks the transcriptional stimulation of interferon-induced genes. This study shows that tyrosine phosphorylation provides a link between the interferon-receptor complex at the plasma membrane and specific activation of gene expression in the nucleus.

Base Sequence↗
PubMed1991

Repression of the interferon signal transduction pathway by the adenovirus E1A oncogene.

The signal transduction pathway initiated by type I interferon (alpha and beta interferons) is inhibited by expression of the adenovirus type 5 E1A oncogene. Cotransfection analyses with the E1A oncogene and an interferon-stimulated reporter gene show that mutations within an amino-terminal domain of the E1A oncoprotein are defective in transcriptional repression. Cotransfection experiments also revealed that the transcriptional repression is mediated through the interferon-stimulated response element (ISRE) found within the promoter of interferon-stimulated genes. Since interferon treatment activates a latent cytoplasmic DNA-binding factor that can recognize the ISRE and subsequently stimulate transcription, the appearance of this factor was analyzed in a cell line that constitutively expresses the E1A oncogene. The DNA binding activity of this transcriptional activator was found to be inhibited in the E1A-expressing cell line. In vitro cytoplasmic mixing experiments with extracts from control and E1A-expressing cells identified a specific component of this multimeric transcription factor to be defective.

Adenovirus Early Proteins↗
PubMed1991

Transmembrane signaling by interferon alpha involves diacylglycerol production and activation of the epsilon isoform of protein kinase C in Daudi cells.

The early events that occur after treatment of the highly interferon alpha (IFN-alpha)-sensitive human lymphoblastoid Daudi cell line with human leukocyte IFN-alpha have been examined. IFN-alpha treatment of Daudi cells results in a rapid and transient increase in the cellular content of diacylglycerol, which occurs in the absence of inositol phospholipid turnover, or an increase in intracellular calcium concentration. Furthermore, IFN-alpha treatment results in a selective, time-dependent activation of the Ca(2+)-independent epsilon isoform of protein kinase C (PKC), while the alpha isoform is unaffected by IFN-alpha treatment. In contrast, IFN-alpha treatment of an IFN-resistant subclone of Daudi cells had no effect on the diacylglycerol content of cells and on the activation of PKC-epsilon. The selective PKC inhibitor staurosporine blocked the transcriptional activation of IFN-alpha-stimulated genes, the cytoplasmic accumulation of mRNAs for these genes, and the induction of antiviral activity by IFN-alpha against vesicular stomatitis virus in IFN-sensitive cells. These observations suggest that transmembrane signaling of IFN-alpha involves diacylglycerol production and activation of PKC-epsilon in Daudi cells.

Alkaloids↗
PubMed1990

Evidence for involvement of protein kinase C in the cellular response to interferon alpha.

Phospholipid/Ca2(+)-dependent protein kinase (protein kinase C; PKC) appears to be involved in the signal-transduction pathway mediated by human leukocyte interferon (IFN) in HeLa cells. IFN treatment results in a rapid increase in [3H]phorbol 12,13-dibutyrate binding to intact cells, indicating an activation of PKC. In addition, inhibitors of PKC (H7 and staurosporine) block the induction of antiviral activity by IFN against vesicular stomatitis virus. PKC inhibitors also block the accumulation of IFN-stimulated mRNAs in the cytoplasm of HeLa cells and suppress the transcriptional induction of IFN-stimulated genes. Activation of IFN-stimulated genes is mediated through a DNA response element that is necessary and sufficient for the transcriptional response to IFN. IFN treatment induces the appearance of several DNA-binding factors that specifically recognize the response element, and the appearance of these factors is suppressed by PKC inhibitors. This observation provides evidence that PKC activity is involved during IFN-stimulated signal transduction. Although activation of PKC appears to be required for the response to IFN, agonists of PKC activity alone do not turn on expression of IFN-stimulated genes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Differential binding of interferon-induced factors to an oligonucleotide that mediates transcriptional activation.

Type I interferons elicit a number of biological responses by rapidly and transiently stimulating the transcriptional expression of a specific set of genes. The promoters of the inducible genes contain an enhancer element which is required for transcriptional activation. A specific oligonucleotide of 18 residues is sufficient for transcriptional induction when positioned in a heterologous promoter. Previous studies have identified three protein factors which can bind to the interferon stimulated enhancer. We show here that the binding of one of the interferon-induced factors requires specific nucleotides flanking the minimum recognition site for the other two factors. The distinct interaction of this factor with the response element is of significant importance since this factor is the sole candidate for a primary transcriptional activator of interferon-induced genes.

Adenoviruses, Human↗
PubMed1984

The amino acid sequence of murine p53 determined from a c-DNA clone.

A c-DNA clone containing the complete sequence information for the murine p53 protein, from embryonal carcinoma cells, has been isolated. The nucleotide sequence of this clone reveals an open reading frame encoding a protein of 390 amino acids with a molecular weight of 43,364 Da. The NH2-terminal domain of this protein is acidic whereas the carboxyl terminus is rich in basic amino acid residues. These terminal domains are separated by a proline-rich, hydrophobic run of amino acids. Proline comprises approximately 10% of the total amino acid residues. Two tryptic peptides, derived from p53 protein radiolabeled with either methionine or proline, were purified and the position of these labeled residues in the peptide was determined. The positions of three methionine and five proline residues in these two peptides matched the amino acid sequence of the predicted open reading frame determined from the c-DNA clone.

Amino Acid Sequence↗
PubMed1983

Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein.

Two hybridoma cell lines were obtained, A1 and B6, which produced monoclonal antibodies reacting with the 44,000-MW C-terminal domain of the adenovirus type 5 DNA-binding protein (DBP). Clone A1 antibodies reacted with the native form of the DBP, but failed to recognize this protein after denaturation (by exposure to sodium dodecylsulfate, or production of the DBP at 39.5 degrees by H5ts107, a temperature-sensitive DBP mutant). Clone B6 antibodies bound to both the native and denatured forms of the DBP. Immunofluorescent staining of wild-type-virus infected cells revealed the DBP located in discrete nuclear patches. A1 and B6 antibodies detected this patched localization of the DBP in nuclei of H5ts107-infected cells grown at 32 degrees. However, at the nonpermissive temperature of 39.5 degrees, A1 antibodies failed to detect the DBP, and B6 antibodies gave a uniform nuclear fluorescent distribution of the DBP. Thus the nuclear pattern of localization for the DBP synthesized by H5ts107 was temperature dependent in this mutant.

Adenoviruses, Human↗
PubMed1983

Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.

Animals↗
PubMed1982

Regulation of the cellular p53 tumor antigen in teratocarcinoma cells and their differentiated progeny.

F9 embryonal carcinoma cells express high levels of a 53,000-molecular-weight cellular tumor antigen called p53. When F9 cell cultures are treated with retinoic acid and dibutyryl adenosine 3',5'-phosphate, they differentiate, predominantly into endoderm-like cells. This differentiation is accompanied by a marked decrease in the levels of p53. The mechanism(s) responsible for this decline in the level of p53 in differentiated cells was investigated. The results demonstrate that the high levels of p53 in F9 cells relative to their differentiated progeny were not due to alterations in the stability or turnover of this protein. Rather, the regulation during differentiation involved a marked decrease in the amount of in vitro translatable p53 mRNA detected in the differentiated cell cultures. This mechanism is unlike the one operating during the simian virus 40 infection or transformation, where the increased levels of p53 are largely due to the increased stability of the p53 protein.

Animals↗
PubMedDate not supplied

Growth regulation of a cellular tumour antigen, p53, in nontransformed cells.

Many transformed cells in culture have been found to express elevated levels of a cellular tumour antigen, termed p53. This protein has also been implicated in the regulation of cellular growth. For these reasons experiments were designed to examine the expression of p53 as quiescent cultures of nontransformed 3T3 fibroblasts were stimulated to reenter the cell cycle. Synchronous populations of cells were obtained by releasing a culture from density-dependent inhibition of growth with the addition of fresh serum. Steady-state levels of p53 protein and mRNA were measured as a function of time after addition of serum to quiescent cultures and the rate of synthesis of p53 protein was analysed at a number of time points. The results, reported here, demonstrate an increase in the synthesis and steady-state levels of p53 protein and mRNA prior to DNA synthesis in late G1, and suggest a role for p53 in the progression of cells from a growth-arrested state to an actively dividing state.

Animals↗