Biomedical subjects

B R Williams

Publications and source records attributed to B R Williams.

At least 127 records · Page 7Linked to original sources

PubMed1992

Basal expression of the gene (TIMP) encoding the murine tissue inhibitor of metalloproteinases is mediated through AP1- and CCAAT-binding factors.

In cultured murine fibroblasts, the TIMP gene (encoding tissue inhibitor of metalloproteinases) is transcribed constitutively, although at low levels. We have used a cell-free system in which nuclear extracts prepared from murine L cells support transcription from TIMP DNA templates in vitro. This system was used to study the role of cis-acting DNA sequences in the constitutive expression of TIMP. Sequences important for expression are located both 5' and 3' (in intron 1) to the major transcription start point and are required to obtain detectable levels of transcription. These regions are specifically recognized by murine nuclear factors and contain DNA motifs whose sequences closely resemble binding sites for known transcriptional activators. In particular, the data strongly suggest a role for CCAAT-binding factor(s) and AP1-binding factors in the basal transcription of TIMP.

Animals↗

PubMed1992

Transcriptional activation of human (2'-5')oligoadenylate synthetase gene expression by the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate in type-I-interferon-treated HL-60 and HeLa cells.

(2'-5')Oligoadenylate [(2'-5')(A)n] synthetase is a key enzyme in the interferon-elicited antiviral response whose controlled expression in interferon-treated cells has been only partially elucidated. In this investigation, we have compared the modulation of the (2'-5')(A)n synthetase gene by interferon alone and by the combination of interferon and a second cellular effector, 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Although TPA alone had no effect on (2'-5')(A)n synthetase, it potentiated the induction of (2'-5')(A)n of synthetase by interferon in HL-60 and HeLa cells by increasing content of its mRNA and an immunoreactive 40-kDa isoenzyme. Since TPA activates protein kinase C (PKC), other PKC-activating phorbol-ester analogues were tested and found to be effective, whereas the PKC inhibitor staurosporine reduced the potentiative activity of TPA. By using the (2'-5')(A)n synthetase gene promoter linked to a reporter gene, chloramphenicol acetyltransferase (CAT), TPA and interferon were found to result in a doubling of CAT activity compared to cells treated with interferon alone. Moreover, when nuclear extracts prepared from control cells or cells treated with TPA and interferon (IFN), separately or together, were incubated with radioactively labeled oligodeoxynucleotides containing the interferon-responsive element (IRE), TPA was shown to down-regulate an IFN-inducible IRE/protein complex. These data further suggest that TPA regulates (2'-5')(A)n synthetase gene expression at the level of transcription.

2',5'-Oligoadenylate Synthetase↗

PubMed1992

Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase.

The double-stranded RNA (dsRNA)-binding domain of the human p68 kinase has been localized to the N-terminal half of the enzyme by using progressive deletion analysis and in vitro binding assays. To further define the domains responsible for binding to dsRNA, we cloned the mouse dsRNA-activated p65 kinase and used sequence alignment to identify conserved domains in the N-terminal region. Deletions in either of two 12-amino-acid-long and arginine- or lysine-rich regions abrogated binding to dsRNA. Moreover, in an in vivo growth inhibition assay in the yeast Saccharomyces cerevisiae, these mutants failed to exhibit a slow-growth phenotype.

Amino Acid Sequence↗

PubMed1992

Loss of heterozygosity mapping in Wilms tumor indicates the involvement of three distinct regions and a limited role for nondisjunction or mitotic recombination.

Loss of heterozygosity (LOH) for polymorphic markers is a frequently occurring event in some tumors, reflecting the role of allele loss in the development of these tumors. We have determined LOH in 38 cases of Wilms tumor for the 2 known loci on chromosome arm 11p and for a newly detected locus on chromosome arm 16q. Only 7 of the 38 tumors studied showed reduction to homozygosity of 11p13 markers. In 4 of these tumors, reduced expression of WT1 and WIT1, genes located at 11p13 and implicated in Wilms tumorigenesis, was noted. However, this was also found in 2 of 7 tumors showing LOH exclusively of 11p15 markers and in 15 of the remaining 24 tumors in which there was no LOH for 11p markers. This suggests that events not involving mitotic recombination or chromosome nondisjunction are the most common mechanisms for mutations at the 11p Wilms tumor locus. We also noted that mitotic recombination involving 11p15 loci occurred in addition to reduced expression of the 11p13 locus genes in 2 tumors, suggesting a possible interaction between these 2 loci. In addition, LOH for 16q markers was observed in 6 tumors. In one case this was coincident with reduction of WT1 and WIT1 gene expression, and in 3 other cases it occurred in addition to 11p LOH. This indicates that an additional locus on 16q is likely to be involved in Wilms tumorigenesis.

Adolescent↗

PubMed1992

Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.

The human p68 kinase is an interferon-regulated enzyme that inhibits protein synthesis when activated by double-stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N-terminus, shown to bind double-stranded RNA in vitro and expression of a mutant form of the alpha-subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha-subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

Amino Acid Sequence↗

PubMed1992

Chemosensitivity and breathing pattern regulation of the coatimundi and woodchuck.

An analysis of breathing pattern regulation was carried out on the coatimundi and woodchuck who represent two different volume-time patterns. It was found that the coati, with a short expiratory time as a fraction of total breath time, TE/TTOT, has a greater sensitivity to CO2 as represented by the slope and threshold of its ventilatory response. Breathing air the coati maintains post-inspiratory inspiratory activity (PIIA) of the posterior cricoarytenoid (PCA) through 51% of expiration, while the woodchuck, who is less sensitive to CO2 and has a long TE/TTOT, exhibits no PIIA of the PCA. The woodchuck also has a greater incidence and duration of end-expiratory pauses (or delayed inspiratory onset). The woodchuck does not demonstrate the usual inverse relationship between VT and TE in response to 5% CO2 and does not recruit PIIA of the PCA at this level of CO2. These data confirm the importance of CO2 chemosensitivity in regulation of TE. It is further demonstrated that interspecific differences in chemosensitivity among three mammals of the same size are reflected in regulation of TE but not in inspiratory 'drive' (as indicated by mean inspiratory flow, VT/TI).

Animals↗

PubMed1992

Ontogenetic modification of the hypercapnic ventilatory response in the zebra finch.

We investigated the effect of exposure to CO2 during development on the adult ventilatory response to CO2 of zebra finches. Developing zebra finches were exposed to chronic hypercapnic respiratory environments as embryos, as nestlings or during both developmental periods. Their responsiveness to CO2 was then measured following a 125-135 day post-fledging deacclimation period. At a FICO2 of 0.06, mean ventilation of adult finches in the three groups was 74.8, 61.3 and 65.8%, respectively, of that of finches reared under normocapnic conditions. These data indicate that adult acute ventilatory responsiveness to CO2 in birds may be in part determined by CO2 exposure during early development and may help explain the observation that fossorial and semifossorial species of birds and mammals, that naturally encounter high CO2 conditions in their burrows, have a blunted hypercapnic ventilatory response.

Animals↗

PubMed1992

Interferon-alpha activates binding of nuclear factors to a sequence element in the c-fos proto-oncogene 5'-flanking region.

Interferon-alpha (IFN-alpha) can regulate the expression of the c-fos proto-oncogene in different cell types. Here we show IFN-alpha-activated binding of murine and human fibroblast nuclear factors to a DNA sequence element located in the 5' upstream region (nucleotides -351/-337) of the c-fos gene. This element, like the conserved enhancer element, the IFN-stimulated response element (ISRE), that mediates transcriptional induction of IFN-alpha-inducible genes, also binds factors in response to platelet-derived growth factor (PDGF) and v-sis-conditioned medium (SCM). The IFN-inducible ISRE shares an 8-bp stretch of sequence homology with the IFN-responsive c-fos SCM element, and competes efficiently for binding of factors to the SCM. Protein-DNA cross-linking experiments with the SCM binding site identified an IFN-modulated nuclear protein of approximately 98 kD. This protein does not appear to be involved in transcription activation, since IFN-alpha failed to stimulate c-fos transcription in nuclear run-off assays, or the c-fos promoter in transient transcription assays of 3T3 fibroblasts. Our data nonetheless suggest the c-fos promoter may be an early target for signal transduction triggered by IFN alpha-receptor interaction.

Base Sequence↗

PubMed1992

Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth.

The cDNA encoding interferon-induced human double-stranded RNA-activated p68 kinase was expressed in murine NIH 3T3 cells by using the pcDNA1/neo vector. Several stable clones were selected which expressed either the wild-type kinase or an inactive mutant possessing a single amino acid substitution in the invariant lysine 296 in the catalytic domain II. The transfected wild-type kinase showed properties similar to those of the natural kinase, such as subcellular ribosomal localization and dependence on double-stranded RNA for autophosphorylation. Upon infection with encephalomyocarditis virus (EMCV), wild-type- but not mutant-expressing clones were found to partially resist virus growth. Such natural antiviral activity was virus specific, since no inhibition was observed in the case of vesicular stomatitis virus infection. In accord with EMCV inhibition, the wild-type p68 kinase was found to be highly phosphorylated during infection. Furthermore, its natural substrate, the small subunit of protein synthesis initiation factor eIF2, was phosphorylated. These results demonstrate that p68 kinase is activated during EMCV infection, leading to reduced virus production.

3T3 Cells↗

PubMed1992

Multiple tumor suppressor genes in multistep carcinogenesis.

One of the most exciting areas of molecular oncology is the convergence of two independent lines of evidence suggesting involvement of multiple tumor suppressor genes in a given type of cancer. First, epidemiology and somatic cell genetics indicate the presence of multiple tumor suppressor genes in each of several malignancies. Second, cancers often lose multiple chromosomal regions during tumor progression. We will use two tumors, colorectal cancer and Wilms tumor, to illustrate the questions that multiple tumor suppressor genes raise.

Colorectal Neoplasms↗

PubMed1992

Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes.

The cloning and molecular characterization of two putative tumor genes, WT1 and WIT1, from the chromosome 11p13 region has provided a means of evaluating their role in the generation of Wilms' tumor heterogeneity. A series of 29 tumors were analyzed for WT1 and WIT1 expression by Northern blot or RNase protection analyses, and results were compared with tumor histopathology. Tumors were scored for the percentage of mesenchymal and epithelial derived tissue components. Homotypic tumors comprised blastema, tubular epithelium, and a fibroblast-like mesenchyme. In addition to these tissue components, the group of tumors designated as heterotypic also contained ectopic cell phenotypes such as muscle and squamous epithelium. The analyses suggest that heterotypic differentiation patterns occur when WT1 and WIT1 expression is low relative to normal fetal kidney. In situ hybridization using antisense RNA probes showed that WT1 and WIT1 were concordantly expressed in normal fetal kidney and in the blastema of tumors. The ratio of WT1:WIT1 expression remained relatively constant in homotypic tumors but deviated significantly in heterotypic tumors. These results suggest that expression patterns of the WT1 and WIT1 genes can be closely correlated to Wilms' tumor histopathology.

Blotting, Northern↗

PubMed1992

Regulation of TIMP gene expression in cell culture and during mouse embryogenesis.

We have identified elements in the 5' region of the murine tissue inhibitor of metalloproteinase (TIMP) gene which control the response to serum, phorbol esters and transforming growth factor beta (TGF-beta) in cell culture. These elements lie between -858 and -601 relative to the translation initiation start site in the gene and are distinct from those that control expression in response to virus induction. The temporal and spatial expression of the TIMP gene was also analysed during mouse embryogenesis using in situ hybridization. A transgenic mouse line was constructed which expressed a TIMP/lac Z fusion gene. Using in situ beta-galactosidase assays we were able to compare the expression of the transgene with the endogenous gene. Thus we concluded that most of the sequences controlling in vivo expression of TIMP were present in the transgene and could be localised to the 5' region of the gene.

3T3 Cells↗

PubMed1991

Transcriptional regulation of interferon-stimulated genes.

Interferons (IFN) stimulate the expression of a number of genes following interaction with specific high-affinity plasma membrane receptors. The products of these genes either singly or coordinately mediate the antiviral, growth inhibitory or immunoregulatory activities attributed to IFN. While the gene products in some cases have been well characterized, other IFN-regulated genes encode proteins whose functions have yet to be elucidated. A feature common to all IFN-stimulated genes characterized thus far is the presence of a DNA element which constitutes an IFN-responsive enhancer, usually present in the 5' upstream region of the genes. This element, termed interferon-stimulated response element (ISRE) binds a nuclear factor(s) translocated from the cytoplasm to the nucleus following IFN-receptor-triggered signal transduction. The binding of these factors to the ISRE represents the initiating event in stimulating RNA-polymerase-II-mediated transcription from IFN-responsive genes. Depending on the nature of the cells responding to IFN and the genes involved, induced transcription may be prolonged or rapidly terminated. The rapid termination of transcription is dependent in some cases on IFN-induced protein synthesis and also involves factor binding to the ISRE. Recent progress in detailing these events will be discussed including IFN-receptor interactions, signal-transduction pathways, comparing and contrasting IFN-regulated genes and elucidation of IFN-regulated factors.

Animals↗

PubMed1991

Enhancement by retinoic acid and dibutyryl cyclic adenosine 3':5'-monophosphate of the differentiation and gene expression of human neuroblastoma cells induced by interferon.

Human neuroblastoma cell lines are induced to differentiate and display neuronal phenotypes when treated with interferon (IFN)-alpha 2, retinoic acid (RA), or dibutyryl cyclic AMP (dbcAMP). We investigated the effects of combinations of these agents in induction-differentiation in the neuroblastoma cell line, NUB-6. The inductive effect of IFN-alpha 2 was markedly enhanced when used in combination with RA or dbcAMP. In parallel, RA or dbcAMP also enhanced the level of 2'-5'-oligoadenylate (2-5A) synthetase, and enzyme induced by IFNs and implicated in their biological action. The levels of another IFN-inducible enzyme, p68 kinase, were not enhanced by the combination treatments. The enhancement effects appeared to be exerted largely at the posttranscriptional level as both RA and dbcAMP stabilized IFN-induced 2-5A synthetase mRNA, resulting in increased enzyme activity. Thus, the 2-5A synthetase system is likely involved in mediating the IFN-alpha 2-induced differentiation of neuroblastoma cells and may also mediate the enhancement effects of RA and dbcAMP on IFN activity in these cells. These results also provide a rational basis for establishing a combination therapeutic approach for the treatment of neuroblastoma.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

Induced factor binding to the interferon-stimulated response element. Interferon-alpha and platelet-derived growth factor utilize distinct signaling pathways.

Interferon-alpha (IFN alpha) and platelet-derived growth factor (PDGF) each rapidly stimulate binding of nuclear factors from Balb/c 3T3 fibroblasts, to a 29-base pair regulatory sequence derived from the 5' upstream region of the murine 2-5A synthetase gene. This regulatory sequence contains a functional interferon-stimulated response element (ISRE) and also functions as a PDGF-responsive sequence. We show that IFN alpha induces binding of a protein of molecular mass 65 kDa to the ISRE. Constitutively expressed ISRE-binding proteins of 98 and 150 kDa are also demonstrated. Binding of inducible factors to the ISRE increases significantly within 15 min of IFN alpha or PDGF treatment. PDGF-induced binding is not mediated by IFN beta. The protein kinase inhibitors, staurosporine and K252a, block PDGF-induced ISRE binding and 2-5A synthetase gene expression. IFN alpha-induced ISRE binding and gene activation are not blocked by these inhibitors. Treatment of cells with 12-O-tetradecanoyl-13-acetate or dibutyryl cyclic AMP does not activate ISRE binding factors or 2-5A synthetase gene expression. PDGF responsiveness of the ISRE in vivo is also sensitive to staurosporine, indicating that inhibition of a protein kinase activity blocks the PDGF-specific transcriptional signal. Our data indicate the signal transduction pathway for IFN alpha-induced, ISRE-dependent transcription is distinct from the PDGF-induced ISRE response and is likely independent of cyclic AMP-dependent protein kinase and protein kinase C activities.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

The murine 2-5A synthetase locus: three distinct transcripts from two linked genes.

The cloning of cDNAs encoding murine 2-5A synthetase has identified three related transcripts, represented by a previously described cDNA clone, L3 and two novel cDNAs, L1 and L2. L1 contains an open reading frame coding for a protein that shows 70% conservation at the amino acid level when compared to the protein predicted to be encoded by L3. L2 recognizes an IFN-induced transcript 600-bp larger than the L3 transcript. These three cDNAs map to a cosmid, cII, containing two murine 2-5A synthetase genes, ME12 and ME5/ME8, situated in a head-to-tail orientation separated by approximately 8 kb. Southern analyses of ME12 and ME5/ME8 using L3, L1-specific and L2-specific probes indicate that these genes have a similar organization. cII was transiently and stably transfected into CV-1 cells. When treated with interferon, the transfected cells produced mature, murine 2-5A synthetase transcripts identified using L3 and L2-specific probes. Thus all transcripts present in IFN-treated mouse cells which are recognized by the available murine 2-5A synthetase cDNA probes map to an approximately 40 kb region of the mouse genome containing two 2-5A synthetase genes.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

Identification of a serum- and phorbol ester-responsive element in the murine tissue inhibitor of metalloproteinase gene.

Tissue inhibitor of metalloproteinase (TIMP) is one of a family of metalloproteinase inhibitors and a major interstitial inhibitor of collagenase. Transcription of the TIMP gene is induced by such diverse agents as viruses, phorbol esters, serum, and growth factors. We have previously assigned the regulatory elements responsible for induction of transcription in response to viruses to the first intron of the murine TIMP gene. Here we have identified a promoter and an enhancer element responsive to serum and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Based on a comparative sequence analysis of the murine and human genes, the enhancer element is part of a 38-base pair conserved sequence. Gel mobility shift assays indicate that this enhancer is a phorbol ester-responsive-like element that likely binds one of a family of AP-1 proteins. Interestingly, the region containing the phorbol ester-responsive-like element is also sufficient to direct a response to transforming growth factor beta 1 in the presence of serum.

Animals↗

PubMed1991

Signal transduction by interferon-alpha through arachidonic acid metabolism.

Molecular mechanisms that mediate signal transduction by growth inhibitory cytokines are poorly understood. Type I (alpha and beta) interferons (IFNs) are potent growth inhibitory cytokines whose biological activities depend on induced changes in gene expression. IFN-alpha induced the transient activation of phospholipase A2 in 3T3 fibroblasts and rapid hydrolysis of [3H]arachidonic acid (AA) from prelabeled phospholipid pools. The phospholipase inhibitor, bromophenacyl bromide (BPB), specifically blocked IFN-induced binding of nuclear factors to a conserved, IFN-regulated enhancer element, the interferon-stimulated response element (ISRE). BPB also caused a dose-dependent inhibition of IFN-alpha-induced ISRE-dependent transcription in transient transfection assays. Specific inhibition of AA oxygenation by eicosatetraynoic acid prevented IFN-alpha induction of factor binding to the ISRE. Treatment of intact cells with inhibitors of fatty acid cyclooxygenase or lipoxygenase enzymes resulted in amplification of IFN-alpha-induced ISRE binding and gene expression. Thus, IFN-alpha receptor-coupled AA hydrolysis may function in activation of latent transcription factors by IFN-alpha and provides a system for studying the role of AA metabolism in transduction of growth inhibitory signals.

5,8,11,14-Eicosatetraynoic Acid↗