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Biomedical subjects

D W Leaman

Publications and source records attributed to D W Leaman.

At least 19 recordsLinked to original sources
PubMed2001

Interferon action against human parainfluenza virus type 3: involvement of a novel antiviral pathway in the inhibition of transcription.

Interferon (IFN)-induced 2'-5' oligoadenylate synthetase (2-5A synthetase)/RNase L, PKR, and Mx proteins are considered to be the principal antiviral protein pathways through which IFN induces an antiviral state. It was previously reported that human parainfluenza virus type 3 (HPIV3) multiplication was inhibited by IFN-alpha in human lung epithelial cells A549 and that MxA was found to contribute to the inhibition process (Zhao et al., Virology 220:330-338, 1996). Viral primary transcription was dramatically inhibited in A549 cells after IFN-alpha treatment, but a step following primary transcription was inhibited in U87-MxA cells constitutively expressing MxA. Here we have investigated the role of MxA, believed to be cell type specific, and other antiviral pathways in the inhibition of viral primary transcription. Our data indicate that a novel IFN-induced pathway(s) is involved in the inhibition of primary transcription. This is based on the following findings: (i) IFN-alpha inhibited viral primary transcription in U87-MxA and other cell types including cells lacking MxA; (ii) cells constitutively expressing 2-5A synthetase had no antiviral effect against HPIV3; and (iii) primary transcription occurred in the absence of protein synthesis, a step of PKR target. The novel antiviral pathway(s) was induced by both IFN-alpha and IFN-gamma to establish an effective antiviral state against HPIV3. By using IFN-alpha-signaling mutant cells, we found that IFN-gamma could elicit antiviral effect against HPIV3 without cross talk with the IFN-alpha-signaling pathway. These data provide the first evidence that a novel antiviral pathway(s) contributes to the antiviral action of IFN against a nonsegmented negative-strand RNA virus by targeting the primary transcription.

Animals↗
PubMed2001

Preferential induction of apoptosis by interferon (IFN)-beta compared with IFN-alpha2: correlation with TRAIL/Apo2L induction in melanoma cell lines.

On the basis of in vitro inhibition of tumor cell growth, IFNs have been generally considered to be antiproliferative proteins. To probe further the potential mechanisms of the antitumor effects of IFNs, we have assessed apoptosis in response to IFN-alpha2 and IFN-beta in cell lines of varied histologies, with a focus on melanomas. Many of the cell lines tested underwent apoptosis in response to IFN-beta, as assessed both by Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. In general, IFN-beta had greater growth inhibitory and proapoptotic effects than IFN-alpha2 on all cell lines. The melanoma cell line WM9, sensitive to growth inhibition by IFNs, had a greater degree of apoptosis than A375 melanoma cells, which were largely resistant to antigrowth effects of IFNs. IFN-beta-induced apoptosis was dependent on activation of the caspase cascade with cleavage of caspases 3, 8, and 9 and of the caspase 3 substrate, poly(ADP-ribose) polymerase. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl keton or benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl keton, inhibited IFN-beta-induced apoptosis. Other changes associated with apoptosis, including the movement of cytochrome c from mitochondria to cytoplasm and DNA fragmentation, were also identified in response to IFN-beta. Apo2L ligand [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)] was one of the early genes induced by IFN-beta in apoptosis-sensitive WM9 cells. Other sensitive melanoma cell lines had a similar IFN-beta-specific induction of TRAIL. Neutralizing antibody to TRAIL inhibited IFN-beta-induced apoptosis in WM9 cells. In resistant A375 cells, IFN-beta did not induce TRAIL/Apo2L expression. Thus, induction of TRAIL by IFNs in some tumor types may initiate the apoptotic cascade. This study offers another mechanism for the antitumor effects of IFNs.

Annexin A5↗
PubMed2000

2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells.

OBJECTIVE: RNase L is converted to an active form upon binding short 2',5'-oligoadenylates (2-5A). To direct RNase L to an RNA target, 2-5A is attached to an antisense oligonucleotide (2-5A antisense). This chimera can be directed against telomerase-an RNA-protein complex that elongates telomeric DNA and is involved in cellular immortalization. Our objective is to investigate the effect of 2-5A antisense by targeting telomerase RNA (hTR) in the ovarian cancer cell line, HEY-1B. METHODS: Baseline RNase L levels and telomerase activities were measured in both HEY-1B and normal ovarian epithelial cells (NOE). Cells were treated daily with chimeric oligonuclotides (ODN) directed against four different hTR sites, or control ODNs including nonchimeric antisense, 2-5A fused to a mismatched sequence, or inactive 2-5A fused to antisense. At 48 h, apoptosis was evaluated using the TUNEL assay. After six daily ODN administrations, telomerase activity was redetermined, and at 7 days viability counts were obtained. RESULTS: Both cell lines expressed similar levels of RNase L. Hey-1B displayed telomerase activity while NOE did not. After 7 days of transfection, 2-5A antisense ODNs caused profound cell death in the HEY-1B cells, but not in the NOE cells. This effect was seen regardless of hTR target site, and ODN controls showed no significant decrease in cell viability in either cell line. HEY1B cells treated with 2-5A antisense against hTR showed a decrease in telomerase activity and a profound induction of programmed cell death. CONCLUSIONS: The results suggest that 2-5A antisense directed against telomerase RNA results in apoptotic cell death in ovarian cancer cells, but not normal ovarian epithelial cells. The 2-5A antisense strategy may hold a considerable advantage over the conventional antisense approach in targeting cancer-causing genes.

Adenine Nucleotides↗
PubMed1999

Catalytically active TYK2 is essential for interferon-beta-mediated phosphorylation of STAT3 and interferon-alpha receptor-1 (IFNAR-1) but not for activation of phosphoinositol 3-kinase.

TYK2, a Janus kinase, plays both structural and catalytic roles in type I interferon (IFN) signaling. We recently reported (Rani, M. R. S., Gauzzi, C., Pellegrini, S., Fish, E., Wei, T., and Ransohoff, R. M. (1999) J. Biol. Chem. 274, 1891-1897) that catalytically active TYK2 was necessary for IFN-beta to induce the beta-R1 gene. We now report IFN-beta-mediated activation of STATs and other components in U1 (TYK2-null) cell lines that were complemented with kinase-negative (U1.KR930) or wild-type TYK2 (U1.wt). We found that IFN-beta induced phosphorylation on tyrosine of STAT3 in U1.wt cells but not in U1.KR930 cells, whereas STAT1 and STAT2 were activated in both cell lines. Additionally, IFN-beta-mediated phosphorylation of interferon-alpha receptor-1 (IFNAR-1) was defective in IFN-beta treated U1.KR930 cells, but evident in U1.wt cells. In U1A-derived cells, the p85/p110 phosphoinositol 3-kinase isoform was associated with IFNAR-1 but not STAT3, and the association was ligand-independent. Further, IFN-beta treatment stimulated IFNAR-1-associated phosphoinositol kinase activity equally in either U1.wt or U1.KR930 cells. Our results indicate that catalytically active TYK2 is required for IFN-beta-mediated tyrosine phosphorylation of STAT3 and IFNAR-1 in intact cells.

Catalysis↗
PubMed1999

Controlling gene expression with 2-5A antisense.

Recent work has demonstrated that the activity of a ubiquitous cellular enzyme, ribonuclease L (RNase L), can be harnessed to cleave targeted RNA species. Activation of RNase L is dependent on the presence of 2',5'-linked oligoadenylates (2-5A), usually produced by cells infected with viruses. By conjugating synthetic 2-5A to specific antisense compounds, it is now possible to selectively degrade RNAs in an RNase L-dependent manner, thereby providing an alternative to RNase H-dependent approaches. In this summary, we provide an updated description of the synthesis procedure for constructing these chimeric 2-5A antisense molecules. Examples of successful applications of the 2-5A antisense strategy are described, along with some of the procedures involved in those studies. Several methods are also provided for optimizing compound uptake and analyzing their effects on cells. Finally, we discuss the current body of evidence that supports the contention that RNase L is indeed the primary mediator of 2-5A antisense effects and the possible implications that this has on the future of this therapeutic approach.

Adenine Nucleotides↗
PubMed1998

A mutant cell line defective in response to double-stranded RNA and in regulating basal expression of interferon-stimulated genes.

Although much progress has been made in identifying the signaling pathways that mediate the initial responses to interferons (IFNs), much less is known about how IFN-stimulated genes (ISGs) are kept quiescent in untreated cells, how the response is sustained after the initial induction, and how ISG expression is down-regulated, even in the continued presence of IFN. We have used the cell sorter to isolate mutant cells with constitutively high ISG expression. A recessive mutant, P2.1, has higher constitutive ISG levels than the parental U4C cells, which do not respond to any IFN. Unexpectedly, P2.1 cells also are deficient in the expression of ISGs in response to double-stranded RNA (dsRNA). Electrophoretic mobility-shift assays revealed that the defect is upstream of the activation of the transcription factors NFkappaB and IFN regulatory factor 1. Analysis of the pivotal dsRNA-dependent serine/threonine kinase PKR revealed that the wild-type kinase is present and is activated normally in response to dsRNA in P2.1 cells. Together, these data suggest that the defect in P2.1 cells is either downstream of PKR or in a component of a distinct pathway that is involved both in activating multiple transcription factors in response to dsRNA and in regulating the basal expression of ISGs.

Cell Line↗
PubMed1997

The antiproliferative and antiviral activities of IFN-tau variants in human cells.

The IFN-tau are type I IFN expressed by the early trophoblast of cattle and sheep but have activity on human cells and have been predicted to have potential therapeutic value. We have compared a series of mutant bovine and ovine IFN-tau with regard to their ability to inhibit the proliferation of Daudi cells and to evoke an antiviral (AV) response in WISH cells. Whereas Daudi cell growth was inhibited by Bo-IFN-tau1 in the 1 nM range, WISH cells were much less responsive, requiring exposure to 150 nM for protection against vesicular stomatitis virus. Replacement of lysines at positions 34, 107, 121, and 132 in Bo-IFN-tau, which are in regions predicted to interact with the type I receptor, led to modest but significant alterations in antiproliferative (AP) and AV activities. Replacement of the lysine residues at 160 and 164 had marked effects on biopotency, with K160 being particularly important. The different IFN-tau were able to activate the transcription factors ISGF3 and AAF (GAF) in Daudi cells at concentrations that correlated reasonably well with their AP potencies. Stat activation occurred in WISH cells in response to approximately 2 nM Bo-IFN-tau1, but ISGF3 formation could not be demonstrated even at the 100-fold higher IFN-tau concentrations that gave viral protection. Pretreatment of WISH cells with Hu-IFN-gamma allowed ISGF3 formation to be observed in response to subsequent treatment with Bo-IFN-tau1 or type I human IFN but did not increase the AV responsiveness of the cells. No evidence was found that IFN-tau elicit uniquely different responses on human cells than type I Hu-IFN, except they are much less potent. The data emphasize the importance of a region near the carboxyl terminus for the functional activity of type I IFN, and that although ISFG3 formation may be necessary, its mere presence is not sufficient to provide an antiviral response.

Amino Acid Sequence↗
PubMed1996

A type I ovine interferon with limited similarity to IFN-alpha, IFN-omega and IFN-tau: gene structure, biological properties and unusual species specificity.

A gene encoding a 195 amino-acid (a.a.) polypeptide with a putative 23 a.a. signal sequence that had about 60% a.a. sequence identity to ovine interferon-omega (OvIFN-omega) and 55% or less identity to BoIFN-tau, OvIFN-tau and all known IFN-alpha and -beta has been identified from an ovine genomic DNA library. Surprisingly, it shared almost complete identity to genes for rabbit IFN-omega within its coding sequence and proximal promoter region, although the two were different in their 3'-ends. This IFN (tentatively termed ovine IFN-omega variant, OvIFN-omegav), purified in recombinant form from E. coli, had normal antiviral activity when tested on sheep fetal tongue and brain cells and rabbit kidney cells, but very low activity towards bovine, goat and human cells. It competed with 125I-labeled BoIFN-tau for binding to IFN receptors on ovine cells. Expression of OvIFN-omegav was not detected by reverse transcription-PCR either in ovine peripheral blood leukocytes infected with Sendai virus, or in any other tissues examined. OvIFN-omegav may represent a previously unrecognized, non-virally inducible type I subtype distinct from IFN-alpha, -beta, -omega and -tau. The presence of a conserved gene in rabbit and sheep could reflect a recent interspecies transfer.

Amino Acid Sequence↗
PubMed1996

Participation of JAK and STAT proteins in growth hormone-induced signaling.

The binding of growth hormone leads to dimerization of its receptor, accompanied by phosphorylation and activation of intracellular tyrosine kinases (JAKs) and the latent cytoplasmic transcriptions factors STAT1, STAT3, and STAT5. Both JAK1 and JAK2 are phosphorylated in response to growth hormone in mouse 3T3 F442A and human HT1080 cells. The roles of JAKs in growth hormone signal transduction were examined by using mutant HT1080 cells missing either JAK1 or JAK2. JAK2 is absolutely required for growth hormone-dependent phosphorylation of the receptor, STAT1 and STAT3, JAK1, and the SH2-containing adaptor molecule Shc. In contrast, JAK1 is not required for any of the above functions. These data indicate that JAK2 is both necessary and sufficient for the growth hormone-dependent phosphorylation events required to couple the receptor both to STAT-dependent signaling pathways and to pathways involving Shc. Furthermore, STAT5 is activated by growth hormone in 3T3 F442A cells, but not in HT1080 cells, revealing that the set of STATs activated by growth hormone can vary, possibly contributing to the specificity of the growth hormone response in different cell types.

3T3 Cells↗
PubMed1996

The interferon-tau genes of the giraffe, a nonbovid species.

Genes for interferon-tau (IFNT) have been cloned from several Bovidae, and the IFNT lineage has been predicted to have arisen from the IFNW, as the divergence of the Ruminantia from other artiodactyls. Southern genomic blotting with probes specific for IFNT identified a gene in the giraffe, a nonbovid member of the Ruminantia. Here this gene has been cloned, sequenced, and expressed. It encodes a 172-amino acid mature protein with antiviral activity on bovine cells. Giraffe IFN-tau lacks the normally conserved Cys99 but has a pair of other cysteines (Cys64 and Cys86) that could provide a disulfide bridge. The giraffe IFNT gene possesses the highly conserved promoter region that distinguishes IFNT from IFNW. It seems likely that IFNT emerged before the divergence of the Giraffidae and Bovidae, which occurred some 24 million years ago.

Amino Acid Sequence↗
PubMed1996

Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor.

The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells.

Base Sequence↗
PubMed1996

Regulation of STAT-dependent pathways by growth factors and cytokines.

Polypeptide growth factors and cytokines elicit their effects by activating specific cell-surface receptors, thereby initiating signaling cascades that culminate in the induction of specific subsets of genes. Initially identified as the primary mediators of interferon-dependent signaling, JAKs and STATs are now known to be utilized by many different extracellular signaling proteins. In this report we describe how JAK-STAT pathways transduce signals initiated by both cytokines and growth factors, focusing on how specificity is achieved through STAT-receptor interactions and on how receptor-associated kinases function in STAT activation. We also summarize current information on interactions between signaling pathways, particularly STAT-Ras cross-talk and the relative importance of these two pathways in regulating the transcription of target genes.

Animals↗
PubMed1994

Multiple regulatory elements are required to direct trophoblast interferon gene expression in choriocarcinoma cells and trophectoderm.

Interferon-tau (IFN tau) is produced exclusively by the trophectoderm during the peri-implantation stage of pregnancy in ruminant ungulate species. Human choriocarcinoma cells (Jar) stably transfected with 1.8 kilobases of promoter from a bovine IFN tau gene ahead of a human GH (hGH) reporter gene constitutively synthesize hGH, but expression is not increased further by exposure to Newcastle disease virus. This and earlier experiments suggest that the transcriptional cues regulating IFN tau expression are distinct from those operating on other type I IFN genes. Transient transfection experiments reveal that two distinct promoter regions are required for full constitutive expression: one proximal (to position -126), which directs basal expression, and a more distal promoter region (positions -280 to -400), which acts as an enhancer. Nuclear extracts prepared from ovine conceptuses during the period of IFN tau expression interact with the proximal promoter region (positions -34 to -126) to form several complexes of high electrophoretic mobility. Although nucleotide sequence motifs potentially capable of binding the transcription factor IRF-1 are present in this region, IRF-1 does not transactivate the IFN tau gene. The distal part of the promoter contains only one region (-322 to -358) that forms a complex with these conceptus nuclear extracts. Both proximal and distal gel shift patterns become dramatically different when IFN tau gene expression ceases, perhaps reflecting the appearance of transcriptional repressors. Together these experiments support the conclusion that the control of IFN tau gene expression is very different from that of other type I IFN genes and that trophoblast-specific expression depends upon distal as well as proximal promoter regulatory elements.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Insulin-like growth-factor binding protein (IGFBP) serum levels and hepatic IGFBP-2 and -3 mRNA expression in diabetic and insulin-treated swine (Sus scrofa).

1. Diabetes had no significant effect on IGFBP-3 message and serum levels however, subsequent insulin treatment caused more than a two-fold increase in both hepatic IGFBP-3 mRNA and serum levels above controls (P < 0.05). 2. The induction of diabetes in pigs significantly increased the steady state levels of IGFBP-2 mRNA in the liver of young swine (P < 0.05). 3. Both liver message and serum IGFBP-2 were reduced to control levels with insulin therapy. 4. We report here that in addition to its affects on IGFBP-2, insulin is involved in the regulation of IGFBP-3 expression.

Animals↗
PubMed1992

Genes for the trophoblast interferons and their distribution among mammals.

The trophoblast interferons (IFN), ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1 respectively), possess many properties in common with other Type I IFN, including antiviral and antiproliferative activities. However, they differ from other IFN subtypes in terms of their specific pattern of expression and poor responsiveness to viral stimulation. We describe here data that suggest that the trophoblast IFN genes are also distinct from other Type I IFN genes with respect to species distribution, promoter organization and transcriptional regulation. These results have important implications in terms of trophoblast IFN gene evolution and add credence to the concept that the trophoblast IFN genes are a distinct IFN gene subtype well suited for their role in early pregnancy.

Animals↗
PubMed1992

Genes for the trophoblast interferons in sheep, goat, and musk ox and distribution of related genes among mammals.

The trophoblast interferons (IFNs) are a family of Type 1 IFN found in domestic ruminants that are most closely related to the little-studied 172-amino-acid IFN-omega. They are produced in massive amounts by the preimplantation conceptus at a time coincident with maternal recognition of pregnancy, and are implicated in playing an important role in this process. Here we report the characterization of four distinct members of the ovine trophoblast IFN (oTP-1) gene family, and demonstrate that they, along with previously characterized bovine trophoblast (bTP-1) genes, possess distinctive promoter sequences when compared to ovine and bovine IFN-omega genes. Genomic Southern blot analysis of numerous mammalian species (zoo blots) indicate that, whereas the IFN-omega are widely distributed among mammals, genes for the trophoblast IFN appear to be limited to ruminant species within the Artiodactyla order. Further polymerase chain reaction (PCR) analysis of trophoblast IFN genes in these ruminant species has permitted isolation of genes for goat and musk ox trophoblast IFN. These data suggest that the trophoblast IFNs are a distinct family of IFN with a limited distribution among mammals.

Animals↗