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S T Crooke

Publications and source records attributed to S T Crooke.

At least 73 records · Page 4Linked to original sources
PubMed1993

Progress toward oligonucleotide therapeutics: pharmacodynamic properties.

Although many issues remain to be resolved, recent progress suggests that oligonucleotides may have therapeutic potential. The first therapeutic oligonucleotide, ISIS 2105, an oligonucleotide designed to treat genital warts, has completed Phase I clinical trials and is undergoing Phase II testing. Conceptually, oligonucleotides may interact with nucleic acid or nonnucleic acid receptors; however, rational design of oligonucleotide drugs to interact with nucleic acid receptors is significantly simpler because the factors that determine affinity and selectivity are better understood. Recent progress demonstrates that this information can be coupled to rapid advances in the medicinal chemistry of oligonucleotides to create new generations of oligonucleotide drugs with dramatically improved properties. The purposes of this review are to place oligonucleotide therapeutics in the context of modern molecular drug discovery and to summarize progress in understanding the pharmacodynamic properties of oligonucleotides designed to bind to nucleic acid receptors. The pharmacokinetic and toxicologic properties of oligonucleotides have been reviewed recently (1-4). The activities of oligonucleotides that bind to nonnucleic acid receptors will be considered in future reviews.

Animals↗
PubMed1993

Regulation of 5-lipoxygenase and 5-lipoxygenase-activating protein expression in HL-60 cells.

5-Lipoxygenase performs the first two enzymic reactions in the biosynthetic pathway for the leukotrienes. We have utilized HL-60 cells to study the mechanisms regulating expression of 5-lipoxygenase and the recently described 18 kDa membrane protein, 5-lipoxygenase-activating protein (FLAP). Differentiation of HL-60 cells into granulocyte-like cells with dimethyl sulphoxide (Me2SO), retinoic acid or dibutyryl cyclic AMP (Bt2-cAMP) resulted in a 2-3-fold increase in 5-lipoxygenase enzyme activity and a 4-fold increase in leukotriene B4 synthesis. Differentiation of HL-60 cells into monocyte-like cells with 1,25-dihydroxyvitamin D3 induced 5-lipoxygenase activity 5-fold, but leukotriene B4 production was only increased 2-fold. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells failed to induce 5-lipoxygenase or leukotriene B4 production. Examination of the levels of the transcript encoding 5-lipoxygenase and FLAP demonstrated that differentiation of HL-60 cells into granulocytes resulted in a co-ordinate induction of 5-lipoxygenase and FLAP mRNA. In contrast, differentiation of HL-60 cells into monocytes resulted in discordant regulation of 5-lipoxygenase and FLAP mRNA. Treatment with 1,25-dihydroxyvitamin D3 resulted in a 6-fold increase in 5-lipoxygenase mRNA and a 1.3-fold increase in FLAP mRNA, while treatment with phorbol ester did not induce 5-lipoxygenase mRNA but did increase FLAP mRNA 2-fold. The transcriptional rate of the 5-lipoxygenase and FLAP genes did not change upon Me2SO or 1,25-dihydroxyvitamin D3 treatment, suggesting that the increase of the mRNA coding for these proteins was not due to transcriptional activation of their respective genes. The mRNA half-life for 5-lipoxygenase did not change significantly upon treatment with Me2SO or 1,25-dihydroxyvitamin D3. The FLAP mRNA half-life increased from approx. 3.5 h to 4.5 h in cells treated with either Me2SO or 1,25-dihydroxyvitamin D3. These data suggest that expression of 5-lipoxygenase and FLAP is controlled by a post-transcriptional event other than stabilization of the mRNA.

5-Lipoxygenase-Activating Proteins↗
PubMed1993

Growth inhibition of human tumor cell lines by antisense oligonucleotides designed to inhibit p120 expression.

The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody (MAbp120) in most human malignant tumors (Freeman et al., Cancer Research, 48, 1244-1251, 1988). Stable transfection of the sense p120 cDNA caused malignant transformation of NIH/3T3 cells in vitro, and the antisense p120 constructs markedly delayed the growth of these transformed cells (Perlaky et al., Cancer Research, 52, 428-436, 1992). Several p120 antisense phosphorothioate oligonucleotides designed to hybridize with different regions of the p120 sequence were screened on human tumor cell lines in vitro. Marked growth inhibition of HeLa, LOX and HRCC cell lines was found, particularly with antisense p120 oligonucleotide ISIS 3466 in combination with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); oligonucleotide ISIS 3466 is complementary to a non-translated region at the 3' end of the molecule. Preliminary in vivo studies on human LOX ascites tumor in nude mice showed marked inhibitory effects on tumor growth by the antisense oligonucleotide ISIS 3466 in the presence of DOTMA when treated on alternate days.

Animals↗
PubMed1992

Stimulation of leukotriene production and membrane translocation of 5-lipoxygenase by cross-linking of the IgE receptors in RBL-2H3 cells.

Recent studies in rat basophilic leukemia cells (RBL-2H3) have shown that two pharmacological agents, ionomycin and thapsigargin, induce leukotriene C4 production and translocation of 5-lipoxygenase from cytosol to membrane, primarily by causing an influx of extracellular calcium. In the present study, we investigate the induction of these events by receptor activation. Cross-linking of high-affinity IgE receptors (Fc epsilon RI) by antigen in RBL-2H3 cells leads to leukotriene C4 production and membrane translocation of 5-lipoxygenase. As in the ionomycin-stimulated cells, leukotriene C4 production in antigen-stimulated cells is calcium-dependent since the amount of leukotriene C4 produced correlates quantitatively with the increase in intracellular free calcium concentration ([Ca2+]i). However, the increase in [Ca2+]i required for equivalent leukotriene C4 production by antigen is not as high as it is using ionomycin. In addition, no threshold [Ca2+]i level is required for leukotriene production by antigen, which is in contrast to the ionomycin stimulation that a [Ca2+]i level of 300-400 nM is required. Furthermore, antigen causes an additive increase in leukotriene C4 production in cells stimulated by the ionomycin. These results suggest that another as yet unidentified intracellular pathway acts in conjunction with Ca2+ for leukotriene synthesis in antigen-stimulated cells. Antigen stimulation causes 20-30% of the total cell 5-lipoxygenase to associate with membranes (compared with 10% in unstimulated cells) as demonstrated by enzyme activity assay and by Western Blot using antibodies to 5-lipoxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Topoisomerase II alpha and topoisomerase II beta genes: characterization and mapping to human chromosomes 17 and 3, respectively.

Human cells contain two topoisomerase II isozymes named topo II alpha and topo II beta. The complementary DNAs for both enzymes have been cloned. The topo II alpha and topo II beta complementary DNAs hybridized to unique sequences of human, rodent, and chicken DNAs in Southern blots. The human topo II alpha gene has previously been mapped to chromosome 17. We confirmed the chromosomal location of topo II alpha and mapped the topo II beta gene to chromosome 3. In addition, topo II beta exhibits genetic polymorphism as has been reported for topoisomerases I and II alpha.

Blotting, Southern↗
PubMed1992

Oligonucleotide therapy.

Rapid progress in oligonucleotide therapeutics has continued over the past year as major programs established in the past four years have grown and begun to be productive. Important advances were reported in the medicinal chemistry of oligonucleotides and in understanding their pharmacodynamic properties. Significant progress was made in understanding the pharmacokinetic and toxicologic properties of first generation analogs, particularly phosphorothioates and one oligonucleotide, ISIS 2105, entered clinical trials. Additionally, combinatorial approaches designed to identify oligonucleotides that may bind to a variety of targets were reported.

Animals↗
PubMed1992

Therapeutic applications of oligonucleotides.

This review focuses strictly on the pharmacodynamic considerations of the use of oligonucleotides designed to interact with nucleic acids as therapeutics. The objectives are to place oligonucleotide therapeutics in the context of modern drug discovery and development and to summarize recent progress.

Base Composition↗
PubMed1991

Cloning of a phospholipase A2-activating protein.

Recently we have described the isolation and biochemical characterization of a phospholipase A2-activating protein (PLAP). We have cloned this protein and found it to be expressed as a 2.5-kilobase mRNA. The steady-state levels of PLAP mRNA are induced in smooth muscle and endothelial cells following treatment with leukotriene D4. The increased message levels coincide with increased amounts of PLAP. Synthetic antisense DNA was used to block the synthesis of PLAP and this treatment effectively blocked the activation of phospholipase A2 and the increased generation of prostanoids in smooth muscle and endothelial cells treated with leukotriene D4.

Amino Acid Sequence↗
PubMed1991

Antisense technology.

During the past 12 months, significant advances have been reported in the medicinal chemistry, and the pharmacodynamic and pharmacokinetic characterization of oligonucleotides. Advances in medicinal chemistry suggest that the scope for additional modifications as a means of developing therapeutic agents is substantial. This is confirmed by our clearer understanding of the pharmacodynamic and pharmacokinetic properties of oligonucleotides and the increasing number of molecular targets against which they have been shown to be active.

Animals↗
PubMed1991

Leukotriene receptors and mechanisms of signal transduction.

To better understand the mechanisms by which leukotriene tone is regulated, we have characterized the mechanisms of genetic regulation of 5-lipoxygenase in HL60 cells induced to differentiate with dimethyl sulfoxide (DMSO) and compared a number of rat and human 5-lipoxygenase introns. We demonstrate that differentiation of HL60 cells with DMSO results in coordinate induction of 5-LO and 5-LO activating protein (FLAP). The production of LTB4, 5-LO protein, 5-LO mRNa and FLAP RNA increased coordinately. However, two approaches demonstrated no increase in the initiation of transcription of 5-LO and FLAP pre-mRNA and no changes in the mRNA half lives. Moreover, cycloheximide inhibits the induction of the mRNAs and proteins. Thus, we suggest that 5-LO and FLAP are coordinately regulated in HL60 cells via mechanisms involving changes in RNA processing. To better understand potential mechanisms involved, we have cloned and sequenced several human 5-LO introns and compared them to analogous rat 5-LO introns. A number of regions of potential regulatory significances are conserved and may be important in controlling the rate of pre-mRNA processing.

5-Lipoxygenase-Activating Proteins↗
PubMed1991

In vitro and in vivo pharmacologic activities of antisense oligonucleotides.

The use of antisense oligonucleotide as pharmacologic agents is a derivative of the central dogma of molecular biology and knowledge of the physical and chemical properties that govern the structure of nucleic acids. Oligonucleotides have been reported to inhibit the growth of a large number of viruses in cell culture, as well as the expression of numerous oncogenes, a variety of normal genes and transfected reporter genes controlled by several regulatory elements. The therapeutic activity of antisense compounds in animal disease models have also been reported. This review provides some general conclusions and trends regarding the pharmacologic action of antisense oligonucleotides, that can be formulated from studies previously reported in the literature. In addition, data is highlighted for two specific examples in which antisense oligonucleotides have demonstrated activity against herpes viruses and intracellular adhesion molecule RNA targets.

Animals↗
PubMed1991

Regulation of topoisomerase I and II activities by cyclic nucleotide- and phospholipid-dependent protein kinases. Effects of interactions between the two transduction pathways.

Incubation of cultured rat aortic smooth muscle cells (A-10) with activators of cyclic nucleotides resulted in transiently increased activity of extractable topoisomerase I or topoisomerase II. ANF, which induces cGMP accumulation, potentiated camptothecin-induced, topoisomerase I linked DNA strand breakage and increased the specific activity of extractable topoisomerase I (maximum activity 5-15 min after treatment), but had no effect on topoisomerase II activity. These effects are similar to those reported for AVP and phorbol esters, activators of protein kinase C. Forskolin and isoproterenol, which induce cAMP accumulation, activated extractable topoisomerase II (maximum 5-15 min after treatment), but not topoisomerase I. Permeable cyclic nucleotide analogs dBcAMP and 8BrcGMP selectively activated extractable topoisomerase II and topoisomerase I activities, respectively. Activation of topoisomerase I by either AVP or PdBu was attenuated by cotreatment with 8BrcGMP or dBcAMP, and activation of topoisomerase II by dBcAMP was attenuated by cotreatment with AVP or PdBU, suggesting that elements of the protein kinase C and the cyclic nucleotide linked signal-transduction pathways can interact to modify nuclear enzymic activity. IBMX, which elevates intracellular cAMP and cGMP, increased the extractable activities of both topoisomerase I and topoisomerase II. Thus, topoisomerase activity in cells may be governed in part by cyclic nucleotide levels.

Animals↗
PubMed1990

Purification and characterization of a soluble phospholipase A2 from guinea pig lung.

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.

Animals↗
PubMed1990

Mobilization of extracellular Ca2+ by prostaglandin F2 alpha can be modulated by fluoride in 3T3-L1 fibroblasts.

Changes in the intracellular concentration of calcium [( Ca2+]i) have been shown to mediate the physiological effects of certain agonists. Ca2+ mobilization occurs through multiple mechanisms which involve both influx and internal release of Ca2+. Prostaglandin F2 alpha (PGF2 alpha) caused a transient mobilization of intracellular Ca2+ in 3T3-L1 fibroblasts. This effect was characterized by fluorescence measurements of trypsin-treated cells loaded with fura-2/AM. In the absence of extracellular Ca2+, the peak amount of Ca2+ mobilized by PGF2 alpha was decreased by 70%, a lag time before the onset of [Ca2+]i increase was observed, and the rate of rise of [Ca2+]i was slowed. Addition of NaF (10 mM) to fura-2-loaded 3T3-L1 cells caused a dose-dependent increase in [Ca2+]i after a brief (approximately 10 s) lag. Maximal effects (approximately 300 nM) were observed at 5-10 mM-NaF. This effect was dependent on the presence of extracellular Ca2+ and appeared to be independent of inositol phosphate production. After reaching a peak at around 40 s after fluoride addition, [Ca2+]i returned to near-baseline within 120 s. This return of [Ca2+]i to near-baseline after fluoride stimulation and the inability of the cells to respond to a subsequent addition of fluoride indicated that the response to fluoride underwent desensitization. Similarly, the pathway used by PGF2 alpha to mobilize Ca2+ underwent desensitization. Exposure of the cells to a maximally effective concentration of fluoride and subsequent addition of PGF2 alpha produced a [Ca2+]i response to PGF2 alpha which was similar in magnitude and kinetics to that seen for PGF2 alpha in the absence of extracellular Ca2+. Conversely, prior exposure of cells to PGF2 alpha diminished the ability of fluoride to mobilize Ca2+. PGF2 alpha also increased inositol phosphate formation, with a time course and dose-response consistent with its ability to increase [Ca2+]i. Prior exposure of cells to fluoride did not change the time course or dose-response characteristics of PGF2 alpha-induced generation of inositol phosphates. These data suggest that PGF2 alpha and fluoride share a common mechanism of activating Ca2+ influx in 3T3-L1 cells.

Animals↗
PubMed1990

A human ubiquitin carboxyl extension protein functions in yeast.

The ability of the human ubiquitin carboxyl extension protein (HUBCEP80) to functionally replace its yeast homolog was determined in a ubi3 mutant of Saccharomyces cerevisiae. Expression of HUBCEP80 in ubi3 mutants resulted in processing of the fusion protein to produce free ubiquitin and extension protein, the latter of which localized specifically with the 40 S ribosomal subunit. Furthermore, expression of the human fusion protein completely alleviated the phenotypic deficiencies found in ubi3 mutants, including slow growth, abnormal ribosomal RNA processing, and correspondingly low levels of 40 S ribosomal subunits. Finally, expression of the extension protein alone was much less efficient in complementing the ubi3 mutant phenotype as compared with expression of the normal ubiquitin-fused extension protein. In the latter case, cells were found to contain at least 5-fold more extension protein, suggesting that ubiquitin either increased translational efficiency of the HUBCEP80 transcript or increased the stability of the processed extension protein.

Chromosome Deletion↗