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1.
Nucleic Acids Res ; 29(8): 1683-9, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11292840

ABSTRACT

2'-O-(2-methoxyethyl) (2'-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2'-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC(50) values of 5-10 nM at 37 degrees C. IC(50) values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.


Subject(s)
Enzyme Inhibitors/metabolism , Oligonucleotides/chemistry , Oligonucleotides/metabolism , RNA, Antisense/chemistry , RNA, Antisense/metabolism , Telomerase/antagonists & inhibitors , Animals , Base Pair Mismatch/genetics , Base Pairing , Base Sequence , DNA, Recombinant/chemistry , DNA, Recombinant/genetics , DNA, Recombinant/metabolism , Enzyme Inhibitors/chemistry , Genetic Engineering , Humans , Inhibitory Concentration 50 , Mice , Mice, Nude , Neoplasm Transplantation , Oligonucleotides/genetics , Oligoribonucleotides , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA, Antisense/genetics , Substrate Specificity , Telomerase/genetics , Telomerase/metabolism , Time Factors , Transfection , Tumor Cells, Cultured
2.
Transplantation ; 70(4): 656-61, 2000 Aug 27.
Article in English | MEDLINE | ID: mdl-10972225

ABSTRACT

BACKGROUND: C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival. METHODS: Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2'-methoxyethyl (ME) substitutions at the 2'-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides. RESULTS: Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos. CONCLUSIONS: C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity.


Subject(s)
Gene Expression Regulation/drug effects , Graft Survival/genetics , Heart Transplantation/physiology , Oligodeoxyribonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogenes , Animals , Base Sequence , Cell Line , Graft Survival/drug effects , Heart Transplantation/immunology , Humans , Mice , Rats , Rats, Inbred ACI , Rats, Inbred Lew , Thionucleotides , Transplantation, Homologous
3.
Proc Natl Acad Sci U S A ; 96(20): 11464-9, 1999 Sep 28.
Article in English | MEDLINE | ID: mdl-10500199

ABSTRACT

A large fraction of pediatric pre-B acute lymphoblastoid leukemias (ALL) consistently contain a t(1;19) chromosomal translocation. The t(1;19) translocation results in the production of a chimeric transcription factor containing the N-terminal transactivation domain of E2A fused to the C-terminal DNA-binding homeodomain of Pbx1. Here, we show that the E2A-Pbx1 fusion protein activates the expression of a novel WNT gene, WNT-16. WNT-16 normally is expressed in peripheral lymphoid organs such as spleen, appendix, and lymph nodes, but not in bone marrow. In contrast, high levels of WNT-16 transcripts are present in bone marrow and cell lines derived from pre-B ALL patients carrying the E2A-Pbx1 hybrid gene. Inhibition of E2A-Pbx1 expression leads to a significant decrease in WNT-16 mRNA levels, suggesting that WNT-16 is a downstream target of E2A-Pbx1. Three putative WNT receptors, FZ-2, FZ-3, and FZ-5, are expressed in cells of the B lineage, including pre-B ALL cells aberrantly expressing WNT-16. We propose that a WNT-16-mediated autocrine growth mechanism contributes to the development of t(1;19) pre-B ALL.


Subject(s)
Homeodomain Proteins/physiology , Oncogene Proteins, Fusion/physiology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins/genetics , Transcription Factors/physiology , Zebrafish Proteins , Amino Acid Sequence , Gene Expression Regulation , Humans , Molecular Sequence Data , Tumor Cells, Cultured , Wnt Proteins
4.
Biochem Biophys Res Commun ; 172(3): 1273-81, 1990 Nov 15.
Article in English | MEDLINE | ID: mdl-2244911

ABSTRACT

Interleukin 1 (IL-1), bacterial lipopolysaccharide (LPS) and tumor necrosis factor (TNF alpha) enhance the adherence properties of endothelial cells (EC) for neutrophils (PMN). This is mediated in part by the up-regulation of Intercellular Adhesion Molecule 1 (ICAM-1) on EC. Phorbol esters, which activate protein kinase c (PKC) and enhance the adherence properties of EC for PMN also up-regulate the ICAM-1 expression on EC. We investigated the effect of PKC inhibitors on ICAM-1 expression of human umbilical vein EC (HUVEC). Staurosporine (STS) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) prevented inflammatory mediator-dependent stimulation of both ICAM-1 expression and PMN adherence by HUVEC (ID50 for STS = 2.7-2.9 microM; for H-7 = 7.6-8.8 microM). Inhibition was dose and time-dependent and was not due to HUVEC toxicity. The STS analog K252a and the H-7 analog W-7 were less potent inhibitors of ICAM-1 up-regulation and adherence promotion. Prolonged exposure of HUVEC to phorbol myristate acetate down-regulated PKC activity and inhibited subsequent ICAM-1 up-regulation by this agent and by IL-1. We conclude that inflammatory mediator induced stimulation of HUVEC expression of ICAM-1 and promotion of adherence properties are mediated in part by activation of PKC.


Subject(s)
Cell Adhesion Molecules/biosynthesis , Endothelium, Vascular/metabolism , Interleukin-1/pharmacology , Lipopolysaccharides/pharmacology , Protein Kinase C/antagonists & inhibitors , Tumor Necrosis Factor-alpha/pharmacology , Cell Adhesion , Cells, Cultured , Dose-Response Relationship, Drug , Down-Regulation , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Humans , Kinetics , Neutrophils/physiology , Umbilical Veins/drug effects , Umbilical Veins/enzymology , Umbilical Veins/metabolism
5.
Proc Natl Acad Sci U S A ; 78(2): 732-6, 1981 Feb.
Article in English | MEDLINE | ID: mdl-6262767

ABSTRACT

Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation.


Subject(s)
Protein Kinases/metabolism , Sterol Esterase/metabolism , Adipose Tissue/enzymology , Animals , Cell Differentiation , Cells, Cultured , Cyclic AMP/pharmacology , Enzyme Activation , Hydrogen-Ion Concentration , Kinetics , Lipid Mobilization , Mice , Substrate Specificity
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