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1.
EMBO J ; 18(19): 5389-98, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10508171

ABSTRACT

We have investigated ligand-dependent negative regulation of the thyroid-stimulating hormone beta (TSHbeta) gene. Thyroid hormone (T3) markedly repressed activity of the TSHbeta promoter that had been stably integrated into GH(3 )pituitary cells, through the conserved negative regulatory element (NRE) in the promoter. By DNA affinity binding assay, we show that the NRE constitutively binds to the histone deacetylase 1 (HDAC1) present in GH(3 )cells. Significantly, upon addition of T3, the NRE further recruited the thyroid hormone receptor (TRbeta) and another deacetylase, HDAC2. This recruitment coincided with an alteration of in vivo chromatin structure, as revealed by changes in restriction site accessibility. Supporting the direct interaction between TR and HDAC, in vitro assays showed that TR, through its DNA binding domain, strongly bound to HDAC2. Consistent with the role for HDACs in negative regulation, an inhibitor of the enzymes, trichostatin A, attenuated T3-dependent promoter repression. We suggest that ligand-dependent histone deacetylase recruitment is a mechanism of the negative-feedback regulation, a critical function of the pituitary-thyroid axis.


Subject(s)
Feedback , Histone Deacetylases/metabolism , Thyrotropin/genetics , Base Sequence , Chromatin/chemistry , Cyclic AMP/pharmacology , DNA , Gene Expression Regulation/drug effects , Genes, Reporter , Ligands , Protein Binding , Regulatory Sequences, Nucleic Acid , Sequence Deletion , Sequence Homology, Nucleic Acid , Thyrotropin/metabolism , Triiodothyronine/pharmacology
2.
Cancer Lett ; 147(1-2): 163-73, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10660102

ABSTRACT

Antisense phosphorothioate oligodeoxynucleotides (ODNs) are increasingly used to target specific proteins for inhibition. Previous reports of antisense inhibition of the inducible nitric oxide synthase (iNOS) gene suggested its utility in defining the role of nitric oxide (NO) in carcinogenesis, as NO is mutagenic and chemical inhibitors of iNOS block neoplastic transformation in C3H 10T1/2 fibroblasts. Treatment with ODNs (0.025-25 microM) directed against 15mer sequences in the iNOS coding region decreased NO production consistent with a reduction of iNOS protein and iNOS mRNA, however, control ODNs (2.5 microM) also showed considerable nonspecific inhibition of NO synthesis. Treatment with both iNOS antisense and missense ODNs during the promotional phase of the C3H10T1/2 transformation assay significantly increased the number of neoplastic foci in 3-methylcholanthrene (MCA) treated cells which corresponded with the ability of the ODN to inhibit NO production. Enhanced neoplastic transformation and non-specific inhibition of NO synthesis resulting from exposure to antisense ODNs suggest limitations to their long-term use in humans at higher doses.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cell Transformation, Neoplastic/drug effects , Fibroblasts/drug effects , Oligonucleotides, Antisense/pharmacology , Thionucleotides/pharmacology , Animals , Base Sequence , Blotting, Western , Carcinogens , Cell Line , Cell Transformation, Neoplastic/chemically induced , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/genetics , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Fibroblasts/cytology , Fibroblasts/enzymology , Interferons/pharmacology , Lipopolysaccharides/pharmacology , Methylcholanthrene , Mice , Mice, Inbred C3H , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Oligonucleotides, Antisense/genetics , RNA, Messenger/metabolism , Thionucleotides/genetics , Transfection , Tumor Stem Cell Assay
3.
Cancer Lett ; 122(1-2): 67-75, 1998 Jan 09.
Article in English | MEDLINE | ID: mdl-9464493

ABSTRACT

Tamoxifen (TAM) is used in the prevention and treatment of breast cancer, however, its mechanisms of therapeutic action as well as its pathologic effects are not fully understood. We report that TAM (10(-7)-10(-5) M) inhibits 3-methylcholanthrene-induced transformation of C3H 10T1/2 murine fibroblasts in a dose-responsive manner. Over this concentration range, TAM (>10(-6) M) potentiates inducible nitric oxide synthase (iNOS) activity in 10T1/2 cells. This increase in NO synthase activity was mediated through an increase in iNOS protein for cells stimulated with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). Significant increases in NO formation were observed when TAM (10(-5)) was added prior to or simultaneously with IFN-gamma/LPS treatment, whereas the addition of TAM 48 h after IFN-gamma/LPS treatment had no effect on NO synthesis. The morphologic changes seen with cells treated with TAM are similar to those observed in cells treated with TGF-beta1. TGF-beta1 inhibited NO production at high doses and slightly enhanced NO formation at low doses in IFN-gamma/LPS-stimulated cells. The transformation inhibitory effects of TAM did not appear to be related to the effects on cellular proliferation of neoplastic cells as TAM did not inhibit the growth of neoplastic cells into foci in the presence of normal confluent C3H 10T1/2 fibroblasts.


Subject(s)
Cell Transformation, Neoplastic/drug effects , Estrogen Antagonists/pharmacology , Nitric Oxide/biosynthesis , Tamoxifen/pharmacology , Animals , Mice , Mice, Inbred C3H
4.
Mol Cell Biol ; 16(8): 4366-77, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8754837

ABSTRACT

Expression of the human thyrotropin beta (hTSHbeta) gene is restricted to thyrotrophs, at least in part, by silencing. Using transient-transfection assays, we have localized a silencer element to a region between -128 and -480 bp upstream of the transcription initiation site. The silencing activity was overcome in a thyrotroph-specific manner by an unknown enhancer located in the sequences at -approximately 10000 to -1200 bp. The ubiquitous POU homeodomain protein Oct-1 recognized the A/T-rich silencer element at multiple sites in gel mobility shift assays and in vitro footprinting analyses. The silencing activity of Oct-1 was localized in its C-terminal alanine-rich domain, suggesting that Oct-1 plays a role in silencing of the hTSHbeta promoter. Further, a significant fraction of Oct-1 was shown to be associated with the nuclear matrix, and the hTSHbeta silencer region was tethered to a nuclear matrix of human cells in vivo, suggesting a possible role of the Oct-1-hTSHbeta silencer region interaction in chromatin organization.


Subject(s)
DNA-Binding Proteins , Homeodomain Proteins/metabolism , Nuclear Matrix/metabolism , Promoter Regions, Genetic , Thyrotropin/genetics , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Cell Compartmentation , Cells, Cultured , Down-Regulation , Gene Expression Regulation , Host Cell Factor C1 , Humans , Male , Mice , Molecular Sequence Data , Octamer Transcription Factor-1 , Oligodeoxyribonucleotides/chemistry , RNA, Messenger/genetics , Rats , Solubility , Structure-Activity Relationship
5.
Hum Gene Ther ; 6(4): 395-405, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7612697

ABSTRACT

We report on an in vivo delivery system that attenuates the growth, in nude mice, of a malignant human breast cancer cell line containing a p53 mutation. Nude mice, inoculated with breast carcinoma cells, were injected every 10-12 days with a liposome-p53 complex via the tail vein. A significant reduction of greater than 60% in primary tumor volume was observed as compared to the control groups. Furthermore, when individual growth patterns of the tumors were assessed, we found that primary tumor size regressed in the majority of p53-treated animals (8/15), whereas only one tumor in the control groups (1/22) regressed. The eight tumors that regressed with the liposome-p53 complex showed no evidence of relapse for 1 month after the cessation of treatment. We also determined that the administration of the liposome-p53 complex reduced the incidence of metastases. The MDA-MB-435 tumor cells, transduced with the lacZ gene, facilitated quantitation of beta-galactosidase activity and tumor burden in the lungs. The number of metastatic cells in the lung was significantly lower in the p53-treated group (0.53 +/- 0.43 x 10(6), p < 0.01) than in either the vector-treated (8.1 +/- 3.7 x 10(6)) or untreated control groups (15.8 +/- 5.9 x 10(6)). Thus, systemic administration of the liposome-p53 complex reduced not only the size of the primary tumors but, more importantly, prevented the relapse and metastases of these tumors.


Subject(s)
Breast Neoplasms/therapy , Carcinoma/secondary , Genes, p53 , Genetic Therapy , Lung Neoplasms/secondary , Animals , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma/prevention & control , Carcinoma/therapy , Disease Progression , Genes, Reporter , Humans , Injections, Intravenous , Liposomes , Lung Neoplasms/prevention & control , Mice , Mice, Nude , Molecular Sequence Data , Neoplasm Transplantation , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Tumor Cells, Cultured/transplantation , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/physiology , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
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