Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 20
Filter
Add more filters











Publication year range
1.
Br J Cancer ; 88(6): 803-7, 2003 Mar 24.
Article in English | MEDLINE | ID: mdl-12644813

ABSTRACT

In this review, we discuss the available experimental evidences supporting the chemopreventive efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) on colorectal cancer and the biological basis for their possible role as anticancer agents. Although the comprehension of the mechanisms underlying the effects of these drugs on colon cancer cells is incomplete, research efforts in identifying the biochemical pathway by which NSAIDs exert their chemopreventive effect have provided a rationale for the potential use of NSAIDs alone or in combination with conventional and experimental anticancer agents in the treatment of colorectal cancer. In this paper, we review three main issues: (i) the role of COX-2 in colon cancer; (ii) the common death pathways between NSAIDs and anticancer drugs; and (iii) the biological basis for the combination therapy with COX-2 selective inhibitors and new selective inhibitors of growth factor signal transduction pathways.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/prevention & control , Isoenzymes/pharmacology , Prostaglandin-Endoperoxide Synthases/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Apoptosis , Chemoprevention , Combined Modality Therapy , Cyclooxygenase 2 , Growth Substances/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , Membrane Proteins , Signal Transduction
2.
Br J Cancer ; 86(9): 1501-9, 2002 May 06.
Article in English | MEDLINE | ID: mdl-11986787

ABSTRACT

Numerous studies demonstrate that the chemopreventive effect of non-steroidal anti-inflammatory drugs on colon cancer is mediated through inhibition of cell growth and induction of apoptosis. For these effects non-steroidal anti-inflammatory drugs have been recently employed as sensitising agents in chemotherapy. We have shown previously that treatments with aspirin and NS-398, a cyclo-oxygenase-2 selective inhibitor, affect proliferation, differentiation and apoptosis of the human colon adenocarcinoma Caco-2 cells. In the present study, we have evaluated the effects of aspirin and NS-398 non-steroidal anti-inflammatory drugs on sensitivity of Caco-2 cells to irinotecan (CPT 11) and etoposide (Vp-16) topoisomerase poisons. We find that aspirin co-treatment is able to prevent anticancer drug-induced toxicity, whereas NS-398 co-treatment poorly affects anticancer drug-induced apoptosis. These effects correlate with the different ability of aspirin and NS-398 to interfere with cell cycle during anticancer drug co-treatment. Furthermore, aspirin treatment is associated with an increase in bcl-2 expression, which persists in the presence of the anticancer drugs. Our data indicate that aspirin, but not NS-398, determines a cell cycle arrest associated with death suppression. This provides a plausible mechanism for the inhibition of apoptosis and increase in survival observed in anticancer drug and aspirin co-treatment.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/drug effects , Aspirin/pharmacology , Enzyme Inhibitors/pharmacology , Nitrobenzenes/pharmacology , Sulfonamides/pharmacology , Topoisomerase Inhibitors , Caco-2 Cells , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Cell Survival , Etoposide/pharmacology , Humans , Irinotecan
3.
Oncogene ; 19(48): 5517-24, 2000 Nov 16.
Article in English | MEDLINE | ID: mdl-11114729

ABSTRACT

Nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer and this effect is mediated in part through inhibition of type 2 prostaglandin endoperoxide synthase/ cyclo-oxygenase (COX-2). In the present study, we demonstrate that COX-2 expression and PGE2 synthesis are up-regulated by an IGF-II/IGF-I receptor autocrine pathway in Caco-2 colon carcinoma cells. COX-2 mRNA and PGE2 levels are higher in proliferating cells compared with post-confluent differentiated cells and in cells that constitutively overexpress IGF-II. Up-regulation of COX-2 expression by IGF-II is mediated through activation of IGF-I receptor because: (i) treatment of Caco-2 cells with a blocking antibody to the IGF-I receptor inhibits COX-2 mRNA expression; (ii) transfection of Caco-2 cells with a dominant negative IGF-I receptor reduces COX-2 expression and activity. Also, the blockade of the PI3-kinase, that mediates the proliferative effect of IGF-I receptor in Caco-2 cells, inhibits IGF-II-dependent COX-2 up-regulation and PGE2 synthesis. Moreover, COX-2 expression and activity inversely correlate with the increase of apoptosis in parental, IGF-II and dominant-negative IGF-I receptor transfected cells. This study suggests that induction of proliferation and tumor progression of colon cancer cells by the IGF-II/IGF-I receptor pathway may depend on the activation of COX-2-related events.


Subject(s)
Caco-2 Cells/metabolism , Dinoprostone/biosynthesis , Insulin-Like Growth Factor II/physiology , Isoenzymes/biosynthesis , Prostaglandin-Endoperoxide Synthases/biosynthesis , RNA, Messenger/biosynthesis , Receptor, IGF Type 1/physiology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Caco-2 Cells/enzymology , Cell Division/physiology , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Disease Progression , Humans , Insulin-Like Growth Factor II/antagonists & inhibitors , Insulin-Like Growth Factor II/biosynthesis , Isoenzymes/genetics , Isoenzymes/metabolism , MAP Kinase Signaling System/physiology , Membrane Proteins , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Nitrobenzenes/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/genetics , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/genetics , Signal Transduction/physiology , Sulfonamides/pharmacology , Transfection , Up-Regulation/physiology
4.
Gastroenterology ; 116(6): 1358-66, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10348819

ABSTRACT

BACKGROUND & AIMS: We have previously shown that autocrine insulin-like growth factor (IGF)-II synthesis through IGF-I receptor stimulates proliferation and inhibits differentiation of Caco-2 cells. To demonstrate whether differentiation of Caco-2 cells is dependent on cell growth status, we analyzed the effect of cell cycle arrest on differentiation of wild-type and IGF-II-overexpressing cells. METHODS: Cells were treated with drugs that inhibit the progression either to S phase (l-b-D-arabinofuranosylcytosine or M phase (nocodazole). Cell differentiation was analyzed by assessing apolipoprotein A-1 and sucrase-isomaltase expression. Cell proliferation and DNA content were assessed by thymidine incorporation and fluorescence-activated cell sorter analysis, respectively. Cell cycle regulatory molecules were analyzed by assessing p21 and retinoplasma protein (pRb) expression and pRb phosphorylation. RESULTS: Cell cycle block at G1-S phase was associated with increased expression of differentiation markers in both parental and IGF-II-transfected cells. On the contrary, cell cycle arrest at G2-M phase correlated with the expression of differentiation markers in parental but not in IGF-II-transfected cells. Constitutive IGF-II-expressing cells actively incorporated thymidine and showed an increase in the proportion of cells with >4N DNA ploidy in the presence of nocodazole. Nocodazole treatment of constitutive IGF-II-expressing cells stimulated p21 expression in the presence of hyperphosphorylated pRb. CONCLUSIONS: The data show that cell cycle arrest increases differentiation of Caco-2 cells. IGF-II-mediated proliferation may prevent cell differentiation through effects on control cell checkpoint proteins.


Subject(s)
G1 Phase/physiology , G2 Phase/physiology , Insulin-Like Growth Factor II/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Anti-Bacterial Agents/pharmacology , Caco-2 Cells , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Division/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Cytarabine/pharmacology , Drug Resistance, Microbial/genetics , Humans , Insulin-Like Growth Factor II/genetics , Neomycin/pharmacology , Nocodazole/pharmacology , Ploidies , Retinoblastoma Protein/metabolism , Transfection
5.
J Biol Chem ; 273(44): 28560-3, 1998 Oct 30.
Article in English | MEDLINE | ID: mdl-9786845

ABSTRACT

Helicobacter pylori has been suggested to play a role in the development of gastric carcinoma in humans. Also, mounting evidence indicates that cyclooxygenase-2 overexpression is associated with gastrointestinal carcinogenesis. We studied the effect of H. pylori on the expression and activity of cyclooxygenase-1 and cyclooxygenase-2 in MKN 28 gastric mucosal cells. H. pylori did not affect cyclooxygenase-1 expression, whereas cyclooxygenase-2 mRNA levels increased by 5-fold at 24 h after incubation of MKN 28 cells with broth culture filtrates or bacterial suspensions from wild-type H. pylori strain. Also, H. pylori caused a 3-fold increase in the release of prostaglandin E2, the main product of cyclooxygenase activity. This effect was specifically related to H. pylori because it was not observed with Escherichia coli and was independent of VacA, CagA, or ammonia. H. pylori isogenic mutants specifically lacking picA or picB, which are responsible for cytokine production by gastric cells, were less effective in the up-regulation of cyclooxygenase-2 mRNA expression and in the stimulation of prostaglandin E2 release compared with the parental wild-type strain. This study suggests that development of gastric carcinoma associated with H. pylori infection may depend on the activation of cyclooxygenase-2-related events.


Subject(s)
Dinoprostone/biosynthesis , Gastric Mucosa/microbiology , Helicobacter pylori/physiology , Isoenzymes/genetics , Prostaglandin-Endoperoxide Synthases/genetics , RNA, Messenger/genetics , Up-Regulation , Adenocarcinoma/microbiology , Cyclooxygenase 2 , Gastric Mucosa/enzymology , Gastric Mucosa/metabolism , Helicobacter pylori/pathogenicity , Humans , Membrane Proteins , Stomach Neoplasms/microbiology , Tumor Cells, Cultured , Virulence
6.
Int J Oncol ; 13(3): 443-7, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9683776

ABSTRACT

Detection of systemic tumor dissemination in colon carcinoma patients might be important for selection of appropriate treatment modalities. It has been previously shown that Apolipoprotein A-I (Apo A-I) is expressed in human intestinal epithelial cells, and in some human colon carcinoma cell lines. We examined the expression of Apo A-I mRNA in 14 human primary colon carcinomas by Northern blot and/or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An Apo A-I specific transcript was found in up to 70% of the colon carcinomas. We developed an RT-PCR assay for Apo A-I transcripts, to identify circulating carcinoma cells in the peripheral blood of colon cancer patients. The Apo A-I RT-PCR assay was optimized using limiting dilution of an Apo A-I positive cancer cell line mixed with peripheral blood from healthy donor. In this system, up to 10 colon carcinoma cells were detected in 5 ml of peripheral blood. We examined Apo A-I mRNA expression in peripheral blood samples from 4 healthy donors, 20 colon carcinoma patients, and 11 individuals with tumor disease other than colon cancer. No Apo A-I mRNA was detected in the healthy donors and in the patients without colon cancer. Two out of 10 patients with metastatic colon carcinoma were positive by this assay, whereas Apo A-I mRNA was not found in any of the blood samples from the 10 radically resected colon carcinoma patients. These data suggest that Apo A-I RT-PCR assay is a highly specific and sensitive assay, although a low number of advanced colon carcinoma patients was found to be positive.


Subject(s)
Apolipoprotein A-I/biosynthesis , Colonic Neoplasms/blood , Colonic Neoplasms/metabolism , Neoplastic Cells, Circulating/metabolism , RNA, Messenger/blood , RNA, Messenger/metabolism , Blotting, Northern , Caco-2 Cells/metabolism , Colon/metabolism , Colonic Neoplasms/pathology , DNA, Neoplasm/genetics , Humans , Intestinal Mucosa/metabolism , Polymerase Chain Reaction , Transcription, Genetic
7.
Int J Cancer ; 73(6): 880-4, 1997 Dec 10.
Article in English | MEDLINE | ID: mdl-9399670

ABSTRACT

Several lines of evidence suggest that long-term treatment with non-steroidal anti-inflammatory drugs may reduce the risk of colon cancer and the size and number of colonic polyps in patients with familial adenomatous polyposis. Aspirin has also been shown to inhibit cell proliferation in human tumor cell lines and to induce apoptosis in colonic mucosa of familial polyposis patients. To elucidate the molecular mechanisms of the antiproliferative action of aspirin, we studied the effects of aspirin on cell growth and differentiation of the human colon carcinoma Caco-2 cell line. These cells represent a useful tool for studying the mechanisms involved in the regulation of cell growth and differentiation of intestinal epithelial cells since they spontaneously differentiate into polarized cells, expressing brush border enzymes. We show in this study that aspirin (0.1-10 mM) induces a profound inhibition of cell replication as assessed either by cell counts or thymidine incorporation. Moreover, aspirin concentrations of 5 and 10 mM induce apoptosis, whereas concentrations of 1 and 2 mM do not. The inhibition of growth is associated with a dose-dependent reduction in insulin-like growth factor II mRNA expression and with an increase in sucrase activity (a brush border enzyme) and apolipoprotein A-I mRNA expression, 2 specific markers of the differentiative status of this cell line. Our data thus show that aspirin-dependent inhibition of cell growth is associated with the enterocyte-like differentiation of Caco-2 cells.


Subject(s)
Adenocarcinoma/pathology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , Colonic Neoplasms/pathology , Adenocarcinoma/metabolism , Apolipoprotein A-I/biosynthesis , Apoptosis , Blotting, Northern , Caco-2 Cells , Cell Differentiation/drug effects , Cell Division/drug effects , Colonic Neoplasms/metabolism , DNA/analysis , Dose-Response Relationship, Drug , Humans , Insulin-Like Growth Factor II/biosynthesis , RNA, Messenger/analysis , S Phase/drug effects , Sucrase/metabolism , Thymidine/metabolism
8.
J Biol Chem ; 271(14): 8108-14, 1996 Apr 05.
Article in English | MEDLINE | ID: mdl-8626497

ABSTRACT

In this study we have examined the role of insulin-like growth factor-II (IGF-II) in the differentiation of the CaCo-2 human colon carcinoma cell line. We have shown previously that IGF-II is an autocrine growth factor for CaCo-2 cells. IGF-II expression is high in proliferating, undifferentiated CaCo-2 cells and markedly decreases when cells become confluent and start to differentiate. To evaluate whether differentiation of CaCo-2 cells depends on an IGF-II related pathway, we treated cells with a blocking antibody to the IGF-I receptor that mediates most IGF-II biological effects. Treatment of preconfluent CaCo-2 cells with this antibody decreased by 40% autonomous cell proliferation and induced differentiation as shown by an increase in sucrase isomaltase activity and apolipoprotein A-I (apoA-I) mRNA levels. To examine the significance of autocrine IGF-II production in CaCo-2 cell differentiation, we generated stable CaCo-2 cell lines that constitutively express rat IGF-II under the control of a Rous sarcoma virus promoter. Sustained expression of IGF-II resulted in: (a) increased proliferative rate; (b) high IGF-I receptor number, even after reaching confluence; (c) increased capability of anchorage-independent growth; (d) inhibition of the expression of apoA-I and SI mRNAs. Analysis of several independent IGF-II-transfected clones showed an inverse correlation between IGF-II mRNA levels and expression of the differentiation markers, the cells expressing the higher levels of the transfected IGF-II being the less differentiated ones. Our data suggest that perturbation of IGF-II-mediated cell proliferation interferes with the enterocyte-like differentiation pathway of CaCo-2 cells.


Subject(s)
Insulin-Like Growth Factor II/physiology , Intestinal Mucosa/cytology , Apolipoprotein A-I/metabolism , Caco-2 Cells , Cell Adhesion , Cell Differentiation , Cell Division , Gene Expression , Humans , RNA, Messenger/genetics , Receptor, IGF Type 1/physiology , Sucrase-Isomaltase Complex/metabolism
9.
Endocrinology ; 136(8): 3179-85, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7628349

ABSTRACT

19S Thyroglobulin (Tg), a dimeric glycoprotein with an M(r) of 660,000, was extracted from rat, bovine, and human (goitrous) thyroid tissues, as well as from culture medium of FRTL5 rat thyroid cells. Subjected to rigorous purification procedures, all Tg preparations showed a protein kinase activity that is able to phosphorylate serine residues in vitro. Further characterization of this enzymatic activity revealed that Tg has the specificities of a protein kinase A when Kemptide was used as specific substrate and after analysis of cAMP-stimulated phosphotransferase activity in all Tg preparations tested. Furthermore, Tg contains a specific and saturable ATP binding site, as evidenced by specific binding with the ATP affinity analog p-fluorosulfonylbenzoyl 5'-p-adenosine (FSO2BzAdo). Limited proteolysis of FSO2[14C]BzAdo-labeled human goiter Tg with clostripain gave rise to a M(r) 64,000 amino terminal polypeptide carrying almost all of the label. The analysis of this fragment revealed two sequences that may play an analogous phosphate-loop (P-loop) function, even though their primary sequences differ slightly from the classical A consensus sequence or P-loop. The ATP binding site of Tg may be functionally associated with the presence of a protein kinase A phosphorylating activity linked to the Tg itself, because the addition of the analog is able to inhibit the enzymatic activity in a dose-dependent fashion. Contamination with non-Tg phosphorylase cannot be ruled out at present, even though the amount of ATP that Tg was able to bind did not significantly change during the various purification steps. Together these findings favor the hypothesis that the Tg molecule contains a protein kinase activity that is capable of autophosphorylation.


Subject(s)
Adenosine Triphosphate/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , Thyroglobulin/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Goiter/metabolism , Humans , Molecular Sequence Data , Molecular Weight , Oligopeptides/metabolism , Phosphorylation , Rats , Thyroglobulin/chemistry , Thyroglobulin/genetics
10.
Cell Growth Differ ; 5(10): 1085-91, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7848910

ABSTRACT

We have studied the expression of insulin-like growth factor type II (IGF-II) and its autocrine role during the proliferation and differentiation of the CaCo-2 colon carcinoma cell line. IGF-II RNA levels were high in proliferating cells and decreased by more than 10-fold when cells ceased to proliferate and differentiated. Immunoreactive IGF-II protein was high in the conditioned media of proliferating cells and decreased 20-fold in the media of differentiated cells. Reduced IGF-II expression was associated with a decrease in IGF-I receptor number that was high in proliferating cells (approximately 80,000 binding sites/cell) and reduced by 4-fold in differentiated cells. Exogenously added IGF-II was able to stimulate proliferation of serum-deprived cells in a dose-dependent fashion. IGF-II acted through the IGF-I receptor, since both basal and IGF-II-stimulated cell proliferation was inhibited by the monoclonal antibody alpha-IR3, which blocks the binding sites of the IGF-I receptor. The inhibition of CaCo-2 basal cell growth by the alpha-IR3 antibody suggests that IGF-II may act as an autocrine growth factor for these cells.


Subject(s)
Colonic Neoplasms/metabolism , Receptor, IGF Type 1/biosynthesis , Receptor, IGF Type 2/biosynthesis , Antibodies, Monoclonal , Basal Metabolism , Base Sequence , Cell Differentiation/physiology , Cell Division/drug effects , Cell Division/physiology , Colonic Neoplasms/pathology , Humans , Insulin-Like Growth Factor II/pharmacology , Molecular Sequence Data , Tumor Cells, Cultured
11.
Cell Growth Differ ; 5(9): 967-73, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7819134

ABSTRACT

We have investigated the expression of protein kinase C (PKC) and protein kinase A (PKA) during the phases of growth and differentiation of the human colon carcinoma Caco-2 cells. We studied whether differentiation correlated with the responsiveness to cAMP and with an increased transport of the catalytic subunit of PKA into the nucleus. Also, we evaluated whether this phenomenon was affected by PKC activity. High levels of activated PKC were found in the plasma membranes of replicating cells. When the cells began to differentiate, plasma membrane-activated PKC decreased, while the cytosolic fraction increased. On the contrary, PKA holoenzyme increased during differentiation, along with the transport of its catalytic subunit into the nucleus. Both types I and II kinase A holoenzymes increased during differentiation, with maximal type II activity found when cells were fully differentiated. In replicating preconfluent cells, the inhibition of PKC by high dose phorbol 12-myristate 13-acetate or sphingosine increased the amount of both PKA catalytic subunit in the nucleus and sucrase activity. During differentiation, 8-Bromo-cAMP increased PKA catalytic subunit in the nucleus and apoliprotein A1 mRNA levels. These effects were inhibited by low-dose phorbol 12-myristate 13-acetate, which activates PKC in the plasma membranes. Our data suggest that PKC is activated in proliferating Caco-2 cells. The inhibition of PKC induces the transport of PKA catalytic subunit into the nucleus and the expression of the differentiation markers. Differentiated Caco-2 cells show a lower activation of PKC and an increased transport of the catalytic subunit of PKA into the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Colonic Neoplasms/pathology , Cyclic AMP-Dependent Protein Kinases/drug effects , Cyclic AMP/pharmacology , Intestinal Mucosa/pathology , Amino Acid Sequence , Biomarkers, Tumor/metabolism , Catalysis , Cell Differentiation/physiology , Cell Division/drug effects , Cell Nucleus/enzymology , Cyclic AMP-Dependent Protein Kinase Type II , Enzyme Activation , Humans , Molecular Sequence Data , Tumor Cells, Cultured
12.
Mol Cell Biol ; 12(12): 5373-85, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1448072

ABSTRACT

Transcription of the human erythropoietin (Epo) gene is stimulated by exposure to hypoxia and/or cobalt in whole animals and in Hep3B cells. We have systematically investigated the promoter and 3' enhancer elements necessary for this induction by transient transfection of Hep3B cells. We define a promoter region of 53 bp and an enhancer region of 43 bp that confer hypoxia and cobalt inducibility. Each element gives rise to a 6- to 10-fold induction alone. In combination they produce a 50-fold induction after stimulation, similar to the 50- to 100-fold induction of the endogenous Epo gene. Two areas of DNA sequence homology are present in these regions. We demonstrate specific DNA-protein interactions in the enhancer and the ability of the promoter element to compete with these interactions in electrophoretic mobility shift assays. DNase I footprinting and methylation interference data further refine the cis-acting element in the 43-bp enhancer to a short region containing a direct repeat of a steroid/thyroid hormone receptor response element half-site separated by a 2-bp gap. Two half-site consensus sequences are also present in the 53-bp promoter. Site-specific mutation of the half-site sequences in the enhancer destroys the functional activity of the enhancer.


Subject(s)
Enhancer Elements, Genetic , Erythropoietin/genetics , Gene Expression Regulation , Oxygen/metabolism , Promoter Regions, Genetic , Receptors, Steroid/metabolism , Base Sequence , Binding, Competitive , Cell Line , DNA/metabolism , Deoxyribonuclease I , Erythropoietin/metabolism , Humans , Methylation , Molecular Sequence Data , Organ Specificity/genetics , Sequence Homology
14.
J Biol Chem ; 262(21): 10304-14, 1987 Jul 25.
Article in English | MEDLINE | ID: mdl-2440862

ABSTRACT

Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.


Subject(s)
Phosphates/analysis , Thyroglobulin/analysis , Animals , Carbohydrates/analysis , Cattle , Cell Line , Chromatography, Gel , Chromatography, High Pressure Liquid , Humans , Mannose/metabolism , Methionine/metabolism , Phosphoserine/analysis , Phosphotyrosine , Pronase/metabolism , Rats , Rats, Inbred F344 , Thyroid Gland/analysis , Thyroid Gland/drug effects , Tunicamycin/pharmacology , Tyrosine/analogs & derivatives , Tyrosine/analysis
15.
Diabetes ; 31(7): 656-8, 1982 Jul.
Article in English | MEDLINE | ID: mdl-6186554

ABSTRACT

Total RNA extracted from a rat liver cell line (BRL 3A) that synthesizes rat insulin-like growth factor II (IGF-II) was translated in a reticulocyte lysate cell-free system. Incubation of the translation products with antisera to mature rat IGF-II (Mr 8700) selectively immunoprecipitated a Mr 21,600 protein. We propose that this protein represents pre-pro-IGF-II.


Subject(s)
Insulin/biosynthesis , Peptide Biosynthesis , Somatomedins/biosynthesis , Animals , Cell Line , Electrophoresis, Polyacrylamide Gel , Immunologic Techniques , Liver , Molecular Weight , RNA/metabolism , Rats
16.
Gene ; 19(1): 117-25, 1982.
Article in English | MEDLINE | ID: mdl-6292044

ABSTRACT

Two plasmids containing rat thyroglobulin cDNA sequences have been constructed and characterized. A plasmid with a 500-bp insert (pRT6) was isolated and identified as thyroglobulin-specific on the basis of the tissue specificity of the inserted sequence and of its ability to retain thyroglobulin mRNA on a nitrocellulose filter. The cDNA insert in pRT6 was subsequently used to screen a rat thyroid cDNA library constructed with large cDNA. A plasmid was found containing a 1700-bp insert. The polarity and the fidelity of the insert is demonstrated by S1 mapping.


Subject(s)
Cloning, Molecular , DNA, Recombinant , Plasmids , RNA, Messenger/genetics , Thyroglobulin/genetics , Animals , Base Sequence , DNA , DNA Restriction Enzymes , DNA Transposable Elements , Male , Nucleic Acid Hybridization , Rats , Rats, Inbred Strains , Thyroid Gland/metabolism
17.
Proc Natl Acad Sci U S A ; 79(5): 1525-9, 1982 Mar.
Article in English | MEDLINE | ID: mdl-6280188

ABSTRACT

Thyrotropin increases the ADP-ribosylation activity of bovine thyroid membranes. Rapid ADP-ribosylation of membrane components is followed by increasing ADP-ribosylation of components in the supernatant of the reaction mixture. One of the major membrane proteins ADP-ribosylated in the thyrotropin-stimulated reaction has an approximate molecular weight of 40,000; this same protein is also a major ADP-ribosylated product of the A promoter of cholera toxin and appears to be related to the G regulatory subunit of the adenylate cyclase complex. The ADP-ribosylated products appearing in the supernatant solution comigrate with thyrotropin and preparations of 125I-labeled alpha subunit of thyrotropin; the alpha subunit, but not the beta subunit, of thyrotropin can be ADP-ribosylated by the membrane ADP-ribosyltransferase activity. NAD can be shown to enhance the ability of thyrotropin to stimulate the adenylate cyclase activity of bovine thyroid membrane preparations and of membrane preparations of a rat thyroid tumor whose adenylate cyclase activity is otherwise unresponsive to thyrotropin. The beta subunit of thyrotropin inhibits thyrotropin stimulation of both the ADP-ribosylation and adenylate cyclase activities of the thyroid membrane.


Subject(s)
Adenylyl Cyclases/metabolism , Nucleotidyltransferases/metabolism , Thyroid Gland/enzymology , Thyrotropin/pharmacology , Animals , Cattle , Cell-Free System , Cholera Toxin/pharmacology , Enzyme Activation/drug effects , NAD/metabolism , Poly(ADP-ribose) Polymerases
18.
C R Seances Soc Biol Fil ; 172(6): 1085-93, 1978.
Article in French | MEDLINE | ID: mdl-159095

ABSTRACT

In vitro, transcription by thyroid chromatin has been compared to that carried out by deproteinized DNA prepared from the same tissue. The chromatin-primed transcripts are represented by 4S and 10S RNAs, whereas the DNA-primed transcript is heterogenous and polydisperse. The sequences expressed in vitro have been analyzed by hydridization of 4S and 10S RNA to the total native thyroid DNA. Most of the sequences transcribed are represented by intermediate repetitive-associated an unique sequence. Furthermore, they are expressed only by chromatin and not by DNA. Thus, thyroid chromatin very likely holds in vitro the same fonctional restriction exhibited in vivo, whereas the transcriptional behaviour of thyroid DNA is totally aspecific.


Subject(s)
Chromatin/physiology , DNA/physiology , Thyroid Gland/metabolism , Transcription, Genetic , Animals , Chromatin/isolation & purification , DNA/isolation & purification , Nucleic Acid Hybridization , RNA/biosynthesis , RNA/isolation & purification , Swine , Thyroid Gland/ultrastructure
19.
Eur J Biochem ; 78(1): 1-10, 1977 Aug 15.
Article in English | MEDLINE | ID: mdl-913390

ABSTRACT

Purified pig thyroid chromatin has been transcribed in vitro with Escherichia coli RNA polymerase. The transcript, analyzed by DNA-RNA hybridization, shows two major kinetic components: 40% of the transcript is copied by repetitive sequences present 100 times per haploid genome; another 25% anneals to DNA with a rate constant Kh 10-4 M - S-1, typical of single-copy sequences. The transcript annealed at cot = 40 M - S to fractions of 2000-nucleotide DNA, when banded in neutral CsCl gradient only hybridizes to the heavy side of the main band. At cot = 3000 M - S, another hybridizing fraction appears on the light side of the main band of the gradient. The reassociation properties of these fractions show that the heavy DNA fraction is reiterated about 100 times per haploid genome, whereas the light DNA appears as a unique sequence, associated to small repetitive elements. The transcript, analyzed by formamide/sucrose gradient, shows two peaks with sedimentation coefficients of 10 S and 4 S, respectively. The 10-S RNA, hybridized to native 2000-nucleotide-length DNA, has a Kh of 10-4 M - S-1 and a cot1/2 of 10(3) M - S, typical of single-copy sequences.


Subject(s)
Chromatin , DNA , Thyroid Gland/metabolism , Transcription, Genetic , Animals , Base Sequence , Chromatin/metabolism , DNA/metabolism , DNA-Directed RNA Polymerases/metabolism , Deoxyribonucleases/metabolism , Kinetics , Molecular Weight , Nucleic Acid Hybridization , Nucleic Acid Renaturation , Swine , Temperature
20.
Nucleic Acids Res ; 3(10): 2491-505, 1976 Oct.
Article in English | MEDLINE | ID: mdl-995641

ABSTRACT

The sequence organization of porcine DNA isolated from thyroid has been analyzed by hydroxylapatite (HAP) chromatography. The reassociation of 0.4 kilobase (Kb) DNA fragments shows, besides the presence of 5% inverted repeat sequences (foldback DNA), that 45% of the genome is represented by high (10%) and intermediate (35%) repetitive components, whereas the remaining 50% is unique sequences. 30% of the unique sequences consists of 1,000 nucleotide fragments interspersed with repetitive elements 400 nucleotides in length. The remaining 20% is longer unique sequences (10,000 nucleotides) apparently not linked to repetitive elements.


Subject(s)
DNA/analysis , Thyroid Gland/analysis , Animals , Base Sequence , Kinetics , Molecular Weight , Nucleic Acid Renaturation , Swine
SELECTION OF CITATIONS
SEARCH DETAIL