Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 103
Filter
Add more filters










Publication year range
1.
Intern Med J ; 46(4): 393-403, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26247919

ABSTRACT

There is an increasing number of inherited disorders in which excessive telomere shortening underlies the molecular defect, with dyskeratosis congenita (DC) being the archetypal short telomere syndrome. DC is classically described as a mucocutaneous triad of oral leukoplakia, nail dystrophy and abnormal skin pigmentation. However, excessive telomere shortening can affect almost any organ system, so the clinical manifestations are protean, including developmental delay, cerebellar hypoplasia, exudative retinopathy, aplastic anaemia, acute myeloid leukaemia, idiopathic pulmonary fibrosis, idiopathic hepatic cirrhosis, head and neck cancer and dental abnormalities, and may be multi-systemic. Undiagnosed patients may be seen by essentially any medical subspecialist. Correct diagnosis is important to ensure appropriate management, and for initiating investigations to identify affected family members. Treatment is often supportive, with transplantation offering cure for pulmonary fibrosis or bone marrow failure. Higher rates of mortality and morbidity with transplantation often require regimen alterations, underscoring the need for correct diagnosis. Short telomeres result from mutations in genes essential for telomere maintenance (e.g. genes encoding subunits of the telomerase enzyme complex). Disease severity reflects not only the severity of the defect, but also the inheritance of short telomeres, giving rise to incomplete penetrance and genetic anticipation. Attendees of the inaugural Australian Short Telomere Syndrome Conference were updated on the current scientific and clinical understanding of these disorders, and discussed the best approach for management of these patients in the Australian context. This review will include recommendations from the conference and aims to increase awareness of short telomere disorders.


Subject(s)
Dyskeratosis Congenita/diagnosis , Dyskeratosis Congenita/genetics , Telomere Homeostasis/physiology , Australia , Congresses as Topic/trends , Dyskeratosis Congenita/therapy , Humans , Syndrome , Telomere/genetics , Telomere/metabolism
2.
Neuroscience ; 229: 49-54, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23159313

ABSTRACT

Stanniocalcin 1 (STC1), originally described as an antihypercalcemic hormone in fish, is highly expressed in differentiated mammalian neurons. Mild hypoxic treatment and focal cerebral ischemia induce upregulation of STC1 in the brain. These findings prompted us to investigate whether STC1 contributes to neuroprotection after ischemia and whether STC1 is required for development of ischemic tolerance. We induced 60 minutes of temporary middle cerebral artery occlusion in wild type (WT) and STC1-deficient mice (STC1(-/-)) with or without prior hypoxic preconditioning (HPC, 8% oxygen for 6 hours followed by reoxygenation for 24 hours). Infarct sizes, neurological scores, and Stc1, Stc2, and Il-6 mRNA brain levels were measured 24 hours after ischemia. Additionally, we examined blood-brain barrier (BBB) integrity (Evans Blue fluorescence) under normal conditions and 0 and 24 hours after hypoxia. STC1(-/-) and WT mice developed brain infarcts of similar size. In both strains, HPC triggered ischemic tolerance with similar reduction in infarct size. However, STC1(-/-) mice had worse neurological scores in both scenarios. HPC induced upregulation of STC1 and STC2 in WT mice and of STC2 in STC1(-/-) mice. Ischemic STC1(-/-) mice showed significantly lower Il-6 mRNA expression than ischemic WT mice. Evans Blue fluorescence levels showed no difference in between WT and STC1(-/-) mice under evaluated conditions, thus BBB integrity is preserved despite STC1 deficiency. STC1 was not crucial for the development of ischemic tolerance triggered by HPC or for preserving BBB integrity but may be involved in functional recovery after stroke.


Subject(s)
Brain Ischemia/metabolism , Brain/metabolism , Glycoproteins/metabolism , Infarction, Middle Cerebral Artery/metabolism , Ischemic Preconditioning , Recovery of Function/physiology , Animals , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/physiopathology , Brain/physiopathology , Brain Ischemia/genetics , Glycoproteins/genetics , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/physiopathology , Intercellular Signaling Peptides and Proteins , Interleukin-6/genetics , Interleukin-6/metabolism , Intracellular Signaling Peptides and Proteins , Mice , Mice, Knockout , Permeability , RNA, Messenger/genetics , RNA, Messenger/metabolism
3.
Oncogene ; 28(29): 2678-89, 2009 Jul 23.
Article in English | MEDLINE | ID: mdl-19483728

ABSTRACT

These investigations demonstrate that expression of the inhibitor of apoptosis family member, survivin, is dramatically increased during immortalization of nontransformed human fibroblasts that were transduced with telomerase reverse transcriptase (hTERT). Expression of survivin in immortalized fibroblasts peaked during G(2)/M phase of the cell cycle. However, the upregulation of survivin was dissociated from the rate of proliferation and proportion of G(2)/M cells. Depletion of survivin from immortal fibroblasts increased sensitivity to stress-induced apoptosis and resulted in an accumulation of cells with 4N DNA content. Conversely, overexpression of survivin in mortal fibroblasts conferred resistance to apoptosis. In contrast, very low levels of survivin in proliferating parental fibroblasts had no bearing on sensitivity to apoptosis. The upregulation of survivin did not appear to be a direct consequence of hTERT transduction. However, repression of hTERT resulted in the rapid downregulation of survivin in telomerase-immortalized fibroblasts and tumor cell lines, but not in cells immortalized via an Alternative Lengthening of Telomeres mechanism. These results have important therapeutic implications, as telomerase and survivin are both broadly expressed in human cancers. Selection during the immortalization process for cells expressing high levels of survivin may account for the abundance of survivin in diverse tumor types.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Microtubule-Associated Proteins/biosynthesis , Telomerase/metabolism , Apoptosis , Cell Line , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Fibroblasts/metabolism , Humans , Inhibitor of Apoptosis Proteins , Survivin , Telomerase/genetics , Transduction, Genetic , Up-Regulation
4.
Oncogene ; 28(18): 1982-92, 2009 May 07.
Article in English | MEDLINE | ID: mdl-19347030

ABSTRACT

Mammalian Stanniocalcin-1 (STC1) is a glycoprotein that has been implicated in various biological processes including angiogenesis. Aberrant STC1 expression has been reported in breast, ovarian and prostate cancers, but the significance of this is not well understood. Here, we report that oxidative stress caused a 40-fold increase in STC1 levels in mouse embryo fibroblasts (MEFs). STC1-/- MEFs were resistant to growth inhibition and cell death induced by H(2)O(2) or by 20% O(2) (which is hyperoxic for most mammalian cells); this is the first phenotype reported for STC1-null cells. STC1-/- cells had higher levels of activated MEK and ERK1/2 than their wild-type (WT) counterparts, and these levels were all reduced by stable expression of exogenous STC1 in STC1-/- cells. Furthermore, pharmacological inhibition by PD98059 or UO126 of MEK and therefore of ERK1/2 activation restored sensitivity of STC1-/- cells to oxidative stress. We also found that H(2)O(2)-induced STC1 expression in WT cells was abolished by inhibition of ERK1/2 activation. Thus, the ERK1/2 signaling pathway upregulates STC1 expression, which in turn downregulates the level of activated MEK and consequently ERK1/2 in a novel negative feedback loop. Therefore, STC1 expression downregulates prosurvival ERK1/2 signaling and reduces survival under conditions of oxidative stress.


Subject(s)
Feedback, Physiological , Glycoproteins/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Oxidative Stress/physiology , Signal Transduction , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Embryo, Mammalian , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/physiology , Hydrogen Peroxide/pharmacology , Mice , Mice, Knockout , Oxidative Stress/drug effects
5.
Oncogene ; 26(32): 4635-47, 2007 Jul 12.
Article in English | MEDLINE | ID: mdl-17297460

ABSTRACT

Telomerase-negative cancer cells can maintain their telomeres by a recombination-mediated alternative lengthening of telomeres (ALT) process. We reported previously that sequestration of MRE11/RAD50/NBS1 complexes represses ALT-mediated telomere length maintenance, and suppresses formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are PML bodies containing telomeric DNA and telomere-binding proteins, and are observed only in a small fraction of cells within asynchronously dividing ALT-positive cell populations. Here, we report that methionine restriction caused a reversible arrest in G0/G1 phase of the cell cycle and reversible induction of APB formation in most cells within an ALT-positive population. We combined methionine restriction with RNA interference to test whether the following proteins are required for APB formation: PML body-associated proteins, PML and Sp100; telomere-associated proteins, TRF1, TRF2, TIN2 and RAP1; and DNA repair proteins, MRE11, RAD50, NBS1 and 53BP1. APB formation was not decreased by depletion of Sp100 (as reported previously) or of 53BP1, although 53BP1 partially colocalizes with APBs. Depletion of the other proteins suppressed APB formation. Because of the close linkage between ALT-mediated telomere maintenance and ability to form APBs, the eight proteins identified by this screen as being required for APB formation are also likely to be required for the ALT mechanism.


Subject(s)
Genetic Techniques , Telomere/genetics , Telomere/metabolism , Acid Anhydride Hydrolases , Antigens, Nuclear/genetics , Antigens, Nuclear/physiology , Autoantigens/genetics , Autoantigens/physiology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/physiology , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Repair Enzymes/antagonists & inhibitors , DNA Repair Enzymes/genetics , DNA Repair Enzymes/physiology , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , G1 Phase , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , MRE11 Homologue Protein , Methionine/deficiency , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Promyelocytic Leukemia Protein , RNA Interference , RNA, Small Interfering/pharmacology , Resting Phase, Cell Cycle , Telomere-Binding Proteins/antagonists & inhibitors , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/physiology , Telomeric Repeat Binding Protein 1/antagonists & inhibitors , Telomeric Repeat Binding Protein 1/genetics , Telomeric Repeat Binding Protein 1/physiology , Telomeric Repeat Binding Protein 2/antagonists & inhibitors , Telomeric Repeat Binding Protein 2/genetics , Telomeric Repeat Binding Protein 2/physiology , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/physiology , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/physiology , Tumor Suppressor p53-Binding Protein 1
6.
Endocr Relat Cancer ; 10(3): 359-73, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14503913

ABSTRACT

Stanniocalcin (STC) is a glycoprotein hormone that is secreted by the corpuscle of Stannius, an endocrine gland of bony fish, and is involved in calcium and phosphate homeostasis. The related mammalian proteins, STC1 and STC2, are expressed in a wide variety of tissues. The ovaries have the highest level of STC1, and this increases during pregnancy and lactation. STC1 is present in breast ductal epithelium, and its expression is induced by BRCA1, a tumor suppressor gene that has an important role in breast and ovarian cancer. The expression of STC2 is induced by estrogen, and there is a positive correlation between the level of expression of estrogen receptor and expression of both STC1 and STC2 in breast cancer. This article reviews the data currently available regarding the mammalian STCs, and discusses the roles they may play in normal physiology and in breast and other cancers.


Subject(s)
Calcium/metabolism , Glycoproteins/physiology , Neoplasms/metabolism , Animals , Hormones , Humans , Intercellular Signaling Peptides and Proteins , Neoplasms/pathology , Paracrine Communication , Receptors, Estrogen/metabolism
7.
Lancet ; 361(9360): 836-8, 2003 Mar 08.
Article in English | MEDLINE | ID: mdl-12642053

ABSTRACT

Despite advances in the molecular pathogenesis of glioblastoma multiforme, no reliable prognostic markers have been identified. We analysed telomerase activity and telomere lengths in glioblastoma multiformes from 77 patients. 19 patients (25%) had tumours with the alternative-lengthening-of-telomere (ALT) phenotype. Median survival for patients with this phenotype was 542 days (95% CI 114-970) compared with 247 days (224-270) for glioblastoma multiformes with normal telomeres (p=0.0003). Cox's regression analysis showed that this association is independent of age. In patients with non-ALT tumours, telomerase activity did not affect survival (median 287 [199-375] vs 236 [230-242] days, p=0.275). We conclude that ALT is a prognostic indicator for patients with glioblastoma multiforme.


Subject(s)
Astrocytoma/enzymology , Glioblastoma/enzymology , Telomerase/metabolism , Telomere/genetics , Adult , Astrocytoma/mortality , Astrocytoma/pathology , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Middle Aged , Phenotype , Prognosis , Survival Analysis
9.
Neoplasia ; 3(2): 110-4, 2001.
Article in English | MEDLINE | ID: mdl-11420746

ABSTRACT

The mouse mot-2 protein was earlier shown to bind to the tumor suppressor protein, p53. The mot-2 binding site of p53 was mapped to C-terminal amino acid residues 312-352, which includes the cytoplasmic sequestration domain. In the present study, we have found that both mot-1 and mot-2 bind to p53 in vitro. By using His-tagged deletion mutant proteins, the p53-binding domain of mot-2 was mapped to its N-terminal amino acid residues 253-282, which are identical in mot-1 and mot-2 proteins. Some peptides containing the p53-binding region of mot-2 were able to compete with the full-length protein for p53 binding. The data provided rationale for in vitro binding of mot-1 and mot-2 proteins to p53 and supported the conclusion that inability of mot-1 protein to bind p53 in vivo depends on secondary structure or its binding to other cellular factors. Most interestingly, the p53-binding region of mot-2 was common to its MKT-077, a cationic dye that exhibits antitumor activity, binding region. Therefore it is most likely that MKT-077-induced nuclear translocation and restoration of wild-type p53 function in transformed cells takes place by a competitional mechanism.


Subject(s)
HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Amino Acids/chemistry , Animals , Binding Sites , COS Cells , Coloring Agents/pharmacology , Cytoplasm/metabolism , DNA, Complementary/metabolism , Gene Deletion , Mice , Mutation , Peptides/chemistry , Precipitin Tests , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Pyridines/pharmacology , Thiazoles/pharmacology
10.
Mol Cell Biol ; 21(12): 3862-75, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11359895

ABSTRACT

It has been shown previously that some immortalized human cells maintain their telomeres in the absence of significant levels of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Cells utilizing ALT have telomeres of very heterogeneous length, ranging from very short to very long. Here we report the effect of telomerase expression in the ALT cell line GM847. Expression of exogenous hTERT in GM847 (GM847/hTERT) cells resulted in lengthening of the shortest telomeres; this is the first evidence that expression of hTERT in ALT cells can induce telomerase that is active at the telomere. However, rapid fluctuation in telomere length still occurred in the GM847/hTERT cells after more than 100 population doublings. Very long telomeres and ALT-associated promyelocytic leukemia (PML) bodies continued to be generated, indicating that telomerase activity induced by exogenous hTERT did not abolish the ALT mechanism. In contrast, when the GM847 cell line was fused with two different telomerase-positive tumor cell lines, the ALT phenotype was repressed in each case. These hybrid cells were telomerase positive, and the telomeres decreased in length, very rapidly at first and then at the rate seen in telomerase-negative normal cells. Additionally, ALT-associated PML bodies disappeared. After the telomeres had shortened sufficiently, they were maintained at a stable length by telomerase. Together these data indicate that the telomerase-positive cells contain a factor that represses the ALT mechanism but that this factor is unlikely to be telomerase. Further, the transfection data indicate that ALT and telomerase can coexist in the same cells.


Subject(s)
RNA , Telomerase/genetics , Telomerase/metabolism , Telomere/metabolism , Cell Line , Chromosomes, Human/genetics , Chromosomes, Human/metabolism , Chromosomes, Human/ultrastructure , DNA-Binding Proteins , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Telomere/genetics , Telomere/ultrastructure , Transfection
11.
Exp Cell Res ; 265(1): 125-34, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11281650

ABSTRACT

Under standard culture conditions, normal human mammary epithelial cells (HMECs) divide a limited number of times before proliferation ceases in a growth-arrested state referred to as selection. Cells that have undergone spontaneous loss of p16(INK4a) expression due to hypermethylation of the p16(INK4a) CpG island emerge from selection and proliferate for an extended, but limited, period before senescence. Here we show, as expected, that selection was bypassed by expression of SV40 large T-antigen proteins containing an intact pRb-binding domain in preselection cells. These cells were immortalized with high efficiency (seven of nine separate cultures). Also as expected, postselection cells were immortalized by expression of the human papillomavirus-16 E6 oncoprotein (four of four cultures), which inactivates p53 protein. In contrast, we found that expression of SV40 large T-antigen protein, which also inactivates p53, was poorly maintained in postselection cultures due to its growth-suppressive effects; consequently, these cells became immortalized at low efficiency (one of 11 cultures). Reexpression of p16(INK4a) in postselection HMECs by the demethylating agent, 5-azacytidine, or transfection of a p16(INK4a) expression plasmid did not restore the ability of these cells to undergo SV40-induced transformation. Postselection HMECs are a widely used in vitro model system, but these observations indicate they have undergone changes in gene expression in addition to loss of p16(INK4a) expression.


Subject(s)
Antigens, Polyomavirus Transforming/genetics , Cell Transformation, Neoplastic , Cyclin-Dependent Kinase Inhibitor p16/genetics , Gene Expression Regulation , Repressor Proteins , Animals , Azacitidine/pharmacology , Breast/cytology , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Humans , Oncogene Proteins, Viral/genetics , Proteins/genetics , Retinoblastoma Protein/metabolism , Simian virus 40/genetics , Transfection , Tumor Suppressor Protein p14ARF
12.
J Biol Chem ; 276(22): 18649-52, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11259404

ABSTRACT

We isolated a 33-kDa protein, Pex19p/HK33/HsPXF, as a p19ARF-binding protein in a yeast two-hybrid screen. We demonstrate here that Pex19p interacts with p19ARF in the cell cytoplasm and excludes p19ARF from the nucleus, leading to a concurrent inactivation of p53 function. Down-regulation of Pex19p by its antisense expression resulted in increased levels of p19ARF, increased p53 function, and a p53/p21WAF1-mediated senescence-like cell cycle arrest. The data demonstrated a novel mechanism of down-regulation of the p19ARF-p53 pathway.


Subject(s)
Cyclins/metabolism , Genes, Tumor Suppressor , Membrane Proteins/metabolism , Membrane Proteins/physiology , Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , Animals , COS Cells , Cell Cycle , Cell Line , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cytoplasm/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Down-Regulation , Genes, Reporter , Haplorhini , Mice , Oligonucleotides, Antisense/metabolism , Phenotype , Plasmids/metabolism , Precipitin Tests , Protein Binding , Tumor Suppressor Protein p14ARF , Two-Hybrid System Techniques
13.
Radiat Res ; 155(1 Pt 2): 194-200, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11121234

ABSTRACT

Activation of a telomere maintenance mechanism appears to be essential for immortalization. In most human tumors and tumor cell lines, the telomere maintenance mechanism involves the activity of telomerase, a reverse transcriptase holoenzyme that synthesizes telomeric repeat DNA. In some cases, telomere maintenance occurs in the absence of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). The development of telomere-targeted anticancer therapies will be facilitated by an understanding of the molecular mechanisms of ALT and of the means whereby ALT is repressed in normal cells.


Subject(s)
Telomere/metabolism , Animals , Cell Line, Transformed , Cell Transformation, Neoplastic , Humans , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/metabolism , Telomerase/metabolism , Telomere/genetics
14.
Nat Genet ; 26(4): 447-50, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11101843

ABSTRACT

Telomeres of eukaryotic chromosomes contain many tandem repeats of a G-rich sequence (for example, TTAGGG in vertebrates). In most normal human cells, telomeres shorten with each cell division, and it is proposed that this limits the number of times these cells can replicate. Telomeres may be maintained in germline cells, and in many immortalized cells and cancers, by the telomerase holoenzyme (first discovered in the ciliate Tetrahymena), which uses an RNA subunit as template for synthesis of telomeric DNA by the reverse transcriptase catalytic subunit. Some immortalized human cell lines and some tumours maintain their telomeres in the absence of any detectable telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Here we show that DNA sequences are copied from telomere to telomere in an immortalized human ALT cell line, indicating that ALT occurs by means of homologous recombination and copy switching.


Subject(s)
Recombination, Genetic , Telomere/genetics , Cell Line , DNA/genetics , Humans , In Situ Hybridization, Fluorescence , Tumor Cells, Cultured
15.
Biochem Biophys Res Commun ; 279(2): 602-6, 2000 Dec 20.
Article in English | MEDLINE | ID: mdl-11118332

ABSTRACT

Mouse mortalin proteins, mot-1 and mot-2, differ by only two amino acid residues in their C-terminus. In previous studies we showed that they differ in their subcellular distributions and interactions with the tumor suppressor protein, p53. By using mot-1 deletion mutants and amino acid substitution constructs, we report here that inability of mot-1 to affect p53 activity in vivo is dependent on the presence of both of the unique mot-1 amino acids and all three of the predicted hsp70, EF hand, and leucine zipper motif regions. The two proteins and their single amino acid mutants showed different mobilities on SDS-polyacrylamide gel presenting an evidence for their different secondary structures. Taken together, the data suggest that each of the two differing amino acids between mot-1 and mot-2 is an important determinant of their secondary structures and in vivo activities.


Subject(s)
HSP70 Heat-Shock Proteins/genetics , Sequence Deletion , Transcription, Genetic , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Amino Acid Substitution , Animals , Carrier Proteins , Genes, Reporter , HSP70 Heat-Shock Proteins/chemistry , Mice , Mice, Knockout , Mice, Transgenic , Models, Molecular , Protein Structure, Secondary , Transfection
16.
Biochem Biophys Res Commun ; 276(2): 439-46, 2000 Sep 24.
Article in English | MEDLINE | ID: mdl-11027494

ABSTRACT

We report the identification and characterization of a novel human mitochondrial RNA species approximately 0.47 kb long that is transcribed from the mtDNA L-strand and is derived from the D-loop. Its expression increases when human cells become immortal, a key event in tumorigenesis. The RNA is therefore designated IDL (Immortalization-associated D-Loop). Sequence and hybrid cell analyses suggest that the increased level of IDL RNA in immortal cells is due to a recessive change, possibly in the activity of a trans-acting factor that controls IDL RNA expression.


Subject(s)
Cell Transformation, Neoplastic/genetics , DNA, Mitochondrial/genetics , Gene Expression Regulation , RNA/isolation & purification , Base Sequence , DNA, Mitochondrial/analysis , Humans , Molecular Sequence Data , Nucleic Acid Conformation , RNA/analysis , RNA/chemistry , RNA, Mitochondrial , Trans-Activators/physiology , Transcription, Genetic , Transcriptional Activation , Up-Regulation
17.
Biochem J ; 350 Pt 2: 453-61, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10947959

ABSTRACT

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with (32)P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the in vivo kinase. STC2 was phosphorylated in vitro by casein kinase II (CK2), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization-time-of-flight MS to be a single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.


Subject(s)
Fibrosarcoma/metabolism , Glycoproteins/metabolism , Hormones/metabolism , Blotting, Western , Casein Kinase II , Culture Media, Conditioned/metabolism , Electrophoresis, Polyacrylamide Gel , Glycoproteins/chemistry , Hormones/chemistry , Humans , Immunohistochemistry , In Situ Hybridization , Intercellular Signaling Peptides and Proteins , Kinetics , Phosphorylation , Protein Kinase C/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Serine/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tumor Cells, Cultured
18.
DNA Res ; 7(3): 229-31, 2000 Jun 30.
Article in English | MEDLINE | ID: mdl-10907855

ABSTRACT

The mouse mortalin proteins Mot-1 and Mot-2 differ by two amino acids in their carboxy-terminus. These proteins are differentially localized in the cell cytoplasm and have contrasting biological activities. The genetic relationship between Mot-1 and Mot-2 was deciphered by mouse family analyses. Mot-1 and Mot-2 segregated in F1 and F2 progeny, providing direct evidence that the two proteins are encoded by two alleles.


Subject(s)
Carrier Proteins/genetics , HSP70 Heat-Shock Proteins/genetics , Alleles , Animals , Cytoplasm/metabolism , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Models, Genetic , Pedigree , Polymorphism, Restriction Fragment Length , Restriction Mapping
19.
J Endocrinol ; 165(2): 457-66, 2000 May.
Article in English | MEDLINE | ID: mdl-10810309

ABSTRACT

We previously isolated a mammalian gene STC1 that encodes a glycoprotein related to stanniocalcin (STC), a fish hormone that plays a major role in calcium homeostasis. However, the mammalian STC1 gene is expressed in a variety of adult tissues in contrast to fish where STC is expressed only in one unique gland, the corpuscles of Stannius. This suggested that STC1 may have wider autocrine/paracrine functions in mammals. In the present study, using immunocytochemistry, we showed that STC1 protein is localized in the developing bone and muscle of the mouse fetus. During endochondral bone formation, STC1 is found principally in prechondrocytes and prehypertrophic chondrocytes. During intramembranous bone formation STC1 is present in the mesenchyme that is about to undergo ossification. STC1 is also found in the myocardiocytes of the developing heart and at all stages of differentiation from myoblasts to myotube formation in developing skeletal muscle. The specific localization of STC1 to chondrocytes and muscle cells suggests a role for this protein in chondrogenic and myogenic differentiation.


Subject(s)
Bone and Bones/embryology , Embryonic and Fetal Development , Glycoproteins/physiology , Hormones/physiology , Muscle, Skeletal/embryology , Animals , Bone and Bones/chemistry , Chondrocytes/chemistry , Fetal Heart/chemistry , Glycoproteins/analysis , Hormones/analysis , Immunohistochemistry , Mesoderm/chemistry , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Muscle, Skeletal/chemistry
20.
Cancer Res ; 60(3): 537-41, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10676632

ABSTRACT

The acquisition of expression of hTERT, the catalytic subunit of the telomerase enzyme, seems to be an essential step in the development of a majority of human tumors. However, little is known about the mechanisms preventing telomerase gene expression in normal and transformed cells that do not express hTERT. Using a methylation-specific PCR-based assay, we have found that the CpG island associated with the hTERT gene is unmethylated in telomerase-negative primary tissues and nonimmortalized cultured cells, indicating that mechanisms independent of DNA methylation are sufficient to prevent hTERT expression. The hTERT CpG island is methylated in many telomerase-negative and telomerase-positive cultured cells and tumors, but the extent of methylation did not correlate with expression of hTERT. Demethylation of DNA with 5-azacytidine in two cell lines induced expression of hTERT, suggesting that DNA methylation can contribute to hTERT repression in some cells. Together, these data show that the hTERT CpG island can undergo cytosine methylation in cultured cells and tumors and that DNA methylation may contribute to the regulation of the hTERT gene, but that CpG island methylation is not responsible for repressing hTERT expression in most telomerase-negative cells.


Subject(s)
CpG Islands , DNA Methylation , RNA , Telomerase/genetics , Azacitidine/pharmacology , Cells, Cultured , DNA-Binding Proteins , Humans
SELECTION OF CITATIONS
SEARCH DETAIL
...