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M Karin

Publications and source records attributed to M Karin.

264 records · Page 15Linked to original sources
PubMed1980

Metallothionein mRNA induction in HeLa cells in response to zinc or dexamethasone is a primary induction response.

Metallothioneins (MTs) are low molecular weight, heavy metal binding proteins unique in their high cysteine content and high affinity for Zn2+, Cd2+, Hg2+, Ag2+ and Cu2+ (refs 1--3). The synthesis of MTs is induced by zinc or cadmium in the liver and kidney and in cultured cells. More recently MT induction by the steroid hormone dexamethasone (Dex) has been demonstrated in HeLa cells and adrenalectomized rats. Because glucocorticoid hormones lead to an intracellular accumulation of zinc, the question arises of whether the induction of MT gene expression by steroids is a 'primary induction response' (ref. 18), or due to elevated intracellular Zn2+. The glucocorticoid-induced transport of Zn2+ is dependent on concurrent protein synthesis. We now show that, in contrast to glucocorticoid-stimulated Zn2+ transport, the Zn2+ and Dex induction of translatable MT-mRNA is independent of concomitant protein synthesis but not RNA synthesis; that is, MT induction by either agent is a primary induction response.

Biological Transport↗
PubMed1980

Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells.

HeLa cells grown in chemically defined medium lacking glucocorticoids synthesize metallothioneins, low molecular-weight heavy-metal binding proteins. Dexamethasone and hydrocortisone increase the rate of metallothionein synthesis five- to ten-fold. Maximal induction is achieved with 10(-8)M dexamethasone and 10 (-7)M hydrocortisone. Half-maximal induction is achieved at 5 X 10(-9)M dexamethasone and 5 X 10(-8)M hydrocortisone. Although carried for many generations in the absence of any glucocorticoids, HeLa cells (clone S) contain 25,000 specific 3H-dexamethasone receptors that translocate into the nucleus after one hour of incubation. 3H-dexamethasone binds to a single class of receptors with an apparent Kd = 18.8 nM. A variety of steroids can be classified into three classes, based on their effect on metallothionein synthesis: (a) full agonists (optimal inducers), (b) intermediate effectors which have either partial agonist or antagonist activities, and (c) inactive steroids. There is a correlation between the effects on metallothionein synthesis of different steroids and their ability to compete with 3H-dexamethasone binding. We conclude that metallothionein is induced in HeLa cells by a glucocorticoid receptor mediated mechanism.

Dexamethasone↗
PubMed1980

Characterization of the metallothioneins induced in HeLa cells by dexamethasone and zinc.

The metallothioneins induced in HeLa cells by dexamethasone and Zn2+ were labeled with [3H]cysteine and [35S]cysteine respectively. These labeled metallothioneins were compared to one another and to metallothioneins from human liver by (a) gel permeation chromatography on Sephadex G-75 columsn, (b) ion-exchange chromatograhy on DEAE-Sephadex A-25 columns, (c) electrophoresis on non-denaturing polyacrylamide gels, and (d) electrophoresis of reduced and alkylated proteins on dodecylsulfate/polyacrylamide gels. The dexamethasone-induced metallothioneins of HeLa cells, the Zn2+-induced metallothioneins of HeLa cells, and the metallothioneins of human liver were indistinguishable by these four criteria.

Chromatography, Ion Exchange↗
PubMed1979

Dexamethasone stimulation of metallothionein synthesis in HeLa cell cultures.

HeLa cells in culture synthesize metallothionein. To investigate the effects of glucocorticoids on metallothionein synthesis we adapted HeLa cells to growth in a defined medium lacking hydrocortisone. In this defined medium, containing 1.5 x 10(-6)M Zn2+, dexamethasone (10(-6)M) caused a five- to sixfold increase in the synthesis of metallothionein. Progesterone (10(-5)M), a known antagonist of glucocorticoids, inhibited this response by 50 percent.

Biological Transport↗
PubMedDate not supplied

Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene.

Deletion experiments have defined two stretches of DNA (genetic elements), lying close to the promoter for a human gene for metallothionein, that separately mediate the induction of the gene by heavy metal ions, particularly cadmium, and by glucocorticoid hormones. The element responsible for induction by cadmium is duplicated, yet a single copy is fully functional; the element responsible for induction by glucocorticoid hormones is coincident with the DNA-binding site for the glucocorticoid hormone receptor.

Base Sequence↗
PubMedDate not supplied

Metallothionein gene cluster is split by chromosome 16 rearrangements in myelomonocytic leukaemia.

The metallothioneins (MTs) are a family of proteins of low relative molecular mass which bind heavy-metal ions. MTs exist in several molecular forms (MT-I, MT-II) and are encoded by a multi-gene family containing at least 14 closely related genes and pseudogenes. These proteins function in the regulation of trace-metal metabolism, the storage of these ions in the liver, and as a protective mechanism against heavy-metal toxicity. Somatic cell hybridization has shown that most MT genes, including the functional MT genes (MT1A, MT1B, MT2A), lie on human chromosome 16. Using in situ hybridization, we have now localized the MT genes to band q22 of chromosome 16. This chromosomal band is also a breakpoint in two specific rearrangements, the inv(16)(p13q22) and t(16; 16)(p13;q22) rearrangements, found in a subgroup of patients with acute myelomonocytic leukaemia (AMML). Hybridization of a MT probe to malignant cells from two patients with an inv(16) showed labelled sites on both arms of the inverted chromosome, indicating that the breakpoint at 16q22 splits the MT gene cluster. Similar results were obtained when this probe was hybridized to metaphase cells from two patients with a t(16; 16). These results suggest that the MT genes or their regulatory regions may function as an 'activating' sequence for an as yet unidentified cellular gene located at 16p13.

Chromosome Inversion↗
PubMedDate not supplied

Phorbol ester induces the transcriptional stimulatory activity of the SV40 enhancer.

Phorbol ester tumour promoters can induce the transcription of a number of genes, including c-myc and c-fos. These genes are part of a group referred to as 'competence' genes because they are expressed very early after quiescent cells are stimulated to enter the cell cycle. The 'competence' genes are coordinately induced by serum and by factors such as platelet-derived growth factor and fibroblast growth factor. These factors, as well as the tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), are thought to exert their action by a mechanism involving the activation of protein kinase C. It is likely that these factors induce the transcription of the 'competence' genes either by activating specific transcription factors or by increasing their intracellular concentration; either mechanism may be mediated by protein kinase C. One approach to identifying such a putative transcription factor is to characterize the cis-acting transcriptional control elements that serve as a target site for the factor. Here we report that, in a human hepatoma cell line, TPA can specifically induce the activity of the simian virus 40 (SV40) transcriptional enhancer element. Since the SV40 enhancer is a thoroughly characterized cis-acting element, this system may facilitate the eventual identification of the trans-acting factor(s) whose activity is modified by TPA treatment.

Acetyltransferases↗
PubMedDate not supplied

Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40.

Genetic analysis of eukaryotic transcriptional promoters has revealed that protein-coding genes often contain a complex array of cis-control elements consisting of upstream activator sequences and enhancer elements. The metallothionein genes provide a useful example for dissecting the action of multiple interspersed control elements that govern both basal level and regulated expression in animal cells. The human metallothionein (hMTIIA) promoter has been analysed in detail and found to contain no less than five distinct control elements in the 5' flanking regions of the gene that mediate specificity and regulation of transcription. These different control elements can be functionally subdivided into two categories: basal and induced elements. There are several distinct basal recognition sequences, which include a TATA-box, a GC-box, and at least two basal level enhancer (BLE) sequences, that function like classical enhancer elements. The hMTIIA gene also responds to induction by heavy metals and by steroid hormones through the action of metal regulatory elements (MRE) and glucocorticoid responsive elements (GRE). Here we report the identification of two cellular DNA-binding proteins that interact selectively with sequences governing the basal level expression of hMTIIA. One of these factors is a novel activator protein (AP1) that interacts with sequences in the BLE of hMTIIA and also binds to a site within the 72-base pair (bp) repeats of the simian virus 40 (SV40) enhancer region. The second protein has been purified to homogeneity and shown to be transcription factor Sp1 which recognizes and binds to a single GC-box element within the hMTIIA promoter.

Base Sequence↗
PubMedDate not supplied

Multiple cis- and trans-acting elements mediate the transcriptional response to phorbol esters.

Protein kinase C is important in the transduction of signals generated at the plasma membrane. The physiological activators of protein kinase C are diacylglycerols, and the tumour-promoting phorbol esters, such as 12-O-tetradecanoyl-phorbol-14 acetate (TPA), constitute another group of specific activators. Many cellular substrates for phosphorylation by protein kinase C have been described, but proteins that directly control transcription in response to protein kinase C activation are yet to be identified. TPA treatment leads to induction of various proto-oncogenes, growth factor genes, and genes encoding secreted proteases. In addition. TPA increases the activity of viral enhancer elements. To identify trans-acting factors that mediate the transcriptional response to TPA we chose the simian virus 40 (SV40) enhancer as a model, because it is known to be composed of several discrete cis-acting elements which are recognized by multiple transacting factors. We report here that the SV40 enhancer contains at least four different TPA responsive elements whose activity is dependent on cell-type. The induction response is likely to involve at least two distinct post-translational steps which modulate the activity of the proteins that recognize these elements.

Base Sequence↗
PubMedDate not supplied

Dissection of functional domains of the pituitary-specific transcription factor GHF-1.

The specific expression of growth hormone (GH) in the somatotrophic cells of the anterior pituitary is largely attributable to a short promoter in the 5' flanking region of the GH gene. This promoter contains two binding sites for the transcription factor GHF-1, the expression of which is also specific to cells of the somatotrophic lineage and correlates with activation of the GH gene in the developing mouse pituitary. Various studies indicate that GHF-1 is the main determinant of cell type-specific expression of the GH gene. GHF-1 is a member of the POU-domain class of proteins that each contain two highly conserved sequence motifs, the homoeodomain and the POU-specific domain. Here we report that the GHF-1 homoeodomain is sufficient for sequence-specific DNA binding, although its activity is stimulated by the POU-specific domain, which does not interact directly with the DNA. Transcriptional activation is mediated by a separate domain rich in hydroxylated amino-acid residues. Similar sequences are present in other cell type-specific transcription factors.

Amino Acid Sequence↗
PubMedDate not supplied

Promoter elements and transcriptional regulation of the acetylcholinesterase gene.

The 5' region of the acetylcholinesterase gene from the electric ray Torpedo californica has been cloned and its cap site identified. The 5' untranslated region is divided into two exons where a small exon extending between bp -22 to -60 is alternatively spliced. Cap sites are defined at two positions, bp -138 and -143. Twenty-one base pairs 5' of the -143 cap site a repeating TATA sequence is found. Further upstream in the gene consensus sequences for Sp1, AP1, and AP2 factors are evident. The promoter region of the acetylcholinesterase gene enhances transcription of a luciferase reporter gene transfected into C2 myoblasts. However, increased transcription was not evident after C2 myoblasts were induced to form myotubes. Cotransfection of this construct with c-Jun (AP1) and AP2 expression vectors shows marked increases of transcription rates in HepG2 and C2 cells. Protein kinase A elicited regulation of expression is also evident in quail fibroblasts. In gel retardation experiments both recombinant c-Jun (AP1) and AP2 proteins bind to the appropriate Torpedo sequences. Cellular extracts from the Torpedo electric organ exhibit AP2 binding activity. Thus, although all facets of specific regulation expected upon differentiation of mammalian muscle cells were not evident, the 5'-flanking region from the Torpedo AChE gene contains consensus sequences and functional promoter elements typical of mammalian nerve and muscle systems.

Acetylcholinesterase↗