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Biomedical subjects

M Szyf

Publications and source records attributed to M Szyf.

At least 55 records · Page 3Linked to original sources
PubMed1995

Regulation of the DNA methyltransferase by the Ras-AP-1 signaling pathway.

Using deletion analysis and site-specific mutagenesis to map the 5' regulatory region of the DNA methyltransferase (MeTase) gene, we show that a 106-bp sequence (at -1744 to -1650) bearing three AP-1 sites is responsible for induction of DNA MeTase promoter activity. Using transient cotransfection chloramphenicol acetyl-transferase assays in P19 cells, we show that the DNA MeTase promoter is induced by c-Jun or Ha-Ras but not by a dominant negative mutant of Jun, delta 9. The activation of the DNA MeTase promoter by Jun is inhibited in a ligand dependent manner by the glucocorticoid receptor. Stable expression of Ha-Ras in P19 cells results in induction of transcription of the DNA MeTase mRNA as determined by nuclear run-on assays and the steady state levels of DNA MeTase mRNA as determined by an RNase protection assay. These experiments establish a potential molecular link between nodal cellular signaling pathways and the control of expression of the DNA MeTase gene. This provides us with a possible molecular explanation for the hyperactivation of DNA MeTase in many cancer cells and suggests that DNA MeTase is one possible downstream effector of Ras.

Animals↗
PubMed1994

DNA methylation properties: consequences for pharmacology.

DNA methylation plays an important role in controlling the profile of gene expression of mammalian cells. The hypothesis presented in this article by Moshe Szyf is that DNA methylation patterns are determined by an interplay between the level of DNA methyltransferase and demethylase activities and site-specific signals. The expression of the DNA methyltransferase gene is regulated with the proliferative state of the cell and it is upregulated by cellular oncogenic pathways, resulting in hypermethylation and repression of tumour-suppressing loci. DNA methyltransferase inhibitors would inhibit the excessive activity of DNA methyltransferase in cancer cells and induce the original cellular programme of tumour suppression. They can also be used to turn on alternative programmes of gene expression. Specific DNA methyltransferase antagonists might provide us with therapeutic agents directed at a nodal point of regulation of genetic information.

DNA↗
PubMed1993

Functional nicotinic receptor expression in mesodermal cells transfected with MyoD cDNA.

Previous studies had shown that MyoD promoted nicotinic acetylcholine subunit gene expression; the present experiments were done to determine whether this subsequently led to the development of functional nicotinic acetylcholine receptors. Transfection of C3H 10T1/2 cells with MyoD cDNA resulted in the appearance of [125I]alpha-bungarotoxin binding sites; radiolabelled alpha-toxin binding was not observed in cells transfected with a plasmid that lacked MyoD cDNA. Receptor development plateaued over a time course of several days with maximal binding seven and 11 days after exposure to fusion medium. [125I]alpha-bungarotoxin binding was of high affinity (Kd = 1 nM), saturable and was inhibited by nicotinic but not muscarinic receptor ligands, with IC50s of 1-3 nM for alpha-bungarotoxin, 1-3 microM for d-tubocurarine and 3-10 microM for nicotine. Not only did the cells exhibit a cell surface nicotinic receptor but they also expressed a nicotinic receptor mediated functional response. Carbachol resulted in uptake of 22Na into the cells at concentrations similar to those required for receptor activation at a muscle type nicotinic receptor; furthermore, the functional response was effectively blocked by nicotinic receptor ligands, including alpha-bungarotoxin (IC50 = 2 to 6 nM) and d-tubocurarine (IC50 = 0.1 to 0.4 microM); muscarinic receptor ligands had no effect. A time course study showed that alpha-bungarotoxin binding and carbachol stimulated 22Na uptake developed in parallel, suggesting that the observed functional response was mediated through an interaction at the alpha-bungarotoxin recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

An element regulating adrenal-specific steroid 21-hydroxylase expression is located within the slp gene.

In this report we demonstrate that a transcriptional regulatory element for one gene lies within a second, seemingly unrelated gene. Specifically, the 3' portion of the murine sex-limited protein (slp) gene, located within the class III region of the major histocompatibility complex, contains an element that regulates expression of the linked steroid 21-hydroxylase gene. A 4.2-kilobase (kb) major histocompatibility complex region, located between -2.2 and -6.4 kb upstream of 21OH-A, is required for expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Two short regions of DNA, located between -5.3 and -6.0 kb, stimulate chloramphenicol acetyltransferase expression in Y1 adrenocortical tumor cells, and both of these active regions lie within the slp gene. A 21-base pair sequence, which is required for activity of the most 3' region, does not contain any of over 100 previously identified transcriptional regulatory elements. This juxtaposition of structural and regulatory elements of otherwise unrelated genes suggests a mechanism by which the evolutionarily conserved genetic linkage of 21OH-A and slp (or the homologous complement component C4) might provide a selective advantage. Analogous genetic arrangements may explain other examples of conserved linkage of disparate genes.

Adrenal Glands↗
PubMed1992

Induction of myogenic differentiation by an expression vector encoding the DNA methyltransferase cDNA sequence in the antisense orientation.

To test the hypothesis that DNA methylation controls the state of differentiation of a mammalian cell, we transfected the stable mesenchymal line 10T1/2 with an expression vector encoding sequences from the DNA methyltransferase (DNA MeTase) cDNA in the antisense orientation. 10T1/2 cells transfected with the antisense construct (pZ alpha M), but not with the vector alone, exhibit morphological changes, convert into multinucleated tubular cells, and express the skeletal myosin heavy chain protein. The conversion to myogenic phenotype is a late event and is dependent on the number of replication events that the cell has undergone, suggesting that induction of myogenesis is a multistep process. Demethylation of sequences that are not involved in the myogenic process is detected at early passages, while demethylation and expression of the MyoD gene is a late event. This report establishes for the first time that demethylation is a very early event in commitment to myogenic differentiation, while demethylation and expression of MyoD is a late event. We suggest that other genes serve as the initial targets for demethylation and commitment of mesenchymal cells to myogenesis. The cell lines described in this report can serve as an important system for identifying these genes.

Animals↗
PubMed1992

The mouse DNA methyltransferase 5'-region. A unique housekeeping gene promoter.

We have cloned and characterized 5'-flanking sequences of the DNA methyltransferase (MeTase) gene. DNA MeTase gene transcription is initiated at a few discrete sites: 343 and 90 base pairs upstream of the translation initiation site as determined by RNase protection and primer extension assays. The promoter sequences that regulate expression of DNA MeTase, as defined by chloramphenicol acetyltransferase assays, reside between position -171 and the transcription start site. The promoter of DNA MeTase does not contain TATAA or CAAT boxes and is unusual because it does not contain the CG-rich elements characteristic of TATAA-less housekeeping genes. The 5'-flanking region of DNA MeTase contains AP-1, AP-2 and glucocorticoid response elements, suggesting possible regulation by cellular signal transduction pathways. The base composition of the DNA MeTase promoter is markedly different from that of other housekeeping genes. Whereas most housekeeping genes are characterized by CG-rich areas in their 5'-flanking regions, the TG dinucleotide is over-represented in DNA MeTase 5'-flanking sequences, including a perfect tandem repeat of T/G between positions -685 and -650. DNA methylation patterns play an important role in the developmental regulation of gene expression in vertebrates. DNA MeTase activity is probably regulated to maintain this pattern of methylation. We suggest that the DNA MeTase promoter represents a new class of housekeeping gene promoters that was designed to ensure high fidelity regulation of gene expression.

3T3 Cells↗
PubMed1992

Status inequality in the social worker-nurse collaboration in hospitals.

An Israeli pilot study among 34 social worker-nurse teams (team members working in the same hospital ward) was aimed at elucidating the conditions for promoting a milieu of collaboration between them. The data suggest that this collaboration is characterized by status-inequality, the nurse's dominance prevailing with respect to meeting the patient's psychosocial needs. Nurses view social workers mainly as fulfilling chores relating to the patients' instrumental needs that emanate from outside the hospital, while social workers, though overtly objecting to the nurses' dominance, still view the milieu as collaborative. Explanations are offered for this apparent contradiction. Possible implications are suggested regarding the consequences both for effectively meeting the patients' psychosocial needs and for the social workers' professional status in hospitals.

Adult↗
PubMed1992

Conditional transformation mediated via a pertussis toxin-sensitive receptor signalling pathway.

To determine whether a cloned receptor coupled to pertussis toxin (PTx)-sensitive G-proteins can induce cell proliferation and oncogenic transformation, as observed for receptors that elicit PTx-insensitive enhancement of phosphatidyl inositol (PI)-specific phospholipase-C (PLC) activity, nontransformed murine BALB/c-3T3 cells were transfected with the rat serotonin-1A (5-HT1A) receptor. The 5-HT1A receptor is coupled to PTx-sensitive G-proteins to induce a cell-specific activation of PLC. While 1 microM 5-HT induced no change in PI turnover or cytosolic free calcium levels ([Ca2+]i) in receptor-negative nontransfected 3T3 cells, 5-HT induced a 2-fold increase in inositol trisphosphate accumulation and a 2.5-fold increase in [Ca2+]i in the 3T3-ZD8 clone, which expressed 0.6 +/- 0.2 pmol/mg protein of specific 5-HT1A binding sites. The stimulatory actions of 5-HT on PI turnover and [Ca2+]i in 3T3ZD8 cells displayed the pharmacology of the 5-HT1A receptor and were abolished by pretreatment with PTx. Thus, BALB/c-3T3 fibroblast cells express the PLC-linked pathway of the 5-HT1A receptor. Overnight treatment with 5-HT (1 microM) enhanced incorporation of [3H]thymidine into DNA extracted from serum-starved 3T3ZD-8 cells, an action that was also blocked by pretreatment with pertussis toxin. Long term (1-2 weeks) exposure to 5-HT in the medium led to phenotypic transformation of the cells, including the formation of foci with 1 microM 5-HT. These actions of 5-HT were not observed in untransformed 3T3 cells. We conclude that the PTx-sensitive PLC-linked pathway of the 5-HT1A receptor expressed in nontransformed BALB/c-3T3 cells, in concert with other serum-derived factors, predisposes the cells to enhanced proliferation and transformation.

3T3 Cells↗
PubMed1991

Growth regulation of mouse DNA methyltransferase gene expression.

The steady state level of DNA methyltransferase mRNA is markedly increased as growth-arrested Balb/c 3T3 cells progress into the S phase of the cell cycle. mRNA abundance is reduced to the basal level before termination of DNA synthesis activity. Maintenance DNA methylation activity in nuclear extracts follows a similar pattern with two exceptions. (a) A small peak of DNA methylation activity is detected in early G1 phase. (b) The extinction of DNA methylation activity lags behind the termination of DNA synthesis. Nuclear runon experiments demonstrate that the gene is transcribed in growth-arrested cells, and expression of the gene is post-transcriptionally regulated. We suggest that this mode of regulation of the DNA methyltransferase gene might play an important role in determining and maintaining DNA methylation patterns.

Animals↗
PubMed1990

A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.

Animals↗
PubMed1990

cis modification of the steroid 21-hydroxylase gene prevents its expression in the Y1 mouse adrenocortical tumor cell line.

The Y1 mouse adrenocortical tumor cell line retains the ability to synthesize and secrete steroids, but does not express steroid 21-hydroxylase (C21) and, therefore, does not produce 21-hydroxylated steroids. In this investigation the mechanisms underlying the loss of C21 activity in the Y1 cell line were explored. A 9-kilobase BglII fragment containing the C21 gene was cloned from the Y1 genome. This genomic clone directed the synthesis of C21 transcripts and 21-hydroxylated steroid products when transfected back into the Y1 cell line. As determined by restriction endonuclease digestions with MspI and HpaII, enzymes that distinguish between unmethylated and methylated CCGG sites, the endogenous C21 gene was extensively methylated in Y1 adrenal cells and in cells from other mouse tissues that do not normally express this gene. In contrast, the C21 gene was hypomethylated in primary cultures of mouse adrenal cells which normally synthesize large amounts of C21. The cloned C21 gene transfected into Y1 cells initially was unmethylated, but became extensively methylated with prolonged culture of the cells; prolonged culture of these transfectants also resulted in a loss of C21 expression. Loss of C21 expression in Y1 transfectants, however, temporally preceded the extensive methylation of the transfected C21 gene. Furthermore, treatment of Y1 cells with 5-azacytidine caused a demethylation of the endogenous C21 gene, but did not result in the recovery of C21 expression. These results indicate that Y1 cells contain a functional C21 gene that has been silenced by a reversible cis-modification event.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Neoplasms↗
PubMed1989

Nucleotide-sequence-specific de novo methylation in a somatic murine cell line.

DNA fragments encoding the mouse steroid 21-hydroxylase (C21 or Cyp21A1) gene are de novo methylated when introduced into the mouse adrenocortical tumor cell line Y1 by DNA-mediated gene transfer. Although CCGG sequences within the C21 gene are de novo methylated, CCGG sites within flanking vector sequences, other mammalian gene sequences driven by the C21 promoter, and the neomycin-resistance gene, which was cotransfected with the C21 gene, do not become methylated. At least two separate signals for de novo methylation are encoded within the gene since three fragments derived from the C21 gene were methylated de novo. Specific de novo methylation of C21-derived sequences does not occur in L cells or Y1 kin8 cells; this suggests that the cellular factors needed for de novo methylation of the C21 gene are not ubiquitous. Most DNA sequences are not de novo methylated when introduced into somatic cells and DNA sequences other than the C21 gene are not de novo methylated when introduced into Y1 cells. Several groups have suggested that de novo methylation occurs in early embryonic cells and that somatic cells strictly maintain their methylation pattern by a semiconservative methyltransferase. Our results suggest that de novo methylation of specific nucleotide sequences can occur in some mammalian somatic cells.

Animals↗
PubMed1989

Regulation of 21-hydroxylase gene expression.

The steroid 21-hydroxylase (21-OH) gene is selectively expressed in the adrenal cortex and is transcriptionally regulated by ACTH. We examined the role of the 5'-flanking sequences of 21-OH in this regulated expression by analyzing their ability to direct the expression of a human growth hormone (hGH) reporter gene upon transfection into Y1 mouse adrenocortical tumor cells. The 330 bp of 5'-flanking sequences directed basal and hormonally-inducible expression of hGH in Y1 cells, but did not direct expression in I-10 mouse testicular Leydig cells. Both constitutive and hormonally-inducible expression required a functional cAMP-dependent protein kinase. These results indicate that the first 330 bp of 5'-flanking sequences of the 21-OH gene contain sufficient information for cell-specific and hormonally regulated expression, and that this expression requires the integrity of cAMP-dependent protein kinase. Markedly lower expression of hGH was seen when 156 bp of 5'-flanking sequences were placed in front of the reporter gene, suggesting that sequences between -330 and -156 are essential for expression. The addition of sequences from -330 to -150 to the p-156GH plasmid, in either the correct or the reverse orientation, restored promoter activity to approximately the level obtained with the 330 bp of 5'-flanking sequences. Moreover, the addition of sequences from -230 to -150 increased by 5-fold the expression of hGH driven by the heterologous thymidine kinase promoter. Based on these results, we conclude that an enhancer element is contained within the sequences from 230 to 150 bp upstream of the transcription initiation site.

Adrenal Cortex Neoplasms↗
PubMed1988

An enhancer element and a functional cyclic AMP-dependent protein kinase are required for expression of adrenocortical 21-hydroxylase.

The steroid 21-hydroxylase (21-OHase) gene is selectively expressed at high levels in cells of the adrenal cortex and is transcriptionally regulated by corticotropin (ACTH). In this study, we examined the contribution of cis-acting nucleotide sequences to the regulated expression of the mouse 21-OHase gene. The 5'-flanking sequences of the mouse 21-OHase gene, extending 330 bp upstream from the transcription initiation site, were placed in front of the human growth hormone (hGH) reporter gene, and expression of the fusion gene was measured following transient transfection in Y1 mouse adrenocortical tumor cells. The 330 bp of 21-OHase flanking sequence directed both basal and ACTH-stimulated expression of hGH in Y1 adrenocortical cells but did not direct hGH expression in I-10 mouse testicular Leydig cells or in mouse fibroblast L cells. The 21-OHase/hGH fusion gene was poorly expressed in Y1 mutants defective in cAMP-dependent protein kinase activity. These results indicate that sequences necessary for adrenal cell-selective and ACTH-regulated expression of the 21-OHase gene reside within the first 330 bp of 5'-flanking DNA and that constitutive expression of the gene requires the integrity of cAMP-dependent protein kinase. The constitutive expression of hGH in Y1 cells was decreased dramatically (40-fold) when the 21-OHase flanking sequences in front of hGH were shortened to 156 bp from the transcription initiation site and was restored when the upstream sequences of the 21-OHase gene, from -330 to -150, were added back; the sequences from -330 to -150 were equally effective in either the correct or reverse orientation. From these observations, we conclude that an enhancer element is contained within the sequences from -330 to -150 bp upstream of the 21-OHase transcription initiation site.

Adrenal Cortex↗
PubMed1986

Characterization of a tumorigenic murine T-lymphoid-cell line spontaneously derived from an IL-2-dependent T-cell line.

The establishment of IL-2-independent T-cell lines spontaneously derived from long-term IL-2-dependent cytotoxic T-cell lines is described. Two lines (cloned and uncloned) studied in detail have shown the following characteristics: (1) Permanent loss of IL-2 dependence. (2) Partial or complete loss of both cytotoxic activity and the IL-2 receptor. (3) Increased expression of T-cell membrane markers (Thy1.2, Lyt1.2) compared with the parental line. (4) Lower level of DNA methylation than in freshly obtained lymphoid cells. (5) Different karyotypic pattern from the parental IL-2-dependent line, with a mean number of 39-40 chromosomes and a resemblance to T leukemic lines. (6) Leukemia caused in normal syngeneic C57BL/6 mice by the uncloned line, in contrast to the cloned IL-2-independent line or the parental dependent line. Unlike established leukemic lines, however, the independent line gave rise to tumors which regressed in some mice within a few days of their appearance. These findings suggest that T-cell lines maintained with IL-2 for prolonged periods of time (greater than 3 months) can undergo transformation and, therefore, should not be utilized for immunotherapeutic purposes.

Animals↗
PubMed1986

Quantitation of single- and double-strand DNA breaks in vitro and in vivo.

This communication describes a rapid and convenient procedure for quantitation of strand breaks in bacterial DNA, both in vitro and in vivo, using agarose gel electrophoresis. The electrophoretic determination of single strand breaks is carried out in alkaline medium, followed by renaturation of the gel and intercalation of the fluorescent dye, ethidium bromide. Double-strand breaks are determined by electrophoresis in neutral medium containing the dye. The distribution of DNA fragment sizes, the determination of the number-average molecular weight, the quantitation of the average number of DNA breaks per molecule, and the ratio between the single- and double-strand breaks are evaluated from microdensitometric scanning of the gels. The application of this analysis to damage caused by a combination of ascorbate and copper is demonstrated.

Ascorbic Acid↗
PubMed1986

Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation.

In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation. In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy[5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-light) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into light-light duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3H]adenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the 3H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.

5-Methylcytosine↗