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A C Cato

Publications and source records attributed to A C Cato.

At least 55 records · Page 3Linked to original sources
PubMed1994

In vivo binding of proteins to stably integrated MMTV DNA in murine cell lines: occupancy of NFI and OTF1 binding sites in the absence and presence of glucocorticoids.

Activation of expression at the mouse mammary tumor virus (MMTV) promoter is thought to be controlled by nucleosome positioning. On stably integrated MMTV DNA, the long terminal repeat (LTR) region is organized in a phased array of nucleosomes which allegedly occludes transcription factors such as NFI from binding. NFI only binds to the promoter region when the ordered nucleosome structure is apparently disrupted by activated steroid hormone receptors in hormone induced transcription. In certain cell lines, binding sites for the transcription factors NFI and OTF1 are however required for hormone-independent expression of MMTV. We have used stably transfected mouse NIH3T3 and GR cells that exhibit detectable MMTV expression in the absence of hormone for in vivo determination of proteins binding to the MMTV promoter. Here, we present in vivo dimethyl sulfate footprinting data that show that the NFI and OTF binding sites are permanently occupied in vivo in these cells. The contacting guanine residues identified in vivo were demonstrated in in vitro methylation interference assays to correspond to binding by NFI and OTF1. These results demonstrate a novel feature of transcription factor occupancy at the MMTV LTR promoter.

3T3 Cells↗
PubMed1993

Characterization of two point mutations in the androgen receptor gene of patients with perineoscrotal hypospadia.

Perineoscrotal hypospadia is a major sign of sexual ambiguity due to inadequate androgen action in genetic and gonadal males. In patients showing these symptoms we have detected two androgen receptor gene mutations. In consequence we characterized the properties of the mutant receptors with respect to hormone-binding, transactivation and DNA-binding. An amino acid substitution alanine-596-->threonine in the D-box of the androgen receptor was detected in 3 and 2 brothers, respectively. This mutant receptor, AR-thr596, bound ligand in a normal fashion. It showed a promoter-dependent defect of transactivation and was unable to induce transcription of a promoter containing one androgen responsive element but showed almost wild-type transactivation of a promoter containing two closely spaced androgen-responsive elements. The complex promoter of the human prostate-specific antigen gene was induced with intermediate efficiency. In electrophoretic mobility shift assays AR-thr596 was unable to form a complex with oligonucleotides containing 1 or 2 androgen responsive elements, however its DNA-binding activity was restored by an anti-androgen receptor antibody in the presence of ligand. A point mutation which caused substitution of serine-703 in the hormone-binding domain with glycine was detected in a new-born male with ambiguous genitalia. This mutant receptor, AR-gly703, showed a reduced ligand affinity. The total amount of specific androgen binding sites in genital fibroblasts of the patient was reduced. Transactivation activity of AR-gly703 was dependent on hormone concentration. It was inactive at low levels of androgens but was fully activated in the presence of high androgen concentrations. The nature of the promoter had no effect on transactivation properties of this mutant androgen receptor. Its DNA-binding activity in gel shift experiments was normal.

Base Sequence↗
PubMed1993

Regulation of expression of mouse mammary tumor virus through sequences located in the hormone response element: involvement of cell-cell contact and a negative regulatory factor.

Mouse mammary tumor virus (MMTV) is a latently oncogenic retrovirus responsible for the neoplastic transformation of mammary epithelial cells. Its expression is regulated by steroids, polypeptide growth factors, and cell-type-specific factors. Using GR mouse mammary cells and NIH 3T3 fibroblasts stably transfected with chimeric constructs of the long terminal repeat region of MMTV, we have demonstrated a novel mechanism of cell-type-specific expression of this virus. In confluent monolayer cultures that permit cell-cell interaction, MMTV long terminal repeat expression is positively regulated by sequences within the hormone response element (HRE) that bind the transcription factors CTF/NFI and OTFI. Although these factors are present in NIH 3T3 cells, MMTV expression in these cells is not regulated by cell density. This is partially due to a negative regulatory factor that binds sequences between -164 and -151 in the HRE. Mutations that destroy the binding site for this factor restored in part the cell density-regulated expression of MMTV to NIH 3T3 fibroblasts. The HRE is thus a central coordinator of regulatory pathways that positively or negatively influence the expression of MMTV.

3T3 Cells↗
PubMed1993

A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors.

Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors. Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.

Alanine↗
PubMed1993

Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone.

Structural changes of the androgen receptor (AR) may contribute to the development of resistance to endocrine therapy in prostatic carcinoma. We have isolated AR cDNA fragments from seven tumor specimens derived from patients with advanced metastatic prostatic tumors. In one specimen obtained from a patient who failed to respond to endocrine and cytotoxic therapy we have detected a point mutation in the hormone-binding domain of the receptor. This AR mutation is a guanine-to-adenine transition at nucleotide 2671 that leads to substitution of methionine for the wild type valine at position 715. It is a somatic mutation because it was not present in the AR genomic DNA fragments isolated from prostatic and testicular tissues of the same patient. The mutant AR was recreated in an expression vector and transiently expressed in COS-7 and CV-1 cells. Hormone-binding assays revealed that the mutant receptor does not differ from the wild type receptor in its ability to bind androgen. The dissociation constant for the synthetic androgen mibolerone was 3 nM for both receptors. There was also no significant difference in binding of other steroids and nonsteroidal antiandrogens as revealed by competition binding assays. However, transfection experiments to determine the trans-activation potential of the mutant receptor produced differences in the action of this receptor compared to the wild type receptor. Dihydrotestosterone and the synthetic androgens methyltrienolone (R1881) and mibolerone were equally proficient in conferring trans-activation activity to both the mutant and wild type receptors. Adrenal androgens such as dehydroepiandrosterone and androstenedione, as well as progesterone mediated a higher trans-activation through the mutant than through the wild type receptor. These data demonstrate that the exchange of a single valine into methionine at position 715 in the AR promoters trans-activation not only by testicular but also by adrenal androgens and progesterone. This pattern of ligand-dependent trans-activation may have significance in the process controlling the progression of prostatic carcinoma.

Adrenal Glands↗
PubMed1992

Interference of pathway specific transcription factors.

A mechanism for reversibility of cellular decisions, e.g., proliferation or differentiation, is mediated by direct interference of pathway-specific transcription factors. Due to the relative activity of the transcription factors, genetic programs are reversibly switched on and off. In the case of the transcription factors AP-1 and the family of steroid hormone receptors (including retinoic acid receptor, thyroid hormone and vitamin D receptors) two different mechanisms account for negative interference. In one case overlapping DNA binding sites are suggestive of mutually exclusive binding of transcription factors to account for repression. In the other case, a DNA bound transcription factor is attacked by another one via protein-protein interaction, without the need of the latter factor binding to DNA. Although it is not excluded, that also in the case of overlapping DNA binding elements protein-protein interaction might in fact be responsible for repression instead of substitution, the two situations can be clearly distinguished by specific mutants in the glucocorticoid receptor and in Jun and Fos. For the interaction mechanism, we favor the interpretation that the binding of one transcription factor to another one interferes with transactivation directly without influence on DNA binding. The fact that at least two different mechanisms are established to guarantee effective interference, demonstrates how important this type of transcription factor crosstalk is.

Amino Acid Sequence↗
PubMed1992

Steroids and growth promoting factors in the regulation of expression of genes and gene networks.

Steroid hormones and growth promoting factors regulate the expression of a number of genes involved in cellular growth and differentiation. In many cases, cellular proliferation and differentiation-specific pathways are mutually exclusive. As an example for the mechanism of mutual exclusion, transcription factors responsible for pathway-specific gene expression interact with each other. Steroid hormone receptors inhibit the action of the transcription factor AP-1 (Fos/Jun) and vice versa. This inhibitory interaction is predominantly effected at the level of protein-protein contact without the need for the interfering transcription factor to bind to DNA. In some cases the two pathways function additively or even synergistically resulting in cooperation of the transcription factors in regulation of gene expression. The examples to be discussed in this text document how elaborate and important cross-talks between signal transduction pathways are. This is particularly demonstrated by the fine tuning and reversibility of these processes.

Animals↗
PubMed1992

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites.

Animals↗
PubMed1991

Two types of antiprogestins identified by their differential action in transcriptionally active extracts from T47D cells.

Transcriptionally active nuclear extracts from human breast carcinoma cells (T47D) were used to compare the action of progestins and several antiprogestins of the 11 beta-aryl substituted steroid series on the DNA-binding properties and the trans-activating potential of progesterone receptor (PR) in vitro. Using the gel-shift assay we identified a novel type of antiprogestin (ZK98299, type I), which in contrast to type II antiprogestins, including RU486, does not induce binding of PR to progesterone response elements (PREs). In competition experiments excess of type I antiprogestin inhibits induction of DNA binding of PR by progestins and type II antiprogestins suggesting that its binding to PR interferes with the formation of stable receptor dimers. Moreover, we demonstrate that the antagonistic action of ZK98299 can be fully mimicked in vitro by using cell-free nuclear extracts from T47D cells and a 'simple' test promoter. In contrast, type II antiprogestins known to induce certain promoters in vivo exert strong agonistic effects on in vitro transcription of the test template used.

Base Sequence↗
PubMed1991

In two genes, synergism of steroid hormone action is not mediated by cooperative binding of receptors to adjacent sites.

Synergistic action of multiple steroid hormone response elements (HREs) has been proposed to be due to cooperative binding of receptors. We have studied the cooperativity of steroid hormone receptor binding to synergistic HREs in two natural genes. In the mouse mammary tumor virus long terminal repeat that contains four progesterone receptor binding sites, no cooperativity in receptor binding was observed between the single distal and the three proximal sites whereas a low level of cooperativity in receptor binding (about 2-fold) was found between the three proximal sites. This contrasted with the very strong synergism of these four HREs in stimulation of transcription. In the chicken vitellogenin II gene upstream sequences, an estrogen and a progestin response elements act synergistically. In this case again, no cooperativity of binding of the estrogen and progesterone receptors to their respective binding sites was observed. We therefore conclude that cooperative receptor binding may not always be required for synergistic action of multiple HREs.

Animals↗
PubMed1991

Co-operation of progestational steroids with epidermal growth factor in activation of gene expression in mammary tumor cells.

The progesterone receptor belongs to a class of ligand binding transcription factors that regulate transcription by interacting with specific DNA sequences on hormone regulated genes. In human mammary tumor T47D cells that contain both progesterone and epidermal growth factor (EGF) receptors, the progestin-induced transactivation at various hormone regulated promoters is enhanced by EGF. The effect of EGF is rapid and does not require new protein synthesis. EGF treatment does not alter the DNA binding activity of the progesterone receptor nor does it affect the total ligand-dependent phosphorylation of this receptor. These results suggest that EGF enhances the transactivation property of the progesterone receptor through mechanisms other than those involving a direct interaction of this receptor with its cognate binding sites.

Base Sequence↗
PubMed1990

Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone.

Glucocorticoid hormones counteract inflammation and phorbol ester tumor promotion and drastically decrease the expression of several extracellular proteases, including collagenase I. Glucocorticoid hormone inhibits basal and induced transcription of collagenase by interfering with AP-1, the major enhancer factor of the collagenase promoter. The mechanism of interference is novel in that it does not require protein synthesis, it depends on the hormone receptor but not its binding to DNA, it occurs at hormone doses one order of magnitude below those required for gene activation, and it involves down-modulation of the trans-activating function of preexisting unbound and DNA-bound AP-1. Coprecipitation experiments suggest direct AP-1-hormone receptor interaction, which also possibly explains the reverse experiment: overexpression of Fos or Jun inhibits the expression of hormone-dependent genes.

Animals↗
PubMed1990

The long terminal repeat region of the mouse mammary tumour virus contains multiple regulatory elements.

Mouse mammary tumour virus (MMTV) is the major aetiologic agent of mouse mammary tumour formation. The expression of this virus is regulated by steroid hormones and cell type specific factors. The nucleotide sequence that controls the steroid hormone response has already been localized between -202 and -59 upstream of the start of transcription in the long terminal repeat (LTR) region of the proviral DNA. Through transfection experiments in three different cultured mouse cell lines (NIH3T3, NMuMG and GR), we have investigated which sequences in the MMTV LTR play a role in the cell type specific expression at the proviral promoter. We have identified two elements on the MMTV LTR from -631 to -560 and from -428 to -364 that have the potential to influence expression at the MMTV LTR promoter. The -631 to -560 element mediated a negative response in all the cell types we studied whereas the -428 to -364 element had negative effects in the mouse fibroblast NIH3T3 and the normal mouse mammary gland cell NMuMG but not in the mouse mammary tumour epithelial GR cells. The -428 to -364 element therefore contributes to the cell type specific expression at MMTV LTR promoter. We have also identified another regulatory element between -1094 and -739 that had a slight positive regulatory effect at the MMTV LTR promoter but greatly enhanced expression at a foreign promoter when present at this promoter in an orientation that is the reverse of its own orientation in the MMTV LTR. This orientation-dependent effect was only observed in the mouse mammary epithelial cells NMuMG and GR but not in mouse fibroblastic cell line NIH3T3. This element may be important in regulating the expression of neighbouring genes in a cell type specific manner. These results show that the MMTV LTR contains multiple regulatory elements necessary for the control of expression at its own promoter and the expression of neighbouring genes.

Animals↗
PubMed1990

Transforming growth factor-beta represses transcription of the mouse mammary tumour virus DNA in cultured mouse mammary cells.

Increased expression of mouse mammary tumour virus (MMTV) is associated with hyperplastic alveolar growth and subsequent development of mammary cancers in the mouse. The expression of this virus is repressed when mammary tumour cells undergo sarcomatous transformation. We have demonstrated that a spontaneous progression of mouse mammary adenocarcinoma cells into highly malignant cells with the transformed phenotype is accompanied by an increased expression of transforming growth factors alpha and beta (TGF alpha and TGF beta), as well as a decreased expression of MMTV. Mouse mammary adenocarcinoma cells transformed with activated ras oncogene also expressed high levels of the transforming growth factors and a low level of MMTV. Thus a reverse correlation exists between the increased expression of the transforming growth factors and a low level of expression of MMTV. Mouse mammary cells that express high levels of MMTV when treated with exogenous TGF alpha and TGF beta 1 showed a down regulation of MMTV expression in response to TGF beta 1 but not to TGF alpha. These results demonstrate that the repression of MMTV expression in mouse mammary tumour progression may be due in part to an increased expression of TGF beta.

Animals↗
PubMed1989

The regulation of expression of mouse mammary tumor virus DNA by steroid hormones and growth factors.

Mouse mammary tumor virus (MMTV) expression is associated with hyperplastic alveolar growth and subsequent development of mammary cancers in the mouse. The expression of this virus is also controlled by factors involved in the normal proliferation and differentiation of the mammary epithelium. During pregnancy when the mammary gland undergoes massive proliferation, MMTV expression is increased. Steroid hormones and growth factors that play an important role in the proliferation of mammary gland cells are responsible for the increased MMTV expression. In sarcomatous transformation of mouse mammary epithelial cells, MMTV expression is repressed. This repression is due to negative control of MMTV expression by transforming growth factor-beta (TGF beta). This growth factor is produced in high amounts when mammary epithelial cells progress into the transformed state. The expression of MMTV is therefore under multiple control by steroid hormones and growth factors.

Animals↗
PubMed1989

Different regions of the estrogen receptor are required for synergistic action with the glucocorticoid and progesterone receptors.

Estrogen and progesterone or estrogen and glucocorticoid receptors functionally cooperate in gene activation if their cognate binding sites are close to one another. These interactions have been described as synergism of action of the steroid receptors. The mechanism by which synergism is achieved is not clear, although protein-protein interaction of the receptors is one of the favorite models. In transfection experiments with receptor expression vectors and a reporter gene containing estrogen and progesterone-glucocorticoid receptor binding sites, we have examined the effects that different portions of the various receptors have on synergism. N-terminal domains of the chicken progesterone and human glucocorticoid receptors, when deleted, abolished the synergistic action of these receptors with the estrogen receptor. Deletion of the carboxy-terminal amino acids 341 to 595 of the estrogen receptor produced a mutant receptor that could not trans-activate on its own. This mutant receptor did not affect the action of the glucocorticoid receptor but functioned synergistically with the progesterone receptor. We therefore conclude that the synergistic action of the receptors for estrogen and progesterone is mechanistically different from the synergistic action of the receptors for estrogen and glucocorticoid.

Animals↗
PubMed1989

Protein-DNA interactions at steroid hormone regulated genes.

This work summarizes binding data that were obtained with partially purified glucocorticoid and progesterone receptors, as well as with a crude nuclear protein extract, to DNA sequences in and around hormonally regulated genes. The sequence recognition by the glucocorticoid receptor at the different defined glucocorticoid regulatory elements (GRE) is discussed and a consensus sequence formulated. A three dimensional representation gives an impression of the mode of interaction between the protein and the double helix of DNA. In the promoters of mouse mammary tomour virus (MTV) and chicken lysozyme overlapping binding sites for both, glucocorticoid and progestine-receptors are found that are responsible for the hormonal inducibility of the genes. In crude extract from rat liver nuclei, a nonhistone protein is found that specifically binds to sequences on the MTV-LTR region overlapping the GRE. The possible implication of this protein in hormonal regulation of transcription is discussed.

Animals↗