Biomedical subjects

L J Gudas

Publications and source records attributed to L J Gudas.

At least 37 records · Page 2Linked to original sources

PubMed1997

Human breast cancer cells and normal mammary epithelial cells: retinol metabolism and growth inhibition by the retinol metabolite 4-oxoretinol.

To understand the signaling and growth-inhibitory effects of retinoids, we have examined the metabolism of [3H]retinol in a number of estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) human breast cancer cell lines. We have also assayed the metabolism of [3H]retinol in normal human mammary epithelial cells. The ER+ breast cancer cell lines MCF-7 and T47D produce [3H]4-oxoretinol from [3H]retinol; the production of [3H]4-oxoretinol is increased by initial culture in the presence of nonradiolabeled retinoic acid (RA) or N-(4-hydroxyphenyl)retinamide, indicating that these drugs enhance [3H]retinol metabolism to [3H]4-oxoretinol. No metabolism of [3H]retinol to [3H]RA in these ER+ tumor lines was detected. ER- breast cancer lines MDA-MB-231, MDA-MB-468, and BT20 do not metabolize [3H]retinol to [3H]4-oxoretinol. In the ER- tumor lines, most of the [3H]retinol remains unmetabolized during the 24-h culture period; MDA-MB-468 and BT20 metabolize some [3H]retinol to [3H]RA. Unlike the majority of the tumor lines, the normal human breast epithelial cell strains AD074 and MCF10A rapidly metabolize [3H]retinol to [3H]retinyl esters. No detectable [3H]RA is produced from [3H]retinol in AD074 and MCF10A cells. Thus, the normal breast epithelial strains, the ER+ tumor lines and the ER- tumor lines differ greatly in their pathways of [3H]retinol metabolism. The levels of cellular retinol binding protein-I mRNA expression are not correlated with the levels or types of various retinol metabolites. Whereas the normal breast epithelial cells and the ER+ tumor lines are growth inhibited by RA, N-(4-hydroxyphenyl)retinamide, and 4-oxoretinol, only the 4-oxoretinol is growth inhibitory in the ER- tumor lines. The cellular retinoic acid-binding protein II mRNA levels are not correlated with the growth inhibition by RA or 4-oxoretinol in the normal and tumor lines.

Breast↗

PubMed1997

Sensitivity to jerky gene dosage underlies epileptic seizures in mice.

Animals with one deleted jerky allele are more susceptible to chemically induced seizures than wild-type mice and display recurrent behavioral seizures. The phenotype of these hemizygotes is characterized by no apparent neurological symptoms other than recurrent seizures reminiscent of human idiopathic epilepsy. The jerky gene encodes a 60 kDa protein resembling a number of DNA-binding proteins. Here, we show that the jerky gene is expressed in all tissues examined, including brain, liver, lung, spleen, testis, and ovary, and study an apparent paradox of how an allelic deletion of the ubiquitously expressed jerky gene can lead to hyperexcitability and seizures but not to other symptoms. We demonstrate that jerky has a dosage-sensitive function (haploinsufficiency) in brain and that this sensitivity to reduced jerky dosage could explain the occurrence of seizures in hemizygotes. However, jerky has a nondosage-sensitive function as well, because the total absence of jerky in homozygotes results in abnormalities of somatic and sexual development. A number of idiopathic epilepsies are dominantly inherited, such as benign familial neonatal convulsions, juvenile myoclonic epilepsy, as well as benign epilepsy with centrotemporal spikes, and the pathomechanism of these epilepsies may be based on haploinsufficiency in the brain.

Aging↗

PubMed1997

Retinoic acid-responsive enhancers located 3' of the Hox A and Hox B homeobox gene clusters. Functional analysis.

Homeobox genes control the spatial identity and differentiation of tissues in the developing vertebrate embryo. Retinoids are signaling molecules involved in the regulation of Hox genes. We previously identified a 3' enhancer called the RAIDR5, which contained a DR5 retinoic acid response element (RARE) and was responsible for the retinoic acid (RA)-associated expression of the murine Hoxa-1 gene in teratocarcinoma cells. We demonstrate that a similar enhancer, which contains a DR5 RARE, is located at a DNase I-hypersensitive site 3' of the murine Hoxb-1 gene. This enhancer, the Hoxb-1 RAIDR5, regulates the RA responsiveness of the Hoxb-1 gene and is different in location and sequence from the RA-regulated 3' Hoxb-1 enhancers previously described. Several DNA elements within the murine Hoxa-1 RA-inducible RAIDR5 enhancer, including the DR5 RARE, conserved element (CE) 1, and CE2, are conserved in the murine Hoxb-1 RAIDR5 enhancer, the human homolog of Hoxa-1, and in the chicken Hoxb-1 gene. Gel shifts show that the CE2 sequence TATTTACTCA binds an RA-inducible factor, while UV cross-linking indicates that a 170-kDa protein binds to this sequence. Thus, the Hoxa-1 and Hoxb-1 genes possess 3' enhancers with similar sequences through which their expression and responsiveness to endogenous retinoids are controlled.

Amino Acid Sequence↗

PubMed1997

The CRABP I gene contains two separable, redundant regulatory regions active in neural tissues in transgenic mouse embryos.

The CRABP I gene is expressed in a spatiotemporal pattern in neural and mesenchymal tissues at the onset of organogenesis. The neural pattern of CRABP I expression includes specific rhombomeres of the hindbrain, neural crest cells and their derivatives the optic stalk, and the central area of the neural retina. We have created transgenic mouse lines with CRABP I 5' and transcribed regions fused to the lacZ structural gene that recapitulate much of this neural pattern of expression. Sequences 5' of the transcription initiation site between -7.8 and -3.2 kb confer beta-galactosidase expression to specific rhombomeres, migrating neural crest cells, trigeminal ganglion, the optic stalk, and the neural retina. We have also defined a region located between exon 1 and exon 8 that confers a portion of this expression pattern, including the mesencephalic projections of the trigeminal ganglion, the inner layer of the neural retina, and the peripheral layer of the posterior hindbrain. CRABP I expression in mesenchyme appears to require sequences in addition to or outside of those examined here.

Animals↗

PubMed1997

Sequences 5' of the basement membrane laminin beta 1 chain gene (LAMB1) direct the expression of beta-galactosidase during development of the mouse testis and ovary.

The murine LAMB1 gene encoding laminin beta 1 is expressed in the developing male and female gonads and mesonephros. To identify the cis-acting elements regulating the expression of LAMB1, murine transgenic lines were generated by fusing regions of the LAMB1 gene to the Eschericia coli lacZ gene. The p3.9LAM beta gal construct contained approximately 4 kb of 5' flanking sequence and directed beta-galactosidase expression in many different organs including the kidney, mammary gland, and the male and female genital systems, the focus of this report. In male embryos, between gestational ages E 14.5 and birth beta-galactosidase was transiently expressed in the prospermatogonia cells of the testis and in the differentiating epithelial cells in the ductus deferens, ductus epididymis, and seminal vesicles. In female embryos, beta-galactosidase was not detected in the ovary until about 1 week after birth; at this time, beta-galactosidase was expressed by oocytes of primary and secondary follicles. In contrast, transgenic mice carrying the first 0.7 kb of LAMB1 fused to the lacZ gene expressed beta-galactosidase only in the prospermatogonia cells of the testis. Thus, the cis-acting element(s) necessary for the expression of the LAMB1 gene in prospermatogonia cells are located in the first 0.7 kb of LAMB1 5' flanking sequence; element(s) required for expression of the LAMB1 gene in oocytes and epithelial cells of the mesonephric ducts, mesonephric tubules, the ductus deferens, ductus epididymis, and seminal vesicles are located with 4 kb 5' of the transcription initiation site.

Animals↗

PubMed1997

Regulation of the laminin beta 1 (LAMB1), retinoic acid receptor beta, and bone morphogenetic protein 2 genes in mutant F9 teratocarcinoma cell lines partially deficient in cyclic AMP-dependent protein kinase activity.

We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA.

Animals↗

PubMed1996

An analysis of retinoic acid-induced gene expression and metabolism in AB1 embryonic stem cells.

Murine embryonic stem cells such as the AB1 cell line undergo differentiation in the presence of retinoic acid (RA) into an extraembryonic epithelial cell type. This results in the activation of genes such as Hoxa-1, Hoxb-1, laminin, collagen IV(alpha1), tissue plasminogen activator, RARbeta, and CRABPII. The CRABPI gene is regulated in an unusual fashion; CRABPI message and protein levels are induced at low concentrations of RA, but induction is diminished at higher concentrations. AB1 cells take up RA rapidly from the medium, and the addition of low, exogenous concentrations of RA to the culture medium results in very high intracellular RA concentrations. For example, AB1 stem cells cultured in 5 nM [3H]RA have an internal [3H]RA concentration of 1-2 microM within the first hour. AB1 cells also metabolize [3H]RA to more polar RA derivatives. The half-life of RA in AB1 cells not previously exposed to RA is about 2-2.5 h versus 40-45 min in cells cultured for 2-3 days in 1 microM exogenous RA. Thus, the enzyme(s) which metabolize RA are induced or activated by RA. Furthermore, the local concentration of RA required to elicit some biological responses may be higher than previously thought.

Actins↗

PubMed1996

4-Oxoretinol, a new natural ligand and transactivator of the retinoic acid receptors.

All-trans-retinoic acid (at-RA) induces cell differentiation in a wide variety of cell types, including F9 embryonic teratocarcinoma cells, and can influence axial pattern formation during embryonic development. We now identify a novel retinoid synthetic pathway in differentiating F9 cells that results in the intracellular production of 4-oxoretinol (4-oxo-ROL) from retinol (vitamin A). Approximately 10-15% of the total retinol in the culture is metabolized to 4-hydroxyretinol and 4-oxo-ROL by the at-RA-treated, differentiating F9 cells over an 18-hr period, but no detectable metabolism of all-trans-retinol to at-RA or 9-cis-retinoic acid is observed in these cells. Remarkably, we show that 4-oxo-ROL can bind and activate transcription of the retinoic acid receptors whereas all-trans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10(-9) or 10(-10 M) can activate the retinoic acid receptors even though, unlike at-RA, 4-oxo-ROL does not contain an acid moiety at the carbon 15 position. 4-oxo-ROL does not bind or transcriptionally activate the retinoid X receptors. Treatment of F9 cells with 4-oxo-ROL induces differentiation without conversion to the acid and 4-oxo-ROL is active in causing axial truncation when administered to Xenopus embryos at the blastula stage. Thus, 4-oxo-ROL is a natural, biologically active retinoid that is present in differentiated F9 cells. Our data suggest that 4-oxo-ROL may be a novel signaling molecule and regulator of cell differentiation.

Animals↗

PubMed1996

Murine laminin B1 gene regulation during the retinoic acid- and dibutyryl cyclic AMP-induced differentiation of embryonic F9 teratocarcinoma stem cells.

Retinoic acid (RA) and cyclic AMP analogs cause the differentiation of F9 embryonic teratocarcinoma stem cells into parietal endoderm, an epithelial cell of the early mouse embryo. Laminin B1 is induced in this differentiation process, but is not transcriptionally activated until 24-48 h after RA addition and is not maximally induced until approximately 72 h. Cyclic AMP analogs enhance this transcriptional activation. Although several DNase I hypersensitive sites (DHSS) were observed in the LAMB1 5 -flanking DNA, one of the sites, DHSS2, was detected only after 72 h of RA treatment. Transient transfections have demonstrated that the DHSS2 region functions as a "late-acting RA-inducible enhancer," and motifs in this enhancer contain the homeobox protein-binding site TTATTAACA. Greater binding is observed at these sites by electrophoretic mobility shift assay when cells are cultured with RA and cyclic AMP analogs versus RA alone, and no binding is seen in extracts from RA-treated F9 RAR gamma-/- cells which lack RAR gamma mRNA and protein. Laminin B1 mRNA is not induced by RA in the RAR gamma-/- cells (Boylan, J. F., Lohnes, D., Taneja, R., Chambon, P., and Gudas, L. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9601-9605). Our data show that these DNA regulatory elements contribute to the transcriptional activation of the LAMB1 gene during the later stages of the differentiation process.

Animals↗

PubMed1996

FGF-2, BMP-2, and BMP-4 regulate retinoid binding proteins and receptors in 3T3 cells.

An Important part of intercellular signalling is the ability of responding cells to regulate multiple signal transduction pathways. Just as retinoic acid exposure alters expression of many peptide growth factors and their receptors, we have found that peptide growth factors alter the expression of cellular retinoic acid binding proteins (CRABPs) and retinoic acid receptors (RARs). FGF-2 (basic fibroblast growth factor) treatment of BALB 3T3 fibroblasts increased the level of CRABP I RNA, whereas bone morphogenetic protein (BMP)-2 and BMP-4 reduced this level as well as the levels of CRABP II and RAR beta 1/beta 3 transcripts. Regulation of the CRABP I gene by FGF-2 occurred posttranscriptionally by increasing RNA stability. However, BMP-2 down-regulated CRABP I message without affecting message stability. Neither of these mechanisms was dominant, with intermediate levels of CRABP I RNA occurring in the presence of both FGF-2 and BMP-2 or BMP-4. These two different modes of regulation thus allow different levels of CRABP I RNA accumulation in the presence of different ratios of FGF-2 and BMP-2 or BMP-4.

3T3 Cells↗

PubMed1995

Reexpression of retinoic acid receptor (RAR) gamma or overexpression of RAR alpha or RAR beta in RAR gamma-null F9 cells reveals a partial functional redundancy between the three RAR types.

Disruption of retinoic acid receptor (RAR) gamma in F9 embryonal carcinoma cells leads to aberrent differentiation and reduced activation of expression of several all-trans-retinoic acid (RA)-induced genes. We have analyzed the expression of several additional RA-responsive genes in RAR alpha- and RAR gamma-null F9 cells. The RA-induced activation of Cdx1, Gap43, Stra4, and Stra6 was specifically impaired in RAR gamma-null cells, supporting the idea that each RAR may regulate distinct subsets of target genes. To further investigate the role of RAR gamma in F9 cell differentiation, "rescue" cell lines reexpressing RAR gamma 2 or overexpressing either RAR alpha 1 or RAR beta 2 were established in RAR gamma-null cells. Reexpression of RAR gamma or overexpression of RAR alpha restored both target-gene activation and the differentiation potential. In contrast, over-expression of RAR beta only poorly restored differentiation, although it could replace RAR gamma for the activation of target genes. Functional redundancy between the various RARs is discussed.

Animals↗

PubMed1995

Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism.

F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.

Animals↗

PubMed1995

The roles of retinoids in vertebrate development.

Several lines of experimentation suggest that endogenous retinoids, metabolites of vitamin A, play a role in the anterior/posterior development of the central body axis and the limbs of vertebrates. High levels of endogenous retinoids have been detected in proximity to these developing axes in a variety of vertebrate fetuses. Teratogenesis studies suggest that both retinoid excess and deficiency are capable of disrupting the development of these axes. Finally, retinoic acid receptors regulate many developmental control genes, including homeobox genes and growth factor genes.

Animals↗

PubMed1994

Retinoids and vertebrate development.

In conclusion, it is obvious that many of the abnormalities in pattern formation and organ formation that result from the exogenous addition of RA during embryogenesis are related at least in part to the ability of RA to change the pattern of expression of the clusters of homeobox genes in the mammalian embryo. The majority of experimental protocols to study RA-induced changes in embryonic development have utilized treatments with excess, exogenous RA, but it has not yet been proven that endogenous RA acts as a key signaling molecule in developing embryo. Thus, we need to develop an understanding of the metabolic enzymes that control the levels of the important endogenous retinoids, including RA, in the developing embryo. The continuing study of retinoids and their receptors will provide us with significant new information about the regulatory programs that control differentiation and development. Moreover, these studies will lead to discoveries relevant to the clinical use of retinoids in the areas of cancer prevention and treatment, and dermatology.

Animals↗

PubMed1994

Activation of keratin 19 gene expression by a 3' enhancer containing an AP1 site.

We previously reported that the human keratin 19 (K19) gene was expressed in nonkeratinizing oral epithelial subtypes, and that the steady state K19 mRNA levels in different normal epithelial subtypes correlated with the levels of the retinoic acid receptor (RAR) beta-mRNA (Hu, L., Crowe, D. L., Rheinwald, J. G., Chambon, P., and Gudas, L. J. (1991) Cancer Res. 51, 3972-3981). To elucidate the mechanisms by which the K19 gene is differentially expressed in various epithelial subtypes, we isolated phage containing human K19 genomic DNA from a human placental library. Through transient transfection assays with various K19/CAT constructs that contain different portions of K19 genomic DNA, an enhancer sequence has been identified in the K19 3'-flanking region. In normal human epithelial cell strains, the activity of this enhancer correlates with K19 mRNA abundance. This enhancer activates both the K19 and TK basal promoters in HeLa cells. A high level of K19/CAT fusion mRNA was detected when this K19 3' enhancer sequence was present at the 3' end of the fusion gene whereas no K19/CAT fusion transcript was detected if this 3' K19 enhancer sequence was absent, suggesting that the 3' K19 enhancer is crucial for the positive regulation of K19 expression. Deletion analysis has permitted the localization of the enhancer to a 19-base pair sequence which contains an AP1 binding site (AGTCATCT). Point mutations within this AP1 site completely abolished K19 enhancer activity in HeLa cells. Co-transfections of a c-jun expression vector with K19/CAT reporter constructs demonstrated that c-jun was able to activate the K19 promoter via the 3' K19 enhancer. Collectively, these data indicate that a cis-acting AP1 element and its associated trans-acting effector proteins regulate expression of lineage-specific genes in epithelial cells.

Base Sequence↗

PubMed1994

Localization of PDGF A and PDGFR alpha mRNA in Xenopus embryos suggests signalling from neural ectoderm and pharyngeal endoderm to neural crest cells.

In situ hybridization analysis of Xenopus laevis embryos reveals that mRNA encoding the platelet-derived growth factor alpha receptor (PDGFR alpha) is expressed in cephalic neural crest masses prior to migration from the future neural tube and during their migration into the visceral arches. The analysis of fluorescently labeled neural crest tissue transplanted to unlabeled host embryos demonstrates that neural crest cells are the only detectable source of PDGFR alpha mRNA within visceral arches. Transcripts encoding PDGF A are present in neural ectoderm, otic vesicle and pharyngeal endoderm. Their location suggests that PDGF A provides a signal, first from the neural epithelium and later from the otic vesicle and pharyngeal endoderm, to cephalic neural crest cells during their migration in the arch region.

Animals↗

PubMed1994

Xenopus laevis cellular retinoic acid-binding protein: temporal and spatial expression pattern during early embryogenesis.

There is increasing evidence that retinoic acid (RA) has a role in establishing normal axial patterns during Xenopus laevis embryo-genesis. Several types of retinoid binding proteins are thought to mediate the effects of RA. We report the isolation of a cDNA, named xCRABP-b, which encodes a X. laevis cellular retinoic acid-binding protein (xCRABP). This cDNA hybridises to a transcript in gastrular stage embryos of approximately 3 kb, much larger than those CRABP transcripts expressed in mice. The expression of the xCRABP mRNA is generally restricted to tissues which are sensitive to the teratogenic effects of excess RA. It is likely, that during normal X. laevis embryogenesis, concentrations of RA in RA-responsive cells are modulated by the xCRABP gene product.

Amino Acid Sequence↗

PubMed1994

Vitamin A, differentiation and cancer.

Retinoids, which are derivatives of vitamin A, have a variety of effects on normal cellular differentiation and on the process of carcinogenesis. A number of novel endogenous retinol metabolites have been identified recently. The response of many cell types to retinoid treatment is mediated by retinoid receptors, and involves changes in gene expression, cell growth and cell differentiation. The gene encoding one of the retinoic acid receptors is disrupted by the chromosome translocations associated with acute promyelocytic leukemia, and the expression of another is altered in epithelial tumors; both of these findings have important implications for the use of retinoids as anti-carcinogenic agents. It has been demonstrated recently that certain homeobox genes are regulated by retinoids; these genes may also prove to be useful agents for anti-carcinogenic therapies.

Animals↗