Biomedical subjects

K J Soprano

Publications and source records attributed to K J Soprano.

At least 37 records · Page 2Linked to original sources

PubMed1997

Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor beta.

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RARbeta mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys228 is necessary to inhibit RA binding when RARbeta is modified by reagents which transfer large bulky groups while both Cys228 and Cys267 must be modified when a small functional group is transferred. These data suggest that both Cys228 and Cys267 but not Cys258 lie in the ligand binding pocket of RARbeta. However, Cys228 lies closer to the opening of the RARbeta ligand binding pocket whereas Cys267 lies more deeply buried.

Binding Sites↗

PubMed1997

Differential expression of c-jun and junD in end-stage human cardiomyopathy.

The proto-oncogenes c-jun and junD are closely related transcriptional factors with opposing actions on cell growth and division. Expression of c-jun rapidly increases as cells enter the cell cycle. Levels of c-jun are also increased in the early stages of experimental cardiac hypertrophy and failure but expression decreases with time. In contrast, junD accumulates in quiescent cells. Expression in end-stage cardiomyopathy has not been studied. Steady-state levels of c-jun and junD mRNA were determined in failing human myocardium (obtained at the time of cardiac transplantation) and in control myocardium from patients who died of noncardiac causes. Relative expression was normalized for glyceraldehyde-3-phosphate dehydrogenase expression. Levels of junD were almost four-fold depressed in myocardium from myopathic hearts (2.1 +/- 0.27, x +/- SE; n = 20) vs. the controls (7.7 +/- 1.1; n = 3). Levels of c-jun were similar in both myopathic and control hearts. Relative expression of beta-myosin heavy chain was the same in both myopathic and control hearts. Levels of junD were still found to be depressed in the myopathic hearts after normalization for myosin heavy chain gene expression. We conclude that c-jun and junD are differentially regulated in end-stage human cardiomyopathy with expression of junD being decreased while relative levels of c-jun mRNA remain unchanged. Further studies are needed to determine the role of junD down-regulation in the development and/or maintenance of the abnormalities present in end-stage heart disease.

Cardiomyopathies↗

PubMed1996

Overexpression of both RAR and RXR restores AP-1 repression in ovarian adenocarcinoma cells resistant to retinoic acid-dependent growth inhibition.

Retinoids including retinoic acid (RA) have been demonstrated to be effective growth inhibitors of a number of human cancer cell lines including ovarian adenocarcinoma cells. To begin to determine the mechanism of action by which RA inhibits the growth of ovarian carcinoma cells, we have examined AP-1 activity in two representative cell lines: CaOV-3 a RA-sensitive cell line and SK-OV-3 a RA-resistant cell line. AP-1 activity was found to be inhibited by 50% upon RA treatment of the RA-sensitive cells while there was no change in AP-1 activity following RA treatment of the RA-resistant cells. Maximal inhibition of AP-1 activity could be achieved in the RA-resistant SK-OV-3 cells by overexpression of any one of the three retinoic acid receptor (RAR) subtypes in conjunction with retinoid X receptor (RXR) alpha. This inhibition of AP-1 activity was nearly comparable to that of the RA-sensitive cells. A similar change in AP-1 complex formation in vitro has also been observed. These results suggest that one mechanism by which RA inhibits growth of RA-sensitive ovarian carcinoma cells is by repressing AP-1 activity. Moreover, in the RA-resistant cells the RAR/RXR signalling pathway leading to inhibition of AP-1 activity is impaired however overexpression of one of the RAR subtypes along with RXR alpha is sufficient to restore this pathway.

Adenocarcinoma↗

PubMed1996

Overexpression of Mxi1 inhibits the induction of the human ornithine decarboxylase gene by the Myc/Max protein complex.

We have previously shown that the Myc/Max protein complex plays a role in the growth-associated expression of the human ornithine decarboxylase gene. Mxi1 and Mad, novel Max-associated proteins have been identified and shown to form heterodimers with Max which bind efficiently to the Myc/Max consensus recognition sequence, CACGTG, in vitro. However, formation of Max/Mxi1 or Max/Mad heterodimers results in a reduction in Myc/Max dependent transcriptional activation of reporter plasmid constructs containing the consensus element. In light of the evidence that ODC is transcriptionally regulated in vitro and in vivo by the Myc/Max protein complex and the potential role of Mxi1 and Mad as antagonists of Myc transactivation activity, we set out to determine if one of these Max associated proteins, Mxi1, could affect the regulation of ODC expression by Myc/Max and if this regulation was correlated to growth status. Our results show that overexpression of Mxi1 does in fact inhibit ODC gene expression in a dose-dependent manner both in vivo and in vitro. In addition, evidence is presented which shows that levels of Mxi1 are up-regulated during long term quiescence and down-regulated following growth stimulation by serum. These results suggest that alterations in the levels of Max-associated proteins such as Mxi1 can modulate critical levels of functional Myc/Max protein complexes. This can alter transcriptional transactivation of Myc-regulated targets and as a consequence affect levels of genes essential for initiation and/or maintenance of growth.

Base Sequence↗

PubMed1995

Activation of a cell-cycle-regulated histone gene by the oncogenic transcription factor IRF-2.

The human histone H4 gene FO108 is regulated during the cell cycle with a peak in transcription during early S phase. The cell-cycle element (CCE) required for H4 histone activation is a sequence of 11 base pairs that binds a protein factor in electrophoretic mobility shift assays that has been designated histone nuclear factor M (HiNF-M). Here we report the purification of HiNF-M, and show it to be a protein of relative molecular mass (M(r)) 48K that is identical to interferon (IFN) regulatory factor 2 (IRF-2), a negative transcriptional regulator of the IFN response. Recombinant IRF-2 (as well as the related protein IRF-1 (ref. 5)) binds the CCE specifically and activates transcription of this H4 histone gene. IRF-2 has been shown to have oncogenic potential, and our results demonstrate a link between IRF-2 and a gene that is functionally coupled to DNA replication and cell-cycle progression at the G1/S phase transition.

Animals↗

PubMed1995

Alteration in the retinoid specificity of retinoic acid receptor-beta by site-directed mutagenesis of Arg269 and Lys220.

Retinoic acid receptor-beta (RAR-beta) specifically binds retinoic acid (RA) and functions as a RA-inducible transcriptional regulatory factor. Simultaneous mutation of Arg269 and Lys220 of RAR-beta to Ala results in a dramatic reduction in both transactivation and affinity for RA along with creating a RA concentration-dependent dominant negative mutant. In this report, we found that mutation of these two amino acid residues singly and simultaneously to Gln results in mutant RAR-beta s, each displaying a more dramatic reduction in transactivation and affinity for RA than their corresponding Ala mutant, with the R269Q more profoundly affected than K220Q. Furthermore, we examined both the Ala and Gln mutants for their ability to transactivate and bind two other retinoids with different functional end groups (all-trans-retinol and all-trans-retinal). Mutation of Lys220 to either an Ala or a Gln favors transactivation and binding of retinal, while mutation of either Lys220 or Arg269 to Gln favors retinol transactivation and binding. Taken together, these results suggest that Arg269 and Lys220 lie within the ligand binding pocket of RAR-beta and Lys220 lie within the ligand binding pocket of RAR-beta and that these two amino acid residues play an important role in determining retinoid specificity most likely by directly interacting with the carboxylate group of RA.

Animals↗

PubMed1995

Arginine 132 of cellular retinoic acid-binding protein (type II) is important for binding of retinoic acid.

Cellular retinoic acid-binding protein type II (CRABP-II) is one of two small molecular weight, cytosolic proteins which specifically bind retinoic acid (RA). Crystallographic and site-directed mutagenesis studies of several related proteins have indicated that either one or two conserved amino acid residues, homologous to positions Arg111 and Arg132 of CRABP-II, are important for the binding of the hydrophobic ligand. In this report we have prepared site-directed mutations of these two positions of CRABP-II, Arg111 and Arg132, as well as Lys82 to determine the role of these residues in the binding of RA. Recombinant wild type and mutant CRABP-II proteins were expressed and purified, and the affinity for retinoids was determined by fluorometric titration and binding of 3H-labeled compounds. K82A displayed an identical Kd for all-trans-RA as wild type CRABP-II and the Kd for all-trans-RA of R111A was only slightly higher. On the other hand, the two Arg132 mutants, R132A and R132Q, of CRABP-II demonstrated undetectable binding of all-trans-RA. Taken together these data demonstrate that Arg132 is a critical amino acid residue for the binding of RA by CRABP-II.

Animals↗

PubMed1995

Tyrosine protein kinase expression in long-term quiescent WI-38 cells following growth factor simulation.

We have used the WI-38 cell long-term quiescent model system to study the regulation of cell cycle progression at the molecular level. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Stimulation of long- and short-term density arrested WI-38 cells with different growth factors or higher concentrations of individual growth factors does not alter the time required by long-term cells to enter S after stimulation. However, the time during the prereplicative period for which these growth factors are needed is different. Long-term quiescent WI-38 cells require EGF to traverse the G-0/G-1 border but do not need and apparently cannot respond to IGF-1 during the first 10 h after EGF stimulation, the length of the prolongation of the prereplicative phase. This suggests that EGF stimulation of long-term quiescent WI-38 cells initiates a series of molecular events which make these cells "competent" to respond to the "progression" growth factor, IGF-1. In light of the well-established role of protein tyrosine kinases in signal transduction, we set out to identify, clone, and analyze the expression of receptor and non-receptor tyrosine kinases which potentially could play a role during the prolongation of the prereplicative phase in making the long-term quiescent WI-38 cells competent to respond to IGF-1. We obtained 49 clones representing 11 different receptor and non-receptor type protein tyrosine kinases. Analysis of expression of these clones revealed a variety of different patterns of expression. However, the most striking pattern was exhibited by IGF-1 receptor. Our results suggest that induction of IGF-1 receptor mRNA by EGF may be an important event in the establishment of competence by EGF in long-term density arrested WI-38 cells.

Amino Acid Sequence↗

PubMed1995

Regulation of human ornithine decarboxylase expression following prolonged quiescence: role for the c-Myc/Max protein complex.

WI-38 cells can remain quiescent for long periods of time and still be induced to reenter the cell cycle by the addition of fresh serum. However, the longer these cells remain growth arrested, the more time they require to enter S phase. This prolongation of the prereplicative phase has been localized to a point early in G1, after the induction of "immediate early" G1 genes such as c-fos and c-jun but before maximal expression of "early" G1 genes such as ornithine decarboxylase (ODC). Understanding the molecular basis for ODC mRNA induction can therefore provide information about the molecular events which regulate the progression of cells out of long-term quiescence into G1 and subsequently into DNA synthesis. Studies utilizing electrophoretic mobility shift assays (EMSA) of nuclear extracts from short- and long-term quiescent WI-38 cells identified a region of the human ODC promoter at -491 bp to -474 bp which exhibited a protein binding pattern that correlated with the temporal pattern of ODC mRNA expression. The presence of a CACGTG element within this fragment, studies with antibodies against c-Myc and Max, the use of purified recombinant c-Myc protein in the mobility shift assay, and antisense studies suggest that these proteins can specifically bind this portion of the human ODC promoter in a manner consistent with growth-associated modulation of the expression of ODC and other early G1 genes following prolonged quiescence. These studies suggest a role for the c-Myc/Max protein complex in regulating events involved in the progression of cells out of long-term quiescence into G1 and subsequently into S.

Base Sequence↗

PubMed1995

Endothelin-1 induces gene expression through stimulation of endothelin type A receptors in normal rat kidney cells.

RNA blots of total cellular RNA isolated from quiescent and endothelin (ET-1)-stimulated normal rat kidney (NRK) cells demonstrated that ET-1 induced the expression of c-jun, jun B, and c-fos mRNA in a time-dependent manner with maximal expression of mRNA by 1 hr after the addition of ET-1. Five hundred picomolar ET-1 was sufficient to induce maximal mRNA expression. These data agreed with saturation experiments which demonstrated that maximal binding of [125I]ET-1 was achieved at concentrations greater than 100 pM. The Kd and Bmax values for [125I]ET-1 binding to NRK membranes were 20.5 pM and 22.2 fmol/mg protein, respectively. Competition experiments for the binding of [125I]ET-1 to NRK membranes demonstrated that ET-1 was a more potent inhibitor (Ki = 0.047 nM) than ET-3 (Ki = 10.8 nM). No specific binding of [125I]ET-3 (40 or 500 pM) to NRK membranes could be observed. The expression of c-jun, jun B, and c-fos mRNA was inhibited by the endothelin type A receptor (ET)-selective antagonist, BQ-123. Thus, these data demonstrate that ET-1 mediates the expression of immediate response gene mRNA in NRK cells via the ETA receptor. ET-1 stimulation of NRK cells also upregulated EGF receptors, providing a possible mechanism for ET-1 complementation of epidermal growth factor (EGF) mitogenicity in NRK cells.

Animals↗

PubMed1995

Effect of 9-cis-retinoic acid on growth and RXR expression in human breast cancer cells.

A number of studies have demonstrated the ability of retinoic acid (RA) to inhibit the growth of estrogen receptor-positive (ER+) human breast cancer cell lines. The precise mechanism of growth inhibition is not known. However, the biological effects of RA in other model systems have been shown to be mediated via the nuclear retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). While several laboratories have examined the expression of RARs in various breast cancer cell lines, no information is available concerning the role of the RXRs and 9-cis-RA, the natural ligand of RXRs, in the response of breast cancer cells to RA. Using a representative panel of breast cancer cell lines, we determined the effect of 9-cis-RA on growth and cell cycle stage distribution, analyzed steady-state mRNA levels of RXR-alpha, -beta, and -gamma, and determined the effect of all-trans-RA and 9-cis-RA on RXR expression. Our results show that: (1) the growth of ER+/RA-sensitive breast cancer cells is inhibited by treatment with 9-cis-RA by blocking entry into S phase; (2) both ER+/RA-sensitive and ER-/RA-resistant breast cancer cell lines express RXR-alpha and RXR-beta mRNAs but not RXR-gamma; however, levels of these transcripts did not correlate with the RA response; and (3) levels of RXR-alpha and RXR-beta mRNA were not significantly altered following treatment with either all-trans-RA or 9-cis-RA. These results suggest that the mechanism responsible for the retinoid sensitivity of breast cancer cells does not involve transcriptional modulation of the RXRs by RA.

Breast Neoplasms↗

PubMed1995

Retinoids as teratogens.

Vitamin A is a necessary nutrient in the diet. However, excessive doses of retinoids by pregnant women result in teratogenesis. In this chapter, we initially discuss the occurrence and characteristics of fetal malformations associated with maternal ingestion of natural and synthetic retinoids in both experimental animals and humans. We then turn to an examination of the pharmacology of teratogenic retinoids, focusing on structure-function relationships and pharmacokinetics. Finally, we review the current literature on the molecular mechanism of action of teratogenic doses of retinoids and the role of the retinoic acid receptors and other target genes in this process.

Abnormalities, Drug-Induced↗

PubMed1995

Modulation of limb bud chondrogenesis by retinoic acid and retinoic acid receptors.

An excess of retinoic acid (RA) in the mouse embryo in utero produces hypochondrogenesis and severe limb bone deformities. Since one of the RA receptors--RAR-beta 2, is specifically induced in the limb bud cells upon treatment of embryos with teratogenic doses of RA, we investigated if this receptor played a role in teratogenesis by regulating the process of chondrogenesis. In micromass cultures of mouse limb bud mesenchymal cells, we found that a downregulation of RAR-beta 2 as well as several other RAR isoforms by supplementation of the culture medium with specific oligodeoxynucleotides stimulated chondrogenesis: cartilage nodule number, sulfated proteoglycans, and synthesis of collagen type IIB were all enhanced in a dose-dependent manner. However, only the antisense RAR-beta 2 probe efficiently prevented the strong inhibitory effects of exogenous RA on chondrogenesis in these cells. The data suggest that the RAR-RA complexes play a role in position-dependent patterning of the limb skeleton in normal development and that, in particular, RAR-beta 2 serves to prevent the mesenchymal cells from expressing their chondrogenic bias. Our results further strengthen the argument that RA-dependent elevation in RAR-beta 2 levels plays a unique role in RA-induced teratogenesis.

Animals↗

PubMed1994

Arg269 and Lys220 of retinoic acid receptor-beta are important for the binding of retinoic acid.

Retinoic acid receptors, RARs, are retinoic acid (RA)-inducible transcriptional regulatory proteins which transduce the RA signal at the level of gene expression via a retinoic acid response element. Three subtypes of RARs have been described termed RAR-alpha, RAR-beta, and RAR-gamma. RARs, like other members of the steroid/thyroid hormone receptor family, are composed of six structurally distinct domains, one of which is responsible for binding RA. No structural information is available concerning the nature of the amino acids which are responsible for binding of RA within the ligand binding domain of any RAR. In this report, the role of 2 positively charged amino acids of RAR-beta for binding of RA, Arg269 and Lys220, was examined using site-directed mutagenesis. When compared with wild type RAR-beta, mutation of either Arg269 or Lys220 singly to the small neutral amino acid Ala had only a small effect on both the EC50 value in all-trans-RA and 9-cis-RA transactivation assays and the apparent Kd for all-trans-RA. However, mutation of both of these positively charged amino acids simultaneously to Ala caused a 500- and 100-fold elevation in the EC50 for all-trans-RA and 9-cis-RA, respectively, compared with that of wild type RAR-beta. Similarly, the apparent Kd for all-trans-RA was increased 580-fold when that of the double mutant was compared with that of the wild type RAR-beta. Furthermore, this double mutant RAR-beta acted as a dominant negative mutant when transfected with wild type RAR-alpha, -beta, or -gamma in a RA concentration-dependent fashion. Taken together these data demonstrate the importance of both Arg269 and Lys220 of RAR-beta for the binding of RA, possibly by interacting with the negatively charged carboxyl group of RA.

Amino Acid Sequence↗

PubMed1994

WI-38 cell long-term quiescence model system: a valuable tool to study molecular events that regulate growth.

A number of cell culture model systems have been used to study the regulation of cell cycle progression at the molecular level. In this paper we describe the WI-38 cell long-term quiescence model system. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Through studies aimed at understanding the cause and molecular nature of the prolongation of the prereplicative phase, we have determined that gene expression plays an important role in establishing growth factor "competence" and that once the cell becomes "competent" there is a defined order to the molecular events that follow during the remainder of G-1. More specifically, we have determined that the prolongation represents a delay in the ability of long term quiescent cells to become fully "competent" to respond to growth factors which regulate progression through G-1 into S. This prolongation appears to occur as a result of changes during long term quiescence in the ability of immediate early G-1 specific genes (such as c-myc) to activate the expression of early G-1 specific genes (such as ornithine decarboxylase). While ODC is the first and thus far only growth associated gene identified as a target of c-myc (and the Myc/Max protein complex), it is likely that further studies in this model system will reveal other early G-1 growth regulatory genes. We anticipate that future follow-up studies in this model system will provide additional valuable information about the function of growth-regulatory genes in controlling growth factor responsiveness and cell cycle progression.

Animals↗

PubMed1994

Induction of c-fos and c-jun mRNA at the M/G1 border is required for cell cycle progression.

The proto-oncogenes c-fos and c-jun have been shown in numerous model systems to be induced within minutes of growth factor stimulation, during the G0/G1 transition. In this report we use the mitotic shake-off procedure to generate a population of highly synchronized Swiss 3T3 cells. We show that both of these immediate-early, competence genes are also induced during the M/G1 transition, immediately after completion of mitosis. While c-fos mRNA levels drop to undetectable levels within 2 hr after division, c-jun mRNA levels are maintained at a basal level which is approximately 30% maximum throughout the remainder of G1. In order to access the functional significance of these patterns of c-fos and c-jun expression, antisense oligodeoxynucleotides specific to c-fos or c-jun were added to either actively growing Swiss 3T3 cells or mitotically synchronized cells, and their ability to inhibit DNA synthesis and cell division determined. Our results show that treatment of Swiss 3T3 cells with either c-fos or c-jun antisense oligodeoxynucleotides, while actively growing, during mitosis, or in early G1, results in a reduction in ability to enter S and subsequently divide. This was also true if Swiss 3T3 cells were treated during mid-G1 with c-jun antisense oligodeoxynucleotides. These results demonstrate that the regulation of G1 progression following mitosis is dependent upon the expression and function of the immediate-early, competence proto-oncogenes c-fos and c-jun.

3T3 Cells↗

PubMed1994

Teratogenesis by retinoic acid analogs positively correlates with elevation of retinoic acid receptor-beta 2 mRNA levels in treated embryos.

Retinoic acid (RA) plays an important role during normal embryogenesis, however high doses of RA are teratogenic. Retinoic acid receptor-beta 2 (RAR-beta 2) mRNA and protein levels were previously demonstrated to undergo rapid elevation in susceptible tissues after treatment with teratogenic doses of RA. In this report we compared the effects of a number of retinoids, which represent a wide variety of chemical structures and which differ in their teratogenic potencies, on RAR-beta 2 mRNA levels in mouse embryos 6 hr after treatment. Retinoid treatments which result in a high incidence of limb defects elevated RAR-beta 2 mRNA levels similarly (10-14 fold in the limb buds, 4-8 fold in the head, and 2-4 fold in the remainder of the body). On the other hand, retinoid treatments which cause a low or no incidence of limb defects resulted in minor changes in RAR-beta 2 mRNA levels in each embryonic region. Therefore, a strong positive correlation was found between the elevation of RAR-beta 2 mRNA levels and the retinoids which produce limb defects. This provides further evidence that an elevation of RAR-beta 2 mRNA levels, and subsequently protein levels, is an important event involved in mediating the effects of RA during dysmorphogenesis.

Abnormalities, Drug-Induced↗

PubMed1994

A sustained elevation in retinoic acid receptor-beta 2 mRNA and protein occurs during retinoic acid-induced fetal dysmorphogenesis.

We have previously shown that oral treatment of pregnant mice with all-trans retinoic acid (RA) at doses which cause 100% fetal dysmorphogenesis results in a rapid elevation in the mRNA of one specific isoform of the RA receptor-beta, RAR-beta 2, in susceptible embryonic regions. To further investigate the involvement of RAR-beta 2 mRNA in teratogenesis, we have examined its expression in mouse embryos exposed to marginal/nonteratogenic and teratogenic dosing regimens of both 13-cis RA and all-trans RA. We have found that the mere elevation in embryonic RAR-beta 2 mRNA levels and free retinoid levels is not sufficient to result in dysmorphogenesis. Rather, retinoid-induced dysmorphogenesis of embryos appears to occur only when RAR-beta 2 mRNA and unbound retinoid levels remain elevated for at least 6-9 h following retinoid treatment resulting in a significant and prolonged elevation in RAR-beta protein levels.

Animals↗