Biomedical subjects

D R Soprano

Publications and source records attributed to D R Soprano.

At least 37 records · Page 2Linked to original sources

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

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

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

Mutagenesis of rat liver arginase expressed in Escherichia coli: role of conserved histidines.

Rat liver arginase has been overexpressed in Escherichia coli using a T7-based expression system. The kinetic properties of the recombinant wild-type protein are essentially identical to those of the native rat liver enzyme. The recombinant wild-type protein contains six Mn(II) ions per trimer, in good agreement with results obtained with the fully active native enzyme. However, in contrast to the native enzyme which loses three Mn(II) per trimer upon extended dialysis, the recombinant protein binds Mn(II) tenaciously, and retains six Mn(II) per trimer even after extensive dialysis. Three histidine residues, corresponding to His101, His126, and His141 in the rat liver enzyme, are highly conserved in arginases from evolutionarily divergent species. The replacement of His101 and His126 with Asn by site-directed mutagenesis produced only modest effects on enzymatic activity when measured in the presence of Mn(II) ions. However, EDTA treatment of these mutant enzymes reduced activity to < 0.2% of that for the wild-type enzyme. The activity of wild-type enzyme and the His141 Asn mutant was unaffected by treatment with EDTA. Thus, His101 and His126 are proposed to be ligands to the binuclear Mn(II) center of the enzyme. The His141 Asn mutation produced an enzyme which, in contrast to the native, wild-type, His101 Asn, and His126 Asn arginases, was not inactivated by diethyl pyrocarbonate. These results suggest a catalytic role for His141.

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

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↗

PubMed1994

Regulation of neuronal thyroid hormone receptor alpha 1 mRNA by hydrocortisone, thyroid hormone and retinoic acid.

The regulation of thyroid hormone receptor alpha 1 (TR alpha 1) mRNA by hydrocortisone (HC), thyroid hormone (T3) and retinoic acid (RA) has been studied in mixed and neuronal primary cultures of cells dissociated from fetal rat cerebra. Steady-state levels of TR alpha 1 mRNA were increased as much as 5-fold at 13 days of development by 0.3 microM HC in both mixed and neuronal-enriched cultures. The regulation of TR alpha 1 mRNA by HC appears to be mainly limited to neurons. This conclusion is based on two observations. First, stimulation by HC occurs in cultures highly enriched in neurons at approximately the same time and extent as that seen in mixed-cell cultures (containing neurons, oligodendroglia and astroglia). Second, the stimulation reaches a peak at 13 days in mixed-cell cultures, an age at which neurons but not glia differentiate. In most cases, neither T3 (20 nM) nor RA (10(-7) M) stimulated an increase in TR alpha 1 mRNA steady-state levels. The exception was that RA increased TR alpha 1 mRNA levels at 13 days in culture, but at no other stage of development. In both types of cultures, RA and T3 separately and together produced as much as a complete inhibition of the stimulation by HC. Regulation by T3, when it occurred, was always negative. The fact that T3 can strongly repress the induction of TR alpha 1 mRNA by HC demonstrates that T3 can function through negative cooperativity as a potent regulator of its own receptor in brain.

Animals↗

PubMed1994

Transcriptional regulation of c-Jun expression during late G1/S in normal human cells is lost in human tumor cells.

Previous results from our laboratory have identified a second peak in steady state levels of c-jun mRNA (in addition to the immediate early induction) which occurs at the G1/S border in WI-38 normal human diploid fibroblasts. The present studies were undertaken in an attempt to determine (1) the molecular mechanism responsible for the expression of c-jun in late G1/S, (2) the relationship between this second peak of c-jun mRNA expression and the induction of DNA synthesis and (3) whether this cell cycle specific c-jun expression is deregulated in transformed cells. Our results show that the second peak in steady state levels of c-jun mRNA is the result of new transcription during late G1 and not altered stability of the c-jun mRNA transcribed during G1. We also show that this second peak of expression still occurs even when DNA synthesis is inhibited by either hydroxyurea or aphidicolin. Thus, the second peak precedes and is independent of DNA synthesis. Finally, we find that while two other normal human fibroblast cell lines exhibit a second peak of c-jun mRNA during late G1/S, c-jun expression is not cell cycle-regulated but rather is constitutively expressed in a number of distinct transformed cell lines. Since events occurring throughout G1 are known to regulate cell growth, our results suggest that the extent of regulation of c-jun expression during G1 may affect molecular events which ultimately lead to altered growth control as a result of cellular transformation.

Cell Transformation, Neoplastic↗

PubMed1993

Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex.

The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

Base Sequence↗

PubMed1993

Epidermal growth factor receptors lose ligand binding ability as WI-38 cells progress from short-term to long-term quiescence.

WI-38 cells, density arrested for short periods of time, can be stimulated to re-enter the cell cycle by epidermal growth factor (EGF) alone. However, cells density arrested for longer periods have a prolonged prereplicative phase when serum stimulated and cannot be stimulated by EGF alone. Radio-ligand binding studies performed on WI-38 cells showed that actively growing cells bind [125I]EG at relatively low levels that increase to a maximum as the cells become contact inhibited. As the cells enter a state of deeper quiescence, EGF binding falls to one-third to one-fifth the short-term growth arrested levels, remaining constant thereafter. The EGF-receptor complexes internalize more slowly in long-term growth arrested cells, and the rate of ligand association to the receptor is lower than short-term growth arrested cells. The amount of EGF receptor protein in lysates of equal numbers of both short- and long-term quiescent cells remains the same. These results suggest that the failure of long-term growth arrested cells to respond to EGF is not due to dramatic changes in the amount of receptor protein during prolonged quiescence but more likely to an alteration in the ability of these receptors to bind ligand and/or activate the EGF signal transduction pathway.

Cell Cycle↗