DES amplification of the c-myc gene

image of C-Myc-DNA_complex

Characterization of murine cell lines from diethylstilbestrol-induced uterine endometrial adenocarcinomas

1992 Study Abstract

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice.

Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormone-dependent lesions in 18-mo.-old mice.

Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear cytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli.

After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen.

Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes.

Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three- to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line.

These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.

Sources and more information
  • Characterization of murine cell lines from diethylstilbestrol-induced uterine endometrial adenocarcinomas, In vitro cellular and developmental biology : journal of the Tissue Culture Association, NCBI PubMed PMID: 1597405, 1992 May.
  • C-Myc-DNA_complex featured image credit wikipedia.

DES compromises the DNA repair system, accumulating lesions in the genome

image of DNA repair

Mutations in DNA polymerase beta mRNA of stilbene estrogen-induced kidney tumors in Syrian hamster

1995 Study Abstract

We report here the alteration(s) in the expression of the DNA repair gene, DNA polymerase beta, in kidney tumors induced by stilbene estrogen (diethylstilbestrol, DES).

RT-PCR, slot blotting, and Northern blotting experiments revealed that expression of DNA polymerase beta (DNA pol beta) was several fold lower in stilbene-estrogen-induced kidney tumors than in age-matched controls. Several mutations were identified in DNA pol beta mRNA from DES-induced kidney tumors, but not in age-matched control kidney.

The mutations in DNA pol beta mainly occurred in the catalytic domain of pol beta, and not in the DNA binding domain. All the mutations produced a stop codon at nucleotide 199 indicating that a protein of aberrant size may be synthesized.

These data suggest that mutation of DNA pol beta coupled with attenuation in expression might compromise the DNA repair system. This in turn may allow a greater error rate during DNA repair and the accumulation of lesions in the genome.

Sources and more information
  • Mutations in DNA polymerase beta mRNA of stilbene estrogen-induced kidney tumors in Syrian hamster, Biochemistry and molecular biology international, NCBI PubMed PMID: 8653081, 1995 Sep.
  • DNA Damage Response featured image credit rndsystems.

DES causal role in p53 expression and in estrogen receptor alterations

Diethylstilbestrol-induced immortalization of human endometrial cells: alterations in p53 and estrogen receptor

1996 Study Abstract

Carcinogenesis is a process requiring multiple steps. Immortalization is one step in this process and may be rate limiting.

To further our understanding of estrogen-induced carcinogenesis, we evaluated diethylstilbestrol (DES)-induced immortalization of human endometrial stromal cells.

This was achieved by assessing at the restrictive temperature the colony-forming efficiency of cells that were conditionally immortalized with a temperature-sensitive simian virus 40 large T antigen.

  • Treatment with DES for 1 wk did not increase the immortalization frequency;
  • however, cultures that were treated for 20 wk had a twofold increase in immortalization frequency,
  • and continued treatment for a total of 44 wk produced a threefold increase in immortalization frequency that was dose dependent.

DES-treated restrictive temperature variants (RTVs) but not spontaneous RTVs lost the temperature-sensitive phenotype. DES-RTVs also had a shorter doubling time than spontaneous RTVs did. p53 expression was increased in DES-RTVs, and its localization within the cell was altered. Conversely, expression of the estrogen receptor was decreased in DES-immortalized cells.

These changes in gene expression often occur in estrogen-related malignancies, and our results are consistent with a causal role for estrogens in these p53 and the estrogen receptor alterations. Immortalization of human cells may be analogous to initiation of rodent cells, and our results suggest that estrogen-induced alterations in p53 or other genes that regulate life span could contribute to estrogen-induced initiation.

Sources and more information
  • Diethylstilbestrol-induced immortalization of human endometrial cells: alterations in p53 and estrogen receptor, Molecular carcinogenesis, NCBI PubMed PMID: 8599578, 1996 Feb.
  • Crystal structure of four p53 DNA binding domains featured image credit wikipedia.

Lactoferrin gene regulation by estrogen receptor

Promoter and species specific differential estrogen-mediated gene transcription in the uterus and cultured cells using structurally altered agonists

1997 Study Abstract

Certain types of estrogenic compounds have been shown to have tissue-specific actions. In addition, some tissues may exhibit differential gene regulation by agonists and antagonists.

Our previous studies using structurally modified estrogenic molecules had indicated differential effects on specific estrogen responses, indicating that the activity of the estrogen receptor protein can be altered depending not only upon the structure of the bound ligand but also the regulated gene itself. The mechanism of differential induction, however, was not determined, and might involve altered binding to the estrogen response element (ERE), altered transcription, or post-transcriptional modification of gene products.

Our previous studies indicated that differential induction by modified diethylstilbestrol (DES) agonists could not be accounted for by differences in ligand affinity for the estrogen receptor (ER) or differential binding of the ER to a consensus vitellogenin A2 (vit A2) ERE.

To determine if this differential hormonal responsiveness was reflected at the level of transcription, we analyzed mouse uterine mRNA of several estrogen-responsive genes, including glucose-6-phosphate dehydrogenase (G6PD), ornithine decarboxylase (ODC) and lactoferrin, by Northern blot following injection with the modified agonists DES, indenestrol A (IA), indenestrol B (IB) and Z-pseudo DES (ZPD).

All compounds induced the G6PD message, although IB and ZPD induced expression only transiently, while DES and IA maintained the message for 24 h. No difference in induction was seen for ODC message, which was induced equally by all the compounds. In contrast, lactoferrin, a highly estrogen-responsive gene, was induced only by DES and IA and not by the other agonists IB or ZPD, showing that the lactoferrin gene was differentially regulated by these compounds.

To determine whether this difference was due to altered transcriptional activity, the mouse lactoferrin estrogen-responsive module (mERM) linked to a chloramphenicol acetyl transferase (CAT) reporter gene was tested in transfected cells. Using the mouse estrogen receptor in RL95 cells, DES and IA induced expression of CAT, but IB did not, confirming the differential response seen in vivo.

To show whether this difference in transcription occurred because of altered binding to the lactoferrin ERE, which is not a perfect consensus ERE a gel shift assay was used to examine DNA binding of ER bound to the agonists. All ligands produced equivalent binding to the lactoferrin ERE suggesting that differential regulation was not a result of altered DNA binding.

Taken together, these observations indicate that the differential induction of lactoferrin by these compounds occurs via altered activation of the transcriptional components unique to lactoferrin and is likely to involve altered interaction with co-activators. Surprisingly, unlike the mouse ER, the human estrogen receptor activated and induced expression of lactoferrin estrogen-responsive module-CAT with all the compounds. Mouse ER is also known to vary from the human ER in its activity with the triphenylethylene estrogen tamoxifen, which has agonist activity with the mouse ER but mixed antagonist/agonist activity with the human ER. The data show that human and mouse estrogen receptors are activated differently by this group of stilbestrol estrogen ligands when assayed on the lactoferrin response element, which is the first description of this type of gene and species specific difference.

Lactoferrin gene regulation by estrogen receptor can be used as a model to study the mechanism of differential gene activation by different estrogen agonists and antagonists using a more physiological situation than commonly used with in vitro gene reporter systems.

Sources and more information
  • Promoter and species specific differential estrogen-mediated gene transcription in the uterus and cultured cells using structurally altered agonists, Journal of molecular endocrinology, NCBI PubMed PMID: 9195474, 1997 Jun.
  • Estrogen receptor (ER-alpha) featured image credit wikipedia.

AMH and AMHRII mRNAs expression levels

Effect of Prenatal Exposure to Diethylstilbestrol on Müllerian Duct Development in Fetal Male Mice

1998 Study Abstract

The clinical use of diethylstilbestrol (DES) by pregnant women has resulted in an increased incidence of genital carcinoma in the daughters born from these pregnancies. Also, in the so-called DES-sons abnormalities were found, mainly, the presence of Müllerian duct remnants, which indicates that fetal exposure to DES may have an effect on male sex differentiation.

Fetal regression of the Müllerian ducts is under testicular control through anti-Müllerian hormone (AMH). In male mice, treated in utero with DES, the Müllerian ducts do not regress completely, although DES-exposed testes do produce AMH.

We hypothesized that incomplete regression in DES-exposed males is caused by a diminished sensitivity of the Müllerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Müllerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was studied.

It was observed that Müllerian duct regression was incomplete at E19 (19 days post coitum), upon DES administration during pregnancy from E9 through E16. Furthermore, analysis of earlier time points of fetal development revealed that the DES treatment had clearly delayed the onset of Müllerian duct formation by approximately 2 days; in untreated fetuses, Müllerian duct formation was complete by E13, whereas fully formed Müllerian ducts were not observed in DES-treated male fetuses until E15.

Using in situ hybridization, no change in the localization of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protection assay, showing an increased expression level of AMH and AMHRII mRNAs at E 13 in DES-exposed male fetuses. Furthermore, the mRNA expression levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development.

Prenatal DES exposure had no effect on Hoxa 11 mRNA expression, indicating that DES did not exert an overall effect on the rate of fetal development. In DES-exposed male fetuses, SF-1 showed a similar increase in mRNA expression as AMH, in agreement with the observations that the AMH gene promoter requires an intact SF-1 DNA binding site for time- and cell-specific expression, although an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increased expression levels of AMH and AMHRII mRNAs do not directly explain the decreased sensitivity of the Müllerian ducts to AMH.

Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Müllerian duct development, which may result in an asynchrony in the timing of Müllerian duct formation, with respect to the critical period of Müllerian duct regression, leading to persistence of Müllerian duct remnants in male mice.

Sources and more information
  • Full study (free access) : Effect of prenatal exposure to diethylstilbestrol on Müllerian duct development in fetal male mice, Endocrinology, NCBI PubMed PMID: 9751506, 1998 Oct;.

Estrogen receptor nature influence whether DES responsive genes are activated

image of ERalpha and ERbeta

Differential activation by diethystilbestrol of ER alpha and ER beta when linked to different response elements

1998 Study Abstract

The discovery of a second estrogen receptor (ER beta) has significant implications for our understanding of the molecular basis for the diverse actions of estrogen.

Here we report the differential activation by natural and xenobiotic estrogens of ER alpha and ER beta when linked to different response elements.

Receptor mediated activation of reporter constructs containing either the estrogen response element (ERE) from the vitellogenin (Vit) gene or from the luteinizing hormone beta (LH) gene were examined in transiently transfected Cos-1 cells.

ER beta preferentially activated the consensus Vit ERE whereas ER alpha showed greater activation at the divergent LH ERE. This differential activation was observed for a number of ligands including estradiol, estrone, bisphenol A, octylphenol and diethystilbestrol.

These findings show that the nature of the ERE, as well as the ratio of ER subtypes in a particular cell/tissue, will influence whether particular estrogen responsive genes are activated in the presence of natural or xenobiotic estrogens.

Sources and more information
  • Differential activation by xenoestrogens of ER alpha and ER beta when linked to different response elements, The Journal of endocrinology, NCBI PubMed PMID: 9846177, 1998 Sep.
  • Estrogen receptor featured image credit slideserve.

DES induces structural chromosome aberrations

image of chromosome aberrations

Isolation of DNA probes specific for rat chromosomal regions 19p, 19q and 4q and their application for the analysis of diethylstilbestrol-induced aneuploidy in binucleated rat fibroblasts

2000 Study Abstract

DNA probes specific for rat chromosomes 19p, 19q and 4q were isolated, characterized and used for the detection and analysis of diethylstilbestrol (DES)-induced aneuploidy.

By denaturing and partially reassociating total genomic DNA a new rat repetitive DNA family was isolated, which was located on chromosome 19p21. Sequencing of a number of subclones from cos76-1 and other clones of this so-called 76-family revealed that the repeat units are interrupted with large areas of other (unique) DNA.

Consequently, after fluorescence in situ hybridization (FISH) the signals in interphase nuclei are large and spread out. The other two probes, cos25 (chromosome 4q) and cos42-47 (chromosome 19q), were isolated by screening cosmid libraries with probes isolated previously in our laboratory.

The repeat unit of cos25 is a 2174 bp long EcoRI unit that contains three Sau3A sites and is tandemly organized.

Sequencing of subclones of cos42-47 revealed that this probe was in fact the 5S RNA gene, located on 19q12.

In order to determine if these probes were suitable probes for aneuploidy detection, two series of dual colour FISH with the combinations cos25/cos76-1 (4q/19p) and cos42-47/cos76-1 (19q/19p) were carried out on slides from an in vitro micronucleus assay with DES.

With all three probes used, an increase in binucleated cells with non-disjunction or chromosome loss was observed in the DES-treated cultures.

Scoring of additional micronucleated cells on slides hybridized with the cos25/cos76-1 (4q/19p) probes revealed that the hybridization signal of probe cos25 (4q) was over-represented in the micronuclei of the control cultures.

The simultaneous use of the 19q and 19p probes is a particularly valuable tool for the detection of aneuploidy, since it allows distinction between aneugenic and clastogenic events in binucleated cells.

Results of this analysis showed that apart from aneuploidy, DES also induced structural chromosome aberrations, although to a lesser extent.

Sources and more information
  • Isolation of DNA probes specific for rat chromosomal regions 19p, 19q and 4q and their application for the analysis of diethylstilbestrol-induced aneuploidy in binucleated rat fibroblasts, Mutagenesis, NCBI PubMed PMID: 10719043, 2000 Mar.
  • Chromosomal Aberrations featured image credit atlasgeneticsoncology.

DES must be considered genotoxic agent

Estrogens as endogenous genotoxic agents–DNA adducts and mutations

2000 Study Abstract

Estrogens induce tumors in laboratory animals and have been associated with breast and uterine cancers in humans. In relation to the role of estrogens in the induction of cancer, we examine

  • formation of DNA adducts by reactive electrophilic estrogen metabolites,
  • formation of reactive oxygen species by estrogens and the resulting indirect DNA damage by these oxidants,
  • and, finally, genomic and gene mutations induced by estrogens.

Quinone intermediates derived by oxidation of the catechol estrogens 4-hydroxyestradiol or 4-hydroxyestrone may react with purine bases of DNA to form depurinating adducts that generate highly mutagenic apurinic sites. In contrast, quinones of 2-hydroxylated estrogens produce less harmful, stable DNA adducts. The catechol estrogen metabolites may also generate potentially mutagenic oxygen radicals by metabolic redox cycling or other mechanisms.

Several types of indirect DNA damage are caused by estrogen-induced oxidants, such as oxidized DNA bases, DNA strand breakage, and adduct formation by reactive aldehydes derived from lipid hydroperoxides.

Estradiol and the synthetic estrogen diethylstilbestrol also induce numerical and structural chromosomal aberrations and several types of gene mutations in cells in culture and in vivo.

In conclusion, estrogens, including the natural hormones estradiol and estrone, must be considered genotoxic carcinogens on the basis of the evidence outlined in this chapter.

Sources and more information
  • Estrogens as endogenous genotoxic agents–DNA adducts and mutations, Journal of the National Cancer Institute, NCBI PubMed PMID: 10963621, 2000.
  • A model of the steps required for checkpoint adaptation after treatment with pharmacological concentrations of genotoxic agents featured image credit semanticscholar.

Ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins

image of ERalpha and ERbeta

Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta

2000 Study Abstract

It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) alpha and/or ERbeta following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ERalpha and/or ERbeta subsequent to receptor binding.

In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ERalpha and ERbeta to recruit TIF2 and SRC-1a in the presence of 17beta-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2′,3′,4′, 5′-tetrachlorobiphenyl-ol, and bisphenol A.

We show that ligand-dependent differences exist in the ability of ERalpha and ERbeta to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ERbeta (over ERalpha) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ERbeta to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2.

We conclude that ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens.

Sources and more information
  • Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta, The Journal of biological chemistry, NCBI PubMed PMID: 10964929, 2000 Nov.
  • Mitochondrial DNA featured image credit zmescience.

DES-induced mitochondrial structural abnormality

Base sequence-specific attack of stilbene estrogen metabolite(s) on the mitochondrial DNA: implications in the induction of instability in the mitochondrial genome in the kidney of Syrian hamsters

2001 Study Abstract

We have demonstrated previously that diethylstilbestrol is metabolized to diethylstilbestrol reactive metabolites by mitochondrial enzymes in vitro. In vitro, these reactive intermediates bind to mitochondrial DNA.

Here we have investigated

  • the in vivo formation of diethylstilbestrol adducts with mitochondrial DNA,
  • the nature of mitochondrial DNA-diethylstilbestrol adducts,
  • and the influence of diethylstilbestrol adduction on in vitro replication of a mitochondrial gene.

Diethylstilbestrol administration to male hamsters produced several adducts in mitochondrial DNA of both kidney and liver. The total relative adduct levels were 5- to 6-fold higher in mitochondrial DNA than in nuclear DNA. The chromatographic mobility of mitochondrial DNA adducts formed in vivo were similar to that of dGMP-DES quinone adducts formed in vitro. The identity of mitochondrial DNA adducts formed in vivo was further confirmed as dGMP-diethylstilbestrol quinone adducts by rechromatography and cochromatography. Using a DNA polymerase arrest assay we found that the DES quinone attack on a mitochondrial respiratory gene, i.e., the gene for subunit III of cytochrome c oxidase (COIII), was specific for guanine residues that were adjacent to cytosine residues. Long-term treatment with diethylstilbestrol produced tumors in the kidney, and the level of COIII transcripts was 5- to 10-fold higher in tumor samples than age-matched control kidneys.

These findings suggest that

  • mitochondrial DNA appears more susceptible to formation of diethylstilbestrol adducts than nuclear DNA,
  • the DNA adducts formed by DES were predominantly with guanines,
  • the adducted bases stopped DNA polymerase-mediated in vitro replication of the COIII gene,
  • and long-term exposure of hamsters to diethylstilbestrol elevated the expression of COIII mRNA.

These results suggest that obstruction of replication of the mitochondrial genes by covalent modifications of the mitochondrial DNA by diethylstilbestrol may produce mitochondrial genomic instability in vivo and may provide an explanation for the DES-induced mitochondrial structural abnormality.

Sources and more information
  • Base sequence-specific attack of stilbene estrogen metabolite(s) on the mitochondrial DNA: implications in the induction of instability in the mitochondrial genome in the kidney of Syrian hamsters, International journal of molecular medicine, NCBI PubMed PMID: 11254879, 2001 April.
  • Mitochondrial DNA featured image credit zmescience.