DES alters vaginal epithelial cell fate through downregulation of RUNX1

image of downregulation of RUNX1

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium

2013 Study Abstract

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise.

Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive.

Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1.

BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5′ sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure.

Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism.

This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.

Sources and more information
  • Full text (free access) : Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium, Journal List, HHS Author Manuscripts PMC3947918, 2013 Jul 4.
  • BMP4/ActA-SMAD/RUNX1 signaling is dispensable for expression of ΔNp63 in differentiated vaginal epithelial cells featured image credit PMC3947918/figure/F5.
DES DIETHYLSTILBESTROL RESOURCES

Do breast tissue of women exposed in utero to DES exhibit genetic abnormalities ?

image of breast-neoplasms

In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium

2006 Study Abstract

BACKGROUND
In 1992, the National Cancer Institute (NCI) established the Continuation of Follow-Up of DES-Exposed Cohorts to study the long-term health effects of exposure to diethylstilbestrol (DES). Genetic effects on human breast tissue have not been examined. The authors investigated whether breast tissue of women exposed in utero to DES might exhibit the genetic abnormalities that characterize other DES-associated tumors.

METHODS
Subjects enrolled in the NCI Cohort were queried about breast biopsies or breast cancer diagnoses. Available tissue blocks were obtained for invasive cancers (IC), in situ cancers (CIS), or atypical hyperplasia (AH). Exposure status was blinded, lesions were microdissected, and their DNA was analyzed for microsatellite instability (MI) and loss of heterozygosity (LOH), or allele imbalance (AI), at 20 markers on 9 chromosome arms.

RESULTS
From 31 subjects (22 exposed, 9 unexposed), 273 samples were analyzed (167 normal epithelium, 16 AH, 30 CIS, 60 IC). Exposed and unexposed subjects exhibited no differences in breast cancer risk factors or demographic characteristics, except for age at diagnosis (exposed vs. unexposed: 43.2 vs. 48.8 years of age, P = .02). The authors found that MI was rare and that AI was common, with frequencies consistent with previous reports. The global age-adjusted relative rate (RR) of AI was 1.3, 95% CI = 0.8-2.4. No statistically significant associations were observed after adjustment for risk factors or after stratification by histology or by chromosome arm.

CONCLUSIONS
In utero DES exposure does not appear to significantly increase genomic instability in breast epithelium, as measured by MI and AI. Breast tissue may respond differently from that of the reproductive tract to in utero DES exposure. Consequences of in utero DES exposure on the breast may be mediated by proliferative effects of estrogen.

Sources and more information
  • In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium, Cancer, NCBI PubMed PMID: 16998936, 2006 Nov.
  • Full text: wiley: DOI: 10.1002/cncr.22223,
    Cancer, Volume 107, Issue 9, pages 2122–2126, 1 November 2006.
  • Breast neoplasms featured image credit vmshashi.
DES DIETHYLSTILBESTROL RESOURCES

Effect of neonatal exposure to DES on testicular gene expression

image of tests gene expression

Toxicogenomic effects of neonatal exposure to diethylstilbestrol on mouse testicular gene expression in the long term: a study using cDNA microarray analysis

2002 Study Abstract

We examined the effect of neonatal exposure to diethylstilbestrol (DES) on mouse testicular gene expression, using in-house mouse fetus (day 14.5) cDNA microarrays.

Newborn male ICR mice were exposed to DES (50 microg/mouse/day) from neonatal day 1 to 5. Differential expression was detected in 14 genes in 4-week-old (day 28) mouse testes by cDNA microarray analysis;

  • 11 genes (AI035263, AU080565, AU080361, AU080678, AI131681, AU080631, AA986882, AI037066, AA986537, AI156816, and AI596237) were up-regulated
  • and three genes (AI131656, AI118968, and AI117606) were down-regulated in DES-treated mouse testes.

Higher expression levels of the former eight genes, out of the up-regulated genes picked-up by the microarray, were also confirmed by reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) analysis. However, the differential expression of other genes could not be confirmed. Real-time RT-PCR analysis also revealed that expression levels of the eight genes were still higher in DES-treated testes at 8 and 12 weeks of age.

Our results suggest that cDNA microarray analysis is a useful method by which a large number of gene expressions are simultaneously detected and changes in gene expression are screened. In addition, our results suggest that these genes, whose expressions are changed in the testes of adult mice by fetal or neonatal exposure to exogenous chemicals, might be candidates for predictive biological markers.

Sources and more information
  • Toxicogenomic effects of neonatal exposure to diethylstilbestrol on mouse testicular gene expression in the long term: a study using cDNA microarray analysis, Molecular reproduction and development, NCBI PubMed PMID : 12211056, 2002 Sep.
  • Featured image credit dev.biologists.
DES DIETHYLSTILBESTROL RESOURCES

Hox-a10 and Hox-a11 expression potently repressed by perinatal DES exposure

image of des and hox genes

Promoter CpG methylation of Hoxa-10 and Hoxa-11 in mouse uterus not altered upon neonatal diethylstilbestrol exposure

2001 Study Abstract

Mouse abdominal B-like Hoxa genes are expressed and functionally required in the developing reproductive tracts. Mice lacking either Hoxa-10 or Hoxa-11, two of the AbdB Hoxa genes, exhibit abnormal uterine development similar to that induced by in utero diethylstilbestrol (DES) exposure.

Indeed, uterine Hoxa10 and Hoxa11 expression is potently repressed by perinatal DES exposure, providing a potential molecular mechanism for DES-induced reproductive tract malformations.

We have shown previously that DES can permanently alter uterine lactoferrin gene expression through modulation of the lactoferrin promoter methylation pattern. Here we ask whether a similar mechanism also functions to deregulate uterine Hoxa-10 or Hoxa-11 expression during neonatal DES exposure.

We mapped the Hoxa-10 promoter by cloning a 1.485 kb DNA fragment 5′ of the Hoxa-10 exon1a. A 5′ rapid amplification of cDNA ends (RACE) experiment revealed a transcription start site for the a10-1 transcript.

Functional analysis of the proximal 200-bp sequences demonstrated significant promoter activity, confirming the location of the Hoxa-10 promoter. Moreover, methylation assays performed on eight CpGs in Hoxa-10 and 19 CpGs in Hoxa-11 proximal promoters demonstrated that all these CpGs were highly unmethylated in both control and DES-dosed mice from postnatal day 5 to day 30. Significant methylation around Hoxa10 and Hoxa11 promoters was only observed in DES-induced uterine carcinomas in 18-mo-old mice.

Our results suggest that DES-induced downregulations of Hox-a10 or Hox-a11 gene expression are not associated with methylation changes in their proximal promoters and that gene imprinting by developmental DES exposure may be a gene-specific phenomenon.

Sources and more information
  • Promoter CpG methylation of Hox-a10 and Hox-a11 in mouse uterus not altered upon neonatal diethylstilbestrol exposure, Molecular carcinogenesis, NCBI PubMed PMID : 11746833, 2001 Dec.
  • Featured image credit .researchgate.
DES DIETHYLSTILBESTROL RESOURCES

DES regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR beta

image of DES and ERRβ

DES interacts with ERRα, ERRβ, and ERRγ to suppress coactivator binding and transcription from a reporter gene. DES controls the differentiation of trophoblast cells in culture and in utero

2001 Study Abstract

The orphan nuclear receptor ERR beta is expressed in undifferentiated trophoblast stem cell lines and extraembryonic ectoderm, and genetic ablation of ERR beta results in abnormal trophoblast proliferation and precocious differentiation toward the giant cell lineage.

Here, we show that the synthetic estrogen diethylstilbestrol (DES) promotes coactivator release from ERR beta and inhibits its transcriptional activity. Strikingly, treatment of trophoblast stem cells with DES led to their differentiation toward the polyploid giant cell lineage. In addition, DES-treated pregnant mice exhibited abnormal early placenta development associated with an overabundance of trophoblast giant cells and an absence of diploid trophoblast.

These results define a novel pathway for DES action and provide evidence for steroidlike control of trophoblast development.

Sources and more information
  • Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR beta, Genes and development, NCBI PubMed PMC312665, 2001 Apr.
  • Effect of DES on the morphology of trophoblast tissues featured image credit NCBI.
DES DIETHYLSTILBESTROL RESOURCES

Effects of neonatal DES exposure on c-fos and c-jun protooncogene expression

image of c-fos mRNA expression

Permanent changes in the expression of estrogen-regulated protooncogenes, c-fos and c-jun genes, by neonatal DES exposure

2001 Study Abstract

Quantitative and cell-type-specific expression of c-fos and c-jun genes after 17beta-estradiol (E2) stimulation, was investigated in the uteri of neonatally diethylstilbestrol (DES)-exposed and ovariectomized adult mice (neoDES-mice), employing Northern blot analysis, immunohistochemistry and in situ hybridization.

The c-fos mRNA level before E2 injection (at baseline) was about 2.2-fold higher in neoDES-mice than in vehicle-treated control mice. In controls, E2 treatment transiently increased c-fos mRNA levels, showing a peak value (15.8-fold relative to the baseline) after 2 hours. In neoDES-mice, c-fos mRNA level reached a peak showing a 2.1-fold increase compared with its baseline value 1 hour after E2 injection.  Immunohistochemistry and in situ hybridization revealed that c-fos protein (Fos) and mRNA are induced in the epithelium and vascular endothelium in both groups. Most uterine epithelia of neoDES-mice revealed low sensitivity to the c-fos expression after E2 administration compared with those of vehicle-treated controls, whereas few epithelia showed high c-fos mRNA expression even at baseline.

The c-jun mRNA concentration in the neoDES-mice uteri at baseline was 70% of that in vehicle-treated controls. At 1 hour after E2 injection, c-jun mRNA levels increased 1.8-fold in controls and 1.3-fold in the neoDES-mice relative to each baseline value. There were no significant differences in the distribution pattern of c-jun protein (Jun) and mRNA in the uteri of either groups; E2 stimulated c-jun mRNA expression in the stromal and myometrial cells but suppressed it in the epithelial cells, whereas intensity of c-jun immunostaining increased in the three cell types.

The permanent changes in the expression of estrogen-regulated protooncogenes, c-fos and c-jun genes, by neonatal DES exposure may be responsible for the wide range of abnormalities in the genital tract of mature animals.

Sources and more information
  • Effects of neonatal diethylstilbestrol exposure on c-fos and c-jun protooncogene expression in the mouse uterus, Histology and histopathology, NCBI PubMedPMID: 11193187, 2001 Jan.
  • Mapping of c-fos mRNA expression featured image credit researchgate.
DES DIETHYLSTILBESTROL RESOURCES

Evaluation of the ER-alpha expression , mRNA and protein after DES prenatal exposure

image of ER-alpha

Octylphenol does not mimic diethylstilbestrol-induced oestrogen receptor-alpha expression in the newborn mouse uterine epithelium after prenatal exposure

2000 Study Abstract

This study examined whether the endocrine disruptor octylphenol (OP) mimics the synthetic oestrogen diethylstilbestrol (DES) in ability to induce oestrogen receptor-alpha (ER-alpha) expression in the newborn mouse uterine epithelium after prenatal exposure.

Pregnant mice were given daily s.c. injections with DES (10 or 100 microgram DES/kg maternal wt) or OP (100 or 250 mg/kg maternal wt) or with vehicle alone from day 11.5 to 16.5 of pregnancy.

ER-alpha expression was evaluated on histological sections by detecting ER-alpha mRNA with the in situ hybridization technique and ER-alpha protein using immunohistochemistry. The immunostaining was quantitated using a microspectrophotometer.

Oestrogen-like activity of the DES and OP batches used for in vivo exposure was confirmed in an in vitro assay based on transient gene expression of an oestrogen-dependent reporter plasmid.

In mice exposed prenatally to vehicle alone, the uterine epithelium did not express either ER-alpha mRNA or protein, while both were highly expressed in the stroma. Exposure to either DES dose induced the expression of both ER-alpha mRNA and protein in the epithelium, whereas it was unchanged in the stroma.

In contrast, neither OP dose induced the expression of ER-alpha mRNA or protein in the epithelium and expression was unchanged in the stroma.

Our data stress the importance of in vivo studies when investigating endocrine disruptors.

Sources and more information
  • Octylphenol does not mimic diethylstilbestrol-induced oestrogen receptor-alpha expression in the newborn mouse uterine epithelium after prenatal exposure, The Journal of endocrinology, doi: 10.1677/joe.0.1670029, 2000 Oct.
  • ER alpha featured image credit novusbio.
DES DIETHYLSTILBESTROL RESOURCES

The emergence of molecular gynecology : homeobox and Wnt genes in the female reproductive tract

image of WNT7A Gene in genomic location

Wnt gene family : data evidence about Wnt-7a deregulation in response to pre-natal exposure to DES

2000 Study Abstract

Reproductive tissues respond to steroid hormones and thus are particularly vulnerable to the effects of exogenous steroid ‘mimic’ compounds (endocrine disrupters).

One such endocrine disrupter, diethylstilbestrol (DES), is linked to gynecological cancers and changes in uterine structure that reduce or completely abrogate reproductive competence.

Until recently, little was known about the identity of target genes and signaling pathways involved in pathologies linked to endocrine disrupters such as DES.

We outline genetic, cellular and molecular roles for patterning genes, with emphasis on homeobox and Wnt genes. There is evidence that changes in the expression of Wnt and homeogenes underlie many of the defects induced by DES.

Data obtained from murine systems will likely apply to a broad spectrum of gynecological pathologies involving abnormal cell behaviors ranging from fibroids to malignant tumors. Knowledge garnered from modern molecular genetics should lead to progress in the emerging field of molecular gynecology.

Sources and more information
  • The emergence of molecular gynecology: homeobox and Wnt genes in the female reproductive tract, BioEssays : news and reviews in molecular, cellular and developmental biology, PMID: 10984716, 2000 Oct.
  • Wnt genes and endocrine disruption of the female reproductive tract: a genetic approach, Mol Cell Endocrinol, PMID: 10630399, 1999 Dec.
  • WNT7A Gene in genomic location: bands according to Ensembl, locations according to GeneLoc (and/or Entrez Gene and/or Ensembl if different) featured image credit genecards.
DES DIETHYLSTILBESTROL RESOURCES

Prenatal DES exposure alters Hox gene expression

image of hox genes

In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system

2000 Study Abstract

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities.

Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract.

DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol.

The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes.

Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.

Sources and more information
  • In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system, FASEB journal, NCBI PubMed, PMID : 10834931, 2000 June.
  • Featured image Hox genes credit cnx.
DES DIETHYLSTILBESTROL RESOURCES

DES prenatal exposure : transient altered expression of ER alpha and expression of the p21 gene

image of p53-regulate-p21

Abnormal cell differentiation and p21 expression of endometrial epithelial cells following developmental exposure to diethylstilbestrol (DES)

2000 Study Abstract

Gene expression relevant to abnormal cell differentiation and altered cell cycle in endometrial epithelial cells was investigated immunohistochemically in developing mouse uteri exposed neonatally to diethylstilbestrol (DES).

Female CD-1 mice were given daily s.c. injections of 2 microg of DES in corn oil or were given corn oil alone (control) at 1-5 days of age and euthanatized at 5, 6, 7, 8, 15, and 22 days of age.

The endometrial epithelial cells of DES-treated mice at 5-8 days of age showed enhanced staining intensity for the estrogen receptor alpha (ER alpha), whereas the stromal cells showed decreased staining reaction; the epithelial cells showed that the protein encoded by the c-fos proto-oncogene, which plays a key role in regulating diverse estrogen-related cellular differentiation patterns, was enhanced. These cells also showed increased expression of lactoferrin, a sensitive protein marker of estrogen exposure, although the staining intensity decreased after exposure ended. The stain for p21 protein, a mitotic inhibitor which suppresses cyclin-dependent kinase activity, showed frequent positively stained cells in DES-treated mice at 5-15 days of age, whereas no accumulation of p53 protein of either wild or mutant type was detected immunohistochemically in these cells.

These results indicate that suppressed cell cycle activity of endometrial epithelial cells and abnormal estrogen-related differentiation at the developmental stage following neonatal DES exposure may be caused, in part, by transient altered expression of ER alpha and expression of the p21 gene, which appears to be induced by a p53-independent mechanism.

Sources and more information
  • Abnormal cell differentiation and p21 expression of endometrial epithelial cells following developmental exposure to diethylstilbestrol (DES), Toxicologic pathology, NCBI PubMed, PMID : 10805141, 2000 Mar-Apr.
  • Featured image Activation of p53 by cellular stress and DNA damage credit themedicalbiochemistrypage.
DES DIETHYLSTILBESTROL RESOURCES