Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice

<p>Abstract</p> <p>Background</p> <p>Uterine temporal and dose-dependent histopathologic, morphometric and gene expression responses to the selective estrogen receptor modulator tamoxifen (TAM) were comprehensively examined to further elucidate its estrogen receptor-mediated effects. These results were systematically compared to the effects elicited by the potent estrogen receptor ligand 17α-ethynylestradiol (EE) to identify pathways similarly and uniquely modified by each compound.</p> <p>Results</p> <p>Three daily doses of 100 μg/kg TAM elicited a dose-dependent increase in uterine wet weight (UWW) in immature, ovariectomized C57BL/6 mice at 72 hrs with concurrent increases in luminal epithelial cell height (LECH), luminal circumference and glandular epithelial tubule number. Significant UWW and LECH increases were detected at 24 hrs after a single dose of 100 μg/kg TAM. cDNA microarray analysis identified 2235 differentially expressed genes following a single dose of 100 μg/kg TAM at 2, 4, 8, 12, 18 and 24 hrs, and at 72 hrs after three daily doses (3 × 24 hrs). Functional annotation of differentially expressed genes was associated with cell growth and proliferation, cytoskeletal organization, extracellular matrix modification, nucleotide synthesis, DNA replication, protein synthesis and turnover, lipid metabolism, glycolysis and immunological responses as is expected from the uterotrophic response. Comparative analysis of TAM and EE treatments identified 1209 common, differentially expressed genes, the majority of which exhibited similar profiles despite a temporal delay in TAM elicited responses. However, several conserved and treatment specific responses were identified that are consistent with proliferation (Fos, Cdkn1a, Anapc1), and water imbibition (Slc30a3, Slc30a5) responses elicited by EE.</p> <p>Conclusion</p> <p>Overall, TAM and EE share similar gene expression profiles. However, TAM responses exhibit lower efficacy, while responses unique to EE are consistent with the physiological differences elicited between compounds.</p

Tamoxifen-elicited uterotrophy: cross-species and cross-ligand analysis of the gene expression program

<p>Abstract</p> <p>Background</p> <p>Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level <it>in vivo</it>.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge.</p> <p>Results</p> <p>A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns.</p> <p>Conclusion</p> <p>Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation.</p

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice-9

<p><b>Copyright information:</b></p><p>Taken from "Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice"</p><p>BMC Genomics 2007;8():151-151.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1914052.</p><p></p>) and achieves maximal induction of approximately 5-fold following three daily doses (3 × 24 hrs) of 100 μg/kg TAM. Significant increases (p < 0.05, n = 5) are denoted by an asterisk (*). In contrast, 100 μg/kg EE (positive control) maximally induced uterine wet weight 11-fold (*, p < 0.05, n = 5) with significant water imbibition (#; p < 0.05, n = 3), while TAM only achieved 50% uterotrophic efficacy and no water imbibition. B) A single dose of 100 μg/kg TAM significantly increased uterine wet weight as early as 24 hrs after administration. No significant water imbibition was observed at any time point

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice-4

<p><b>Copyright information:</b></p><p>Taken from "Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice"</p><p>BMC Genomics 2007;8():151-151.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1914052.</p><p></p>lear staining, indicating greater levels of Pcna protein expression, in agreement with the histological assessment and changes in gene expression associated with cell proliferation. Increased Pcna expression is more pronounced in the luminal and glandular epithelium, and stroma (arrows). Tissues were counter-stained with hematoxylin. Images are representative of four biological replicates. Bars represent 20 μm

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice-8

<p><b>Copyright information:</b></p><p>Taken from "Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice"</p><p>BMC Genomics 2007;8():151-151.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1914052.</p><p></p>s non-responsive counterpart. These examples further illustrate that the filtering conditions used were adequate to identify differential responses by TAM and EE

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice-1

<p><b>Copyright information:</b></p><p>Taken from "Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice"</p><p>http://www.biomedcentral.com/1471-2164/8/151</p><p>BMC Genomics 2007;8():151-151.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1914052.</p><p></p> EE treatment induced increases in luminal epithelial cell height. Luminal circumference is increased to a greater degree by EE than TAM. Bars represent 20 μm

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice-2

<p><b>Copyright information:</b></p><p>Taken from "Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice"</p><p>BMC Genomics 2007;8():151-151.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1914052.</p><p></p>expression response especially in comparison to EE elicited gene expression [16] is speculated to be due to the delayed absorption of TAM. Inset numbers indicate the number of features represented by each cluster. Black pseudolines indicate the general profile represented within each cluster

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice-3

<p><b>Copyright information:</b></p><p>Taken from "Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice"</p><p>BMC Genomics 2007;8():151-151.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1914052.</p><p></p>poral patterns of expression. Overall, there was good correlation (average ρ = 0.8) between microarray (lines) and QRT-PCR (bars) data. Examples for six of the genes are illustrated. Statistically significant QRT-PCR differences (p < 0.05, n = 4) due to treatment are denoted by an asterisk (*)