Functional Analysis of Haplotypes and Promoter Activity at the 5’ Region of the ADH7 Gene

Abstract Background: The function of the 5’ regulatory region and the role of the different SNP loci have not been well characterized. This study investigated the effect of several ADH7 haplotypes on the regulation of gene expression in vitro and the functional sequences in the 5’ regulatory region of ADH7. Three SNPs (rs17537595, rs2851028, and rs2654847) and four different haplotypes (T-C-T, T-T-T, C-T-T, T-C-A) were identified by cloning and sequencing. Methods: Effects of 4 different haplotypes and 8 truncated fragments of 5’ regulatory region on ADH7 gene expression were detected using a dual-luciferase reporter assay system. All recombinant plasmids were transfected into HEK-293, U87, and SH-SY5Y cells, respectively, and their relative fluorescence intensity was measured.Results: In HEK-293, U87, and SH-SY5Y cell lines, the relative fluorescence intensity of haplotype T-C-T was significantly higher than that of haplotype T-T-T, C-T-T, and T-C-A. Additionally, we found that regions from -83 to -310bp (ATG, +1), -560 to -768bp , and -987bp to -1203bp up-regulated gene expression. In contrast, the region from -768 to -987bp down-regulated gene expression. The gene expression of regions from -1203 to -1369bp and -1369 to -1626bp was down-regulated in U87 and SH-SY5Y cell lines, but the trend was opposite in HEK-293 cell line. The region from -310 to -560bp up-regulated gene expression in SH-SY5Y cell line, but down-regulated gene expression in HEK-293 and U87 cell lines.Conclusions: This study has shown that the polymorphisms of ADH7 5’ regulatory region play an important role in the regulation of gene expression.

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Functional analysis of haplotypes and promoter activity at the 5' region of the DRD2 gene and associations with schizophrenia.

10.21203/rs.3.rs-15812/v1 ◽

2020 ◽

Author(s):

Xicen Zhang ◽

Mei Ding ◽

Yi Liu ◽

Yongping Liu ◽

Jiaxin Xing ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Promoter Region ◽

Regulatory Region ◽

Gene Promoter ◽

Luciferase Reporter ◽

Drd2 Gene ◽

Functional Regions

Abstract Background: In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing. rs7116768 and rs1799978 were found to be slightly associated with schizophrenia. This study investigated the effects of haplotypes consisted of the four SNPs on protein expression level in vitro and identified the functional sequence in the 5’ regulatory region of DRD2 gene which has a potential link with schizophrenia.Methods: Recombinant plasmids with haplotypes, SNPs and 13 recombinant vectors containing deletion fragments from the DRD2 gene 5' regulatory region were transfected into HEK293 and SK-N-SH cell lines. Relative luciferase activity of the haplotypes, SNPs and different sequences was compared using a dual luciferase reporter assay system.Results: Haplotype H4(G-C-InsC-G) could significantly increase the gene expression in SK-N-SH cell lines. Allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate the gene expression. There were 5~7 functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.Conclusion: We cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.

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Functional polymorphisms and transcriptional analysis in the 5' region of the human serotonin receptor 1B gene (HTR1B) and their associations with psychiatric disorders

10.21203/rs.3.rs-21296/v3 ◽

2020 ◽

Author(s):

Xi Xia ◽

Mei Ding ◽

Jin-feng Xuan ◽

Jia-xin Xing ◽

Jun Yao ◽

...

Keyword(s):

Gene Expression ◽

Behavioral Disorders ◽

Association Studies ◽

Regulation Of Gene Expression ◽

Regulatory Region ◽

Transcriptional Analysis ◽

Luciferase Reporter ◽

Genome Wide Association Studies ◽

Functional Polymorphisms ◽

Mental And Behavioral Disorders

Abstract Background The 5-hydroxytryptamine 1B receptor (5-HT1B) plays an essential role in the serotonin (5-HT) system and is widely involved in a variety of brain activities. HTR1B is the gene encoding 5-HT1B. Genome-wide association studies have shown that HTR1B polymorphisms are closely related to multiple mental and behavioral disorders; however, the functional mechanisms underlying these associations are unknown. This study investigated the effect of several HTR1B haplotypes on regulation of gene expression in vitro and the functional sequences in the 5' regulatory region of HTR1B to determine their potential association with mental and behavioral disorders.MethodsSix haplotypes consisting of rs4140535, rs1778258, rs17273700, rs1228814, rs11568817, and rs130058 and several truncated fragments of the 5' regulatory region of HTR1B were transfected into SK-N-SH and HEK-293 cells. The relative fluorescence intensities of the different haplotypes and truncated fragments were detected using a dual-luciferase reporter assay system.Results Compared to the major haplotype T-G-T-C-T-A, the relative fluorescence intensities of haplotypes C-A-T-C-T-A, C-G-T-C-T-A, C-G-C-A-G-T, and C-G-T-A-T-A were significantly lower, and that of haplotype C-G-C-A-G-A was significantly higher. Furthermore, the effects of the rs4140535T allele, the rs17273700C-rs11568817G linkage combination, and the rs1228814A allele made their relative fluorescence intensities significantly higher than their counterparts at each locus. Conversely, the rs1778258A and rs130058T alleles decreased the relative fluorescence intensities. In addition, we found that regions from -1587 to -1371 bp (TSS, +1), -1149 to -894 bp, -39 to +130 bp, +130 to +341 bp, and +341 to +505 bp upregulated gene expression. In contrast, regions -603 to -316 bp and +130 to +341 bp downregulated gene expression. Region +341 to +505 bp played a decisive role in gene transcription.Conclusions HTR1B 5' regulatory region polymorphisms have regulatory effects on gene expression and potential correlate with several pathology and physiology conditions. This study suggests that a crucial sequence for transcription is located in region +341~+505 bp. Regions -1587 to -1371 bp, -1149 to -894 bp, -603 to -316 bp, -39 to +130 bp, and +130 to +341 bp contain functional sequences that can promote or suppress the HTR1B gene expression.

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Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene

Biochemical Journal ◽

10.1042/bj20050973 ◽

2005 ◽

Vol 393 (1) ◽

pp. 321-329 ◽

Cited By ~ 7

Author(s):

Antonella De Luca ◽

Paolo Sacchetta ◽

Carmine Di Ilio ◽

Bartolo Favaloro

Keyword(s):

Cell Lines ◽

Transcription Initiation ◽

Promoter Region ◽

Promoter Activity ◽

Regulatory Region ◽

Initiation Site ◽

Luciferase Reporter ◽

Mcf7 Cells ◽

Luciferase Reporter Gene ◽

Hek 293

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5′ promoter region of the human msrA gene. Using 5′-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank® accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between −209 and −212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.

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Functional polymorphisms and transcriptional analysis in the 5' region of the human serotonin receptor 1B gene (HTR1B) and their associations with psychiatric disorders

10.21203/rs.3.rs-21296/v4 ◽

2020 ◽

Author(s):

Xi Xia ◽

Mei Ding ◽

Jin-feng Xuan ◽

Jia-xin Xing ◽

Jun Yao ◽

...

Keyword(s):

Gene Expression ◽

Behavioral Disorders ◽

Association Studies ◽

Regulation Of Gene Expression ◽

Regulatory Region ◽

Transcriptional Analysis ◽

Luciferase Reporter ◽

Genome Wide Association Studies ◽

Functional Polymorphisms ◽

Mental And Behavioral Disorders

Abstract Background The 5-hydroxytryptamine 1B receptor (5-HT1B) plays an essential role in the serotonin (5-HT) system and is widely involved in a variety of brain activities. HTR1B is the gene encoding 5-HT1B. Genome-wide association studies have shown that HTR1B polymorphisms are closely related to multiple mental and behavioral disorders; however, the functional mechanisms underlying these associations are unknown. This study investigated the effect of several HTR1B haplotypes on regulation of gene expression in vitro and the functional sequences in the 5' regulatory region of HTR1B to determine their potential association with mental and behavioral disorders. Methods Six haplotypes consisting of rs4140535, rs1778258, rs17273700, rs1228814, rs11568817, and rs130058 and several truncated fragments of the 5' regulatory region of HTR1B were transfected into SK-N-SH and HEK-293 cells. The relative fluorescence intensities of the different haplotypes and truncated fragments were detected using a dual-luciferase reporter assay system. Results Compared to the major haplotype T-G-T-C-T-A, the relative fluorescence intensities of haplotypes C-A-T-C-T-A, C-G-T-C-T-A, C-G-C-A-G-T, and C-G-T-A-T-A were significantly lower, and that of haplotype C-G-C-A-G-A was significantly higher. Furthermore, the effects of the rs4140535T allele, the rs17273700C-rs11568817G linkage combination, and the rs1228814A allele made their relative fluorescence intensities significantly higher than their counterparts at each locus. Conversely, the rs1778258A and rs130058T alleles decreased the relative fluorescence intensities. In addition, we found that regions from -1587 to -1371 bp (TSS, +1), -1149 to -894 bp, -39 to +130 bp, +130 to +341 bp, and +341 to +505 bp upregulated gene expression. In contrast, regions -603 to -316 bp and +130 to +341 bp downregulated gene expression. Region +341 to +505 bp played a decisive role in gene transcription. Conclusions HTR1B 5' regulatory region polymorphisms have regulatory effects on gene expression and potential correlate with several pathology and physiology conditions. This study suggests that a crucial sequence for transcription is located in region +341~+505 bp. Regions -1587 to -1371 bp, -1149 to -894 bp, -603 to -316 bp, -39 to +130 bp, and +130 to +341 bp contain functional sequences that can promote or suppress the HTR1B gene expression.

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Expression-Based Screen Identifies the Calcium Channel Antagonist Bepridil as a Notch1 Modulator in T-ALL.

Blood ◽

10.1182/blood.v114.22.366.366 ◽

2009 ◽

Vol 114 (22) ◽

pp. 366-366 ◽

Cited By ~ 3

Author(s):

Giovanni Roti ◽

Kenneth N. Ross ◽

Adolfo A. Ferrando ◽

Stephen C Blacklow ◽

Jon Aster ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Calcium Channel ◽

Cell Line ◽

Cell Lines ◽

Small Molecule ◽

Lymphoblastic Leukemia ◽

Receptor Activation ◽

Luciferase Reporter ◽

Small Molecule Library

Abstract Abstract 366 Gain of function mutations in Notch1, which encodes a signaling protein that is converted into a transcription factor upon activation, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Although inhibiting Notch1 activity represents a potential therapeutic opportunity, the discovery of new Notch1 pathway antagonists poses a difficult challenge. Traditional small molecule library screening approaches have not been amenable to modulating transcription factor abnormalities. In order to overcome this challenge, we applied Gene Expression-based High-throughput Screening (GE-HTS) to discover new Notch1 modulators. GE-HTS uses gene expression signatures as surrogates for different biological states. We derived a 32-gene Notch1 expression signature from genome-wide microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a Notch1 inactivating γ-secretase inhibitor (GSI; Notch off) and screened a small molecule library for compounds inducing the Notch1 off state in DND41 mutant Notch1 T-ALL cells. Among numerous ion flux modulators validated to induce the Notch1 off signature, one of the top hits was the FDA-approved calcium channel blocker, bepridil, used to treat patients with cardiac disease. In multiple mutant Notch1 T-ALL cell lines, bepridil induced the Notch1 off signature. We next confirmed that bepridil indeed targets Notch signaling by demonstrating its inhibitory effects on a Notch-sensitive luciferase reporter gene in heterologous U2OS cells expressing a mutated form of Notch1. Similar to the phenotypic effects of GSI, bepridil induced a G0/G1 cell cycle arrest, inhibited cellular viability, and decreased cell size in multiple T-ALL cell lines, including the GSI-resistant cell line PF382. Next, in order to confirm dependency of the induced phenotype on inhibition of Notch, we utilized the 8946 T-ALL cell line. This murine line depends on a doxycycline-repressible human c-myc transgene for growth and can be rescued from transgene withdrawal with activated Notch1, which upregulates the endogenous c-myc gene. In these cells, the phenotypic effect of bepridil on viability is also dependent on Notch1 inhibition because cells rescued from transgene withdrawal with activated Notch1 were more sensitive to the effects of the drug than were those cells still dependent on the c-myc transgene. Finally, we asked whether bepridil altered the level of active Notch1 protein in T-ALL cell lines. As with GSI, bepridil treatment results in decreased levels of intracellular Notch (ICN1). In contrast to GSI, however, bepridil treatment decreased levels of the furin-processed extracellular and transmembrane forms of Notch1 while the full length Notch1 precursor form accumulated upon bepridil treatment. One hypothesis is that by altering ER/Golgi compartment calcium, bepridil prevents the folding of newly synthesized Notch1 polypeptides, leading to its retention in the ER/Golgi and a failure to traffic to cellular compartments where receptor activation occurs. Consistent with this hypothesis co-localization studies in U2OS cell lines expressing the L1601P mutant Notch1 suggest retention of Notch1 in the ER/Golgi. An alternative hypothesis under investigation is that bepridil affects the activity of furin, a calcium-dependent protease that is required for processing of Notch receptors. In summary, we have identified an FDA-approved drug with Notch1 modulating activity in T-ALL by a mechanism unique from GSI. These studies have potential for rapid translation to clinical testing. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

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Transcription Factor CEBPB Inhibits the Expression of the Human HTR1A by Binding to 5’ Regulatory Region in Vitro

Genes ◽

10.3390/genes10100802 ◽

2019 ◽

Vol 10 (10) ◽

pp. 802

Author(s):

Liu ◽

Wu ◽

Meng ◽

Ding ◽

Xu ◽

...

Keyword(s):

Transcription Factor ◽

Fluorescence Intensity ◽

Mrna Level ◽

Regulatory Region ◽

Receptor Expression ◽

Relative Fluorescence Intensity ◽

Mobility Shift ◽

Hek 293 ◽

Endogenous Expression

This study identified a transcription factor that might bind to the 5’ regulatory region of the HTR1A and explored the potential effect on 5-HT1A receptor expression. Based on JASPAR predictions, the binding of the transcription factor was demonstrated using the electrophoretic mobility shift assay (EMSA). Vectors over-expressing the transcription factor were co-transfected into HEK-293 and SK-N-SH cells with the recombinant pGL3 vector, and relative fluorescence intensity was measured to determine regulatory activity. Additionally, the qRT-PCR and Western blot were also used to identify whether the transcription factor modulated the endogenous expression of 5-HT1A receptor. The results suggest that the transcription factor CCAA/T enhancer binding protein beta (CEBPB) likely binds to the -1219 to -1209 bp (ATG+1) region of the HTR1A. Two sequences located in the -722 to -372 bp and -119 to +99 bp were also identified. Although the effect of CEBPB on endogenous 5-HT1A receptor expression was not significant, it exhibited the strong inhibition on the relative fluorescence intensity and the mRNA level of HTR1A. CEBPB inhibited the human HTR1A expression by binding to the sequence -1219 - -1209 bp. This is useful and informative for ascertaining the regulation of 5-HT1A receptor and mental diseases.

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Functional polymorphisms and transcriptional analysis in the 5′ region of the human serotonin receptor 1B gene (HTR1B) and their associations with psychiatric disorders

BMC Psychiatry ◽

10.1186/s12888-020-02906-4 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Xi Xia ◽

Mei Ding ◽

Jin-feng Xuan ◽

Jia-xin Xing ◽

Jun Yao ◽

...

Keyword(s):

Gene Expression ◽

Behavioral Disorders ◽

Association Studies ◽

Regulation Of Gene Expression ◽

Regulatory Region ◽

Transcriptional Analysis ◽

Luciferase Reporter ◽

Genome Wide Association Studies ◽

Functional Polymorphisms ◽

Mental And Behavioral Disorders

Abstract Background The 5-hydroxytryptamine 1B receptor (5-HT1B) plays an essential role in the serotonin (5-HT) system and is widely involved in a variety of brain activities. HTR1B is the gene encoding 5-HT1B. Genome-wide association studies have shown that HTR1B polymorphisms are closely related to multiple mental and behavioral disorders; however, the functional mechanisms underlying these associations are unknown. This study investigated the effect of several HTR1B haplotypes on regulation of gene expression in vitro and the functional sequences in the 5′ regulatory region of HTR1B to determine their potential association with mental and behavioral disorders. Methods Six haplotypes consisting of rs4140535, rs1778258, rs17273700, rs1228814, rs11568817, and rs130058 and several truncated fragments of the 5′ regulatory region of HTR1B were transfected into SK-N-SH and HEK-293 cells. The relative fluorescence intensities of the different haplotypes and truncated fragments were detected using a dual-luciferase reporter assay system. Results Compared to the major haplotype T-G-T-C-T-A, the relative fluorescence intensities of haplotypes C-A-T-C-T-A, C-G-T-C-T-A, C-G-C-A-G-T, and C-G-T-A-T-A were significantly lower, and that of haplotype C-G-C-A-G-A was significantly higher. Furthermore, the effects of the rs4140535T allele, the rs17273700C-rs11568817G linkage combination, and the rs1228814A allele made their relative fluorescence intensities significantly higher than their counterparts at each locus. Conversely, the rs1778258A and rs130058T alleles decreased the relative fluorescence intensities. In addition, we found that regions from − 1587 to − 1371 bp (TSS, + 1), − 1149 to − 894 bp, − 39 to + 130 bp, + 130 to + 341 bp, and + 341 to + 505 bp upregulated gene expression. In contrast, regions − 603 to − 316 bp and + 130 to + 341 bp downregulated gene expression. Region + 341 to + 505 bp played a decisive role in gene transcription. Conclusions HTR1B 5′ regulatory region polymorphisms have regulatory effects on gene expression and potential correlate with several pathology and physiology conditions. This study suggests that a crucial sequence for transcription is located in region + 341 ~ + 505 bp. Regions − 1587 to − 1371 bp, − 1149 to − 894 bp, − 603 to − 316 bp, − 39 to + 130 bp, and + 130 to + 341 bp contain functional sequences that can promote or suppress the HTR1B gene expression.

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Role of Cdx-2 in insulin and proglucagon gene expression: a study using the RIN-1056A cell line with an inducible gene expression system

Journal of Endocrinology ◽

10.1677/joe.1.06152 ◽

2005 ◽

Vol 186 (1) ◽

pp. 179-192 ◽

Cited By ~ 3

Author(s):

Yi Zhao ◽

Tao Liu ◽

Nina Zhang ◽

Fenghua Yi ◽

Qinghua Wang ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

Expression System ◽

Insulin Gene ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Gene Expressions ◽

Insulin Gene Expression

Although the homeobox gene Cdx-2 was initially isolated from the pancreatic β cell line HIT-T15, no examination of its role in regulating endogenous insulin gene expression has been reported. To explore further the role of Cdx-2 in regulating both insulin and proglucagon gene expression, we established an ecdysone-inducible Cdx-2 expression system. This report describes a study using the rat insulinoma cell line RIN-1056A, which abundantly expresses both insulin and proglucagon (glu), and relatively high amounts of endogenous Cdx-2. Following the introduction of the inducible Cdx-2 expression system into this cell line and the antibiotic selection procedure, we obtained novel cell lines that displayed dramatically reduced expression of endogenous Cdx-2, in the absence of the inducer. These novel cell lines did not express detectable amounts of glu mRNA or the glucagon hormone, while their insulin expression was not substantially affected. In the presence of the inducer, however, transfected Cdx-2 expression was dramatically increased, accompanied by stimulation of endogenous Cdx-2 expression. More importantly, activated Cdx-2 expression was accompanied by elevated insulin mRNA expression, and insulin synthesis. Cdx-2 bound to the insulin gene promoter enhancer elements, and stimulated the expression of a luciferase reporter gene driven by these enhancer elements. Furthermore, Cdx-2 and insulin gene expressions in the wild-type RIN-1056A cells were stimulated by forskolin treatment, and forskolin-mediated activation on insulin gene expression was attenuated in the absence of Cdx-2. We suggest that Cdx-2 may mediate the second messenger cAMP in regulating insulin gene transcription.

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Histone Deacetylase Inhibitors and Microtubule Inhibitors Induce Apoptosis in Feline Luminal Mammary Carcinoma Cells

Animals ◽

10.3390/ani11020502 ◽

2021 ◽

Vol 11 (2) ◽

pp. 502

Author(s):

Filipe Almeida ◽

Andreia Gameiro ◽

Jorge Correia ◽

Fernando Ferreira

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

Mammary Carcinoma ◽

Human Breast Cancer ◽

Histone Deacetylase Inhibitors ◽

Trichostatin A ◽

Human Breast ◽

Antitumor Effects

Feline mammary carcinoma (FMC) is the third most common type of neoplasia in cats, sharing similar epidemiological features with human breast cancer. In humans, histone deacetylases (HDACs) play an important role in the regulation of gene expression, with HDAC inhibitors (HDACis) disrupting gene expression and leading to cell death. In parallel, microtubules inhibitors (MTIs) interfere with the polymerization of microtubules, leading to cell cycle arrest and apoptosis. Although HDACis and MTIs are used in human cancer patients, in cats, data is scarce. In this study, we evaluated the antitumor properties of six HDACis (CI-994, panobinostat, SAHA, SBHA, scriptaid, and trichostatin A) and four MTIs (colchicine, nocodazole, paclitaxel, and vinblastine) using three FMC cell lines (CAT-MT, FMCp, and FMCm), and compared with the human breast cancer cell line (SK-BR-3). HDACis and MTIs exhibited dose-dependent antitumor effects in FMC cell lines, and for all inhibitors, the IC50 values were determined, with one feline cell line showing reduced susceptibility (FMCm). Immunoblot analysis confirmed an increase in the acetylation status of core histone protein HDAC3 and flow cytometry showed that HDACis and MTIs lead to cellular apoptosis. Overall, our study uncovers HDACis and MTIs as promising anti-cancer agents to treat FMCs.

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Comparative activity of pulsed or continuous estradiol exposure on gene expression and proliferation of normal and tumoral human breast cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0280165 ◽

2002 ◽

Vol 28 (3) ◽

pp. 165-175 ◽

Cited By ~ 14

Author(s):

V Cavailles ◽

A Gompel ◽

MC Portois ◽

S Thenot ◽

N Mabon ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Hormone Replacement ◽

Primary Cultures ◽

Continuous Treatment ◽

Luciferase Reporter ◽

Breast Cancer Cell Lines ◽

Human Breast ◽

Kinetic Profile ◽

Breast Cells

Intranasal administration of hormone replacement therapy presents an original plasma kinetic profile with transient estrogen levels giving rise to the concept of pulsed therapy. To further understand the molecular effects of this new therapy, we have compared the effects of pulsed and continuous estradiol treatments on two critical aspects of estradiol action: gene expression and cell proliferation. Cells were stimulated with estradiol as 1-h pulsed or 24-h continuous treatments at concentrations such that the 24-h exposure (concentration x time) was identical in both conditions. In MCF7 cells, the transcriptional activity of estrogen receptors (ER) on a transiently transfected responsive estrogen response element-luciferase reporter construct was shown to be drastically (approximately 10-fold) and similarly stimulated after both treatments. Moreover, the increased mRNA expression of three representative estradiol-sensitive genes (pS2, cathepsin D, progesterone receptor), evaluated by Northern blot, was identical after 1-h pulse with 7 nM estradiol or continuous treatment with 0.29 nM estradiol with the same kinetic profile over 48 h. Proliferation was quantified by a histomorphometric method on primary cultures of human normal breast cells from reduction mammoplasties and using a fluorescence DNA assay in six human breast cancer cell lines which were ER positive or negative. After a 7-day treatment period, estradiol had no effect on the proliferation of the three ER negative cell lines (BT20, MDA MB231, SK BR3) but significantly stimulated the proliferation of the normal cells and of the three tumoral hormone-sensitive cell lines (MCF7, T47D, ZR 75-1); both hormone treatments producing the same increases in cell growth. In conclusion, we have shown that the genomic or proliferative effects of estradiol were identical with pulsed or continuous treatments, thus indicating that estrogenic effects are not strictly related to concentrations but rather to total hormone exposure.

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