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Biochemistry and Cell Biology =... 19941,25-Dihydroxycholecalciferol D3 (1,25(OH)2D3), the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. We have... (Review)
Review
1,25-Dihydroxycholecalciferol D3 (1,25(OH)2D3), the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. We have shown that MCF-7 cells treated with 100 nM 1,25(OH)2D3 exhibit characteristic apoptotic morphology (pyknotic nuclei, chromatin and cytoplasmic condensation, nuclear matrix protein reorganization) within 48 h. In the experiments reported here, we examined the interactions between 1,25(OH)2D3 and the antiestrogen 4-hydroxytamoxifen (TAM), which also induces apoptosis in MCF-7 cells. Our data suggest that TAM significantly potentiates the reduction in cell number induced by 1,25(OH)2D3 alone. Combined treatment with 1,25(OH)2D3 and TAM enhances the degree of apoptosis assessed using morphological markers that identify chromatin and nuclear matrix protein condensation. We have selected a subclone of MCF-7 cells resistant to 1,25(OH)2D3 (MCF-7D3Res). These cells express the vitamin D receptor and exhibit doubling times comparable to the parental MCF-7 cells, even when grown in 100 mM 1,25(OH)2D3. Treatment of both parental and resistant MCF-7 cells with TAM induces apoptosis and clusterin. These data emphasize that apoptosis can be induced in MCF-7 cells either by activation of vitamin-D-mediated signalling or disruption of estrogen-dependent signalling.
Topics: Apoptosis; Breast Neoplasms; Calcitriol; Cell Survival; Clusterin; Drug Interactions; Estrogen Antagonists; Female; Glycoproteins; Humans; Molecular Chaperones; Receptors, Calcitriol; Tamoxifen; Tumor Cells, Cultured
PubMed: 7654327
DOI: 10.1139/o94-072 -
Nucleic Acids Research Mar 2018Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly...
Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes.
Topics: CRISPR-Cas Systems; Estrogen Receptor beta; Gene Editing; Genomics; Humans; Mutation; Reproducibility of Results; Tamoxifen; Transcriptional Activation
PubMed: 29237052
DOI: 10.1093/nar/gkx1222 -
Biochimie Feb 1982Synthetic antiestrogens such as Tamoxifen (Nolvadex) are currently used to treat postmenopausal breast cancer. Their mechanism of action is reviewed at the cellular and... (Review)
Review
Synthetic antiestrogens such as Tamoxifen (Nolvadex) are currently used to treat postmenopausal breast cancer. Their mechanism of action is reviewed at the cellular and molecular level, through data of the literature and the current view of the author. Synthetic antiestrogens are mostly acting directly on breast cancer cells by interacting with the estrogen receptor (RE). They prevent estrogen action by competing with estrogens on the cytosol RE. The resulting complex is partially activated resulting in its nuclear localisation and a partial and dissociated stimulation of the expression of estrogen responsive genes. The explanation of the antitumoral effect of these drugs is more controversial, since the regulation of cell proliferation is not only due to estrogens but also to other hormones and factors. The antiestrogens can therefore block cancer cell growth by inhibiting estrogen action, or by a RE medicated cytotoxic effect and/or by any other mechanism involving indirect effects, or binding to other proteins than the RE.
Topics: Animals; Binding, Competitive; Biological Transport; Breast Neoplasms; Cell Division; Cell Line; Cell Nucleus; Chromatin; Cytoplasm; Estrogen Antagonists; Estrogens; Female; Humans; Models, Biological; Receptors, Estrogen; Tamoxifen
PubMed: 7039690
DOI: 10.1016/s0300-9084(82)80411-2 -
Nature Methods Dec 2011The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models...
The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.
Topics: Animals; Gene Expression; Gene Knockdown Techniques; Green Fluorescent Proteins; Humans; Integrases; Intestinal Mucosa; Mice; Organoids; Receptors, G-Protein-Coupled; Receptors, Notch; Retroviridae; Stem Cells; Tamoxifen; Transduction, Genetic
PubMed: 22138822
DOI: 10.1038/nmeth.1802 -
Journal of the American Chemical Society Mar 2012Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an...
Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an experimental strategy to regulate the intracellular concentration of any protein of interest by using an engineered destabilizing protein domain and a cell-permeable small molecule. Destabilizing domains have general utility to confer instability to a wide range of proteins including integral transmembrane proteins. This study reports a destabilizing domain system based on the ligand binding domain of the estrogen receptor that can be regulated by one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen.
Topics: Animals; Bacterial Proteins; Binding Sites; Humans; Ligands; Luminescent Proteins; Mice; Models, Molecular; Molecular Structure; Mutation; NIH 3T3 Cells; Protein Engineering; Receptors, Estrogen; Structure-Activity Relationship; Tamoxifen
PubMed: 22332638
DOI: 10.1021/ja209933r -
Biochemistry. Biokhimiia Jul 2021Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism....
Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism. To study functions of the essential genes, as well as the processes of cell differentiation, the method of induced knockout is widely used. The CreERT2/loxP system allows obtaining an inducible knockout in cells expressing tamoxifen-inducible Cre recombinase (CreERT2) and containing loxP sites flanking the target gene by adding 4-hydroxy tamoxifen to the culture medium. However, the rat ESC lines expressing CreERT2 are absent. In this work, we tested three CRISPR/Cas systems for introduction of double-strand breaks into the Rosa26 locus in the rat ESCs and inserted tamoxifen-dependent Cre recombinase into this locus using the CRISPR/Cpf1 system. It was shown that the obtained transgenic rat ESC lines retained the characteristics of pluripotent cells. Tamoxifen-inducible Cre recombinase activity was analyzed using a reporter vector.
Topics: Animals; CRISPR-Cas Systems; Embryonic Stem Cells; Gene Editing; Gene Knockout Techniques; Integrases; Rats; Rats, Transgenic; Tamoxifen
PubMed: 34284709
DOI: 10.1134/S0006297921070051 -
Oncogene Oct 2014Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the...
Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified oestrogen receptor (ER(TAM)) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ER(TAM) fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterized ER(TAM) fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long-term activation of ER(TAM) fusion proteins in vivo.
Topics: Administration, Oral; Animals; Antineoplastic Agents; Genes, Reporter; Injections, Intraperitoneal; Islets of Langerhans; Kinetics; Mice; Rats; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; Transcriptional Activation; Tumor Suppressor Protein p53
PubMed: 24662815
DOI: 10.1038/onc.2014.78 -
International Journal of Molecular... Jan 2022Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of ,-dialkylaminoethyl substituent, which plays an...
Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of ,-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the ,-dialkylaminoethyl substituent as a privileged functional group for SERMs.
Topics: Azulenes; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Development; Drug Synergism; Estradiol; Estrogen Receptor Modulators; Female; Humans; MCF-7 Cells; Models, Molecular; Molecular Structure; Protein Binding; Protein Conformation; Receptors, Estrogen; Sesquiterpenes, Guaiane; Tamoxifen
PubMed: 35163039
DOI: 10.3390/ijms23031113 -
Methods in Molecular Biology (Clifton,... 2013Recent advances have led to several systems to study transcription from defined loci in living cells. It has now become possible to address long-standing questions...
Recent advances have led to several systems to study transcription from defined loci in living cells. It has now become possible to address long-standing questions regarding the interplay between the processes of DNA damage repair and transcription-two disparate processes that can occur on the same stretch of chromatin and which both lead to extensive chromatin change. Here we describe the development of a system to create enzymatically induced DNA double-strand breaks (DSBs) at a site of inducible transcription and methods to study the interplay between these processes.
Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Cell Line; Chromatin; Chromatin Assembly and Disassembly; DNA Breaks, Double-Stranded; DNA Repair; Deoxyribonucleases, Type II Site-Specific; Doxycycline; Humans; Lac Operon; Lasers; Microscopy, Fluorescence; Tamoxifen; Transcription, Genetic
PubMed: 23980011
DOI: 10.1007/978-1-62703-526-2_16 -
Journal of Clinical Oncology : Official... Jan 2013
Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cardenolides; Cytochrome P-450 CYP2D6; Female; Humans; Polymorphism, Genetic; Postmenopause; Saponins; Tamoxifen
PubMed: 23091108
DOI: 10.1200/JCO.2012.44.6625