-
Scientific Reports Jan 2022The aim of the study was to compare 3 blood sampling methods, including capillary blood sampling, for determining Tamoxifen (TAM), Z-endoxifen (END), and... (Clinical Trial)
Clinical Trial Comparative Study
The aim of the study was to compare 3 blood sampling methods, including capillary blood sampling, for determining Tamoxifen (TAM), Z-endoxifen (END), and 4-hydroxytamoxifen (4HT) concentrations. High performance liquid chromatography-mass spectrometry was used to quantify concentrations of TAM, END, and 4HT in plasma, venous blood, and capillary blood samples of 16 participants on TAM therapy for breast cancer. The rhelise kit was used for capillary sampling. Calibration curves using C-labeled analogs of TAM, END, and 4HT as internal standards were used for quantifications. A capillary sampling kit was used successfully for all participants. Mean TAM concentrations did not differ significantly in the 3 types of samples. Mean END and 4HT concentrations did differ significantly between capillary and venous blood samples, possibly related to photodegradation in the internal standards prior to use or degradation products with chromatographic retention times similar to the metabolites. TAM, END, and 4HT concentrations were relatively stable when stored for 14 days at 8 °C and 20 °C. Therapeutic drug monitoring of TAM using an innovative kit and capillary blood sampling is feasible. Preliminary data from this study will aid in developing a multicenter, randomized clinical trial of personalized TAM dose monitoring and adjustments, with the goal of enhancing the quality-of-life and outcomes of patients with breast cancer.Clinical Trial Identification: EudraCT No 2017-000641-44.
Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Capillaries; Chromatography, High Pressure Liquid; Drug Monitoring; Estrogen Antagonists; Feasibility Studies; Female; Humans; Mass Spectrometry; Middle Aged; Pilot Projects; Predictive Value of Tests; Reagent Kits, Diagnostic; Reproducibility of Results; Sweden; Tamoxifen
PubMed: 35102224
DOI: 10.1038/s41598-022-05443-0 -
Hippocampus Jun 2016Recent studies have focused on the identification and manipulation of memory traces in rodent models. The two main mouse models utilized are either a CreER(T2) /loxP...
Recent studies have focused on the identification and manipulation of memory traces in rodent models. The two main mouse models utilized are either a CreER(T2) /loxP tamoxifen (TAM)- or a tetracycline transactivator/tetracycline-response element doxycycline-inducible system. These systems, however, could be improved to label a more specific population of activated neurons corresponding to behavior. Here, we sought to identify an improved selective estrogen receptor (ER) modulator (SERM) in which we could label an individual memory trace in ArcCreER(T2) mice. We found that 4-hydroxytamoxifen (4-OHT) is a selective SERM in the ArcCreER(T2) × Rosa26-CAG-stop(flox) -channelrhodospin (ChR2)-enhanced yellow fluorescent protein (eYFP) mice. The half-life of 4-OHT is shorter than TAM, allowing for more specificity of memory trace labeling. Furthermore, 4-OHT allowed for context-specific labeling in the dentate gyrus and CA3. In summary, we believe that 4-OHT improves the specificity of memory trace labeling and will allow for refined memory trace studies in the future. © 2015 Wiley Periodicals, Inc.
Topics: Animals; Bacterial Proteins; Cell Count; Channelrhodopsins; Conditioning, Psychological; Estradiol; Fear; Fulvestrant; Hippocampus; Immunohistochemistry; Luminescent Proteins; Memory; Mice, Transgenic; Microscopy, Confocal; Models, Animal; Neurons; RNA, Untranslated; Raloxifene Hydrochloride; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Tamoxifen
PubMed: 26662713
DOI: 10.1002/hipo.22556 -
Nature Communications Sep 2017The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis...
The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis of function and proliferation. Here we show that the pancreatic beta-cells in zebrafish exhibit different growth-promoting and functional properties, which in part reflect differences in the time elapsed since birth of the cells. Calcium imaging shows that the beta-cells in the embryonic islet become functional during early zebrafish development. At later stages, younger beta-cells join the islet following differentiation from post-embryonic progenitors. Notably, the older and younger beta-cells occupy different regions within the islet, which generates topological asymmetries in glucose responsiveness and proliferation. Specifically, the older beta-cells exhibit robust glucose responsiveness, whereas younger beta-cells are more proliferative but less functional. As the islet approaches its mature state, heterogeneity diminishes and beta-cells synchronize function and proliferation. Our work illustrates a dynamic model of heterogeneity based on evolving proliferative and functional beta-cell states.Βeta-cells have recently been shown to be heterogeneous with regard to morphology and function. Here, the authors show that β-cells in zebrafish switch from proliferative to functional states with increasing time since β-cell birth, leading to functional and proliferative heterogeneity.
Topics: Animals; Animals, Genetically Modified; Cell Lineage; Cell Proliferation; Cytological Techniques; Embryo, Nonmammalian; Glucose; Insulin-Secreting Cells; Islets of Langerhans; Tamoxifen; Urocortins; Zebrafish
PubMed: 28939870
DOI: 10.1038/s41467-017-00461-3 -
PloS One 2018The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported...
The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.
Topics: Adaptive Immunity; Animals; Autophagosomes; Autophagy; CD4-Positive T-Lymphocytes; Cell Line; GTP Phosphohydrolases; HEK293 Cells; Humans; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Microtubule-Associated Proteins; Mitochondria; Phosphorylation; Protein Serine-Threonine Kinases; Sequestosome-1 Protein; Tamoxifen
PubMed: 29718959
DOI: 10.1371/journal.pone.0196504 -
Nature Oct 2009Growing evidence supports the notion that proteasome-mediated destruction of transcriptional activators can be intimately coupled to their function. Recently, Nalley et...
Growing evidence supports the notion that proteasome-mediated destruction of transcriptional activators can be intimately coupled to their function. Recently, Nalley et al. challenged this view by reporting that the prototypical yeast activator Gal4 does not dynamically associate with chromatin, but rather 'locks in' to stable promoter complexes that are resistant to competition. Here we present evidence that the assay used to reach this conclusion is unsuitable, and that promoter-bound, active Gal4 is indeed susceptible to competition in vivo. Our data challenge the key evidence that Nalley et al. used to reach their conclusion, and indicate that Gal4 functions in vivo within the context of dynamic promoter complexes.
Topics: Binding, Competitive; Chromatin Immunoprecipitation; DNA-Binding Proteins; Estradiol; Galactokinase; Promoter Regions, Genetic; Protein Binding; Receptors, Estrogen; Reproducibility of Results; Research Design; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tamoxifen; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation
PubMed: 19812621
DOI: 10.1038/nature08406 -
Journal For Immunotherapy of Cancer Jan 2021Despite the remarkable benefits associated with the interventional treatment of melanomas (and other solid cancers) with immune checkpoint and Braf inhibitors (Brafi),...
BACKGROUND
Despite the remarkable benefits associated with the interventional treatment of melanomas (and other solid cancers) with immune checkpoint and Braf inhibitors (Brafi), most patients ultimately progress on therapy. The presence of multifocal/disseminated disease in patients increases their mortality risk. Hence, the development of novel strategies to effectively treat patients with melanomas that are resistant to anti-PD1 mAb (αPD1) and/or Brafi, particularly those with multifocal/disseminated disease remains a major unmet clinical need.
METHODS
Mice developing induced/spontaneous Braf/Pten melanomas were treated by cutaneous immunization with a DNA vaccine encoding the melanoma-associated antigen TRP2, with Brafi or αPD1 alone, or with a combination of these treatments. Tumor progression, tumor-infiltration by CD4 and CD8 T cells, and the development of TRP2-specific CD8 T cells were then monitored over time.
RESULTS
Vaccination led to durable antitumor immunity against PD1/Brafi-resistant melanomas in both single lesion and multifocal disease models, and it sensitized PD1-resistant melanomas to salvage therapy with αPD1. The therapeutic efficacy of the vaccine was associated with host skin-resident cells, the induction of a systemic, broadly reactive IFNγCD8 T cell repertoire, increased frequencies of CD8 TIL and reduced levels of PD1CD8 T cells. Extended survival was associated with improved TIL functionality, exemplified by the presence of enhanced levels of IFNγCD8 TIL and IL2CD4 TIL.
CONCLUSIONS
These data support the use of a novel genetic vaccine for the effective treatment of localized or multifocal melanoma refractory to conventional αPD1-based and/or Brafi-based (immune)therapy.
Topics: Animals; CD8-Positive T-Lymphocytes; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Immune Checkpoint Inhibitors; Immunization; Intramolecular Oxidoreductases; Male; Melanoma; Mice; Mutation; PTEN Phosphohydrolase; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Tamoxifen; Treatment Outcome; Vaccines, DNA; Xenograft Model Antitumor Assays
PubMed: 33408093
DOI: 10.1136/jitc-2020-001179 -
Experimental Hematology Sep 2020Hematopoietic stem cells (HSCs) are multipotent cells that form the entire blood system and have the potential to cure several pathogenic conditions directly or...
Hematopoietic stem cells (HSCs) are multipotent cells that form the entire blood system and have the potential to cure several pathogenic conditions directly or indirectly arising from defects within the HSC compartment. Pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs) can give rise to all embryonic cell types; however, efficient in vitro differentiation of HSCs from PSCs remains challenging. HoxB4 is a key regulator orchestrating the differentiation of PSCs into all cells types across the mesodermal lineage, including HSCs. Moreover, the ectopic expression of HoxB4 enhances the in vitro generation and expansion of HSCs. However, several aspects of HoxB4 biology including its regulatory functions are not fully understood. Here, we describe the role of HoxB4 in indirectly inhibiting the emergence of mature CD45 HSCs from iPSCs in vitro. Forced activation of HoxB4 permitted long-term maintenance of functional hematopoietic stem and progenitor cells (HSPCs), which efficiently reconstituted hematopoiesis upon transplantation. Our method enables an easy and scalable in vitro platform for the generation of HSCs from iPSCs, which will ultimately lead to a better understanding of HSC biology and facilitate preparation of the roadma for producing an unrestricted supply of HSCs for several curative therapies.
Topics: Animals; Cell Differentiation; Cell Proliferation; Cellular Reprogramming; Gene Expression Regulation; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Homeodomain Proteins; Humans; Induced Pluripotent Stem Cells; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutant Chimeric Proteins; Primary Cell Culture; Receptors, Estrogen; Stem Cell Factor; Tamoxifen; Thrombopoietin; Transcription Factors; Whole-Body Irradiation
PubMed: 32795499
DOI: 10.1016/j.exphem.2020.08.003 -
Molecules (Basel, Switzerland) Oct 2022Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic...
Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.
Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Electrons; Female; Humans; Reactive Oxygen Species; Receptors, Estrogen; Structure-Activity Relationship; Tamoxifen
PubMed: 36235291
DOI: 10.3390/molecules27196758 -
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 -
Cancer Science Nov 2011Patients with estrogen receptor (ER)-positive breast cancers have a better prognosis than those with ER-negative breast cancers, but often have low sensitivity to... (Comparative Study)
Comparative Study
Combination treatment with fulvestrant and various cytotoxic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) has a synergistic effect in estrogen receptor-positive breast cancer.
Patients with estrogen receptor (ER)-positive breast cancers have a better prognosis than those with ER-negative breast cancers, but often have low sensitivity to chemotherapy and a limited survival benefit. We have previously shown a combination effect of taxanes and fulvestrant and suggested that this treatment may be useful for ER-positive breast cancer. In this study, we evaluated the effects of combinations of hormone drugs and chemotherapeutic agents. In vitro, the effects of combinations of five chemotherapeutic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) and three hormone drugs (fulvestrant, tamoxifen, and 4-hydroxytamoxifen) were examined in ER-positive breast cancer cell lines using CalcuSyn software. Changes in chemoresistant factors such as Bcl2, multidrug resistance-associated protein 1, and microtubule-associated protein tau were also examined after exposure of the cells to hormone drugs. In vivo, tumor sizes in mice were evaluated after treatment with docetaxel or doxorubicin alone, fulvestrant alone, and combinations of these agents. Combination treatment with fulvestrant and all five chemotherapeutic agents in vitro showed synergistic effects. In contrast, tamoxifen showed an antagonistic effect with all the chemotherapeutic agents. 4-Hydroxytamoxifen showed an antagonistic effect with doxorubicin and 5-fluorouracil, but a synergistic effect with taxanes and vinorelbine. Regarding chemoresistant factors, Bcl2 and microtubule-associated protein tau were downregulated by fulvestrant. In vivo, a combination of fulvestrant and docetaxel had a synergistic effect on tumor growth, but fulvestrant and doxorubicin did not show this effect. In conclusion, fulvestrant showed good compatibility with all the evaluated chemotherapeutic agents, and especially with docetaxel, in vitro and in vivo.
Topics: Animals; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma, Ductal, Breast; Docetaxel; Doxorubicin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Fluorouracil; Fulvestrant; Humans; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Paclitaxel; Random Allocation; Tamoxifen; Taxoids; Tumor Cells, Cultured; Vinblastine; Vinorelbine; Xenograft Model Antitumor Assays
PubMed: 21801281
DOI: 10.1111/j.1349-7006.2011.02050.x