Reproducible specificity and efficiency of tamoxifen-mediated cre induction across ages
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PROVIDER: PRJNA1008727 | ENA |
REPOSITORIES: ENA
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Project description:Temporally controlling cre recombinase through tamoxifen (TAM) induction has many advantages for biomedical research studies. While most projects use TAM induction of mice at early post-natal or adolescence (<2m.o.) ages, age-related neurodegeneration and aging studies can require experimental designs with cre induction in older mice (>12m.o.). While anecdotally reported to be problematic, there are no published comparisons of TAM mediated cre induction at young and older ages. Cx3cr1creERT2 mice for studying microglial were crossed to a floxed NuTRAP reporter to compare cre induction at young (3m.o.) and old (18m.o) ages. Specificity and efficiency microglial labeling was identical at 24m.o. in mice induced with TAM at 3m.o. or 18m.o. of age. Age-related microglial translatomic changes were nearly identical regardless of TAM induction age. While each cre and flox mouse line should be validated before use, these findings demonstrate that TAM induction of cres can be performed even into older mouse ages.
2023-12-14 | GSE241574 | GEO
Project description:Temporally controlling Cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report early post-natal/juvenile (<2 m.o.) Tam induction, but age-related neurodegeneration and aging studies can require Cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam-mediated Cre induction at early and late ages. Here, microglial-specific Cx3cr1creERT2 mice were crossed to a floxed NuTRAP reporter to compare Cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at early and late ages. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each Cre and flox mouse line should be independently validated, however, these findings demonstrate that Tam-mediated Cre induction can be performed even into older mouse ages and should be generalizable to other inducible Cre models.
| S-EPMC10696116 | biostudies-literature
Project description:Tamoxifen, an antagonist to estrogen receptor (ER), is a first line drug used in breast cancer treatment. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying the resistance to tamoxifen, we established a tamoxifen-resistant cell line by treating the MCF7 breast cancer cell line with tamoxifen for over 6 months. We showed that this cell line exhibited resistance to tamoxifen both in vitro and in vivo. In order to quantify the phosphorylation alterations associated with tamoxifen resistance, we performed SILAC-based quantitative phosphoproteomic profiling on the resistant and vehicle-treated sensitive cell lines where we identified >5,600 unique phosphopeptides. We found phosphorylation levels of 1,529 peptides were increased (>2 fold) and 409 peptides were decreased (<0.5-fold) in tamoxifen resistant cells compared to tamoxifen sensitive cells. Gene set enrichment analysis revealed that focal adhesion pathway was the top enriched signaling pathway activated in tamoxifen resistant cells. We observed hyperphosphorylation of the focal adhesion kinases FAK1 and FAK2 in the tamoxifen resistant cells. Of note, FAK2 was not only hyperphosphorylated but also transcriptionally upregulated in tamoxifen resistant cells. Suppression of FAK2 by specific siRNA knockdown could sensitize the resistant cells to the treatment of tamoxifen. We further showed that inhibiting FAK activity using the small molecule inhibitor PF562271 repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 significantly associated with short metastasis-free survival of ER-positive breast cancer patients treated with tamoxifen-based hormone therapy. Our studies suggest that FAK2 is a great potential target for the development of therapy for the treatment of hormone refractory breast cancers.
2016-05-25 | PXD001812 | Pride
Project description:The primary objective is, first, the comparison of tamoxifen and anastrozole and, second, the comparison of zoledronate added to standard adjuvant therapy with controls according to disease-free survival (DFS) in premenopausal patients with non-metastatic breast cancer treated with tamoxifen or anastrozole. To assess whether zoledronate added to standard adjuvant therapy can decrease or even prevent bone loss in patients treated with hormonal blockade combined with an antiestrogen or aromatase inhibitor.
| 2026652 | ecrin-mdr-crc
Project description:Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing how Cre distinguishes between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6,000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the flexibility of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations elucidated the mechanism behind this specificity switch. These findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.
2024-07-25 | GSE254392 | GEO
Project description:Aging progress is distinctly characterized by systematic and progressive decline of physiological functions with increasing age in virtually all tissues or organs. Addressing the patterns of molecular changes in different tissues and how different tissues interact with each other during aging are an important question in aging. This study sampled seven tissues at four ages in rats to measure genome-scale gene expression, which allows global insight into coordination, specificity and/or commonness among different tissues with aging progress.
2011-09-01 | GSE23328 | GEO
Project description:The molecular explanation for tamoxifen serving as a breast cancer treatment but displaying partial estrogenic in the uterus is not known. Previously, we reported that differential promoter context and cofactor recruitment contribute to the tissue specificity of tamoxifen. Here, we investigated the genomic basis for the partial oestrogenic activity of tamoxifen in the endometrium. We showed that tamoxifen not only affects the rate of transcription of oestrogen target genes but also targets a unique set of genes. Since oestrogen and tamoxifen are both able to bind to oestrogen receptors (ERs) and because both promote endometrial carcinogenesis, we hypothesized that the molecular effectors for ERs in endometrial carcinogenesis most likely reside in genes that are commonly targeted by oestrogen and tamoxifen. Among those target genes, we identified a paired-box gene PAX2 that is critically involved in cell proliferation and carcinogenesis in the endometrium. Our experiments also demonstrated that PAX2 is activated by oestrogen and tamoxifen in endometrial carcinomas but not in normal endometrium, and this activation is associated with cancer-linked hypomethylation of the PAX2 promoter. Keywords: actions of tamoxifen
2005-07-29 | GSE3013 | GEO
Project description:Resistance to tamoxifen is a major challenge in the treatment of estrogen receptor positive breast cancer. Acquired resistance to drug involves multilayered genetic and epigenetic regulation . The oncogene EZH2 plays significant role in the development of resistance against tamoxifen, widely used in the treatment of breast cancer. Inhibition of EZH2 has proven to reverse the tamoxifen resistance breast cancer cells back to the sensitive state. The molecular mechanism through which EZH2 inhibition triggers its effects are not known.This study was conducted to understand the global change in proteome profile of tamoxifen resistant MCF-7 breast cancer cells as a result of effect of EZH2 knockdown. Label Free Quantitative proteomics revealed a large number of proteins altered in acquired tamoxifen resistant cells compared to the sensitive cells. A total of 286 proteins were identified with normalized RT for each m/z out of which 86 proteins were upregulated by more than 1.3 fold and 98 proteins were down regulated by more than 1.3 fold in MCF-7 tamoxifen resistant breast cancer cells in comparison to the sensitive breast cancer cells. Upon EZH2 knockdown in tamoxifen resistant cells, a total of 115 proteins were found to be altered with 20 proteins upregulated by more than 1.3 fold and 49 proteins down regulated by more than 1.3 fold. Among the top upregulated proteins were L-lactate dehydrogenase A chain, Alpha and Gamma-enolase, Calreticulin, heat shock protein HSP-90-beta, Alpha-actinin-4, Elongation factor 1-alpha, Vimentin, Protein S100A6, Putative protein FAM10A5, Heterogeneous nuclear ribonucleoprotein A1 and Keratin 1. In addition, 15 proteins were found to be down regulated in EZH2si transfected tamoxifen sensitive cells which otherwise were highlyup regulated in resistant cells in the presence of normal level of EZH2. This indicates a possible regulation of these molecules by EZH2 leading to loss of resistance. Our data unveils important molecular players downstream to EZH2 knockdown leading to regain of sensitivity to tamoxifen in acquired tamoxifen resistance.Thus, EZH2 seems to exert its effects through regulation of metabolism, epithelial to mesenchymal transition and protein synthesis & folding. Hence, targeting EZH2 or the molecules down the cascade might be helpful in reacquiring sensitivity to tamoxifen intamoxifen-resistant cells.
2019-09-30 | PXD012609 | Pride
Project description:Engineering spacer specificity of the Cre/loxP system
| PRJNA1070297 | ENA
Project description:Commercially available histone antibodies were analyzed using peptide microarrays to assess specificity. For two H3K27me3 antibodies from different vendors, we performed ChIP-seq in WT and EED-null murine ES cells to assess specificity. Cell lines were generated from e3.5 blastocysts of EED fl/fl animals and transfected with plasmid CAG-CRE-ERT2. Null cells were generated by single cell clones from tamoxifen-treated parent cell line.
2015-06-21 | GSE66902 | GEO