Abstract
IntroductionLineage tracing using inducible genetic labeling has emerged to be a powerful method for interrogating the developmental fate of cells in intact tissues. A common induction mechanism is the use of tamoxifen-dependent Cre recombinase (CreER and CreERT2), but the effects of tamoxifen at doses normally used in lineage-tracing studies on normal adult mammary gland homeostasis are not known.MethodsWe used flow cytometry and immunostaining of intact glands to determine whether varying doses of tamoxifen skew the distribution and the apoptosis and proliferation status of different types of mammary epithelial cells in vivo. We also examined how tamoxifen influences the number of progenitor and mammary repopulating units (MRUs).ResultsOur results indicate that ≥5 mg/25 g body weight of tamoxifen induces a transient increase in cell proliferation and in the number of basal cells in the adult mammary epithelium up to 7 days after tamoxifen administration. However, in the medium term (3 weeks), all doses of tamoxifen ≥1 mg/25 g body weight result in a decrease in the number of basal and EpCAM+CD49b− luminal cells and a decrease in progenitor cell function. Tamoxifen at doses ≥5 mg/25 g body weight induced a transient increase in caspase-3-mediated apoptotic cell death within the mammary epithelium. However, mammary epithelial cell numbers in all subpopulations were restored to their original levels by 8 weeks. No long-lasting effects of tamoxifen on MRU numbers or on pubertal ductal development were observed.ConclusionTamoxifen can skew the distribution of mammary cell types in a dose-dependent manner, and thus caution must be taken when interpreting lineage-tracing studies using high doses of tamoxifen, particularly when short-duration analyses of a quantitative nature are being performed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0411-0) contains supplementary material, which is available to authorized users.
Highlights
Lineage tracing using inducible genetic labeling has emerged to be a powerful method for interrogating the developmental fate of cells in intact tissues
Considering that tamoxifen is an estrogen-receptor (ER) antagonist and is a commonly used drug in the treatment of breast cancer to inhibit the proliferation of estrogen receptor+ (ER+) breast cancer cells [10], we investigated the effects of a range of doses of tamoxifen on the distribution and cell-division kinetics of the different mammary cell populations in the intact adult mouse mammary gland
Tamoxifen induces short-term cell proliferation and cell death in the mammary epithelium Given the change in basal and luminal cell numbers after tamoxifen application, we examined the effect on proliferation of mammary epithelial cells
Summary
Lineage tracing using inducible genetic labeling has emerged to be a powerful method for interrogating the developmental fate of cells in intact tissues. A common induction mechanism is the use of tamoxifen-dependent Cre recombinase (CreER and CreERT2), but the effects of tamoxifen at doses normally used in lineage-tracing studies on normal adult mammary gland homeostasis are not known. Lineage tracing has emerged to become a powerful method for establishing hierarchical relations between stem and progenitor cells within epithelial tissues [1]. One common lineage-tracing approach involves the use of a specific promoter to direct expression of Cre recombinase to defined subsets of cells, such that these. One assumption of lineage tracing is that the inducing agent itself (that is, tamoxifen) does not affect homeostasis of the tissue under study. We examined the influence of tamoxifen on pubertal mammary gland development
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