Defining the contribution of Troy-positive progenitor cells to the mouse esophageal epithelium.

Progenitor cells adapt their behavior in response to tissue demands. However, the molecular mechanisms controlling esophageal progenitor decisions remain largely unknown. Here, we demonstrate the presence of a Troy (Tnfrsf19)-expressing progenitor subpopulation localized to defined regions along the mouse esophageal axis. Lineage tracing and mathematical modeling demonstrate that Troy-positive progenitor cells are prone to undergoing symmetrical fate choices and contribute to esophageal tissue homeostasis long term. Functionally, TROY inhibits progenitor proliferation and enables commitment to differentiation without affecting fate symmetry. Whereas Troy expression is stable during esophageal homeostasis, progenitor cells downregulate Troy in response to tissue stress, enabling proliferative expansion of basal cells refractory to differentiation and reestablishment of tissue homeostasis. Our results demonstrate functional, spatially restricted progenitor heterogeneity in the esophageal epithelium and identify how dynamic regulation of Troy coordinates tissue generation.

Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells.

Compact chromatin is linked to a poor tumour prognosis and resistance to radiotherapy from photons. We investigated DNA damage induction and repair in the context of chromatin structure for densely ionising alpha radiation as well as its therapeutic potential. Chromatin opening by histone deacetylase inhibitor trichostatin A (TSA) pretreatment reduced clonogenic survival and increased γH2AX foci in MDA-MB-231 cells, indicative of increased damage induction by free radicals using gamma radiation. In contrast, TSA pretreatment tended to improve survival after alpha radiation while γH2AX foci were similar or lower; therefore, an increased DNA repair is suggested due to increased access of repair proteins. MDA-MB-231 cells exposed to fractionated gamma radiation (2 Gy × 6) expressed high levels of stem cell markers, elevated heterochromatin H3K9me3 marker, and a trend towards reduced clonogenic survival in response to alpha radiation. There was a higher level of H3K9me3 at baseline, and the ratio of DNA damage induced by alpha vs. gamma radiation was higher in the aggressive MDA-MB-231 cells compared to hormone receptor-positive MCF7 cells. We demonstrate that heterochromatin structure and stemness properties are induced by fractionated radiation exposure. Gamma radiation-exposed cells may be targeted using alpha radiation, and we provide a mechanistic basis for the involvement of chromatin in these effects.

Increased gene translation stringency in mammalian cells by nonsense suppression at multiple permissive sites with a single noncanonical amino acid.

The considerable potential of engineered cells compels the development of strategies for the stringent control of gene expression. A promising approach is the introduction of a premature stop codon (PTC) into a selected gene that is expressed only in the presence of noncanonical amino acids through nonsense suppression. Here, different strategies of amber PTC readthrough in mammalian cells were tested. The use of a tRNA synthetase together with a TAG codon-specific tRNA achieved PTC readthrough depending on the addition of a noncanonical amino acid (4-benzoyl-L-phenylalanine; Bpa). While single TAG codon incorporation exhibited detectable expression of the reporter protein even in the absence of Bpa, the use of a double PTC enabled virtually leakage-free functional gene translation. The introduction of an additional 5'-PTC, therefore, represents a generally applicable strategy to increase stringency in gene translation.