Unraveling the regulatory connections between two controllers of breast cancer cell fate.

Estrogen receptor alpha (ERα) expression is critical for breast cancer classification, high ERα expression being associated with better prognosis. ERα levels strongly correlate with that of GATA binding protein 3 (GATA3), a major regulator of ERα expression. However, the mechanistic details of ERα-GATA3 regulation remain incompletely understood. Here we combine mathematical modeling with perturbation experiments to unravel the nature of regulatory connections in the ERα-GATA3 network. Through cell population-average, single-cell and single-nucleus measurements, we show that the cross-regulation between ERα and GATA3 amounts to overall negative feedback. Further, mathematical modeling reveals that GATA3 positively regulates its own expression and that ERα autoregulation is most likely absent. Lastly, we show that the two cross-regulatory connections in the ERα-GATA3 negative feedback network decrease the noise in ERα or GATA3 expression. This may ensure robust cell fate maintenance in the face of intracellular and environmental fluctuations, contributing to tissue homeostasis in normal conditions, but also to the maintenance of pathogenic states during cancer progression.

The effects of PVP(Fe(III)) catalyst on polymer molecular weight and gene delivery via biodegradable cross-linked polyethylenimine.

PURPOSE: Crosslinked, degradable derivatives of low-molecular-weight polyethylenimine (PEI) are relatively efficient and non-cytotoxic gene delivery agents. To further investigate these promising materials, a new synthetic approach was developed using a poly(4-vinylpyridine)-supported Fe(III) catalyst (PVP(Fe(III))) that provides more facile synthesis and enhanced control of polymer molecular weight. METHODS: Biodegradable polymers (D.PEI) comprising 800-Da PEI crosslinked with 1,6-hexanediol diacrylate and exhibiting molecular weights of 1.2, 6.2, and 48 kDa were synthesized utilizing the PVP(Fe(III)) catalyst. D.PEI/DNA polyplexes were characterized using gel retardation, ethidium bromide exclusion, heparan sulfate displacement, and dynamic light scattering. In vitro transfection, cellular uptake, and cytotoxicity of the polyplexes were tested in human cervical cancer cells (HeLa) and human breast cancer cells (MDA-MB-231). RESULTS: D.PEIs tightly complexed plasmid DNA and formed 320- to 440-nm diameter polyplexes, similar to those comprising non-degradable, 25-kDa, branched PEI. D.PEI polyplexes mediated 2- to 5-fold increased gene delivery efficacy compared to 25-kDa PEI and exhibited 20% lower cytotoxicity in HeLa and no toxicity in MDA-MB-231. In addition, 2- to 7-fold improved cellular uptake of DNA was achieved with D.PEI polyplexes. CONCLUSIONS: PVP(Fe(III)) catalyst provided a more controlled synthesis of D.PEIs, and these materials demonstrated improved in vitro transfection efficacy and reduced cytotoxicity .

Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake.

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery.