Loss of the acinar-restricted transcription factor Mist1 accelerates Kras-induced pancreatic intraepithelial neoplasia.

BACKGROUND & AIMS: Invasive pancreatic ductal adenocarcinoma is thought to originate from duct-like lesions called pancreatic intraepithelial neoplasia (PanIN). PanINs progress from low grade (PanIN-1) to high grade (PanIN-3) as the cells attain molecular alterations to key regulatory genes, including activating mutations in the KRAS protooncogene. Despite a well-documented progression model, our knowledge of the initiator cells of PanINs and the transcriptional networks and signaling pathways that impact PanIN formation remains incomplete. METHODS: In this study, we examined the importance of the acinar-restricted transcription factor Mist1 to KrasG12D-induced mouse PanIN (mPanIN) formation in 3 different mouse models of pancreatic cancer. RESULTS: In the absence of Mist1 (Mist1KO), KrasG12D-expressing mice exhibited severe exocrine pancreatic defects that were rescued by ectopic expression of Mist1 in acinar cells. mPanIN development was greatly accelerated in Mist1KO/KrasG12D/+ pancreata, and in vitro assays revealed that Mist1KO acinar cells were predisposed to convert to a ductal phenotype and activate epidermal growth factor receptor (EGFR) and Notch-signaling pathways. CONCLUSIONS: We propose that convergence of EGFR, Notch, and Kras pathways in acinar cells lacking Mist1 leads to enhanced mPanIN formation.

Effects of the incorporation of a hydrophobic middle block into a PEG-polycation diblock copolymer on the physicochemical and cell interaction properties of the polymer-DNA complexes.

One-component homopolymers of cationic monomers (polycations) and diblock copolymers comprising poly(ethylene glycol) (PEG) and a polycation block have been the most widely used types of polymers for the formulation of polymer-based gene delivery systems. In this study, we incorporate a hydrophobic middle block into the conventional PEG-polycation architecture and investigate the effects of this hydrophobic modification on the physicochemical and cell-level biological properties of the polymer-DNA complexes that are relevant to gene delivery applications. The ABC-type triblock copolymer used in this study consists of (A) PEG, (B) hydrophobic poly( n-butyl acrylate) (PnBA), and (C) cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) component polymers. The properties of the triblock copolymer/DNA complexes are compared with those of two other more conventional DNA carriers derived, respectively, using a PDMAEMA homopolymer and a PEG-PDMAEMA diblock copolymer that had comparable molecular weights for individual blocks. In aqueous solution, the PEG-PnBA-PDMAEMA polymer forms positively charged spherical micelles. The electrostatic complexation of these micelles with plasmid DNA molecules results in the formation of stable small-sized DNA particles that are coated with a micelle monolayer, as confirmed by agarose gel electrophoresis, dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Proton nuclear magnetic resonance ( (1)H NMR) spectroscopy measurements indicate that the whole micelle-DNA assembly (named "micelleplex" for convenience) is shielded predominantly by the PEG chains. DLS and optical microscopy imaging measurements indicate that compared with PDMAEMA-DNA polyplexes, the micelleplexes have a significantly lower tendency to aggregate under physiological salt concentrations and show reduced interactions with negatively charged components in serum such as albumin and erythrocytes. While the micelleplexes are comparable to the PEG-PDMAEMA-based DNA polyplexes in terms of their stability against aggregation under high salt concentrations and in the presence of the albumin protein, they have a slightly higher tendency to interact with erythrocytes than the diblock copolymer polyplexes. Agarose gel electrophoresis measurements indicate that relative to the PEG-PDMAEMA polyplexes, the micelleplexes provide better protection of the encapsulated DNA from enzymatic degradation and also exhibit greater stability against disintegration induced by polyanionic additives; in these respects, the PDMAEMA homopolymer-based polyplexes show the best performance. In vitro studies in HeLa cells indicate that the PDMAEMA polyplexes show the highest gene transfection efficiency among the three different gene delivery systems. Between the micelleplexes and the PEG-PDMAEMA polyplexes, a higher gene transfection efficiency is observed with the latter system. All three formulations show comparable levels of cytotoxicity in HeLa cells.

Identification of a basic helix-loop-helix transcription factor expressed in mammary gland alveolar cells and required for maintenance of the differentiated state.

The development of mammary glands relies on complicated signaling pathways that control cell proliferation, differentiation, and apoptotic events through transcriptional regulatory circuits. A key family of transcription factors used in mammary gland development is the helix-loop-helix/basic helix-loop-helix (HLH/bHLH) protein family. In this study, we identify Mist1 as a tissue-restricted Class II bHLH transcription factor expressed in lactating mammary glands. Mouse and human mammary glands accumulated Mist1 protein exclusively in secretory alveolar cells, and Mist1 transcripts were differentially expressed in mouse SCp2 cells induced to differentiate by addition of lactogenic hormones. Mist1 null (Mist1(KO)) lactating mammary glands were defective in normal lobuloalveolar organization, exhibiting shedding of cells into the alveolus lumen and premature activation of the signal transducer and activator of transcription 3 signaling pathway. These cells also failed to maintain expression of the gap junction proteins connexin26 and connexin32, leading to the loss of gap junctions. Our findings suggest that loss of Mist1 impairs the maintenance of the fully differentiated alveolar state and, for the first time, places Mist1 within the hierarchy of known HLH/bHLH proteins that control mammary epithelial cell development.