Live-Cell Sender-Receiver Co-cultures for Quantitative Measurement of Paracrine Signaling Dynamics, Gene Expression, and Drug Response.

Paracrine signaling is a fundamental process regulating tissue development, repair, and pathogenesis of diseases such as cancer. Herein we describe a method for quantitatively measuring paracrine signaling dynamics, and resultant gene expression changes, in living cells using genetically encoded signaling reporters and fluorescently tagged gene loci. We discuss considerations for selecting paracrine "sender-receiver" cell pairs, appropriate reporters, the use of this system to ask diverse experimental questions and screen drugs blocking intracellular communication, data collection, and the use of computational approaches to model and interpret these experiments.

Interaction of Ras Binding Domain (RBD) by chemotherapeutic zinc oxide nanoparticles: Progress towards RAS pathway protein interference.

Zinc oxide (ZnO) NP is considered as a nanoscale chemotherapeutic. Thus, the drug delivery of this inorganic NP is of considerable importance. Ras mutations are common in cancer and the activation of this signaling pathway is a hallmark in carcinoma, melanoma and many other aggressive malignancies. Thus, here we examined the binding and delivery of Ras binding domain (RBD), a model cancer-relevant protein and effector of Ras by ZnO NP. Shifts in zeta potential in water, PBS, DMEM and DMEM supplemented with FBS supported NP interaction to RBD. Fluorescence quenching of the NP was concentration-dependent for RBD, Stern-Volmer analysis of this data was used to estimate binding strength which was significant for ZnO-RBD (Kd < 10-5). ZnO NP interaction to RBD was further confirmed by pull-down assay demonstrated by SDS-PAGE analysis. The ability of ZnO NP to inhibit 3-D tumor spheroid was demonstrated in HeLa cell spheroids-the ZnO NP breaking apart these structures revealing a significant (>50%) zone of killing as shown by light and fluorescence microscopy after intra-vital staining. ZnO 100 nm was superior to ZnO 14 nm in terms of anticancer activity. When bound to ZnO NP, the anticancer activity of RBD was enhanced. These data indicate the potential diagnostic application or therapeutic activity of RBD-NP complexes in vivo which demands further investigation.

High Risk α-HPV E6 Impairs Translesion Synthesis by Blocking POLη Induction.

High risk genus α human papillomaviruses (α-HPVs) express two versatile oncogenes (α-HPV E6 and E7) that cause cervical cancer (CaCx) by degrading tumor suppressor proteins (p53 and RB). α-HPV E7 also promotes replication stress and alters DNA damage responses (DDR). The translesion synthesis pathway (TLS) mitigates DNA damage by preventing replication stress from causing replication fork collapse. Computational analysis of gene expression in CaCx transcriptomic datasets identified a frequent increased expression of TLS genes. However, the essential TLS polymerases did not follow this pattern. These data were confirmed with in vitro and ex vivo systems. Further interrogation of TLS, using POLη as a representative TLS polymerase, demonstrated that α-HPV16 E6 blocks TLS polymerase induction by degrading p53. This doomed the pathway, leading to increased replication fork collapse and sensitivity to treatments that cause replication stress (e.g., UV and Cisplatin). This sensitivity could be overcome by the addition of exogenous POLη.

Systems-Level Properties of EGFR-RAS-ERK Signaling Amplify Local Signals to Generate Dynamic Gene Expression Heterogeneity.

Intratumoral heterogeneity is associated with aggressive tumor behavior, therapy resistance, and poor patient outcomes. Such heterogeneity is thought to be dynamic, shifting over periods of minutes to hours in response to signaling inputs from the tumor microenvironment. However, models of this process have been inferred from indirect or post-hoc measurements of cell state, leaving the temporal details of signaling-driven heterogeneity undefined. Here, we developed a live-cell model system in which microenvironment-driven signaling dynamics can be directly observed and linked to variation in gene expression. Our analysis reveals that paracrine signaling between two cell types is sufficient to drive continual diversification of gene expression programs. This diversification emerges from systems-level properties of the EGFR-RAS-ERK signaling cascade, including intracellular amplification of amphiregulin-mediated paracrine signals and differential kinetic filtering by target genes including Fra-1, c-Myc, and Egr1. Our data enable more precise modeling of paracrine-driven transcriptional variation as a generator of gene expression heterogeneity. A record of this paper's transparent peer review process is included in the Supplemental Information.

ß-HPV 8E6 Attenuates ATM and ATR Signaling in Response to UV Damage.

Given the high prevalence of cutaneous genus beta human papillomavirus (ß-HPV) infections, it is important to understand how they manipulate their host cells. This is particularly true for cellular responses to UV damage, since our skin is continually exposed to UV. The E6 protein from ß-genus HPV (ß-HPV E6) decreases the abundance of two essential UV-repair kinases (ATM and ATR). Although ß-HPV E6 reduces their availability, the impact on downstream signaling events is unclear. We demonstrate that ß-HPV E6 decreases ATM and ATR activation. This inhibition extended to XPA, an ATR target necessary for UV repair, lowering both its phosphorylation and accumulation. ß-HPV E6 also hindered POLη accumulation and foci formation, critical steps in translesion synthesis. ATM's phosphorylation of BRCA1 is also attenuated by ß-HPV E6. While there was a striking decrease in phosphorylation of direct ATM/ATR targets, events further down the cascade were not reduced. In summary, despite being incomplete, ß-HPV 8E6's hindrance of ATM/ATR has functional consequences.

Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection.

The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these factors in vivo remain poorly characterized. Notably, increasing research has focused on the interplays between Ly6/uPAR proteins and viral pathogens, the results of which have provided new insight into viral entry and virus-host interactions. While LY6E (lymphocyte antigen 6 family member E), one key member of the Ly6E/uPAR-family proteins, has been extensively studied, other members have not been well characterized. Here, we summarize current knowledge of Ly6/uPAR proteins related to viral infection, with a focus on uPAR and CD59. Our goal is to provide an up-to-date view of the Ly6/uPAR-family proteins and associated virus-host interaction and viral pathogenesis.

Comparative functional dynamics studies on the enzyme nano-bio interface.

INTRODUCTION: Biomedical applications of nanoparticles (NPs) as enzyme inhibitors have recently come to light. Oxides of metals native to the physiological environment (eg, Fe, Zn, Mg, etc.) are of particular interest-especially the functional consequences of their enzyme interaction. MATERIALS AND METHODS: Here, Fe2O3, zinc oxide (ZnO), magnesium oxide (MgO) and nickel oxide (NiO) NPs are compared to copper (Cu) and boron carbide (B4C) NPs. The functional impact of NP interaction to the model enzyme luciferase is determined by 2-dimensional fluorescence difference spectroscopy (2-D FDS) and 2-dimensional photoluminescence difference spectroscopy (2-D PLDS). By 2-D FDS analysis, the change in maximal intensity and in 2-D FDS area under the curve (AUC) is in the order Cu~B4C>ZnO>NiO>>Fe2O3>MgO. The induced changes in protein conformation are confirmed by tryptic digests and gel electrophoresis. RESULTS: Analysis of possible trypsin cleavage sites suggest that cleavage mostly occurs in the range of residues 112-155 and 372-439, giving a major 45 kDa band. By 2-D PLDS, it is found that B4C NPs completely ablate bioluminescence, while Cu and Fe2O3 NPs yield a unique bimodal negative decay rate, -7.67×103 and -3.50×101 relative light units respectively. Cu NPs, in particular, give a remarkable 271% change in enzyme activity. Molecular dynamics simulations in water predicted that the surfaces of metal oxide NPs become capped with metal hydroxide groups under physiological conditions, while the surface of B4C becomes populated with boronic acid or borinic acid groups. These predictions are supported by the experimentally determined zeta potential. Thin layer chromatography patterns further support this conception of the NP surfaces, where stabilizing interactions were in the order ionic>polar>non-polar for the series tested. CONCLUSION: Overall the results suggest that B4C and Cu NP functional dynamics on enzyme biochemistry are unique and should be examined further for potential ramifications on other model, physiological or disease-relevant enzymes.

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Engineering the RNA-Nanobio Interface.

RNA nanotechnology is attracting a great deal of attention recently. As the multiple roles that RNA plays in molecular biology and physiological regulation become clearer, there are many opportunities for engineering RNA-Nanoparticle Complexes (RNA-NPCs). The high "engineerability" of RNA-NPCs comes from the ability to modify the RNA and NP chemistry. For example, the NP can be derived from materials with anticancer activity and the RNA delivered by it, designed to target cell signaling pathways that contribute to the molecular basis of these diseases. Despite this rapid advancement and the availability of new quantification and characterization techniques, a key challenge is to develop a better understanding of the RNA-nanobio interface; that is, the interactions of RNA with NP (RNA-nanobio interface) and how that impacts the structure, function, delivery, and activity of the RNA. Here, we attempt to summarize the state-of-the-art in this new and exciting field, and to lay out potential directions for bioengineering research on RNA-NPCs.