NHR-23 activity is necessary for C. elegans developmental progression and apical extracellular matrix structure and function.

Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells after NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.

The CLAMP GA-binding transcription factor regulates heat stress-induced transcriptional repression by associating with 3D loop anchors.

In order to survive when exposed to heat stress (HS), organisms activate stress response genes and repress constitutive gene expression to prevent the accumulation of potentially toxic RNA and protein products. Although many studies have elucidated the mechanisms that drive HS-induced activation of stress response genes across species, little is known about repression mechanisms or how genes are targeted for activation versus repression context-specifically. The mechanisms of heat stress-regulated activation have been well-studied in Drosophila, in which the GA-binding transcription factor GAF is important for activating genes upon heat stress. Here, we show that a functionally distinct GA-binding transcription factor (TF) protein, CLAMP (Chromatin-linked adaptor for MSL complex proteins), is essential for repressing constitutive genes upon heat stress but not activation of the canonical heat stress pathway. HS induces loss of CLAMP-associated 3D chromatin loop anchors associated with different combinations of GA-binding TFs prior to HS if a gene becomes repressed versus activated. Overall, we demonstrate that CLAMP promotes repression of constitutive genes upon HS, and repression and activation are associated with the loss of CLAMP-associated 3D chromatin loops bound by different combinations of GA-binding TFs.

Sex-specific splicing occurs genome-wide during early Drosophila embryogenesis.

Sex-specific splicing is an essential process that regulates sex determination and drives sexual dimorphism. Yet, how early in development widespread sex-specific transcript diversity occurs was unknown because it had yet to be studied at the genome-wide level. We use the powerful Drosophila model to show that widespread sex-specific transcript diversity occurs early in development, concurrent with zygotic genome activation. We also present a new pipeline called time2Splice to quantify changes in alternative splicing over time. Furthermore, we determine that one of the consequences of losing an essential maternally deposited pioneer factor called CLAMP (chromatin-linked adapter for MSL proteins) is altered sex-specific splicing of genes involved in diverse biological processes that drive development. Overall, we show that sex-specific differences in transcript diversity exist even at the earliest stages of development..

Lipid droplets modulate proteostasis, SQST-1/SQSTM1 dynamics, and lifespan in C. elegans.

In several long-lived Caenorhabditis elegans strains, such as insulin/IGF-1 receptor daf-2 mutants, enhanced proteostatic mechanisms are accompanied by elevated intestinal lipid stores, but their role in longevity is unclear. Here, while determining the regulatory network of the selective autophagy receptor SQST-1/SQSTM1, we uncovered an important role for lipid droplets in proteostasis and longevity. Using genome-wide RNAi screening, we identified several SQST-1 modulators, including lipid droplets-associated and aggregation-prone proteins. Expansion of intestinal lipid droplets by silencing the conserved cytosolic triacylglycerol lipase gene atgl-1/ATGL enhanced autophagy, and extended lifespan. Notably, a substantial amount of ubiquitinated proteins were found on lipid droplets. Reducing lipid droplet levels exacerbated the proteostatic collapse when autophagy or proteasome function was compromised, and significantly reduced the lifespan of long-lived daf-2 animals. Altogether, our study uncovered a key role for lipid droplets in C. elegans as a proteostatic mediator that modulates ubiquitinated protein accumulation, facilitates autophagy, and promotes longevity.

Monomethyl auristatin antibody and peptide drug conjugates for trimodal cancer chemo-radio-immunotherapy.

Locally advanced cancers remain therapeutically challenging to eradicate. The most successful treatments continue to combine decades old non-targeted chemotherapies with radiotherapy that unfortunately increase normal tissue damage in the irradiated field and have systemic toxicities precluding further treatment intensification. Therefore, alternative molecularly guided systemic therapies are needed to improve patient outcomes when applied with radiotherapy. In this work, we report a trimodal precision cytotoxic chemo-radio-immunotherapy paradigm using spatially targeted auristatin warheads. Tumor-directed antibodies and peptides conjugated to radiosensitizing monomethyl auristatin E (MMAE) specifically produce CD8 T cell dependent durable tumor control of irradiated tumors and immunologic memory. In combination with ionizing radiation, MMAE sculpts the tumor immune infiltrate to potentiate immune checkpoint inhibition. Here, we report therapeutic synergies of targeted cytotoxic auristatin radiosensitization to stimulate anti-tumor immune responses providing a rationale for clinical translational of auristatin antibody drug conjugates with radio-immunotherapy combinations to improve tumor control.

Establishment of Patient-Derived Succinate Dehydrogenase-Deficient Gastrointestinal Stromal Tumor Models for Predicting Therapeutic Response.

PURPOSE: Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract, with mutant succinate dehydrogenase (SDH) subunits (A-D) comprising less than 7.5% (i.e., 150-200/year) of new cases annually in the United States. Contrary to GISTs harboring KIT or PDGFRA mutations, SDH-mutant GISTs affect adolescents/young adults, often metastasize, and are frequently resistant to tyrosine kinase inhibitors (TKI). Lack of human models for any SDH-mutant tumors, including GIST, has limited molecular characterization and drug discovery. EXPERIMENTAL DESIGN: We describe methods for establishing novel patient-derived SDH-mutant (mSDH) GIST models and interrogated the efficacy of temozolomide on these tumor models in vitro and in clinical trials of patients with mSDH GIST. RESULTS: Molecular and metabolic characterization of our patient-derived mSDH GIST models revealed that these models recapitulate the transcriptional and metabolic hallmarks of parent tumors and SDH deficiency. We further demonstrate that temozolomide elicits DNA damage and apoptosis in our mSDH GIST models. Translating our in vitro discovery to the clinic, a cohort of patients with SDH-mutant GIST treated with temozolomide (n = 5) demonstrated a 40% objective response rate and 100% disease control rate, suggesting that temozolomide represents a promising therapy for this subset of GIST. CONCLUSIONS: We report the first methods to establish patient-derived mSDH tumor models, which can be readily employed for understanding patient-specific tumor biology and treatment strategies. We also demonstrate that temozolomide is effective in patients with mSDH GIST who are refractory to existing chemotherapeutic drugs (namely, TKIs) in clinic for GISTs, bringing a promising treatment option for these patients to clinic.See related commentary by Blakely et al., p. 3.

Reduction of electron deficient guanine radical species in plasmid DNA by tyrosine derivatives.

Guanine bases are the most easily oxidized sites in DNA and therefore electron deficient guanine radical species are major intermediates in the direct effect of ionizing radiation (ionization of the DNA itself) on DNA as a consequence of hole migration to guanine. As a model for this process we have used gamma-irradiation in the presence of thiocyanate ions to generate single electron oxidized guanine radicals in a plasmid target in aqueous solution. The stable species formed from these radicals can be detected and quantified by the formation of strand breaks in the plasmid after a post-irradiation incubation using a suitable enzyme. If a tyrosine derivative is also present during irradiation, the production of guanine oxidation products is decreased by electron transfer from tyrosine to the intermediate guanyl radical species. By using cationic tyrosine containing ligands we are able to observe this process when the tyrosine is electrostatically bound to the plasmid. The driving force dependence of this reaction was determined by comparing the reactivity of tyrosine with its 3-nitro analog. The results imply that the electron transfer reaction is coupled to a proton transfer. The experimental conditions used in this model system provide a reasonable approximation to those involved in the radioprotection of DNA by tightly bound proteins in chromatin.

Structure reactivity relationship in the reaction of DNA guanyl radicals with hydroxybenzoates.

In DNA, guanine bases are the sites from which electrons are most easily removed. As a result of hole migration to this stable location on guanine, guanyl radicals are major intermediates in DNA damage produced by the direct effect of ionizing radiation (ionization of the DNA itself and not through the intermediacy of water radicals). We have modeled this process by employing gamma irradiation in the presence of thiocyanate ions, a method which also produces single electron oxidized guanyl radicals in plasmid DNA in aqueous solution. The stable products formed in DNA from these radicals are detected as strand breaks after incubation with the FPG protein. When a phenolic compound is present in solution during gamma irradiation, the formation of guanyl radical species is decreased by electron donation from the phenol to the guanyl radical. We have quantified the rate of this reaction for four different phenolic compounds bearing carboxylate substituents as proton acceptors. A comparison of the rates of these reactions with the redox strengths of the phenolic compounds reveals that salicylate reacts ca. 10-fold faster than its structural analogs. This observation is consistent with a reaction mechanism involving a proton coupled electron transfer, because intra-molecular transfer of a proton from the phenolic hydroxyl group to the carboxylate group is possible only in salicylate, and is favored by the strong 6-membered ring intra-molecular hydrogen bond in this compound.

Precision Chemoradiotherapy for HER2 Tumors Using Antibody Conjugates of an Auristatin Derivative with Reduced Cell Permeability.

The most successful therapeutic strategies for locally advanced cancers continue to combine decades-old classical radiosensitizing chemotherapies with radiotherapy. Molecular targeted radiosensitizers offer the potential to improve the therapeutic ratio by increasing tumor-specific kill while minimizing drug delivery and toxicity to surrounding normal tissue. Auristatins are a potent class of anti-tubulins that sensitize cells to ionizing radiation damage and are chemically amenable to antibody conjugation. To achieve tumor-selective radiosensitization, we synthesized and tested anti-HER2 antibody-drug conjugates of two auristatin derivatives with ionizing radiation. Monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) were attached to the anti-HER2 antibodies trastuzumab and pertuzumab through a cleavable linker. While MMAE is cell permeable, MMAF has limited cell permeability as free drug resulting in diminished cytotoxicity and radiosensitization. However, when attached to trastuzumab or pertuzumab, MMAF was as efficacious as MMAE in blocking HER2-expressing tumor cells in G2-M. Moreover, MMAF anti-HER2 conjugates selectively killed and radiosensitized HER2-rich tumor cells. Importantly, when conjugated to targeting antibody, MMAF had the advantage of decreased bystander and off-target effects compared with MMAE. In murine xenograft models, MMAF anti-HER2 antibody conjugates had less drug accumulated in the normal tissue surrounding tumors compared with MMAE. Therapeutically, systemically injected MMAF anti-HER2 conjugates combined with focal ionizing radiation increased tumor control and improved survival of mice with HER2-rich tumor xenografts. In summary, our results demonstrate the potential of cell-impermeable radiosensitizing warheads to improve the therapeutic ratio of radiotherapy by leveraging antibody-drug conjugate technology.

Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin.

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.

Redirecting extracellular proteases to molecularly guide radiosensitizing drugs to tumors.

Patients with advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and treatment side effects severe. Drugs sensitizing tumors to radiotherapy have been developed to improve cell kill, but tumor specificity remains challenging. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting using peptide-based drug conjugates. We attached an inhibitor of the DNA damage response to antibody or cell penetrating peptides. Antibody drug conjugates honed in on tumor overexpressed cell surface receptors with high specificity but lacked efficacy when conjugated to the DNA damage checkpoint kinase inhibitor AZD7762. As an alternative approach, we synthesized activatable cell penetrating peptide scaffolds that accumulated within tumors based on matrix metalloproteinase cleavage. While matrix metalloproteinases are integral to tumor progression, they have proven therapeutically elusive. We harnessed these pro-tumorigenic extracellular proteases to spatially guide radiosensitizer drug delivery using cleavable activatable cell penetrating peptides. Here, we tested the potential of these two drug delivery platforms targeting distinct tumor compartments in combination with radiotherapy and demonstrate the advantages of protease triggered cell penetrating peptide scaffolds over antibody drug conjugates to deliver small molecule amine radiosensitizers.

An in vitro study for the dosimetric and radiobiological validation of respiratory gating in conventional and hypofractionated radiotherapy of the lung: effect of dose, dose rate, and breathing pattern.

Stereotactic body radiotherapy (SBRT) of the lung has become a standard of care for early-stage inoperable non-small cell lung cancer (NSCLC). A common strategy to manage respiratory motion is gating, which inevitably results in an increase in treatment time, especially in irregularly-breathing patients. Flattening-filter free (FFF) beams allow for delivery of the treatment at a higher dose rate, therefore counteracting the lengthened treatment time due to frequent interruption of the beam during gated radiotherapy. In this study, we perform our in vitro evaluation of the dosimetric and radiobiological effect of gated lung SBRT with simultaneous integrated boost (SIB) using both flattened and FFF beams. A moving thorax-shaped phantom with inserts and applicators was used for simulation, planning, gated treatment delivery measurements and in vitro tests. The effects of gating window, dose rate, and breathing pattern were evaluated. Planned doses represented a typical conventional fractionation, 200 cGy per fraction with SIB to 240 cGy, flattened beam only, and SBRT, 800 cGy with SIB to 900 cGy, flattened and FFF beams. Ideal, as well as regular and irregular patient-specific breathing patterns with and without gating were used. A survival assay for lung adenocarcinoma A549 cell line was performed. Delivered dose was within 6% for locations planned to receive 200 and 800 cGy and within 4% for SIB locations. Time between first beam-on and last beam-off varied from approximately 1.5 min for conventional fractionation, 200/240 cGy, to 10.5 min for gated SBRT, 800/900 cGy doses, flattened beam and irregular breathing motion pattern. With FFF beams dose delivery time was shorter by a factor of 2-3, depending on the gating window and breathing pattern. We have found that, for the most part, survival depended on dose and not on dose rate, gating window, or breathing regularity.

Gefitinib inhibition of drug resistance to doxorubicin by inactivating ABCG2 in thyroid cancer cell lines.

OBJECTIVE: To investigate the regulation of the breast cancer resistance protein ABCG2/BRCP1 drug transporter by epidermal growth factor receptor (EGFR) kinase activity, and to determine whether gefitinib, an EGFR small molecule inhibitor, will modulate the effects of doxorubicin hydrochloride by inhibiting its extrusion from thyroid cancer cells. DESIGN: Extrusion assays using flow cytometry analysis were used to determine the ability of thyroid cancer cells to extrude the chemotherapy drug, doxorubicin, via the ABCG2 drug transporter in the presence or absence of gefitinib. Immunofluorescence was employed to determine the cellular expression of ABCG2. The ABCG2 expression in ARO and WRO cell lines was analyzed by Western blot analysis. Inactivation of EGFR kinase by gefitinib was analyzed by Western blot analysis and immunofluorescence. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay was performed to demonstrate ABCG2-mediated apoptosis in the presence of doxorubicin. Colony formation assays were performed to determine the effect of gefitinib on thyroid cancer cell survival in response to gefitinib, doxorubicin, or the combination of both drugs. RESULTS: Inhibition of EGFR kinase activity by gefitinib causes the translocation of the ABCG2 drug transporter away from the plasma membrane, resulting in a concomitant decrease in doxorubicin extrusion in thyroid cancer cell lines. Both ARO and WRO demonstrated differential ABCG2 expression, whereas both were sensitized to doxorubicin-induced apoptosis on ABCG2 knockdown with short interfering RNA. The addition of gefitinib increases doxorubicin-induced cell death in thyroid cancer cells as measured by colony formation assay. CONCLUSIONS: Epidermal growth factor receptor regulates the function of the drug transporter ABCG2/BCRP1 and correlates with ABCG2 protein expression levels. Inactivation of the EGFR kinase by gefitinib potentiates the cytotoxic effect of doxorubicin in thyroid cancer, most likely by decreasing the ability of the cell to extrude doxorubicin. The expression of ABCG2 may explain in part the ineffectiveness of doxorubicin as a single modality treatment for anaplastic thyroid cancer or for treatment of metastatic follicular thyroid cancer. Use of this combination treatment of gefitinib and doxorubicin may be a promising therapy for anaplastic thyroid and metastatic follicular thyroid cancer and needs to be investigated further.

A novel 3D-printed phantom insert for 4D PET/CT imaging and simultaneous integrated boost radiotherapy.

PURPOSE: To construct a 3D-printed phantom insert designed to mimic the variable PET tracer uptake seen in lung tumor volumes and a matching dosimetric insert to be used in simultaneous integrated boost (SIB) phantom studies, and to evaluate the design through end-to-end tests. METHODS: A set of phantom inserts was designed and manufactured for a realistic representation of gated radiotherapy steps from 4D PET/CT scanning to dose delivery. A cylindrical phantom (φ80 × 120 mm) holds inserts for PET/CT scanning. The novel 3D printed insert dedicated to 4D PET/CT mimics high PET tracer uptake in the core and low uptake in the periphery. This insert is a variable density porous cylinder (φ44.5 × 70.0 mm), ABS-P430 thermoplastic, 3D printed by fused deposition modeling an inner (φ11 × 42 mm) cylindrical void. The square pores (1.8 × 1.8 mm2 each) fill 50% of outer volume, resulting in a 2:1 PET tracer concentration ratio in the void volume with respect to porous volume. A matching cylindrical phantom insert is dedicated to validate gated radiotherapy. It contains eight peripheral holes and one central hole, matching the location of the porous part and the void part of the 3D printed insert, respectively. These holes accommodate adaptors for Farmer-type ion chamber and cells vials. End-to-end tests were designed for imaging, planning, and dose measurements. RESULTS: End-to-end test were performed from 4D PET/CT scanning to transferring data to the planning system, target volume delineation, and dose measurements. 4D PET/CT scans were acquired of the phantom at different respiratory motion patterns and gating windows. A measured 2:1 18F-FDG concentration ratio between inner void and outer porous volume matched the 3D printed design. Measured dose in the dosimetric insert agreed well with planned dose on the imaging insert, within 3% for the static phantom and within 5% for most breathing patterns. CONCLUSIONS: The novel 3D printed phantom insert mimics variable PET tracer uptake typical of tumors. Obtained 4D PET/CT scans are suitable for segmentation and treatment planning and delivery in SIB gated treatments. Our experiments demonstrate the feasibility of this set of phantom inserts serving as end-to-end quality-assurance phantoms of SIB radiotherapy.

Gleevec suppresses p63 expression in head and neck squamous cell carcinoma despite p63 activation by DNA-damaging agents.

OBJECTIVES: The objectives of this study were to determine the effects of Gleevec on p63 expression in head and neck squamous cell carcinoma (HNSCC) cell lines and to investigate the role of Gleevec in regulating p63 stabilization under DNA-damaging conditions. METHODS: Immunohistochemical staining was performed to determine p63 expression in HNSCC tissue. Annexin V staining was used to assess the effects of p63 on early apoptosis. Immunoblotting was used to examine the effects of Gleevec on p63 protein levels in HNSCC cell lines in response to DNA damage. Immunofluorescence staining was performed to study the expression pattern of p63 and c-Abl. RESULTS: In HNSCC, p63 protein levels are induced by DNA-damaging agents, including ionizing radiation, doxorubicin, and ultraviolet light. We demonstrate that Gleevec reduces p63/DeltaNp63 expression in a dose-dependent manner in HNSCC and overrides the protein induction by DNA-damaging agents. Overexpression of c-Abl in the absence of Gleevec results in higher levels of p63 than those treated with Gleevec, implicating c-Abl kinase activity as a regulator of p63 protein stability. CONCLUSIONS: Gleevec downregulates p63/DeltaNp63 levels in HNSCC in a dose-dependent manner under both normal and DNA-damaging conditions. This downregulation can be explained by Gleevec's inhibition of c-Abl, which destabilizes p63. Based on our data, treating cancers with high expression of TAp63 with Gleevec may result in the unfavorable inhibition of a tumor suppressor, whereas downregulation of DeltaNp63 would be advantageous. Further development of antibodies that can discriminate between TAp63 and DeltaNp63 will be needed to determine the specific effects of Gleevec on p63 in HNSCC.

EGFR regulates the side population in head and neck squamous cell carcinoma.

OBJECTIVE: To identify the presence of side population (SP) cells in established head and neck squamous carcinoma cell (HNSCC) lines and to determine the role of EGFR in the regulation of the side population of these cells. METHODS: SP cells were identified using flow cytometry analysis by the ability of these cells to extrude the Hoechst 33342 dye via the drug transporter BCRP1/ABCG2. Effect of EGFR on the side population was determined also by difference in Hoechst extrusion and by immunofluorescence. Immunohistochemical staining was performed to show the presence of the BCRP1/ABCG2 transporter and the phosphorylated form of EGFR in HNSCC tissue. RESULTS: SP cells are present in HNSCC cell lines. With the Hoechst 33342 extrusion assay, SP cells were found to comprise an average of 0.69% of the UMSCC10B cells and 0.91% of HN12 cells. Addition of the EGF ligand increased the SP population while inactivation of the EGFR kinase by Iressa significantly decreased SP. CONCLUSION: In established head and neck squamous cell carcinoma cell lines, SP cells were found using methods that determine expression and function of the drug transporter BCRP1/ABCG2. Activation of EGFR, a gene implicated in tumorigenesis in HNSCC leads to increased SP, and conversely, inhibition of EGFR leads to decrease in SP. This finding could help explain the role of EGFR in regulating cancer stem cells and thus tumorigenesis in HNSCC.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines.

OBJECTIVES: Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. MATERIALS AND METHODS: HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. RESULTS: E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. CONCLUSION: E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed.

Kinase-independent role for CRAF-driving tumour radioresistance via CHK2.

Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

Use of a coumarin-labeled hexa-arginine peptide as a fluorescent hydroxyl radical probe in a nanoparticulate plasmid DNA condensate.

Coumarin derivatives have found application as probes for the hydroxyl radical because one of the products of the reaction between them is a highly fluorescent umbelliferone. We have examined the interaction in aqueous solution between a cationic coumarin-labeled hexa-arginine peptide ligand and plasmid DNA, and compared after gamma irradiation the yields of products derived from both of them. At low ionic strengths, the ligand binds very tightly to the plasmid. Compared with the structurally similar 4-methylumbelliferone (phenolic pK(a) = 7.8), the fluorescent product derived from gamma irradiation of the coumarin labeled cationic peptide is significantly more acidic (pK(a) = 6.1), making it a very convenient probe for solutions of pH in the physiological range. The yield of this product is generally in excellent agreement over a wide range of conditions with that of the single strand break product produced by the reaction of the hydroxyl radical with the plasmid. Thus coumarin-labeled peptide ligands offer promise as hydroxyl radical probes for locations in close proximity to DNA.

Characterization of condensed plasmid DNA models for studying the direct effect of ionizing radiation.

We have examined the changes in physical properties of aqueous solutions of the plasmid pUC18 that take place on the addition of the cationic oligopeptide penta-arginine. An increase in sedimentation rate and static light scattering, and changes in the nucleic acid CD spectrum all suggest that this ligand acts to condense the plasmid. Dynamic light scattering suggests the hydrodynamic radii of the condensate particles are a few micrometers, ca. 50-fold larger than that of the monomeric plasmid. Condensation of the plasmid also produces a ca. 100-fold decrease in the strand break yield produced by gamma irradiation. This extensive protection against reactive intermediates in the bulk of the solution implies that condensed plasmid DNA may offer a model system with which to study the direct effect of ionizing radiation (ionization of the DNA itself). The use of peptide ligands as condensing agents in this application is attractive because the derivatives of several amino acids (particularly tryptophan and tyrosine) have been shown to modify the radiation chemistry of DNA extensively.