Computation of the conditions for anti-angiogenesis and gene therapy synergistic effects: Sensitivity analysis and robustness of target solutions.

Both anti-angiogenesis and gene therapy involve complex processes depending on non-point parameters belonging to a space of values. To successfully overcome the challenges involved in their therapeutic approaches, there is a need to analyze the sensitivity of these parameters. In this paper, a new mathematical model that combines immune system stimulations, inflammatory processes associated with tumor development, and gene therapy aimed at enhancing the efficacy of both treatments are explored. Using the global sensitivity methods of Sobol and Morris, the most important parameters are estimated. Estimation of the sensitivity variance revealed a strong interdependence between the parameters. Also, determinations of the conditions for effective therapy lead to a target of reducing the cancer cell numbers by at least 50%. This opened the way for delimiting the parameter spaces making it possible to reach the treatment target in addition to enhancing the estimation of the minimum time of remission. The combination of therapies and sensitivity analysis have demonstrated the robustness of therapy success.

Molecular Determinant of DIDS Analogs Targeting RAD51 Activity.

RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.

Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions.

Tauopathies such as Alzheimer's Disease (AD) are neurodegenerative disorders for which there is presently no cure. They are named after the abnormal oligomerization/aggregation of the neuronal microtubule-associated Tau protein. Besides its role as a microtubule-associated protein, a DNA-binding capacity and a nuclear localization for Tau protein has been described in neurons. While questioning the potential role of Tau-DNA binding in the development of tauopathies, we have carried out a large-scale analysis of the interaction of Tau protein with the neuronal genome under physiological and heat stress conditions using the ChIP-on-chip technique that combines Chromatin ImmunoPrecipitation (ChIP) with DNA microarray (chip). Our findings show that Tau protein specifically interacts with genic and intergenic DNA sequences of primary culture of neurons with a preference for DNA regions positioned beyond the ±5000 bp range from transcription start site. An AG-rich DNA motif was found recurrently present within Tau-interacting regions and 30% of Tau-interacting regions overlapped DNA sequences coding for lncRNAs. Neurological processes affected in AD were enriched among Tau-interacting regions with in vivo gene expression assays being indicative of a transcriptional repressor role for Tau protein, which was exacerbated in neurons displaying nuclear pathological oligomerized forms of Tau protein.

Rapid Diminution in the Level and Activity of DNA-Dependent Protein Kinase in Cancer Cells by a Reactive Nitro-Benzoxadiazole Compound.

The expression and activity of DNA-dependent protein kinase (DNA-PK) is related to DNA repair status in the response of cells to exogenous and endogenous factors. Recent studies indicate that Epidermal Growth Factor Receptor (EGFR) is involved in modulating DNA-PK. It has been shown that a compound 4-nitro-7-[(1-oxidopyridin-2-yl)sulfanyl]-2,1,3-benzoxadiazole (NSC), bearing a nitro-benzoxadiazole (NBD) scaffold, enhances tyrosine phosphorylation of EGFR and triggers downstream signaling pathways. Here, we studied the behavior of DNA-PK and other DNA repair proteins in prostate cancer cells exposed to compound NSC. We showed that both the expression and activity of DNA-PKcs (catalytic subunit of DNA-PK) rapidly decreased upon exposure of cells to the compound. The decline in DNA-PKcs was associated with enhanced protein ubiquitination, indicating the activation of cellular proteasome. However, pretreatment of cells with thioglycerol abolished the action of compound NSC and restored the level of DNA-PKcs. Moreover, the decreased level of DNA-PKcs was associated with the production of intracellular hydrogen peroxide by stable dimeric forms of Cu/Zn SOD1 induced by NSC. Our findings indicate that reactive oxygen species and electrophilic intermediates, generated and accumulated during the redox transformation of NBD compounds, are primarily responsible for the rapid modulation of DNA-PKcs functions in cancer cells.

Nuclear magnetic resonance spectroscopy characterization of interaction of Tau with DNA and its regulation by phosphorylation.

The capacity of endogenous Tau to bind DNA has been recently identified in neurons under physiological or oxidative stress conditions. Characterization of the protein domains involved in Tau-DNA complex formation is an essential first step in clarifying the contribution of Tau-DNA interactions to neurological biological processes. To identify the amino acid residues involved in the interaction of Tau with oligonucleotides, we have characterized a Tau-DNA complex using nuclear magnetic resonance spectroscopy. Interaction of an AT-rich or GC-rich 22 bp oligonucleotide with Tau showed multiple points of anchoring along the intrinsically disordered Tau protein. The main sites of contact characterized here correspond to the second half of the proline-rich domain (PRD) of Tau and the R2 repeat in the microtubule binding domain. This latter interaction site includes the PHF6* sequence known to govern Tau aggregation. The characterization was pursued by studying the binding of phosphorylated forms of Tau, displaying multiple phosphorylation sites mainly in the PRD, to the same oligonucleotide. No interaction of phospho-Tau with the oligonucleotide was detected, suggesting that pathological Tau phosphorylation could affect the physiological function of Tau mediated by DNA binding.

DNA-PKcs Ser2056 auto-phosphorylation is affected by an O-GlcNAcylation/phosphorylation interplay.

BACKGROUND: DNA dependent Protein Kinase (DNA-PK) is an heterotrimeric complex regulating the Non Homologous End Joining (NHEJ) double strand break (DSB) repair pathway. The activity of its catalytic subunit (DNA-PKcs) is regulated by multiple phosphorylations, like the Ser2056 one that impacts DSB end processing and telomeres integrity. O-GlcNAcylation is a post translational modification (PTM) closely related to phosphorylation and its implication in the modulation of DNA-PKcs activity during the DNA Damage Response (DDR) is unknown. METHODS: Using IP techniques, and HeLa cell line, we evaluated the effect of pharmacological or siOGT mediated O-GlcNAc level modulation on DNA-PKcs O-GlcNAcylation. We used the RPA32 phosphorylation as a DNA-PKcs activity reporter substrate to evaluate the effect of O-GlcNAc modulators. RESULTS: We show here that human DNA-PKcs is an O-GlcNAc modified protein and that this new PTM is responsive to the cell O-GlcNAcylation level modulation. Our findings reveal that DNA-PKcs hypo O-GlcNAcylation affects its kinase activity and that the bleomycin-induced Ser2056 phosphorylation, is modulated by DNA-PKcs O-GlcNAcylation. CONCLUSIONS: DNA-PKcs Ser2056 phosphorylation is antagonistically linked to DNA-PKcs O-GlcNAcylation level modulation. GENERAL SIGNIFICANCE: Given the essential role of DNA-PKcs Ser2056 phosphorylation in the DDR, this study brings data about the role of cell O-GlcNAc level on genome integrity maintenance.

Comparative Advantages and Limitations of Quantum Dots in Protein Array Applications.

Antibody microarrays have become a powerful tool in multiplexed immunoassay technologies. The advantage of microarray technology is the possibility of rapid analysis of multiple targets in a single sample with a high sensitivity, which makes them ideal for high throughput screening. Usually these microarrays contain biological recognition molecules, such as full-size antibodies, antigen-binding fragments, and single-domain antibodies, and a label for detection. Organic fluorophores are the most popular labels, but they suffer from low sensitivity and instability due to their photodegradation. Here, we describe a protocol for fabricating an antibody microarray with highly fluorescent semiconductor nanocrystals or quantum dots (QDs) as the source of fluorescent signals, which may significantly improve the properties of microarrays, including their sensitivity and specificity. Our approach to analyte detection is based on the use of sandwich approach with streptavidin-biotin to assess and monitor the fluorescence signal instead of direct labeling of samples, which helps improve the reproducibility of results and sensitivity of the microarrays. The antibody microarray developed has been tested for its capacity of detecting DNA-PKcs in glial cell lines and measuring cell protein phosphorylation changes caused by camptothecin-induced DNA damage with different protein kinase inhibitors in HeLa cells.

Binding of YY1 to the proximal region of the murine beta interferon promoter is essential to allow CBP recruitment and K8H4/K14H3 acetylation on the promoter region after virus infection.

Virus-induced activation of the beta interferon (IFN-beta) gene requires orderly recruitment of chromatin-remodeling complexes and time-regulated acetylation of histone residues K8H4 and K14H3 on the promoter region. We have previously shown that transcription factor Yin Yang 1 (YY1) binds the murine IFN-beta promoter at two sites (-122 and -90) regulating promoter transcriptional capacity with a dual activator/repressor role. In this work we demonstrate that both YY1 -122 and -90 sites are required for CBP recruitment and K8H4/K14H3 acetylation to take place on the IFN-beta promoter region after virus infection. A single point mutation introduced at either one of these two sites inhibiting YY1 binding completely disrupted CBP recruitment and K8H4/K14H3 acetylation independently of HMGI or IRF3 binding to the promoter. We have previously demonstrated that YY1 represses the transcriptional capacity of the IFN-beta promoter through its -90 site via histone deacetylation. Here we demonstrate that, in vivo, the binding of YY1 to the -90 site is constant all through virus infection whereas the binding of YY1 to the -122 site is activated after infection. We discuss here the capacity of YY1 to either repress (through histone deacetylase recruitment) or activate (through CBP recruitment) IFN-beta gene expression according to the occupancy of either only its -90 site or both its -122 and -90 sites.

Assessment of DNA-PKcs kinase activity by quantum dot-based microarray.

Therapeutic efficacy against cancer is often based on a variety of DNA lesions, including DNA double-strand breaks (DSBs) which are repaired by homologous recombination and non-homologous end joining (NHEJ) pathways. In the past decade, the functions of the DNA repair proteins have been described as a potential mechanism of resistance in tumor cells. Therefore, the DNA repair proteins have become targets to improve the efficacy of anticancer therapy. Given the central role of DNA-PKcs in NHEJ, the therapeutic efficacy of targeting DNA-PKcs is frequently described as a strategy to prevent repair of treatment-induced DNA damage in cancer cells. The screening of a new inhibitor acting as a sensitizer requires the development of a high-throughput tool in order to identify and assess the most effective molecule. Here, we describe the elaboration of an antibody microarray dedicated to the NHEJ pathway that we used to evaluate the DNA-PKcs kinase activity in response to DNA damage. By combining a protein microarray with Quantum-Dot detection, we show that it is possible to follow the modification of phosphoproteomic cellular profiles induced by inhibitors during the response to DNA damage. Finally, we discuss the promising tool for screening kinase inhibitors and targeting DSB repair to improve cancer treatment.

Bioconjugated fluorescent organic nanoparticles targeting EGFR-overexpressing cancer cells.

The field of optical bioimaging has considerably flourished with the advent of sophisticated microscopy techniques and ultra-bright fluorescent tools. Fluorescent organic nanoparticles (FONs) have thus recently appeared as very attractive labels for their high payload, absence of cytotoxicity and eventual biodegradation. Nevertheless, their bioconjugation to target specific receptors with high imaging contrast is scarcely performed. Moreover, assessing the reality of bioconjugation represents high challenges given the sub-nanomolar concentrations resulting from the commonly adopted nanoprecipitation fabrication process. Here, we describe how the combination of a magnetic shell allows us to easily generate red-emitting FONs conjugated with the epidermal growth factor ligand (EGF), a small protein promoting cancer cell proliferation by activating the EGF receptor (EGFR) pathway. Dual color fluorescence correlation spectroscopy combined with immunofluorescence is originally harnessed in its time trace mode to unambiguously demonstrate covalent attachment between the FON and EGF at sub-nanomolar concentrations. Strong asymmetric clustering of EGF-conjugated FONs is observed at the membrane of MDA-MB-468 human breast cancer cells overexpressing EGF receptors using super-resolution fluorescence microscopy. Such high recruitment of EGF-conjugated FONs is attributed to their EGF multivalency (4.7 EGF per FON) which enables efficient EGFR activation and subsequent phosphorylation. The large hydrodynamic diameter (DH ∼ 301 nm) of EGF-conjugated FONs prevents immediate engulfment of the sequestered receptors, which provides very bright and localized spots in less than 30 minutes. The reported bioconjugated nanoassemblies could thus serve as ultra-bright probes of breast cancer cells with EGFR-overexpression that is often associated with poor prognosis.

Transcription factor YY1 associates with pericentromeric gamma-satellite DNA in cycling but not in quiescent (G0) cells.

Pericentromeric gamma-satellite DNA is organized in constitutive heterochromatin structures. It comprises a 234 bp sequence repeated several thousands times surrounding the centromeric sequence of all murine chromosomes. Potential binding sites for transcription factor Yin Yang 1 (YY1), a repressor or activator of several cellular and viral genes, are present in pericentromeric gamma-satellite DNA. Using gel retardation and chromatin immunoprecipitation, we demonstrate in this work that YY1 specifically interacts in vitro and in vivo with gamma-satellite DNA. Using immunoFISH and confocal microscopy we show that YY1 specifically co-localizes with pericentromeric gamma-satellite DNA clusters organized in constitutive heterochromatin in murine L929 and 3T3 fibroblasts cell lines. Immunoelectron microscopy experiments further confirmed YY1 localization in heterochromatic areas. Overall, our results demonstrate for the first time that a fraction of YY1 is directly associated with constitutive heterochromatin structures. This association appears physiologically relevant since the association of YY1 with pericentromeric gamma-satellite DNA observed in cycling 3T3 fibroblasts strongly diminished in quiescent (G0) 3T3 fibroblasts. We discuss the implications of these results in the context of heterochromatin formation as well as with regard to the YY1-induced repression of euchromatic genes.

Tuning the architectural integrity of high-performance magneto-fluorescent core-shell nanoassemblies in cancer cells.

High-density nanoarchitectures, endowed with simultaneous fluorescence and contrast properties for MRI and TEM imaging, have been obtained using a simple self-assembling strategy based on supramolecular interactions between non-doped fluorescent organic nanoparticles (FON) and superparamagnetic nanoparticles. In this way, a high-payload core-shell structure FON@mag has been obtained, protecting the hydrophobic fluorophores from the surroundings as well as from emission quenching by the shell of magnetic nanoparticles. Compared to isolated nanoparticles, maghemite nanoparticles self-assembled as an external shell create large inhomogeneous magnetic field, which causes enhanced transverse relaxivity and exacerbated MRI contrast. The magnetic load of the resulting nanoassemblies is evaluated using magnetic sedimentation and more originally electrospray mass spectrometry. The role of the stabilizing agents (citrate versus polyacrylate anions) revealed to be crucial regarding the cohesion of the resulting high-performance magneto-fluorescent nanoassemblies, which questions their use after cell internalization as nanocarriers or imaging agents for reliable correlative light and electron microcopy.

Loss of Tau protein affects the structure, transcription and repair of neuronal pericentromeric heterochromatin.

Pericentromeric heterochromatin (PCH) gives rise to highly dense chromatin sub-structures rich in the epigenetic mark corresponding to the trimethylated form of lysine 9 of histone H3 (H3K9me3) and in heterochromatin protein 1α (HP1α), which regulate genome expression and stability. We demonstrate that Tau, a protein involved in a number of neurodegenerative diseases including Alzheimer's disease (AD), binds to and localizes within or next to neuronal PCH in primary neuronal cultures from wild-type mice. Concomitantly, we show that the clustered distribution of H3K9me3 and HP1α, two hallmarks of PCH, is disrupted in neurons from Tau-deficient mice (KOTau). Such altered distribution of H3K9me3 that could be rescued by overexpressing nuclear Tau protein was also observed in neurons from AD brains. Moreover, the expression of PCH non-coding RNAs, involved in PCH organization, was disrupted in KOTau neurons that displayed an abnormal accumulation of stress-induced PCH DNA breaks. Altogether, our results demonstrate a new physiological function of Tau in directly regulating neuronal PCH integrity that appears disrupted in AD neurons.

Are Fluorescent Organic Nanoparticles Relevant Tools for Tracking Cancer Cells or Macrophages?

Strongly solvatochromic fluorophores are devised, containing alkyl chains and enable to self-assemble as very bright fluorescent organic nanoparticles (FONs) in water (Φf = 0.28). The alkyl chains impart each fluorophore with strongly hydrophobic surroundings, causing distinct emission colors between FONs where the fluorophores are associated, and their disassembled state. Such color change is harnessed to assess the long-term fate of FONs in both cancer cells and monocytes/macrophages. Disintegration of the orange-emitting FONs by monocytes/macrophages is evidenced through the formation of micrometer green-yellowish emitting vesicles. By contrast, cancer cells retain longer the integrity of organic nanoparticles. In both cases, no significant toxicity is detected, making FONs as valuable bioimaging agents for cell tracking with weak risks of deleterious accumulation and low degradation rate.

Mathematical modeling of glioma therapy using oncolytic viruses.

Diffuse infiltrative gliomas are adjudged to be the most common primary brain tumors in adults and they tend to blend in extensively in the brain micro-environment. This makes it difficult for medical practitioners to successfully plan effective treatments. In attempts to prolong the lengths of survival times for patients with malignant brain tumors, novel therapeutic alternatives such as gene therapy with oncolytic viruses are currently being explored. Based on such approaches and existing work, a spatio-temporal model that describes interaction between tumor cells and oncolytic viruses is developed. Conditions that lead to optimal therapy in minimizing cancer cell proliferation and otherwise are analytically demonstrated. Numerical simulations are conducted with the aim of showing the impact of virotherapy on proliferation or invasion of cancer cells and of estimating survival times.