Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO₂-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.     

Effect of APE1 T2197G (Asp148Glu) Polymorphism on APE1, XRCC1, PARP1 and OGG1 Expression in Patients with Colorectal Cancer

It has been hypothesized that genetic variation in base excision repair (BER) might modify colorectal adenoma risk. Thus, we evaluated the influence of APE1 T2197G (Asp148Glu) polymorphism on APE1, XRCC1, PARP1 and OGG1 expression in normal and tumor samples from patients with colorectal cancer. The results indicate a downregulation of OGG1 and an upregulation of XRCC1 expression in tumor tissue. Regarding the anatomical location of APE1, OGG1 and PARP-1, a decrease in gene expression was observed among patients with cancer in the rectum. In patients with or without some degree of tumor invasion, a significant downregulation in OGG1 was observed in tumor tissue. Interestingly, when taking into account the tumor stage, patients with more advanced grades (III and IV) showed a significant repression for APE1, OGG1 and PARP-1. XRCC1 expression levels were significantly enhanced in tumor samples and were correlated with all clinical and histopathological data. Concerning the polymorphism T2197G, GG genotype carriers exhibited a significantly reduced expression of genes of the BER repair system (APE1, XRCC1 and PARP1). In summary, our data show that patients with colorectal cancer present expression changes in several BER genes, suggesting a role for APE1, XRCC1, PARP1 and OGG1 and APE1 polymorphism in colorectal carcinogenesis.     

On the capacity of the state‐dependent interference relay channel

This paper considers the state‐dependent interference relay channel (SIRC) in which one of the two users may operate as a secondary user and the relay has a noncausal access to the signals from both users. For discrete memoryless SIRC, we first establish the achievable rate region by carefully merging Han‐Kobayashi rate splitting encoding technique, superposition encoding, and Gelfand‐Pinsker encoding technique. Then, based on the achievable rate region that we derive, the capacity of the SIRC is established in many different scenarios including (a) the weak interference regime, (b) the strong interference regime, and (c) the very strong interference regime. This means that our capacity results contain all available known results in the literature. Next, the achievable rate region and the associated capacity results are also evaluated in the case of additive Gaussian noise. Additionally, many numerical examples are investigated to show the value of our theoretical derivations.     

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa-Perturbed Nucleotides

The formation of artificial metal base pairs is an alluring and versatile method for the functionalization of nucleic acids. Access to DNA functionalized with metal base pairs is granted mainly by solid-phase synthesis. An alternative, yet underexplored method, envisions the installation of metal base pairs through the polymerization of modified nucleoside triphosphates. Herein, we have explored the possibility of using thiolated and pK_a-perturbed nucleotides for the enzymatic construction of artificial metal base pairs. The thiolated nucleotides S2C, S6G, and S4T as well as the fluorinated analogue 5FU are readily incorporated opposite a templating S4T nucleotide through the guidance of metal cations. Multiple incorporation of the modified nucleotides along with polymerase bypass of the unnatural base pairs are also possible under certain conditions. The thiolated nucleotides S4T, S4T, S2C, and S6G were also shown to be compatible with the synthesis of modified, high molecular weight single-stranded (ss)DNA products through TdT-mediated tailing reactions. Thus, sulfur-substitution and pK_a perturbation represent alternative strategies for the design of modified nucleotides compatible with the enzymatic construction of metal base pairs.     

Additive-Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C-Nucleosides at the Reaction Points

We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D * , T * , G * , and C * , corresponding—from a viewpoint of hydrogen-bonding patterns—to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5′-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5′-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3′-natural ⋅ 5′-artificial, 3′-artificial ⋅ 5′-natural, and 3′-artificial ⋅ 5′-artificial terminal combination at the nicks.     

Controlling Pyrene Association in DNA Duplexes by B- to Z-DNA Transitions

B- to Z-DNA transitions play a crucial role in biological systems and have attracted the interest of researchers for their applications in DNA nanotechnology. DNA and DNA analogues have also been used as templates to construct helical chromophore associations with π interactions. In this work, the B- to Z-DNA transition-induced switching of pyrene in an association manner was evaluated using DNA duplexes with non-nucleosidic pyrene residues in the middle of d(CG) repeat sequences. One of the pyrene-labeled DNAs was shown to exhibit inverted exciton coupled circular dichroism signals upon pyrene association through a B- to Z-DNA transition. This observation indicates that pyrene association switches the DNA conformation from right- to left-handed. Interestingly, the fluorescence of the pyrene-labeled DNA duplex also dynamically changed upon switching of the pyrene in an association-based manner. Taken together, these studies demonstrate that pyrene-labeled DNA shows promise as a chiroptical molecular switch.