Network modeling of the interconnections among the crucial factors involved in metabolic flow and signaling pathways of the Warburg effect to “protect” the cancer cells.

<p>(A) Cancer cells make use of their nutrient-rich environment by taking in glucose and converting it into molecular precursors by aerobic glycolysis (shown in yellow). This is mediated by activation of proto-oncogenes such as AKT and MYC and other genes in important growth factor signaling pathways. Moreover, RAS-mediated activation of HIF1 induces adaptation to hypoxic environments and promotes “niches” that are conducive to cancer cells. In addition, the use of antioxidants and recycling of NADPH as defense mechanisms to sequester ROS favor the survival of cancer cells. (B) Oxidative phosphorylation (OXPHOS) impairment leads to crippled mitochondrial respiration. The dysfunctional TCA cycle generates fewer reactive oxygen species that may or may not induce DNA damage, and this subsequently leads to fewer mitochondrial DNA mutations. (C) Efficient repair of mtDNA leads to fewer mutations and less mitochondrial dysfunction. (D) In contrast, normal, low proliferative cells utilize OXPHOS and generate ROS that induce mtDNA damage and increase mutation frequency (shown in green).</p

Lambda-loop, 5-Ala and Loop1- are mutators in the missing base primer extension assay in the absence of dCTP

<p><b>Copyright information:</b></p><p>Taken from "Loop II of DNA polymerase beta is important for polymerization activity and fidelity"</p><p></p><p>Nucleic Acids Research 2007;35(9):2924-2935.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888816.</p><p>© 2007 The Author(s)</p> Quantification of the products for all loop II variants was performed using Phosphoimager and ImageQuant software as described in the Materials and methods section. () nucleotide pool missing A; () nucleotide pool missing T; () nucleotide pool missing C; () nucleotide pool missing G. The -axis lists the loop mutants. 1 is 9-Ala, 2 is Loopless, 3 is 5-Ala, 4 is 4-Ala, 5 is 3-Ala, 6 is 2-Ala, 7 is 1-Ala, 8 is Loop1-, 9 is lambda-loop and 10 is Loop2-. Loop mutants are organized in groups of 10 for each graph with the incubation time points of 2, 5 and 10 min listed underneath. -axis is the fold-increase of loop II variant product over wild-type product. The results are the average of three experiments. Error bars represent the standard deviation of the mean

Example of the five base-pair missing base primer-extension fidelity assay

<p><b>Copyright information:</b></p><p>Taken from "Loop II of DNA polymerase beta is important for polymerization activity and fidelity"</p><p></p><p>Nucleic Acids Research 2007;35(9):2924-2935.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888816.</p><p>© 2007 The Author(s)</p> Reaction products formed during the gap-filling assay when dCTP is missing from the nucleotide pool are shown. Purified enzymes were incubated with gapped DNA substrate that was radiolabeled at the 5′ end of the primer () and a pool of three dNTPs missing dCTP for 2, 5 and 10 min at 37°C as described in the Materials and methods section. The products resulting from incorporation of nucleotides into the primer were resolved on a denaturing acrylamide gel and visualized on a Phosphorimager. Lane 1 is wild type, lane 2 is lambda-loop, lane 3 is 5-Ala loop, lane 4 is 4-Ala loop and P is the primer strand. The length of incubation time is listed underneath each group. The sequence of the template is listed on the side of each group

A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

<div><p>Several germline single nucleotide polymorphisms (SNPs) have been identified in the <em>POLB</em> gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the <em>POLB</em> gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the <em>POLB</em> germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.</p> </div

Chromosomal aberrations in P242R-expressing cells.

<p>Representative metaphase spread of MCF10A expressing (A.) WT or (B.) P242R Pol β. Chromosomal fusions are shown with the gray arrow and fragments are shown with black arrows. C. Number of aberrations per metaphase. A total of at least 50 metaphases were scored for each cell line.</p

Accumulation of BER intermediates in MCF10A cells expressing P242R Pol β.

<p>A. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 minutes and allowed to recover for 0, 30, or 60 min and single-strand breaks (SSBs) were analyzed by comet assay. The percentage of tail DNA is plotted on the Y-axis. B. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 min and allowed to recover for 0, 30, or 60 min, stained with γH2AX antibody, and analyzed by flow cytometry. Cells were treated for 120 min as a positive control. C–D. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 2 h and allowed to recover for 0 or 2 hours. Cells were stained with γH2AX antibody and propidium iodide to assess the levels of double-strand breaks (DSBs) and the cell cycle phase, respectively, and analyzed by flow cytometry. Data are plotted as the mean ± SEM. Data are plotted as the mean ± SEM (n = 3). A and C. ** and *** denote <i>p</i><0.01 and 0.001, respectively. ∧ denotes <i>p</i><0.05 comparing 0 vs 2 h recovery within each cell line. ∧∧∧ denotes <i>p</i><0.001 comparing 30 or 60 min recovery to 0 recovery. D. *** denotes <i>p</i><0.001 comparing WT+MMS to P242R+MMS in each phase of the cell cycle. ∧ and ∧∧∧ denote <i>p</i><0.05 and 0.001, respectively, comparing WT+MMS+recovery to P242R+MMS+recovery in each phase of the cell cycle.</p

The P242R germline variant of Pol β is slow and binds DNA tightly.

<p>A. Representative results from a presteady-state burst assay. Results for the WT are shown as filled squares fit with a solid curve. Results for the P242R are shown as open triangles fit with a dashed curve. The assay was repeated four times for each protein. B. Representative results from a gel electrophoretic mobility shift assay. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003052#s2" target="_blank">Results</a> for the different proteins are shown as in A.</p

P242R Pol β confers slight sensitivity to MMS compared to WT.

<p>Clonogenic survival assays were conducted with (A.) Pol β^−/− MEFs, (B.) Pol β^+/+ MEFs, or (C.) MCF10A pools expressing WT or P242R Pol β. Filled circles represent results from pools expressing empty vector, filled squares represent pools expressing WT Pol β, and filled triangles represent pools expressing P242R Pol β. Data are plotted as the mean ± SEM (n = 3).</p

KIF5B suppresses centrosome amplification.

<p>A–C) Control cells during different stages of cell division with one and two centrosomes. D–F) Representative images of cells with <i>Kif5b-shRNA-</i>mediated knockdown with more than two centrosomes. The DNA is stained with DAPI (blue), and centrosomes are stained with γ-tubulin (green). The arrow indicates the centrosome in each cell. G) The representative number of cells analyzed for centrosomes in control cells (n = 119) and <i>Kif5b-shRNA</i> knockdown cells (n = 175). Cells with greater than two centrosomes were significantly enriched by <i>Kif5b-shRNA-</i>mediated knockdown (*** P<0.0001). The Graph Pad Prism program was used for analysis of the data and employed the Mann Whitney test.</p

KIF5B is required for chromosome segregation and cytokinesis.

<p>A) Examples of control cells with normal anaphase chromosome separation (n = 50). B) Anaphase bridges shown with arrow in <i>Kif5b</i> downregulated cells (n = 40). Note that *** represents P<0.0001 statistically significant differences between control cells that are transfected with scrambled shRNA and <i>Kif5b</i> knockdown cells in chromosome arrangements at anaphase stage. C) Representative images of cells with normal and abnormal cytokinesis. D) The percentage of cells with cytokinesis failure is significantly increased (* P<0.01) in <i>Kif5b-shRNA-</i>mediated knockdown cells (n = 77) compared to control cells transfected with scrambled shRNA cells (n = 109). Note that the white arrow points to an example of aberrant cytokinesis, red represents DNA, and green is α- tubulin.</p