Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts <i>S</i>-[2-(<i>N</i>⁷-Guanyl)ethyl]glutathione, <i>S</i>-[2-(<i>N</i>²-Guanyl)ethyl]glutathione, and <i>S</i>-[2-(<i>O</i>⁶-Guanyl)ethyl]glutathione^†

The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding

Synthesis of Oligonucleotides Containing the Ethylene Dibromide-Derived DNA Adducts <i>S</i>-[2-(<i>N</i>⁷-Guanyl)ethyl]glutathione, <i>S</i>-[2-(<i>N</i>²-Guanyl)ethyl]glutathione, and <i>S</i>-[2-(<i>O</i>⁶-Guanyl)ethyl]glutathione at a Single Site^†

The carcinogen ethylene dibromide (EDB) is activated by enzymatic conjugation with GSH to form S-(2-bromoethyl)GSH, which reacts with DNA via an episulfonium ion. The major DNA adduct derived from EDB was previously characterized as S-[2-(N7-guanyl)ethyl]GSH, and S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH are minor adducts [Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., and Guengerich, F. P. (1992) J. Biol. Chem. 267, 6672−6679]. S-[2-(N7-Guanyl)ethyl]GSH has been incorporated at the G* site in d(5‘-TGCTG*CAAG-3‘), a site previously found to show GC to AT transitions following treatment of M13 phage with S-(2-chloroethyl)GSH, and the desired product was separated by HPLC. This was ligated to d(5‘-GGTACCGAG-3‘) to yield d(5‘-TGCTG*CAAGGGTACCGAG-3‘). S-[2-(N2-Guanyl)ethyl]GSH was incorporated into the G* site of the oligonucleotide in d(5‘-TGCTG*CAAGGGTACCGAG-3‘) by reacting S-(2-aminoethyl)GSH with an oligomer containing 2-fluoro-O6-[(trimethylsilyl)ethoxy]deoxyinosine at the target site. The 5‘-(dimethoxytrityl)-N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl] derivative of S-[2-(O6-deoxyguanosyl)ethyl]GSH dimethyl ester was synthesized by Mitsunobu alkylation of 5‘-(dimethoxytrityl)-N2-(phenoxyacetyl)deoxyguanosine with N-[(fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH dimethyl ester, modified to form the phosphoramidite derivative, and incorporated at the G* site of d(5‘-TGCTG*CAAGGGTACCGAG-3‘). The protective groups were removed with 0.10 N NaOH to give the modified oligonucleotide containing S-[2-(O6-guanyl)ethyl]GSH. Although the overall yields were low, the synthesis of a single set of target site oligonucleotides containing these three known guanyl adducts allows for in vitro site-specific misincorporation studies

Metformin enhanced radiosensitivity by reducing p53-related HR repair proteins <i>in vitro</i>, especially in p53-deficient cells.

<p>HCT116 p53^+/+ and p53^-/- cells were pretreated with 2.5 mM metformin for 24 h, and then irradiated with 6 Gy. Cells from both groups were immunoblotted with p53-related HR repair proteins such as MRE11-Rad50-p95/NBS1 complex, BRCA1, BRCA2, Rad51, Rad52, and ERCC1 24 h after ionizing radiation (IR). Densitometric quantification was normalized to <i>β</i>-actin. HR, homologous recombination; MRE11, meiotic recombination 11; NBSI, nijmegen breakage syndrome protein 1; BRCA, breast cancer early onset; ERCC 1, excision repair cross-complementation group 1.</p

Metformin delayed repair of IR-induced DNA damage in p53-deficient cells compared with p53 wild-type cells.

<p>HCT116 p53^+/+ and p53^-/- cells were pretreated with 2.5 mM metformin for 24 h, and then irradiated with 6 Gy. (A) Immunofluorescence staining at 6 and 24 h after IR, showed γ-H2AX, a marker for DNA damage; Rad51, a marker of DNA repair; and nuclear DNA stained with DAPI using image analysis in three (green/red/blue) fluorescence channels. (B) For quantitative analysis, γ-H2AX or Rad51 foci-positive cells were counted in at least 100 cells from randomly captured images. Values are mean ± S.E.M, *<i>p</i> < 0.001. IR, ionizing radiation; γ-H2AX, γ-H2A histone family, member X.</p

Platform TCE model simulation of two-dimensional contour plots for the effects of TCEs’ equilibrium dissociation constants (kds) on TAAs and CD3s on immune synapse (TAA-TCE-CD3 trimer) formation in blood, bone marrow, lymph node, spleen, and other tissue compartment after subcutaneous TCE (an IgG format, 150 kDa) administration once weekly in patients.

Other parameters were the same as those for PF-06863135.</p

Summary of parameters related to bispecific binding, two pore theory and systemic elimination of shed BCMAs, AMG420, AMG211 and PF-06863135 in patients.

Summary of parameters related to bispecific binding, two pore theory and systemic elimination of shed BCMAs, AMG420, AMG211 and PF-06863135 in patients.</p

Schematic computational platform model structures for TCEs.

(A) Mechanistic bispecific binding model of TCEs where bispecific TCEs bind either to tumor-associated antigens (TAAs) on tumor cells or to CD3s on T cells to form TCE-TAA or TCE-CD3 dimers, subsequently binding with other binding targets, either CD3s or TAAs, respectively, to form immune synapses (TAA-TCE-CD3 trimer). Shed targets competitively inhibit TCEs or TCE-CD3s binding to TAAs. (B) Two-pore theory biodistribution model. TCEs and shed targets are transported from vascular space to tissue interstitial space via two groups of pores (small pores: ~4.44 nm, large pores: ~22.9 nm) by diffusion and convection in a molecular size-dependent manner. Q, Q-L, and L represent an arterial blood flow, a venous blood flow, and a lymphatic flow in each tissue, respectively. PS, J, and Jiso represent diffusion, convection, and insogravimetric flows, respectively. Subscript L and S represent large and small pores. (C) Integrated physiologically-based platform model for TCEs. The biodistribution processes of TCEs and shed targets into and out of each tissue were characterized by integrated rate constants accounting for perfusion, diffusion, convection, and lymphatic flow rate constants in a molecular size-dependent manner based on the two-pore theory. Extravasation and biodistribution of T cells and multiple myeloma cells are incorporated into the model. In each tissue and blood compartment, bispecific TCE binding interactions between TAAs, CD3s, and shed targets were considered. Q’ and (Q-L)’ represent integrated biodistribution rate constants in and out of tissue interstitial spaces, respectively. L and CL represent lymphatic flows and a systemic clearance of TCEs or shed targets, respectively. Subscript BM, SP, LN, and Tis represent bone marrow, spleen, lymph node, and other tissue, respectively.</p

Metformin prevented cell cycle progression by significantly prolonging IR-induced G2/M arrest in HCT116 p53^-/- cells.

<p>HCT116 p53^+/+ and p53^-/- cells were pretreated with 2.5 mM metformin for 24 h, and then irradiated with 6 Gy. Cell cycle measured by flow cytometry (A) 24 and (B) 48 h after IR. (C) Expression of G2/M checkpoint regulators including cyclin B1, phosphorylated cdc2 (Tyr15), phosphorylated histone H3 (Ser10), and phosphorylated Chk2 (Thr68) were measured using immunoblotting in HCT116 p53^+/+ and p53^-/- cells 48 h after irradiation. Densitometric quantification was normalized to <i>β</i>-actin. Values are mean ± SEM. of three experiments, *<i>p</i> < 0.001. IR, ionizing radiation</p

Summary of physiological parameters in patients.

Summary of physiological parameters in patients.</p

Metformin decreased clonogenic survival in p53-deficient compared with p53 wild-type cells.

<p>HCT116 p53^+/+ and p53^-/- cells were treated with 1–10 mM metformin for 48 h, cultured and used in (A) clonogenic and (B) WST-1 (cell viability) assays; *<i>p</i> < 0.001 for HCT116 p53^+/+ cells, ^†<i>p</i> < 0.001 for HCT116 p53^-/- cells. ^§<i>p</i> < 0.001, between HCT116 p53^+/+ and p53^-/- cells, compared to control. WST, water-soluble tetrazolium.</p