Supplementary Figures 1 - 9 from Hypoxic Stress Facilitates Acute Activation and Chronic Downregulation of Fanconi Anemia Proteins

PDF file - 1436KB, S1. FANCD2 is ubiquitinated and transcriptionally down-regulated upon treatment with DFX in MCF7 cells. S2. FANCD2 is ubiquitinated and transcriptionally down-regulated upon treatment with DFX in A549 cells. S3. FANCD2 and FANCI ubiquitination does not occur at less severe levels of hypoxia. S4. siRNA depletion of ATR, but not ATM, inhibits FANCD2 and FANCI ubiquitination induced by hypoxia and DFX. S5. FANCD2 and FANCI ubiquitination upon DFX treatment is independent of HIF-1. S6. FANCD2 protein down-regulation by DFX is not due to altered protein stability and transcriptional down-regulation may be related to p130 dephosphorylation and HIF-2. S7. FANCD2 protein level decreases in hypoxia and is restored by HPV16-E7 overexpression. S8. FANCI transcriptional down-regulation upon prolonged exposure to DFX or hypoxia is mitigated by HPV16-E7 overexpression. S9. Increased chromosomal aberrations induced by hypoxia in the absence of FANCD2 are not detected by cytogenetic analysis.</p

Supplementary Figure 3 from miR-155 Overexpression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-Prone DSB Repair

Confirmation of polymerase delta mRNA down-regulation.</p

Supplementary Figure Legends from miR-155 Overexpression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-Prone DSB Repair

Legends for all 4 supplemental figures</p

Supplementary Figure 4 from miR-155 Overexpression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-Prone DSB Repair

NHEJ activity is unchanged upon FOXO3a knockdown in AV16.</p

Supplemental Figure 1 from miR-155 Overexpression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-Prone DSB Repair

Mutation pattern remains unchanged upon miR-155 over-expression in vivo.</p

Supplementary Figure 2 from miR-155 Overexpression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-Prone DSB Repair

Validation of DSB repair assays.</p

Mechanism of Action Studies of Lomaiviticin A and the Monomeric Lomaiviticin Aglycon. Selective and Potent Activity Toward DNA Double-Strand Break Repair-Deficient Cell Lines

(−)-Lomaiviticin A (<b>1</b>) and the monomeric lomaiviticin aglycon [aka: (−)-MK7-206, (<b>3</b>)] are cytotoxic agents that induce double-strand breaks (DSBs) in DNA. Here we elucidate the cellular responses to these agents and identify synthetic lethal interactions with specific DNA repair factors. Toward this end, we first characterized the kinetics of DNA damage by <b>1</b> and <b>3</b> in human chronic myelogenous leukemia (K562) cells. DSBs are rapidly induced by <b>3</b>, reaching a maximum at 15 min post addition and are resolved within 4 h. By comparison, DSB production by <b>1</b> requires 2–4 h to achieve maximal values and >8 h to achieve resolution. As evidenced by an alkaline comet unwinding assay, <b>3</b> induces extensive DNA damage, suggesting that the observed DSBs arise from closely spaced single-strand breaks (SSBs). Both <b>1</b> and <b>3</b> induce ataxia telangiectasia mutated- (ATM-) and DNA-dependent protein kinase- (DNA-PK-) dependent production of phospho-SER139-histone H2AX (γH2AX) and generation of p53 binding protein 1 (53BP1) foci in K562 cells within 1 h of exposure, which is indicative of activation of nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. Both compounds also lead to ataxia telangiectasia and Rad3-related- (ATR-) dependent production of γH2AX at later time points (6 h post addition), which is indicative of replication stress. <b>3</b> is also shown to induce apoptosis. In accord with these data, <b>1</b> and <b>3</b> were found to be synthetic lethal with certain mutations in DNA DSB repair. <b>1</b> potently inhibits the growth of breast cancer type 2, early onset- (BRCA2-) deficient V79 Chinese hamster lung fibroblast cell line derivative (VC8), and phosphatase and tensin homologue deleted on chromosome ten- (PTEN-) deficient human glioblastoma (U251) cell lines, with LC₅₀ values of 1.5 ± 0.5 and 2.0 ± 0.6 pM, respectively, and selectivities of >11.6 versus the isogenic cell lines transfected with and expressing functional BRCA2 and PTEN genes. <b>3</b> inhibits the growth of the same cell lines with LC₅₀ values of 6.0 ± 0.5 and 11 ± 4 nM and selectivities of 84 and 5.1, for the BRCA2 and PTEN mutants, respectively. These data argue for the evaluation of these agents as treatments for tumors that are deficient in BRCA2 and PTEN, among other DSB repair factors

Supplemental Figures from DNA Polymerase Beta Germline Variant Confers Cellular Response to Cisplatin Therapy

S1. Dose-response curve of MCF7 cells. S2. Western blotting analysis of Pol beta in cell lines. S3. Pol beta and XPA interact in vitro. S4. The effect of E295K Pol beta expression on survival following cisplatin treatment. Table S1. Difference in viability of crosslinking agents from high-throughput screen.</p

Tumor-Targeted, Cytoplasmic Delivery of Large, Polar Molecules Using a pH-Low Insertion Peptide

Tumor-targeted drug delivery systems offer not only the advantage of an enhanced therapeutic index, but also the possibility of overcoming the limitations that have largely restricted drug design to small, hydrophobic, “drug-like” molecules. Here, we explore the ability of a tumor-targeted delivery system centered on the use of a pH-low insertion peptide (pHLIP) to directly deliver moderately polar, multi-kDa molecules into tumor cells. A pHLIP is a short, pH-responsive peptide capable of inserting across a cell membrane to form a transmembrane helix at acidic pH. pHLIPs target the acidic tumor microenvironment with high specificity, and a drug attached to the inserting end of a pHLIP can be translocated across the cell membrane during the insertion process. We investigate the ability of wildtype pHLIP to deliver peptide nucleic acid (PNA) cargoes of varying sizes across lipid membranes. We find that pHLIP effectively delivers PNAs up to ∼7 kDa into cells in a pH-dependent manner. In addition, pHLIP retains its tumor-targeting capabilities when linked to cargoes of this size, although the amount delivered is reduced for PNA cargoes greater than ∼6 kDa. As drug-like molecules are traditionally restricted to sizes of ∼500 Da, this constitutes an order-of-magnitude expansion in the size range of deliverable drug candidates