Journal: EBioMedicine

Article Title: The Mechanism by Which MYCN Amplification Confers an Enhanced Sensitivity to a PCNA-Derived Cell Permeable Peptide in Neuroblastoma Cells

doi: 10.1016/j.ebiom.2015.11.016

Figure Lengend Snippet: MYCN amplification is associated with high Chk1 signaling and RS. a) Total RNA extracted from MYCN -amplified NB cell lines (blue: SK-N-DZ, SK-N-BE(2)c, BE(2)c, CHP-212, and IMR-32), non- MYCN -amplified NB cell lines (green: SK-N-AS, SK-N-SH, and SH-SY5Y), and non-malignant human cell lines (red: HCN1-A and hBM-MSCs) were analyzed by microarray analysis. The Log2 transformed Chk1 (X-axis) and MYCN (Y-axis) mRNA levels in these cell lines were graphed. The correlation between relative Chk1 and MYCN mRNA levels in these cell lines were analyzed by linear regression (r = 0.886). b) Total cell extracts from the indicated cell lines were analyzed by Western blotting to determine the levels of p-Chk1, Chk1, and Rad17. c) A microarray dataset of NB patient specimens (ArrayExpress: E-MTAB-179 ) was analyzed. The relative expression of Chk1 mRNA in patient specimens with or without MYCN amplification was graphed with the bottom and top ends of the whiskers representing the 5th and 95th percentiles of the expression levels respectively. “All” represents all non- MYCN -amplified NB samples. The “low-risk” subset includes patients with stage 1, 2, or 4S non- MYCN -amplified NB and under the age of 18 months. The “high-risk” subset includes patients with stage 4 non- MYCN -amplified tumors and above the age of 18 months. The p-value was determined by an unpaired t-test. Serial sections made from d) patient NB specimens and e) PTX NB tumors were stained with the indicated antibodies, as described in the Materials and Methods. The left two columns in each panel box are serial sections made from two MYCN -amplified tumors and the right two columns are sections made from two non- MYCN -amplified tumors.

Article Snippet: Antibodies specific to Chk1 and phosphorylated Rad17 were purchased from LifeSpan Biosciences.

Techniques: Amplification, Microarray, Transformation Assay, Western Blot, Expressing, Staining

Journal: EBioMedicine

Article Title: The Mechanism by Which MYCN Amplification Confers an Enhanced Sensitivity to a PCNA-Derived Cell Permeable Peptide in Neuroblastoma Cells

doi: 10.1016/j.ebiom.2015.11.016

Figure Lengend Snippet: R9-caPep works synergistically with Chk1 inhibitors. Sk-N-BE(2)c (Panels a and c), SK-N-DZ (Panel b), and SH-SY5Y (Panel d) NB cells were treated with 2-fold serial dilutions of R9-caPep and UCN-01 (triangles in panels a, b, and d) or R9-caPep and MK-8776 (triangles in panel c) at a fixed concentration ratio. Cells treated with the same concentrations of R9-caPep (squares), UCN-01 (circles), or MK-8776 (circles) alone were used as control. Cells were cultured in the presence of the reagents for 3 days and their growth was determined by a CellTiter-Glo assay (Promega). Top: the relative abundance of cells in triplicates under each treatment conditions was averaged and graphed ± S.D. Bottom: conservative isobolograms were graphed and CI values were calculated for each study by CalcuSyn. Blue: ED 90 ; green: ED 75 ; and red: ED 50 .

Article Snippet: Antibodies specific to Chk1 and phosphorylated Rad17 were purchased from LifeSpan Biosciences.

Techniques: Concentration Assay, Cell Culture, Glo Assay

Journal: EBioMedicine

Article Title: The Mechanism by Which MYCN Amplification Confers an Enhanced Sensitivity to a PCNA-Derived Cell Permeable Peptide in Neuroblastoma Cells

doi: 10.1016/j.ebiom.2015.11.016

Figure Lengend Snippet: Dysregulation of replication initiation and induction of DNA damage and apoptosis by R9-caPep and Chk1 inhibitors. b) SK-N-BE(2) cells or c) SH-SY5Y cells were incubated for 12 h with 7.8 nM UCN-01, 30 μM R9-caPep, or 7.8 nM UCN-01 and 30 μM R9-caPep for 12 h. Untreated corresponding cells were used as controls. Cells were sequentially labeled with CldU and IdU for 15 min each. Following incubation with the nucleotide analogs, the cells were stained by fluorophore-coupled monoclonal antibodies to visualized CIdU and IdU incorporation. The relative distance between two neighboring replication origins was measured as illustrated in panel a. The distances between two neighboring origins from at least 15 DNA segments from cells under each treatment condition were graphed. d) SK-N-BE(2)c (left) or SH-SY5Y (right) cells were treated by 7.8 nM UCN-01, 30 μM R9-caPep, or both for 12 h. The intracellular levels of the indicated proteins were determined by Western blot.

Article Snippet: Antibodies specific to Chk1 and phosphorylated Rad17 were purchased from LifeSpan Biosciences.

Techniques: Incubation, Labeling, Staining, Western Blot

Journal: EBioMedicine

Article Title: The Mechanism by Which MYCN Amplification Confers an Enhanced Sensitivity to a PCNA-Derived Cell Permeable Peptide in Neuroblastoma Cells

doi: 10.1016/j.ebiom.2015.11.016

Figure Lengend Snippet: A working model for exploiting RS in MYCN -amplified or MYC-expressing NB cells. Cell cycle checkpoints play a critical role in maintaining genome integrity. Members of MYC family proto-oncogenes promote cell growth by dysregulating the G1/S checkpoint and inhibiting G1 arrest after DNA damage. Therefore, cells overexpressing MYC family proteins are more likely to enter S-phase with unrepaired DNA damages and to experience RS. R9-caPep induces additional RS by interfering with replication fork extension and by blocking HR-mediated DSB repair , making cells dependent on ATR/Chk1-mediated DDR to prevent the collapse of stalled replication forks and to resolve DNA lesions. By inducing RS and inhibiting DDR, R9-caPep and Chk1 inhibitors work synergistically in causing DSB and apoptosis. Such a synergism may provide an effective way of treating MYCN -amplified NB and other high-risk NB that expresses an augmented level of MYC.

Article Snippet: Antibodies specific to Chk1 and phosphorylated Rad17 were purchased from LifeSpan Biosciences.

Techniques: Amplification, Expressing, Blocking Assay