Journal: bioRxiv

Article Title: Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations

doi: 10.1101/2023.03.17.533211

Figure Lengend Snippet: (A) Illustration of the hypothesis that escapees mount a replication stress response to manage aberrant S phases and tolerate DNA lesions. (B) Western blot analysis of γ-H2AX in the indicated cell lines after 96 h treatments. β-tubulin is used as a loading control. Bar plot displays quantification of γ-H2AX band intensity normalized to respective loading control band, and values are reported as fold change of control. (C) Representative immunofluorescence images of five human MAPK-mutant cancer cell lines after 72 h indicated treatments, stained for DNA content, EdU incorporation, and γ-H2AX. (D) Quantification of γ-H2AX nuclear fluorescence intensity after 72 h indicated treatments in EdU − and EdU + cells, displayed as split violins; p -values determined by Mann-Whitney U tests of MAPK-inhibited EdU + cells compared with DMSO-treated EdU + cells. (E) Top: Representative immunofluorescence images of phospho-CHK1 (S317) in A375 cells escaping BRAF inhibition (cycling status displayed by EdU intensity). Bottom: Histograms of phospho-CHK1 (S317) nuclear intensity in S phase cells (EdU + ) in the indicated cell lines after 96 h MAPK inhibition. (F) Distribution of EdU intensity in escapees (after 100 nM Osimertinib or 1 μM Encorafenib) co-treated with ATR inhibition (100 nM AZ20 or 100 nM AZD6738). 96 h treatments. Line drawn at the mean value; n = 190 cells plotted per condition; p -values determined by Mann-Whitney U test. (G) Quantification of cycling cells (determined by phospho-Rb S807/811 immunofluorescence) after 96 h of indicated treatment combinations with ATR inhibitors. AZ20 doses: 100 nM, 1 μM. AZD6738 doses: 100 nM, 1 μM. Mean ± std of 3 replicates; p -value determined by unpaired t-test.

Article Snippet: Primary antibodies used in this study include: phospho-Rb (S807/811) (D20B12) rabbit mAb (1:500, Cell Signaling Technology #8516); phospho-Rb (S780) mouse mAb (1:1000, BD Biosciences #558385); phospho-Histone H3 (S10) (D2C8) rabbit mAb (1:400, Cell Signaling Technology #3377); phospho-Histone H3 (S10) (6G3) mouse mAb (1:400, Cell Signaling Technology #9706); phospho-H2AX (S139) (20E3) rabbit mAb (1:400 for standard immunofluorescence, 1:100 for FFPE tissue staining, Cell Signaling Technology #9718); p21 (12D1) rabbit mAb (1:400, Cell Signaling Technology #2947); phospho-ATR (T1989) (D5K8W) rabbit mAb (1:250, Cell Signaling Technology #30632); phospho-CHK1 (S317) rabbit mAb (1:250, Abcam #ab278717); FANCD2 rabbit pAb (1:500, Novus Biologicals #100-182); Polκ mouse mAb (1:500, Abcam #ab57070; mouse mAb is discontinued and #ab115625 goat pAb is recommended by manufacturer as a suitable replacement); β-tubulin (D3U1W) mouse mAb (1:1000, Cell Signaling Technology #86298); Ki67 sheep pAb (1:200, R&D Systems #AF7617).

Techniques: Western Blot, Immunofluorescence, Mutagenesis, Staining, Fluorescence, MANN-WHITNEY, Inhibition

Journal: bioRxiv

Article Title: Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations

doi: 10.1101/2023.03.17.533211

Figure Lengend Snippet: (A) Top: Long-term culture of MAPK-mutant cell lines with indicated MAPK inhibitors and clinical ATR inhibitor (5 μM AZD6738). Cell count was quantified longitudinally by imaging H2B-mIFP fluorescence. Unfettered proliferation in control groups causes contact inhibition within first 2 weeks of imaging. Mean ± std of 4 replicate wells; p -value determined by unpaired t-test of final time point. Bottom: Representative wells from the final day of imaging. Inverted H2B-mIFP fluorescence shown. (B) Formalin-fixed paraffin-embedded (FFPE) tissue sections longitudinally biopsied before and after treatment from patients with BRAF V600E melanoma (see for patient and tumour details). Immunofluorescence of Ki67 (proliferation marker) and γ-H2AX (DNA damage marker) displayed. (C) Quantification of all cells (shown in blue coloring) and Ki67-positive cells (shown in red coloring) in FFPE sections with elevated γ-H2AX intensity. Line drawn at the mean; n > 235 cells scored before Ki67 gating; p-value determined by Mann-Whitney U test. (D) Kaplan-Meier survival analyses based on data generated from the Harmonized Cancer Datasets within the Genomic Data Commons (GDC) research database (as of April 28, 2022; https://portal.gdc.cancer.gov ). Patients were included with tumours harboring ≥ 1 EGFR, KRAS, or BRAF mutation and ≥ 1 TP53 mutation. Cohort comparisons were made between tumour subgroups containing wild-type or mutant forms of the indicated genes. Left-most plot examines a cohort that has ≥ 1 mutation among a list of FA-associated genes (see for FA gene list). Displayed p -values were determined by log-rank test.

Article Snippet: Primary antibodies used in this study include: phospho-Rb (S807/811) (D20B12) rabbit mAb (1:500, Cell Signaling Technology #8516); phospho-Rb (S780) mouse mAb (1:1000, BD Biosciences #558385); phospho-Histone H3 (S10) (D2C8) rabbit mAb (1:400, Cell Signaling Technology #3377); phospho-Histone H3 (S10) (6G3) mouse mAb (1:400, Cell Signaling Technology #9706); phospho-H2AX (S139) (20E3) rabbit mAb (1:400 for standard immunofluorescence, 1:100 for FFPE tissue staining, Cell Signaling Technology #9718); p21 (12D1) rabbit mAb (1:400, Cell Signaling Technology #2947); phospho-ATR (T1989) (D5K8W) rabbit mAb (1:250, Cell Signaling Technology #30632); phospho-CHK1 (S317) rabbit mAb (1:250, Abcam #ab278717); FANCD2 rabbit pAb (1:500, Novus Biologicals #100-182); Polκ mouse mAb (1:500, Abcam #ab57070; mouse mAb is discontinued and #ab115625 goat pAb is recommended by manufacturer as a suitable replacement); β-tubulin (D3U1W) mouse mAb (1:1000, Cell Signaling Technology #86298); Ki67 sheep pAb (1:200, R&D Systems #AF7617).

Techniques: Mutagenesis, Cell Counting, Imaging, Fluorescence, Inhibition, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Marker, MANN-WHITNEY, Generated

Journal: International Journal of Molecular Sciences

Article Title: Asparagine and Glutamine Deprivation Alters Ionizing Radiation Response, Migration and Adhesion of a p53 null Colorectal Cancer Cell Line

doi: 10.3390/ijms24032983

Figure Lengend Snippet: ɣH2AX analysis for Caco-2 cells exposed to different doses of X rays (0, 1, 3 and 5 Gy), without (w/o) or with 1 U/mL EcAII (w/EcAII). Panels ( A , B ), illustrative fluorescence microscopy images for the different irradiation and treatment conditions obtained with Hoechst (blue for nuclear DNA, left) and ɣH2AX (red for DNA damage foci, right) of the sample fixed 30 min and 1 h after irradiation, respectively. Images were acquired using a 100× magnification. Cells pre-treated with EcAII for 72 h before radiation exposure. Panels ( C , D ), mean fluorescence intensity (MFI) per cell of the ɣH2AX signal (a.u.). ( E , F ) The count of H2AX foci/nucleus was obtained using open software ImageJ v 1.53 for the sample harvested 30 min and 1 h after irradiation, respectively. ( G ) Cells treated with EcAII after radiation exposure. Illustrative Western blot images of ɣH2AX (S139) and pan-H2AX. The densitometric analysis was performed by evaluating the p-H2AX/total H2AX ratio with changes expressed relative to the unirradiated and untreated control at each time point (% vs. CTRL NT). Samples in the absence of EcAII and with EcAII were harvested at 6 h ( H ), 24 h ( I ) and 48 h after irradiation ( J ). Data reported are mean ± SD, obtained from at least three independent experiments. Statistical significance (Student’s t -test) was calculated comparing the w/o and w/EcAII conditions for each dose and time point (Student’s t -test) and is as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. In all charts black bars represent samples w/o EcAII and gray bars represent samples w/EcAII.

Article Snippet: At 30 min and 1 h after exposure, samples were fixed in 4% formaldehyde and permeabilized in 70% ethanol diluted in ddH 2 O. Coverslips were incubated with blocking solution (1% BSA in PBT) for 30 min at RT, then with the primary antibody anti-phospho H2AX (S139) (dilution 1:400, Cell Signaling Technologies (RRID:AB_2118010)) for 1 h at RT and the secondary antibody goat anti-rabbit IgG 555 (dilution 1:200, Molecular Probes (RRID:AB_2535851)) for 30 min at RT.

Techniques: Fluorescence, Microscopy, Irradiation, Software, Western Blot

Journal: BMC Molecular Biology

Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

doi: 10.1186/1471-2199-13-7

Figure Lengend Snippet: Down-regulation of CK2 results in persistent γ-H2AX signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.

Article Snippet: M059K cells grown on cover slips were incubated with rabbit polyclonal anti-γ-H2AX (p-S139) antibody (#2577, Cell Signaling Technology, Denmark) followed by incubation with biotinylated swine anti-rabbit IgG (Dako, Denmark) and streptavidin-conjugated fluorescein-isothiocyanate (Dako) as previously described [ ].

Techniques: Transfection, Incubation, Labeling, Staining, Software, Standard Deviation

Journal: BMC Molecular Biology

Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

doi: 10.1186/1471-2199-13-7

Figure Lengend Snippet: CK2 co-localizes with γ-H2AX to sites of DNA damage . A . Association between CK2α' and γ-H2AX was revealed by in situ PLA. Cells expressing or lacking CK2α' were left untreated or incubated with 0.5 μg/ml NCS for 1 hour before fixation and labeling with primary antibodies directed against the indicated proteins. Control, refers to cells transfected with si-CK2α' and stained with secondary antibodies. Cell nuclei were revealed by Hoechst staining. B . Quantification of the number of signals/cell as distinct fluorescent red spots was performed by computer-assisted image analysis as described in Experimental Procedures. Mean values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant difference.

Article Snippet: M059K cells grown on cover slips were incubated with rabbit polyclonal anti-γ-H2AX (p-S139) antibody (#2577, Cell Signaling Technology, Denmark) followed by incubation with biotinylated swine anti-rabbit IgG (Dako, Denmark) and streptavidin-conjugated fluorescein-isothiocyanate (Dako) as previously described [ ].

Techniques: In Situ, Expressing, Incubation, Labeling, Transfection, Staining

Journal: BMC Molecular Biology

Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

doi: 10.1186/1471-2199-13-7

Figure Lengend Snippet: Cells overexpressing CK2 exhibit a prompt response to induction of DNA double-strand break . A . M059K cells transiently overexpressing CK2α'-DsRed or DsRed-fluorescent protein were incubated with 0.5 μg/ml NCS for the indicated times. Subsequently, cells were fixed and labeled with anti-γ-H2AX antibody. Cell nuclei were revealed by DAPI staining. Arrows: cells overexpressing CK2α'-DsRed are more efficient in DNA damage repair as compared to cells that do not overexpress the kinase. One representative experiment is shown. B . Average γ-H2AX foci number per red fluorescent cell as described in A . Mean values +/- SD from three independent experiments are shown. More than 100 cells have been analyzed per experiment. *P < 0.001 denotes statistically significant difference. NS, not significant.

Article Snippet: M059K cells grown on cover slips were incubated with rabbit polyclonal anti-γ-H2AX (p-S139) antibody (#2577, Cell Signaling Technology, Denmark) followed by incubation with biotinylated swine anti-rabbit IgG (Dako, Denmark) and streptavidin-conjugated fluorescein-isothiocyanate (Dako) as previously described [ ].

Techniques: Incubation, Labeling, Staining

Journal: PLoS ONE

Article Title: STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G 2 Arrest in the Absence of DNA Damage

doi: 10.1371/journal.pone.0053908

Figure Lengend Snippet: (A) G 2 /M transition regulated proteins expression. HeLa cells were treated with STK295900 1 or 5 µM, Camptothecin 10 µM, etoposide 10 µM, or nocodazole 200 ng/ml. After 24 h incubation, cell lysates were prepared for immunoblot analyses with antibodies against phospho-Cdk1 (T161), phospho-Cdk1 (T14), phospho-Cdk1 (Y15), Cdk1, Wee1, and Cdc25C. GAPDH was used as a loading control. (B) DNA damage-checkpoint related proteins. The same lysates used in (A) were subjected to immunoblot analyses with antibodies against phospho-ATM (S1981), ATM, phospho-ATR (S428), ATR, phospho-Chk1 (S345), Chk1, phospho-Chk2 (T68), Chk2, p53, and p21. β-actin was used as a loading control. (C) Immunofluorescence staining for γ-H2A.X. HeLa cells were treated with 1, 5, or 10 µM of STK295900 or 10 µM of ICRF-193, etoposide, and camptothecin for 24 h. Treated cells were then fixed and stained with anti-γ-H2A.X (middle panel). DNA from ICRF-193-, etoposide-, and camptothecin-treated cells was stained with Hoechst 33342 (bottom panel). Images were analyzed on a fluorescence microscope.

Article Snippet: Phospho-Cdk1 (T14), phospho-Cdk1 (Y15), phospho-Cdk1 (T161), Cdk1, cyclin B1, phospho-ATM (S1981), ATM, phospho-ATR (S428), ATR, phospho-Chk1 (S345), Chk1, phospho-Chk2 (T68), Chk2, phospho-Histone H3 (S10), Histone H3, and γ-H2A.X (S139) antibodies were purchased from Cell Signaling Technology (Denvers, MA).

Techniques: Expressing, Incubation, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G 2 Arrest in the Absence of DNA Damage

doi: 10.1371/journal.pone.0053908

Figure Lengend Snippet: Supercoiled DNA relaxation assay for (A) topoisomerase 1 (Top 1) and (B) topoisomerase 2 (Top 2). Supercoiled pBR322 plasmid DNA was incubated at 37°C for 30 min with Top 1 enzyme (A) or Top 2α (B) in the presence of various concentrations of indicated compounds. DNA samples were separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized by UV light. (−), supercoiled DNA alone; oc, open circular; sc, supercoiled. (C) Antagonistic effect of STK295900 in camptothecin-induced DNA damage. HeLa cells were pretreated with 10 µM of STK295900 for 30 min and then incubated with the indicated concentrations of camptothecin for 1 h. Treated cells were lysed and subjected to immunoblot analyses with antibody against γ-H2A.X. β-actin was used as a loading control. (D) Antagonistic effect of STK295900 on etoposide-induced DNA damage. HeLa cells were pretreated with STK295900 at 10, 20, 30, or 50 µM or ICRF-193 at 10 µM for 30 min. Cells were incubated with 10 µM of etoposide for another 1 h. Treated cells were then lysed and subjected to immunoblot analyses with antibody against γ-H2A.X. β-actin was used as a loading control.

Article Snippet: Phospho-Cdk1 (T14), phospho-Cdk1 (Y15), phospho-Cdk1 (T161), Cdk1, cyclin B1, phospho-ATM (S1981), ATM, phospho-ATR (S428), ATR, phospho-Chk1 (S345), Chk1, phospho-Chk2 (T68), Chk2, phospho-Histone H3 (S10), Histone H3, and γ-H2A.X (S139) antibodies were purchased from Cell Signaling Technology (Denvers, MA).

Techniques: Plasmid Preparation, Incubation, Electrophoresis, Agarose Gel Electrophoresis, Staining, Western Blot