A5000421260- Glioma Diagnosis and Treatment
- Cancer-related molecular mechanisms research
- Medicinal Plant Pharmacodynamics Research
- Extracellular vesicles in disease
- CAR-T cell therapy research
- Protein Degradation and Inhibitors
- Ubiquitin and proteasome pathways
- Signaling Pathways in Disease
- Cancer-related gene regulation
- Cancer Cells and Metastasis
- Cancer Mechanisms and Therapy
Hospital for Sick Children
2019
SickKids Foundation
2019
Glioblastoma is the most common primary brain tumor in adults. While introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid recurrence remains universal. The transcriptional regulatory protein, inhibitor DNA-binding-1 (ID1), a key regulator cell phenotype cancer. We show that CRISPR-mediated knockout ID1 glioblastoma cells, breast adenocarcinoma melanoma cells dramatically reduced progression all three cancer systems through downregulation...
<div>Abstract<p>Glioblastoma is the most common primary brain tumor in adults. While introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid recurrence remains universal. The transcriptional regulatory protein, inhibitor DNA-binding-1 (ID1), a key regulator cell phenotype cancer. We show that CRISPR-mediated knockout ID1 glioblastoma cells, breast adenocarcinoma melanoma cells dramatically reduced progression all three cancer...
<p>(A) Flow chart of patient sample analysis (B) Representative images IHC staining for ID1 expression in a TMA cohort. Images weak and strong intensity tumor tissues are shown. (C) qPCR expresssion U251 cells. Cells were treated with 100uM TMZ indicated timepoints. *P>0.1</p>
<p>(A) TMZ IC50 curves for U251 control and U251.TR cells. (B) cells colony forming assay at different concentrations of TMZ. (C) Western blot analysis EGFR p-EGFR-1068 from lysates U251and cells.</p>
<p>Measurement of ID1 half-life by cycloheximide chase assays. U251 cells were pretreated with or without TMZ for 3 days, followed treatment the indicated times. The cell lysates then analyzed Western blots.</p>
<p>(A) Spearman correlation of ID1 vs EGFR gene expression in all glioblastoma patients. (B) the Classical subtype. (C) and EGFR_pY1173 RPPA (D) GBM patients with available data.</p>
<p>(A) Western blot analysis for the identification of two clones lacking ID1 protein expression, consistent with successful gene knockout by CRISPR. (B) DNA sequence chosen clones. Red boxes mark sgRNA target and denote predicted cut-site. (C) Cell viability was measured using Alamar Blue in a time-dependent manner. (D) Cells were cultured 48h followed staining propidium iodide flow cytometric analysis. Bar chart shows quantification cell cycle distribution.</p>
<p>(A) Representative H&E images of control and ID1 KO mouse brains. (B) mice demonstrated greater accumulation TMEM within the brain tissues compared to animals. TMEM-positive cells were diffusely distributed throughout brain, while in group. The boxes indicate areas shown at higher magnification. (C) Quantification positive relative area. **P<0.005 Western blot analysis shows successful gene knockout clone for protein expression (D) MDA-MB231 (E) A375 cells.</p>
<p>(A) From left: Lane 1-5 U251 CTR. -. Negative control. +. Positive D+. Diluted positive 7-10 ID1-/-U251.1. 11-14 ID1-/-U251.2. (B) PCR method.</p>
<p>(A) Spearman correlation of ID1 vs EGFR gene expression in all glioblastoma patients. (B) the Classical subtype. (C) and EGFR_pY1173 RPPA (D) GBM patients with available data.</p>
<p>(A) Western blot analysis for the identification of two clones lacking ID1 protein expression, consistent with successful gene knockout by CRISPR. (B) DNA sequence chosen clones. Red boxes mark sgRNA target and denote predicted cut-site. (C) Cell viability was measured using Alamar Blue in a time-dependent manner. (D) Cells were cultured 48h followed staining propidium iodide flow cytometric analysis. Bar chart shows quantification cell cycle distribution.</p>
<p>Measurement of ID1 half-life by cycloheximide chase assays. U251 cells were pretreated with or without TMZ for 3 days, followed treatment the indicated times. The cell lysates then analyzed Western blots.</p>
<p>(A) Flow chart of RNA-seq analysis. (B) RT-qPCR validation confirmed a reduction in EGF transcript, with similar trends to data. ****P<0.0001</p>
<p>(A) TMZ IC50 curves for U251 control and U251.TR cells. (B) cells colony forming assay at different concentrations of TMZ. (C) Western blot analysis EGFR p-EGFR-1068 from lysates U251and cells.</p>
<p>(A) Flow chart of patient sample analysis (B) Representative images IHC staining for ID1 expression in a TMA cohort. Images weak and strong intensity tumor tissues are shown. (C) qPCR expresssion U251 cells. Cells were treated with 100uM TMZ indicated timepoints. *P>0.1</p>
<p>(A) Representative H&E images of control and ID1 KO mouse brains. (B) mice demonstrated greater accumulation TMEM within the brain tissues compared to animals. TMEM-positive cells were diffusely distributed throughout brain, while in group. The boxes indicate areas shown at higher magnification. (C) Quantification positive relative area. **P<0.005 Western blot analysis shows successful gene knockout clone for protein expression (D) MDA-MB231 (E) A375 cells.</p>
<p>(A) Flow chart of RNA-seq analysis. (B) RT-qPCR validation confirmed a reduction in EGF transcript, with similar trends to data. ****P<0.0001</p>
<p>(A) From left: Lane 1-5 U251 CTR. -. Negative control. +. Positive D+. Diluted positive 7-10 ID1-/-U251.1. 11-14 ID1-/-U251.2. (B) PCR method.</p>
<div>Abstract<p>Glioblastoma is the most common primary brain tumor in adults. While introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid recurrence remains universal. The transcriptional regulatory protein, inhibitor DNA-binding-1 (ID1), a key regulator cell phenotype cancer. We show that CRISPR-mediated knockout ID1 glioblastoma cells, breast adenocarcinoma melanoma cells dramatically reduced progression all three cancer...