Journal: Frontiers in Oncology

Article Title: Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

doi: 10.3389/fonc.2021.612354

Figure Lengend Snippet: HSP90i by NW457 leads to downregulation of DNA damage response factors, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in human glioblastoma cells. (A) Transcriptomic profiling of regulators of the DNA damage response (DDR) in LN229 and T98G cells. mRNA expression levels were determined by qRT-PCR, normalized to a matrix of 3 reference genes (18S rRNA, δ-amino-laevulinate-synthase, and β2-microglobulin), and calibrated to the results of untransformed human astrocytes. For both cell lines, three replicates were analyzed and are displayed as x-fold log2-values. (B) Time course analysis of DDR regulator protein expression in LN229 and T98G cells upon HSP90i by 10 nM NW457. Arrowheads indicate the bands that were used for quantification. Protein levels were normalized to a matrix comprising vinculin and α-tubulin and are depicted as x-fold log2-values compared to the 0 h controls. (C) Immunofluorescence microscopy of γH2AX and 53BP1 DNA damage repair foci in LN229 and T98G cells upon irradiation at 2 Gy ± HSP90i by NW457. Cells were treated with NW457 (10 nM) or DMSO for 24 h, irradiated, and fixed at the indicated times. Cells were stained for γH2AX, 53BP1, and DNA and subjected to deconvolution immunofluorescence microscopy. Scale bar depicts 10 µm. (D) Quantification of DNA damage repair kinetics from (C) . γH2AX and 53BP1 double-positive foci in at least 20 randomly picked nuclei were counted by hand. Individual data points with superimposed means ± 95% confidence intervals are displayed, and overall curve comparison was performed by two-way ANOVA. (E) Clonogenic survival of LN229 and T98G cells upon irradiation at 0–8 Gy ± HSP90i by NW457. Cells were pre-treated with 10 nM NW457 or DMSO for 24 h followed by irradiation at the indicated doses, and colony formation was allowed for 13 d ± continuous NW457 treatment. Individual data points of 4 independent experiments are shown, linear-quadratic regression lines are superimposed, and overall curve comparison was performed by two-way ANOVA.

Article Snippet: Cells were stained with monoclonal mouse anti-γH2AX (Merck Millipore) and polyclonal rabbit anti-53BP1 (Bio-Techne, Wiesbaden, Germany) antibodies diluted in 3% isotonic bovine serum albumin and 0.1% Triton X-100 for 2 h at room temperature.

Techniques: Irradiation, Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining, Comparison