FIGURE 4 from DNA2 Nuclease Inhibition Confers Synthetic Lethality in Cancers with Mutant p53 and Synergizes with PARP Inhibitors
Year: 2023
Type: other
Abstract: Targeting DNA2 with d16 inhibits ATR function and enhances cisplatin sensitivity in cancer cells harboring mutp53. A, H1299 stable cells expressing an empty vector, WT p53, mutp53-R273H or mutp53-R175H were treated for 72 hours with increasing concentrations of d16. Cell proliferation was measured by MTT assay. Top panels are immunoblots showing the expression of WT p53, mutp53, and GAPDH, respectively. B, MDAH-2774, BT549, and C33A cells stably expressing either a scrambled shRNA (shScr) or an shRNA against mutp53 (shp53) were treated with d16 (5 µmol/L for BT549 and MDAH-2774, 2.5 µmol/L for C33A) for 72 hours. Cell viability was then measured by MTT assay. The data shown represent mean ± SD (n = 3 biological replicates). P values are *, P < 0.05; **, P < 0.01; and ***, P < 0.001 indicating statistically significant difference (two-tailed t test). Right: Immunoblots of mutp53 and GAPDH, respectively. C, H1299 cells transiently expressing an empty vector or mutp53-R273H were treated with d16 (10 µmol/L) for 24 hours, followed by HU (2 mmol/L) treatment for 0, 2, or 4 hours. Cell lysates were subjected to Western blotting. The right panel shows the quantification of pChk1 protein levels normalized by Chk1 protein; *, P < 0.05. D, A2780cis cells were treated with vehicle control or d16 (5 µmol/L) with increasing doses of cisplatin for 72 hours. Cell proliferation was then measured by MTT assay. CI analysis was performed using CompuSyn software. Fa (Fraction affected)-CI plot and CI values (in brackets) were calculated and shown in the right. CI < 1 indicates synergism. E, Clonogenic survival assay was performed by treating A2780cis cells with d16 (2.5 µmol/L), cisplatin (2 µmol/L), or both for 24 hours. After PBS washing, cells were cultured for 10 days. The data shown represent mean ± SD (n = 3 biological replicates). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 indicating statistically significant difference (two-tailed t test).
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4387687527