Fluorescent Biosensor for Detection of the R248Q Aggregation‐Prone Mutant of p53

Abstract

The p53 tumour suppressor and guardian of the genome undergoes missense mutations that lead to functional inactivation in 50 % of human cancers. These mutations occur mostly in the DNA‐binding domain of the protein, and several of these result in conformational changes that lead to amyloid‐like protein aggregation. Herein, we describe a fluorescent biosensor that reports on the R248Q mutant of p53 in vitro and in living cells, engineered through conjugation of an environmentally sensitive probe onto a peptide derived from the primary aggregation segment of p53. This biosensor was characterised both in vitro and by means of fluorescence microscopy following facilitated delivery into cultured cells. It is shown that this biosensor preferentially reports on the p53 R248Q mutant in the PC9 lung cancer cell line compared with other lung cancer cell lines harbouring either wild‐type or no p53.

Ethaverine and Papaverine Target Cyclin-Dependent Kinase 5 and Inhibit Lung Cancer Cell Proliferation and Migration

“…To this aim, the H1299 cell line which is p53 −/− and the PC9 cell line which harbors the p53 R248Q mutation were treated with roscovitine, ethaverine, and papaverine. 26 While the efficacy of ethaverine and papaverine in H1299 and PC9 was quite similar to that observed in A549 cells with an average 30% inhibition after 24 h treatment, roscovitine was much less efficient with only 14 ± 12.3 and 23 ± 12% inhibition in H1299 and PC9, respectively, compared to 32 ± 6.1% in A549 cells (Figure 3, Table 2,Supplementary Table S4). In line with this reduced efficacy of Roscovitine in H1299 cells, we observed that the combined treatment of roscovitine with either ethaverine or papaverine only inhibited H1299 cell proliferation by 36 ± 11.3 and 35 ± 13.8%, respectively, after 24h compared to 48 ± 5.3 and 45 ± 6% inhibition in A549 cells.…”

“…Since A549 cells are p53 +/+, we asked whether ethaverine and papaverine exhibited the same inhibitory efficacy in other NSCLC cell lines, in which p53 was either deleted or mutated. To this aim, the H1299 cell line which is p53 –/– and the PC9 cell line which harbors the p53 R248Q mutation were treated with roscovitine, ethaverine, and papaverine . While the efficacy of ethaverine and papaverine in H1299 and PC9 was quite similar to that observed in A549 cells with an average 30% inhibition after 24 h treatment, roscovitine was much less efficient with only 14 ± 12.3 and 23 ± 12% inhibition in H1299 and PC9, respectively, compared to 32 ± 6.1% in A549 cells (Figure , Table ,Supplementary Table S4).…”

Neoantimycin F, a Streptomyces-Derived Natural Product Induces Mitochondria-Related Apoptotic Death in Human Non-Small Cell Lung Cancer Cells

“…We hypothesize that the differences in the effect of NAT-F on cell cycle arrest are primarily determined by the genetics of the cell lines. Indeed, the NSCLC cells used in our study have different TP53 genotype ( Supplementary Table 1 ), as reported in the literature (Mukhopadhyay and Roth, 1993; Deng et al, 2007; Tyagi et al, 2013; Pellerano et al, 2019). For example, the human lung cancer cell line PC9 harbors mutant TP53, whereas the cell line H1299 is a TP53-null lung cancer model (Yonesaka et al, 2006; Allen et al, 2015; Song et al, 2019).…”