Flow Cytometric Analyses of p53-Mediated Cell Cycle Arrest and Apoptosis in Cancer Cells.

Phenotypic analysis of the effects of a gene of interest may be limited because stable expression of some genes leads to adverse consequences in cell survival, such as disturbance of cell cycle progression, senescence, autophagy, and programmed cell death. One of the best examples is tumor suppressor p53. p53 functions as a tumor suppressor by inducing cell cycle arrest and apoptosis in response to genotoxic and environmental insults. The choice and timing of either pathways induced by p53 depend on cellular context, cell types, and the degree of cellular/genomic damage (For review, see (Chen J, Cold Spring Harb Perspect Med 6:a026104, 2016)). The uncertainty makes the studies on the long-term effects of p53 in cells challenging. This chapter describes a method of flow cytometric analysis of ectopic expression of p53 to better quantify cell cycle distribution and apoptosis in cells treated with DNA damaging agents. The method can be easily adapted to other genes of interest to study their contributions to the fate of variety of cell types in response to endogenous or exogenous stresses.

In silico screening identifies a novel small molecule inhibitor that counteracts PARP inhibitor resistance in ovarian cancer.

Poly ADP-ribose polymerase (PARP) inhibitors are promising targeted therapy for epithelial ovarian cancer (EOC) with BRCA mutations or defective homologous recombination (HR) repair. However, reversion of BRCA mutation and restoration of HR repair in EOC lead to PARP inhibitor resistance and reduced clinical efficacy of PARP inhibitors. We have previously shown that triapine, a small molecule inhibitor of ribonucleotide reductase (RNR), impaired HR repair and sensitized HR repair-proficient EOC to PARP inhibitors. In this study, we performed in silico screening of small molecule libraries to identify novel compounds that bind to the triapine-binding pocket on the R2 subunit of RNR and inhibit RNR in EOC cells. Following experimental validation of selected top-ranking in silico hits for inhibition of dNTP and DNA synthesis, we identified, DB4, a putative RNR pocket-binding inhibitor markedly abrogated HR repair and sensitized BRCA-wild-type EOC cells to the PARP inhibitor olaparib. Furthermore, we demonstrated that the combination of DB4 and olaparib deterred the progression of BRCA-wild type EOC xenografts and significantly prolonged the survival time of tumor-bearing mice. Herein we report the discovery of a putative small molecule inhibitor of RNR and HR repair for combination with PARP inhibitors to treat PARP inhibitor-resistant and HR repair-proficient EOC.