Absence of REV3L promotes p53-regulated cancer cell metabolism in cisplatin-treated lung carcinoma cells

Abstract

Lung cancer is one of the deadliest cancers in the world because of chemo-resistance to the commonly used cisplatin-based treatments. The use of low fidelity DNA polymerases in the translesional synthesis (TLS) DNA damage response pathway that repairs lesions caused by cisplatin also presents a mutational carcinogenic burden on cells that needs to be regulated by the tumor suppressor protein p53. However, there is much debate over the roles of the reversionless 3-like (REV3L) protein responsible for TLS and p53 in regulating cancer cell metabolism. In this study, the fluorescence lifetime of the metabolic coenzyme NADH reveals that the absence of REV3L can promote the p53-mediated upregulation of oxidative phosphorylation in cisplatin-treated H1299 lung carcinoma cells and increases cancer cell sensitivity to this platinum-based chemotherapy. These results demonstrate a previously unrecognized relationship between p53 and REV3L in cancer cell metabolism and may lead to improvements in chemotherapy treatment plans that reduce cisplatin resistance in lung cancer.

Keywords: Cisplatin; Fluorescence lifetime imaging microscopy (FLIM); Oxidative phosphorylation (oxphos); Reduced nicotinamide adenine dinucleotide (NADH); Reversionless 3-like protein (REV3L); p53.

Figure 1. p53 promotes oxidative phosphorylation

(A) The fluorescence lifetime histogram on the phasor plot shows the distribution of lifetimes of autofluorescent NADH. A color spectrum indicates the bound fraction of NADH. (B) Fluorescence intensity images of GFP of an EGFP cell and p53-GFP cell (left panels). Normalized fluorescence intensity images of NADH (middle panels) and pseudo-colored FLIM images (right panels) of H1299 lung cancer cells transfected with EGFP and p53-GFP. Pseudo-coloring corresponds to the phasor position according to the color spectrum in Figure 1A. Scale bar = 10μm. (C) Bar graphs quantifying the bound fraction of NADH in the nucleus (left) and cytoplasm (right). (D) Percent change in bound fraction of NADH in the nucleus (left) and cytoplasm (right) after treatment with DMSO, rotenone and antimycin A (R+AA), or 2-deoxy-D-glucose and dichloroacetate (DG+DCA). * p<0.05, *** p<0.001, N>7.

Figure 2. REV3L is necessary for metabolic regulation by p53

(A) The fraction of bound NADH in H1299 cells after manipulation of p53 by transfection and REV3L by shRNA. (B) The fraction of bound NADH after transfection of wild type p53-GFP (p53 wt) or p53-GFP with the R175H mutation (p53 mut) and depletion of REV3L by siRNA. (C) The fraction of bound NADH before cisplatin treatment and with 12 hours of continuous exposure to cisplatin in the nucleus (left) and in the cytoplasm (right). * p<0.05, ** p<0.01, *** p<0.001, N>7.

Figure 3. The absence of REV3L increases p53-regulated oxphos following cisplatin treatment

(A) The percent change in the fraction of bound NADH after manipulation of p53 by transfection and REV3L by shRNA with 24 hours of 20μM cisplatin treatment. (B) The percent change in the fraction of bound NADH after transfection of p53-GFP with the R175H mutation and depletion of REV3L by siRNA with 24 hours of 20μM cisplatin treatment. * p<0.05, ** p<0.01, N>7.

Figure 4. Cancer cell sensitivity to cisplatin increases with p53 expression and REV3L depletion

(A) Brightfield images of cells after manipulation of p53 by transfection and REV3L by shRNA with 24 hours of 20μM cisplatin treatment. Scale bar = 10μm. (B) Bar graph of the cell survival rate of H1299 cells. (C) A proposed model for the role of REV3L and p53 in cancer cell sensitivity to cisplatin. * p<0.05, ** p<0.01.