Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease
Géraldine Gentric, … , Séverine Celton-Morizur, Chantal Desdouets
Géraldine Gentric, … , Séverine Celton-Morizur, Chantal Desdouets
Published January 26, 2015
Citation Information: J Clin Invest. 2015;125(3):981-992. https://doi.org/10.1172/JCI73957.
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Research Article Hepatology

Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease

Abstract

Polyploidization is one of the most dramatic changes that can occur in the genome. In the liver, physiological polyploidization events occur during both liver development and throughout adult life. Here, we determined that a pathological polyploidization takes place in nonalcoholic fatty liver disease (NAFLD), a widespread hepatic metabolic disorder that is believed to be a risk factor for hepatocellular carcinoma (HCC). In murine models of NAFLD, the parenchyma of fatty livers displayed alterations of the polyploidization process, including the presence of a large proportion of highly polyploid mononuclear cells, which are rarely observed in normal hepatic parenchyma. Biopsies from patients with nonalcoholic steatohepatitis (NASH) revealed the presence of alterations in hepatocyte ploidy compared with tissue from control individuals. Hepatocytes from NAFLD mice revealed that progression through the S/G2 phases of the cell cycle was inefficient. This alteration was associated with activation of a G2/M DNA damage checkpoint, which prevented activation of the cyclin B1/CDK1 complex. Furthermore, we determined that oxidative stress promotes the appearance of highly polyploid cells, and antioxidant-treated NAFLD hepatocytes resumed normal cell division and returned to a physiological state of polyploidy. Collectively, these findings indicate that oxidative stress promotes pathological polyploidization and suggest that this is an early event in NAFLD that may contribute to HCC development.

Authors

Géraldine Gentric, Vanessa Maillet, Valérie Paradis, Dominique Couton, Antoine L’Hermitte, Ganna Panasyuk, Bernard Fromenty, Séverine Celton-Morizur, Chantal Desdouets

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Figure 4

NAFLD hepatocytes preferentially undergo an altered cell cycle.

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NAFLD hepatocytes preferentially undergo an altered cell cycle. Experime...
Experiments were carried out in hepatocytes isolated from WT or ob/ob livers (4 independent cultures). (A) Experimental scheme of culture. T12H, time 12 hours after plating. (B) Immunostaining of primary hepatocytes with anti-BrdU (green) and Hoechst (blue) at 60 hours after plating (scale bar: 20 μm) and quantitative analysis of BrdU labeling (percentage of BrdU+ hepatocytes). Data represent the mean ± SEM. ***P < 0.001, Student’s t test. (C) Double immunostaining of primary hepatocytes at 60 hours after plating, with anti-PHH3 (green) and Hoechst (blue) (scale bar: 20 μm) and quantitative analysis of G2-labeling index (percentage of PHH3+ nuclei). Data represent mean ± SEM. ***P < 0.001, Student’s t test. (D) RNA extracted from WT (black circle) and ob/ob (gray square) primary hepatocytes (n = 5) and Ccna2 and Ccnb1 mRNAs were analyzed by quantitative real-time PCR. *P < 0.05, **P < 0.01, Student’s t test. (E) CCNB1 and phosphorylated CDK1 (Tyr15) protein levels were analyzed in WT and ob/ob cultures during the time-course experiment. γ-Tubulin was used as a loading control. The CCNB1 blot was derived from parallel samples run on a separate gel. The Western blot is from the same experiment as the Figure 6 and Supplemental Figure 4 and is representative of 4 different cultures. Lanes were run on the same gel but were noncontiguous, as indicated by the black line. (F) Soluble chromatin was prepared from culture at 60 hours, and MCM7 was analyzed by Western blotting. Lamin A-C was used as an indicator of fraction purity. Tot, total fraction; Chro, chromatin fraction. The Western blot is representative of 4 different cultures. Lanes were run on the same gel but were noncontiguous.